Control of Innate and Adaptive Immune Responses During Infectious Diseases

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Control of Innate and Adaptive Immune Responses during Infectious Diseases

Julio Aliberti Editor

Control of Innate and Adaptive Immune Responses during Infectious Diseases

Editor Julio Aliberti Associate Professor Divisions of Molecular Immunology and Pulmonary Medicine Cincinnati Children’s Hospital Medical Center and School of Medicine University of Cincinnati Cincinnati, OH, USA [email protected]

ISBN 978-1-4614-0483-5 e-ISBN 978-1-4614-0484-2 DOI 10.1007/978-1-4614-0484-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011936972 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identiﬁed as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Upon infection, pathogen and host perform a complex interaction that ultimately aims to achieve elimination of the invading microbe with the least amount of damage to host tissues and organs. Interestingly, both sides of this equation co-evolved several mechanisms that mediate pathogen recognition, initiation and expansion of immune responses, neutralization of toxic elements and elimination of replicating organisms and ﬁnally healing and remodeling of damaged tissues. On one side pathogens evolved mechanisms to evade recognition and killing, while on the other side, host express numerous (sometimes redundant) mechanisms of recognition and elimination of the pathogen. Nonetheless, it is clear that an absolute successful strategy on the pathogen side would be lethal to both host and pathogen. Therefore, several evasion mechanisms are seen among several microbes. The most successful ones are not necessarily the most abundantly found within the host, but those that can achieve transmission. On the other hand, hosts need a robust and extended immune response in order to expand memory cells. This critical balance is where the co-evolution between host and pathogens lies. This book covers several aspects of induction, control and evasion of host immune response during infectious diseases. Multiple aspects are covered and each chapter focuses on one prominent infectious agent. Cincinnati, OH

Julio Aliberti

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Contents

1

Resolution of Inﬂammation During Toxoplasma gondii Infection ........ Julio Aliberti

2

Mechanisms of Host Protection and Pathogen Evasion of Immune Response During Tuberculosis .............................. Andre Baﬁca and Julio Aliberti

3

NKT Cell Activation During (Microbial) Infection ............................... Jochen Mattner

4

Regulation of Innate Immunity During Trypanosoma cruzi Infection ....................................................... Fredy Roberto Salazar Gutierrez

5

B Cell-Mediated Regulation of Immunity During Leishmania Infection ................................................................... Katherine N. Gibson-Corley, Christine A. Petersen, and Douglas E. Jones

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23 39

69

85

6

Control of the Host Response to Histoplasma Capsulatum.................... George S. Deepe, Jr.

99

7

Modulation of T-Cell Mediated Immunity by Cytomegalovirus .......... 121 Chris A. Benedict, Ramon Arens, Andrea Loewendorf, and Edith M. Janssen

8

T Cell Responses During Human Immunodeﬁciency Virus (HIV)-1 Infection ............................................................................ 141 Claire A. Chougnet and Barbara L. Shacklett

Index ................................................................................................................. 171

vii

Contributors

Julio Aliberti, Ph.D. Associate Professor, Divisions of Molecular Immunology and Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center and School of Medicine, University of Cincinnati, Cincinnati, OH, USA [email protected] Ramon Arens Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Andre Baﬁca, M.D., Ph.D. Assistant Professor, Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis, SC, Brazil andre.baﬁ[email protected] Chris A. Benedict Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA [email protected] Claire A. Chougnet Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA [email protected] George S. Deepe. Jr, M.D. Professor, Veterans Affairs Hospital, Cincinnati, OH, USA; Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA [email protected] Katherine N. Gibson-Corley Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Fredy Roberto Salazar Gutierrez, M.D., Ph.D. Assistant Professor, School of Medicine, Antonio Nariño University, Bogotá, Colombia [email protected]

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Contributors

Edith M. Janssen Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH, USA [email protected] Douglas E. Jones Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA [email protected] Andrea Loewendorf Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Jochen Mattner, M.D. Professor of Molecular Microbiology and Infection Immunology, University Hospital of Erlangen, Microbiology Institute – Clinical Microbiology, Immunology and Hygiene, Erlangen, Germany [email protected] Christine A. Petersen Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Barbara L. Shacklett Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA, USA

Chapter 1

Resolution of Inﬂammation During Toxoplasma gondii Infection Julio Aliberti

Abstract Upon Toxoplasma gondii host infection, a powerful immune response takes place in order to contain dissemination of the parasite and prevent mortality. Once parasite proliferation is contained by IFN-J-dependent responses, nevertheless, parasite immune escape prevents complete clearance characterizing the onset of the chronic phase of infection, with a continuous (and powerful) cell-mediated immunity. Such potent responses are kept under tight control by several, non-redundant mechanisms that control pro-inﬂammatory mediators. Including cytokines, such as members of the IL-10 family, TGF-beta, the membrane receptors, ICOS, CTLA4 and a class of anti-inﬂammatory eicosanoids, the lipoxins. In this chapter we address the host strategies that keep pro-inﬂammatory responses under control during chronic disease. On the other hand, we approach the perspective of the pathogen, which pirates the host’s machinery to its own advantage as a part of the pathogen’s immuneescape mechanisms.

1.1

Introduction

Toxoplasmosis is caused by the protozoan parasite, Toxoplasma gondii. The pathogen can be found worldwide and is particularly prevalent in the United States, where it is estimated that more than 60 million people may be infected. Among those who are infected, few develop symptoms due to healthy immune system that usually prevents the parasite from causing illness. Nevertheless, within the high risk group are pregnant women and individuals with compromised immune systems.

J. Aliberti (*) Divisions of Molecular Immunology and Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center and School of Medicine, University of Cincinnati, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_1, © Springer Science+Business Media, LLC 2012

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Ingestion of oocysts

predation

Feces Congenital transmission Ingestion of infected raw meat or water/food contaminated with oocysts

Fig. 1.1 Toxoplasma gondii life cycle. Cats become infected with T. gondii through predation of infected mice or rats. After cysts or oocysts are ingested the organisms are released and spread throughout the small intestine and then form oocysts, which are excreted and can potentially survive for long periods in the environment. Human acquire infection via in several routes: ingestion of infected food containing Toxoplasma cysts; ingestion of oocysts from contaminated hands or food; organ transplantation or blood transfusion from infected humans; transplacental transmission from an infected mother; and accidental inoculation of tachyzoites

Felines, including the house cat are deﬁnitive hosts in which it is observed the sexual stages of T. gondii and thus, are considered to be the main parasite reservoirs. Cats become infected with T. gondii by carnivorism (Fig. 1.1). After tissue cysts or oocysts are ingested, viable organisms are released and invade epithelial cells of the small intestine, where they undergo an asexual cycle followed by a sexual cycle and then form oocysts, which can be excreted. The unsporulated oocyst takes 1–5 days after excretion to sporulate (become infective). Although cats shed oocysts for only 1–2 weeks, large numbers may be shed. Oocysts can survive in the environment for several months and are remarkably resistant to disinfectants, freezing, and drying, but are killed by heating to 70°C for 10 min. The persistency of oocysts in the environment may enhance the infectious potential of the parasite. Humans may acquired T. gondii via different routes (Fig. 1.1): (a) Ingestion of: raw or undercooked and infected meat containing Toxoplasma cysts; oocysts from fecally contaminated hands or food; (b) Organ transplantation or blood transfusion from infected humans; (c) Transplacental transmission from an infected mother; and (d) Accidental inoculation of tachyzoites.

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Toxoplasma gondii, a protozoan apicomplexa parasite is highly virulent and can potentially invade and subsequently replicate within any nucleated host cell. Under natural conditions infection occurs by ingestion of parasite oocyst-contaminated food or water. Oocysts are complex structures formed in the digestive tract of the deﬁnitive host – felines which protect the parasites from heat and dehydration and can remain infective within the environment for long periods of time. Once ingested, oocyst rupture occurs within the host digestive system and the released parasites enter host cells through an active process mediated by the apical complex (Morisaki et al. 1995). Host cells include epithelial cells, resident macrophages and dendritic cells (Fig. 1.1, Life Cycle). Once intracellular, the parasites (tachyzoites) quickly replicate. Although deﬁnitive evidence is still required, it is proposed that circulating infected host cells (probably macrophages or DCs) might mediate spread of the parasite to several organs, including the liver. One current hypothesis proposes that the acute phase of infection resolves when the remaining fast-replicating parasites switch, probably as a response to immune attack, to a slow replicating form known as bradyzoites and seclude themselves in cysts in certain tissues, such as the central nervous system (CNS) and the retina (known as chronic or persistent infection) (Black and Boothroyd 2000). For a long time it was widely accepted that cysts containing bradyzoites were latent, biologically inactive structures that eventually died off or in some cases re-activated parasite replication. Today, however this concept has been challenged as it has been shown that cysts are dynamic structures, where parasites convert to tachyzoites. The conclusion is that this “dripping” effect in which tachyzoites are slowly released, continuously stimulating immune response. Therefore, when immune suppression caused by drugs or other infections, such as HIV, can lead to reversion from bradyzoites back to the fast replicating tachyzoites, which rupture cysts causing local tissue necrosis, thus characterizing the main pathology resulting from this infection. If reactivation occurs in the CNS, it is often lethal. During the early years of the AIDS epidemic, encephalitis due to reactivation of chronic T. gondii infection was one of the most relevant pathologies affecting immuno-depressed patients (Martinez et al. 1995). In nature, the main route for T. gondii transmission is through predation (i.e. felines preying on rodents), therefore an evolutionary advantage would be among pathogens that populate the host and simultaneously provide conditions to protect the host to carry as many parasites without killing it. In other words, this means to proliferate while promoting host survival. To achieve this, the parasite has evolved several mechanisms to induce a powerful immune response by the host, which prevents host death by controlling parasite growth. However, to avoid the potential collateral damage of such powerful pro-inﬂammatory reaction, the pathogen subverts the immune system allowing it to persist through the chronic phase of the disease, which can last for many years (Hay and Hutchison 1983). Herein, we discuss the immune response triggered by T. gondii and how hosts and pathogens make use of immune-regulatory pathways to promote host survival, which increases the probability of parasite transmission.

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1.2 1.2.1

J. Aliberti

Experimental T. gondii Infection Microbial Recognition and IL-12 Induction

A balanced interrelationship between host and parasite is highly dependent on the early induction of immune response after infection. Too much immune response and pathogen is swiftly cleared without causing disease. On the other hand, the absence of a proper timely host response may lead to uncontrolled pathogen replication and spread, often leading to the death of the host. Nevertheless, this is an over-simpliﬁcation of the rather complex scenarios that take place during T. gondii infection. Although signiﬁcant protection is achieved after infection, a relevant proportion of invading parasites evade immune effector mechanisms, i.e. tachyzoites turn infected cells incapable to secrete pro-inﬂammatory mediators (Walker et al. 2008), bradyzoites, hidden within tissue cysts populate immune privileged sites, such as the retina or the CNS. Therefore, T. gondii parasites can persist in the host even in the presence highly powerful immune response. To add further complexity to this interaction, several lines of evidence indicate that without innate immune responses, such as following NK-cell depletion, the initial IFN-J-dependent control of parasite replication is compromised and, in the case of NK-cell-depletion of T-cell-deﬁcient mice, host resistance is lost resulting in host death, which indicates an important role for NK cells in the induction of a response (Sher et al. 1993; Hunter et al. 1994). IL-12 is a cytokine produced during pathogen recognition that is essential to trigger both NK cell as well as T cell-derived IFN-J production during T. gondii infection. The biological relevance of this cytokine was evidenced by the ﬁnding that IL-12-deﬁcient animals are extremely susceptible to T. gondii infection (Gazzinelli et al. 1994). B cells, macrophages, neutrophils and DCs are known to produce IL-12 in vitro and in vivo (Denkers 2003). During T. gondii infection, macrophages, neutrophils and DCs can all produce detectable amounts of IL-12 after T. gondii infection (Denkers 2003). However, DCs – abundant producers of IL-12 in vivo – are the most relevant cell population for the development of a parasite-speciﬁc type 1 immune response. Reis e Sousa and colleagues observed that splenic mouse CD8D+ DCs produce IL-12 in response to T. gondii in the absence of co-stimulatory signals (Reis e Sousa et al. 1997). While macrophages require a cognate priming signal, i.e. IFN-J and neutrophil IL-12 production levels are relatively low when compared to DCs. In summary, DCs can either activate the immune system by recognition of parasitederived molecules or can harbor initial replication of the intracellular parasites. A cellular homogenate from culture-derived tachyzoites (STAg) was used in order to decipher which are the parasite components and their respective host receptors involved in DC IL-12 induction by T. gondii. Such approach seemed feasible since STAg was capable to induce markedly higher levels of IL-12 from in vitro stimulated splenic DCs than when the same cell populations are exposed to several other microbial products. Although the mechanisms underlying such responses are

1 Resolution of Inﬂammation During Toxoplasma gondii Infection

5

not completely understood, one potential interpretation came from studies showing that the chemokine receptor CCR5 plays an important role in the induction of IL-12 synthesis following stimulation with T. gondii STAg (Aliberti et al. 2000). The biological relevance of the unusual requirement for a chemokine receptor to participate in microbial recognition by DCs is supported by the fact that decreased IL-12 production is observed during acute infections in CCR5-deﬁcient animals although defects in cell migration could also contribute to this susceptible phenotype. The phenotype of CCR5-deﬁcient mice infected with T. gondii cannot be solely explained by the IL-12 production defects, other studies indicated that NK cells show defective migration patterns after oral infection, leading to a weaker initial NK-derived IFN-J secretion, resulting in susceptibility to infection (Khan et al. 2006). In summary, it seems clear that CCR5 plays several critical roles for the development of innate immunity after T. gondii infection. Both at the DC IL-12 induction level as well as inducing NK cell migration to infection foci. In the pursuit to identify the ligands with IL-12-inducing activity from T. gondii, a thorough analysis of this activity was performed in fractionated suspensions of secreted parasite proteins. Such analysis identiﬁed cyclophillin-18 (C-18) as one such component. T. gondii C-18 is a secreted prolyl-isomerase that can bind avidly to the immunosuppressant cyclosporine, which was therefore initially pursued as a potential therapeutic target for the treatment of toxoplasmosis (Aliberti et al. 2003b). C-18 was found to bind directly to human and mouse CCR5 with afﬁnities comparable to its prototype ligand, the chemokine CCL4 (MIP-1E). Indeed, C-18 competed with the natural ligand CCL4 for binding to CCR5. It has been shown that endogenous CCR5 ligands can trigger IL-12 production. Nevertheless, the low levels of cytokine secretion observed under these conditions indicate that it may not have a critical inﬂuence on determining resistance to infection. Given that CCR5 is a coreceptor for HIV invasion, further studies showed that toxoplasma C-18 could inhibit the infection of monocytes by CCR5-tropic primary and laboratory HIV isolates (Golding et al. 2003, 2005). However, despite the evident stimulating activity of C-18 triggering IL-12 production by murine DCs, the resulting IL-12 levels observed after stimulation of DCs with C-18 are consistently lower than those seen after stimulation with whole parasite lysate or with a pool of tachyzoite-secreted proteins, indicating that pathways other than those initiated by CCR5/C-18 might also be important for IL-12 production by DCs (Aliberti et al. 2003b). Toll-like receptors (TLRs) have been investigated as a likely candidate for such cytokine induction. Mice deﬁcient for the TLR adaptor protein MyD88 were found to have a pronounced defect in IL-12 production in response to STAg stimulation in vivo and in vitro. Moreover, upon T. gondii infection, MyD88-deﬁcient hosts had high mortality due to a lack of protective IFN-J-mediated immunity (Scanga et al. 2002). Suggesting that the residual IL-12 produced in response to CCR5 was clearly not sufﬁcient to provide any level of protection after infection. Obviating the predominant role of TLR’s in initiating innate IL-12 production in the presence of parasite derived molecules. TLR2 has been found to be involved in the development of resistance to infection with a large inoculum of T. gondii cysts (300 cysts/mouse) (Mun et al. 2003).

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Apparently, the defect seen in TLR2-deﬁcient mice is related to inefﬁcient activation of microbicidal functions, as a defect in nitric oxide production by macrophages was reported, whereas no defect in the production of IL-12 or any other pro-inﬂammatory cytokines, which are typical of innate microbial recognition, was seen (Mun et al. 2003). In an attempt to identify the TLR ligands present in STAg, a CCR5-independent IL-12-inducing activity was puriﬁed from parasite preparations, further analysis indicated the cytoplasmic protein proﬁllin was the key molecule inducing CCR5independent, MyD88-sensitive DC IL-12 production in mice (Yarovinsky et al. 2005). In fact, a thorough evaluation of a panel of TLR-responsive elements in a cell reporter assay allowed for the identiﬁcation of mouse TLR11 as the receptor of proﬁllin (Yarovinsky et al. 2005). In the absence of TLR11, DC’s showed major reduction in IL-12 responses. Interestingly, no residual IL-12 was detected under these conditions, something seen previously with STAg-stimulated MyD88-deﬁcient cells. Although the effects are dominant and TLR11-deﬁciency completely abolishes resistance to infection, some points remain unanswered, including the identity of the human TLR involved in T. gondii recognition – given that the human TLR11 homolog is a pseudogene. Moreover, proﬁllin is not a secreted protein it is released only after tachyzoite rupture, suggesting that it may not necessarily be present at the initial stages where the killing mechanisms are still not active. Taken together, the recognition steps that lead to full IL-12 responses is a rather complex interaction that assures the host to produce vigorous DC-derived IL-12 when in the presence of parasite molecules. Such responses are essential for the development of protective adaptive immunity. The biochemical basis for the induction of the IL-12 genes has been studied extensively (for review see (Trinchieri 2003)). However, the transcription factors that are directly involved in IL-12 induction during in vivo infection with intracellular parasites, including T. gondii it is still not completely elucidated. IRF-8deﬁcient mice cannot produce IL-12 during infection with T. gondii and fail to develop resistance to infection (Scharton-Kersten et al. 1997). This observation would directly implicate IRF-8, an interferon-inducible transcription factor that binds to interferon consensus sequences and promotes gene transcription, in the induction of IL-12 gene expression, but recent reports have pointed out that besides their IL-12-induction defect, IRF-8-deﬁcient mice fail to develop the major DC subset involved in IL-12 production in response to STAg stimulation, the CD8D+ subset (Aliberti et al. 2003a; Tsujimura et al. 2003). Furthermore, the remaining DC subsets also had severe defects in response to microbial stimuli. Further studies are required to clarify whether the defect in IL-12 production in these mice precludes the DC developmental defect. NF-kB family members have been studied during T. gondii infection models, it is clear that NF-kB activation is a required step for the development of protective immune response to infection (Tato et al. 2006). However, the developmental abnormalities seen in animals genetically deﬁcient of NF-kB make dendritic cell speciﬁc responses rather complex due to environmental and developmental deﬁciencies. It has been reported that p38 MAP kinases are required for macrophage IL-12 responses to STAg stimulation (Mason et al. 2004). On the other hand, JNK family

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of MAP kinases while have been associated with induction of IL-12 by some (Sukhumavasi et al. 2007), while other studies indicate that the play a negative, inhibitory role (Sukhumavasi et al. 2010). A more comprehensive analysis of this enzyme activity, targets and function remains to be reported. The unusual combination of receptors triggered by T. gondii molecules, i.e. CCR5 and TLR11 suggests that the transcriptional machinery might be unique, which could explain, at least in part, the unusually high IL-12 levels seen after exposure of DCs to STAg. An interesting paradox has been unveiled while studying cytokine responses in infected cells. In other words, both dendritic cells and macrophages failed to produce signiﬁcant levels of IL-12 when exposed to live tachyzoites (Butcher et al. 2001). Such studies led to the hypothesis that dendritic cells may serve as a parasite shuttle during in vivo infection, providing protection while migrating throughout the host (Denkers and Butcher 2005). This set of reports support a scenario in which microbial recognition takes place through released proteins from live free-ﬂoating parasites or from infected cells. The mechanistic that leads to inhibition of infected cell responsiveness to microbial stimulation is still not clear, but it has been shown that intracellular parasites disrupt the cytoskeleton of the host cells, specially membrane proximal structures. It is possible that such disruption decouples the biochemical signaling apparatus that is associated with the recognition receptors. Another possibility is that infected cells produce an autocrine suppressive factor, although this possibility has not been fully investigated.

1.2.2

IFN-g, Th1 Cells and Microbicidal Activity

Once present in the infected host, IL-12 activates NK cells to produce IFN-J and drives the proliferation of type 1 CD4+ and CD8+ T cells, which produce even more IFN-J. IFN-J-producing cells is a central component to induce and maintain control of parasite proliferation and dissemination during both acute and chronic infection (Yap and Sher 1999). Several factors are driven by IFN-J activation that have been to shown to be involved in controlling intracellular parasite growth. Macrophages harboring intracellular parasites and activated with IFN-J can produce nitric oxide, which, in turn, is responsible for microbicidal/microbiostatic control of intracellular parasite growth. Intriguingly, even though IFN-J-induced microbicidal mechanisms are potent, the machinery is not 100% effective at eliminating parasites (Yap and Sher 1999). During the chronic phase, some parasites evade immunity and survive within the host for long periods, despite the continuing survey of the immune system in search of released parasites. Interestingly, among the genetic clusters upon which the strains of T. gondii are grouped, some present extremely high virulence, leading to rodent host death prior to opportunity for viable transmission. Such parasite strains have shown weaker innate immune activating properties. Suggesting that for the lack of some evolutionary pressure, those strains present little to no adaptation to mouse hosts.

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With the onset of the chronic phase parasites make use of two major mechanisms to evade immune responses: 1. Parasites become less susceptible to host microbicidal activity; and/or, 2. Parasites induce immunosuppressive factors that dampen immune effector activity, including the production of pro-inﬂammatory mediators. As an example of an evasion of immune response mechanism, it is well-known that a several microbes escape effector immunity through the actions of membrane receptors or cytoplasmic enzymes that inactivate or neutralize effector molecules, including a complement factors (Karp and Wills-Karp 2001), superoxide or nitric oxide. As an example, during T. gondii infection, complement receptors are activated inducing expression of peroxiredoxins (Son et al. 2001). It seems obvious to speculate that those evasion strategies may increase the frequency of persisting parasites within the host, despite ongoing potent immunity. As an alternative, modulatory or anti-inﬂammatory factors could be selectively enhanced by the pathogen leading to inhibition of the migration, proliferation or differentiation of effector cells at the infection foci, favoring the evasion of the pathogen from protective immunity and progression towards the development of chronic disease.

1.3

1.3.1

Pro-resolution Strategies as a Mechanism to Prevent Immunopathology Resolution Phase of the Inﬂammatory Response

In general, protective pro-inﬂammatory response ultimately clears the tissues of both the cause and consequences of tissue injury that can accompany host defense (Cotran and Pober 1990). If unresolved, acute inﬂammation may lead to chronic inﬂammation, scarring and eventual loss of function (Majno and Joris 1995). A growing list of reports indicated that, in addition to classic diseases associated with inﬂammation, for example psoriasis, periodontal disease and arthritis, uncontrolled inﬂammation governs the pathogenesis of many widely prevalent diseases including infectious, cardiovascular and cerebrovascular disease, cancer, obesity and Alzheimer’s disease (Libby 2002; Calder 2006) (Van Dyke and Serhan 2003). Prostaglandins and leukotrienes are initially produced locally at the inﬂammation site and are key in promoting the cardinal signs of inﬂammation. Of interest, another class of arachidonic acid-derived mediators, the lipoxins (LXs) and aspirin-triggered lipoxins (ATLs), are mediators recently recognized to perform both endogenous anti-inﬂammatory and pro-resolving actions (Serhan 2005, 2007). In the recent years, multiple previously unknown enzymatic pathways were identiﬁed to be present during the resolution phase. Those are derived from the precursors EPA and DHA. Both EPA and DHA are major n-3 fatty acids also widely known as the omega-3 PUFA or ﬁsh oils. The new mediators are biosynthesized during the

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evolution of locally contained inﬂammatory exudates. They possess potent actions in controlling the resolution (Serhan et al. 2000, 2002; Hong et al. 2003). Resolvins are endogenous, local-acting mediators that carry potent anti-inﬂammatory and immunoregulatory signals (Serhan et al. 2002). These include novel actions that are targeted to promote resolution, namely reducing neutrophil inﬁltration and regulating the cytokine-chemokine axis and reactive oxygen species and stimulating the uptake and clearance of apoptotic PMN as well as lowering the magnitude of the inﬂammatory response and associated pain (Serhan et al. 2000, 2002; Svensson et al. 2007). Protectins are biosynthesized in many organs and perform potent anti-inﬂammatory (Hong et al. 2003) as well as protective actions demonstrated for the novel and potent DHA-derived 10,17-docasatriene in animal models of stroke (Marcheselli et al. 2003) and Alzheimer’s disease (Lukiw et al. 2005). Both families, the resolvins and protectins, are potent local-acting agonists of endogenous anti-inﬂammation and promote resolution speciﬁc processes (Serhan 2007). IFN-J-dependent immune and its derived pro-inﬂammatory responses are potentially extremely toxic to the host. As an example, during chronic inﬂammatory diseases such as arthritis or Crohns’ disease, sustained or uncontrolled type 1 cytokine responses has been shown to cause serious damage to host tissues and organs. In order to prevent that potential damage, several host mediators and receptors have evolved to counter host-damaging responses. The homeostasis of the immune response is absolutely dependent on the presence of such counter regulatory pathways. This complex network of anti-inﬂammatory pathways, given its actions, has been seized by pathogens and used to their own beneﬁt to prevent parasite eradication.

1.3.2

Interleukin-10

IL-10 is one of the most biologically active cytokine with anti-inﬂammatory properties besides TGF-E and IL-35. It can be produced by a growing list of activated immune cells, in particular monocytes/macrophages and T cell subsets including Tr1, Treg, and Th1 cells. It acts via activation of a transmembrane receptor complex, which is composed of IL-10R1 and IL-10R2, and controls the activities of several immune cells. In monocytes/macrophages, IL-10 inhibits the production of proinﬂammatory mediators and antigen processing and presentation. Furthermore, IL-10 plays a relevant role in the differentiation/proliferation of B and T cells. In general, its physiological relevance lies in the preventing over-whelming immune responses and, consequently, of tissue damage. Simultaneously, IL-10 enhances the “scavenger”-like activities. To highlight its relevance in inhibiting immune responses during infectious diseases, IL-10 is used by pathogens to evade the development of protective immunity. Viruses, such as EBV, encode a viral IL-10-homologue that can initiate the signaling cascade as the one triggered by the mammalian cytokine (Salek-Ardakani et al. 2002). Poxviruses carry genes that encode IL-10 receptor homologues; therefore, cells expressing such receptors when in the presence of IL-10 become refractory to

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IL-12 NK

tachyzoite

IFN-

DC 4

C-18

8

NO M

IFN-

TNF

Fig. 1.2 Induction of pro-inﬂammatory responses during T. gondii infection. Immediately after infection, host dendritic cells (DCs) produce IL-12 in response to products secreted by T. gondii, via the CCR5-binding cyclophillin-18 and the TLR11-activating proﬁllin. IL-12 promotes the differentiation and proliferation of type 1, IFN-J-producing T cells (CD4+ and CD8+) and NK cells. In turn, IFN-J triggers host cells, including macrophages (MM) to exert microbicidal activity, such as the production of nitric oxide, expression IDO and tryptophan depletion or autophagy

pro-inﬂammatory signals (Haig 1998). Moreover, IL-10 gene transfer has been shown to have anti-inﬂammatory actions in various pathologies associated with increased IFN-J, IL-1 or TNF production (van de Loo and van den Berg 2002, Wille et al. 2001). Importantly, neutralization of IL-10 during chronic toxoplasmic encephalitis leads to increased leukocyte inﬁltration in the CNS, indicating a role for this cytokine in controlling CNS inﬂammation (Deckert-Schluter et al. 1997). IL-10-deﬁcient mice show uncontrolled hyper-inﬂammatory reaction and fail to transition to chronic disease succumbing to T. gondii infection in the earlier to mid-acute phase. The animals show severe inﬁltration of leukocytes and hepatic necrotic lesions in the liver as well as focal necrosis in small intestines, concomitant to high levels of IFN-J and TNF production (Suzuki et al. 2000, Gazzinelli et al. 1996) (Fig. 1.2). IL-10 was hypothesized to mediate immune evasion during T. gondii infection, given its immune modulatory activities. Conversely, IL-10 has not been found to be directly related to the elements that potentially contribute to the mechanisms of T. gondii virulence. Furthermore, the over-production of IL-10 has been shown to perform no clear role in the pathways involved in driving persistence of T. gondii in the chronic stage (Wille et al. 2001). In summary, it is clear that, besides IL-10, several other mechanisms of immune modulation/evasion used by the parasite and by the host are relevant to allow mutual survival (host and pathogen) for enough time to permit successful transmission and, ultimately for parasite survival as a species (Fig. 1.3).

1.3.3

TGF-b

TGF-E, alongside with IL-10, is one of the most relevant cytokines that control effector function of the immune system. It is one of the main soluble factors released by regulatory T cells and plays an essential role in providing a suppressive environment that is essential for homeostasis of the mucosal surfaces, including the gut.

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11

M

IL-10

DC C-18

IL-12 4 8

IFN-

NO M TNF

Fig. 1.3 Central role of IL-10 in controlling pro-inﬂammatory responses during acute phase of T. gondii infection. The potential cytotoxic effects of such activity are controlled, during the midto-late acute phase, by IL-10 production at sites of high-level parasite replication, such as the liver and spleen. IL-10 down-regulates pro-inﬂammatory cytokine and chemokine expression, as well as the microbicidal activities of DCs, T cells, NK cells and macrophages

During the interaction with macrophages, T. gondii tachyzoites expose phosphatidyl serine leading to the release of active TGF-E by infected macrophages (Seabra et al. 2004). TGF-E is a well-known macrophage deactivator, including inhibition of NOS2 expression. Neutralization of TGF-E abolished the inhibition of NO production, thus reducing the persistence of intracellular T. gondii in activated macrophages. Furthermore, the up-regulation of Smad 2 and 3 in infected macrophages conﬁrms that a TGF-E autocrine effect was caused by the T. gondii infection. It is clear that TGF-E is present during host/parasite interaction and that affects key aspects of host immune protective responses. The peroral route T. gondii infection – an experimental model for the mucosal host/pathogen interation that is characterized by ileitis mediated both by parasitemediated tissue destruction as well as by the host immune responses. Buzoni-Gatel et al. 2001 showed that intraepithelial lymphocytes present in the gut mucosa are the main producers of TGF-E (Butcher et al. 2001). CD8+ T cells differentiate into TGF-E-producing cells. The presence of this cytokine inhibits CD4+ T lymphocyte inﬁltration, macrophage activation and tissue destruction mediated by excessive IFN-J production (Mennechet et al. 2004). Consequently, the hallmarks of TGF-E exposure are indeed present in lamina propria-resident CD4+ T cells, the up-regulation of Smad2 and Smad3 (Fig. 1.4).

1.3.4

IL-22

IL-22 is a member of the IL-10 cytokine family and signals through a heterodimeric receptor composed of the common IL-10R2 subunit and the IL-22R subunit. IL-10 and IL-22 both activate the STAT3 signaling pathway. Unlike IL-10, which is produced by a variety of cell types, IL-22 is made by only a subset of activated immune cells, preferentially by Th17 T cells, but also by Th1 cells and conventional

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b

a

c IL-22

NO

M

8

TGF-

Gut Lumen

Th17

Gut Lumen

TGF4

Fig. 1.4 The role of TGF-E and IL-22 in controlling gut mucosal immune responses after oral infection with T. gondii. (a) During the interaction with macrophages, T. gondii tachyzoites expose phosphatidyl serine leading to the release of active TGF-E by infected macrophages. (b) Gut mucosa intraepithelial lymphocytes produce TGF-E that inhibits CD4+ T lymphocyte inﬁltration, macrophage activation and tissue destruction mediated by excessive IFN-J production. (c) IL-22 is made preferentially by Th17 T cells. The receptor for IL-22 is distinct and consists of IL-22R, which is expressed by epithelial cells and the ubiquitous IL-10R2. IL-22 mediates protection against mucosal damage during oral infection with T. gondii

NK cells, JG CD3+ T cells, noncytolytic NK cells, lymphoid tissue inducer cells, and skin-homing IL-13+ T cells. The receptor for IL-22 is distinct and consists of IL-22R, which most studies indicate is restricted to the surfaces of epithelial cells, keratinocytes, and some ﬁbroblasts, and IL-10R2, which is ubiquitous. In fact, T. gondii oral infection triggers IL-22-mediated protection of the gut mucosal surfaces. Suggesting an additional mediator that provides an immune suppressive environment to control gut inﬂammation (Wilson et al. 2010).

1.3.5

IL-27

IL-27 is a heterodimeric cytokine composed of Epstein-Barr virus–induced gene 3 (EBI3) and p28. It is known to signal through a receptor complex composed of the IL-27 receptor and gp130. Expression of IL-27R is conﬁned to immune cells, its partner gp130, a shared receptor component of several cytokines including IL-6, is widely expressed both in and out of the immune system. While IL-27 was initially known to promote T cell proliferation and the development of TH1 responses; it was subsequently indicated that it could suppress TH1 and TH2 responses during various parasitic infections. In agreement with this observation, IL-27R-deﬁcient mice develop exaggerated T helper cell responses during the acute stages of toxoplasmosis, Chagas disease and leishmaniasis and after helminth challenge. Moreover, it was found that IL-27R-deﬁcient mice develop exuberant CD4 + T cell responses in the CNS during chronic toxoplasmosis, in which the majority of the cells are expressing the pro-inﬂammatory cytokine IL-17 (Stumhofer et al. 2006). In fact, IL-27 could inhibit in vitro differentiation of naïve T cells into the Th17 subset. Thus, unveiling a new mechanism to control inﬂammation in a very sensitive environment, the central nervous system (Fig. 1.5).

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4

IL-27 4

IL-6 TGFTh17

8 Fig. 1.5 IL-27 and suppression of immune pathogenic cells in the central nervous system during chronic toxoplasmosis. Naive T cells suppressed the development Th17 cells mediated by IL-6 + TGF-E after exposure to IL-27

1.3.6

Lipoxin A4

“DC paralysis” was a phenomenon in which protection against T. gondii infection was conferred to IL-10-deﬁcient mice by injection with STAg 24 h before T. gondii challenge, which downregulated IL-12 production by DCs (Reis e Sousa et al. 1999). The injection of STAg was found to trigger the endogenous release of an eicosanoid known as lipoxin A4 (LXA4). This mediator inhibited STAg-induced DC migration and IL-12 production in vivo and in vitro (Aliberti et al. 2002a). Lipoxins have been show to have potent anti-inﬂammatory properties in several disease models (Samuelsson 1991; Goh et al. 2003; Van Dyke and Serhan 2003; Kieran et al. 2004). Their actions include inhibition of leukotriene function, NK-cell function, leukocyte migration, TNF-induced chemokine production, NF-NB translocation, and chemokine receptor and adhesion molecule expression (Ramstedt et al. 1985; Clish et al. 1999; Hachicha et al. 1999; Bandeira-Melo et al. 2000; Ohira et al. 2004). Lipoxins are known to bind to two main receptors — a seven-transmembrane G-protein coupled receptor, ALX/FPRL-1 (Maddox et al. 1997), and a nuclear receptor, AhR (Schaldach et al. 1999). Evidence indicating that ALX is mediating some, if not all, of the anti-inﬂammatory actions of lipoxins in vivo came from observations reporting that mice over-expressing human ALX have shorter and less severe inﬂammatory responses (Devchand et al. 2003). Despite intense investigation, it is not yet clear which of the two receptors are most important for the triggering of lipoxin-derived anti-inﬂammatory responses. There is evidence of a role for suppressors of cytokine signaling (SOCS) molecules in the induction of the antiinﬂammatory effects seen after lipoxin exposure (Leonard et al. 2002). The SOCSfamily proteins, SOCS-1, -2 and -3, are thought to mediate their actions by binding to the intracellular domains of cytokine or hormone receptors, thereby blocking activation of downstream signaling pathways (Alexander and Hilton 2004). On the other hand, these proteins may act as part of a ubiquitin ligase molecular complex that lead to proteasome-dependent degradation of transcription factors via their poly-ubiquitinylation (Kile et al. 2002; Alexander and Hilton 2004). The molecular basis for lipoxin-induced SOCS expression and the control of pro-inﬂammatory responses is poorly understood (Fig. 1.6).

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M LXA4

DC C-18

IL-12

4

8

TNF M

IFN-

NO

Fig. 1.6 Lipoxins and control of pro-inﬂammatory cytokine production during chronic toxoplasmosis. With the onset of chronic disease, LXA4 is produced and controls pro-inﬂammatory cytokine responses, mostly at sites where parasite replication might be occurring, such as the CNS, without interfering with the microbicidal activity of macrophages

The biosynthetic cascades for the generation of lipoxin involve several complex trans-cellular pathways and therefore, it is unlikely to be only one cellular source for this mediator. Nevertheless, production of LXA4 seen upon STAg stimulation is completely dependent of 5-lipoxygenase, indicating that the biosynthetic pathways involving this enzyme were crucial in this experimental setting for the production of LXA4 (Aliberti et al. 2002b). 5-lipoxygenase is produced as a pro-peptide that is activated by cleavage, however, low levels of active 5-lipoxygenase are found in different cell types, including macrophages, platelets, DCs and neutrophils (Funk et al. 2002). The expression of a 5-lipoxygenase-activating protein (FLAP) seems to be the key signal for induction of 5-lipoxygenase activity. Although, at the moment, it is not completely clear which cells are the source of lipoxygenase activity in vivo during T. gondii infection, it is evident that 5-lipoxygenase is required for biosynthesis of LXA4. During T. gondii infection, serum levels of LXA4 were found to steadily increase over most of the acute phase, and plateau at high levels through chronic disease (Aliberti et al. 2002b). 5-lipoxygenase-deﬁcient animals succumbed to T. gondii infection at the early onset of chronic disease. Immune responses against the parasite were found to be increased in the absence of 5-lipoxygenase, with signiﬁcantly less brain cyst formation than in control animals. By contrast, excessive pro-inﬂammatory cytokine secretion and massive brain inﬂammatory cell inﬁltration was found. The excessive pro-inﬂammatory response in the brain ultimately caused the death of the 5-lipoxygenase-deﬁcient hosts (Aliberti et al. 2002b).

1.3.7

Redundancy and Control of Inﬂammation

IL-10, IL-27, TGF-E and lipoxins share several biological functions in terms of controlling inﬂammation. Although it is tempting to suggest that they might play redundant roles, in vitro and in vivo evidence suggest otherwise. For example, the treatment of T. gondii-infected 5-lipoxygenase-deﬁcient mice with IL-10 was

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able to control some of the inﬂammation, but concomitant reactivation of parasite proliferation resulted in failure to rescue animals from mortality (Aliberti et al. 2002b). In fact, it was also observed that IL-10 but not LXA4, effectively inhibited the microbicidal activity of macrophages (Aliberti et al. 2002b). Another interesting apparent discrepancy between IL-10 and LXA4 biological actions was associated with the pathological ﬁndings seen during T. gondii infections of IL-10- versus 5-lipoxygenase-deﬁcient mice. While the former showed generalized lymphocytic inﬁltration and massive hepatic necrosis after infection, with little to no inﬂammation in the brain; both liver and CNS inﬁltration was observed during infection of 5-lipoxygenase-deﬁcient mice, indicating that these two anti-inﬂammatory mediators are released in a time and organ controlled fashion and play differentiated intracellular inhibitory pathways. The biochemical pathways involved in inhibition of pro-inﬂammatory responses during infection also support the concept that regulatory mechanisms follow distinct intracellular targets. On one hand, while lipoxins trigger expression of the regulatory protein SOCS2, IL-10 mediate their inhibition via up-regulation of SOCS1/ SOCS3 and IL-27 do so via up-regulation of STAT3 (Machado et al. 2006). TGF-E, on the other hand, seems to mediate its functions via Smad proteins, including Smad2 and Smad3. It is likely that the particular aspects associated with each regulatory mediator, i.e. induction of tolerance, apoptosis, suppression of chemotaxis, can be traced back to their respective biochemical signature.

1.3.8

Induction of Endogenous LXA4 as an Pathogen Evasion Pathway

Several lines of evidence show that the anti-inﬂammatory actions of lipid mediators are used by pathogens, including fungi and helminths. The modulation of host immune responses is the desired effect. The 5-lipoxygenase activity after T. gondii infection was known to be associated with splenic macrophages (Aliberti et al. 2002b), the 15-lipoxygenase-expressing cell population was not known. In order to identify the cell populations involved in mediating 15-lipoxygenase activity after T. gondii infection, Bannenberg and colleagues isolated an enzymatic activity in tachyzoites exposed to calcium ionophore in the presence of arachidonic acid in vitro (Bannenberg et al. 2004). Moreover, proteomics analysis of tachyzoitederived lysates revealed the presence of peptides homologous to plant-derived type 1 lipoxygenases (Bannenberg et al. 2004). Therefore, it seems that the induction of lipoxin biosynthesis by T. gondii has been selected through the carrying of a plant-like lipoxygenase gene, which together with the actions of host-derived 5-lipoxygenase results in lipoxin production. High levels of lipoxin, subsequently, suppress immune responses providing hosts the ability to control parasite proliferation without suffering the damaging consequences of exuberant inﬂammation or tissue necrosis. The molecular basis for 5-lipoxygenase induction after parasite stimulation has not been clariﬁed, it is known that this enzyme can be induced after leukocyte exposure to a

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General pathways for LXA4and Resolvins biosynthesis DHA AA

Pathogen-dependent LXA4 biosynthetic pathway

17S -Resolvins

HOOC COOH

COOH

(O)OH

5-LO

AA

OH

COOH

HO OH

5-LO COOH

COOH

12-LO

OOH OOH

COOH

O(O)H

15-LO

OH OH HO

COOH

LXA4

OOH

15-LO OOH

COOH

O(O)H

LXA4 OH OH

COOH

HO

Fig. 1.7 General and pathogen-dependent LXA4 biosynthetic pathways. (a) General pathways for LXA4 biosynthesis. Arachidonic acid (AA), which is released in response to inﬂammatory stimuli, is catalysed by 5-lipoxygenase (LO) to generate LTA4. This compound, secreted by leukocytes, is captured by neighboring platelets or endothelial cells, and, through the actions of 12- or 15-LO, respectively, is converted to LXA4. (b) Pathogen-dependent LXA4 biosynthetic pathway. After the generation of LTA4 in a 5-LO-dependent manner, it is catalyzed by pathogen-secreted 15-LO into LXA4, which is then secreted by the infected cell

variety of stimuli, including PGE2 (Levy et al. 2001). The interplay between these mediators, the induction of 5-lipoxygenase and the control of immune responses in vivo await further investigation. Another interesting observation that supports the argument for a role of T. gondii 15-lipoxygenase in immune evasion is the presence of such enzymatic activity in an organism that does not have lipids that could serve as substrates for lipoxygenases. Therefore, the substrate has to come from infected host cells. Pseudomonas aeruginosa 15-lipoxygenase-like enzyme is another example of a pathogen carrying an enzyme whose substrate is only present in host cells (Vance et al. 2004). P. aeruginosa is most commonly associated with chronic lung infections in patients with cystic ﬁbrosis. It is possible that the bacteria may use the 15-lipoxygenase pathway leading to lipoxin biosynthesis to promote suppression of inﬂammation and persist throughout chronic disease. However, patients with cystic ﬁbrosis fail to generate lipoxins in the lungs and the continuing proliferation of bacteria results in uncontrolled accumulation of activated neutrophils that ultimately lead to serious tissue damage with organ failure (Karp et al. 2004). This constitutes the major pathology for the lung form of cystic ﬁbrosis. The relevance of pathogenderived 15-lipoxygenase given the lack of lipoxin generation in the lungs of patients with cystic ﬁbrosis and the severity of disease still remains to be elucidated (Fig. 1.7). Mycobacterium tuberculosis is another example of a lung-invading bacterium that leads to a chronic disease, human tuberculosis is among the top infectious diseases worldwide with enormous public health relevance. M. tuberculosis infection is usually asymptomatic, granulomatous reaction in the lungs contain the bacilli and

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prevents its spreading, and a readily-detectable cell-mediated immunity is usually found in exposed patients (Chan and Flynn 2004). However, mycobacterial growth increases and transmission of viable bacilli occurs, along with granuloma disruption and organ function is compromised whenever the immunological status of the hosts is suppressed (Flynn and Chan 2003). Initial pulmonary colonization by M. tuberculosis is a latent process with very little reaction occurring in the organ. With the pathogen slowly establishing into the organ and with almost no intervention from the host innate immune system (Flynn and Chan 2003). Interestingly, it has been shown that in the absence of endogenously generated LXA4, mice become more resistant to infection, with longer survival rates, lower bacterial counts and higher type 1 cellmediated immunity against the bacilli (Baﬁca et al. 2005). The mechanisms involved in control of the immune response during tuberculosis is approached in detail in another chapter of this book. When comparing the outcomes of infections by T. gondii versus M. tuberculosis in 5-lipoxygenase-deﬁcient animals a clear discrepancy becomes evident. This indicates a protective versus a host detrimental role for endogenously produced lipoxins, respectively. While T. gondii – a fast-replicating pathogen – that depends on keeping the host alive so that transmission can occur through predation, M. tuberculosis – slow growing, silent pathogen – requires high proliferation rates in lungs of infected hosts for transmission to occur. Nevertheless, both cases indicate that lipoxindependent inhibition of pro-inﬂammatory type 1 responses provides a favorable environment for pathogen transmission. Therefore, both the host and the pathogen rely on driving a well-balanced immune response.

