The Immune Response to Infection
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The Immune Response to Infection edited
by
Stefan H. E.Kaufmann
Max Planck Institute for Infection Biology Berlin, Germany
Barry T. Rouse
College of Veterinary Medicine, University of Tennessee Knoxville, Tennessee
David L. Sacks
Laboratory of Parasitic Diseases, National Institutes of Health Bethesda, Maryland
W A S H I N G T O N ,
D C
Front cover: Macrophage in the process of engulfing Mycobacterium tuberculosis organisms. Back cover: Left (red): Helicobacter pylori; right (blue): Neisseria gonorrhoeae attached to epithelial cells. All three photos courtesy of Volker Brinkmann and Stefan H.E. Kaufmann (Max Planck Institute for Infection Biology, Berlin, Germany).
Copyright © 2011
ASM Press American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904
Library of Congress Cataloging-in-Publication Data The immune response to infection / edited by Stefan H.E. Kaufmann, Barry T. Rouse, David L. Sacks. p. ; cm. Includes bibliographical references and index. ISBN 978-1-55581-514-1 (alk. paper) 1. Immune response. I. Kaufmann, S. H. E. (Stefan H. E.) II. Rouse, Barry T. III. Sacks, David Lawrence. [DNLM: 1. Adaptive Immunity—immunology. 2. Immunity, Innate—immunology. 3. Host-Pathogen Interactions—immunology. 4. Infection—immunology. QW 541 I326 2011] QR186.I445 2011 616.07'9—dc22 2010019964 All Rights Reserved Printed in the United States of America 10
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Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, U.S.A. Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, U.S.A. Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 Email:
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Contents
8 Regulation of Antimicrobial Immunity / 109
Contributors / ix Preface / xv
yASMINE BELKAID, SHARVAN SEHRAWAT, AND BARRy T. ROUSE
The Immune Response to Infection: Introduction / 1
STEFAN H. E. KAUFMANN, BARRy ROUSE, AND DAVID SACKS
9
SECTIon I
SECTIon II
HoST DEFEnSE: GEnERAL / 5 1
Memory and Infection / 121
DAVID MASOPUST AND MARK K. SLIFKA
THE PATHoGEnS / 131
Invertebrate Innate Immune Defenses / 7
LAURE EL CHAMy, CHARLES HETRU, AND JULES HOFFMANN
10
overview of Viral Pathogens / 133
11
overview of Parasitic Pathogens / 143
12
overview of Bacterial Pathogens / 155
13
overview of Fungal Pathogens / 165
14
Prionoses and the Immune System / 173
JONATHAN W. yEWDELL AND JACK R. BENNINK
2 The ontogeny of the Cells of the Innate and the Adaptive Immune System / 21
RICK L. TARLETON AND EDWARD J. PEARCE
FRITz MELCHERS
3 The Evolutionary origins of the Adaptive Immune System of Jawed Vertebrates / 41
PHILIPPE J. SANSONETTI AND ANDREA PUHAR
JIM KAUFMAN
AxEL A. BRAKHAGE AND PETER F. zIPFEL
4 Host Defense (Antimicrobial) Peptides and Proteins / 57 LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK
JüRGEN A. RICHT AND ALAN yOUNG
5 Reactive oxygen and Reactive nitrogen Intermediates in the Immune System / 69
SECTIon III
InnATE IMMUnITY To MICRoBIAL InFECTIonS / 183
CHRISTIAN BOGDAN
6
Complement in Infections / 85
15
WILHELM J. SCHWAEBLE, yOUSSIF MOHAMMED ALI, NICHOLAS J. LyNCH, AND RUSSELL WALLIS
7
Innate Immunity to Viruses / 185
AKIKO IWASAKI
16 natural Killer Cell Response against Viruses / 197
Immune Defense at Mucosal Surfaces / 97
MARIAN R. NEUTRA AND JEAN-PIERRE KRAEHENBUHL
JOSEPH C. SUN AND LEWIS L. LANIER v
vi
17
Contents
Innate Immunity against Bacteria / 209
THOMAS ARESCHOUG, ANNETTE PLüDDEMANN, AND SIAMON GORDON
18 Innate Immunity to Parasitic Infections / 225
CHRISTOPHER A. HUNTER AND ALAN SHER
SECTIon IV
ACQUIRED IMMUnITY To MICRoBIAL InFECTIonS / 237 19 Acquired Immunity against Virus Infections / 239