1.4

Conclusions

In summary, mechanisms that contain the breadth, intensity and duration of proinﬂammatory functions of the immune system play apparent redundant roles. Nevertheless, animal knockout models indicate otherwise. A large body of evidence clearly establishes that TGF-E is a key player in modulating immune speciﬁc responses in the gut mucosal surfaces during oral infection with T. gondii. Another organ highly sensitive to the presence of inﬂammatory reactions is the brain, targeted by the parasite during chronic infection. In this case, evidence point to the protective effects of IL-27 by inhibiting the differentiation of pathogenic Th17 cells in the nervous tissue. While these mediators seem to play a localized role, several reports indicate that IL-10 plays a systemic role, with animals lacking IL-10 or its receptor showing widespread leukocyte inﬁltration, liver necrosis and mortality after T. gondii infection. Although the presence of lipoxins in the serum is preceded by IL-10, there is no evidence that the two mediators cross-regulate each other. Moreover, their anti-inﬂammatory actions are overlapping but not redundant. The emerging body of evidence including lipoxins as immune-regulatory mediators, and the potential use of their inhibitory effects for pathogen survival and replication, is still a poorly understood area of research. Relevant questions including the nature of

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the pathogen-derived signal (s) that induces lipoxin production, or whether the anti-inﬂammatory actions of lipoxins play a role in modulating the balance between Th1, Th2, Th17 and regulatory T cell responses await to be answered. And, in addition, the use of the lipoxygenase system and its mediators by pathogenic microbes as a general mechanism for evasion and manipulation of immune responses as well as pathogen persistence. The further investigation on the roles played by all of these mediators, their cellular sources, biochemical and cellular targets may provide the basis the development of novel therapeutic intervention strategies to enhance weak or inhibit undesirable pro-inﬂammatory immune responses in vivo. Acknowledgements Julio Aliberti is funded by grants from NIH (AI075038 and AI078969).

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Serhan, C. N. (2007). “Resolution phase of inﬂammation: novel endogenous anti-inﬂammatory and proresolving lipid mediators and pathways.” Annu Rev Immunol 25: 101–37. Serhan, C. N., C. B. Clish, et al. (2000). “Novel functional sets of lipid-derived mediators with antiinﬂammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinﬂammatory drugs and transcellular processing.” J Exp Med 192(8): 1197–204. Serhan, C. N., S. Hong, et al. (2002). “Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinﬂammation signals.” J Exp Med 196(8): 1025–37. Sher, A., I. P. Oswald, et al. (1993). “Toxoplasma gondii induces a T-independent IFN-gamma response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-alpha.” J Immunol 150(9): 3982–9. Son, E. S., K. J. Song, et al. (2001). “Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii.” Korean J Parasitol 39(2): 133–41. Stumhofer, J. S., A. Laurence, et al. (2006). “Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inﬂammation of the central nervous system.” Nat Immunol 7(9): 937–45. Sukhumavasi, W., C. E. Egan, et al. (2007). “Mouse neutrophils require JNK2 MAPK for Toxoplasma gondii-induced IL-12p40 and CCL2/MCP-1 release.” J Immunol 179(6): 3570–7. Sukhumavasi, W., A. L. Warren, et al. (2010) “Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii.” Exp Parasitol 126(3): 415–20 Suzuki, Y., A. Sher, et al. (2000). “IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii.” J Immunol 164(10): 5375–82. Svensson, C. I., M. Zattoni, et al. (2007). “Lipoxins and aspirin-triggered lipoxin inhibit inﬂammatory pain processing.” J Exp Med 204(2): 245–52. Tato, C. M., N. Mason, et al. (2006). “Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma.” Int Immunol 18(4): 505–13. Trinchieri, G. (2003). “Interleukin-12 and the regulation of innate resistance and adaptive immunity.” Nat Rev Immunol 3(2): 133–46. Tsujimura, H., T. Tamura, et al. (2003). “ICSBP/IRF-8 retrovirus transduction rescues dendritic cell development in vitro.” Blood 101(3): 961–9. van de Loo, F. A. and W. B. van den Berg (2002). “Gene therapy for rheumatoid arthritis. Lessons from animal models, including studies on interleukin-4, interleukin-10, and interleukin-1 receptor antagonist as potential disease modulators.” Rheum Dis Clin North Am 28(1): 127–49. Van Dyke, T. E. and C. N. Serhan (2003). “Resolution of inﬂammation: a new paradigm for the pathogenesis of periodontal diseases.” J Dent Res 82(2): 82–90. Vance, R. E., S. Hong, et al. (2004). “The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase.” Proc Natl Acad Sci USA 101(7): 2135–9. Walker, M. E., E. E. Hjort, et al. (2008). “Toxoplasma gondii actively remodels the microtubule network in host cells.” Microbes Infect 10(14-15): 1440–9. Wille, U., E. N. Villegas, et al. (2001). “Interleukin-10 does not contribute to the pathogenesis of a virulent strain of Toxoplasma gondii.” Parasite Immunol 23(6): 291–6. Wilson, M. S., C. G. Feng, et al. (2010) “Redundant and pathogenic roles for IL-22 in mycobacterial, protozoan, and helminth infections.” J Immunol 184(8): 4378–90. Yap, G. S. and A. Sher (1999). “Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function.” Immunobiology 201(2): 240–7. Yarovinsky, F., D. Zhang, et al. (2005). “TLR11 activation of dendritic cells by a protozoan proﬁlinlike protein.” Science 308(5728): 1626–9.

Chapter 2

Mechanisms of Host Protection and Pathogen Evasion of Immune Response During Tuberculosis Andre Baﬁca and Julio Aliberti

Abstract An integrated response of the host is essential in health and disease. Upon microbial exposure, infected hosts strictly regulate immune responses to both contain pathogen dissemination and modulate immunopathology-associated effects, thus preventing mortality. In addition to a variety of molecules, such potent responses are kept under tight control by a class of anti-inﬂammatory eicosanoids, the lipoxins. Lipoxins are induced following exposure to several infectious agents and can function as immuno-modulatory molecules. A number of observations made in animal models of infection and human studies indicate that such lipid mediators play a critical role in controlling early as well as chronic immune responses. This chapter summarizes the role of cytokines and lipoxins in regulating innate immune responses to a major human pathogen, Mycobaterium tuberculosis.

2.1

Introduction

Despite more than 100 years of research, tuberculosis is still the most important bacterial infection worldwide. The discovery of Mycobacterium tuberculosis as the causative agent of TB was announced by Robert Koch in 1882 (Koch 1891). In his lecture Koch, who received the 1905 Nobel Prize for his discoveries, reminded his audience that one in seven human beings died of tuberculosis. Every year, it is estimated that tuberculosis causes around 1.5 million mortalities with 8 million new cases reported (Barber et al. 2009). In general, most infections are controlled by the host’s immune system, leading to latency with persistent/dormant bacteria. The World Health Organization estimates that one-third of the world population

A. Baﬁca (*) Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis, SC, Brazil e-mail: andre.baﬁ[email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_2, © Springer Science+Business Media, LLC 2012

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carries the bacilli however only 10% of them will develop clinical disease. Acquired immunodeﬁciency syndrome (AIDS) and other immune-compromising conditions greatly increases the risk of developing active tuberculosis, supporting the observation that protective immunity suppress M. tuberculosis infection. The only vaccine against tuberculosis, M. bovis Bacillus Calmette Guerin (BCG), has proven to be of low efﬁcacy against the most frequent outcome of tuberculosis, lung infection in adults (Fine 1995). Furthermore, with the current treatment requiring up to three drugs and a high degree of patient compliance, the number of multi drug resistant (MDR) isolates is on the rise in many areas of the world. The urgency to develop more effective vaccines or immunotherapies requires potent immune activating strategies at the interface of innate and adaptive immunity.

2.2 2.2.1

Infection and Innate Immunity Neutrophils

In the presence of pro-inﬂammatory stimulus, neutrophils are among the ﬁrst innate immune cells to migrate from the blood to the foci. Neutrophils are professional phagocytes and express a range of receptors that can recognize opsonized and nonopsonized microbes, which are rapidly killed upon fusion of the phagosome with lysosomal compartments and specialized cytoplasmic granules that contain a vast arsenal of antimicrobial effector molecules including D-defensins, proteases as well as iron and siderophore-binding molecules, i.e. lactoferrin and lipocalin, respectively (Segal 2005; Appelberg 2007). In addition to direct microbicidal activity, neutrophils drive cell migration via production of chemokines as well as produce pro-inﬂammatory cytokines in response to microbial pattern recognition receptor stimulation. Neutrophillic inﬁltrates had been reported during acute pulmonary tuberculosis both in clinical studies and experimental infections (non-aerosol infection models) (Appelberg 1992; Condos et al. 1998; Schluger and Rom 1998; Lasco et al. 2004). However, the role of neutrophils during tuberculosis is still controversial. The lack of a selective neutrophil deﬁcient model has stiﬂed the studies in this area. However, investigators have used antibody-mediated depletion models to question the role of neutrophils during tuberculosis. Pedrosa and colleagues studied BALB/c infected with a high intravenous dose of M. tuberculosis Erdmann and the effect of early (days 0, 2 and 4 post infection) and late (days 16, 18 and 20 post infection) depletion of neutrophils (Pedrosa et al. 2000). While later neutrophil depletion did not show a quantiﬁable effect on M. tuberculosis proliferation in the lungs, earlier neutrophil depletions impaired development of protection against mycobacterial challenge. Further evidence in favor of an early protective role for neutrophils is provided by the observation that intra-tracheal injection of pre-activated neutrophils protected Fischer rats from infection with M. tuberculosis Kurono (Sugawara et al. 2004). On the other hand, other studies failed to show any protective effect when C57Bl/6

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mice were challenged via the aerosol route (Seiler et al. 2000), suggesting that neutrophils do not play an essential part in the early control of infection with M. tuberculosis. Yet in another report, the investigators compared the mouse strains genetically resistant (A/Sn) versus susceptible (I/St) to M. tuberculosis and concluded that upon aerosol infection with M. tuberculosis H37Rv, neutrophils contribute to pathology rather than protection (Eruslanov et al. 2005). This controversy may be partially explained by genetic differences among strains of experimental animals as well as different laboratory strains of M. tuberculosis. Another approach to the potential role of neutrophils during tuberculosis has been raised by a series of studies where it has been shown that apoptotic neutrophils can modulate the induction of acquired immunity by dendritic cells (Aleman et al. 2002, 2005, 2007; Tan et al. 2006), or provide “help” to M. tuberculosis-infected macrophages in vitro (Tan et al. 2006), via delivery of antimicrobial contents of neutrophil granules to M. tuberculosis-containing phagosomes shortly after uptake of apoptotic neutrophils, leading to improved killing of mycobacteria mediated by the neutrophil D-defensin HNP-1.

2.2.2

T Cells

The in vivo mouse model of low-dose aerosol M. tuberculosis exposure produces a very low cellular response, especially when comparing to those responses seen after some viral or other fast-replicating bacteria. These differences in robustness of responses may reﬂect an immune-modulatory activities triggered by the pathogen or may simply result from the inoculum route and dose. During natural as well as during experimental aerosol exposure, M. tuberculosis enters the lung in droplets of 3–5 Pm of diameter (typically generated by a cough of a diseased individual), only a few bacilli might be found within these droplets. Furthermore, those droplets will most likely settle within the alveolar space. It should also be noted that M. tuberculosis is relative slow grower, with estimated duplication time of approximately 28 h to double in vivo (Dunn and North 1995). Therefore, the optimal conditions for an invading bacilli to initiate the cascade that lead to powerful cellular immune response are extremely low and greatly dependent on the inoculum dose as well as the potential to reach professional antigen-presenting cells and secondary lymphoid tissues, such as the draining lymph nodes. Consistent with this observation is the fact that it is possible to detect early T-cell activation in the lymph nodes draining the lung but not within the lung itself (Chackerian et al. 2001). In order to investigate mycobacteria antigen-speciﬁc T cell activation, T-cell receptor transgenic (TCRTg) T cells speciﬁc for an IAb-restricted epitope of the early secreted antigenic target 6 kDa protein (ESAT-6) or for an IAb-restricted epitope of the essential mycolyl transferase of Mtb, Ag85The activation of naive T cells has been had been used. The published data show a signiﬁcant up-regulation of an early marker of T cell activation (CD69) as well as proliferation of naïve T cell in the draining nodes of the lung, which is concomitant to the arrival of the bacilli in the node (Reiley et al. 2008;

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Wolf et al. 2008). The lag between bacterial proliferation in the lungs and the growth and differentiation of effector T cells constitute the basis for the apparent slow cellular immune response during M. tuberculosis.

2.2.3

Dendritic Cells

Fluorescent bacteria delivered by aerosol can be seen within dendritic cells (DCs) and cannot be detected in the lymph node (Wolf et al. 2007). More importantly, if the chemotactic signals responsible for DC migration towards the lymph node are absent, the appearance of M. tuberculosis in that organ is inhibited (Wolf et al. 2007). It has recently been shown that appearance of microbially activated (or infected) DCs in the lymph node is dependent on the presence of homodimers of IL-12p40 (Khader et al. 2006), previously supposed to act as an endogenous antagonist for the Th1promoting cytokine IL-12p70. This was supported by the ﬁndings that while mice that deﬁciency of IL-12p70 or IL-23 could induce accumulation of activated CD41 T cells in the lung 21 days after infection, mice that lacked IL-12p40 could not (Khader et al. 2006). The investigators found that this IL-12p40(2) was very rapid (under 3 h) and drove DC migration via up-regulation of responsiveness to CCL19/CCL21 (CCR7 ligands). If DC did not produce IL-12p40, then migration in response to CCL19 and CCL21 remained at the level of the non-activated DC, despite presence of M. tuberculosis (Khader et al. 2006). On the other hand, DCs lacking IL-12p35, IL-23p19, or both of these cytokine components respond to CCL19 and CCL21 when incubated with M. tuberculosis (Khader et al. 2006). The mechanisms by which IL-12p40(2) mediated up-regulation of CCR7 responsiveness is not yet understood. DC accumulation in the lymph node provides environmental cues to the immune system. Notably, bacterially activated DCs will drive the naïve T cell activation, growth and differentiation. In agreement with this observation, it was shown that M. tuberculosis-exposed DCs adoptively transferred to the lung could provide the signals to drive T-helper 1 cell development (Bhatt et al. 2004). These data also support the paradigm that the mechanisms that modulate the appearance of functional effector T cells depend directly upon the DC activation status and subset and that there are no redundancies that could compensate for by the intact host cells within the node.

2.2.4

Natural Killer Cells

Natural killer (NK) cells are innate granular lymphocytes (Lodoen and Lanier 2006; Newman and Riley 2007; Moretta et al. 2008) known to play a role in mediating in allograft rejection and killing of transformed and virus-infected cells. Additionally, NK cells secrete pro-inﬂammatory cytokines, most prominently IFN-J. NK cell activity is controlled by cytokines (IL-12, IL-18 and IFN-D in particular) and a complex repertoire of activating (e.g. NKG2D, natural cytotoxicity receptors) and inhibitory receptors (e.g. CD94-NKG2A).

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It has become evident that NK cells are capable of mounting a vigorous response to M. tuberculosis. Human NK cells express granulysin within their intra-cytoplasmic granules and it has been shown that this peptide could directly kill M. tuberculosis (Stenger et al. 1999). Moreover, human NK cells are known to directly lyse M. tuberculosis-infected monocytes and macrophages in vitro (Denis 1994; Vankayalapati et al. 2002). The activation occurs via triggering of NKG2D and NKp46 that bind to the stress-induced ligands UL16-binding protein 1 (ULPB1) and vimentin, respectively (Vankayalapati et al. 2002, 2005; Garg et al. 2006). Human NK cells can also actively inhibit mycobacterial growth via induction of apoptotic cell deaths (Brill et al. 2001; Millman et al. 2008). During experimental infections with M. tuberculosis it was found that NK cells accumulate start to accumulate in the lungs and secrete IFN-J after approximately 2 weeks after low dose aerosol infection with M. tuberculosis Erdmann (JunqueiraKipnis et al. 2003). NK cell depletion showed no effect on control of mycobacterial growth. Feng and colleagues provided evidence for this hypothesis when they demonstrated that NK cells are the principal source of IFN-J in T cell-deﬁcient RAG2−/− animals after low-dose M. tuberculosis H37Rv infection. Most importantly, that NK cell-derived IFN-J was essential for controlling the pathology (Feng et al. 2006). In the absence of both NK and T cells (RAG2−/− Jc−/−), the control of mycobacterial growth was severely abolished, indicating that NK cells can drive cellular responses against mycobacteria.

2.2.5

Regulatory T Cells

Regulatory T cells (T regs) were found to accumulate in the lungs of mice during experimental M. tuberculosis infection and bacterial control is improved after their depletion (Scott-Browne et al. 2007). In humans, it was shown that mannose-capped lipoarabinomannan (ManLAM) drove expansion of CD4 + CD25 + FoxP3+ cells in samples from tuberculin responders but not from naïve individuals (Garg et al. 2008). ManLAM is thought to trigger macrophages to produce PGE2, which in turn allows for further Treg expansion (Garg et al. 2008). Interestingly, TB patients show higher frequency of Treg cells than in healthy tuberculin responders (Garg et al. 2008). During anti-viral therapy, AIDS/TB patients, show an increase in both effector T cells and T regs, however the regulatory activity of the T regs is defective (Seddiki et al. 2009).

2.3

Granuloma Formation and Containment of Bacilli

Granuloma is an organized collection of immune cells, including a large proportion of macrophages, within a tissue as a result of chronic unresolved/persistent inﬂammatory stimulus. The purpose of the granuloma for the host is two-fold.

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Firstly, through a ﬁbrous capsule is thought to provide a physical containment of the infected area while preventing pathogen spread. Secondly, it aggregates a collection of immune effector cells including APCs, lymphocytes and granulocytesa crucial step during the development of immunity to infection. The tuberculous granuloma has been described by Ghon in 1912, however only in recent years an accurate depiction of primary human granulomas has allowed a better understanding of their role during tuberculosis. Frequently, an area of necrosis if found at the inner region of the tuberculosis granuloma. This is thought to be a consequence from previous extensive macrophage infection and killing. However, the granuloma also allows the chronic maintenance of M. tuberculosis in infected macrophages. Notably, while the core of the granuloma carries few antigen-presenting cells containing mycobacterial antigens, the periphery of the granuloma is enriched with organized aggregates of APC and proliferating lymphocytes and are thought to be a site of active immunity (Ulrichs and Kaufmann 2006). In summary, tuberculosis granuloma can be considered both an essential player in the protective immune response to the pathogen and a facilitator in the development of latency during chronic disease, which is hard for the immune system to tackle and is notoriously difﬁcult to treat by conventional methods.

2.3.1

TNF

TNF has extensively studied during experimental mouse M. tuberculosis infections. It has been shown that it is central for the development of protective immune response both during acute and chronic disease, notably from the poor granuloma formation observed in vivo as well as defective macrophage activity (Flynn et al. 1995; Mohan et al. 2001; Algood et al. 2005). Other experimental M. tuberculosis infection models, such as zebraﬁsh and nonhuman primate have shown that TNF is relevant for the mechanisms involved in overcoming acute infection and to prevent disease reactivation, however, granuloma formation is not affected by TNF depletion (Lin et al. 2010; Clay et al. 2008). More importantly, genetic polymorphism of the TNF receptor in humans has been associated with increased susceptibility to active TB in Africa (Moller et al.), which is strongly supported by the increased frequency of re-activated tuberculosis among patients treated with TNF antagonists (Keane 2004). Despite the majority of these cases were from tuberculosis reactivation, there is increased concern over the risk of fulminant acute tuberculosis within highly endemic areas. TNF is a pleiotropic cytokine that can affect several arms of the immune system. During early tuberculosis infection, it has been associated with the induction of adhesion molecules (Windish et al. 2009) and chemokines (Peters et al. 2001; Scott and Flynn 2002; Algood et al. 2004), which can perform potentially protective roles during the initiation and expansion of immune responses. Another possible aspect that can be affected by TNF is programmed cell death, as a mediator of apoptosis, TNF can be directly detrimental to the survival of mycobacteria within macrophages.

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Human TNF production by M. tuberculosis infected alveolar macrophages triggers apoptotic cell death, thereby reducing intracellular bacterial burden (Keane et al. 1997). Moreover, attenuated M. tuberculosis strains were found to increase apoptosis and, consequently induce stronger CD8 T cell protective responses, indicating that apoptosis is associated with a better outcome of infection (Hinchey et al. 2007).

2.3.2

IFN-g

IFN-J is a central cytokine in the initiation and effector function of cellular immune responses. During experimental M. tuberculosis infection, the absence of IFN-J leads to uncontrolled bacilli growth and mortality (Cooper et al. 1993). The downstream IFN-J-inducible enzymes required for generation of nitrogen and oxygen radicals, nitric oxide synthase 2 (NOS2) and p47phox, are also relevant for control of immunopathology (Cooper et al. 2002).

2.3.3

Lipoxins

We have reported evidence for the role of a pathway involving the 5-LO–dependent production of lipoxins that dampens M. tuberculosis–driven pro-inﬂammatory immune responses and regulates bacterial growth. Lung tissue from 5-LO-deﬁcient animals display higher production of IL-12 and other pro-inﬂammatory cytokines, suggesting that 5-LO-dependent eicosanoids counter-balance such secreted proteins. Since 5-LO is required for both leukotriene and lipoxin biosynthesis, reconstitution experiments were performed to more directly assess the role of the latter group of eicosanoids in the regulation of mycobacterial growth in vivo. Importantly, administration of the stable lipoxin analog – ATLa2 to M. tuberculosis-infected 5-LO-deﬁcient mice, restored both pulmonary mycobacterial loads and pro-inﬂammatory cytokine production. These observations demonstrated that deﬁciency in lipoxins is sufﬁcient to explain the effects on bacterial growth and host response seen in the infected 5-LO–deﬁcient animals. Interestingly, granuloma formation in 5-LO-deﬁcient mice was found to be altered suggesting that lipoxins regulate chemotaxis of inﬂammatory monocytes to the site of infection and/or activation of macrophages in vivo (Baﬁca et al. 2005). Nevertheless, whether lipoxins directly control inﬂux/efﬂux of lung antigen presenting cells is still unclear. A role for products derived from 5-LO, encoded by ALOX5 gene, in pulmonary tuberculosis was further supported in human association studies. In humans, ALOX5 gene comprises 14 exons and 13 introns approximately 82 kb on chromosome ten (10q11.2). ALOX5 promoter is GC-rich, and the region between 79 and 56 bp is essential for gene expression (Herb et al. 2008). In that region, a variable number of tandem repeats (VNTR) has been identiﬁed, consisting of [5c-GGGCGG-3c]2–8, which are targets for binding of the transcription factor Sp1. Insertions or deletions

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of the Sp1-binding motif have created variant alleles of two to eight repeats and depending on the number of repeats, Sp1 binding and subsequent gene expression is altered (Cohn et al. 2001; Wang et al. 2008). On the basis of the above mentioned results found in mice, Herb et al. hypothesized that naturally occurring ALOX5 variants might also be relevant to the phenotype of M. tuberculosis infection in humans (Herb et al. 2008). The authors found a strong association between low 5-LO activity genotypes and decreased TB risk, consistent with the observation that 5-LO deﬁciency in mice was associated with relative protection from TB. However, it remains to be demonstrated whether lipoxins directly regulate human immune responses during TB. While a number of questions need to be answered from these studies, such ﬁndings can be considered as an example of a gene regulating in vivo M. tuberculosis infection in both human and mice.

2.3.4

IL-10

IL-10 is a powerful inhibitor of the IL-12/IFN-J cellular immunity pathway. However, the role of this cytokine in TB is not well established. In humans, an increased macrophage IL-10 production upon TLR-stimulation such as LPS is associated with faster development of primary TB (Awomoyi et al. 2002). In mice, the genetic deletion of IL-10 causes an accelerated accumulation of lung DC migration in the draining lymph nodes after mycobacterial infection (Demangel et al. 2002). M. tuberculosis infection in a susceptible mouse strain – CBA – leads to increased lung macrophage IL-10 production, something that was not found in C57Bl/6 (resistant) mouse strain (Turner et al. 2002). Conversely, inhibition of IL-10 in CBA mice enhances control of bacterial burden during the chronic TB (Beamer et al. 2008).

2.3.5

TGF-b

It has been reported that an excess of TGF-E is produced during tuberculosis and its message is abundantly up-regulated during active M. tuberculosis infection. Consistent with that T-cell responses from active TB patients are signiﬁcantly improved after TGF-E neutralization. TGF-E modulates multiple cells of the immune system, including T cells (activation and cytokine production), macrophage (migration, microbicidal activity, cytokine production). In agreement with this set of observations, the presence of TGF-E leads to increased growth of intracellular M. tuberculosis (Hirsch et al. 1994). On the other hand, its neutralization (Hirsch et al. 1994) as well as the addition of natural TGF-E inhibitors (Hirsch et al. 1997) caused a reduction in the growth of intracellular mycobacteria. Given its correlation with disease severity, it is likely that TGF-E-mediated macrophage deactivation is severe in patients carrying higher loads of bacilli.

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2.4 2.4.1

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Evasion of Immune Response Cell Wall Components

Mycobacteria are Gram positive and cannot be decolorized by acid alcohol and are therefore classiﬁed as acid-fast bacilli. Acid fastness is mostly due to higher amounts of mycolic acids (long chain cross-linked fatty acids) and other cell-wall lipids in the cell wall (Daffe and Draper 1998). Mycolic acid and other lipids are linked to underlying arabinogalactan and peptidoglycan (Daffe and Draper 1998). A variety of unique lipids, such as lipoarabinomannan (LAM), trehalose dimycolate and phthiocerol dimycocerate, anchor noncovalently with the cell membrane and have been shown to mediate the virulence of M. tuberculosis (Glickman and Jacobs 2001). Disruption of the gene involved in mycolic acid cyclopropanation caused a reduction in virulence (Glickman et al. 2000). The majority of exported proteins and protective antigens of M. tuberculosis are a cluster of related gene products termed the antigen 85 complex, each having ﬁbronectin binding capacity and thus an important role in disease pathogenesis (Belisle et al. 1997). LAM is one of the major components of mycobacterial cell wall. It triggers TNF production from the macrophages (Chatterjee et al. 1992). Moreover, LAM can scavenge for potentially cytotoxic oxygen free radicals, inhibit protein kinase C activity and block the tran- scriptional activation of gamma interferon inducible genes in human macrophages. Thus, potentially contributing to the persistence of mycobacteria within mononuclear phagocytes (Chan et al. 1991).

2.4.2

Inhibition of Phagolysosome Fusion

Phagosome-lysosome fusion is a key event in killing intracellular pathogens (Moulder 1985). Upon engulfment by macrophages, most bacilli are directed to phagolysosomes (McDonough et al. 1993). Interestingly, however, individual M. tuberculosis bud out from the fused phagolysosomes into vacuoles refractory to fusion with the secondary lysosomes thus escape intracellular killing. M. tuberculosisderived sulfatides (anionic trehalose glycolipids) were shown to have such antagonistic fusion activity (Goren 1977). Virulent M. tuberculosis culture supernatants are enriched for ammonium, which were shown to inhibit phagolysosomal fusion by increasing the alkalinity within the intralysosomal environment (Gordon et al. 1980; Hart et al. 1983).

2.4.3

Mycobacterial Dormancy

M. tuberculosis can enter dormancy within host cells for extended times while remaining potentially active. During dormancy/latency of M. tuberculosis infection,

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the bacterium escapes the activity host immune cells. Experimental M. tuberculosis infection in mice is dependent on the glyoxylate shunt enzyme – isocitrate lyase, essential for fatty acid metabolism (McKinney et al. 2000). Its genetic disruption resulted in increased bacterial persistence as well as virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. Hypoxia was also found to be a major factor in the induction of non-replicating persistent mycobacterium (Wayne and Sohaskey 2001).

2.4.4

Modulation of Host Cell Signaling

Upon infection, host proteins are targeted by phosphatases and kinases of several pathogenic bacteria thereby modulating and potentially contributing to the establishment of the disease. The phagocytosis of M. tuberculosis by macrophages is associated with a several early signaling events, including the activation of Src protein tyrosine kinase family members. Such activation leads to the enhanced tyrosine phosphorylation of multiple target proteins and phospholipase D activation (Kusner et al. 1996). LAM derived from virulent M. tuberculosis strains can modulate intracellular signaling associated to bacterial survival via phosphorylation of proapoptotic protein downstream from the phosphatidylinositol 3-kinase-dependent pathway (Maiti et al. 2001). A major anti-phosphotyrosine reactive protein is present only in strains belonging to M. tuberculosis complex (Chow et al. 1994). Also, it has been shown that M. tuberculosis has two functional secreted tyrosine phosphatases that may interfere with the host cells signaling machinery (Koul et al. 2000).

2.5

Disease Reactivation

Presently, the drug regimen for the treatment of latent TB infection is of 9 months of isoniazid (2000), a drug for which the efﬁcacy is dependent upon the synthesis of mycolic acid, which occurs only during active replication. Based on epidemiologic studies, the reactivation of latent TB may derive from: HIV, malnutrition, tobacco smoke, indoor air pollution, alcoholism, silicosis, insulin dependent diabetes, renal failure, malignancy, and immune-suppressive therapy (i.e., glucocorticoids) (Horsburgh 2004; Jick et al. 2006; Lonnroth and Raviglione 2008). AIDS and inhibitors of TNF are well-characterized mechanisms of disease reactivation. An increased incidence of TB was noted among patients upon treatment with TNF inhibitors (Keane 2004; Wallis 2009). Similar observations were made after TNF neutralization in non-human primates during latent tuberculosis (Lin et al. 2000). Importantly, although a high rate of reactivation (~65%) was noted in those animals, not all monkeys showed TB reactivation after short-term anti-TNF treatment (Keane 2004), indicating that TNF is an relevant but not the sole component in maintaining TB latency. AIDS and HIV infection is a central component in the resurgence of TB as a global health threat. HIV infection has been, by far, the most common risk for

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TB reactivation, almost tenfold higher among HIV patients rather than non-HIV patients (Horsburgh 2004). In contrast to the TB epidemiology from the pre-HIV era, an extremely higher rate of disseminated/extrapulmonary TB has been reported among AIDS patients (Small et al. 1994). Although CD4 T cell counts are directly associated with risk of TB reactivation, anti-retroviral therapy had no signiﬁcant effect (Meintjes et al. 2008; Lawn et al. 2009). Moreover, optimal conditions CD4 T cell counts do not necessarily lower the risk of TB reactivation (Badri et al. 2002; Lawn et al. 2005), indicating that mere CD4 T cell counts may not necessarily reﬂect immune ﬁtness to ﬁght TB reactivation. In non-human primates, infection with SIV led to TB reactivation (Diedrich et al. 2010). The synergic effect between HIV and M. tuberculosis involve: HIV-induced loss of mycobacterial speciﬁc CD4 T cells, M. tuberculosis-induced increases in HIV load in serum and macrophages, shift from Th1 to Th2 response via alterations in IL-10, regulatory T cells, IL-12, IL-4, and TNF, loss of granuloma integrity, and alterations in apoptotic mechanisms (Djoba Siawaya et al. 2007).

2.6 Vaccines, Chemotherapy in the Interface with the Immune System BCG is currently the only vaccine available for TB, however it is well accepted that its efﬁcacy, albeit low, is restricted to acute pulmonary disease. Several strains of BCG differ in immunogenicity (Aguirre-Blanco et al. 2007). It is known that BCG differs from M. tuberculosis due to the lack of RD-1 gene (Billeskov et al. 2007; Woodworth et al. 2008). Systemic delivery of BCG induce antigen-speciﬁc cells, thereby triggering protection, which is characterized by the appearance of IFN-J-producing T cells in the lung at day 14. Suggesting that timing for accumulation of antigen-speciﬁc cells in the lung that is key to protection (Mittrucker et al. 2007; Cooper 2009). Strategies aiming at improving protection after BCG vaccination focused on speciﬁc delivery of antigen(s) alongside the original vaccine. The route of this boost is important as BCG vaccinated mice boosted intranasally (but not intradermally) using a recombinant adenovirus expressing Ag85, show better levels of protection and infection control when compared to BCG alone. Moreover, the introduction of a boost to the BCG regimen with a vaccinia virus expressing M. tuberculosis Ag85A caused a signiﬁcant expansion of long-lived memory T cells expressing IFN-J TNF, and IL-2 (Beveridge et al. 2007). Along these lines, BCG engineered to express the DC stimulating factor, GM-CSF was shown to improve cellular immunity after vaccination (Triccas et al. 2007). Furthermore, genetic deletion of the BCG SecA2 made host cells more susceptible to apoptosis induction and resulting in improved CD8 T cell induction (Hinchey et al. 2007). On the other hand, Flt3-ligand-expressing BCG had no effect in improving protection (Triccas et al. 2007). Interestingly, T reg depletion improved vaccine induced cellular responses, however, it failed to enhance protection levels (Quinn et al. 2008).

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In the mouse model, administration of deﬁned antigens and adjuvants has been shown to result in the early T cell recruitment to the lungs upon challenge, resulting in better control of infection. Earlier T-cell responses are desirable effect in order to act to limit M. tuberculosis growth and positively affect the outcome of the infection, by limiting steady-state bacterial infection in the lungs in a timely manner.

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Glickman, M. S., J. S. Cox, et al. (2000). “A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis.” Mol Cell 5(4): 717–27. Glickman, M. S. and W. R. Jacobs, Jr. (2001). “Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline.” Cell 104(4): 477–85. Gordon, A. H., P. D. Hart, et al. (1980). “Ammonia inhibits phagosome-lysosome fusion in macrophages.” Nature 286(5768): 79–80. Goren, M. B. (1977). “Phagocyte lysosomes: interactions with infectious agents, phagosomes, and experimental perturbations in function.” Annu Rev Microbiol 31: 507–33. Hart, P. D., M. R. Young, et al. (1983). “Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. A combined microscopic and computer study.” J Exp Med 158(2): 477–92. Herb, F., T. Thye, et al. (2008). “ALOX5 variants associated with susceptibility to human pulmonary tuberculosis.” Hum Mol Genet 17(7): 1052–60. Hinchey, J., S. Lee, et al. (2007). “Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis.” J Clin Invest 117(8): 2279–88. Hirsch, C. S., J. J. Ellner, et al. (1997). “In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta.” Proc Natl Acad Sci USA 94(8): 3926–31. Hirsch, C. S., T. Yoneda, et al. (1994). “Enhancement of intracellular growth of Mycobacterium tuberculosis in human monocytes by transforming growth factor-beta 1.” J Infect Dis 170(5): 1229–37. Horsburgh, C. R., Jr. (2004). “Priorities for the treatment of latent tuberculosis infection in the United States.” N Engl J Med 350(20): 2060–7. Jick, S. S., E. S. Lieberman, et al. (2006). “Glucocorticoid use, other associated factors, and the risk of tuberculosis.” Arthritis Rheum 55(1): 19–26. Junqueira-Kipnis, A. P., A. Kipnis, et al. (2003). “NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection.” J Immunol 171(11): 6039–45. Keane, J. (2004). “Tumor necrosis factor blockers and reactivation of latent tuberculosis.” Clin Infect Dis 39(3): 300–2. Keane, J., M. K. Balcewicz-Sablinska, et al. (1997). “Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis.” Infect Immun 65(1): 298–304. Khader, S. A., S. Partida-Sanchez, et al. (2006). “Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection.” J Exp Med 203(7): 1805–15. Koch, R. (1891). “A Further Communication on a Remedy for Tuberculosis.” Br Med J 1(1568): 125–127. Koul, A., A. Choidas, et al. (2000). “Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.” J Bacteriol 182(19): 5425–32. Kusner, D. J., C. F. Hall, et al. (1996). “Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages.” J Exp Med 184(2): 585–95. Lasco, T. M., O. C. Turner, et al. (2004). “Rapid accumulation of eosinophils in lung lesions in guinea pigs infected with Mycobacterium tuberculosis.” Infect Immun 72(2): 1147–9. Lawn, S. D., L. G. Bekker, et al. (2005). “How effectively does HAART restore immune responses to Mycobacterium tuberculosis? Implications for tuberculosis control.” AIDS 19(11): 1113–24. Lawn, S. D., L. Myer, et al. (2009). “Short-term and long-term risk of tuberculosis associated with CD4 cell recovery during antiretroviral therapy in South Africa.” AIDS 23(13): 1717–25. Lin, P. L., A. Myers, et al. (2010). “Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.” Arthritis Rheum 62(2): 340–50.

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Lodoen, M. B. and L. L. Lanier (2006). “Natural killer cells as an initial defense against pathogens.” Curr Opin Immunol 18(4): 391–8. Lonnroth, K. and M. Raviglione (2008). “Global epidemiology of tuberculosis: prospects for control.” Semin Respir Crit Care Med 29(5): 481–91. Maiti, D., A. Bhattacharyya, et al. (2001). “Lipoarabinomannan from Mycobacterium tuberculosis promotes macrophage survival by phosphorylating Bad through a phosphatidylinositol 3-kinase/Akt pathway.” J Biol Chem 276(1): 329–33. McDonough, K. A., Y. Kress, et al. (1993). “Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages.” Infect Immun 61(7): 2763–73. McKinney, J. D., K. Honer zu Bentrup, et al. (2000). “Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase.” Nature 406(6797): 735–8. Meintjes, G., S. D. Lawn, et al. (2008). “Tuberculosis-associated immune reconstitution inﬂammatory syndrome: case deﬁnitions for use in resource-limited settings.” Lancet Infect Dis 8(8): 516–23. Millman, A. C., M. Salman, et al. (2008). “Natural killer cells, glutathione, cytokines, and innate immunity against Mycobacterium tuberculosis.” J Interferon Cytokine Res 28(3): 153–65. Mittrucker, H. W., U. Steinhoff, et al. (2007). “Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis.” Proc Natl Acad Sci USA 104(30): 12434–9. Mohan, V. P., C. A. Scanga, et al. (2001). “Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology.” Infect Immun 69(3): 1847–55. Moller, M., F. Flachsbart, et al. “A functional haplotype in the 3’untranslated region of TNFRSF1B is associated with tuberculosis in two African populations.” Am J Respir Crit Care Med 181(4): 388–93. Moretta, A., E. Marcenaro, et al. (2008). “NK cells at the interface between innate and adaptive immunity.” Cell Death Differ 15(2): 226–33. Moulder, J. W. (1985). “Comparative biology of intracellular parasitism.” Microbiol Rev 49(3): 298–337. Newman, K. C. and E. M. Riley (2007). “Whatever turns you on: accessory-cell-dependent activation of NK cells by pathogens.” Nat Rev Immunol 7(4): 279–91. Pedrosa, J., B. M. Saunders, et al. (2000). “Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice.” Infect Immun 68(2): 577–83. Peters, W., H. M. Scott, et al. (2001). “Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis.” Proc Natl Acad Sci USA 98(14): 7958–63. Quinn, K. M., F. J. Rich, et al. (2008). “Accelerating the secondary immune response by inactivating CD4(+)CD25(+) T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosis.” Eur J Immunol 38(3): 695–705. Reiley, W. W., M. D. Calayag, et al. (2008). “ESAT-6-speciﬁc CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes.” Proc Natl Acad Sci USA 105(31): 10961–6. Schluger, N. W. and W. N. Rom (1998). “The host immune response to tuberculosis.” Am J Respir Crit Care Med 157(3 Pt 1): 679–91. Scott, H. M. and J. L. Flynn (2002). “Mycobacterium tuberculosis in chemokine receptor 2-deﬁcient mice: inﬂuence of dose on disease progression.” Infect Immun 70(11): 5946–54. Scott-Browne, J. P., S. Shaﬁani, et al. (2007). “Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis.” J Exp Med 204(9): 2159–69. Seddiki, N., S. C. Sasson, et al. (2009). “Proliferation of weakly suppressive regulatory CD4+ T cells is associated with over-active CD4+ T-cell responses in HIV-positive patients with mycobacterial immune restoration disease.” Eur J Immunol 39(2): 391–403. Segal, A. W. (2005). “How neutrophils kill microbes.” Annu Rev Immunol 23: 197–223.

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Seiler, P., P. Aichele, et al. (2000). “Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.” J Infect Dis 181(2): 671–80. Small, P. M., P. C. Hopewell, et al. (1994). “The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods.” N Engl J Med 330(24): 1703–9. Stenger, S., J. P. Rosat, et al. (1999). “Granulysin: a lethal weapon of cytolytic T cells.” Immunol Today 20(9): 390–4. Sugawara, I., T. Udagawa, et al. (2004). “Rat neutrophils prevent the development of tuberculosis.” Infect Immun 72(3): 1804–6. Tan, B. H., C. Meinken, et al. (2006). “Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens.” J Immunol 177(3): 1864–71. Triccas, J. A., E. Shklovskaya, et al. (2007). “Effects of DNA- and Mycobacterium bovis BCGbased delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis.” Infect Immun 75(11): 5368–75. Turner, J., M. Gonzalez-Juarrero, et al. (2002). “In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice.” J Immunol 169(11): 6343–51. Ulrichs, T. and S. H. Kaufmann (2006). “New insights into the function of granulomas in human tuberculosis.” J Pathol 208(2): 261–9. Vankayalapati, R., A. Garg, et al. (2005). “Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium.” J Immunol 175(7): 4611–7. Vankayalapati, R., B. Wizel, et al. (2002). “The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium.” J Immunol 168(7): 3451–7. Wallis, R. S. (2009). “Infectious complications of tumor necrosis factor blockade.” Curr Opin Infect Dis 22(4): 403–9. Wang, S., M. Wang, et al. (2008). “A novel variable number of tandem repeats (VNTR) polymorphism containing Sp1 binding elements in the promoter of XRCC5 is a risk factor for human bladder cancer.” Mutat Res 638(1–2): 26–36. Wayne, L. G. and C. D. Sohaskey (2001). “Nonreplicating persistence of mycobacterium tuberculosis.” Annu Rev Microbiol 55: 139–63. Windish, H. P., P. L. Lin, et al. (2009). “Aberrant TGF-beta signaling reduces T regulatory cells in ICAM-1-deﬁcient mice, increasing the inﬂammatory response to Mycobacterium tuberculosis.” J Leukoc Biol 86(3): 713–25. Wolf, A. J., L. Desvignes, et al. (2008). “Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs.” J Exp Med 205(1): 105–15. Wolf, A. J., B. Linas, et al. (2007). “Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo.” J Immunol 179(4): 2509–19. Woodworth, J. S., S. M. Fortune, et al. (2008). “Bacterial protein secretion is required for priming of CD8+ T cells speciﬁc for the Mycobacterium tuberculosis antigen CFP10.” Infect Immun 76(9): 4199–205.

Chapter 3

NKT Cell Activation During (Microbial) Infection Jochen Mattner

Abstract Invariant Natural Killer T (iNKT) cells constitute an innate-like lymphocyte population endowed with powerful immunomodulatory functions. Unlike conventional T cells, iNKT cells predominantly express a conserved semiinvariant T cell receptor (TCR), VD14-JD18/VE2, 7, 8 in mice and VD24-JD18/ VE11 in humans. These canonical TCRs in both species do not recognize peptides, but glycosphingolipid (GSL) patterns presented by CD1d on antigen presenting cells (APCs). The natural mechanisms for iNKT cell activation were unclear prior to the recent identiﬁcation of their endogenous and exogenous GSL ligands. Microbes can employ two alternative strategies for iNKT cell activation as exemplarily shown here for Gram-negative bacteria: (a) recognition of endogenous GSLs – by-products of the complex mammalian GSL metabolic pathways – and the presence of interleukin-12 (IL-12), triggered by Toll-like receptor (TLR) signaling of infected APCs, are required for the early secretion of IFN-J by iNKT cells in response to Gram-negative, LPS-positive bacteria. Whereas iNKT cells are secondary to APC-mediated effects in infections with these bacteria, (b) iNKT cells accelerate the clearance of Gram-negative LPS-negative alphaproteobacteria due to the cognate recognition of GSLs in the cell wall of these alphaproteobacteria. Thus, the iNKT cell population represents a major innate recognition pathway for these LPSnegative, GSL-positive alphaproteobacteria that senses infection at sites where iNKT cells accumulate, such as the liver. In this context, iNKT cell activation upon microbial encounter may not only contribute to bacterial clearance, but may be even deleterious for the host, providing innate signals that break peripheral tolerance and unleash autoimmune effector cells.

J. Mattner (*) University Hospital of Erlangen, Microbiology Institute–Clinical Microbiology, Immunology and Hygiene, Erlangen, Germany e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_3, © Springer Science+Business Media, LLC 2012

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3.1

J. Mattner

Introduction

T cells play a central role in the microbial immune defense. Conventional CD8+ and CD4+ T cells recognize peptide antigens presented by classical major histocompatibility complex (MHC) class I and II molecules. Upon antigen encounter, their T cell receptor (TCR) can re-arrange and shape the TCR repertoire due to the clonal expansion of the T cell population with the most suitable TCR for the presented antigen. In contrast to these conventional T cells, the speciﬁcity of invariant Natural Killer T (iNKT) cells is directed against glycosphingolipid (GSL) antigens presented by CD1d, a non-classical MHC class I molecule expressed on dendritic cells (DCs), macrophages and B cells. However, while classical MHC class I molecules are loaded within the endoplasmatic reticulum and present intracellular cytosolic peptides to cytotoxic CD8+ T lymphocytes, CD1d is loaded within the late endosome or the lysosome and presents endogenous or exogenous GSL antigens to iNKT cells. Like classical MHC class I, CD1d associates with E-2-microglobulin (E2m). Unlike classical MHC class I, CD1d molecules as all other CD1 molecules (see below) are nonpolymorphic and present GSLs rather than peptides to iNKT cells (summarized in (Bendelac et al. 2007)). Current models suggest that the hydrophobic tails of the GSL antigens bind into the groove of CD1d via hydrophobic interactions, while the polar head groups contact the TCR (Moody et al. 1997) and subsequently trigger iNKT cell activation. iNKT cells display a canonical VD14 TCR in mice and a VD24 TCR in humans in combination with a limited set of VE chains (VE8, VE7 and VE2 in mice and VE11 in humans) – that does not re-arrange upon antigen encounter – and release immediately copious amounts of cytokines and chemokines upon engagement of their TCR. Although iNKT cells are inherently autoreactive due to the recognition of endogenous GSL antigens, they react also to deﬁned GSL structures in the cell wall of microbes (summarized in (Bendelac et al. 2007)). Thus, iNKT cells recognize distinct self and microbial GSLs and their TCR acts more like a pattern recognition receptor, identifying iNKT cells as an innate (−like) lymphocyte population. These characteristics establish the CD1/NKT system as another line of antimicrobial defense next to MHC class I and II as well as to the diverse pattern recognition receptors of the innate immune system that alternatively recognizes deﬁned GSL antigens in addition to peptide antigens, lipoproteins, nucleic acids or polysaccharides (Brigl and Brenner 2004). These CD1d-restricted iNKT cells expressing the VD14 TCR can be distinguished from CD1a-, CD1b- and CD1c-restricted T cells as well as from CD1d-restricted T cells that express non-invariant TCRs and from a variety of other non-CD1drestricted T cells that express lineage receptors of NK cells (Eberl et al. 1999; Godfrey et al. 2004). Although non-VD14 CD1d-restricted T cells have been recently implicated in various diseases, this book chapter focuses mainly on canonical iNKT cells by providing an overview over GSL antigen presentation and iNKT cell function and summarizing the different GSLs recognized by iNKT cells during microbial infection as well as the functional consequences of this recognition for the host.

3

NKT Cell Activation During (Microbial) Infection

3.2

41

NKT Cells and the CD1 System

NKT cells form a heterogeneous cell population, which respond to (glyco-) lipid and/or GSL antigens presented by CD1. Although the CD1 heavy chain associates with E2 microglobulin similar as classical MHC class I, it contains in contrast to classical MHC class I several hydrophobic channels that bind the lipid portion of GSLs or other lipid-rich antigens (see also Sect. 3.4 – CD1d mediated presentation of glycolipids). The CD1 genes that diverged from the MHC gene complex several 100 million year ago (Kasahara 1997) can be divided into Group I (CD1a, b, c), Group II (CD1d), and Group III (CD1e) genes based on sequence similarities and their organization in the locus. While group I and II CD1 molecules are expressed on the cell surface and present GSL and/or (glyco-) lipid antigens, the ﬁfth human CD1 molecule, CD1e, facilitates the loading of certain lipid antigens onto CD1b (de la Salle et al. 2005). While humans express all ﬁve CD1 genes, their expression is not conserved in all mammalian species: mice, for example, have been shown to express only CD1d (summarized in (Bendelac et al. 2007)). Deﬁned by the recognition of distinct antigens presented by these CD1 groups, NKT cells can be distinguished into the following three categories:

3.2.1

Group I CD1 Molecules Recognizing NKT Cells (CD1a, b, c)

Because of its lipid-rich cell wall, Mycobacterium tuberculosis has been the prime candidate pathogen for studying (glyco-) lipid – reactive T cells in vitro. The ﬁrst study published in this context described a T cell line speciﬁc for mycolic acids that are presented by CD1b (Beckman et al. 1994). Mycolic acids are not only found in Mycobacterium tuberculosis, but also are also constituents of the cell wall of Actinomyces, Corynebacteria, and Nocardia species (Brennan and Nikaido 1995; Moody et al. 2002). During subsequent studies, additional group I CD1 antigens in the cell wall of Mycobacterium tuberculosis have been identiﬁed: didehydroxymycobactin, presented by CD1a (Moody et al. 2004; Rosat et al. 1999), Ac2SGL, a sulfoglycolipid presented by CD1b (Gilleron et al. 2004), and hexosyl -1 – phosphoisoprenoids, presented by CD1c (Rosat et al. 1999; Beckman et al. 1996; Moody et al. 2000). Although Mycobacteria are a productive source for studying group I CD1-restricted T cell responses in vitro, the functional consequences of group I CD1 restricted T cell activation in vivo due to the lack of a suitable animal model is not understood. Interestingly, preferentially animals susceptible to infections with Mycobacteria like cattle express functional group I CD1 genes. Ongoing studies in cows may explain in the future the evolution of these group I CD1 – restricted T cell populations and their respective roles during mycobacterial infections (Kasmar et al. 2009).

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Group II CD1 Molecules Recognizing iNKT Cells (CD1d)

Group II CD1 genes have been preferentially studied in the mouse. CD1d is constitutively expressed on DCs, macrophages, and B cells (Roark et al. 1998; Brossay et al. 1997), particularly marginal zone B cells; CD1d is also expressed on cortical thymocytes, where its expression is essential for the development of iNKT cells (Bendelac 1995a, b, c), hepatocytes, hepatic Kupffer cells and endothelial cells lining liver sinusoids (Geissmann et al. 2005). Although CD1d is upregulated on microglial cells during inﬂammation (Busshoff et al. 2001), only relatively modest changes in CD1d expression occur upon TLR activation, infection or exposure to inﬂammatory cytokines on DCs, macrophages or B cells (Skold et al. 2005). Similar to the MHC class II system, most other solid tissue cells with the exception of the liver, where iNKT cells are abundantly found and non-antigen-presenting hematopoietic cells express low or undetectable levels of CD1d.

3.2.2.1

CD1d: Restricted Invariant NKT Cells (iNKT Cells, Type I NKT Cells)

iNKT cells constitute a population of T lymphocytes that predominantly use a conserved semi-invariant TCR with speciﬁcity for CD1d combined with glycosphingolipid lipid (GSL) ligands (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005). Reactivity towards alpha-GalactosylCeramide (D-GalCer), the prototypical iNKT cell ligand isolated from marine sponges distinguishes iNKT (or type I NKT) cells (Kawano et al. 1997) from non-classical (also summarized as type II) NKT cells (see Sect. 3.2.2.2). The iNKT cell TCR utilizes a canonical D-chain (VD14JD18 in mice and VD24-JD18 in humans) in combination with a limited set of VE chains (VE8, VE7 and VE2 in mice and VE11 in humans). Analysis of CD1d- and MHC-deﬁcient mice demonstrated that iNKT cells (also called type I NKT cells) represent the majority of CD1d-restricted T cells (Kawano et al. 1997; Bendelac et al. 1994, 1995a, b, c). Their conserved TCR structure, their memory/effector differentiation – as indicated by the combined expression of CD44 and CD69 even in the absence of exogenously added GSL antigens (Bendelac et al. 1992) -, and their expression of a panoply of NK receptors suggest modalities of activation that are distinct from conventional T cells. The conserved semi-invariant structure of their TCR is remarkably similar to that of B-1 B cells and JG T cells (Bendelac et al. 2001) and transgenic expression of the antigen receptor of each of these cell-types was found to instruct the corresponding lineage, suggesting that interactions with self ligands dictate lineage commitment and differentiation. Developmental studies used D-GalCer loaded CD1d tetramers (Benlagha et al. 2000; Matsuda et al. 2000) to follow the iNKT precursors and demonstrated that their TCR is intrinsically autoreactive, inducing a phase of clonal expansion in the thymus prior to migration into peripheral organs and secondary acquisition of NK cell receptors (Bendelac et al. 2007; Benlagha et al. 2002).

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Using D-GalCer loaded CD1d tetramers iNKT cells have been identiﬁed in mice, rats, rhesus macaques, chimpanzees and humans (Matsuura et al. 2000; Pyz et al. 2006; Liu et al. 2006). The distribution of iNKT cells has been well characterized in mice and differs from the one of conventional MHC I- or MHC II- restricted T cells. iNKT cells represent only 0.2–0.5% of the T cell population in the blood, the thymus and the peripheral lymph nodes, 1–2% of lymphocytes in the spleen, mesenteric, and pancreatic lymph nodes, and up to 30% of T cells in the liver. Mechanisms causing that unique distribution have remained elusive. Whereas the precise distribution of iNKT cells within the lymphoid organs is still unknown, they have been shown to patrol along the liver sinusoids (Geissmann et al. 2005; Ohteki and MacDonald 1994) providing intravascular immune surveillance: most likely they crawl along a CXCL16 gradient as they express high levels of CXCR6. Upon engagement of their TCR by potent GSL antigens like alpha-GalCer, however, iNKT cells immediately arrest. Although the expression of CXCR6 on iNKT cells matches the expression of CXCL16 on endothelial cells lining the liver sinusoids, CXCR6 deﬁciency resulted in reduced survival rather than altered migration of iNKT cells. Like NK cells, iNKT cells constitutively express mRNA but not protein for IFN-J, a hallmark of their poised effector stage (Stetson et al. 2003). Unlike NK cells, however, iNKT cells also produce interleukin four and 13 (IL-4 and IL-13) and the contribution of both Th1 and Th2 cytokines has been demonstrated in vivo, in conditions where iNKT cells either improved or aggravated infectious, malignant or autoimmune diseases (Terabe et al. 2000). Like conventional memory T cells iNKT cells do not absolutely require classical costimulatory signals to secrete cytokines following TCR engagement (Uldrich et al. 2005). Indeed, using CD1d0 or Ja180 deﬁcient mice that allow to distinguish iNKT cells from non-classical NKT cells, iNKT cells have been suggested to regulate various infectious, malignant and autoimmune diseases (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005; Terabe et al. 2000; Van Kaer 2005; Mars et al. 2004). The mechanisms of iNKT cell activation and the role of speciﬁc iNKT cell populations under these various conditions have remained unclear. Recent studies have, however, identiﬁed endogenous and microbial GlycoSphingoLipid (GSL) ligands that are critical for the activation of iNKT cells during infection with Gram-negative bacteria (see also Sect. 3.7) and begun to elucidate the functional consequences of this activation.

3.2.2.2

CD1d–Restricted Diverse NKT Cells (Type II NKT Cells, Non-classical NKT Cells)

Whereas iNKT (type I NKT) cells are deﬁned by their reactivity to D-GalCer, there exist other populations of CD1d-restricted NKT cells that do not respond to D-GalCer. This heterogeneous group of cells are summarized as type II NKT cells; they supposedly recognize (glyco-) lipids as well and are characterized by a more diverse TCR repertoire than iNKT cells (Terabe and Berzofsky 2008; Behar et al. 1999b; Cardell et al. 1995). Although D-ManCer has been suggested as a potential ligand for a subpopulation of these type II NKT cells expressing a VD19-JD33 TCR

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(Shimamura et al. 2006, 2007, 2008), another study could not conﬁrm this observation (Huang et al. 2008). As these non D-GalCer – reactive NKT cells form a very heterogeneous group of different cells and constitute at most organs sites for less than 1% of the T cells, the identiﬁcation of single cell populations within these type II NKT cells is likely to be very problematic and may explain at least in part some of the discrepant results. Nonetheless, type II NKT cells have been described as critical for the promotion of liver pathology in a hepatitis B virus model (Vilarinho et al. 2007) and may play a role in the suppression of tumor immunity (Terabe and Berzofsky 2008). Although some progress has been made in characterizing and classifying these diverse NKT cell populations, their functional role and physiological ligands have remained unknown so far.

3.2.3

Group III CD1 Molecules (CD1e)

CD1e is present in many different animal species (De Libero and Mori 2006). CD1e protein is non-covalently associated with E2-microglobulin and shares an overall structure similar to that of the other CD1 molecules. However, rather than presenting antigens to TCRs, CD1e allows the processing and loading of microbial antigens with a large carbohydrate component onto CD1b as shown for phosphatidylinositol-mannosides (PIM6) (de la Salle et al. 2005). Thus, CD1e is indispensable for processing of glycolipids and lipoglycans to become optimally immunogenic and to ensure effective protective immunity to microbes expressing these antigens.

3.2.4

Species Distribution of CD1 Molecules

The CD1 system is highly conserved (Porcelli and Modlin 1999; Beckman and Brenner 1995) with CD1 analogues found in almost all mammalian species (Bendelac et al. 2007; Dascher and Brenner 2003) and in chicken (Miller et al. 2005; Salomonsen et al. 2005). The existence of CD1 in birds suggests that CD1 or its immediate ancestor was present in an ancient version of the MHC locus that arose several 100 million years ago as part of the evolving, early adaptive immune system. It is suspected that even jawed ﬁsh may contain CD1 (Dascher 2007). Different CD1 isotypes specialize in sampling partially overlapping sets of lipids in different cellular compartments, including the endosome and lysosome through tyrosine containing cytoplasmic motifs that bind adaptor proteins for clathrinmediated endocytosis. CD1 isoforms are also very diversely distributed in mammalians (Barral and Brenner 2007) and the functional consequences of this diverse distribution for the single species are not known; for example, humans contain one CD1a, b, c, d and e allele. Guinea pigs instead possess an extended family of CD1 genes (Dascher et al. 1999), where more than 11 potential CD1 genes including four CD1B and three CD1C orthologs were identiﬁed, whereas CD1d is the only

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representative in mice (Brigl and Brenner 2004). Rabbits show another pattern of distribution for CD1 expression: two CD1A genes and one CD1E gene (Hayes and Knight 2001) as well as orthologs for CD1B and CD1D have been reported (Calabi et al. 1989). Although iNKT cells, based on genomic and functional studies of CD1d, may be absent in cows (Van Rhijn et al. 2006), group I CD1 genes are detected predisposing this animal species for the analysis of Mycobacteria-infection and group I CD1 restricted T cells in vivo. In addition, the expression of the CD1 isoforms on different immune cells in humans is very diverse: CD1a is widely used as a key marker of Langerhans cells, although several DC subsets express CD1a as well (Crawford et al. 1989; Fithian et al. 1981; Sholl et al. 2007). Human Langerhans cells typically lack CD1d in situ, while dermal DCs and monocyte-derived DCs express CD1d (Caux et al. 1992; Gerlini et al. 2001; Nestle et al. 1993; Ochoa et al. 2008). The distribution of CD1 isoform expression and the accumulation of distinct immune cells at the site of infection/inflammation may therefore have important implications for the protection/susceptibility to certain pathogens, malignant diseases or inﬂammatory/ autoimmune disorders. As iNKT cells are the best characterized cell population and their biology can be studied in vivo in mice, we will focus in the following paragraphs exclusively on iNKT cells.

3.3

iNKT Cell Function

Our understanding of the function of iNKT cells is largely based on disease studies in iNKT cell-deﬁcient CD1d0 or JD180 mice (Swann et al. 2004). iNKT cells play a role in the detection of bacteria and viruses as different as Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella typhimurium, Mycobacterium tuberculosis, Listeria monocytogenes, Borrelia burgdorferi, Chlamydia spp., inﬂuenza, encephalomyocarditis, coxsackie B3 virus or cytomegalovirus (Nieuwenhuis et al. 2002; Kawakami et al. 2003; Brigl et al. 2003; Behar et al. 1999a; Szalay et al. 1999; Ranson et al. 2005; Kinjo et al. 2006; Kumar et al. 2000; Joyee et al. 2007, 2008; De Santo et al. 2008; Ilyinskii et al. 2006; Huber et al. 2003; Broxmeyer et al. 2007). iNKT cells were also shown to be key players in the natural rejection of chemicallyinduced primary sarcomas (Crowe et al. 2002), as well as transplanted tumors, either by enhancing or suppressing their immune rejection through the release of Th1 or Th2 cytokines, respectively. Again, neither the mechanism of iNKT cell activation nor the bias towards Th1 or Th2 cytokines in the respective mouse models has been elucidated so far. A role of iNKT cells in non-infectious inﬂammatory processes such as type I diabetes (T1D) (Gombert et al. 1996; Falcone et al. 1999; Godfrey et al. 1997; Hong et al. 2001; Sharif et al. 2001), allergic asthma (Akbari et al. 2003; Lisbonne et al. 2003; Meyer et al. 2007, 2008), systemic lupus erythematodes (SLE) (Zeng et al. 1998, 2000, 2003; Forestier et al. 2005) and ulcerative colitis (Fuss et al. 2004; van Dieren et al. 2007) has been reported as well.

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The involvement of iNKT cells in such diverse conditions is perplexing because it is unclear what common antigens could be induced and what the mechanisms of iNKT cell activation are. However, iNKT cells have been shown to be inherently autoreactive (Bendelac 1995a, b, c) and their autoreactivity is under the control of inhibitory NK receptors suggesting a model whereby induction of the self antigen(s) of iNKT cells combined with the loss of inhibitory MHC I ligands would result in their net activation. In addition, the fortuitous discovery of an agonist iNKT cell ligand, alpha-GalactosylCeramide (D-GalCer) (Kawano et al. 1997) and its use in vivo to characterize the consequences of iNKT cell activation, have shed some light on the cellular circuits and molecular mechanisms involved. D-GalCer was isolated from marine sponge extracts based on their ability to elicit potent rejection

a

Hydroxyl groups Anomericity

a-GalCer

HO

a-D-Galactose

OH O

HO

HN

OH

HO O

Linkage

Ceramide portion

Sphingomonas/Novosphingobium GSLs Glucuronosyl Series

HO

OH

HO HO

Sphingosine base

OH

Sugar moiety

b

Acyl chain

O

COOH O

O HO

HN

HO

HO O OH

c

OH

O HN

O OH

HO

iGb3

Galacturonosyl Series COOH O

HO

OH O HO

OH

O

OH O

O OH HO

O OH O

HN O

HO

iGb3

OH

Gal a1,3 Gal b1,4 Glc b1,1 Ceramide

Fig. 3.1 Glycosphingolipid (GSL) antigens of iNKT cells. (a) Marine sponge derived A-GalCer (KRN 7000), the prototypical iNKT cell ligand with a detailed description of its single components. (b) GSL-1 of Sphingomonas/Novosphingobium spp. has structural analogies with KRN 7000; GSL-1 containing either one glucose or one galactose head (alpha-glucuronylceramide or alphagalacturonylceramide) is a potent stimulator of cytokine production by iNKT cells compared to GSL-3 and GSL-4 that are not immunogenic. The rearrangement of their cell wall GSL composition may be used by Sphingomonas/Novosphingobium spp. to evade their recognition by the innate immune system. (c) Mammalian isoglobotrihexosylceramide (iGb3), or GalA1, 3GalB1, 4GlcB1, 1Cer, one of the endogenous physiological iNKT cell ligands. Note that the proximal glucose of iGb3 has a beta-anomeric linkage to ceramide, in contrast with the alpha-branched galactose or glucose of A-GalCer or Sphingomonas/Novosphingobium GSLs. However, the terminal sugar in the alpha-linkage appears to be predominantly responsible for the antigenicity of iGb3

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G IFN-

-G IFN

-G

MO

CD8+ N IF

NK 40 /CD L 40 CD

CD4+ DC

iNKT IL-12 ? Gr

47

B IL-4

?

CD4+ CD25+

Fig. 3.2 Innate-like iNKT cell functions and the cellular and molecular network activated downstream of iNKT cell stimulation. Cellular and molecular networks activated by iNKT cells upon recognition of A-GalCer by the iNKT cell TCR. DCs and macrophages/Kupffer cells are the center of a cellular network of cross-activation, starting with the presentation of A-GalCer by CD1d to the iNKT cell TCR and the subsequent upregulation of CD40L on iNKT cells upon A-GalCer recognition, secretion of Th1 and Th2 cytokines and chemokines, and DC mediated priming of adaptive CD4 and CD8 T cell responses. iNKT cells provide also direct help to B cells for antibody production or indirect help via the activation of T helper cells; iNKT cells also rapidly trans-activate NK cells. Although interactions of iNKT cells with granulocytes and Tregs are suspected, no experimental evidence for the underlying mechanisms for these interactions has been provided to date

of B16 melanoma cells in mice (Kobayashi et al. 1995; Morita et al. 1995). D-GalCer consists of a sugar head group and a ceramide tail that can be divided in an acyl chain and a sphingosine base (Fig. 3.1). In vivo, it was shown using systemic administration of D-GalCer that iNKT cells participate in a prompt and widespread cellular and molecular activation cascade (Fig. 3.2). The sequence initially involves cognate interaction with, and cross-activation of CD1d expressing dendritic cells (DCs) and macrophages through CD40L/CD40 interactions, explosive release of cytokines and chemokines and powerful trans-activation of natural killer (NK) cells within 30–60 min (Ishikawa et al. 2005b; Fujii et al. 2003, 2004; Carnaud et al. 1999). Likewise, B cells, which also express CD1d, are activated to upregulate their costimulatory properties (Galli et al. 2003a, b) and antibody production referentially releasing IgG2a antibodies (Mattner et al. 2008). Besides these cognate interactions (Galli et al. 2003a, b; Mattner et al. 2008; Leadbetter et al. 2008), iNKT cells can help B cells also indirectly, via the enhancement of T helper (Th)-B-cell

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interactions (Galli et al. 2007; Tonti et al. 2009) and/or the release of cytokines or chemokines. iNKT cell ligands have been shown to constitute one of the most efﬁcient adjuvant available (Fujii et al. 2003; Gonzalez-Aseguinolaza et al. 2002), particularly for antibody production and for priming cytotoxic T lymphocytes (CTLs) against non-replicating antigens, and are widely used in clinical trials (Giaccone et al. 2002; Nieda et al. 2004; Ishikawa et al. 2005a; Chang et al. 2005). In summary, the anti-microbial activity of iNKT cells could be due to direct elimination of CD1d expressing infected antigen presenting cells (APCs), cross-activation of NK cells, priming of MHC class I restricted anti-pathogen CTLs, and/or indirect cytokine/chemokine effects on the infected tissue or cell populations. The respective mechanisms involved in defending deﬁned bacteria, viruses, parasites and fungi need to be elucidated in the respective infection models in the future.

Fig. 3.3 Loading of CD1d with GSLs within the late endosome/lysosome. Newly biosynthesized CD1d molecules reach the plasma membrane and are internalized through an AP-2/AP-3 clathrindependent pathway and trafﬁcked to late endosomal/lysosomal compartments, where the exchange of GSLs is performed by lipid transfer proteins (LTPs). One important group of LTPs constitute the saposins that facilitate also the loading of CD1d with GSL antigens. CD1d extensively recycles between lysosome and plasma membrane, allowing further GSL exchange. Exogenous lipids bound to lipoproteins may enter the cell with VLDL (very low density lipoprotein) particles through the LDL receptor pathway, whereas microbial lipids can be released in the lysosome after fusion with the microbial phagosome and digestion of the microbe. The endogenous ligand iGb3 is produced through lysosomal degradation of iGb4 by B-hexosaminidase. Upon recognition if the sugar head of GSL antigens by the iNKT cell TCR, iNKT cells rapidly release copious amounts of Th1 and Th2 cytokines and chemokines

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3.4

49

CD1d Mediated Presentation of GSLs

CD1d is a conserved family member of the MHC like CD1 molecules expressed on hematopoietic cells and APCs, whose general function is to capture cellular and microbial GSLs for presentation to the iNKT cell TCR (Bricard and Porcelli 2007). The CD1d lipid binding groove is composed of connected hydrophobic channels where the fatty acid (=ceramide) portion of the GSL is buried, whereas the polar sugar head is exposed for recognition by the iNKT cell TCR (Brigl and Brenner 2004; Zajonc et al. 2005; Koch et al. 2005; Borg et al. 2007). Mouse and human CD1d collect GSLs from the late endosome and lysosome, and their antigen presenting capacities are tightly dependent on lysosomal functions (Joyce and Van Kaer 2003). Endosomal trafﬁcking is required for CD1d to acquire the natural ligands of iNKT cells in APCs (Chiu et al. 2002), and recently characterized lysosomal saposins and microsomal triglyceride transfer protein (MTP) as the lipid transfer proteins (LTPs) promote the editing of the GSL antigens (Zhou et al. 2004a; Winau et al. 2004; Kang and Cresswell 2004; Brozovic et al. 2004; Dougan et al. 2005, 2007) (Fig. 3.3). Saposins are also critical for the presentation of the natural endogenous iNKT cell ligand(s) by CD1d on the cell surface, as shown by the absence of iNKT cell activation in co-culture with saposin-deﬁcient DCs in autoreactivity assays (Bendelac et al. 2007).

3.5

Self-GSL Antigens for CD1d Molecules

Reactivity against CD1d molecules in the absence of exogenous ligands like D-GalCer is a hallmark of peripheral iNKT cells and a requirement for their thymic selection and differentiation (Bendelac 1995a, b, c; Chen et al. 1997). Presentation of endogenous GSLs by CD1d underlies this autoreactivity of iNKT cells. As no experimental evidence has been reported to date that mammalian species can synthesize alphalinked GSLs, beta-linked GSLs residing within the late endosomes or lysosomes were considered as potential endogenous candidate antigens (Chiu et al. 1999, 2002; Bendelac et al. 1995; Roberts et al. 2002; Park et al. 1998; Spada et al. 1998; Stanic et al. 2003). Although being controversially discussed to date (Speak et al. 2007; Porubsky et al. 2007; Burrows et al. 2009), isoglobotrihexosylceramide (iGb3) has been identiﬁed as one of the physiological endogenous ligands underlying the autoreactivity and selection of iNKT cells (Zhou et al. 2004b; Li et al. 2009) (Fig. 3.1c). iGb3 stimulates human and murine iNKT cells, albeit its stimulatory capacity is lower than the one of D-GalCer (Zhou et al. 2004b; Xia et al. 2006). The activation of iNKT cells by iGb3 is blocked by the absence of CD1d in mice and by the application of anti-CD1d antibodies in humans and the autoreactivity of VD14 expressing iNKT cell hybridomas is lost when stimulated with thymocytes from littermates lacking the E-hexosaminidase B (=Hexb) enzyme. This E-hexosaminidase B enzyme is required for the generation of iGb3 by the mammalian GSL degradation pathways.

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However, iGb3 is also synthesized by the iGb3 synthase in the lysosome. Thus, the generation of GSLs is tightly controlled and the existence of two iGb3 generation pathways emphasizes the importance of the endogenous GSL supply for iNKT cells. These dual pathways may have more broad implications outside iNKT cell immunology and may be also important for the maintenance of some other likely evolutionary conserved functions as the GSL metabolism can be regulated at multiple levels, including through the control of enzyme expression, post-translational modiﬁcations and allosteric mechanisms (Bendelac et al. 2007). Both enzymes E-hexosaminidase B and iGb3 synthase are ubiquitously expressed in different tissues of mice (Speak et al. 2007). In the lysosome/late endosome of APCs, iGb3 can be loaded into CD1d and presented to the iNKT cell TCR as CD1d is constantly cycling between the late endosome/lysosome and the cell surface. Selflipid antigens like iGb3 may be also important in the folding and stable assembly of CD1d molecules. In some cases these endogenous antigens can be replaced by microbial antigens or other self-antigens while they trafﬁc through the endocytic system. In general, the self-lipids reported to date are weaker agonists than the microbial antigens (see below) or D-GalCer (Bendelac et al. 2007; Zhou et al. 2004b; Gumperz et al. 2000). In addition, changes in the self-GSL repertoire in APCs upon microbial exposure have been found to skew towards more stimulatory self-lipids (De Libero et al. 2005; Paget et al. 2007) which may enhance autoreactivity of iNKT cells, although accessory signals are likely required for the full activation of iNKT cells (see below).

3.6

Activation of iNKT Cells During Bacterial Infection

Although iNKT cells are activated during infection with many different microbes including bacteria, viruses, parasites and fungi (summarized in (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005)), the mechanisms of iNKT cell activation have often not been elucidated. However, two alternative strategies for iNKT cell activation by Gram-negative bacteria have been described that induce functionally different iNKT cell responses. These two pathways that distinguish between the activation of iNKT cells by endogenous and exogenous GSL antigens likely have broader implications for iNKT activation by other pathogens:

3.6.1

Indirect, Bystander Activation of iNKT Cells by Gram-Negative, LPS-Positive Bacteria

Autoreactivity induced by endogenous GSL ligands like iGb3 and the presence of interleukin-12 (IL-12), triggered by Toll-like receptor (TLR) signaling of infected APCs, are required for the early secretion of IFN-J by iNKT cells during

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immune responses against Gram-negative, LPS-positive bacteria (Brigl et al. 2003; Mattner et al. 2005) (Fig. 3.4a). TLR signaling on APCs may be thereby not only important for the induction of inﬂammatory cytokines, but also for the accumulation of endogenous GSL antigens that may enhance the autoreactivity of iNKT cells (De Libero et al. 2005; Paget et al. 2007). In this context, it has been reported that Toll-like receptor (TLR) signaling inhibited alpha-Gal-A activity in APCs triggering subsequent accumulation of GSL antigens including iGb3 in the lysosome and enhanced iNKT cell activation (Darmoise et al. 2010). However, microbial invasion is detected by TLRs expressed on APCs, and iNKT cells function downstream of this primary event. iNKT cells are also just one of several IFN-J-producing cell-types recruited during infection with Gram-negative, LPS-positive bacteria like Salmonella (Brigl et al. 2003). This may explain why iNKT-deﬁcient mice do not appear to be particularly susceptible to Salmonella.

Fig. 3.4 Dual recognition of self and microbial GSLs during microbial infections. (a) Infection by Gram-negative, LPS-positive bacteria like Salmonella activates TLR4 through LPS and induces IL-12, augmenting iNKT cell autoreactivity induced by endogenous GSL ligands like iGb3 (indirect microbial recognition). (b) Infection by Gram-negative, LPS-negative Sphingomonas/ Novosphingobium spp. induces the direct activation of iNKT cells through recognition of microbial cell wall alpha-glucuronylceramides or alpha-galacturonylceramides. Subsequent DC activation through CD40/CD40L interactions and IL-12 release augments the activation of iNKT cells

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3.6.2

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Direct, Cognate Recognition of GSL Antigens in the Cell Wall of Gram-Negative LPS-Negative Alphaproteobacteria

Another pathway of iNKT cell activation involves the direct recognition of microbial GSLs in the cell wall of Gram-negative alphaproteobacteria (Mattner et al. 2005; Kinjo et al. 2005; Sriram et al. 2005) that substitute for lipopolysaccharide (LPS) (Kawahara et al. 1999; Kawahara et al. 2001). Speciﬁcally, alphagalacturonylceramides or alphaglucuronylceramides in the cell wall of Sphingomonas/ Novosphingobium spp. have been identiﬁed as strong iNKT cell antigens (Mattner et al. 2005, 2008; Kinjo et al. 2005) (Fig. 3.1b). Thereby, only the alphagalacturonylceramides or alphaglucuronylceramides (summarized as GSL-1) that express one sugar were stimulatory (Long et al. 2007). Alphagalacturonyl- or -glucuronylceramides containing three or four sugars (summarized as GSL-3 and GSL-4) did not stimulate iNKT cells in co-culture with DCs and are likely not truncated into GSL-1 as the glycosidase necessary to cleave glucosamine from GSL-3 and 4 might be absent or inactive in the lysosom. Although most Sphingomonas/Novosphingobium spp. express preferentially GSL-1, changes in the ratio of GSL-1 to GSL-3 and - 4 might be exploited by bacteria to avoid detection under certain circumstances. Nonetheless, iNKT cell-deﬁcient CD1d−/− and JD18−/− mice clear an infection with Sphingomonas/Novosphingobium and other LPS-negative alphaproteobacteria like Ehrlichia (Lin and Rikihisa 2003) slower than wild type mice (Mattner et al. 2005; Kinjo et al. 2005; Sriram et al. 2005; Stevenson et al. 2008). Thus, iNKT cells and CD1d represent a major innate recognition pathway for this class of bacteria. As both bacterial species are ubiquitously found in the environment (Barbeau et al. 1996; Brodie et al. 2007; Cavicchioli et al. 1999; Selmi et al. 2003; Shi et al. 2001) and as TLRs appear to be partially (Mattner et al. 2005, 2008) or completely (von Loewenich et al. 2004) dispensable for their detection, it is tempting to speculate that iNKT cells may have evolved due to microbial pressure to speciﬁcally target alphaproteobacteria lacking cell-wall ligands for TLRs. Intravenous injection of high doses of Sphingomonas causes also septic shock in wild type, but not iNKT cell-deﬁcient mice highlighting the importance of iNKT cells in promoting pathological cytokine storms (Mattner et al. 2005). Although some of the remaining TLR ligands in Sphingomonas may induce DC activation, its cell wall GSLs subsequently induce DC activation, probably through CD40L/CD40 interaction with iNKT cells (Fig. 3.3) and subsequent IL-A2 production, as reported for D-GalCer (Fujii et al. 2003, 2004) suggesting that preferentially the iNKT cell signal is critical for the activation of the innate immune response. These distinct innate detection pathways for the two groups of bacteria may not only affect early immune responses, but also inﬂuence the adaptive immune reaction. This discovery of GSL antigens in LPS-negative alphaproteobacteria may even have broader implications: it is known that extracts from the marine sponge Agelas mauritianus have different immunological properties depending on the season and their location. As this marine sponge was the original source where D-GalCer was identiﬁed from and as these sponges are often colonized by D-proteobacterial

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symbionts, particularly by Sphingomonas spp. (Dieckmann et al. 2005) which have been also described as coral pathogens (Cavicchioli et al. 1999; Rosenberg and Ben-Haim 2002; Miller et al. 2003), the marine sponge D-GalCer may in fact have originated from bacterial symbionts. The presence of iNKT cell antigens within the marine environment supports the suspicion that CD1 may be even present in jawed ﬁsh (Dascher 2007). Alpha-galactosyldiacylglycerols expressed by Borrelia burgdorferi (Kinjo et al. 2006), the Gram-negative LPS-negative agent of Lyme disease, as well as puriﬁed phosphatidylinositolmannoside (PIM4) of Mycobacterium tuberculosis (Fischer et al. 2004) have been also described as microbial iNKT cell ligands.

3.7 3.7.1

Activation of iNKT Cells During Non-bacterial Infection Viral Infections

Several reports have also addressed the role of iNKT cells during viral infections: for example, iNKT cells accelerate the clearance of inﬂuenza A (De Santo et al. 2008), encephalomyocarditis (Ilyinskii et al. 2006) or coxsackie B3 virus (Huber et al. 2003), whereas the viral burden and the subsequent course of infection remains unaffected in the absence of iNKT cells during infections with lymphocytic choriomeningitis virus (Roberts et al. 2004), mouse cytomegalovirus (Broxmeyer et al. 2007), and vaccinia virus (Renukaradhya et al. 2005) despite iNKT cell activation. Although these studies implicate a role of iNKT cells in the detection of these viral pathogens, other viruses have developed mechanisms to evade the immune recognition by iNKT cells: HSV-1 speciﬁcally inhibits the recycling of CD1d from the lysosome to the cell surface (Yuan et al. 2006), an essential pathway for GSL antigen presentation to iNKT cells. The HIV proteins Nef and gp120 downregulate the expression of CD1d on the cell surface as well (Chen et al. 2006; Cho et al. 2005; Hage et al. 2005), and the infection of T cells expressing CD4 may contribute to reduced iNKT cell numbers in HIV patients, although no studies have investigated that question and its functional consequences to date. However, absence of iNKT cells may contribute to the lethal outcome of infections with Epstein-Barr virus in patients with X-linked lymphoproliferative (XLP) immunodeﬁciency syndrome due to SAP and XIAP mutations (Pasquier et al. 2005; Rigaud et al. 2006; Nichols et al. 2005). Although SAP-Fyn signaling is important for the development of iNKT cells (Griewank et al. 2007) and although SAP deﬁcient mice do not as efﬁciently clear gammaherpesvirus 68, the equivalent of EBV as wild type mice (Yin et al. 2003), patients with XLP suffer from additional immune defects affecting preferentially CD8+ T and NK cell responses that may contribute to the susceptibility to EBV. Those defects need to be evaluated in the context of iNKT cell deﬁciency. The abundance of iNKT cells in the liver may on the other hand contribute to the antiviral effects of D-GalCer in a transgenic Hepatitis B virus (HBV) model (Kakimi et al. 2000);

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however, the application of D-GalCer as monotherapy for patients with chronic hepatitis B infection resulted in a strong decrease of iNKT cells and did not clearly affect HBV DNA and alanine aminotransferase levels (Woltman et al. 2009). The nature of the iNKT cell ligand(s) and the mechanism(s) of iNKT cell activation involved in these infectious conditions remain to be determined. It also needs to be elucidated which of these effects reﬂect a speciﬁc viral evasion or an immune defense strategy (Bendelac et al. 2007).