EVA SzOMOLANyI-TSUDA, MICHAEL A. BREHM, AND RAyMOND M. WELSH
20 Immune Responses to Persistent Viruses / 255
E. JOHN WHERRy AND PAUL KLENERMAN
21 Acquired Immunity: Acute Bacterial Infections / 269 DENNIS W. METzGER
22 Acquired Immunity: Chronic Bacterial Infections / 279
ANDREA M. COOPER AND RICHARD ROBINSON
23 Acquired Immunity: Fungal Infections / 289 LUIGINA ROMANI
24 Acquired Immunity to Intracellular Protozoa / 301 PHILLIP SCOTT AND ELEANOR M. RILEy
25 Acquired Immunity to Helminths / 313
29 Pathology and Pathogenesis of Malaria / 361
CHANAKI AMARATUNGA, TATIANA M. LOPERA-MESA, JEANETTE G. TSE, NEIDA K. MITA-MENDOzA, AND RICK M. FAIRHURST
30 Pathology and Pathogenesis of Virus Infections / 383 CARMEN BACA JONES AND MATTHIAS VON HERRATH
SECTIon VI
EVASIon AnD SUPPRESSIon oF THE AnTIMICRoBIAL HoST RESPonSE / 391 31 Viral Immune Evasion / 393
LILA FARRINGTON, GABRIELA O’NEILL, AND ANN B. HILL
32 Growing old and Immunity to Viruses / 403 JANKO NIKOLICH-ŽUGICH AND MARCIA A. BLACKMAN
33 Growing old and Immunity to Bacteria / 413 JOANNE TURNER
34 Bacterial Strategies for Survival in the Host / 425
ANNA D. TISCHLER AND JOHN D. McKINNEy
35 Suppression of Immune Responses to Protozoan Parasites / 441 DAVID L. SACKS
36
Immune Evasion by Parasites / 453
JOHN M. MANSFIELD AND MARTIN OLIVIER
DAVID ARTIS AND RICK M. MAIzELS
SECTIon VII SECTIon V
PATHoLoGY AnD PATHoGEnESIS / 325
GEnETICS oF THE AnTIMICRoBIAL HoST RESPonSE / 471
26 Pathology and Pathogenesis of Bacterial Infections / 327
37 Genetics of Antibacterial Host Defenses / 473
WARWICK J. BRITTON AND BERNADETTE M. SAUNDERS
27 Helicobacter pylori: the Role of the Immune Response in Pathogenesis / 337
STEVEN M. HOLLAND
38 Immunogenetics of Host Response to Parasites in Humans / 483 JENEFER M. BLACKWELL
KAREN ROBINSON AND JOHN C. ATHERTON
28 Pathogenesis of Helminth Infections / 347
THOMAS A. WyNN AND JUDITH E. ALLEN
39 Immunogenetics of Virus Pathogenesis / 491
SEAN WILTSHIRE, DAVID I. WATKINS, EMIL SKAMENE, AND SILVIA M. VIDAL
Contents
SECTIon VIII
AUToIMMUnITY AnD CAnCER / 509 40 Viruses, Autoimmunity, and Cancer / 511 MEGHANN TEAGUE GETTS, LIES BOGAERT, W. MARTIN KAST, AND STEPHEN D. MILLER
41 The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity / 521 STEFAN EHLERS AND GRAHAM A. W. ROOK
42 Theileria-Induced Leukocyte Transformation: an Example of oncogene Addiction? / 537 MARIE CHAUSSEPIED AND GORDON LANGSLEy
SECTIon IX
IMMUnE InTERVEnTIon / 547 43 Systems Vaccinology: Using Functional Signatures To Design Successful Vaccines / 549 TROy D. QUEREC AND BALI PULENDRAN
44 Meeting the Challenge of Vaccine Design To Control HIV and other Difficult Viruses / 559 BARNEy S. GRAHAM AND CHRISTOPHER WALKER
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45 Immune Intervention Strategies against Tuberculosis / 571
PETER ANDERSEN AND STEFAN H. E. KAUFMANN
46
Immune Intervention in Malaria / 587
CAROLE A. LONG AND FIDEL P. zAVALA
47 Targeting Components in Vector Saliva / 599
MARy ANN McDOWELL AND SHADEN KAMHAWI
SECTIon X
THE MAJoR KILLERS (CLInICS, EPIDEMIoLoGY, AnD IMMUnE PARAMETERS) / 609 48 AIDS Vaccines: the Unfolding Story / 611 STEPHEN NORLEy
49 Tuberculosis / 623
GERHARD WALzL, PAUL VAN HELDEN, AND PHILIP R. BOTHA
50 Malaria: Clinical and Epidemiological Aspects / 633 ANDREA A. BERRy, MyAING M. NyUNT, AND CHRISTOPHER V. PLOWE
51 The Epidemiology and Immunology of Influenza Viruses / 643 RAFAEL A. MEDINA, IRENE RAMOS, AND ANA FERNANDEz-SESMA
Index / 653
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Contributors
yOUSSIF MOHAMMED ALI
ANDREA A. BERRy
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201
MARCIA A. BLACKMAN
The Trudeau Institute, Saranac Lake, Ny 12983
JUDITH E. ALLEN