3.7.2

Parasitic, Helminth and Fungal Infections

Glycosylphosphatidylinositol (GPI)-anchored surface antigens of Plasmodium have been reported as iNKT cell ligands (Schoﬁeld et al. 1999). GPI-activated iNKT cells promote the production of IgG antibodies to the circumsporozoite antigens of malaria, one of the key components of protective immune responses in humans. However, additional studies could not conﬁrm the role of GPIs in iNKT cell activation and the CD1d dependency of antibody responses (Molano et al. 2000; Romero et al. 2001). iNKT cells are also activated during infections with other parasites including different Trypanosoma, Leishmania and Schistosoma spp.; however, the functional relevance of this activation appears to be species speciﬁc and the anti-parasite responses may be controlled by different iNKT cell subsets (Duthie and Kahn 2005, 2006; Duthie et al. 2005a, b; Procopio et al. 2002; Stanley et al. 2008; Wiethe et al. 2008; Campos-Martin et al. 2006; Mattner et al. 2006; Ishikawa et al. 2000; Mallevaey et al. 2006, 2007). It is believed that TLR-induced IL-12 or other inﬂammatory cytokines augment the CD1d-mediated iNKT cell autoreactivity during most of these infections (indirect, by-stander iNKT cell activation – see also Sect. 3.6.1). However, a subset of liver iNKT cells recognizes lipophosphoglycan of Leishmania donovani (Amprey et al. 2004) (direct, cognate recognition of iNKT cell antigens – see also Sect. 3.6.2) and the seize of the pool for this iNKT cell subset may control parasitic replication. iNKT cells also contribute to the clearance of Leishmania spp. in visceral organs (Mattner et al. 2006); however, parasite elimination at the peripheral sites during cutaneous Leishmaniasis may be mainly mediated by iNKT cell-independent mechanisms (Mattner et al. 2006) considering the very low iNKT cell numbers in peripheral lymph nodes (<0.2%) compared to spleen (1%) and liver (up to 30%). Although CD4+ NK1.1+ T lymphocytes have been identiﬁed as a cellular source for protective IL-2 during early Toxoplasma gondii infections (Denkers et al. 1996), it remained unclear if these cells were identical with CD1d-restricted iNKT cells. DCs pulsed with Schistosoma mansoni eggs induced cytokine release by iNKT cells in a MyD88-independent manner, suggesting alternative pathways of recognition similarly as suggested during infections with the fungus Cryptococcos neoformans (Cohen et al. 2009). The contribution of iNKT cells to immunity against other signiﬁcant fungal pathogens such as Candida albicans and Aspergillus fumigatus remains to be assessed (Bendelac et al. 2007).

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55

Human Studies Correlation of iNKT Cell Numbers with Susceptibility to Infection

The relative numbers of VD24 iNKT cells in humans are lower in all tissues compared to mice, although humans contain a large cellular fraction of NK cell receptor expressing NKT cells (particularly CD161) that may not be CD1d-restricted. However, CD1d-restricted iNKT cells are also preferentially found in the liver in humans and the overall distribution of iNKT cells appears to be comparable to mice. The range of frequencies of VD24 NKT cells, however, found in human blood varies by up to 500-fold between individuals and iNKT cell numbers may be under strict genetic control, as shown by identical twin studies (Lee et al. 2002) and/or under the inﬂuence of environmental factors. No convincing association of iNKT cell numbers with a particular disease (Chan et al. 2009) or the susceptibility to a speciﬁc infection has been reported. Although absence of iNKT cells may be involved in the lethal outcome of infections with Epstein-Barr virus in patients with X-linked lymphoproliferative (XLP) immunodeﬁciency syndrome due to SAP and XIAP mutations (Pasquier et al. 2005; Rigaud et al. 2006; Nichols et al. 2005), other immunological defects may contribute to this fatal outcome as particularly NK cell and CD8+ T cell numbers/functions are also impaired. Most interestingly, patients with Primary Biliary Cirrhosis (PBC), a chronic cholestatic liver disease, but not healthy individuals reveal a striking redistribution of VD24 iNKT cells from the blood to the livers and an aberrant expression of CD1d on bile duct epithelial cells (Kita et al. 2002; Harada et al. 2003; Tsuneyama et al. 1998). Considering the fact that they are abundantly found in the liver, iNKT cells may have important implications in detecting bacteria like Sphingomonas/Novosphingobium spp. and their GSL antigens that circulate through the liver (see below). iNKT cell activation in this context may not only accelerate bacterial clearance, but may also augment tissue-speciﬁc inﬂammation and assist in the propagation and/or expansion of auto-reactive lymphocyte populations.

3.8.2

Infections with Novosphingobium/Sphingomonas Spp. in Clinical Settings

Novovosphingobium/Sphingomonas spp. form a gram-negative bacterial genus of the Sphingomonodaceae family that is found ubiquitously in the environment (Barbeau et al. 1996; Brodie et al. 2007; Cavicchioli et al. 1999; Shi et al. 2001), at mucosal surfaces and in the feces of humans (Selmi et al. 2003). Strains of Sphingomonas/Novosphingobium exhibit xenobiotic-metabolizing properties and degrade a wide variety of environmentally hazardous compounds, including

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polycyclic aromatics (Pinyakong et al. 2003), dioxine compounds (Halden et al. 1999) and chlorinated phenols (Ederer et al. 1997). Although the number of clinical case reports describing nosocomial infections and septic shock associated with Sphingomonas spp. is limited (Kilic et al. 2007; Hsueh et al. 1998; Perola et al. 2002), one has to keep in mind that Sphingomonas/Novosphingobium spp. had to be renamed because of their unique cell wall composition (Yabuuchi et al. 1990). Preferentially Sphingomonas paucimobilis – formerly Pseudomonas paucimobilis – has been implicated in a variety of community-acquired and nosocomial infections, including bacteremia, catheterrelated sepsis, meningitis, peritonitis, pneumonia, cutaneous infections, visceral abscesses, urinary and biliary tract infections, adenitis, and diarrheal disease (Glupczynski et al. 1984; Morrison and Shulman 1986; Peel et al. 1979; Reina et al. 1991; Salazar et al. 1995; Saltissi and Macfarlane 1994; Southern and Kutscher 1981; Martino et al. 1996). The lack of a typical lipopolysaccharide (LPS) constituent of the cellular membrane of Sphingomonas/Novosphingobium spp., with the accompanying deﬁciency of endotoxin activity, may explain the lack of deaths attributed to this organism (Kawahara et al. 1999, 2001).

3.8.3

Association of Primary Biliary Cirrhosis with Novosphingobium Aromaticivorans

3.8.3.1

Role of GSL Recognition from Novosphingobium spp. by iNKT Cells in the Pathogenesis of Primary Biliary Cirrhosis (PBC)

The outer membrane of Gram-negative bacteria contains characteristically lipopolysaccharide (LPS), which consists of lipid A, core polysaccharide, and O antigen outside the peptidoglycan layer. Although belonging to the group of Gramnegative bacteria, alpha-proteobacteria like Sphingomonodaceae (Kawahara et al. 1999, 2001) or Ehrlichia spp. lack LPS (Lin and Rikihisa 2003). Sphingomonodaceae use instead GSLs to build their cell wall (Kawahara et al. 1999, 2001), which are speciﬁcally recognized by iNKT cells. iNKT cells target Sphingomonodaceae and, in the absence of TLR4 activation by LPS, dominate the innate immune response (Mattner et al. 2005; Kinjo et al. 2005). A latent, unrecognized infection with Novosphingobium spp. or related alphaproteobacteria may therefore account for the increased iNKT cell numbers and the enhanced expression of CD1d on biliary epithelial cells that are observed in the livers of PBC patients (Kita et al. 2002; Harada et al. 2003; Tsuneyama et al. 1998).

3.8.3.2

Pathogenesis of Primary Biliary Cirrhosis (PBC)

PBC is a chronic inﬂammatory liver disease that usually progresses to liver failure and death unless liver transplantation is performed. Its pathophysiologic hallmarks include the immune-mediated destruction of small bile ducts by lymphocytes and

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the production of autoantibodies to mitochondrial antigens (AMAs) that bind the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). These subunits are both the dominant autoreactive B cell epitopes and the main antigens recognized by liver-inﬁltrating, autoreactive T cells (Selmi et al. 2004; Gershwin et al. 2000). However, it is not understood to date, why these mitochondrial antigens are speciﬁcally targeted and why the immune reaction is restricted to the liver, although the auto-antigen is ubiquitously expressed in all tissues.

3.8.3.3

Speciﬁc Recognition of PDC-E2 Homologues in Novosphingobium aromaticivorans by Sera of PBC Patients

Several Sphingomonas/Novosphingobium strains are xenobiotics, a feature that may impact on steroid metabolism and autoimmunity in humans. Notably, there is compelling evidence that patients with Primary Biliary Cirrhosis (PBC) express antibodies against various lipoylated enzymes of Novosphingobium aromaticivorans including the bacterial homolog of the mitochondrial enzyme PDC-E2, the major PBC antigen (22). In fact, seropositivity for Novosphingobium is found in seronegative PBC patients and is therefore more closely associated with disease than the presence of anti-PDCE2 antibodies. Seropositivity for Novosphingobium highly speciﬁc of PBC and not found in healthy subjects or patients with other diseases. Analysis of mitochondrial encoded genes and their genomic organization/distribution imply that mitochondrial genomes are derived from an alphaproteobacterium (−like) ancestor that invaded an Archea-type host more than 1.5 billion years ago (Dyall et al. 2004). This evolutionary association of alphaproteobacterial and mammalian mitochondrial antigens might be critical for the development of PBC (molecular mimicry) and the target speciﬁcity of the immune attack to mitochondrial enzymes. Although these anti-PDC-E2 antibodies reﬂect the hallmark of PBC, their role in the pathogenesis of PBC has remained unknown. Similar as in many other autoimmune diseases, there has not been established an association between the titers and the severity/progression of disease and the organ-speciﬁc autoimmune attack despite ubiquitous autoantigen expression. Based on these clinical reports it is therefore tempting to speculate that iNKT cells may break B and/or T cell tolerance to crossreactive self antigens.

3.9

Mouse Model of Infection-Induced Primary Biliary Cirrhosis

Notably, we have replicated these clinical reports in mice establishing a model where infection of mice with Novosphingobium aromaticivorans induces antiPDC-E2 IgG responses and autoimmune biliary liver lesions resembling PBC in humans (Mattner et al. 2008). Although iNKT cells signiﬁcantly accelerate the clearance of Novosphingobium aromaticivorans, their activation upon microbial encounter appears to be deleterious for the host, providing innate signals that

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contribute to the breakdown of tolerance by autoimmune effector cells. The greater persistence of Novosphingobium in the liver after infection than in other organs and the activation of iNKT cells, which are particularly abundant in the liver of mice and redistributed to the livers of PBC patients, by Novosphingobium-GSLs likely explain the liver speciﬁc pathology. Although iNKT cells may directly damage liver tissue as indicated by mechanisms involving CD1d-targeted killing, as reported in tumor models (Song et al. 2009; Nicol et al. 2000), iNKT cells more likely promote the expansion of autoreactive T and/or B cells that are attracted to the site of inﬂammation in the liver and subsequently confer tissue damage. In contrast, TLR signaling alone, in the case of many other bacteria, fails to provide similar helper signals as suggested by the drastically reduced anti-PDC-E2 IgG titers in Novosphingobiuminfected CD1d-deﬁcient compared to wild-type mice. Direct cognate interactions between iNKT and B cells are required to produce anti-PDC-E2 IgG2a responses, the subclass of auto-antibodies that is in particular considered pathogenic. Once disease is established in the mouse model, liver lesions can be adoptively transferred by conventional CD4+ and CD8+ T cells from wild type, but not iNKTdeﬁcient mice after bacterial clearance into irradiated congenic recipient mice. This illustrates the importance of early microbial activation of iNKT cells in initiating chronic autonomous, organ-speciﬁc autoimmunity. This model does not only unleash an innate immune mechanism for the organ speciﬁc development of (auto-) immune responses to ubiquitously expressed antigens, but also reﬂects the situation in humans that is characterized by the redistribution of iNKT cells from the blood to the liver. The unique innate signals associated with Novosphingobium infection may promote the expansion of PDC-E2 reactive B and T cells (that may even be only present at low frequency) as iNKT cell mediated help for co-administered protein antigens is a well-established concept (Ishikawa et al. 2005a, b; Fujii et al. 2003; Gonzalez-Aseguinolaza et al. 2002; Giaccone et al. 2002; Nieda et al. 2004; Chang et al. 2005). Although the inherent autoreactivity to self GSL antigens may well underlie the role of iNKT cells in some forms of autoimmunity, the discovery of microbial GSL ligands of iNKT cells suggests an alternative, complementary scenario whereby unrecognized infection by alphaproteobacteria may drive autoimmunity. This hypothesis, which challenges the idea that autoreactivity is the “be all and end all” pressure for iNKT cells, is particularly relevant in the context of our infection induced PBC model that replicates the recent clinical reports associating human PBC with a striking antibody response against Sphingomonas/Novosphingobium and with tissue redistribution of iNKT cells to the liver.

3.10

Overall Summary of iNKT Cell Activation

Invariant NKT cells play a critical role in alerting the immune system of microbial infections. Whereas their activation contributes in some cases to the elimination of the pathogens, their activation upon microbial encounter might be sometimes

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detrimental for the host as exemplarily shown for Novosphingobium-infection in the mouse model of autoimmune biliary disease. The identiﬁcation of microbial iNKT cell antigens considerably reinforces the view that iNKT cells and their canonical mouse VD14-JD18 or human VD24-JD18 TCRs evolved to recognize conserved ligands and to perform innate-like rather than adaptive functions. Based on their potent adjuvant functions, vaccination strategies with iNKT cells ligands may provide new tools for the prevention of infectious diseases, considering the fact that some of the pathogens re-emerge due to altered migration features of the population. However, the consequences of iNKT cell activation under these circumstances and in the respective infectious models need to be carefully evaluated and elucidated in further detail. This will help also to determine in which situations the extremely powerful adjuvant properties of NKT cells can be utilized in anti-microbial vaccination strategies without inducing side effects as exemplarily shown in the Novosphingobium – infection model of PBC.

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Chapter 4

Regulation of Innate Immunity During Trypanosoma cruzi Infection Fredy Roberto Salazar Gutierrez

Abstract Chagas Heart disease is caused by the infection with T. cruzi. The mechanisms of disease progression remain largely unknown although it has been reported that parasite persistence as well as the intensity of the inﬂammatory immune response are determinants for the clinical manifestations of the disease. Through a long co-evolutionary history, both the human immune system and the pathogen have acquired diverse mechanisms to interact, guaranteeing their mutual survival. Even though inﬂammation is indispensable for host defense and tissue repair, when deregulated or disproportionate, it can contribute to continuous tissue injury, organ dysfunction, and disease. Thus, the immune system has acquired a great complexity in order to maintain the host’s integrity while it is able to arrest the proliferation of pathogens as soon as detected. This chapter aims to review the regulatory mechanisms involved in the control of the effectors mechanisms of the innate immunity during experimental T. cruzi infection and Chagas disease. It provides a comprehensive revision of the immunologic mechanisms triggered by the interaction of the parasite and the host cells during acute phase of the infection, as well as the possible implications for the design of therapeutic or diagnostic approaches.

4.1

Introduction

Trypanosoma cruzi is the etiological agent of Chagas’ disease, and is transmitted by haematophagous insects belonging to the Triatominae subfamily. This condition constitutes a serious problem to public health in Latin America, affecting about 18 millions of people, mostly in the poorest territories, due to the persistence of the conditions that keep humans and vectors living together. F.R.S. Gutierrez (*) School of Medicine, Antonio Nariño University, Bogotá, Colombia e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_4, © Springer Science+Business Media, LLC 2012

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Fig. 4.1 Life cycle of T. cruzi. Haematophagous Triatominae insects acquire the protozoan parasite while they take a blood from infected wild mammals with circulating trypomastigotes (not shown). The parasite replicates in the midgut of the insect by binary fusion (a). The bug usually defecates when biting a mammalian host (in this case a human being), resealing the infective metacyclic trypomastigotes, which can invade the skin through the wound made by the Triatominae, or through intact mucosae or conjunctivae (b). T. cruzi is either internalized by active invasion or phagocytized, and proliferates inside cells from host’s tissues, including skeletal muscle and myocardium, leading to intense inﬂammation (c). After rupture of the parasitized cells, new trypomastigotes are released to the blood, invading new cells. The chronic disease is characterized by dilated cardiomyopathy and mega syndromes of the digestive system. The exact mechanism that determine the disease progression and different manifestations of the disease remains largely unknown, although parasite persistence and immune system deregulation appear to be involved in the pathogenesis of Chagas’ disease

Human beings (and several other mammalian hosts) naturally acquire the disease when the infective forms of the protozoan are deposited on the feces of an infected haematophagous insect, immediately after a blood meal. As a result, the parasite gains access to host tissues trough any continuity lesion on the skin, or trough intact mucosae or conjunctivae (Fig. 4.1). The infection is characterized by a silent acute phase, or with unspeciﬁc symptoms. Afterwards, the infected individual may develop dilated cardiomyopathy or “mega” syndromes involving esophagus and/or colon, usually after staying asymptomatic for decades. Its clinical features impede the prompt diagnosis of the infection. Moreover, the socio economic conditions of the regions where it is endemic

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favor the persistence of infected people without veriﬁcation of the possibility of evolution to cardiomyopathy. Moreover, current therapies are extremely toxic and the parasite is acquiring resistance to the compounds (benzimidazols). The precise physiopathological mechanisms leading to the development of cardiac or digestive damages by the disease remain elusive despite of decades of biomedical research. Of note, it has been repeatedly reported that the persistence of the parasite as well as the intensity of the inﬂammatory immune response are largely responsible for the clinical manifestations of the disease. Hence, the study of the mechanisms involved in the regulation of the immune response during the infection is crucial for understanding pathogenesis and to the development of novel approaches for treatment and prognosis of Chagas’ disease. This chapter reviews recent reports on the mechanisms regulating the immune response during the acute phase of Chagas’ disease and aims to provide insights to potential strategies to the development of novel therapeutic agents.

4.2

Recognition of T. cruzi by the Immune System

Interaction of T. cruzi with host’ cells starts with the recognition of the pathogen by specialized receptors of the innate immune system. This recognition is in theory important for the establishment of the adequate parasite killing machinery, but its uncontrolled activation could lead to the establishment of a strong inﬂammatory response which may destroy host tissues. Through evolution, T. cruzi has acquired numerous mechanisms to subvert the host’s immune response and get access to the interior of a variety of host cell types, including phagocytes. The study of the ligands and receptors as well as signaling pathways involved in recognition of T. cruzi can provide novel strategies that would help in the development of vaccines and therapeutics for Chagas’ disease. For a detailed review on early interaction of T. cruzi with host’s cells, the readers could see (de Souza et al. 2010; Epting et al. 2010). The external surfaces of trypanosomatid parasites are coated with large amounts of glycoinositolphospholipids (GIPL), which can be found as GIPL anchors for glycoproteins and polysaccharides, or as free GIPL containing the identical core structure of GIPL. This molecular coat provides a changing surface which is essential for immune evasion. The sequences of oligosaccharide and lipids present on the major GIPL from T. cruzi have been already determined. Recognition of pathogen-associated molecular patterns (PAMP) occurs by innate immune receptors, such as Toll-like receptors (TLR) (Medzhitov 2001). Among them, TLR2, TLR4 and TLR9 have been found to be involved in T. cruzi recognition (Gazzinelli and Denkers 2006; Tarleton 2007). In addition, we recently described that the NOD2 receptor participates in the activation of macrophages by cytoplasmic T. cruzi (Silva et al. 2010) (Fig. 4.2).

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Fig. 4.2 Mechanisms of innate immune system recognition of T. cruzi in macrophages. The main molecules described to act as PAMP of T. cruzi are GIPL, Tc52 and CpG. These molecules bind to their receptors (TLR2, TLR4 and TLR9 respectively) and activate the recruitment of the MyD88 adaptor, which in turns recruit TRAF6 and induce the phosphorylation of MAPK cascades, ending in the translocation of JNK and NF-kB to the nucleus, which induces the expression of TNF-alpha, IL-12, NO, and costimulatory molecules. In addition, the ﬁgure shows the theoretical activity of the cytoplasmic sensors of PAMPS, NLR. NOD1 and NOD2 can activate NF-kB after cleavage of IL-1b, IL-33 and IL-18 in response to T. cruzi amastigotes present in the cytoplasm

4.3

Recognition of Glycoinositolphospholipids (GIPL) and Tc52 from T. cruzi by TLR2

The evidence that T. cruzi triggers TLR2 activation came from experiments conducted on transgenic cells expressing CD25 exclusively upon activation of TLR2. These cells were activated by T. cruzi in vitro. Moreover, the addition of soluble tGIP triggered the expression of CD25 in a dose-dependent manner. These ﬁndings were conﬁrmed by the absence of activation of macrophages from TLR2−/− mice, as demonstrated by absence of up-regulation of cytokine production. These data demonstrated that TLR2 is important for the initial activation of phagocytes by glycoinositolphospholipids, an essential constituent of parasite surface (Campos et al. 2001). In addition to GIPL, The protein Tc52 from T. cruzi has also been shown to stimulate the production of pro-inﬂammatory cytokines by macrophages in a TLR2 dependent manner (Ouaissi et al. 2002).

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Nonetheless, as an intracellular parasite which invades several cell types, including hepatocytes, adipocytes and cardiomyocytes, T. cruzi is able to cause an infection that usually occurs absolutely silent to the host. One of the reasons for that is the fact that the surface structures that are recognized by TLR2 trigger regulatory pathways upon detection by phagocytes and antigen presenting cells (APC), leading to arrested activation of these cells (Camargo et al. 1997; Almeida et al. 2000; Almeida and Gazzinelli 2001). In fact, the exposure of DC to trypomastigotes of T. cruzi induced a tolerogenic state of these cells, characterized by the increased production of TGF-B and IL-10, as well as reduced production of IL.12p70 and expression of MHC-II (Poncini et al. 2008). Interestingly, it has also been reported that the cooperative signaling via TLR4 and TLR2 induces the production of anti-inﬂammatory cytokines in murine dendritic cells (DC) (Hirata et al. 2008). As both pathways are triggered by the parasite, it is a possible mechanism to grant parasite survival within these cells. Moreover, excessive activation of TLR could lead to pathological conditions (Akira et al. 2006). Indeed, individuals with well recognized differential susceptibilities to T. cruzi show differential expression of the TLR2, TLR4 and TLR9 (Carrera-Silva et al. 2008). However, the infection of TLR2−/− mice with T. cruzi lead to production of amounts of pro-inﬂammatory cytokines and Nitrogen reactive species (RNS) that are similar to that observed in WT mice, and no signiﬁcant differences in terms of mortality curve or parasite burden were observed when comparing TLR2−/− to WT mice (Campos et al. 2004). Nonetheless, the infection of MyD88−/− mice (an essential signaling molecule of most TLR) revealed that these mice show increased parasitemia and mortality than WT mice after T. cruzi infection. Thus, while MyD88 is essential for activation of innate immunity to T. cruzi, it is evident that more than one TLR (or PRR) is involved in this process.

4.4

Recognition of Glycoinositolphospholipids Residues of T. cruzi Containing Ceramides (GIPL-Ceramides) by TLR4

Subsequent studies demonstrated that in addition to TLR2 and MyD88, the innate immune response to T. cruzi also needs the activation of TLR4 signaling. TLR4−/− mice showed impaired production of IFN-gamma, TNF-alpha and nitric oxide (NO) after infection with T. cruzi (Oliveira et al. 2004; Medeiros et al. 2007). Although eventual contamination of preparations with LPS could lead to the observed activation by GIPL, the treatment with polymyxin B was able to block the expression of the reporter transgene CD25 only when the transfected cells were stimulated with LPS but not with GIPL. Moreover, in vitro assays using MD-2-mutant CHO reporter cells demonstrated the requirement of functional TLR4 expression for GIPLinduced NF-kB activation. As expected, TLR4 deﬁciency leads to increased parasite burden. Of note, the invasion rate of T. cruzi was not signiﬁcantly altered by the

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deﬁciency of TLR4 in macrophages (Oliveira et al. 2004, 2010). Nonetheless, each of these pathways have not found to be individually essential for the generation of the adaptive immune response against T. cruzi, indicating that TLR4 signaling is partially required to host defense against T. cruzi.

4.5

Recognition of T. cruzi DNA by TLR9

In addition to surface components of the parasite, the DNA from T. cruzi is also able to trigger the activation of innate immune response. Indeed, TLR9−/−mice exhibited increased parasite burden and decreased survival when compared to WT mice. Of note, the infection of double deﬁcient (Tlr2−/−Tlr9−/−) mice induced levels of parasitemias equivalents to that observed on MyD88−/− mice, proving the hypothesis that the fully competent signaling through more than one TLR is required to host resistance against T. cruzi. Conversely, these double knockout mice did not show the acute mortality displayed by MyD88−/− animals (Baﬁca et al. 2006). Further evidence for the involvement of TLR9 in the recognition of T. cruzi DNA came from studies in gene reporter cells that were transfected with human TLR. These cells show luciferase activity when NFkB is activated, which is restricted to the activation of TLR9. Unmethylated CpG motifs similar to that of the B class found in bacteria, were able to induce translocation of TLR9 to the lysosomes of dendritic cells, suggesting that the degradation of parasites inside the lysosomes provides a ligand for TLR9 pathway and a potent activator of pro-inﬂammatory responses during T. cruzi infections (Bartholomeu et al. 2008). Thus, depending on the dominant molecule that is being recognized, the signaling pathways triggered by surface molecules from T. cruzi end mainly with the production of anti-inﬂammatory cytokines. The manipulation of pharmacological ligands for TLR could provide strong adjuvants to be potentially applied in the design of vaccines or other therapies against T. cruzi infection.

4.6

Recent Advances in the Study of Innate Immune Recognition of T. cruzi: Participation of NLR

As a cytoplasmic pathogen, T. cruzi is able to evade the above described systems for pathogen detection. Thus, the ultimate scenery where the immune system would be able to contain the parasite proliferation growth is by means of the activation of the inﬂammasome (a cytoplasmic system responsible for the activation of proinﬂammatory caspases) (Tschopp et al. 2003). These intracellular PRR include the family of nucleotide-binding oligomerization domain (Nod)-like receptor proteins (NLR) that detect bacterial infection. A recent study demonstrated that NOD1 but

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not NOD2 deﬁcient mice are susceptible to experimental T. cruzi infection, exhibiting increased parasite load and reduced production of pro-inﬂammatory cytokines during the acute phase of disease (Silva et al. 2010). However, the parasite structure responsible for the possible activation of NOD1 remains to be elucidated. Further research on the potential activation of the components of the inﬂammasome will make available novel therapeutic targets against T. cruzi infection, and will provide additional understanding about the pathogenesis of Chagas disease.

4.7

Regulation of Innate Immunity to T. cruzi by Nitric Oxide

Oxidation of L-arginine into L-citrulline by a family of enzymes (the nitric oxide synthases, or NOS that are dependent of NADPH) leads to production of nitric oxide (NO), which plays a pivotal role in several physiological processes. NO is able to react with the redox forms of oxygen, thiols, amines and transition metals. These properties enable NO to be involved in many biological functions, from neurotransmission to microbicidal activity. After activation in the presence of T. cruzi parasites, murine macrophages produce enormous amounts of nitric oxide (NO), which is crucial for parasite killing. Consequently, mice deﬁcient in the inducible isoform of nitric oxide synthase (NOS2) are extremely susceptible to T. cruzi, showing massive parasitemias and succumbing at the early stages of the infection (Silva et al. 2003; Gutierrez et al. 2009) (Fig. 4.3). Nonetheless, nitric oxide is also largely responsible for the induction of tissue damage to the myocardium, through S-nitrosylation or nitration of some amino acids of proteins that are crucial to tissue structure homeostasis and cell signaling. Nitric oxide is important for the regulation of innate and adaptive response mechanisms. Depending on its concentrations, it can down regulate or induce the production of cytokines like IL-1, IL-6, IL-8, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha, TGF-beta, G-CSF, M-CSF, VEGF, and chemokines like MIP-1alpha, MIP-2 and MCP-1. All these mediators have been found to be critical for the host response against T. cruzi. Furthermore, NO can also regulate the expression of adhesion molecules, or induce apoptosis of APC and T cells. In addition, it has been recently demonstrated that NO is able to regulate the induction and activity of Regulatory T cells (Treg) and TH17 cells. (Nitric oxide modulates TGF-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development (Lee et al. 2011) and regulation of type 17 helper T-cell function by nitric oxide during inﬂammation (Niedbala et al. 2011). These data demonstrate that NO constitutes one of the most versatile immune mediators produced during T. cruzi infection, which exert pleiotropic effects over the innate immune system. Indeed, nitric oxide donors are being proposed as promising agents to therapeutics of Chagas disease, mostly with the objective of design compounds that can release NO in a controlled manner directly to the inﬂammatory lesions, reducing collateral tissue damage (Silva et al. 2009; Guedes et al. 2010).

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Fig. 4.3 Overview of the impact of deﬁciency/blockade of selected genes on the parasite burden during acute experimental infection by T. cruzi. The importance of mediators of immune response in the control of parasitemia is depicted based on the ﬁndings by reviewed studies. The Cytokines Il-12, IL-10, IFN-gamma, the recombination activating genes (RAG) and the PAMP receptors TLR and NLR are shown. The ﬁgure describes clearly how the recognition of the pathogen and regulation of immune response are crucial processes for host resistance to this infection

4.8

Regulation of Phagocytic Cells

Activation of macrophages is one crucial step in controlling the infection. It is classically induced by the cytokine IFN-gamma, produced by NK cells in response to the parasite (Cardillo et al. 1996; Antunez and Cardoni 2000). The most remarkable evidence for the role of macrophage activation by IFN-gamma in T. cruzi infection was demonstrated in a recent study involving transgenic mice animals which macrophages are insensitive to IFN-gamma (MIIG), because the selective deﬁciency of IFN-gamma inducible receptor on macrophages. It was demonstrated that the absence of macrophage responsiveness to IFN-gamma leads to increased parasite burden, late control of parasitemia and early mortality, compared with WT mice (Lykens et al. 2010) (Fig. 4.3).

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Fig. 4.4 Overview of the central role of macrophages in the immune response during T. cruzi infections. The killing of the parasite by macrophages (red, center) requires the presence of IFN-gamma, produced mainly by NK and tissue cells. After T. cruzi is detected mainly though TLR, macrophages produce cytokines (mainly TNF-alpha, IL-12), inﬂammatory mediators such as NO, as well as several chemokines. These mediators are able to induce migration and activation of inﬂammatory cells to the site of infection, which include monocytes, NK cells and T lymphocytes. In the presence of activated professional APC like DC, the recruited T lymphocytes will be mainly driven to differentiation into Th1, Th17 and/or Treg. Chemokines and IL-12 induce NK cells and T lymphocytes to produce IFN-J, resulting in activation of macrophages. Other cytokines such as IL-10 and TGF-beta are also produced and regulate macrophages activation and the whole local inﬂammatory response, which is strategic for the parasite proliferation (up, brown)

Activation of macrophages triggers intracellular signaling cascades that induce the transcription of genes involved in the production of cytokines TNF-alpha, IL-12, NO and other pro-inﬂammatory mediators, in order to amplify the immune response, recruiting and activating new cells to the site of the infection (Fig. 4.4). It has been shown that GIP-anchored mucin-like glycoproteins puriﬁed from T. cruzi trypomastigotes (tGIP-mucin) can directly trigger the phosphorylation of different mitogen-activated protein kinases (MAPKs), like ERK-1/ERK-2, SKK-1/MKK-4, and SAPK-2/p38, as well as IkB shortly after stimulation of macrophages in culture (Ropert et al. 2001) (Fig. 4.2). Noteworthy, the activation of these signaling pathways by GIP leads to production of TNF-alpha, but not IL-12 or NOS2 expression,

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indicating that additional signaling is required for the complete activation of innate immune effectors responses to the parasite. During the phase of intracellular replication, the parasite inhibits a number of intracellular processes which otherwise would end in its killing. One of the mechanisms by which this occurs has been recently elucidated (Epting et al. 2010; Kayama and Takeda 2010) particularly during the inﬂammatory reaction at myocardium, where the phagocytosis of apoptotic cells in the absence of TLR signaling leads to the induction of tolerogenic responses by DC. These mechanisms have been already described as a strategy actively used by T. cruzi to subvert the immune system of the host (Freire-de-Lima et al. 2000; DosReis et al. 2005).

4.9

Regulation of Innate Immunity to T. cruzi by Cytokines

Cytokines can potentiate or regulate the effectors mechanisms of the innate and adaptive immune response. In fact, one of the proposed mechanisms that determine the development of Chagas’ disease is the excessive production of some cytokines by the host. Though, one remarkable difference of T. cruzi infection with other intracellular parasites is that the production of IFN-gamma and the inﬂammatoryderived tissue damage is independent on the Th-1 or Th-2 genetic proﬁle of the host (Fig. 4.3). Macrophages activation leads to the production of pro-inﬂammatory cytokines, such as IL-12, TNF-alpha, as well as NO (Fig. 4.4). All these mediators induce and maintain the activation of other cells of the innate immunity, including APC, neutrophils and NK cells. IL-12 is a central cytokine in the activation of innate immunity to T. cruzi. It was demonstrated in experiments conducted on infected mice that were treated with blocking antibodies to IL-12, leading to increased parasite burden (Aliberti et al. 1996). Accordingly, IL12−/− mice exhibited increased parasitemia and succumb early to experimental infection (Une et al. 2003) (Fig. 4.3). The activity of innate immune system is mostly regulated by the production of IL-10. After T. cruzi infection, mice deﬁcient in IL-10 developed reduced parasitemias than wild type (WT) mice (Fig. 4.3), which can be reverted by the treatment with recombinant IL-10. In addition to IL-10, TGF-beta is also able to block the activation and NO-dependent parasite killing of macrophages in vitro (Gazzinelli et al. 1992) (Fig. 4.4). Moreover, TGF-beta modulates the development of myocardiopathy in Chagas disease because it favors: (1) invasion of cardiac ﬁbroblasts and myocytes; (2) intracellular parasitism; (3) regulation of inﬂammatory response; (4) ﬁbrosis and remodeling of myocardium during the acute and chronic phases of the disease (Silva et al. 1991; Araujo-Jorge et al. 2008). Therefore, TGF-beta is a promising molecule in the development of immunomodulatory therapeutic strategies against Chagas’ heart disease.

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Regulation of Cell Migration

Cell migration is a crucial process during inﬂammatory diseases, and for the control of infections. It is orchestrated by the production of cytokines, chemokines and the expression of their receptors in cells of innate and adaptive immunity (Bachmann et al. 2006). The migration of inﬂammatory cells to the myocardium is a crucial process that may be determinant in the induction of heart pathology during T. cruzi infection (Aliberti et al. 1999; Teixeira et al. 2002). It has been described that CCR5 is required for cell migration and regulation of inflammatory response in the myocardium, which leads to proper control of parasite proliferation. In fact, CCR5−/− mice exhibit enormous parasite nests in their myocardium, with absence of inﬂammatory inﬁltrates, and subsequently succumb to the acute phase of infection, in comparison with WT animals (Machado et al. 2005). Furthermore, it was demonstrated that the CCR5 59029-A/G genotype was signiﬁcantly increased in asymptomatic with respect to cardiomyopathy patients (p = 0.02; OR = 0.33, 95% CI 0.10–0.94). In addition, the presence of the CCR5 59029-G allele was increased in asymptomatics when compared with cardiomyopathics (p = 0.02; OR = 0.35, 95% CI 0.12–0.96) in a cohort of 85 seropositive patients (53 of them asymptomatic and 32 with cardiomyopathy), suggesting that the polymorphism of CCR5 59029 promoter determines differential susceptibility to chagasic cardiomyopathy (Calzada et al. 2001). One of the most important cytokine in the regulation of innate immune response is macrophage migration inhibitor factor (MIF) which is expressed in a variety of immune and non immune cells. Its signaling triggers the activation of ERK1/ERK2, up-regulation of TLR4 expression and suppression of p53 activity (Calandra and Roger 2003). The infection of MIF−/− mice with T. cruzi lead to increased parasite burden and reduced survival and the failure to produce cytokines like TNF-alpha, IL-1beta and IL-18 (Reyes et al. 2006). One striking fact about MIF is the recent identiﬁcation of functional promoter polymorphisms in the gene, that are associated with different manifestations of different diseases. Accordingly, it has been reported that the −173 C allele confers a risk effect in chagasic patients (pooled OR = 1.75, 95% CI = 1.30–2.33, p = 0.0002). In addition, the MIF −173 C allele was also associated to increased risk of cardimyopathy (pooled OR =4.01, 95% CI =1.25–12.85, p = 0.004), suggesting that the polymorphism in MIF −173 G/C determines susceptibility to Chagas disease in a cohort of Chagas’ disease patients from Colombia and Peru (Torres et al. 2009). Although some studies have been performed in cytokines and their receptors, additional investigations focusing on polymorphisms and mutations of immune regulatory genes are currently required in order to gain further insight on the immune regulatory mechanisms that induce host protection from dilated cardiomyopathy.

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4.11

Regulation of Immune Response to T. cruzi by Matrix Metalloproteinases (MMP)

Besides cytokines, chemokines and NO, MMP are up-regulated during inﬂammatory responses, thus they are being frequently reported as candidates to markers of inﬂammation, and are involved in the modulation of cell motility, cytokine and chemokine activation among other functions (Manicone and McGuire 2008). The fact that MMP possess a wide variety of substrates has been associated to the induction of pathology, mostly when a strong inﬂammatory response is mounted, leading to uncontrolled MMP activation. That could be the case of T. cruzi infection. We showed that MMP-2 and MMP-9 are expressed in the myocardium of infected mice, and remarkably, that their inhibition favors host survival mostly because diminish the tissue damage secondary to inﬂammation, without affecting tissue parasitism (Gutierrez et al. 2008). These data indicate that the eventual development of speciﬁc MMP inhibitors to be used on chronically infected individuals may be promising as an approach to stop the disease evolution. However, clinical studies are required to gain further comprehension of the participation of this family of enzymes during the pathogenesis of T. cruzi induced dilated cardiomyopathy.

4.12

Concluding Remarks and Future Perspectives

An urgent necessity exists for the implementation of novel, efﬁcient strategies for the diagnostic, prognostic and treatment of Chagas’ disease evolution. The response to this could come from translation of basic research onto clinic studies, aiming to understand the mechanisms of innate immune recognition of T. cruzi, and the mechanisms of immune regulation triggered during the infection, which would favor the design of immunomodulators or vaccines against this protozoan parasite. The recent description of the genome from T. cruzi has provided an great source of biological information which is starting to be used as a tool to explore the interaction between the parasite and the host’s immune response. (El-Sayed et al. 2005). In addition, the availability of new high throughput systems for screening of pharmacologic interactions will help ﬁnding novel candidate compounds for therapeutic agents (Canavaci et al. 2010). Genome-wide expression analyses are also providing experimental data that would accelerate the understanding of many mechanisms of innate immunity. A recent study compared the transcriptional response of macrophages after in vitro infection with T. cruzi, Leishmania sp, or their stimulation with LPS. This study demonstrated that differently from the response to LPS, the response to T. cruzi is more complex, and has not a well-deﬁned pattern in terms of physiological response. Most of the T. cruzi induced genes were induced after 24 h of cell infection, corresponding with the escape of the parasite from parasitophorus vacuole. In addition, most of the genes induced by T. cruzi have totally unknown functions at

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the moment (Zhang et al. 2010). This clearly indicates that T. cruzi is able to inhibit the activation of phagocytes, and also indicates that our understanding of the mechanisms by which this happens is very limited. Cells from the phagocytic lineage such as macrophages are able to respond to several stimuli once they recognize molecular patterns that are more probably associated to pathogens. They do so through specialized receptors, mainly TLR. Macrophages are able to express different activation programs and consequently perform a variety of effectors functions, depending on the microenvironment where they ﬁnd the pathogen, and the signaling pathways that are activated afterwards (Mosser and Edwards 2008). It is becoming clear that the participation of cells of the innate immunity plays a central role in the generation of pathological responses, and in the maintenance of homeostasis of host’s immune system. A metabolic process which is acquiring relevance during immune responses is autophagy. It is the major mechanism used by eukaryotic cells to the ﬁnal dispose and recycling of proteins and organelles. Some studies have found that autophagy pathways are strongly up-regulated during differentiation between developmental stages of T. cruzi, a process that is essential for the propagation of the parasite (Alvarez et al. 2008a, b). The advances in the understanding of the autophagy pathways from the host will also provide novel insight on the interaction of this parasite with its mammalian host.

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Chapter 5

B Cell-Mediated Regulation of Immunity During Leishmania Infection Katherine N. Gibson-Corley, Christine A. Petersen, and Douglas E. Jones

Abstract Intracellular pathogens cause a wide spectrum of clinical disease ranging from mild self-limiting infections to chronic diseases and even acute life-threatening illnesses. T cell-mediated immunity is central to effective immune control of Leishmania infection and the role that B cells and antibodies play in promoting immunopathology during disease associated with chronic Leishmania infection is becoming more clear. However, the role that B cells and antibodies play during a successful immune response may be subtle and range from protecting the host from a robust cell-mediated response to enhancing immune effectiveness. The B cell response may therefore be a signiﬁcant component of both ineffective and effective immunity to Leishmania infection.

5.1

Introduction

Intracellular pathogens cause a wide spectrum of clinical disease ranging from mild self-limiting infections to chronic diseases and even acute life-threatening illnesses (Casadevall 1998; Igietseme et al. 2004). Intracellular pathogens pose an interesting problem for host immunity as the immune response must strike an appropriate balance between sparing host cells while targeting the organism inside. Cellmediated immunity (CMI) is the primary productive host defense against intracellular pathogens such as Mycobacterium tuberculosis, Leishmania major and Listeria monocytogenes (Casadevall 1998; Igietseme et al. 2004). This arm of the immune system involves T-helper type-1 CD4+ (Th1) cells that activate macrophages and natural killer cells enabling them to destroy intracellular invaders (Casadevall 1998).

D.E. Jones (*) Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_5, © Springer Science+Business Media, LLC 2012

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The alternate arm of host defense, humoral immunity, involves speciﬁc antibodies produced by B cells which can directly neutralize viral pathogens or toxins and opsonize extracellular organisms to promote phagocytosis or complement deposition. The contribution of humoral immunity to the host immune response during intracellular pathogen infection is often overlooked as the role of antibodies is currently understood to function largely outside of the host cell via direct recognition of relevant extracellular or membrane bound epitopes. Many studies using passive immunization have demonstrated that antibodies had little effect on intracellular pathogens such as Mycobacterium tuberculosis (reviewed in (Casadevall 2003)). In fact, there is a long list of intracellular pathogens that are negatively impacted by antibodies (reviewed in (Casadevall 1998)). However, antibodies can aid in phagocytosis, complement activation, toxin neutralization, cytotoxicity and a productive CMI response. It is therefore important to determine the role of the humoral immune system during intracellular pathogen infection to potentially enhance vaccine design and immunotherapy.

5.2

B Cell Regulation of Immunity to Intracellular Pathogens

Resolution of intracellular pathogens primarily requires effectors of CMI. These effectors lead to activation of phagocytes, generation of reactive oxygen and nitrogen intermediates, tryptophan degradation and iron deprivation (Igietseme et al. 2004). Although CMI, as deﬁned by Th1 CD4+ cells, is the primary immune response elicited by the majority of intracellular organisms, induction of a concomitant antibody response can be important for a productive CMI response (Igietseme et al. 2004). Known mechanisms by which antibodies can inﬂuence CMI is by pathogen opsonization during ongoing infection, enhancing antigen presentation by increased antigen internalization, endosomal transport and delivery to the lysosome for processing and presentation and promotion of dendritic cell maturation and antigen presentation to T cells (Igietseme et al. 2004). Manca et al. showed in vitro that antigens could be targeted to macrophages by antibodies and this led to differential activation of T cells (Manca et al. 1988). Much of the work demonstrating the role of antibodies in modulating T cell responses during intracellular infection has been performed using experimental Chlamydia infection (Igietseme et al. 2004). Chlamydial genital infections in mice require CMI for control of the primary infection but require B cells and antibodies for control of re-infection (Igietseme et al. 2004). An adequate and rapid Th1 immune response is required for clearance of re-infection but Fc gamma receptors (FcJR), the antibody scavenger receptors that recognize the common chain of IgG molecules that is present on macrophages, are also required (Moore et al. 2003). These results indicate that the role of antibodies is to bind FcJR and deliver antigen to the phagocyte for rapid antigen processing and presentation to memory T cells (Moore et al. 2002). Studies focused on the facultative intracellular pathogen Salmonella have demonstrated an important role

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for antibodies in controlling this infection in both mice and humans (Gondwe et al. 2010; Uppington et al. 2006). Likewise, antibodies opsonize an extracellular phase of the intracellular bacteria Ehrlichia chaffeensis and are required for immune control in an experimental mouse model of infection (Yager et al. 2005). Recent studies have also demonstrated that antibodies can play a signiﬁcant role in immune control of the intracellular bacterium Legionella via a mechanism independent of complement. Antibodies targeted the pathogen to lysosomes, with or without direct opsonization (Joller et al. 2010). These studies support the idea that both CMI and humoral immunity promote pathogen removal through synergistic mechanisms, indicating that these two arms of the immune system are not mutually exclusive and the latter study suggests we have more to learn about how these two aspects of immunity interact.

5.2.1

IgG Isotypes: Proinﬂammatory Versus Anti-inﬂammatory

Complicating our ability to understand the role of B cells and antibodies during intracellular infections is the fact that the antibody response itself has a vast array of phenotypes ranging from non-speciﬁc polyclonal activation to speciﬁc antibody responses. These speciﬁc antibody responses include IgG isotypes that are associated with Th2 immunity to those associated with Th1 immunity (Hjelm et al. 2006; Nimmerjahn and Ravetch 2008). Furthermore, antibody function can be signiﬁcantly affected by the avidity of the antibody itself and the consequences of varying antibody avidity are essentially unexplored with respect to intracellular pathogens, such as Leishmania, that do not have a deﬁned virulence factor or toxin (Goodnow et al. 2010). However it is clear that certain antibody characteristics can and do help skew the immune response towards either a pro-inﬂammatory or antiinﬂammatory response (Nimmerjahn and Ravetch 2008). As a population of effector molecules antibodies can act broadly and depend upon in part, by either lowering the threshold of cellular activation by activating immunoreceptor tyrosine-based activating motifs (ITAM) or increasing the threshold of cellular activation by activating immunoreceptor tyrosine-based inhibitory motifs (ITIM) (Nimmerjahn and Ravetch 2008). For example, immune complexes (ICs) are well known for their ability to promote a pro-inﬂammatory response either by themselves or in combination with complement. Through this mechanism, ICs or opsonized bacteria readily generate superoxide via FcJR- Phosphatidylinositol 3-kinases (PI3K) mediated activation of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase. The pro-inﬂammatory nature of the immune complexes is largely determined by the isotype as well as the pattern of FcJR expression on target cells and tissues (Hjelm et al. 2006; Nimmerjahn and Ravetch 2008). Recent studies have demonstrated that the sialylation patterns on the antibodies themselves can play a signiﬁcant role determining the pro-inﬂammatory vs. anti-inﬂammatory responses. Increased sialylation promotes an anti-inﬂammatory response (Nimmerjahn and Ravetch 2010). Consequences of different antibody sialylation patterns during the

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immune response to an infectious disease are unknown. ICs have been routinely isolated from Leishmania infected animals, including humans, and IC deposition in various organs lead to classic histopathologic lesions associated with chronic, lethal visceral leishmaniasis (Elshaﬁe et al. 2007; Kharazmi et al. 1982; Mary et al. 1993). This consistent ﬁnding reinforces the perception that the B cell response during Leishmania infection is a pathologic response although evaluation of mouse models of Leishmania infection suggest the relationship between the B cell and antibodies and the immunopathology of Leishmania infection is more complex.

5.3

B Cell Mediated Immuno-Regulation

B cells produce many pro-inﬂammatory and anti-inﬂammatory cytokines which may modulate the immune response. Both interleukin (IL)-1 and IL-6 have been shown to be produced by B cells (Pistoia and Corcione 1995). IL-1 induces fever and macrophage activation. IL-6 will also produce fever, along with acute phase proteins and stimulate T and B cell growth and differentiation. B cells are reported to produce IL-12 when cultured in a Th1 environment (Harris et al. 2000). A relatively newly described B regulatory cell type also produces anti-inﬂammatory cytokines (Mizoguchi and Bhan 2006) such as IL-10, a potent suppressor of macrophage functions (Fillatreau et al. 2002; Palanivel et al. 1996) and transforming growth factor (TGF)-E, another anti-inﬂammatory cytokine (Parekh et al. 2003). Therefore, B cells are not only important for antibody production, but can also play a role in both cytokine production and polarization of immune responses and broader immune regulation.

5.3.1

Macrophage Activation and Antibodies

Macrophages are the cell type primarily infected by Leishmania parasites. Classically-activated macrophages produce nitric oxide (NO) which functions to kill intracellular pathogens. Inducible nitric oxide synthase (iNOS) is the enzyme that generates NO and is primarily expressed in macrophages and neutrophils (Hostetter et al. 2005). Following production of NO, which interacts with water and oxygen, there is generation of multiple reactive nitrogen intermediates, including NO2−, NO3−, N2O2 and in the presence of superoxide, peroxynitrite (Bogdan 2001; Hostetter et al. 2005). These reactive intermediates are directly toxic to intracellular pathogens, including Mycobacterium and Leishmania major (Hostetter et al. 2005; Linares et al. 2001). NO functions to kill pathogens via multiple mechanisms including S-nitrosylation of proteins, tyrosine nitration, deprivation of iron, inhibition of DNA synthesis, DNA oxidation, DNA degradation, and oxidation of lipids (reviewed in (Bogdan 2001)). iNOS is activated by multiple triggers, including lipopolysaccharide (LPS) and mycobacterial lipoarabinomanins (Bogdan 2001;

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Hostetter et al. 2005). iNOS is also strongly activated by Th1 cytokines, speciﬁcally interferon (IFN)-J (Chen et al. 2003). The other arm of macrophage effector molecules is superoxide and antibodies can efﬁciently promote the production of superoxide. This reactive oxygen species is generated in macrophages primarily by NADPH oxidase assembly and activation which is particularly relevant to antibody opsonization and FcJR-mediated phagocytosis (Underhill and Ozinsky 2002). As mentioned above superoxide and nitric oxide produced in the same vicinity create the opportunity to produce peroxynitrite a particularly potent effector molecule that has been demonstrated to be required for macrophage antimicrobial responses for a variety of pathogens such as Rhodococcus equi and some species of Leishmania (Darrah et al. 2000; Linares et al. 2001).

5.4

Leishmania as an Intracellular Pathogen

Leishmaniasis is a disease that affects humans and other mammalian species; dogs and rodents are often the primary reservoirs for human disease in endemic regions (Desjeux 2001). There are 21 classiﬁed species of Leishmania that can cause disease and infection with the parasite can lead to four main categories of disease; cutaneous leishmaniasis, diffuse cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (David and Craft 2009; Mitropoulos et al. 2010).

5.4.1

Th1 Versus Th2

The murine model of infection has been successfully used to characterize the immune response of both susceptible and resistant strains of mice during L. major infection. The majority of inbred mouse strains, such as C3H/He, CBA, C57Bl/6 and 129Sv/Ev, are considered resistant to infection and develop local cutaneous lesions that spontaneously resolve over time. The healing phenotype associated with L. major infection is characterized by a polarized CD4+ Th1 immune response, which activates infected macrophages to kill the intracellular pathogen (Sacks and Noben-Trauth 2002). Dendritic cells from resistant mouse strains produce IL-12 after infection which induces L. major-speciﬁc CD4+ Th1 cells to produce IFN-J. IFN-J will in turn activate the inducible nitric oxide synthase in macrophages to produce microbicidal NO (Gumy et al. 2004; Louis et al. 1998). There are a few murine strains considered susceptible to L. major infection, such as BALB/c mice that develop progressively large, non-healing lesions (Gumy et al. 2004; Louis et al. 1998). The BALB/c mice develop a polarized Th2 immune response with high levels of IL-4, which is associated with susceptibility to disease. Not all species of Leishmania are controlled by a classically deﬁned Th1 immune response. Infection of L. major-resistant strains of mice with L. amazonensis does not produce either a

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polarized Th1 or Th2 immune response and CD4+ T cells have been shown to disparately promote disease progression and pathology (Ji et al. 2002; Soong et al. 1997). The severity and chronicity of disease caused by L. amazonensis, as well as other New World L. mexicana complex of parasites, is generally linked to a poor T cell response during infection (Afonso and Scott 1993; Ramer et al. 2006; Vanloubbeeck et al. 2004). A clear Th1/Th2 immune response has also not been well-characterized during Leishmania donovani infection in both mice and humans (Alexander et al. 1999).

5.4.2

B Cell Response During Clinical Leishmania Infection

5.4.2.1

Clinical Leishmaniasis

Many studies over the previous three decades have documented the defects in the human T cell compartment during clinical leishmaniasis (Carvalho et al. 1981, 1985, 1988, 1994; Nylen and Akuffo 2009; Nylen et al. 2007). Although there have been several studies focused on the appearance of Leishmania speciﬁc antibodies and their isotypes during the development of clinical disease in humans the relationship between antibodies and protection vs. pathology remains unclear (Saha et al. 2006). Many studies have recognized the role of parasite antigens in precipitating immune complex-mediated disease (Caravaca et al. 1991; ElShaﬁe et al. 2006; Marcussen et al. 1989; Dutra et al. 1985; Weisinger et al. 1978). Prior to chronic disease, antigen-speciﬁc B cell responses are associated with disease progression, not immune efﬁcacy, and this response is useful for some diagnostics (Saha et al. 2006). A recent study of visceral leishmaniasis in humans described an association between antibodies against L. infantum chagasi in Brazil and increased risk of clinical disease after immunosuppression, highlighting the complexity between the B cell response and protective vs. pathologic immunity (Souza et al. 2009). It has also been demonstrated that circulating immune complexes decrease after successful treatment (Makni et al. 1989). The clinical data as well as experimental data implicate the B cell response as a primary phenomena leading to broader immunosuppression although, as discussed later, the B cell response is thought to be shaped primarily by the T cell response (Igietseme et al. 2004). Data from experimental infection of mice, discussed below, clearly demonstrate the capacity for antibodies to promote a non-healing phenotype. There has been no strong evidence that a particular Leishmania speciﬁc antibody isotype is associated with immune control (Saha et al. 2006). Leishmaniasis is often a zoonotic disease and dogs can serve as an important domestic reservoir to human leishmaniasis (Petersen 2009). Although there are many serological tests which appear to work both in human and canine disease, there are distinct antigen recognition patterns in each species (Goto and Prianti 2009; Saha et al. 2006). Immune complexes, or antigen-antibody complexes, have been shown to be associated with visceral leishmaniasis in humans and dogs and

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analysis of IgG subclasses have shown that all classes of IgG are markedly increased with clinical disease (Anam et al. 1999; Ghosh et al. 1995). A study in dogs determined that levels of IgG2 were higher in dogs that were parasite positive via PCR ampliﬁcation than those that were negative (Quinnell et al. 2003). A separate study found that dogs naturally infected with L. infantum had higher IgG2 reactivity than those experimentally infected (Leandro et al. 2001). A more recent study looking at the antibody responses to recombinant L. infantum proteins, as compared to whole parasite antibody responses, indicated that responses to speciﬁc antigen decreased much earlier than those targeted to crude whole parasite lysates. Furthermore, decreases against one of the recombinant parasite proteins (rKMPII) occurred earlier in dogs that resolved clinical disease than those that remained clinically ill after treatment with glucantime and allopurinol (Todoli et al. 2010). These ﬁndings suggest speciﬁc antibodies and antibody isotypes may be important during Leishmania infection, but little is known about the function of these antibodies during clinical disease. A recent comparison of monoclonal and polyclonal antibodies for detection of IgG1 and IgG2 did not ﬁnd a correlation between antibody isotype and clinical or parasitological outcomes of L. infantum infection (Carson et al. 2010). Similarly, in dogs likely infected via vertical transmission, there does not seem to be a clear pattern of antibody isotype corresponding with progression to disease (Boggiatto et al. 2010). Although there is clear evidence that late in canine disease antibodies contribute to renal failure and other systemic problems, little is known regarding the role of antibodies in earlier or non-symptomatic periods of infection with Leishmania.

5.4.2.2

Murine Models of Infection

The role of antibodies and B cells during experimental murine leishmaniasis has been explored using techniques that deplete B cells prior to infection (via anti-IgM antibodies or gene deletion technologies). Across all studies the results demonstrate that there is variability in the role of B cells and antibodies, which appears dependent, to a large extent, on the species of parasite. For Old World species of Leishmania that cause cutaneous disease, studies using mouse strains that spontaneously heal this infectious challenge demonstrate that the B cell response is important for the efﬁcient generation of the initial immune response. Therefore, although B cells and the production of protective antibodies are classically considered part of a productive Th2 immune response, these studies indicate there is a protective role for B cells and antibodies during Leishmania infection. Scott et al. showed that deleting B cells in neonatal C3H mice with an anti-IgM antibody impaired T cell-mediated immune responses following L. major infection (Scott et al. 1986). More recently, B cell production of antibody has been shown to be important for phagocytosis of L. major by dendritic cells. Without antibodies, infected mice developed larger lesion sizes, higher parasite loads, lower IFN-J production, and a decreased T cell response (Woelbing et al. 2006). This is consistent with an active T cell response

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being important for an effective pro-inﬂammatory antimicrobial immune response and the early B cell response primarily supporting T cell activation either through enhancing APC function via parasite opsonization or the B cells themselves acting as APCs. After L. major infection, once an effective antigen-speciﬁc T cell response is generated the B cell response is dispensable (Scott et al. 1986). However, although B cells are not required they may contribute to immune homeostasis during a chronic L. major infection. A supporting role for the B cell response is even evident upon reinfection where B cell-deﬁcient mice had a reduced inﬂammatory response and decreased IFN-G production after re-challenge when compared to immunocompetent WT mice (DeKrey et al. 2003). In contrast, there is evidence that CD4+ T cells alone are not sufﬁcient for resolution of L. amazonensis infection (Ji et al. 2002, 2003; Mukbel et al. 2007). Both CD4+ T cells and B cells were necessary for limiting L. amazonensis infection within macrophages and a poor B cell response is associated with the inability of C57Bl/6 mice to heal a co-infection with both L. amazonensis and L. major (Gibson-Corley et al. 2010; Mukbel et al. 2006). During L. amazonensis infection these B and T cells are likely to trigger intracellular signaling cascades which activate both superoxide and nitric oxide production in the infected macrophage to kill the parasite (Bogdan 2001; Underhill and Ozinsky 2002). This supporting role for the B cell response is in contrast to the relationship between B cells and non-healing lesions associated with several New World species of the Leishmania mexicana complex or the old world parasite L. major in susceptible mice strains. For these parasites and/or hosts, antibodies exacerbate disease. For the L. mexicana complex, the antibodies in association with FcJR are important in maintaining the non-healing phenotype and appear to play a less signiﬁcant role during the immune response of the initial infection (Buxbaum 2008). During these infections the T cell response appears compromised by the normal B cell response, as determined by larger lesions and higher parasite loads of wild-type mice when compared to antibody deﬁcient mice (Kima et al. 2000; Colmenares et al. 2002; Chu et al. 2010). Miles et al. demonstrated that when IgG-negative BALB/c mice were infected with L. major they had smaller lesions with fewer parasites compared to infected mice with IgG (Miles et al. 2005). A more recent study showed L. amazonensisinfected mice that lacked functional B cells, and therefore antibodies, had delayed onset of disease and developed smaller lesions (Wanasen et al. 2008). It has also been described that there were limited infections with both L. amazonensis and L. pifanoi in the absence of circulating antibodies, and infection of FcJR knockout mice resulted in similarly limited lesions (Kima et al. 2000; Colmenares et al. 2002; Chu et al. 2010). When FcJRIII knockout mice were infected with L. mexicana they also produced smaller lesions. These mice also produced higher levels of IFN-J, indicating there is a negative immunoregulatory effect when antibodies bind FcJRIII during L. mexicana infection (Chu et al. 2010). IgG1 is the dominant antigen-speciﬁc isotype in L. mexicana or L. amazonensis infections in mice and via interaction with at least FcJRIII generate IL-10 mediated suppression of the immune response (Buxbaum 2008; Gibson-Corley et al. 2010; Chu et al. 2010). In the absence of antibodies the T cell defect appears to be conﬁned primarily to the sight of infection such that the T cell response at the draining lymph node is comparable to immune intact mice,

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suggesting that antibody and FcJR mediated uptake by macrophages at the site of infection are an important mechanism to maintain persistent infections for those parasites that establish a chronic high parasite load (Colmenares et al. 2002). Like the New World parasites, B cells appear to restrain an effective immune response during L. donovani infection. Therefore, in the absence of B cells a pro-inﬂammatory response dominates, creates enhanced granuloma-associated pathology with a quickly resolving infection (Deak et al. 2010; Smelt et al. 2000). Although clinically during leishmaniasis the B cell response is detected via antigenspeciﬁc antibodies relatively late during infection, murine studies demonstrate an acute polyclonal B cell response is generated early during infection and in models of visceral leishmaniasis this B cell activation plays a signiﬁcant role in determining susceptibility (Deak et al. 2010). That is, the polyclonal B cell response contributes to a signiﬁcantly higher parasite load. Although non-speciﬁc, the contribution to susceptibility is dependent on the antibody response and less on any immunoregulatory role that the B cell itself may play either in antigen presentation or IL-10 production as a potential B regulatory cell (Deak et al. 2010). Within the New World group of parasites that cause chronic cutaneous disease it is clear that antibodies, and IgG1 in particular, limit the ability of the immune response to effectively activate macrophages to promote parasite killing (Kima et al. 2000; Colmenares et al. 2002; Chu et al. 2010). This effectively restrains the immune response from developing a beneﬁcial pro-inﬂammatory environment. In this case immunopathology is associated with chronic infection, high parasite load and unresolved lesions, some of which is mediated by the antibody/FcJR relationship that promotes IL-10 production and decreased immune function (Buxbaum 2008; Miles et al. 2005; Thomas and Buxbaum 2008; Wanasen et al. 2008). It is interesting that there are such varied relationships between the B cell response and disease with these different strains and species of Leishmania given the fact that these different outcomes have been assessed in only a few strains of inbred mice. Therefore the bulk of our understanding of the host-parasite relationship represents only a few genotypes in one species of host animal. Given this high variability in experimental disease outcomes and relatively low genetic variability of the experimental animals, the actual contribution of the B cell response to long term immune homeostasis in outbred populations and across different host species may be highly variable.

5.5

Conclusions

The most consistent conclusion about the B cell response during clinical leishmaniasis across different host and parasite species is that in the absence of effective immunity the B cell response is a contributing factor to the poor host response. The ineffective B cell response appears to manifest itself primarily as an ineffective antibody response, as opposed to other potential immunoregulatory mechanisms. These antibodies promote cellular acquisition of the parasite via opsonization or

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uptake of parasite antigens as part of ICs into local macrophages and induce IL-10 via FcJR-mediated phagocytosis. The signaling pathways triggered by this uptake signiﬁcantly limit the ability of the macrophage to upregulate an effective antimicrobial response. In an ineffective feedback loop, higher concentrations of IC assembly further suppress the macrophage response (Gallo et al. 2010). These phenomena bring up an important point that has not adequately been answered; does the B cell response determine susceptibility or simply reﬂect an ineffective host-response, which it then ampliﬁes? If the immune response were effective there would not be opportunity for detrimental IC formation or parasite opsonization. Clearly as this poor response is ampliﬁed the host can be compromised by IC deposition and secondary organ failure as a result of high levels of ICs leading to severe clinical disease and death. Murine studies suggest that the B cells may initiate clinical disease, but again they would need sufﬁciently high levels of antigen to form detrimental ICs or continued presence of parasites to be opsonized, suggesting that in these experimental systems the T cell response does not limit parasite load below a threshold in which a detrimental feedback mechanism is initiated. Although there is little data describing the B cell response during early clinical leishmaniasis, using experimental mouse infection it appears that an early B cell response signiﬁcantly affects local immunopathology. During a host response to cutaneous leishmaniasis that naturally resolves, immunoglobulins appear to support that effective response early during infection or re-infection. In contrast, a polyclonal antibody response during visceral leishmaniasis leads to higher parasite loads but protects tissue structure by limiting granuloma formation. Because experimental murine infections with L. major in resistant mouse strains do not require B cells for a long-term resolution, it is difﬁcult to demonstrate a positive role for these cells or their antibodies during established chronic, but resolved, infections. Our knowledge of this role is limited by the fact that the bulk of experimental data is centered on L. major infection in C57BL/6 mice. L. major is relatively susceptible to the mouse immune response and the C57BL/6 mouse has known deﬁciencies in the B cell response including a reduced ability to generate high avidity antibodies and a poor response to antibody-mediated immune enhancement (Gustavsson et al. 1998, 1999; Petty et al. 1972). Basic immunology suggests that the immune response to intracellular pathogens is not independent of the B cell antibody response but the B cell response is a function of the T cell response, which during leishmaniasis is determined by a myriad of factors including parasite species and strain as well as the immune response of the host itself. This would suggest that the B cell response simply reﬂects the T cell response and is a secondary event, an effective T cell response leads to an effective B cell response whereas an ineffective T cell response leads to an ineffective B cell response. If this relationship is as straightforward as it appears, it would be useful to identify the properties of a B cell response that can be effective against Leishmania. Future treatment strategies could focus on inhibiting a detrimental B cell response and promoting a beneﬁcial B cell response or mimicking any positive aspects of the B cell and antibody response for post-infection therapeutics.

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Kima PE, Constant SL, Hannum L, Colmenares M, Lee KS, Haberman AM, Shlomchik MJ, McMahon-Pratt D (2000) Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis. J Exp Med Leandro C, Santos-Gomes GM, Campino L, Romao P, Cortes S, Rolao N, Gomes-Pereira S, Rica Capela MJ, Abranches P (2001) Cell mediated immunity and speciﬁc IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis. Vet Immunol Immunopathol Linares E, Giorgio S, Mortara RM, Santos CX, Yamada AT, Augusto O (2001) Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation. Free Radic Biol Med Louis J, Himmelrich H, Parra-Lopez C, Tacchini-Cottier F, Launois P (1998) Regulation of protective immunity against Leishmania major in mice. Curr Opin Immunol Makni S, Ayed K, Ben Said M, Ben Rachid MS (1989) Study of circulating immune complexes during the evolution of visceral Mediterranean leishmaniasis. Ann Trop Med Parasitol Manca F, Fenoglio D, Kunkl A, Cambiagg Ci, Sasso M, Celada F (1988) Differential activation of T cell clones stimulated by macrophages exposed to antigen complexed with monoclonal antibodies. A possible inﬂuence of paratope speciﬁcity on the mode of antigen processing. J Immunol Marcussen N, Vetner M, Kristensen HM (1989) Interstitial nephritis and glomerulonephritis in visceral leishmaniasis in a dog. A case report. Apmis Mary C, Ange G, Dunan S, Lamouroux D, Quilici M (1993) Characterization of a circulating antigen involved in immune complexes in visceral leishmaniasis patients. Am J Trop Med Hyg Miles SA, Conrad SM, Alves RG, Jeronimo SM, Mosser DM (2005) A role for IgG immune complexes during infection with the intracellular pathogen Leishmania. J Exp Med Mitropoulos P, Konidas P, Durkin-Konidas M (2010) New World cutaneous leishmaniasis: updated review of current and future diagnosis and treatment. J Am Acad Dermatol Mizoguchi A, Bhan AK (2006) A case for regulatory B cells. J Immunol. 176:705–710 Moore, T., G.A. Ananaba, J. Bolier, S. Bowers, T. Belay, F.O. Eko, and J.U. Igietseme. 2002. Fc receptor regulation of protective immunity against Chlamydia trachomatis. Immunology Moore T, Ekworomadu CO, Eko FO, MacMillan L, Ramey K, Ananaba GA, Patrickson JW, Nagappan PR, Lyn D, Black CM, and Igietseme JU (2003) Fc receptor-mediated antibody regulation of T cell immunity against intracellular pathogens. J Infect Dis Mukbel R, Petersen CA, Jones DE (2006) Soluble factors from Leishmania major-speciﬁc CD4+T cells and B cells limit L. amazonensis amastigote survival within infected macrophages. Microbes Infect Mukbel RM., Patten C., Gibson K, Ghosh M, Petersen C, Jones DE (2007) Macrophage killing of Leishmania amazonensis amastigotes requires both nitric oxide and superoxide. Am J Trop Med Hyg Nimmerjahn F, Ravetch JV (2008) Fcgamma receptors as regulators of immune responses. Nat Rev Immunol Nimmerjahn F, Ravetch JV (2010) Antibody-mediated modulation of immune responses. Immunol Rev Nylen S, Akuffo H (2009) Tracing immunity to human leishmaniasis. Future Microbiol Nylen S, Maurya R, Eidsmo L, Manandhar KD, Sundar S, Sacks D (2007) Splenic accumulation of IL-10 mRNA in T cells distinct from CD4+CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis. J Exp Med Palanivel V, Posey C, Horauf AM, Solbach W, Piessens WF, Harn DA (1996) B-cell outgrowth and ligand-speciﬁc production of IL-10 correlate with Th2 dominance in certain parasitic diseases. Exp Parasitol Parekh VV, Prasad DV, Banerjee PP, Joshi BN, Kumar A, Mishra CG (2003) B cells activated by lipopolysaccharide, but not by anti-Ig and anti-CD40 antibody, induce anergy in CD8+ T cells: role of TGF-beta 1. J Immunol Petersen CA (2009) Leishmaniasis, an emerging disease found in companion animals in the United States. Top Companion Anim Med

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Chapter 6

Control of the Host Response to Histoplasma Capsulatum George S. Deepe, Jr.

Abstract The mammalian host response to fungal pathogens is complex and varies with the different species in part as a result of differences in biochemical and genetic composition, portals of entry, and morphology. Among the fungal pathogens, Histoplasma capsulatum is one of the few that behaves both as a primary pathogen and as an opportunist, i.e., causes serious life-threatening infection in both immunocompetent and immunocompromised hosts. This fungus is found in the soil in many continents but does manifest a geographic restriction. The highly endemic areas are the midwestern and southeastern regions of the United States and large segments of Central and South America. The portal of entry for this fungus is the lungs. Infection of mammals is coincidental with the disruption of the soil. Most infections resolve spontaneously although the organism establishes a dormant state. Numerous mediators and cell populations must cooperate to effect a successful resolution of infection. Primary among them are T cells, macrophages, tumor necrosis factor-D and interferon-J. However, these constituents constitute only a fraction of the mediators and cells that contribute to host regulation of invasion. The chapter herein will explore the elements of the immune response and how each contributes to regulation. We also will endeavor to deﬁne intersections among cell populations and soluble mediators.

6.1

Introduction

Thousands of fungi exist in nature, yet relatively few are known to infect humans. Among those Histoplasma capsulatum is soil-based and found principally in the temperate southeastern and midwestern United States and South America. Of the primary G.S. Deepe, Jr. ( ) Veterans Affairs Hospital, Cincinnati, OH, USA Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_6, © Springer Science+Business Media, LLC 2012

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pathogenic fungi, H. capsulatum is the most frequent cause of respiratory infection. In the US, it is estimated to cause as many as 500,000 new infections annually (Deepe 2010). H. capsulatum exists in nature as a mycelium with macro- and microconidia, and the latter are suspected to be the infectious form since they possess the appropriate size to reach the terminal bronchioles of lungs. Upon settling, the conidia undergo a remarkable genetic, biochemical, and morphological transformation into yeast cells that cause the clinical and pathological features of histoplasmosis (Hwang et al. 2003; Deepe 2010). Often, there is no apparent clinical illness or the manifestations mimic those of an inﬂuenza-like illness. It is the exception that primary infection produces a protracted symptomatic infection characterized by shortness of breath, fever, malaise, and chest pain. The clinical manifestations, if they develop, typically resolve within a week or two. During the ﬁrst 2 weeks, the fungus replicates within macrophages (MI) and monocytes and like Mycobacterium tuberculosis is transported presumably within these cells to draining lymph nodes that subsequently form a Ghon-like complex. The regional lymph nodes are not the ﬁnal destination of yeast cells; they travel to distant visceral organs such as spleen and liver that contain abundant resident mononuclear phagocytes. Cellular immunity, which is essential for halting the progression of histoplasmosis, is typically activated within 2–4 weeks after exposure. However, the immune system fails to sterilize tissues, and a latent state is established. Dormant fungi reactivate when immunological controls are disrupted either by immunosuppressive pharmacological agents or by diseases such as AIDS. This chapter will detail the mechanisms by which the host successfully defends against Histoplasma and deﬁne the holes in immunity that permit the fungal escape.

6.2

6.2.1

Mobilization and Organization of the Inﬂammatory Response to H. Capsulatum Characterization of the Inﬂammatory Response to H. Capsulatum

Eradication of microbial invaders including H. capsulatum depends on recruitment of the proper inﬂammatory cells to sites of infection. Characterization of the inﬂammatory response in lungs has been conducted principally in mice because in humans it is nearly impossible to identify a precise date of infection, and the risk of invasive procedures is not trivial. Numerous lymphoid and myeloid cell populations are mobilized to enter the lung. The early response (d7 days) is characterized by a large inﬂux of neutrophils, monocytes and dendritic cells (DC). Optimal ingress of these populations is dependent on signaling through chemokine receptors CCR2 and CCR5 (Cain and Deepe 1998; Szymczak and Deepe 2009; Kroetz and Deepe 2010). In their absence, the numbers, but not necessarily the proportions, of inﬂammatory cells, are dramatically reduced with the exception of neutrophils. These cells are strikingly elevated in CCR2−/− mice at both days 3 and 7 of infection. Although these cells inhibit

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intracellular replication of H. capsulatum (Brummer et al. 1991; Newman et al. 1993) the excess numbers do not prevent the death of CCR2−/− mice. This ﬁnding contrasts with the report that elevated neutrophil numbers is genetically linked to enhanced resolution of infection (Mayﬁeld et al. 2010). Alveolar MM are presumed to be the initial contact between host and fungus since they are resident within lungs and do not require movement from the circulation. By 24 h, DC are the dominant infected population, and in early infection, they act as guardians since they constitutively kill yeast cells and prevent conversion of conidia to yeasts (Gildea et al. 2001; Newman et al. 2010). A population of DC that are CD11c+, CD205+, Ly6C+, and CD11b+ preferentially harbor H. capsulatum (Deepe et al. 2008). Among MM, yeast cells are principally found in Ly6C+ and/or CD62L+ inﬂammatory monocytes. The proportion of infected alveolar MM wanes over time as waves of monocytes migrate into lungs and ingest yeast cells. A portion of infected monocytes likely set up residence in lungs, but another fraction exit lungs, carry viable yeast cells to other organs, and initiate infection. Migration of T and B cells develops on or after day 7 at which time the fungal burden peaks in models of self-limited histoplasmosis. This inﬂux of lymphocytes coincides with the onset of the cellular immune response. Among T cells, CD4+ cells constitute a higher proportion and number than CD8+ T cells. Proportionally T cells peak between weeks 2 and 3 of infection. The movement of a sufﬁcient number of T cells, particularly CD4+ cells, is critically important to a successful outcome (Gomez et al. 1988; Allendorfer et al. 1999). Much less is known about the importance of B cells in host control although the data suggest they are dispensable (Allendorfer et al. 1999). In fact, they may actually dampen efﬁcient clearance by preventing the inﬂux of neutrophils into lungs (Mayﬁeld et al. 2010).

6.2.2

The Granuloma and H. Capsulatum

Granulomatous inﬂammation has long been considered a hallmark of an effective cellular immune response for intracellular pathogens. The granuloma is a highly organized structure consisting of multiple cell populations that are strategically located to communicate with one another (Kindler et al. 1989). Accumulated evidence suggests that the granuloma is a compromise between the host and the pathogen. Granulomas protect the host by limiting spread, and the tradeoff is that the organism ﬁnds a niche in which to survive (Cosma et al. 2004). Histoplasma, like other intracellular pathogens, induces granulomas. These are formed as early as 2 days after infection is established in tissues thus supporting the contention that these aggregates are not dependent on adaptive immunity for their formation (Heninger et al. 2006). Moreover, this early development may be a means of spreading the infection (Davis et al. 2002; Davis and Ramakrishnan 2009). The size of H. capsulatum granulomas peaks at 10 days and thereafter contracts probably as a consequence of interleukin (IL)-10 (Heninger et al. 2006; Davis and Ramakrishnan 2009). The central portion of the granuloma contains yeast cells and apoptotic 0M that are likely to be a target for ingestion by surrounding 0M. Uptake of apoptotic cells

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prompts IL-10 generation (Kim et al. 2004). CD4+ and CD8+ T cells are found within the granuloma, and based on T cell receptor repertoire analysis these cells are heterogeneous. This ﬁnding contrast with the oligoclonality found among T cells in the lungs of infected mice (Gomez et al. 1998). Granuloma T cells are the source of both IL-17 and IFN-J, two cytokines that contribute to host defenses (see below). Neutrophils are rarely found, and DC are sparse. Their contribution to the formation and maintenance of the granuloma is not known, although it is speculated they may recruit additional T cells or migrate to distant sites to activate T cells (Heninger et al. 2006).

6.3 6.3.1

The Intracellular Lifestyle of H. Capsulatum Residence Within the Mf

Among phagocytes, 0I are the ideal haven for H. capsulatum. They provide a niche in which the saprobic form germinates into yeast cells and these forms replicate (Bullock and Wright 1987; Newman et al. 2010). The centrality of these cells in host defenses has been buttressed by the ﬁnding that individuals with congenital monocytopenia are susceptible to disseminated histoplasmosis (Vinh et al. 2009). Yeasts and conidia utilize the CD11/CD18 adhesin receptors for gaining entrance into 0I (Bullock and Wright 1987; Newman et al. 1990). CD11/CD18 recognizes heat shock protein (hsp) 60 on the surface of yeast cells (Long et al. 2003) (Fig. 6.1). CD11b (complement receptor 3) in conjunction with the E chain, CD18, appears crucial for delivering post-binding signals and for phagocytosis. Yeast cells attach to the lectin binding domain of CD11b, and this interaction relies on sialic acids on 0M. Other receptors known to be important in pathogen attachment including dectin-1, mannose receptor, or toll-like receptors two and four do not initiate phagocytosis of H. capsulatum. However, dectin-1 and CD11b cooperate to induce the production of IL-6 and TNF-D (Lin et al. 2010). The ﬁnding that Histoplasma yeast cells utilize dectin-1 to promote cytokine responses may vary with the speciﬁc isolate. A few genotypes possess D glucan on their surface that serves to mask E glucan in the cell wall (Rappleye et al. 2007). Removal of D glucan exposes E glucan in the cell wall permitting these yeast cells to attach via dectin-1 (Marion et al. 2006). Differences have been reported concerning the intracellular residence of yeast cells. These forms grow within phagolysosomes of a murine 0M line, but this has not been observed in human 0M or in other murine 0M (Eissenberg et al. 1988; Strasser et al. 1999). These disparate results have not been reconciled, but they denote that phagolysosomal fusion varies with the tissue source of 0M. Nevertheless, yeast cells thrive in phagolysosomes and resist the oxidative burst (Bullock 1993). Yeast cells thwart the antifungal machinery of 0M by raising the pH of phagosomes to ~ 6.2–6.4. This pH prevents activation of acidic proteinases that damage the fungus, yet it is sufﬁciently acidic for the fungus to acquire iron which is necessary for its growth (Eissenberg et al. 1993; Newman et al. 1994; Strasser et al. 1999).

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Fig. 6.1 Binding of H. capsulatum to DC or MM. The fungus attaches to the VLA5 integrin on the surface of dendritic cells. This receptor recognizes cyclophilin A on the surface of the yeast cells. Yeast cells are killed (denoted by an X) by the DC upon internalization. On the other, yeast cells enter MM through the CD11/CD18 integrin family that recognizes hsp 60 on the surface of the yeast. The fate of yeast cells entering this phagocyte is differs considerably than that found in DC. Yeast cells thrive in this environment until the MM is activated by cytokines. CR3 = complement receptor 3 or CD11b; CypA = cyclophilin A; Hc = H. capsulatum; HSP 60 = heat shock protein 60, and VLA5 = very late antigen-5 (D5E1) integrin

Growth inhibition of H. capsulatum by 0M requires exogenous stimulation with cytokines. Differences are apparent between murine and human cells. In murine 0M, IFN-J, GM-CSF, and M-CSF activate several sources of 0M although splenic 0M need priming before they respond to IFN-J (Lane et al. 1993; Brummer and Stevens 1994; Winters et al. 2010). Two murine 0M lines and human MM do not respond at all to IFN-J (Wu-Hsieh and Howard 1989; Fleischmann et al. 1990; Newman and Gootee 1992). Human cells activated with CSFs inhibit growth (Newman and Gootee 1992). Murine 0M mediate growth inhibition by production of nitric oxide (NO) that nitrosylates iron thus rendering unusable by the fungus (Lane et al. 1994). Zinc deprivation as a consequence of GM-CSF or IFN-J activation of murine MM is considered another mechanism by which the host starves the fungus (Winters et al. 2010).

6.3.2

Neutrophils

Although neutrophils bind to yeast cells via the same integrin family as MM, the former inhibit intracellular growth of H. capsulatum. Antifungal defenses exerted by neutrophils are dependent on the contents of azurophilic granules (Newman et al. 1993, 2000). Human defensins are partially responsible for limiting growth, but these molecules are not the only ones since murine neutrophils exert identical activity and these cells do not contain defensins (Kurita et al. 1991; Newman et al. 1991; Eisenhauer and Lehrer 1992; Couto et al. 1994). In vivo studies describe the

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necessity of murine neutrophils for host survival against H. capsulatum (Zhou et al. 1998; Sa-Nunes et al. 2006). The interpretation of these studies may be in question since they employed an antibody that not only depletes murine neutrophils but also activated monocytes (Serbina et al. 2008). Therefore, the conclusion that neutrophils are requisite in vivo should be construed with caution since monocytes and neutrophils were depleted.

6.3.3

DC

These cells bind and ingest H. capsulatum through a ﬁbronectin receptor, very late antigen-5 or D5E1 integrin (Gildea et al. 2001) (Fig. 6.1). This surface receptor recognizes cyclophilin A on the surface of yeast cells (Gomez et al. 2008). DC, unlike neutrophils or 0M, exhibit an intrinsic capacity to kill yeast cells. Destruction is exceptionally rapid with structural alterations apparent within 30 min to an hour. Killing requires phagolysosomal fusion (Gildea et al. 2001, 2005). The fact that yeast cells enter through a different receptor-ligand interaction might explain why the fate of the organism differs so much from those that enter MM or neutrophils. Not only do DC kill, but these cells present immunogenic peptides through the class II MHC pathway or through cross-presentation to trigger antigen-speciﬁc CD4+ and CD8+ T cells (Gildea et al. 2001; Lin et al. 2005). Entrance into the cross-presentation pathway proceeds when DC ingest apoptotic infected MM (Lin et al. 2005). Apoptosis, therefore, exerts a beneﬁcial effect in controlling histoplasmosis. On the other hand, much of the literature reports that apoptosis leads to exacerbation of a variety of infections because pathogens induce apoptosis of T cells that are necessary for elimination of the pathogen (Thompson 1995; Hirsch et al. 1999; Hotchkiss et al. 2001; Welsh et al. 2004). In histoplasmosis however, apoptosis is necessary for clearance since interfering with this process leads to a fatal outcome (Allen and Deepe 2005).

6.4 6.4.1

Lymphoid Cell Control of Progression of Infection Inﬂuence of T Cells

Activation of cellular immunity is generally considered to be the primary means by which the host restricts growth of the fungus. T cells are critical for eradication and in most mouse models, this coincides with accumulation and activation of T cells (Artz and Bullock 1979; Wu-Hsieh 1989; Zhou et al. 1995; Allendorfer et al. 1999; Lin and Wu-Hsieh 2004). In primary infection, a hierarchy exists in the function of CD4+ and CD8+ cells. The loss of the former results in uncontrolled infection whereas depletion of the latter only modestly increases the severity of infection

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(Deepe 1994; Zhou et al. 1998; Allendorfer et al. 1999). Mice that have a congenital lack of CD4+ cells as a result of genetic excision of MHC II antigens establish a chronic infection that remains static for up to 16 weeks (Lin et al. 2005). In these mice, CD8+ T cells are enabled to constrain Histoplasma growth but cannot sterilize tissues. The import of CD8+ T cells as mediators of protection has been bolstered by vaccination studies. In mice vaccinated with live yeast cells, the loss of CD4+ or CD8+ cells does not blunt fungal eradication, but depletion of both does (Allendorfer et al. 1999; Wuthrich et al. 2003). Progressive infection develops in lymphoid tissue but not lungs (Allendorfer et al. 1999). The ability of lungs to control infection in a T cell-independent manner relies on TNF-D production whereas in lymphoid tissue the lack of T cells abolishes cytokine generation (Allendorfer et al. 1999).

6.4.2

The T Cell Receptor (TCR) Repertoire Response

The TCR is pivotal in dictating the biological activities of T cells. It binds to MHC molecules loaded with peptide antigen (Acha-Orbea et al. 1988; Zamvil et al. 1988; Ruberti et al. 1991). Two receptors have been described on CD3+ T cells: D/E and J/G. The former are the principal cells involved in adaptive immunity, and the latter engage in innate immune functions; both produce IFN-J, IL-17, and a host of other cytokines (Ishikawa et al. 1993; Hamada et al. 2008). The focus of Histoplasma studies has been on the D/E TCR since no data exist that J/G T cells participate in immunity. The TCR response to pulmonary infection is skewed. Coincident with the onset and expression of the adaptive immune response in lungs of mice, an oligoclonal VE4 family arises but dissipates by day 21. These cells contribute to the control of infection since their elimination blunts but does not account for all the antifungal activity expressed by T cells (Gomez et al. 1998). On the other hand, others have reported the TCR repertoire in histoplasmosis is not biased (Lin and Wu-Hsieh 2004). The disparate ﬁndings may be attributed to the use of different Histoplasma strains. In addition, the latter study examined exclusively IFN-J-producing cells whereas the former analyzed all T cells. Skewing of the VE4 cells is mouse strain speciﬁc (Deepe et al. 2002). In a memory T cell response, one might expect that VE4+ cells would be conserved and expand again upon re-exposure to the fungus. Indeed, primary and memory T cells manifest an identical TCR repertoire in murine listerosis (Busch et al. 1998). In contradistinction, secondary histoplasmosis is associated with an expansion of both VE6+ and VE4+ cells. Protective immunity relies on collaboration between VE6+ and VE4+, cells (Gomez et al. 2001). The antigen, hsp 60, confers protection in a model of histoplasmosis, and its effect is dependent on T cells (Gomez et al. 1995; Deepe and Gibbons 2002). VE8+ cells emerge as the dominant T cell population that recognizes hsp 60. This population is necessary for protection conferred by this vaccine (Scheckelhoff and Deepe 2002). The VE8 cells that mediate protection is characterized as Th1 that reacts with a

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protective fragment of hsp 60 and bears a speciﬁc complementarity determining region sequence (Scheckelhoff and Deepe 2002). The ﬁne speciﬁcity of protective immunity induced by T cells is quite remarkable, yet it raises serious issues when one extrapolates to the human condition. The possibility that any given individual may not express a particular TCR family raises the possibility that holes in the TCR repertoire may predispose them either to not respond to a vaccine or to be more susceptible to histoplasmosis.

6.4.2.1

T Cells and Reactivation Histoplasmosis

After primary infection, much of the disease ascribed to H. capsulatum is considered to be reactivation as latent organisms escape conﬁnement from within granulomas. Little is known about the breakdown in immunity that facilitates the breakout of the fungus. In mice, reactivation requires the elimination of both CD4+ and CD8+ T cells. Under these experimental conditions, fungi are detected by 14 days and the infection persists, but does not progress. Depletion of CD4+ cells induces reactivation in B cell knockout mice suggesting there is interplay between these two populations. A progressive, fatal reactivation develops when CD4+ and CD8+ -depleted mice are given anti-Thy1.2. This progression is not duplicated by treatment with anti-Thy-1.2 alone. A double negative (CD4−CD8−) T cell prevents reactivation from escalating to a lethal infection, and its effect is only unearthed when CD4+ and CD8+ cells are absent.

6.4.3

T Cell Function

How do T cells promote immunity? The most likely mechanism is by production of cytokines that enhance protective immunity. T cells are rich sources of IFN-J and TNF-D that are necessary for control of infection. Both CD4+ and CD8+ T cells generate them during active infection (Lin and Wu-Hsieh 2004). T cells, especially CD4+, but also CD8+, cells produce IL-17 that modestly contributes to control of infection in wild-type mice (Deepe and Gibbons 2009). The protective action of CD4+ T cells requires TNF-D (Deepe and Gibbons 2006). H. capsulatum-speciﬁc CD8+ cytotoxic T cells recognize and lyse infected 0M. The upshot is that yeast cells presumably decorated with host membrane are ingested by DC that shuttle these bodies into the cross-presentation and the class II MHC pathway thereby activating both CD4+ and CD8+ cells (Lin et al. 2005). The surface molecule, programmed death (PD) 1, delivers negative signals to T cells upon engagement by PD ligands, and by doing so, is necessary for self tolerance. PD 1 is highly expressed on “exhausted” T cells that are found in chronic infections; blockade of the PD 1 –PD ligand interaction restores their vitality (Keir et al. 2007; Franceschini et al. 2009). In murine histoplasmosis, PD ligand is upregulated on MM which may suppress T cell responsiveness. Interrupting the PD 1-PD ligand interaction rescues mice given a lethal challenge (Lazar-Molnar et al. 2008).

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Regulatory T Cells

Regulatory T cells constitute one of the major lineages of T cells. These cells express a number of activities; they suppress T cell growth, inhibit cytokine production, and depress effector T cell ingress into tissues (Belkaid et al. 2002, 2003; Mendez et al. 2004; Mills 2004; Belkaid and Rouse 2005; Shaﬁani et al. 2010). These cells are classically deﬁned as CD4+CD25+ and express the transcription factor Foxp3. Other populations with regulatory function have been identiﬁed, thus this particular population is not the exclusive regulatory T cell. An abundance of regulatory T cells is accompanied by depression in the vigor of cellular immunity. This perturbation favors replication of an organism. Regulatory T cells or suppressor cells have been observed in murine and human histoplasmosis (Stobo et al. 1976; Artz and Bullock 1979; Nickerson et al. 1981; Watson and Bullock 1982). CD4+CD25+ Foxp3+ cells are detected in the lungs of mice infected with H. capsulatum, but their numbers are sharply increased when TNF-D is neutralized (Deepe and Gibbons 2008). Expansion of this population contributes to the deleterious effects of anti-TNF-D in histoplasmosis, and their immune suppressive activity is mediated by IL-10 (Deepe and Gibbons 2008).

6.4.5

B Cell and Antibody Contribution to Immunity

The inﬂuence of B cells and antibody in limiting the growth of H. capsulatum appears limited. Transfer of monoclonal antibodies that react with various antigens of the fungus reduces fungal burden (Nosanchuk et al. 2003; Guimaraes et al. 2009; Lopes et al. 2010). The fact that serum cannot transfer immunity (Saslaw and Schaefer 1956) would suggest that antigen-speciﬁc antibodies are probably insufﬁciently produced to exert a meaningful impact on the course of infection. In vitro these antibodies do facilitate uptake of the fungus, cause agglutination, and enhance killing by 0M (Guimaraes et al. 2010).

6.5

The Impact of Cytokines and Chemokines in Host Control

6.5.1

Cytokines that Promote Immunity

6.5.1.1

IL-12/IFN-g Axis

Non-lethal challenges with H. capsulatum evoke a strong Th1 response in mice, and integrity of the IL-12-IFN-J axis is essential to protection in primary, but not secondary, histoplasmosis (Zhou et al. 1995, 1997; Allendoerfer and Deepe 1997; Cain and Deepe 2000). The bulk of IFN-J is produced by T cells, and CD4+ cells appear

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to the major source of this cytokine (Allendorfer et al. 1999). Depletion of CD4+ T cells sharply reduces IFN-J levels by at least 80–90%. Production of IFN-J requires perforin but not CD40 or CD40 ligand (Zhou and Seder 1998; Zhou et al. 2001; Wheat et al. 2002). Humans require IFN-J for host control; a genetic deﬁciency in IFN-J receptor 1 is associated with disseminated histoplasmosis (Zerbe and Holland 2005). Only a few explanations account for the failure of mice lacking IFN-J to control infection. These include depressed levels of NO (Zhou et al. 1995), and a shift in the Th1/Th2 balance (Allendoerfer et al. 1997). In contrast to the abundance of literature regarding the impact of IL-12/IFN-J axis on host resistance, there is little known about the role of IFN-D/E. Bone marrow-derived 0M synthesize this gene in response to conidia but not yeast cells suggesting that the saprobic phase, and not the pathogenic phase, contains antigens that trigger this pathway. The source or sources of IFN-D/E in vivo have not been identiﬁed, thus it is not known if alveolar or inﬂammatory 0M produce this cytokine following infection. The absence of IFN-D/E signaling inﬂuences the course of infection. IFN-D/E receptor knockout mice challenged with conidia manifest accelerated clearance but not before 2 weeks of infection (Inglis et al. 2010). Enhanced elimination transpires when yeast cells are exclusively present, thus leading to the conclusion that IFN-D/E negatively impacts T cell-dependent, but not innate immunity.

6.5.1.2

GM-CSF

Control of primary infection requires endogenous GM-CSF. Its absence in vivo produces depressed levels of IFN-J, TNF-D, and NO and elevated levels of IL-4 and IL-10 (Deepe et al. 1999). In secondary histoplasmosis, neutralization of GM-CSF impairs fungal elimination, but animals do not succumb to overwhelming histoplasmosis. The mechanism underlying this effect is unknown but is not caused by a decrement in IFN-J, TNF-D, or NO (Deepe et al. 1999).

6.5.1.3

IL-1

The inability to signal through the IL-1 receptor or a deﬁciency in IL-1E exacerbates infection in association with increases in IL-4 and IL-10 (Deepe and McGuinness 2006). The absence of IL-1 signaling produces a transient reduction in NO that may contribute to the failure of the host to control infection. Interestingly, a deﬁciency in IL-1 signaling is accompanied by a decrease in CD4+ cells in the lungs of mice. These results suggest that engagement of IL-1/IL-1 receptor engagement is key in promoting the inﬂux of this T cell population. Since these studies were conducted prior to knowledge regarding the inﬂuence of IL-1 on IL-17 production (Weaver et al. 2007), it is not known if IL-1 promotes IL-17 release in histoplasmosis.

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IL-17 and Th17

In wild-type mice, both IL-17A and IL-17 F are induced during the course of primary infection with H. capsulatum. CD4+ and CD8+ T cells are the major, but not exclusive, source of IL-17A, and the former are numerically more abundant. One of the primary cytokines that promote IL-17 production is IL-6 (Kimura et al. 2007; Zhou et al. 2007). Surprisingly, IL-17 generation by CD4+ and CD8+ cells is largely independent of IL-6 in this infectious diseases model. Although IL-17 production is independent of IL-6, the immunological activity of IL-17 requires the presence of IL-6 and IL-10. Despite robust production, neutralization of IL-17A in murine histoplasmosis produces only a modest to moderate dampening of control of infection. The slight impairment in protective immunity is puzzling since there is a 60–70% reduction in the number of inﬂammatory cells in the lungs. This result indicates that the host does not require an optimal number of inﬂammatory cells to clear infection (Deepe and Gibbons 2009). A more vigorous inﬂuence of IL-17 is uncovered in mice lacking the p35 chain of IL-12. These mice produce IL-23 but not IL-12 and they manifest prolonged survival against a challenge with H. capsulatum. The prolonged survival in the p35 knockout mice is attenuated when anti-IL-17A is given (Deepe and Gibbons 2009).

6.5.1.5

TNF-D

TNF-D is one of the most inﬂuential cytokines involved in immunity to this fungus. This assertion stems from the observation that it is the only cytokine that abolishes immunity in primary and secondary infection. Even mice with pre-existing immunity succumb as rapidly as naïve mice to infection when anti-TNF-D is administered (Smith et al. 1990; Wu-Hsieh et al. 1992; Allendoerfer and Deepe 1998; Zhou et al. 1998). The signiﬁcance of this cytokine in control of histoplasmosis has extended to the clinical realm. A portion of patients with a variety of inﬂammatory diseases such as rheumatoid arthritis, Crohn’s disease, and psoriasis receive treatment with one of several TNF-D antagonists. A major complication associated with these antagonists is infection, often with intracellular pathogens including H. capsulatum (Wood et al. 2003; Wallis et al. 2004; Saliu et al. 2006). Neutralization of TNF-D perturbs immunity in primary and secondary infection by different means. In primary histoplasmosis, NO levels are diminished and in secondary infection, IL-4 and IL-10 are increased (Allendoerfer and Deepe 1998; Deepe 2007).. The elevation in both is biologically meaningful since neutralization of both recues mice from the deleterious effects of anti-TNF-D. Other disturbances caused by antagonism of TNF-D include irreversible impairment in T cell function and expansion of regulatory CD4+CD25+ T cells (Deepe and Gibbons 2006; Deepe and Gibbons 2008). The biological actions of TNF-D are mediated by signaling through TNF receptor 1 (p55) and TNF receptor 2 (p75). TNF receptor 1−/− or 2−/− mice succumb to primary histoplasmosis. The failure to control infection in TNF receptor 1−/− mice is

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associated with poor mobilization of inﬂammatory cells whereas in TNF receptor 2−/− mice there is a profound reduction in IFN-J levels (Allendoerfer and Deepe 2000). Recombinant IFN-J restores protective immunity in the TNF receptor 2−/− mice. Unlike the ﬁndings in primary infection, TNF receptor 2−/− mice resist secondary infection if exposed initially to a smaller number of yeast cells to induce immunity.

6.5.2

Cytokines that Exacerbate Infection

IL-4 and IL-10 are the two cytokines that are currently known to cause disease exacerbation. Elevated IL-4 and/or IL-10 are associated with a number of immunosuppressive conditions including neutralization of TNF-D, GM-CSF, or the absence of IL-1 receptor signaling. IL-4 and/or IL-10 dampen immunity since neutralization of one or both latter leads to a recovery of anti-Histoplasma immunity (Allendoerfer and Deepe 1998; Deepe et al. 1999). Injection of goat anti-mouse IgD to mice enhances the numbers of IL-4 and IL-10-producing cells, decreases the number of IFN-J+ cells, and blunts fungal clearance (Peng et al. 2005); eventually, however, the numbers of IFN-J+ cells recover, and Histoplasma is eliminated. This reversal may be a result of counter-regulatory mechanisms that turn off the signals that cause excessive numbers of IL-4+ and IL-10+ cells. Another explanation is that the activity of anti-IgD may have waned as a result of catabolism.

6.5.2.1

IL-4

It is not necessary for IL-4 to be elevated in order to dampen protective immunity. A decrement in IL-12 or IFN-J changes the balance between IFN-J and IL-4, and this alteration produces a dominance of IL-4 bioactivity and enhances severity of infection (Allendoerfer et al. 1997). IL-4 lung transgenic mice manifest impaired clearance, but these mice eventually recover suggesting that overproduction of IL-4 only may not abolish immunity (Gildea et al. 2003). Alternatively, these mice exhibit elevated numbers of neutrophils prior to infection, and this increase may prevent progression of the infection. Despite the negative impact of IL-4, IL-4−/− mice do not manifest enhanced immunity as one might predict (Szymczak and Deepe 2009). The mechanisms by which IL-4 hinders protective immunity have not been deﬁned. This cytokine alternatively activates 0M and these cells are more permissive for intracellular growth (Winters et al. 2010). In vivo, alternatively activated 0M are detected in H. capsulatum-infected mice with elevated IL-4, but whether these cells are actually more permissive for intracellular growth is not known (Szymczak and Deepe 2009).

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IL-10

IL-10−/− mice exhibit accelerated clearance in both primary secondary histoplasmosis (Deepe and Gibbons 2003). The effect of the absence of IL-10 is not observed until T cell-mediated immunity is activated (>7 days). These results indicate that innate immunity remains intact despite the absence of IL-10. Although the lack of IL-10 promotes more rapid fungal clearance, it does not afford protection from the deleterious effects of neutralization of TNF-D or IFN-J (Deepe and Gibbons 2003). In IL-10−/− mice, memory T cells are increased in number and the potency of their protective activity is enhanced.

6.5.3

Chemokines and Chemokine Receptors

Forty members comprise the chemokine superfamily. They are divided into four (D to G) families based on the spacing of the ﬁrst two conserved cysteines at the NH2 terminus. Another categorization is the inducible (i.e., inﬂammatory) or the constitutive chemokines. The former are produced in response to pathogens and mediate a number of effector properties including cell trafﬁcking and T cell differentiation (Rossi and Zlotnik 2000; Sallusto et al. 2000; Mellado et al. 2001; Rot and von Andrian 2004). Chemokine receptors and chemokines manifest promiscuity which creates redundancy in chemokine actions (Sallusto et al. 2000).

6.5.3.1

CCR2 and Its Ligands

Mice lacking the chemokine receptor CCR2 are more susceptible to infection with H. capsulatum. These mice manifest several immunological defects including a decrement in the number of inﬂammatory cells recruited to the lungs. The principal reduction is in inﬂammatory 0M and DC. Progression of infection in CCR2−/− mice is caused by elevation of IL-4 that is produced by several cell populations including 0M and DC (Szymczak and Deepe 2009). This increase in IL-4 is also associated with an increase in signatures of alternatively activated MM including arginase 1, FIZZ1, Ym1, and transferrin receptor but not a decrement in NO, which is a typical consequence of an increase in arginase. A role for arginase in impairing immunity has been suggested since administration of L-arginine improves fungal elimination (Fig. 6.2). As previously mentioned, IL-4 alone may lead to death of animals that are otherwise intact. Since the lungs of CCR2−/− mice contain far fewer cells, especially phagocytes, it is likely that MM will encounter excessive IL-4 and thus become alternatively activated (Fig. 6.3). Consequently, their anti-Histoplasma activity is impaired. CCR2 binds four chemokines, and CCL2 exclusively binds CCR2. The murine CCL2-CCR2 axis is exceedingly important in shaping the development of type 1 or 2 immunity by altering generation of IL-12 or by promoting the production of IL-4 by activated T cells (Luther and Cyster 2001). The lack of CCL2 does not mimic the

Fig. 6.2 Treatment with L-arginine but not D-arginine modiﬁes the fungal burden of CC2−/− mice infected intranasally with H. capsulatum. Wild-type (WT) or CCR2−/− mice were infected with 2 × 106 yeast cells intranasally and on day 6 they were given 7.5 mg of L- or D-arginine intraperitoneally. They were sacriﬁced on day 7, and the number of colony-forming units (CFU) from lungs was quantiﬁed. The results represent mean ± Standard error the mean (SEM) of six mice. * = p < 0.05

Fig. 6.3 The network operative in CCR2−/− mice. The inability of CCL2 and CCL7 to signal leads to increased production of IL-4 by a number of cell populations including CD4+, macrophages, and dendritic cells. IL-4 can act as a paracrine or in a autocrine fashion. One of the major effects of this excessive IL-4 is to alternatively activate macrophages. Thus, these cells upregulate several genes including arginase 1, FIZZ1, Ym1, and transferrin receptor. The increase in arginase 1 likely impairs killing, and elevated numbers of transferrin receptors promotes uptake of iron that facilitates growth. Arg 1 = arginase 1; IL-4 = interleukin 4, and TR = transferrin receptor

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absence of CCR2 in murine histoplasmosis since the CCL2−/− mice efﬁciently eradicate infection. Only the absence of CCL2 and CCL7 mirrors what is found in CCR2−/− mice (Szymczak and Deepe 2009). Hence, CCL2 and CCL7 cooperate to constrain IL-4 generation. The increase in IL-4 in CCR2−/− mice is in part caused by the paucity of DC migrating into lungs. Supplementation of lungs with antigenloaded DC suppresses IL-4 but not the fungal burden. The effect of DC is dependent on the presence of MHC class II and expression of CD40.The increased IL-4 levels and fungal burden in CCR2−/− mice are restored to that of wild-type when DC are transferred and CD4+ cell are eliminated prior to infection (Szymczak and Deepe 2010). This ﬁnding strongly suggests that a regulatory T cell population exists in Histoplasma-infected CCR2−/− mice.

6.5.3.2

CCR5 and Its Ligands

CCR5 is best known as a co-receptor for the human immunodeﬁciency virus (Oppermann 2004), but it is a mediator that is crucial for attracting a number of cell populations including regulatory T cells. Unlike the progressive infection in CCR2−/− mice, CCR5−/− manifest an accelerated elimination of fungus during the time when T cell-mediated immunity is activated. By contrast the infection in knockouts is slightly worse between days 3 and 7. The heightened severity during the innate response is probably a result of fewer inﬂammatory cells in lungs thus creating a suboptimal protective response. Accelerated elimination of Histoplasma at day 14 and beyond is caused by a shift in the regulatory T cell/Th17 balance. The numbers of the former are sharply depressed in lungs while there is a concomitant increase in Th17 cells (Fig. 6.4). Examination of the cytokine proﬁle of the lungs provides an insight of why Th17 cells are increased. There is higher amounts of IL-6 and IL-23 and lower quantities of transforming growth factor (TGF)-E, three factors critical in shaping the emergence of the Th17 cells (Bettelli et al. 2007). Neutralization of IL-17A does increase fungal burden but only to the level of that observed in wild-type and does not cause a progressive infection (Kroetz and Deepe 2010). This effect of IL-17A neutralization is strikingly distinct from the modest effect observed in wild-type mice. Taken together these ﬁndings imply in murine histoplasmosis that a decrement of regulatory T cells promotes IL-17 but the converse is not true. Thus, regulatory T cells appear to be the dominant driving force in dictating that balance.

6.5.4

Leukotrienes

Leukotrienes are important to H. capsulatum elimination (Medeiros et al. 1999). Inhibition of leukotriene synthesis is associated with a decrease in IFN-J which may explain in part the impaired immunity. The absence of leukotriene synthesis hampers

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Fig. 6.4 CCR5 dictates the balance between regulatory T cells and Th17 cells. In wild-type mice there is a balance between these two populations. When IL-17A neutralized, there is not an expansion of regulatory T cells, but the balance is shifted. However, there is only a slight change in the fungal burden in these animals. In the absence of CCR5, the balanced is more dramatically tipped and favors Th17. The associated decrease in regulatory T cells is marked by increases in IL-6, IL-23, and IL-17A and a concomitant decrease in TGF-E. These changes are not observed in wildtype mice given anti-IL-17A. IL-6 = interleukin 6; IL-23 = interleukin 23; IL-17A = interleukin 17A; TGF-E = transforming growth factor-E, and Treg = regulatory T cells

the recruitment of memory T cells into lungs of mice. Moreover, the lack of leukotrienes is accompanied by an upregulation of IL-10, which is known to depress immunity, and TGF-E (Medeiros et al. 2008). It is tempting to speculate that the increase in IL-10 and TGF-E means that regulatory T cells may be activated in mice unable to synthesize leukotrienes.

6.6

Conclusions

This chapter summarizes the current ﬁndings regarding host control of the protective immune response to the pathogenic fungus, H. capsulatum. It highlights the many mediators and the cells that must cooperate to effect optimal clearance. Although much is known, there are still major gaps in our knowledge regarding molecular and cellular control of infection. Answers to the many unsolved questions will likely have a clinical impact as patients receive newer immunotherapies that may be beneﬁcial but also may induce serious infectious complications. The knowledge gained from pursuit of these studies may facilitate the creation of biologicals that are more precise in modulating immunity.

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Wu-Hsieh, B. and D. H. Howard (1989). “Macrophage cell lines P388D1 and IC-21 stimulated with gamma interferon fail to inhibit the intracellular growth of Histoplasma capsulatum.” Infect Immun 57(9): 2903–5. Wu-Hsieh, B. A., G. S. Lee, et al. (1992). “Early activation of splenic macrophages by tumor necrosis factor alpha is important in determining the outcome of experimental histoplasmosis in mice.” Infect Immun 60(10): 4230–8. Wuthrich, M., H. I. Filutowicz, et al. (2003). “Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 help in exogenous antigen presentation to CD8+ T cells: implications for vaccine development in immune-deﬁcient hosts.” J Exp Med 197(11): 1405–16. Zamvil, S. S., D. J. Mitchell, et al. (1988). “Predominant expression of a T cell receptor V beta gene subfamily in autoimmune encephalomyelitis.” J Exp Med 167(5): 1586–96. Zerbe, C. S. and S. M. Holland (2005). “Disseminated histoplasmosis in persons with interferongamma receptor 1 deﬁciency.” Clin Infect Dis 41(4): e38–41. Zhou, P. and R. A. Seder (1998). “CD40 ligand is not essential for induction of type 1 cytokine responses or protective immunity after primary or secondary infection with Histoplasma capsulatum.” J Exp Med 187(8): 1315–24. Zhou, P., M. C. Sieve, et al. (1995). “IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-J.” J Immunol 155(2): 785–95. Zhou, P., M. C. Sieve, et al. (1997). “Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasma capsulatum.” Infect Immun 65(3): 936–42. Zhou, P., G. Miller, et al. (1998). “Factors involved in regulating primary and secondary immunity to infection with Histoplasma capsulatum: TNF-D plays a critical role in maintaining secondary immunity in the absence of IFN-J.” J Immunol 160(3): 1359–68. Zhou, P., B. L. Freidag, et al. (2001). “Perforin is required for primary immunity to Histoplasma capsulatum.” J Immunol 166(3): 1968–74. Zhou, L., Ivanov, II, et al. (2007). “IL-6 programs Th-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways.” Nat Immunol 8(9): 967–74.

Chapter 7

Modulation of T-Cell Mediated Immunity by Cytomegalovirus Chris A. Benedict, Ramon Arens, Andrea Loewendorf, and Edith M. Janssen

7.1

Introduction

The herpesviruses have coevolved with their vertebrate hosts for over one hundred million years (McGeoch et al. 2000), resulting in a ﬁnely tuned equilibrium with the immune system. Consequently, all herpesviruses employ a multitude of strategies to modulate the host immune response, facilitating the establishment of lifelong latency and/or persistence in the face of a robust innate and adaptive immune response. Cytomegalovirus (CMV, a E-herpesvirus) is the largest of the herpesviruses, with a genome of ~230 kB in size encoding >200 open reading frames (orfs). Approximately ~60% of the encoded genes are not essential for replication of virus in tissue culture where there is no selective pressure from the host immune system and are predicted to perform immunomodulatory functions and facilitate establishment of latency (Murphy et al. 2003; Brocchieri et al. 2005). CMV directly targets dendritic cells (DC) and exploits the DC’s crucial role in the regulation of innate and adaptive anti-viral immune responses to promote replication and establish latency while preventing host pathology.

C.A. Benedict (*) Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA e-mail: [email protected] E.M. Janssen (*) Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_7, © Springer Science+Business Media, LLC 2012

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CMV Epidemiology and Pathology

Human (H)CMV is highly prevalent throughout all geographic locations and socioeconomic groups and infects –depending on the country- between 50% and 90% of a population. HCMV is usually acquired early in life as an asymptomatic, subclinical infection (Zanghellini et al. 1999). However, if primary HCMV infection occurs in the developing fetus or neonate (before full immune system development) consequences can be severe, with HCMV being the most common infectious cause of congenital birth defects (Gaytant et al. 2002). HCMV establishes a latent infection in many cell lineages, including monocyte precursors and stromal cells, and persists for life in its host. In healthy, persistently infected individuals huge numbers of CMV-speciﬁc T cells accumulate over time (i.e. “memory inﬂation”), a process recently associated with the immune risk proﬁle and immunosenescence in the elderly (Pawelec et al. 2005). Reactivation of HCMV and subsequent systemic HCMV viremia can occur upon immunosuppression (e.g. organ transplant recipients, patients receiving immunosuppressive drugs, and HIV-infected patients) (Rubin 2001; Steininger et al. 2006), and these cases represent signiﬁcant causes of morbidity and mortality if not controlled by antiviral drug therapy. Importantly, studies identify CMV infection as risk factor for increased arterial blood pressure, and a co-factor in aortic atherosclerosis (Streblow et al. 2008; Cheng et al. 2009). Recently, CMV infection in humans has been suggested to be associated with several human malignancies like glioblastoma. It has been proposed that CMV may directly infect tumor cells, or tumor stem cells, potentially increasing their malignancy through the process of oncomodulation (Michaelis et al. 2009).

7.3

Immune Response to CMV

Over the last decades, many cellular components, including DCs, NK cells, macrophages, B cells and CD8 and CD4 T cells have been identiﬁed to participate in the anti-CMV immune response. Although CMV replication is largely species restricted all CMVs show signiﬁcant genomic homology and exhibit similar tissue tropism, pathogenesis and temporal regulation of gene expression (Rawlinson et al. 1996; Vink et al. 2000; Davison et al. 2003; McGregor et al. 2004; Powers and Fruh 2008a, b). As a consequence, research of CMV infection in animal models has accelerated the dissection of the molecular mechanisms that carefully balance CMV infection and host immune responses (Brocchieri et al. 2005).

7.3.1

DCs in CMV Infection

DCs have multiple key regulatory roles role in CMV infection. DCs play a crucial role in the initiation and regulation of the immune response, both through their

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direct production of key cytokines (e.g. Type I IFND/E and IL12) as well as through their interaction with NK cells. In addition, DCs are critical regulators of the adaptive T cell response to CMV. DCs not only activate naïve CD4 and CD8 T cells, they also dictate the acquisition of T cell effector functions and confer the capacity for T cell survival, homeostasis, and memory formation. Importantly, CMV also directly infects DCs and myeloid lineage cells, and these cells have been shown to be reservoirs of latent CMV infection (Sinclair 2008a, b; Rolle and Olweus 2009). Perhaps not surprisingly given their central role in promoting anti-viral immune responses, many viruses have evolved strategies to speciﬁcally modulate DC phenotype and/or function. CMV directly infects DCs in vivo, and encodes several gene products that speciﬁcally interfere with various aspects of DC function (Raftery et al. 2001) (Table 7.1).

Table 1 CMV immune modulatory gene products Gene

Primate CMV homologues

Antigen presentation MCMV m04 (gp34) MCMV m06 (gp48) MCMV m152 HCMV US2 (gpUS2)

RhCMV 182

HCMV US3 (gpUS3)

HCMV US6 (gpUS6) HCMV US10 (gpUS10) HCMV US11 (gpUS11) HCMV UL82 (pp71) HCMV UL83 (pp65) HCMV UL40 HCMV UL142 (gpUL142) RhCMV Rh178 Co-stimulation MCMV m138 (fcr-1) MCMV m147.5/modB7-2 HCMV UL144 (HVEM) homolog

RhCMV 184 RhCMV 189 RhCMV 112 (pp65-2)

Cellular target/interacts with Binding of MHC I, Complex with H2-Dk is recognized by Ly49P Reduces surface expression of MHC I Reduces surface expression of MHC I MHC I heavy chain degradation. HLA-DRA, HLA-DMA degradation. Assembly of MHC I. Reduces association of MHC II A/B complexes with the invariant chain. Inhibition of TAP MHC I MHC I heavy chain degradation MHC I Binding and inactivation of NKp30, Accumulation of MHC II Increases HLA-E, recognized by HLA-E restricted TCRAB+ CD8+ CTLs (MHC)–class-I–chain–related A (MICA), a ligand for NKG2D MHC I heavy chain translation Fc-portion of mIgG, B7-1, MULT-1, H60, Rae-1varepsilon B7-2 Binds BTLA; Activates NFKB (continued)

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Table 1 (continued) Gene Cytokine homologues HCMV UL111a (cmvIL-10); LAcmvIL-10 (alt. splicing)

Primate CMV homologues

Cellular target/interacts with

RhCMV IL-10, BaCMV IL-10, AGCMV IL-10

Binds hIL-10R. Inhibits IFN-G, TNF-A, MHC I and II and NFKB. Stimulates STAT 3. Induces IDO.

NK cell recognition MCMV m157 LY49h RhCMV: Rhesus CMV; BaCMV: Baboon CMV; AGCMV: African Green Monkey CMV

7.3.2

NK and NKT Cell Response to CMV, the “Innate-to-Adaptive Transition”

NK cells play a critical role in the innate or “early” immune control of CMV infection. Humans lacking NK cells cannot control HCMV infection (Biron et al. 1989). Similarly, mice lacking NK cells or the Ly49H NK cell receptor that recognizes the MCMV m157 protein show enhanced virus production in the spleen, lung and liver during the peak of infection and are more susceptible to CMV-induced death at highdose infection (Bukowski et al. 1984; Andrews et al. 2005; Andoniou et al. 2006). Recent studies show that optimal activation of NK cells requires interaction between DC and NK cells via NKG2D activating receptor and cytokine production (IL-12, IL-18, type I IFN) by DCs. In turn, activated NK cells accelerate the induction of anti-viral CD8 T cell responses through early control of the virus and prevention of overproduction of anti-viral type I IFN that would otherwise ablate DC populations and thereby delay the priming of CD8 T cells (Andoniou et al. 2005; Walzer et al. 2005; Robbins et al. 2007). In addition, NKT cells are also activated early during MCMV infection in an IL-12 and TLR9 dependent fashion, independently of antigen presentation by CD1d (Tyznik et al. 2008). As NKT cells promote both NK cell and T cell responses in various settings, this suggests a potential role for NKT cells in promoting the adaptive immune response to CMV infection (van Dommelen et al. 2003; Wesley et al. 2008).

7.3.3

CMV Speciﬁc CD8 and CD4 T Cell Responses

Although NK cells are important cellular players for early innate defense against MCMV, both CD8 and CD4 T cells play a critical role and are absolutely required for the eventual control and clearance of MCMV infection (Holtappels et al. 2008). The contribution of CD8 T cells was originally suggested by the inability of Balb/c SCID mice to control MCMV infection (Welsh et al. 1991), and that adoptively transferred CD8 T cells restrict systemic MCMV replication in irradiated

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Balb/c mice MCMV (Reddehase et al. 1985). Interestingly, B6 RAG−/− mice (lacking T and B lymphocytes) control MCMV replication effectively for the ﬁrst ~25 days of infection, but eventually succumb to infection shortly thereafter due to in vivo selection of viral variants with mutations in m157 that circumvent NK-mediated control (French et al. 2004). Studies using a mouse model mimicking CMVreactivation and viral-induced pneumonia following bone marrow transplantation (a serious clinical complication associated with HCMV infection) indicate that CD8 T cells are strictly required for resolution of this disease, with CD4 T cells not being able to substitute in this particular model (Podlech et al. 1998). In humans, the presence of HCMV-speciﬁc CD8 T cells is associated with protection from virusinduced disease, and forms the basis for prophylactic cellular immunotherapy procedures for prevention of HCMV disease (Riddell and Greenberg 1997). HCMV-speciﬁc CD4 T cells correlate strongly with disease protection in patients. (Jonjic et al. 1989, 1990, 1994). Convincing data for a role of CD4 T cells in immune control of HCMV has come from the ubiquitous cases of HCMV-induced disease in AIDS patients, especially before the advent of highly active antiretroviral therapy (HAART) (Steininger et al. 2006). In mice, CD4 T cells can contribute to control of primary, systemic CMV infection, and are necessary to restrict persistent replication in select tissues and promote antibody responses (Walton et al. 2008). CD4 T cells are absolutely required for the control of MCMV replication in the acinar epithelial cells of the salivary gland, and can functionally substitute for immune defense to MCMV in mice depleted of CD8 T cells (Jonjic et al. 1989, 1990). Recent work has revealed that the apparent inability of CD8 T cells to control persistent MCMV replication at select anatomical sites (e.g. the salivary gland) is due, at least in part, to the efﬁcient inhibition of MHCI-mediated antigen presentation by three separate immune modulatory genes (m04, m06 and m152) (Lu et al. 2006). There is an intertwined relationship for CMV-speciﬁc CD4 and CD8 T cell responses; Priming of CD8 T cell responses in the presence of CD4 T cell “help” promotes the generation and/or maintenance of speciﬁc subsets of MCMV-speciﬁc CD8 memory T cells (Janssen et al. 2003; Sun and Bevan 2003; Snyder et al. 2009). In line with this observation in the mouse, the long term maintenance of HCMVspeciﬁc CD8 T-cells in cellular immunotherapy procedures is greatly enhanced by the co-infusion of HCMV-speciﬁc CD4 T-cells (Greenberg and Riddell 1999).

7.3.3.1

Subsets of CMV-Speciﬁc T Cells

In HCMV infected individuals, gradual increase (“memory inﬂation”) a large number of oligoclonal HCMV-speciﬁc of distinctive memory CD4 and CD8 T cell subsets exists with diverse speciﬁcities for many viral antigens, and these T cell populations display an almost exclusive characterized by the surface marker phenotype of CCR7-CD27-CD28 (van Lier et al. 2003; van Leeuwen et al. 2004; Vescovini et al. 2007).Memory T cells of this speciﬁc phenotype are not observed nearly to this extent for other chronic virus-speciﬁc T-cell populations including HIV, EpsteinBarr virus and hepatitis C virus (van Lier et al. 2003). Both mouse and rhesus CMV

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also induce characteristic CD8 and CD4 T cell memory subsets that undergo memory inﬂation and exhibit a similar “effector memory” phenotype. (Pitcher et al. 2002; Karrer et al. 2003; Munks et al. 2006; Arens et al. 2008). During MCMV infection, distinct antigen-speciﬁc CD8 T cell responses develop that do not or minimally undergo contraction (T cell contraction is typically observed during viral infections). These unique CMV-speciﬁc responses increase to signiﬁcant numbers over the course of the ensuing months and are maintained at relatively high numbers during the lifespan of the host (Sierro et al. 2005; Munks et al. 2006). Thus far it is unknown what determines the development of these phenotypically unique T cell responses during CMV infections. The immunomodulatory effects of CMV on DCs (as discussed later), the balance of CMV latency and reactivation and CMV’s cellular tropism might all directly or indirectly contribute. Importantly, the phenotype of these CMV-speciﬁc inﬂationary T cells does not correlate with that of “exhausted” memory T cells seen in models of chronic LCMV or HCV infection (Barber et al. 2006; Boni et al. 2007). It is quite likely that the “smoldering” persistent phase of CMV replication primes/restimulates T cells at a much lower level than does these other chronic viral infections, perhaps due to both viral immune modulation and a more modest antigen load.

7.4

Induction of Adaptive T Cell Responses

Adequate activation of T cells that results in proliferation and acquisition of effector functions has been shown to require multiple positive signals from the DC to the T cell. The ﬁrst signal, which is antigen-speciﬁc, is provided through the TCR which interacts with peptide-MHC molecules on the DCs. The additional signals, the costimulatory signals, are antigen nonspeciﬁc and are provided by the interaction between co-stimulatory molecules expressed by the DC and the T cell.

7.4.1

Requirement for MHC-TCR Interactions

During infection DCs present viral antigens via their MHC class I molecules to CD8 T cells and via their MHC class II molecules to CD4 T cells. There are two basic pathways by which DCs can process and present viral antigens: the exogenous pathway where viral antigens originate from the uptake of dying infected cells, and the endogenous pathway, that requires direct infection of the DC (Mellman 2005; Trombetta and Mellman 2005; Vyas et al. 2008). Thus, CMV-infected DCs fall victim to the immune-evasive machinations of the virus, while uninfected DCs are able to present viral antigens solely from exogenous sources. In the classic exogenous antigen presentation pathway, the infected material will be shuttled through endosomal and lysosomal compartments where it is cleaved/proteolyzed by various enzymes. MHC class II molecules are synthesized

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in the endoplasmic reticulum (ER) and transported from the ER via the Golgi compartments to endosomal vesicles. These vesicles fuse with the vesicles in which antigen degradation occurs to form a specialized compartment where peptide loading onto MHCII molecules takes place. Subsequently, the peptide-MHC class II complexes are transported to the cell surface for presentation to T cells. In the non-classical exogenous pathway, peptides derived from extracellular antigens are presented by MHC class I molecules in a process called cross-presentation or cross-priming; the viral proteins are cleaved into peptide fragments by the multicatalytic proteasome complexes in the cytosol. The fragments are transported to the TAP transporter and passed to the endoplasmatic reticulum (ER) where peptide loading onto class I molecules occurs. The MHC class I – peptide complex is then transported to the cell surface via the Golgi apparatus and secretory pathway (Cresswell et al. 2005; Guermonprez and Amigorena 2005). The classical endogenous antigen presentation pathway predominantly involves MHC class I molecules and normally presents peptides derived from intracellular (self) antigens. Upon infection, CMV utilizes the protein synthesis machinery of the infected cell to replicate and as a consequence viral antigens have the capacity to be targeted for MHC class I presentation and to a lesser degree for MHC class II presentation.

7.4.2

Costimulation in T Cell Priming

Although MHC-peptide recognition by the T cell receptor (TCR) on the T cell is crucial for initial activation, it will lead to anergy or non-responsiveness without appropriate additional costimulation. Costimulation can be provided by: (i) B7.1 (CD80) and B7.2 (CD86) molecules on the DC that ligate CD28 on the T cell (Greenwald et al. 2005); (ii) TNF (receptor) family members (CD40-CD40L, CD27-CD70, CD134 (OX40)CD134L and CD137 (4-1BB)-CD137L, CD30-CD30L) (Greenwald et al. 2005); and (iii) soluble mediators such as cytokines and chemokines that support the survival of the T cells and inﬂuence the phenotype of the T cells. Importantly, DCs also express molecules that provide negative signaling and can prevent T cell activation or inhibit ongoing T cell responses which is crucial to maintain peripheral tolerance and prevent pathology associated with lymphoproliferative aberrations. Binding of B7 family members B7.1 and B7.2 to cytotoxic T lymphocyte antigen (CTLA)-4 on activated T cells has been shown to be critical in the inhibition of T cell responses. In addition, binding of the B7 family members programmed death ligand (PD-L1, CD274; B7-H1) and PD-L2 (CD273/B7-DC) to PD-1 on T and B cells, and TNF receptor family member herpesvirus entry mediator (HVEM), to B and T lymphocyte attenuator (BTLA) have been shown to negatively regulate T cell activation. Besides cell-associated inhibitory molecules, DC express various soluble molecules like interleukin (IL)-10, Tumor Growth Factor (TGF)-E, and indoleamine 2,3-dioxygenase (IDO) that have been shown to negatively affect T cell activation, and inhibit ongoing T cell responses (Greenwald et al. 2005).

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The current models view the activation of naive T cells as a sum of positive and negative signals dictated by the relative expression of ligands and receptors on T cells and DC. T cells receiving a “positive net signal” from DC will undergo proliferation and acquire effector functions. Based on the type of signals, CD4 T cells will develop into T-helper (Th) 1 cells, producing IFN-J, TNF-D, and IL-2, Th2 cells that produce IL-4, -5, and −13, regulatory T cells that have been shown to secrete IL-10 and TGFE, Th17 cells secreting IL-17, or follicular helper cells (Tfh) that secrete IL-21. CD8 T cells predominantly develop into IFN-J, TNF-D, and IL-2 producing T-cytotoxic (Tc)1 cells (Woodland and Dutton 2003),and to a lesser degree into Tc2 cells (IL-4, IL-5), IL17 producing Tc cells, or Tc with regulatory capacity. In contrast, T cells receiving a “negative net signal” from the DC will be deleted or become anergic/nonresponsive.

7.5

CMV Modiﬁcation of T Cell Activating Molecules

Evidence is emerging that CMV alters the DC cosignaling rheostat, shifting the balance of positive and negative signals delivered to the T cells (Benedict et al. 2008; Sinclair 2008a, b; Rolle and Olweus 2009). Infection of DC by MCMV leads to the induction of a so-called “paralyzed” phenotype, characterized by the down-regulation of MHC class I and II, costimulatory molecules (e.g. B7 molecules) and proinﬂammatory cytokines (Andrews et al. 2001). As a consequence these infected DC are unable to promote mixed lymphocyte reactions (MLR) or activate T cells (Andrews et al. 2001; Mathys et al. 2003). Similar negative effects on T cell activation have been reported for HCMV infection of DCs or monocytes (Odeberg and Soderberg-Naucler 2001; Moutaftsi et al. 2002; Hertel et al. 2003). Importantly, recent studies show that infected DCs not only passively prevent T cell activation by restricting positive cosignals, but also actively suppress T cells by enhancing negative cosignals, potentially leading to very different biological consequences and subsequent fates for virus-speciﬁc T cells upon interaction with these virally manipulated DCs (Fig. 7.1).

7.5.1

Viral Modiﬁcation of MHC Class I Expression

As MHC-peptide expression is an absolute requirement for the activation of CD4 and CD8 T cells, it is not unexpected that all CMVs have been found to encode multiple gene products that target MHC antigen presentation (Yewdell and Hill 2002). MCMV encodes three known genes that interfere with antigen-presentation through the MHC class I pathway: m04, m06, and m152 (Pinto, Munks et al. 2006). m152 retains MHC class I molecules in the ER-Golgi intermediate compartment, while m06 redirects MHC class I molecules to the lysosmes (Gold et al. 2002). Expression of either one or both of these gene products results in

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CMV infection DC

T cell TCR CD8

MHC B7.1 B7.2

+

CD28

+

+

BTLA

UL144 MHC

TCR CD8

B7.1 B7.2

CD28

CD40

CD40L

CD40

CD40L

PD-L1

PD-1

-

PD-L1

PD-1

+ IFN- IL-12

cytokine receptor

positive net signal

proliferation effector function memory

-

cytokine receptor

+

-

-

IL-10 IDO

negative net signal

anergy tolerance deletion

Fig. 7.1 CMV mediated modulation of MHC class I/II and positive and negative cosignaling molecules on infected DC

downregulation of MHC class I expression on infected cells (Pinto et al. 2006). The precise mechanism by which m04 functions to restrict T cell recognition of MHCI-peptide remains unknown, as m04-MHCI complexes are cotransported to the surface of infected cells, but recent evidence indicates a key role for m04 in inhibiting NK cell-mediated recognition of infected cells through its interaction with H-2DK (Kleijnen et al. 1997; Kavanagh et al. 2001; Kielczewska et al. 2009). Comparably, HCMV encodes 4 glycoprotein products, US2, US3, US6, and US11 that interfere with MHC class I expression (van der Wal et al. 2002). US2 and US11 relocate the heavy chain of the MHC class I the cytosol where it is targeted for proteasomal degradation (Jones and Sun 1997; Lee et al. 2005). US3 has been shown to interfere with the MHC class I assembly pathway, while US6 inhibits peptide transport through the TAP pore preventing the loading of peptide into the MHC groove (Jones et al. 1996; Ahn et al. 1997). Recently Rhesus CMV has been shown to encode a novel inhibitor of MHC class I expression not encoded by HCMV or MCMV, Rh178. Unlike the MHC class I modulating genes m06, m152, US2, US3, US6 and US11 that affect existing MHC molecules, Rh178 targets the MHC class I heavy chain signal peptide and interferes with heavy chain translation (Powers and Fruh 2008a, b). Interestingly, infection of C57Bl/6 mice with an MCMV mutant virus lacking three “immunoevasion” genes that regulate MHC class I expression (m04/m06/ m152) did not alter the immunodominance hierarchy and magnitude of the CD8 T-cell response, and it has been hypothesized that this may be because CD8 T cell responses to CMV are largely generated/primed by DC via cross-presentation/

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exogenous pathways (Gold et al. 2004; Munks et al. 2007). It may also be that although CMV effectively reduces the expression of MHC class I on the surface of infected DC, this reduction is not complete, and is likely sufﬁcient to lower the strength-of-signal that CMV-speciﬁc T cells receive as opposed to completely inhibiting it. In support of this, studies have indicated that sufﬁcient MHC/peptide complexes remain expressed on the surface of CMV-infected DC to allow for the initiation of proximal events leading to T cell priming/activation, but restrict the full differentiation of these T cells to a full-blown effector phenotype (Benedict et al. 2008).

7.5.2

Viral Regulation of MHC Class II Expression

Like MHC class I, CMV infection has also been shown to reduce MHC class II expression. However, the molecular mechanisms that confer this phenomenon are not well understood. HCMV infected DC show delayed and decreased MHC class II biosynthesis (Lee et al. 2006; Kessler et al. 2008). When overexpressed US2 can promote the degradation of human leukocyte antigen (HLA)-DR- and -DM- (Rehm et al. 2002). Similarly, overexpression of US3 results in its binding to class II /ß complexes in the endoplasmic reticulum (ER), reducing their association with the invariant chain, preventing efﬁcient trafﬁcking of to the class II loading compartment and resulting in signiﬁcantly reduced peptide-loaded class II complexes (Hegde et al. 2003). However, more recent experiments in both monocyte derived DC and Langerhans DC suggest that US2/US3 may not function similarly to inhibit MHCII in HCMV infected cells (Kavanagh et al. 2001; Lee et al. 2006). CMV has also been shown to target molecules involved in the proteolytic degradation process. HCMV was reported to decrease expression of MHC II-associated proteases cathepsins S, Z, B, H and L and asparagine-speciﬁc endopeptidase (AEP) which correlated with lessefﬁcient proteolytic degradation of peptide substrates by HCMV-infected DCs in vitro (Kessler et al. 2008). Besides these direct mechanisms affecting antigen presentation, CMV has also been described to exploit indirect pathways to affect MHC expression. Various studies have indicated that virally encoded IL-10 reduces both MHC class I and class II expression and may impede anti-viral T cell responses both during acute and latent infection (Cheung et al. 2009; Slobedman et al. 2009).

7.5.3

Regulation of B7 Costimulation

T cells require costimulatory signals for optimal activation and acquisition of effector functions (Wang and Chen 2004; Greenwald et al. 2005). The B7-CD28 costimulatory pathway consists of B7.1 and B7.2 that both bind to the activating

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receptor CD28 and the inhibiting receptor CTLA-4. High levels of B7.1 and B7.2 expression are restricted to APCs. B7.2 is constitutively expressed at low levels and rapidly upregulated, whereas B7.1 is inducibly expressed. CD28 is constitutively expressed on the surface of T cells while CTLA-4 expression is rapidly upregulated following T cell activation. Engagement of CD28 on naïve T cells by either B7.1 or B7.2 ligands concomitant with TCR signaling provides a potent positive costimulatory signal to T cells, resulting in the induction of IL-2 transcription, expression of CD25 and entry into the cell cycle (Sharpe 2009). Importantly, TCR interaction in the absence of CD28 engagement leads to T cell tolerance, anergy or deletion (Wang and Chen 2004; Greenwald et al. 2005). The MCMV m138 gene product, originally identiﬁed as an Fc receptor homologue, facilitates the down-regulation of B7.1 (Mintern et al. 2006), while m147.5 inhibits expression of B7.2 (Loewendorf et al. 2004). Both of these viral proteins target the B7 proteins at a post-transcriptional level, similar to CMV targeting of MHC expression. Human CMV also down-regulates B7/1/B7.2 from the surface of infected DC -although the responsible gene product(s) are yet to be identiﬁed. The relevance of B7-CD28 interactions in promoting CMV-speciﬁc T cell responses was recently shown by our group. Mice lacking B7.1/B7.2 or CD28 showed marked reductions in the initial expansion of MCMV-speciﬁc CD8 and CD4 T cells, and never controlled persistent MCMV replication in the salivary gland (Arens et al. 2011a, b). Interestingly, MCMV-speciﬁc inﬂationary memory CD8T cell responses did reach levels equivalent to those seen in wild-type mice by ~ 100 days after infection, suggesting the regulation of these populations is different from the “stable” memory pool of MCMV-speciﬁc CD8 T cells (Snyder et al. 2008), but even these populations eventually crashed in mice lacking B7-CD28 signaling by day ~ 200 after infection. Importantly, using a mutant of MCMV incapable of restricting B7.1 and B7.2 expression resulted in selective increases in MCMV-speciﬁc CD4 and CD8 T cell responses when compared to wild-type virus, proving that CMV modulation of this costimulatory pathway does dampen virus-speciﬁc T cell responses in vivo. It is becoming apparent that other positive cosignaling pathways can enhance, alter or substitute for the requirement for B7-CD28 signaling. Interestingly, CMV has also been shown to impinge upon some of these pathways by downregulating CD40 and ICAM-1 (Moutaftsi et al. 2002; Benedict et al. 2008). In addition, HCMV infection has been reported to induce the shedding of CD83 from infected DC, also resulting in the inhibition of T cell proliferation (Senechal et al. 2004).

7.5.4

Modulation of Soluble Positive Cosignals

CMV has been shown to affect the production of DC-derived cytokines that play roles in the maturation of DC, activation of NK cells and the priming, survival and effector function acquisition of T cells. Although the molecular mechanisms of the inhibition of cytokine production has not been identiﬁed, various studies have shown that HCMV and/or MCMV inhibit production of IL-2, IL-6, IL-8, IL-12, and TNFD in infected

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DCs (Moutaftsi et al. 2002; Beck et al. 2003). Importantly, this reduced cytokine production is maintained when infected DC are exposed to activated T cells or inﬂammatory stimuli like LPS. Most reports indicate that only infected DCs show decreased capacity for cytokine production while other DC that encounter virus through other pathways show increased cytokine production (Andrews et al. 2001; Tyznik et al. 2008). However, various studies, using total DCs from individuals suffering from HCMV induced hepatitis or mononucleosis, indicate that disregulation of cytokine production can be observed in both infected and bystander DC (Varani et al. 2009).

7.6

Viral Modiﬁcation of T Cell-Inhibitory Signals

To further tip the balance from a “positive net signal”, to a “negative net signal”, CMV has evolved many strategies to modulate molecules that provide negative cosignals in the priming of T cells.

7.6.1

Regulation Via the PD-1/PDL-1 Pathway

Recently the B7 family has been expanded with PD-L1 (CD274/B7-H1) and PD-L2 (CD273/B7-DC) that have been show to provide negative signals that limit, terminate and/or attenuate T cell responses through its receptor PD-1 (CD279) (Wang and Chen 2004; Greenwald et al. 2005). PD-L1 is constitutively expressed on many cells types of hematopoietic and non-hematopoietic origin, whereas PD-L2 is transiently induced on DCs and macrophages upon activation. PD-1 is inducibly expressed upon activation on T, B and NK-T cells and confers a negative signal when engaged simultaneously with the TCR or BCR (Sharpe 2009). Early studies reported that PD-1 signaling resulted in cell cycle arrest, whereas later studies indicate that PD-1 signaling promotes death, either through the direct engagement of death pathways or indirectly by down-regulating survival signals and growth factors (Latchman et al. 2001; Dong et al. 2002; Petrovas et al. 2006; Keir et al. 2008). Our recent study showed that MCMV-infected DCs actively upregulate PD-L1 while down regulating MHC class I/II and positive costimulatory molecules (Benedict et al. 2008). T cells activated by the infected DC had a stunted phenotype characterized by poor survival, poor proliferation and lack of acquisition of effector functions. Blocking PD-L1 on the DC or PD-1 on the T cells using antibodies signiﬁcantly improved survival, proliferation and effector functions, illustrating the importance of “negative cosignaling” in the induction CMV-speciﬁc T cell responses (Benedict et al. 2008). Interestingly, in B7.1/B7.2 double deﬁcient mice which show a signiﬁcantly compromised initial expansion of MCMV-speciﬁc T cell responses, CD8 T cell responses can be restored to wild-type levels at day 8 by blocking PD-1 signaling, while PD-1/PD-L1 blocking studies in WT mice resulted in very modest or no increases in CD8 T cell responses (Arens et al. in press). In total, these results

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strongly suggest that the B7-CD28 pathway is closely intertwined and counterbalanced by the PD-L1/2-PD-1 pathway, both in the case of cross-presented CMV antigen and in DC directly infected by CMV. Besides the direct effect on the T cells, it has been suggested that reverse signaling through PD-L1 on DC by PD-1 reduces the expression of CD40, CD80 and CD86 and increases IL-10 production, thereby reinforcing the immune-suppressive or “paralyzed” DC phenotype (Blocki et al. 2006; Kuipers et al. 2006; Van Keulen et al. 2006).

7.6.2

Regulation of HVEM/BTLA Interactions

CMV also exploits additional negative cosignaling pathways by targeting the recently identiﬁed B and T cell attenuator (BTLA), another Ig superfamily member with homology to CTLA-4 and PD-1 (Ware 2008). The ligand for BTLA was recently found to be a member of the TNF receptor superfamily, the herpes virus entry mediator (HVEM/HveA), establishing a new paradigm in cross-family ligand receptor interactions (Ware 2008). All primate CMVs encode an orthologue of HVEM in their genome, the canonical member being the ul144 orf in HCMV (Lurain et al. 1999), which binds to BTLA and is a potent inhibitor of CD4 T cell proliferation. Interestingly, UL144 has lost its ability to bind to LIGHT, the TNFfamily cytokine that binds HVEM and functions as a positive costimulatory system for T cells. This highlights the coevolution of CMV with its host, usurping the negative cosignaling functions of HVEM-BTLA interaction, while “losing” the positive aspects of HVEM-LIGHT signaling in the UL144 protein (Benedict et al. 1999; Ware 2008).

7.6.3

Immune Regulation via Cytokines

Besides membrane-associated molecules, CMV has been shown to affect the induction, production and sensitivity to various cytokines and/or chemokines that interfere with the induction of adaptive responses. A prime example of this is the CMV vIL-10, which has been shown to suppress the induction of the immune responses. HCMV encodes a biological active IL-10 (UL111a) that has low sequence similarity to other vIL-10 molecules, and similar IL-10 orthologues are contained within all the primate CMV genomes (Lockridge et al. 2000). CmvIL-10 has been shown to decrease production of proinﬂammatory cytokines and chemokines involved in anti-viral responses. A recent study demonstrated that cmvIL-10, like IL-10, can directly suppress synthesis of type I interferons (IFN) in plasmacytoid dendritic cells (pDC). In addition, cmvIL-10 has been shown to prevent the upregulation of costimulatory molecules and MCH class I and II molecules (Muller et al. 1998; Chang et al. 2004). Besides the prevention of the induction of activating molecules,

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cmvIL-10 has been suggested to actively downregulate MHC class I and class II proteins while upregulating indoleamine 2,3-dioxygenase (IDO), a molecule with potent immunoregulatory activity (Raftery et al. 2004). Both IDO and IL-10 have been described to inhibit T cell activation, proliferation and acquisition of effector functions, while promoting the induction of regulatory T cells. Although all of these pathways would be excellent means of viral sabotage, the in vivo evidence that CMV usurps these pathways to shape the antiviral immune response is still sparse. Importantly, cmvIL-10 is transcribed during latency in myeloid cells, and is currently the only boniﬁed HCMV latency-associated gene where a functional consequence has been demonstrated. A HCMV mutant with a UL111a deletion was compromised in its ability to establish, maintain and reactivate from experimental latency. Additionally, MHC class II levels increased signiﬁcantly on the surfaces of cells infected with the deletion virus, indicating a functional role for cmvIL-10 in regulating antigen presentation in infected cells. Finally, HCMV-speciﬁc CD4 T cells responded more vigorously to myeloid progenitors infected with the UL111a deletion virus, demonstrating that viral IL-10 expression during latency plays an important role in the regulation of T cell responses. (Cheung et al. 2009; Slobedman et al. 2009).

7.7

Biological Consequences

The direct infection of DCs by CMV demonstrates the ingenious approach the virus has evolved to manipulate the anti-viral immune response in the host. Naïve virus-speciﬁc T cells encountering infected DC receive suboptimal MHC and positive costimulation, while receiving ampliﬁed levels of negative signals. These virus-speciﬁc T cells will undergo apoptosis, become anergic or non-responsive, or may develop a regulatory or suppressive phenotype. As a result, the virus has eliminated or modiﬁed a signiﬁcant proportion of the virus-speciﬁc T cell population that could have been adequately activated by DC that present viral antigens from exogenous sources. Moreover, the production of immune-suppressive factors by infected DC may instill a less than optimal antigen-presenting capacity in noninfected DC, providing an additional mechanism for the ﬁne-tuning of the quality, quantity and kinetics of the anti-viral response. CMV has shown to interfere with apoptosis pathways in the infected DC (Miller-Kittrell and Sparer 2009), allowing for extended persistence of the infected DC thereby maximizing the transient immune suppression during acute infection in the host. However, the immunesuppressive machinations of CMV may not only affect the induction of anti-CMV response; the possibility exists that these strategies may impose a more profound and/or generalized inhibition of immune responses. Although there is no direct evidence that individuals infected with HCMV show compromised immunity, there has been much recent speculation regarding the potential role that lifelong, persistent infection with CMV may have on promoting immune senescence in the aging population (Pawelec et al. 2005).

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Chapter 8

T Cell Responses During Human Immunodeﬁciency Virus (HIV)-1 Infection Claire A. Chougnet and Barbara L. Shacklett

Abstract The deﬁning features of the acquired immunodeﬁciency are the “persistent and profound selective decrease in the function as well as number of T lymphocytes of the helper/inducer subset and a possible activation of the suppressor/cytotoxic subset”, as described in 1982 (Mildvan, D., U. Mathur, et al. (1982). “Opportunistic infections and immune deﬁciency in homosexual men.” Ann Intern Med 96(6 Pt 1): 700–4). Nowadays, although depletion of CD4+ T-cells remains a hallmark of Human Immunodeﬁciency Virus (HIV) infection, the multifactorial nature of the disease provoked by infection by HIV-1 or Simian Immunodeﬁciency Virus (SIV) in “non-natural” hosts is generally acknowledged, in that no unique immune alteration has been identiﬁed that can fully explain the plethora of dysregulation associated with the development of pathogenic HIV and SIV infection. This review will focus on what we know (or do not know) about T cell responses during HIV infection. This choice reﬂects the main expertise of the authors, and the major theme of this book. However, other aspects of adaptive and innate immunity should not be overlooked. Notably, HIV-speciﬁc antibodies, including neutralizing antibodies, are an important defense of the adaptive immune system, although HIV appears to quickly evade the effect of these antibodies. Recent studies have also highlighted the role of innate immunity in protection against HIV/SIV. In addition, several cellular antiretroviral restriction factors, either constitutively expressed or induced by interferons, have been identiﬁed, which provide considerable resistance to retroviral infection. For more on these topics, we refer readers to recent reviews summarizing these crucial aspects of the virus/host interaction

C.A. Chougnet (*) Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_8, © Springer Science+Business Media, LLC 2012

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(Kuritzkes, D. R. and B. D. Walker (2007). HIV-1 Pathogenesis, Clinical Manifestations and Treatment. Fields Virology. D. Knipe and P. M. Howley. Philadelphia, PA, Lippincott Williams & Wilkins. 2: 2187–2214; Levy, J. A. (2007). HIV and the Pathogenesis of AIDS. Washington, D.C., ASM Press; Zwick, M. B. and D. R. Burton (2007). “HIV-1 neutralization: mechanisms and relevance to vaccine design.” Curr HIV Res 5(6): 608–24; Alter, G. and M. Altfeld (2009). “NK cells in HIV-1 infection: evidence for their role in the control of HIV-1 infection.” J Intern Med 265(1): 29–42; Neil, S. and P. Bieniasz (2009). “Human immunodeﬁciency virus, restriction factors, and interferon.” J Interferon Cytokine Res 29(9): 569–80; Stamatatos, L., L. Morris, et al. (2009). “Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine?” Nat Med 15(8): 866–70; Strebel, K., J. Luban, et al. (2009). “Human cellular restriction factors that target HIV-1 replication.” BMC Med 7: 48; Altfeld, M., L. Fadda, et al. (2011). “DCs and NK cells: critical effectors in the immune response to HIV-1.” Nat Rev Immunol 11(3): 176–86).

8.1 8.1.1

Background HIV Life Cycle

HIV-1 is a primate lentivirus that infects cells of the human immune system, predominantly CD4-expressing cells, i.e. CD4+ T cells, macrophages and dendritic cells, although in vitro infection of non-classical CD4− cells, such as hepatocytes or astrocytes, has also been reported (Lopez-Herrera et al. 2005; Xiao et al. 2008). From the 5c- to 3c-ends, the HIV-1 genome includes three structural genes: the gag (group-speciﬁc antigen), pol (polymerase), and env (envelope) genes (reviewed in (Freed and Martin 2007)). The Gag and Pol proteins are produced as a Gag-Pol precursor protein whose synthesis requires a (−1) ribosomal frameshift at the junction between the two overlapping translational reading frames of Gag and Pol (Jacks et al. 1988). This frameshift is directed by a highly conserved RNA secondary structure. The Gag-Pol precursor protein is then cleaved autocatalytically by its own Protease (PR) domain (Kramer et al. 1986). The Gag polyprotein is cleaved into the MA (matrix, p17), CA (capsid, p24), NC (nucleocapsid, p7) and several smaller proteins (Freed and Martin 2007). The Pol polyprotein is cleaved into the viral enzymes PR (protease, p10), RT (reverse transcriptase and RNAse-H, p51/66) and IN (integrase, p32) (di Marzo Veronese et al. 1986). The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor, gp160, which is processed by a cellular protease during Env trafﬁcking to the cell surface (Decroly et al. 1994). Gp160 processing results in the generation of the surface Env glycoprotein (SU, gp120), which interacts with the cellular receptor and coreceptor for HIV (Sattentau and Weiss 1988) (Lifson et al. 1986), and the transmembrane glycoprotein (TM, gp41) (Veronese et al. 1985). In addition to these structural genes, HIV-1 also encodes a number of regulatory and accessory proteins. These include Tat

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(transactivator of transcription) (Arya et al. 1985), which is critical for transcription from the HIV-1 LTR (Selby et al. 1989)(Starcich et al. 1985); Rev (regulator of virion) (Sodroski et al. 1986), which plays a role in the transport of viral RNAs from the nucleus to the cytoplasm (Malim et al. 1989); Nef (negative factor) (Franchini et al. 1986; Terwilliger et al. 1986); Vif (virion infectivity factor) (Strebel et al. 1987); Vpr (viral protein R); and Vpu (viral protein U) (Strebel et al. 1988) (Cohen et al. 1988). Very brieﬂy, HIV replication proceeds in a series of stages: following interaction with its receptor (CD4) and co-receptor (a chemokine receptor, mainly CCR5 or CXCR4) (Bleul et al. 1996; Dragic et al. 1996; Oberlin et al. 1996), and after fusion with the host cell membranes, the virion capsid is released into the cytoplasm. From there, it undergoes a series of still-controversial steps leading to uncoating and nuclear import of the viral nucleic acid (reviewed in (Arhel 2010)). Once inside the nucleus, the viral RNA is reverse transcribed into DNA, which is integrated into the host cell chromosome. Messenger RNAs encoding viral accessory and structural proteins are transcribed from integrated proviral DNA. “Early” regulatory proteins Tat and Rev are the ﬁrst viral proteins synthesized. Tat positively regulates transcription from the viral promoter, and Rev facilitates transport of mRNAs encoding the structural proteins (Gag, Pol, Env) from the nucleus to the cytoplasm. “Late” structural proteins are then synthesized and assembled along with the viral genomic RNA into infectious progeny virions that bud from the cell membrane (reviewed in (Cullen 1991)(Freed and Martin 2007)). All steps of the viral life cycle have become targets for therapy. Inhibitors of viral reverse transcriptase and protease are usually combined to form the regimen called “Highly Active Anti-Retroviral Therapy” (HAART). Inhibitors of virus entry (chemokine receptor inhibitors or Enfuvirtide, a fusion inhibitor) and viral integrase are added to HAART if it is failing (Eggink et al. 2010; Zolopa et al. 2010 ; Gilliam et al. 2011).

8.1.2

Experimental Models to Study HIV Infection

Immune cells from rodents are generally resistant to HIV infection, because of barriers to infection at the level of entry (i.e., differences between human and rodent CD4 and CCR5) and viral gene expression (i.e., differences in factors required for HIV gene expression) (Gofﬁnet et al. 2007; Tervo et al. 2008; Michel et al. 2009). Therefore, most of the knowledge we possess on HIV infection comes from ex vivo studies of cells from HIV-infected subjects, as well as from the experimental model of progressive infection that Asian macaques developed after infection by SIV. SIV infection of Rhesus macaques (RM) exhibits many features similar to that of progressive HIV infection in humans (Chakrabarti et al. 1987; Franchini et al. 2002; Pandrea et al. 2009), and this model remains the best choice for pathogenesis and vaccination studies. SIV naturally infects African nonhuman primates (referred to as SIV “natural hosts”), and is generally non-pathogenic in these species

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(Hahn et al. 2000). These SIV natural hosts however can develop AIDS, and when they do, the spectrum and pathogenesis of the disease are very similar to those encountered in humans or rhesus macaques with AIDS (reviewed in (Pandrea et al. 2009)). Obviously, the usefulness of SIV/RM model is limited by its use of heterologous viruses, particularly for vaccine studies. Many groups have thus invested considerable effort in developing humanized mice as models of infection (for review, see (Denton and Garcia 2009; Legrand et al. 2009; Van Duyne et al. 2009)). An increasing number of studies are performed with these systems; however, whether they will constitute a suitable alternative to the SIV/RM model to conduct pathogenesis studies is very much debated. We have therefore focused this review on what we have learned from studies of HIV-infected individuals and SIV-infected hosts, RM or natural hosts.

8.1.3

A Brief Summary of HIV Transmission

Despite our advances in dissecting the HIV life cycle, the identity of the “founder” cell following mucosal HIV infection is not yet clearly ascertained. Following mucosal exposure of RM to high doses of SIV, the virus crosses the mucosal epithelial barrier within hours and establishes a small founder population of infected cells, mainly “resting” CD4+ T cells (reviewed in (Haase 2010)). This founder population undergoes local expansion during the ﬁrst week of infection, before propagating systemically, throughout the secondary lymphoid organs. Analysis of HIV envelope sequences in people with acute infection with clade B virus shows that the great majority of these subjects had evidence of productive infection by a single virus (Keele et al. 2008). Phenotypic analysis of transmitted or early founder envelopes revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to fusion inhibitors and broadly neutralizing antibodies compared with those from chronically infected subjects (Keele et al. 2008). On the other hand, dendritic cells with detectable viral antigens reach the draining lymph nodes much earlier – 18–24 h after exposure (Hu et al. 2000). Many researchers have thus suggested that dendritic cells are the ﬁrst cells interacting with HIV. These cells could then transmit the virus to CD4+ T cells, through cis- or trans-infection (Teleshova et al. 2003; Wu and KewalRamani 2006; Cavrois et al. 2008). However, arguing against the latter model, the draining lymph nodes are not the site where productive infection is ﬁrst detected (Zhang et al. 1999). Although models of mucosal infection in RM have been informative, they have some important weaknesses that need to be remembered because they contribute to the lack of precise knowledge about the founder cells. First, the dose of virus used to obtain a reliable infection of RM is very high compared to the doses that humans are usually exposed to, although many investigators have started using repeated low doses of viruses to infect macaques, a model that better mimics human transmission

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(Alpert et al. 2010; Liu et al. 2010; Reynolds et al. 2010). Second, free viruses, not cell-associated viruses, are used in RM infection, another major difference with the human situation. Third, results are likely to differ depending on the route of transmission (vaginal, rectal, penile). Fourth, it is difﬁcult to reproduce in RM the complexity of human transmission, particularly the changes due to concomitant mucosal infections.

8.2 8.2.1

CD4+ T Cells and HIV Is HIV/AIDS a Disease of Depletion?

CD4 depletion is a hallmark of progressive HIV/SIV infection (Fig. 8.1). Loss of CD4+ T cells from peripheral blood during the chronic phase of infection is quite slow in HIV-infected humans or SIV-infected RM (Mellors et al. 1997), although this loss is closely associated with HIV morbidity and mortality. Opportunistic infections generally start developing when CD4 blood counts plummet under 200/ ul (Crowe et al. 1991). However, seminal studies performed by R. Veazey and colleagues more than 10 years ago showed that CD4 depletion in the gastrointestinal (GI) tract was both more severe and earlier than that observed in the blood (Veazey et al. 1998). These ﬁndings in SIV-infected RM were later conﬁrmed in HIVinfected patients (Brenchley et al. 2004; Mehandru et al. 2004). As most transmitted viruses use CCR5 for entry, activated/memory CD4+ T cells, which express CCR5, are preferential targets of HIV/SIV infection compared to naïve (CCR5− ) CD4+ T cells. Accordingly, studies of SIV-infected RM have shown a high level of infection in GI tract CCR5+ memory CD4+ T cells, which are subsequently depleted in less than 3 weeks after infection (Mattapallil et al. 2005). GI devastation leads to microbial translocation, although additional failure of liver macrophages appears to be necessary to lead to elevated plasma LPS levels (Hofer et al. 2010), which are associated with immune activation and poor outcome (Brenchley et al. 2006). However, the contribution of the CD4 depletion occurring in the GI tract to HIV pathogenesis must be interpreted with caution. Indeed, acute loss of GI CD4+ T cells also occurs during nonpathogenic SIV infection, although it is less dramatic than it is in progressive HIV/SIV infection, and mucosal CD4+ T cells somewhat recover in these natural hosts during the chronic phase of infection (Gordon et al. 2007; Milush et al. 2007; Pandrea et al. 2007). Similarly, although CD4 depletion in the GI tract occurs very early after HIV infection, the majority of HIV-infected individuals do not develop AIDS for years. Taken together, these studies suggest that devastation of the GI tract is an important, but not unique, contributor to HIV pathogenesis. Moreover, a different dynamic between virus and immune system is likely at play in the “natural” versus “non-natural” hosts (reviewed in (Pandrea et al. 2009; Sodora et al. 2009; Brenchley et al. 2010)). Reasons for decreased CD4 numbers caused by HIV infection remain under intense scrutiny. Direct cytopathic effects of the virus have been implicated

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Fig. 8.1 CD4+ T cells are the primary target cells for HIV-1 (macrophages and dendritic cells, not shown, may also be infected). HIV-speciﬁc CD4+ T-cells may be preferentially targeted (Douek et al. 2002). CD4+ T-cells are rapidly depleted from the gut, either through direct infection (Mattapallil et al. 2005), or bystander apoptosis (Li et al. 2005). CD4+ T-cell dysfunction is apparent in early infection (Shearer et al. 1991; Rosenberg et al. 2000), with important implications for the ‘help’ normally provided to B-cells and CTL. HIV Nef mediates downregulation of MHC class I molecules, protecting infected cells from CTL surveillance (Collins et al. 1998). Meanwhile, cytotoxic T-cells (CTL) progressively exhibit the characteristics of exhaustion or functional senescence (Wherry et al. 2007); in addition, high-avidity CTL responses drive viral sequence ‘escape’ (Borrow et al. 1997). B-cell polyclonal activation is a hallmark of HIV disease, also beginning in early infection (Levesque et al. 2009). Neutralization escape is another important mechanism by which HIV circumvents immune control (Frost et al. 2005; Frost et al. 2008). Finally, systemic effects of HIV infection, not reviewed in detail here, include gut epithelial impairment, thymic dysfunction, generalized immune activation, and lymphoid tissue ﬁbrosis. For additional detail on these topics, see (McCune et al. 2000), (Brenchley and Douek 2008)(Hunt 2007)

(Mattapallil et al. 2005), although they do not appear to account for all HIV-induced cell death. Instead, increased susceptibility of “bystander” CD4+ T cells (i.e. cells that do not exhibit markers of active HIV replication) to apoptosis is well described (Li et al. 2005). Apoptosis is always difﬁcult to visualize in vivo, but expression of molecules associated with T cell death is increased in lymphoid tissues of HIVinfected and SIV-infected progressors (Badley et al. 1998; Herbeuval et al. 2005), strongly supporting the involvement of apoptosis in HIV pathogenesis. Increased apoptotic death of CD4+ T cells follows cross-linking of CD4 by HIV gp120 (Banda et al. 1992), which appears to be mediated by ligand-receptor systems of the tumor necrosis factor superfamily (principally FasL/Fas and TRAIL/DR).

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CD4 cross-linking induces FasL expression not only on T cells but also on monocytes/macrophages, which could indirectly contribute to the apoptotic death of uninfected CD4+ T cells (Oyaizu et al. 1997). Furthermore, CCR5+ CD4+ T cells preferentially express the receptor for IFN-D/E, and may therefore be particularly susceptible to the pro-apoptotic effect of type I IFNs (Boasso et al. 2008). Other HIV proteins have also been implicated in cell death, including Nef through membrane permeabilization (Laforge et al. 2007) and Tat, through alteration of the Egr1-PTEN-Akt and p53 pathways (Dabrowska et al. 2008). Importantly, a recent study reported that the death of “bystander” CD4+ T cells also involves abortive HIV infection, as inhibitors of HIV entry or fusion, but not of reverse transcription, prevented their death. Incomplete viral reverse transcripts were shown to accumulate in the cytoplasm of the cells, and to kill them through activation of a caspasedependent pyroptotic response (Doitsh et al. 2010).

8.2.2

Is HIV/AIDS a Disease of Immune Dysregulation?

CD4+ T cells are functionally abnormal in the majority of HIV-infected individuals and SIV-infected RM. These defects start occurring early in the course of HIV infection, prior to the decline of circulating CD4+ T cell numbers (Shearer et al. 1991, 1998; Rosenberg et al. 2000). Moreover, loss of in vitro CD4+ T cell responses and of in vivo delayed-type hypersensitivity were predictive of disease progression and time to death in HIV-1-infected individuals before the advent of HAART (Miedema et al. 1994; Dolan et al. 1995). Many CD4+ T-cell abnormalities have been deﬁned ex vivo, and can be summarized as follows: (1) decreased polyfunctionality, particularly of HIV-speciﬁc T cells, in that activated CD4+ T cells exhibit decreased proliferation and production of IL-2, but not IFN-J and TNF-D (Palmer et al. 2004; Sun et al. 2005), and defective upregulation of some activation markers, such as CD40 ligand, but not CD69 or OX40 (Zhang et al. 2004); (2) upregulation of the inhibitory molecules CTLA-4 (Kaufmann et al. 2007), PD-1 (Day et al. 2006; D’Souza et al. 2007) and Tim-3 (Kassu et al. 2010). Many HIV-speciﬁc CD4+ T cells from untreated subjects coexpress PD-1, CTLA-4, and Tim-3, in contrast to cytomegalovirus- or varicella-zoster virus-speciﬁc CD4+ T cells in these patients. Coexpression of all three inhibitory receptors on HIV-speciﬁc CD4+ T cells is more strongly correlated with viral load than with the expression of each receptor individually (Kassu et al. 2010); (3) increased expression of the apoptosis markers TRAIL DR5 and increased percentage of CD4+ T cells entering apoptosis (Herbeuval et al. 2005); (4) blunted T cell signaling induced by TCR cross-linking, IL-2 or PMA/ionomycin (Cayota et al. 1994; Bostik et al. 2001; Schweneker et al. 2008; Nyakeriga et al. 2009). Interestingly, there may be a genetic basis underlying maintenance of strong polyfunctional CD4+ T-cell responses, as HIV-infected controllers with the strongest HIV-speciﬁc CD4 responses in the gut mucosa also possessed HLA-DRB1*13 and/or HLA-DQB1*06, two class II HLA alleles previously associated with non-progression (Ferre et al. 2010a).

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Notably, there is a discrepancy between the ex vivo picture, in which CD4+ T cells from HIV-infected patients exhibit increased expression of many activation markers, including increased basal level of kinase phosphorylation and CD40L, and the in vitro picture, in which CD4+ T cells from patients respond poorly to stimulation (Zhang et al. 2004; Schweneker et al. 2008). These ﬁndings suggest a model of chronic “tickling” of the adaptive immune system, which precludes its full response to pathogens. Of interest, binding and cross-linking of CD4, besides their induction of apoptosis, also affect the function of conventional CD4+ T cells from normal uninfected individuals, inhibiting the up-regulation of activation markers (Chirmule et al. 1995; Tamma et al. 1997; Zhang et al. 2004) and the formation of the immunological synapse (Nyakeriga et al. 2009). These data provide a potential mechanism for the decreased response to TCR stimulation of CD4+ T cells in HIVinfected patients. HIV-CD4+ T cell interaction also involves an activated form of the heterodimeric integrin D4E7, normally associated with mucosal localization of lymphocytes, inducing increased LFA-1 expression on CD4+ T cells, which favors the formation of the virus-cell synapse (Arthos et al. 2008). However, whether the D4E7-gp120 interaction and subsequent signaling play a role in the dysfunction of CD4+ T cells or of other immune cells is still unknown. Finally, interactions with other HIV proteins have been implicated in CD4 dysfunction: for example, HIV Nef can directly inhibit CD4+ T cell function by disturbing tyrosine phosphorylation at the immunological synapse (Thoulouze et al. 2006).

8.2.3

Does HIV Particularly Affects Certain CD4+ T Cell Subsets?

Th1/Th2 cells: Based on the fact that production of IL-2 and T cell proliferation, but not IL-10 responses, were lost during chronic infection, Mario Clerici and Gene Shearer proposed in 1993 that the imbalance in the “type-1” versus “type-2” responses was a key factor in the immune dysregulation associated with HIV infection (Clerici and Shearer 1993), and this viewpoint was vigorously debated at the time. As mentioned above, the HIV ﬁeld has since moved on to the concept of decreased polyfunctionality, particularly of HIV-speciﬁc T cells, in that activated CD4+ T cells exhibit decreased proliferation and production of IL-2, but somewhat preserved IFN-J and TNF-D production, data that do not encompass the traditional Th1/Th0/Th2 concepts. In terms of susceptibility to HIV infection, early work showed that HIV-1 spreads better through cultures of Th2 cells than Th1 cells (Maggi et al. 1994; Tanaka et al. 1997), although these results were not conﬁrmed by all studies (Mikovits et al. 1998). Although cell surface density of CCR5 molecules was higher in Th1 versus Th2 subsets, preferential infection and entry of Th1 cells by R5 HIV-1 was not associated with preferential replication, as eventually the R5-virus replicated to a higher level in Th2 cells (Moonis et al. 2001). Th2 subsets

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expressed higher levels of CXCR4 than Th1 subsets and were more susceptible to HIV infection by X4-viruses (Moonis et al. 2001). Interestingly, new studies reported higher levels of two cellular antiretroviral restriction factors, APOBEC3G and APOBEC3F, in Th1 cells than in Th2 cells, and HIV-1 produced from Th1 cells had decreased infectivity, compared to virions produced from Th2 cells (Vetter et al. 2009), data that provide a mechanistic insight on why Th2 are more susceptible to in vitro infection than Th1 cells. Th17 cells produce IL-17, as well as other cytokines such as IL-21 and IL-22, in response to stimulation through the TCR and play an important role in antibacterial responses, promoting recruitment of neutrophils, as well as inducing proliferation of enterocytes and production of antibacterial defensins (Korn et al. 2009). Remarkably, IL-17 production induced by Salmonella typhimurium infection in the ileum was inhibited in SIV-infected macaques, leading to a much-exacerbated bacterial disease (Raffatellu et al. 2008). Early depletion of Th17 cells is observed in the gut mucosa of SIV-infected RM that progress to disease, but not in nonpathogenic SIV-infection of sooty mangabeys nor, importantly, in elite controller SIV-infected RM (Brenchley et al. 2008; Cecchinato et al. 2008). In highly viremic RM, IFN-J-producing cells predominated over IL-17-producing cells and the frequency of Th17 cells at mucosal sites was negatively correlated with plasma virus level (Cecchinato et al. 2008). During chronic infection, the frequency of IL-17-producing cells was severely decreased in all lymphoid tissues in pathogenic as compared to non-pathogenic SIV infection (Favre et al. 2009). These ﬁndings were recently conﬁrmed in humans, as progressive HIV disease was associated with the loss of Th17 cells both in peripheral blood and rectosigmoid biopsies (Favre et al. 2010). Systemic immune activation (as evidenced by increased Ki67 expression) also correlated with the loss of IL-17–producing cells from the intestine (Gordon et al. 2010). Such alteration likely contributes to the disruption of the mucosal barrier during pathogenic SIV/HIV-1 infection, leading to increased microbial translocation (Estes et al. 2010), but the underlying mechanisms of this loss remain unclear. Although it is clear that Th17 cells are susceptible to SIV infection both in vitro and in vivo, whether they are preferentially infected in vivo compared to Th1 cells is debated, with discrepant data coming from different studies (Cecchinato et al. 2008; Gosselin et al. 2010). Of note, a recent study implicated a tryptophan catabolite in the Th17 loss (Favre et al. 2010). Regulatory T cells (“Treg cells”, deﬁned here as CD3+CD4+FOXP3+ cells) are essential for maintaining host homeostasis, as evidenced by the catastrophic autoimmunity developed by mice or humans in which FoxP3 is not functional. However, Treg cells also dampen effector responses to pathogens, and may thus hamper the capacity of the hosts to control chronic infections (Li et al. 2008; Feuerer et al. 2009; Josefowicz and Rudensky 2009). Comparison of progressor and nonprogressor HIV-infected patients and SIV-infected RM showed an association between increased frequency of Treg cells in lymphoid tissues and high viral loads or reduced anti-viral cytotoxic T cell activity (Andersson et al. 2005; Epple et al. 2006;

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Estes et al. 2006; Nilsson et al. 2006; Boasso et al. 2007). The Th17/Treg cell balance in peripheral blood and in rectosigmoid biopsies is severely altered in progressors (Favre et al. 2010). However, it should be noted that highly pathogenic SIV infection (in nemestrina or fascicularis macaques) leads to decreased Treg cell frequency in tissues, particularly in the gut (Chase et al. 2007; Qin et al. 2008), suggesting either inter-species variations or altered Treg cell-virus interactions during rapid progression. Inhibitory functions of Treg cells appear to be preserved in HIV-infected patients and SIV-infected RM (Hryniewicz et al. 2006; Kinter et al. 2007; Thorborn et al. 2010). However, Treg cells likely play a complex balancing role during HIV-1 infection. They also limit non-speciﬁc immune activation (Eggena et al. 2005; Chase et al. 2007; Ndhlovu et al. 2008), and likely limit HIV infection/replication in susceptible target cells (Liu et al. 2009) (Moreno Fernandez et al. 2011), which could play a beneﬁcial role during early HIV infection when the effector immune cells are not yet activated. In support of this hypothesis, ﬁrst, Treg cells are activated very early following SIV infection in African green monkeys, which could limit harmful generalized activation and allow for this infection to remain non-pathogenic (Kornfeld et al. 2005); second, in utero activation of Treg cells in HIV-exposed uninfected children has been postulated to contribute to the lack of vertical transmission by reducing T cell activation (Legrand et al. 2006); and third, CTLA-4 blockade during acute primary SIV infection in rhesus macaques reduced Treg cell numbers and increased viral replication at mucosal sites (Cecchinato et al. 2008). Treg cells could also maintain some protective role during chronic infection by limiting HIV infection/replication, although their dampening effect on HIV/SIV speciﬁc responses (Aandahl et al. 2004; Weiss et al. 2004; Hryniewicz et al. 2006; Kinter et al. 2007), as well as their production of ﬁbrosis-inducing TGF-E1 (Estes et al. 2007), may tilt the balance towards a detrimental role during chronic infection. Like many other immune processes, Treg cells thus behave as a double-edged sword during HIV/SIV infection. Mechanisms underlying increased Treg cell frequency in tissues during HIV/SIV infection are not well understood, and are likely multiple. HIV and SIV can infect Treg cells both in vivo and in vitro, but Treg cells appear to be relatively less frequently infected than non-Treg memory T cells in vivo (Estes et al. 2006; Allers et al. 2010), which is in agreement with our data showing that Treg cells were less susceptible than non-Treg to in vitro infection by R5 viruses (Moreno-Fernandez et al. 2009). Binding and cross-linking of CD4 may have different effect on Treg cells than on other subsets: although CD4+ T cells generally show increased susceptibility to apoptosis upon CD4 cross-linking (see above), the same pathway promotes in vitro survival of the Treg cell subset (Nilsson et al. 2006). Increased proliferation of Treg cells may also promote their relative accumulation, as Treg cells express high levels of the cycling marker Ki67, compared to other non-Treg CD4+ T cells, and this difference is exacerbated in HIV-infected individuals (Chougnet et al., in preparation). Finally, the role of peripheral conversion in Treg cell accumulation has not yet been thoroughly examined, but HIV-exposed plasmacytoid dendritic cells (PDC) induced a higher rate of in vitro FOXP3 induction in naïve T cells than unexposed PDC (Manches et al. 2008).

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8.3 The CD8+ T-cell Response to HIV 8.3.1

CD8+ T Cells Contribute Signiﬁcantly to Immune Control of HIV

One of the body’s major defense mechanisms against viral infection is the CD8+ T-cell. These cells may act through a combination of mechanisms including direct cytolysis, mediated by cytolytic granules that speciﬁcally target and eliminate virally infected host cells (Bangham 2009), and through non-cytolytic mechanisms termed “viral suppression” (Freel et al. 2010)(Killian et al. 2011). Several ﬁndings are frequently cited as strong evidence that CD8+ T-cells contribute signiﬁcantly to immune control of HIV: ﬁrst, during acute HIV infection, the emergence of HIVspeciﬁc CD8+ T-cells coincides temporally with the decline in peak viremia (Koup et al. 1994); second, viral escape mutations have been documented in response to certain HIV/SIV-speciﬁc CD8+ T-cell responses (Borrow et al. 1997; Goulder and Watkins 2004; Leslie et al. 2004); third, experimental removal of circulating CD8+ T-cells in SIV-infected rhesus macaques leads to a rapid surge in viremia (Jin et al. 1999; Schmitz et al. 1999); fourth, it is clear that infected individuals differ widely in their ability to control HIV, and recent ﬁndings support a role for MHC class I-restricted, CD8+ T-cell responses in the establishment and maintenance of control in the elite controllers (discussed below) (Fellay et al. 2007; Pereyra et al. 2010). Nevertheless, although HIV-speciﬁc CD8 responses may be quite robust, they ultimately fail to contain the infection in the vast majority of infected individuals. Cytolytic CD8+ T cells (CTL) have traditionally been deﬁned by their ability to induce death in MHC class I-compatible target cells expressing foreign peptides, an outcome quantiﬁed in vitro by measuring 51Cr released by dying target cells into a culture supernatant. This approach has numerous shortcomings, and has been largely replaced by surrogate assays, such as Elispot and intracellular ﬂow cytometry, that measure the production of various cytokines, chemokines and cytolytic granule constituents by the CD8+ T-cells upon TCR stimulation (Doherty 1998; McMichael and O’Callaghan 1998; Maecker 2009). However, in the absence of well-deﬁned correlates of protection from HIV infection and/or disease progression, it remains unclear which T-cell functions are most desirable or most strongly associated with a positive outcome (reviewed in (Appay et al. 2008; Makedonas and Betts 2011)). Candidates for “most valuable correlate of CD8+ T-cell immunity”, based upon studies of chronic HIV infection, long-term nonprogression, and in some cases vaccine trials, have included: “polyfunctionality”, i.e., the capacity to respond to TCR stimulation by producing multiple effector molecules (Betts et al. 2006), proliferative capacity (Migueles et al. 2002), production of perforin, granule loading and/or degranulation (Migueles et al. 2008; Hersperger et al. 2010, 2011), non-cytolytic suppression of HIV replication (Freel et al. 2010, 2011), T-cell avidity (Almeida et al. 2007, 2009), localization of HIV-speciﬁc T-cells to mucosal tissues (Critchﬁeld et al. 2007, 2008; Ferre et al. 2009, 2010a, b), and the predominance of speciﬁc memory subsets (Letvin et al. 2006; Hansen et al. 2009; Chattopadhyay and Roederer 2010).

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Lessons from HIV Controllers

In the absence of clear-cut correlates of protection revealed by vaccine studies, many clinical studies have attempted to identify correlates of disease non-progression in the small subset of infected persons known as “HIV Controllers”. These rare individuals, likely representing less than 1–2% of all infected persons, are able to control viral replication to levels undetectable by standard assays, without antiretroviral therapy. Over the past 5 years, several genome-wide association studies (GWAS) of HIV controller cohorts have attempted to identify genetic correlates of HIV non-progression (Fellay et al. 2007, 2009; Dalmasso et al. 2008; Pelak et al. 2010; Pereyra et al. 2010). To date, the consensus from these studies is that polymorphisms localized to the MHC class I region of chromosome 6, notably the alleles HLA-B57 and B27, are most strongly associated with HIV control (Pereyra et al. 2010). Protection associated with certain MHC class II alleles (i.e., DRB1*01, DRB1*0701, DRB1*1303, and the haplotype DRB1*1301/2-DQB1*06), acting either independently or in combination with protective MHC class I alleles, was reported earlier (Malhotra et al. 2001; Ferre et al. 2010a; Vingert et al. 2010; Julg et al. 2011), but these associations have not been conﬁrmed in larger scale GWAS. Interestingly, synergy of certain combinations of MHC class I and II alleles was suggested by studies in rhesus macaques (Giraldo-Vela et al. 2008), and further investigation of this issue is warranted (Bashirova et al. 2011). What properties conferred by “protective” MHC class I alleles might be responsible for their unique association with HIV control? First, B57 and B27 alleles may recognize highly conserved HIV epitopes that cannot be mutated without signiﬁcant loss of viral ﬁtness (Schneidewind et al. 2007, 2008; Miura et al. 2009a, b). Several reports suggest that Gag-speciﬁc CD8+ T-cell responses are associated with relative immune control, while Env-speciﬁc responses are not (Kiepiela et al. 2004; Ferre et al. 2010b). In the case of HLA-B27, a single immunodominant epitope (KK10) is located in a structurally constrained region involved in interactions between the virion capsid protein (p24) and the host protein cyclophilin A (Schneidewind et al. 2007, 2008). Viral escape from KK10-speciﬁc CTL frequently occurs late in infection and is associated with loss of immune control. In the case of HLA-B57, four immunodominant epitopes are located within p24; of these, the TW10 epitope is targeted by CTL early in infection. TW10 escape mutations occur rapidly and have a negative impact on viral ﬁtness during the early phase of infection; however, many escape variants also elicit strong CTL responses (Brockman et al. 2007). Thus, the combination of strong CTL responses and reduced viral ﬁtness likely contributes to immune control in this case. Intriguingly, GWAS have implicated another MHC class I region in control of HIV viral load, as indicated by a single nucleotide polymorphism (SNP) located 35 kb upstream of the HLA-C gene (reviewed in (Bashirova et al. 2011)). HLA-C proteins present antigenic peptides to CTL, but can also serve as ligands for killer

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immunoglobulin-like receptors (KIR) that regulate natural killer (NK) cell function. Thus, protective effects of HLA-C polymorphisms could be mediated via CTL and/ or NK cells, and this topic is an important area for future study. In contrast, certain MHC class I polymorphisms, notably HLA-B*35 alleles, have been associated with rapid progression to AIDS (Gao et al. 2001; Bashirova et al. 2011). The HLA-B*35 family of alleles has been subdivided into two groups according to peptide-binding speciﬁcity and disease association. B*35-PY allotypes, including B*3501, bind peptides with the amino acids proline and tyrosine at positions 2 and 9, respectively, and are not associated with a rapid disease course. In contrast, B*35-Px allotypes, which include B*3502, B*3503, B*3504, and B*5301, prefer residues other than tyrosine at position 9, and are associated with accelerated progression to AIDS. There remains some debate as to the mechanistic basis for these associations, as in vitro binding studies have failed to ﬁnd strong differences in peptide-binding afﬁnity between the two variants (Huang et al. 2009). At least part of the HLA effect on HIV control is thought to be due to interactions between killer immunoglobulin-like receptors (KIR) and MHC class I molecules (reviewed in (Carrington et al. 2008)). Most studies to date agree that there is an association of KIR3DL1/S1 and certain HLA-B alleles with disease outcome. KIR3DL1, an inhibitory KIR, binds HLA-B allotypes containing the Bw4 epitope, particularly those with an isoleucine rather than a threonine at position 80 (i.e., Bw480I), such as HLA-B*57 (Martin et al. 2007). The stimulatory receptor KIR3DS1, which is highly similar to KIR3DL1, may not interact directly with HLA-Bw4 (Gillespie et al. 2007); however, the combination of KIR3DS1 and HLA-Bw4-80I has been associated with slow disease progression in some (Qi et al. 2006; Martin et al. 2007) but not all cohorts (Barbour et al. 2007). Thus, HIV control may be inﬂuenced by interactions between MHC class I molecules and NK activating/inhibitory receptors, although the ﬁne details of these interactions remain to be elucidated. A recent study made use of a novel in silico model for thymic selection to predict the outcome of thymic selection when the repertoire of self-reactive peptides was varied in diversity (Kosmrlj et al. 2010). The model was used to calculate the number of peptides from the human proteome (i.e., “self” peptides) that could be bound by the different HLA molecules. Strikingly, two MHC class I molecules associated with protection, human HLA-B*5701 and rhesus macaque Mamu-B*17, were predicted to recognize far fewer self-peptides than alleles not associated with protection, such as human HLA-B*0701 or macaque Mamu-A*02. Previous studies in mice have demonstrated that T cells that develop with exposure to a limited selfpeptide repertoire are signiﬁcantly more cross-reactive than T-cells that develop with exposure to the full range of self-peptides (Huseby et al. 2005). Accordingly, the work of Kosmrlj et al. suggests that, by encountering fewer self-peptides during thymic selection, T-cells restricted by HLA-B*5701 or Mamu-B*17 should be capable of recognizing a broader range of variant peptides once in the periphery. This model also provides a potential explanation for the puzzling link between HLA-B*57 and a predisposition to autoimmune psoriasis and hypersensitivity reactions (Bhalerao and Bowcock 1998; Chessman et al. 2008).

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CD8+ T-cell Responses During Acute/Early HIV Infection

Recent studies have shed additional light on the breadth and speciﬁcity of the acute phase T-cell response and its relationship to the establishment of the viral load set point (Altfeld et al. 2006; Streeck et al. 2007, 2008, 2009). The viral load set point is a strong prognostic indicator (Mellors et al. 1996). In a study involving more than 400 patients with acute/early infection, CD8+ T-cell responses were mapped to the epitope level (Streeck et al. 2009). In this study, those patients whose acute/earlyphase CD8+ T-cell responses were strongly focused towards immunodominant epitopes had lower viral load set points than those whose responses were distributed across a variety of epitopes, without a clear immunodominance hierarchy. This was true whether or not patients with protective MHC class I alleles (i.e., HLA-B27 and B57) were included in the analysis. Notably, the association between immunodominance hierarchy and plasma viral load was lost during chronic infection (Streeck et al. 2009). The ability of HIV/SIV-speciﬁc CD8+ T-cells to localize to tissue sites of viral replication during acute/early infection may also be critical in establishing the host-pathogen balance. In RM infected intravaginally, SIV-speciﬁc CD8+ T-cell responses in cervicovaginal and GI mucosal tissues remained weak for at least 1–2 weeks following initial infection (Reynolds et al. 2005). This pre-immune “window” allowed viral replication and mucosal CD4+ T-cell depletion to occur. More recently, a comparative study of acute lymphocytic choriomeningitis virus (LCMV) infection of mice and SIV infection of RM, visualizing both viral mRNA and MHC class I tetramer staining at multiple time points post-infection (Li et al. 2009), showed that control of viral replication is directly related to the ratio of virusspeciﬁc CD8+ T-cells to virus-infected cells in tissues. Taken together, these studies suggest that in addition to response speciﬁcity and magnitude, the localization of HIV/SIV-speciﬁc T-cells proximal to tissue foci of viral replication is critical in determining outcome.

8.3.4

CD8+ T-cell Function, and Dysfunction, During Chronic HIV Infection

Much work has been devoted to characterizing the cytolytic functions of CD8+ T-cells, as well as their ability to release cytokines, chemokines, and induce non-cytolytic suppression of viral replication. As with CD4+ T-cells, CD8+ T-cell dysfunction and ‘exhaustion’ during chronic infection appear to be major contributors to the failure to control HIV. The phenomenon of immune exhaustion has been well documented in mice infected with LCMV (Wherry and Ahmed 2004). In this model, T-cell functions are progressively lost, beginning with proliferative capacity and IL-2 production, followed by loss of TNF-D production, whereas IFN-J production persisted until the ﬁnal stages of exhaustion (Wherry et al. 2003, 2007).

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The contribution of CD4+ T-cell loss/dysfunction to CD8+ T-cell dysfunction is not well understood, but several recent studies started tackling the issue. Notably, help of CD4+ T cells appears critical to HIV-speciﬁc CD8 proliferation and control of autologous virus replication (Lichterfeld et al. 2004; Chevalier et al. 2011), and this effect may be mainly linked to IL-21-producing CD4+ T cells (Chevalier et al. 2011). Other CD4 factors likely contribute to CD8 function, including Treg cell control of CD8 effector cells (see Treg cell section above). Premature “ageing” of the immune system has been invoked as a signiﬁcant contributor to HIV-speciﬁc CD8+ T-cell dysfunction (Appay and Rowland-Jones 2002). High levels of antigenic stimulation appear to drive the induction of a “senescent” phenotype, characterized by expression of cell surface antigens such as PD-1, lymphocyte activation gene-3 (LAG-3), Tim-3 and CD57, as well as telomere shortening and decreased responsiveness to TCR stimulation (Day et al. 2006; Petrovas et al. 2006; Trautmann et al. 2006; Jones et al. 2008). Several inhibitory receptors play a role in the contraction of the immune response following clearance of infection (Blackburn et al. 2009). Blocking these pathways in vivo can lead to increased CD8+ T-cell function, with potential implications for HIV immunotherapy (Barber et al. 2006; Freeman et al. 2006; Velu et al. 2009). However, expression of a single inhibitory receptor such as PD-1 does not necessarily indicate a senescent phenotype, as recent studies indicate that up to seven different receptors participate in complex pathways of negative regulation during chronic viral infection (Blackburn et al. 2009).

8.4

8.4.1

Effect of Anti-retroviral Therapy on T Cell Responses: Successes and Limitations HAART-Mediated Reconstitution of the Immune System

HAART has transformed a diagnosis of HIV infection from a likely death sentence to a prospect of a life-long, serious, but mostly treatable, disease. Current US and European guidelines recommend starting therapy in individuals with CD4 counts less than 350/PL (http://www.aidsinfo.nih.gov/ ContentFiles) and (Clumeck et al. 2008). A meta-analysis of more than 20,000 patients starting HAART showed that deferring therapy until CD4 counts were lower than 350/PL was associated with higher rates of development of AIDS and death than starting therapy in the 351–450/PL range (Sterne et al. 2009). In terms of CD4 reconstitution post HAART, a recent study of the AIDS Clinical Trials Group showed an initial phase (~8 weeks) characterized by expansion and redistribution of memory CD4+ T cells, followed by a second phase with reconstitution of both naïve and memory CD4+ T cells, as well as reduction of CD4 and CD8 activation (Robbins et al. 2009), data that are in agreement with the results of previous small-scale studies (reviewed in (Lederman 2001)). If delayed, HAART never restores CD4 counts to levels found in healthy volunteers

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(Robbins et al. 2009). In addition to these quantitative differences, qualitative differences were also noted. In particular, patients starting with lower baseline CD4 counts exhibit modest increase in CD4+ naive cells and greater increase in CD4+ memory cells. Functional amelioration of CD4+ T cells occurs post-HAART, but remains limited. First, HAART generally reconstitutes immune responses against prevalent microbial antigens, but HIV-1-speciﬁc responses remain largely dysfunctional (Rinaldo et al. 1999; Chougnet et al. 2001), which explains why most people with chronic HIV-1 infection cannot control viremia after HAART interruption (reviewed in (Lederman 2001)). Second, although expression of TRAIL and FasL is reduced in circulating and tonsil CD4+ T-cells from HAART-treated patients, tissue expression of DR5 and Fas remains higher in treated patients than in HIV-uninfected subjects (Herbeuval et al. 2009). Third, T-cell activation (deﬁned as a high percentage of CD4+CD38+HLA-DR+) persists among treated patients, whatever their baseline CD4 counts. Fourth, we found that recruitment of lck and actin to the immunological synapse was ameliorated in the CD4+ T cells of HAART-treated patients, but responses remained lower than those measured in uninfected subjects (Nyakeriga et al. 2009). Fifth, suppressive HAART was associated with low Treg cell frequency in lymphoid tissues compared to untreated patients (Andersson et al. 2005), as well as decreased Treg cell frequency in the blood (Montes et al. 2011), but this frequency remained higher than that measured in uninfected subjects (Lim et al. 2007; Kolte et al. 2009). Intestinal biopsies from HIV-infected patients who were on long-term HAART showed that when effective CD4+ T cell restoration (>50% compared to uninfected controls) was achieved, enhanced Th17 responses and, in general increased polyfunctionality of anti-HIV cellular responses, were also found, but these functional ameliorations did not occur if reconstitution was more limited (Macal et al. 2008). Incomplete CD4+ T-cell repopulation of lymphoid tissue, including the gut, has been linked to collagen deposition and ﬁbrosis mediated by TGF-E secretion (Schacker et al. 2005; Estes et al. 2008). Although HAART can be very effective at limiting viral replication, HIV-speciﬁc CD8+ T-cell responses generally wane in patients on HAART (Kalams et al. 1999; Ogg et al. 1999; Spiegel et al. 1999), suggesting that maintenance of these responses is to some extent antigen-driven. In patients interrupting HAART, viral load rebound is accompanied by increased CD8+ T-cell responses (Ortiz et al. 2001). In crosssectional studies, CD8+ T-cells from blood and mucosal tissues of patients on HAART secreted fewer cytokines in response to HIV peptide stimulation as compared to T-cells from HAART-naïve patients, suggesting that HAART leads to a decline in “polyfunctionality” as well as response magnitude (Critchﬁeld et al. 2008). The reasons for persistent immunological defects and incomplete CD4+ T-cell repopulation despite prolonged suppressive treatment are still unclear despite multiple studies, but potential mechanisms include ongoing low-level HIV replication, perhaps in speciﬁc tissue reservoir sites, irreversible structural changes in the lymphoid compartments, and incomplete reconstitution of the gut mucosal integrity leading to persistent low levels of bacterial translocation.

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In Search of Efﬁcient Immune Based Therapies

Searching for interventions that could ameliorate immune functional reconstitution, alone or combined with HAART, is still of high priority in HIV research. The goal of such interventions is to transform progressors into non-progressors, and thus delay or prevent starting HAART (if given alone), or to enhance control of HIV infection in patients undergoing HAART, and thus lower the viral set-point after treatment removal, or prevent failure of HAART. Several strategies have been tested or are currently tested. Broadly, they include cytokine supplementation, antibodies against negative regulators (CTLA-4, PD-1/PD-L1), and therapeutic vaccinations. In terms of cytokines, IL-2 was ﬁrst evaluated. Despite a substantial and sustained increase in CD4 cell counts, addition of IL-2 to HAART has yielded no clinical beneﬁt in either of two large international studies. On the contrary, IL-2 therapy increased the incidence of life-threatening clinical events in patients with the highest baseline CD4 counts (Abrams et al. 2009). The mechanisms underlying these deleterious effects are unclear but could be related to the induction of CD4+ T cells with characteristics of Treg cells (Velilla et al. 2008; Weiss et al. 2010) and/or that IL-2 had pronounced pro-inﬂammatory effects in patients with higher numbers of CD4+ T cells at baseline. IL-7 therapy appears more promising (Levy et al. 2009), and is currently being tested in Phase I/II randomized placebo clinical trials. Blocking anti-CTLA-4 combined with ART was not efﬁcient in chronically SIVinfected RM. Despite a modest improvement in CD4 and CD8 anti-viral responses and a slight decrease in viral RNA in tissues of animals receiving anti-CTLA-4 antibody plus HAART compared to HAART alone-animals, anti-CTLA-4 antibody did not bring signiﬁcant functional improvement (Hryniewicz et al. 2006). Moreover, treatment of RM with the same blocking anti-CTLA-4 antibody during the acute phase of infection was detrimental, inducing higher viral loads and accelerated mucosal CD4+ depletion (Cecchinato et al. 2008). Blockade of the PD-1/PD-L1 pathway is generating considerable interest. Velu and colleagues presented the ﬁrst in vivo study to show enhancement of SIV-speciﬁc immune response using a blocking PD-1 Ab (Velu et al. 2009). In vivo blockade of the PD1/PDL1 pathway appeared safe and led to prolonged survival of SIV-infected RM. However, viral loads returned to pre-blockade levels within a few weeks. These studies highlight the problems associated with immune-based therapies in HIV/SIV infection: blockade of one inhibitory pathway will likely be insufﬁcient to provide sustained beneﬁts, but combined blockade of several regulatory pathways may provoke severe auto-immune reactions in treated patients. In terms of therapeutic vaccines, there are currently a number of clinical trials using dendritic cells, with the goal of enhancing control of HIV infection in patients undergoing HAART. We refer the reader to a recent review written by C. Rinaldo, which summarizes the strategies currently developed, and the difﬁculties researchers face when designing such treatments (Rinaldo 2009). Another tested approach was the combination of several types of HIV vaccines to early HAART (the QUEST trial). Results were very disappointing, as vaccinated subjects did not control HIV-1

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after treatment discontinuation better than those receiving the placebo, despite displaying signiﬁcantly increased IFN-J expression by HIV-speciﬁc CD4+ and CD8+ T-cells (Kinloch-de Loes et al. 2005).

8.5

Concluding Remarks

Since the discovery of HIV more than 25 years ago, the ﬁeld has come a long way despite many setbacks. We now clearly understand that HIV is a master at evading and impairing all arms of the immune system, while using basic mechanisms of T cell activation to its advantage. However, we also understand better its “Achilles’ heel”, revealed by the study of elite controllers, both humans and RM. This knowledge is currently being applied to design better vaccine strategies, better immunebased therapies, and better use of HAART, although most HIV researchers have stopped believing that we will ever ﬁnd a “silver bullet”. Beyond this progress, HIV researchers also have been forced to develop better ways to probe the immune systems of humans and nonhuman primates, as appropriate rodent models were not available. The past and current studies of CD4/CD8 function, lymphocyte homeostasis and cell-cell interactions conducted in HIV-infected populations are providing signiﬁcant insights regarding the human immune system, insights that will continue to be useful to many other research ﬁelds. Acknowledgments The authors thank the members of their laboratories and their colleagues for useful discussions, as well as their mentors without whom they would not have joined the HIV research ﬁeld. CAC wants to particularly thank Dr Gene Shearer. CAC is supported by the National Institutes of Health (R01 AI068524, R01 AG033057 and U01 HL101800). BLS is supported by the National Institutes of Health (R01 AI057020, R01 DE021273, P01 AI083050, and R21 NS069219) and the American Foundation for AIDS Research (AmFAR 107854-RGRL).

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Index

A Aliberti, J., 1, 23–34 Anti-retroviral therapy and T cell responses HAART AIDS and death, 155 CD4 count levels and healthy volunteers, 155–156 CD4+ T cells amelioration, 156 diagnosis, HIV infection, 155 immunological defects and interruption, 156 limiting viral replication, 156 Treg cell activation and frequency, 156 immune based therapies blocking anti-CTLA–4, 157 goal and IL–2 effects, 157 PD–1/PD-L1, 157 vaccines, 157–158 Arens, R., 121

B Bacillus Calmette Guerin (BCG), 24 Bacterial and non-bacterial infection, iNKt cells bystander indirect activation description, 50–51 microbial infections, dual recognition, 51 Salmonella and TCR signaling, 51 direct recognition, microbial GSLs Agelas mauritianus, 52–53 alphaglucuronylceramides, 52 description, 52 lyme disease, 53 Sphingomonas/Novosphingobium, 52

gram-negative bacteria, 50 parasitic, helminth and fungal infections Candida albicans and Aspergillus fumigatus, 54 control parasitic replication, 54 Plasmodium, 54 role and anti-parasite responses, 54 viral infections detection, viral pathogens, 53 Hepatitis B virus (HBV) model, 53–54 immune defense strategy, 54 iNKT role, 53 lethal outcome, Epstein-Barr virus, 53 role and SAP-Fyn signaling, 53 Baﬁca, A., 23–34 B and T lymphocyte attenuator (BTLA), 133 B cell-mediated regulation, Leishmania antibodies, 86 arm, immune system, 85–86 CMI, 85 humoral immunity, 86 IL–1 and IL–6, 88 intercellular pathogen clinical infection, 90–91 description, 89 murine models, 92–93 Th1 vs. Th2, 89–90 intracellular pathogen antibodies role, 86–87 CMI effects, 86 immune control, Ehrlichia chaffeensis, 87 proinﬂammator vs. anti-inﬂammatory, 87–88 Th1 response, FcJR, 86 macropages activation and antibodies

J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2, © Springer Science+Business Media, LLC 2012

171

172 B cell-mediated regulation, Leishmania (cont.) functions, 88–89 NADPH oxidase, 89 nitric oxide (NO) production, 88 superoxide production, 89 Benedict, C.A., 121 BTLA. See B and T lymphocyte attenuator Buzoni-Gatel, D., 11

C CD1 system and NKT cells description, 41 group II (CD1d) description, 42 in mouse, 42 restricted diverse type II iNKT cells, 43–44 restricted type I iNKT cells, 42–43 group III (CD1e), 44 group I (CD1a, b, c), mycobacterium tuberculosis, 41 mediated presentation, GSLs, 49 MHC gene complex, 41 self-GSL antigens endogenous presentation, 49 E-hexosaminidase B, 50 isoglobotrihexosylceramide (iGb3), 49–50 regulation, 50 self-lipid antigens, 50 species distribution birds, 44 existence, 44 human and guinea pigs, 44–45 isoforms, 44 mycobacteria-infection, 45 CD4+ T cells and HIV affecting subsets regulatory T cells, 149–150 Th17 cells, 149 Th1/Th2 cells, 148–149 AIDS and depletion disease cell loss, morbidity and mortality, 145 cross-linking, 146–147 cytopathic effects, virus, 145–146 GI loss, 145 IFNs effects, 147 increases “bystander” cells, 146 “natural” vs. “non-natural” hosts, 145 primary target cells, 146 immune dysregulation abnormalities, 147

Index chronic “tickling”, 148 defects, 147heterodimeric integrin D4E7, 148 in vitro CD4+ T cells, 147 CD8+ T cells responses, HIV acute/early infection, 154 function and dysfunction, chronic infection ageing, immune system, 155 characterization, 154 contribution, 155 immune exhaustion and LCMV, 154 immune control, 151 lessons and HIV controllers antiretroviral therapy, 152 GWAS, 152 HLA-B*35 alleles and AIDS, 153 human HLA-B*5701, Mamu-B*17, 153 in silico model, 153 MHC class I and II alleles, 152 natural killer (NK) cell function, 152–153 Cell mediated immunity (CMI), 85–87 Chemokine receptors CCR2 binds CCL2, 111, 113 inability, CCL2 and CCL7, 112 L-arginine, fungal elimination, 111, 112 CCR5 IL–17A neutralization, 113 role, 5 T and Th17 cells, 113, 114 Chougnet, C.A., 141 CMI. See Cell mediated immunity (CMI) CMV. See Cytomegalovirus Culture-derived tachyzoites (STag), 4, 13 Cytomegalovirus (CMV) adaptive T cell responses costimulation, 127–128 MHC-TCR interactions, 126–127 apoptosis pathways, 134 CD8+ and CD4+ T cells HCMV, 125 immunomodulatory effects, DCs, 126 MCMV replication, 124–125 memory inﬂation, 125–126 DCs, 122–124 epidemiology and pathology HCMV infection, 122 risk factor, 122 herpesviruses, 121 immunosuppressive machinations, 134 modiﬁcation, T cells B7 costimulation, 130–131 cytokines, 133–134 HVEM/BTLA interactions, 133

Index MHC expressions, 128–130 PD–1/PDL–1 pathway, 132–133 soluble positive cosignals, 131–132 NK and NKT cells, 124

D Debbabi, H., 11 Deepe, G.S. Jr., 99 Dendritic cells (DCs) CMV cytokines, 122–123, 131–132 HCMV infection, 126 immune modulatory gene products, 123–124 MCMV, 128, 132 MHC molecules, 126 negative net signal, 128, 129 NK cells, 124 PD-L1, 132 positive net signal, 128, 129 cross-presentation pathway, DC, 104 cytomegalovirus (CMV), 122–124 Histoplasma capsulatum bind and ingest, 103, 104 cross-presentation pathway, 104 population, 101 yeast cells, 103, 104 immunomodulatory effects, 126 intracellular lifestyle, 104 microbial recognition, 4 neutrophils, 104 paralysis, 13 production, DC, 131–132

G Gibson-Corley, K.N., 85–93 Glycosphingolipid (GSL) bacterial and non-bacterial infection Agelas mauritianus, 52–53 alphaglucuronylceramides, 52 description, 52 lyme disease, 53 Sphingomonas/Novosphingobium, 52 CD1 system and NKT cells endogenous presentation, 49 E-hexosaminidase B, 50 isoglobotrihexosylceramide (iGb3), 49–50 mediated presentation, GSLs, 49 regulation, 50 self-lipids reportoire, 50 iNKT cell activation GSL antigens and structures, 40

173 in humans, 56 self-GSL antigens, 49–50 Gram-negative, LPS-positive bacteria, 50–53 Granuloma formation and bacilli tuberculosis chronic maintenance, 28 description, 27–28 ﬁbrous capsule, 28 IFN-J, 29 IL–10, 30 lipoxins, 29–30 TGF-E, 30 TNF, 28–29 Gutoerrez, F.R.S., 69–81

H HAART. See Highly Active Anti-Retroviral Therapy (HAART) HCMV. See Human cytomegalovirus Hepatitis B virus (HBV) model, 53–54 Herpesvirus entry mediator (HVEM), 133 Highly Active Anti-Retroviral Therapy (HAART), 143, 155–156 Histoplasma capsulatum characterization, inﬂammatory response MI, 101 neutrophils, 100–101 T and B cells, 101 chemokines and chemokine receptors CCR2, 111–113 CCR5, 113 cytokines GM-CSF, 108 IL–1, 108 IL–4, 110 IL–10, 111 IL–17 and Th17, 109 IL–12/IFN-J axis, 107–108 TNF-D, 109–110 description, 99–100 granuloma intracellular pathogens, 101 T cells, 102 intracellular lifestyle DC, 104 MI, 102–103 neutrophils, 103–104 leukotrienes, 113–114 lymphoid cell, infection control B cells, 107 T cells, 104–107 TCR, 105–106

174 Human cytomegalovirus (HCMV) CD4 and CD8 T cells, 125 glycoprotein products, 129 IL–10, 133–134 NK cells, 124 Human immunodeﬁciency virus (HIV)–1 anti-retroviral therapy effect HAART, 155–156 search immune based therapies, 157–158 CD4+ T cells depletion disease, 145–147 immune dysregulation, 147–148 regulatory T cells, 149–150 Th17 cells, 149 Th1/Th2 cells, 148–149 CD8+ T-cells function and dysfunction, chronic infection, 154–155 immune control, 151 lessons and controllers, 152–153 responses, acute/early infection, 154 cell-cell interaction, 158 discovery, 158 experimental models limitation, heterologous viruses, 144 rodents, 143 SIV and Rhesus macaques (RM), 143–144 immune system, humans and honhumans, 158 life cycle CD4 T cells, 142 gag, pol and env genes, 142 HAART, 143 mRNA encoding, 143 replication, 143 Tat and Rev, transport RNAs, 142–143 T cell activation, 158 transmission cis-/trans-infection, 144 clade B virus, 144 draining lumph nodes, 144 mucosal exposure, 144 phenotypic analysis, 144 route and mucosal infections, 145 virus dose and human transmission, 144–145 HVEM. See Herpesvirus entry mediator

I IL. See Interleukin Immunopathology mechanism, Toxoplasma gondii

Index IL–27 and suppression, 12–13 IL–22 role, 11–12 inﬂammation redundancy and control, 14–15 interleukin–10 central role, 11 mediators and antigen processing, 9 modulatory activities, 10 neutralization, 10 pathogens and poxviruses, 9–10 pro-inﬂammatory responses, 10 TGFE and IL–35, 9 lipoxin A4 control, 14 “DC paralysis”, 13 injection, STAg, 13 Mycobacterium tuberculosis, 16–17 pathogen evasion, 15–16 production, 14 receptors and evidence role, 13 resolution phase cardinal signs, 8 controlling and promoting, 9 homeostasis, 9 omega–3 PUFA/ﬁsh oils, 8–9 tissue injury, cause and consequences, 8 TGF-E description, 10 macrophage deactivator, 11 mucosal host/pathogen interaction, 11 role, 12 iNKT cells, humans CD1d-restricted, 55 infections, Novosphingobium/ Sphingomonas spp.. osocomial and septic shock, 56 xenobiotic-metabolizing properties, 55–56 PBC association GSL recognition role, 56 pathogenesis, 56–57 PDC-E2 homologues, 57 primary biliary cirrhosis (PBC), 55 VD24 iNKT cells, 55 Innate immunity regulation, Trypanosoma cruzi cardiomyopathy, 70–71 cell migration, 79 Chagas’ disease, 69 characterization, 70 cytokines, 78 description, 69 development, cardiac damages, 71 diagnosis, 70

Index GIPL and Tc52 activation, 73 intracellular parasite, 73 production, TGF-E and IL–10 and IL.12p70, 73 TLR2 activation, 72 TLR2-/-and MyD88-/-mice, 73 GIPL recognition, 73–74 glycoinositolphospholipids (GIPL), 71 in human beings, 70 interaction and evolution, 71 life cycle, 70 mechanisms, innate immune system, 72 MMP, 80 nitric oxide (NO) APC and T cells and induce apoptosis, 75 deﬁciency impact, 76 donors and control, 75 production and role, 75 response mechanisms, 75 versatile immune mediators, 75 NLR participation, 74–75 phagocytic cells intracellular replication, 78 intracellular signaling, 77–78 macrophages activation, 76 mechanisms, 78 parasite killing, 77 TLR, 71 TLR9 involvement, 74 Interleukin (IL) cmvIL–10, 133–134 HCMV, IL–10, 133–134 Histoplasma capsulatum IL–1, 108 IL–4, 110 IL–10, 111 IL–17 and Th17, 109 IL–12/IFN-J axis, 107–108 IL–1 receptor, 108 IL–17A neutralization, 113 IL–12 induction, inﬂammation resolution (see Toxoplasma gondii infection) immune based therapies, IL–2 effects, 157 Leishmania, IL 1 and 6, 88 prevent immunopathology mechanism IL–22, 11–12 IL–27, 12–13 Toxoplasma gondii IL–10, 9 IL–27 and suppression, 12–13 IL–12 induction (see Toxoplasma gondii and inﬂammation resolution) IL–22 role, 11–12

175 Trypanosoma cruzi IL–10, 73 IL.12p70, 73 tuberculosis, IL–10, 30 Invariant Natural Killer T (iNKT) cell activation, microbial infection bacterial infection bystander indirect activation, 50–51 cognate recognition, GSL antigens, 52–53 CD1 presentation, 49 CD1 system division, 41 formation, 41 group I (CD1a, b, c), 41 group II (CD1d), 42–44 group III, (CD1e), 44 species distribution, 44–45 function antibody production, 48 anti-microbial activity, 48 autoreactivity, 46 bacteria and virus detection, 45 circuits and molecular mechanisms, 46 deﬁcient CD1d0/JD180 mice, 45 interactions, 47–48 mechanisms, 48 natural and immune rejection, 45 systemic administration, 46–47 Th1 or Th2 cytokines, 45 trans-activation, 47 type I diabetes, 45–46 GSL antigens and structures, 40 in human correlation, 55 Novovosphingobium/Sphingomonas spp., 55–56 primary biliary cirrhosis, 56–57 MHC class I, 40 mouse model infection, 57–58 non-bacterial infection parasitic, helminth and fungal infections, 54 viral infections, 53–54 role, 40 self-GSL antigens, 49–50 TCR, cell population, 40 V124 TCR, human, 40 V114 TCR, mice, 40

J Janssen, E.M., 121 Jones, D.E., 85–93

176 K Koch, R., 23

L Lipoxins M. tuberculosis in humans, ALOX5, 29–30 5-LO-dependent, 29 risk, 30 role, 29 Toxoplasma gondii control, 14 “DC paralysis”, 13 injection, STAg, 13 mycobacterium tuberculosis, 16–17 vs. Mycobacterium tuberculosis, 17 pathogen evasion, 15–16 production, 14 receptors and evidence role, 13 Loewendorf, A., 121

M Macrophages (MM), Histoplasma capsulatum CD11/CD18 adhesin receptors, 102, 103 CD8+ cytotoxic T cells, 106 cross-presentation pathway, DC, 104 granuloma, 101 growth inhibition, 103 IL–4, 110 neutrophils, 103 yeast cells, 102, 103 Major histocompatibility complex (MHC) CD1 system and NKT cells, 41 CD8+ T cells responses, HIV, 152 class I expression antigen-presentation, 128–129 glycoprotein products, 129 “immunoevasion” genes, 129–130 class II expression antigen presentation, 130 proteolytic degradation process, 130 CmvIL–10, 133–134 cytomegalovirus (CMV), 126–130 dendritic cells, 126 iNKT cell activation, 40 MCMV, 128–129 TCR interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 Matrix Metalloproteinases (MMP), 80

Index Mattner, J., 31–58 MCMV. See Murine cytomegalovirus MHC. See Major histocompatibility complex Mixed lymphocyte reactions (MLR), 128 MM. See Macrophages MLR. See Mixed lymphocyte reactions Mouse model infection, 57–58 Murine cytomegalovirus (MCMV) CD8 and CD4 T cells, 124–125 m138 gene, 131 MHC class I pathway, 128–129 m157 protein, 124

N NADPH oxidase, 87, 89 Natural killer (NK) cells absence, 27 accumulation, 27 CMV DCs, 124 MCMV m157 protein, 124 depletion, 27 role, 26 Natural vs. non-natural hosts, 145 Neutorphils Histoplasma capsulatum DC, 104 human defensins, 103–104 IL–4, 110 yeast cells, MM, 103 tuberculosis acute pulmonary tuberculosis, 24 deﬁnition, 24 depletion, 24 mouse strains, 25 potential role, 25 protection, 24–25 Nitric oxide (NO) Leishmania, 88 Trypanosoma cruzi deﬁciency impact, 76 donors and control, 75 production and role, 75 response mechanisms, 75 versatile immune mediators, 75 NK cells. See Natural killer cells

P PDL–1. See Programmed death ligand Petersen, C.A., 85–93 Programmed death ligand (PDL–1), 132–133 Proinﬂammator vs. anti-inﬂammatory, 87–88

Index S Shacklett, B.L., 141 Superoxide production, 89

T T cell receptor (TCR) cell population V124 TCR, human, 40 V114 TCR, mice, 40 Histoplasma capsulatum CD3+, 105 protective immunity, 105–106 pulmonary infection, 105 reactivation histoplasmosis, 106 interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 lymphoid cell, infection control, 105–106 T cells B7 costimulation CD28 and CTLA–4, 130–131 MCMV m138 gene, 131 positive cosignaling pathways, 131 costimulation B7 family, 127 positive and negative net signals, 128, 129 TNF receptor and soluble mediators, 127 cytokines cmvIL–10 and IDO, 133–134 HCMV, 134 production, DC, 131–132 Histoplasma capsulatum CCR5, 113, 114 CD4+ and CD8+ cells, 104–105 granuloma, 102 IL–1 receptor, 108 PD ligands, 106 promote immunity, 106 receptors, CD3+, 105 regulatory, 107 TNF-D production, 105, 109 HVEM/BTLA interactions, 133 MHC expressions, 128–130 MHC-TCR interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 MLR, 128 PD–1/PDL–1 pathway B7-CD28 pathway, 132–133

177 cell cycle arrest, 132 negative cosignaling, 132 TCR. See T cell receptor Th1 vs. Th2, 89–90 TNF-D. See Tumor necrosis factor-alpha Toll-like receptors (TLRs) microbial recognition, 5–6 signaling, 51 Trypanosoma cruzi TLR, 71 TLR2 activation, 72 TLR2-/-and MyD88-/-mice, 73 TLR9 involvement, 74 Toxoplasma gondii and inﬂammation resolution cysts and bradyzoites, 3 description, 1 felines, cat, 2 in human, 2 IFN-J, Th1 cells and microbicidal activity activation factors and parasite strains, 7 components, 7 immune responses mechanisms, 8 investigation, 18 life cycle, 2 microbial recognition and IL–12 induction biochemical signaling, 7 CCR5 role, 5 complexity and protection, 4 cyclophillin–18, 5 cytoplasmic protein proﬁllin, 6 hypothesis, dendritic cells, 7 IFN-J, 4 immune response and pathogen, 4 IRF–8, mice, 6 macrophages, neutrophils and DCs, 4 p38 MAP kinases, 6–7 TLRs, 5–6 transcription factors, 6 use STAg, 4–5 natural conditions infection, 3 parasite replication, 3 prevent immunopathology mechanism endogenous LXA4, 15–17 IL–22, 11–12 IL–27, 12–13 inﬂammation redundancy and control, 14–15 interleukin–10, 9–10 lipoxin A4, 13–14 resolution phase, 8–9 TGF-B, 10–11 protozoan apicomplexa parasite, 3 survival, oocysts, 2, 3

178 Toxoplasma gondii and inﬂammation resolution (cont.) symptoms development and risk, 1 tachyzoites replication and “dripping” effect, 3 transmission, 3 Toxoplasma gondii vs. Mycobacterium tuberculosis, 17 Tuberculosis, host protection and pathogen evasion BCG and treatment, 24 BCG vaccine, 33–34 disease reactivation AIDS and TNF, 32 drug and treatment, 32 effects, 33 epidemiology, 32 HIV, 32–33 granuloma formation and bacilli (see Granuloma formation and bacilli) history, 23 immune response cell wall components, 31 host cell signalling, 32 mycobacterial dormancy, 31–32 phagosome-lysosome fusion, 31

Index infection and innate immunity dendritic cells, 26 natural killer cells, 26–27 neutorphils, 24–25 regulatory T cells, 27 T cells, 25–26 risk, 24 WHO, 23–24 Tumor necrosis factor-alpha (TNF-D), Histoplasma capsulatum primary and secondary infection, 109 T cells, 106 TNF receptors, 109–110

V Vaccines BCG, 33 HIV, 33 Viral infections detection, viral pathogens, 53 Hepatitis B virus (HBV) model, 53–54 immune defense strategy, 54 iNKT role, 53 lethal outcome, Epstein-Barr virus, 53 role and SAP-Fyn signaling, 53