Institutes of Evolution, Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, UK
JENEFER M. BLACKWELL
Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Subiaco, Western Australia, Australia
CHANAKI AMARATUNGA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
LIES BOGAERT
Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Department of Surgery and Anesthesiology of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, B-9820, Belgium
PETER ANDERSEN
Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
THOMAS ARESCHOUG
Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 22362 Lund, Sweden
CHRISTIAN BOGDAN
Microbiology Institute Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University Erlangen, Nuremberg, and University Clinic of Erlangen, Wasserturmstraße 3/5, D-91054 Erlangen, Germany
DAVID ARTIS
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-4539
PHILIP R. BOTHA
Division of Infectious Diseases, Department of Medicine Tygerberg Academic Hospital, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
JOHN C. ATHERTON
Nottingham Digestive Diseases Centre Biomedical Research Unit, University Hospital, Nottingham, NG7 2UH, United Kingdom
AxEL A. BRAKHAGE
CARMEN BACA JONES
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany
yASMINE BELKAID
MICHAEL A. BREHM
Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 Mucosal Immunology Unit, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Health, 4 Center Drive B1-28, Bethesda, MD 20892
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
WARWICK J. BRITTON
JACK R. BENNINK
Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209
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contributors
MARIE CHAUSSEPIED
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France
ANDREA M. COOPER
Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny
STEFAN EHLERS
Microbial Inflammation Research, Research Center Borstel, Parkallee 1, D-23845 Borstel, Germany, and Molecular Inflammation Medicine, Institute of Experimental Medicine, Christian-Albrechts-University, Arnold-Heller-Str. 3, D-24105 Kiel, Germany
LAURE EL CHAMy
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
RICK M. FAIRHURST
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
LILA FARRINGTON
Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
ANA FERNANDEz-SESMA
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
MEGHANN TEAGUE GETTS
Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
SIAMON GORDON
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, Ox1 3RE, United Kingdom
BARNEy S. GRAHAM
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
ROBERT E. W. HANCOCK
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada
CHARLES HETRU
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
ANN B. HILL
Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
JULES HOFFMANN
CHRISTOPHER A. HUNTER
Department of Pathobiology School of Veterinary Medicine, University of Pennsylvania, Rm 313, Hill Pavilion, 380 South University Avenue, Philadelphia, PA 19104-4539
AKIKO IWASAKI
Department of Immunobiology, yale University School of Medicine, New Haven, CT 06520
SHADEN KAMHAWI
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD 20852
W. MARTIN KAST
Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI 96822
JIM KAUFMAN
University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom
STEFAN H. E. KAUFMANN
Department of Immunology, Max Planck Institute for Infection Biology, Chapritéplatz 1, D 10117 Berlin, Germany
PAUL KLENERMAN
Nuffield Dept of Medicine and NIHR Biomedical Research Centre Programme, Peter Medawar Building, University of Oxford, Oxford Ox1 3Sy, UK
JEAN-PIERRE KRAEHENBUHL
Health Sciences eTraining (HSeT) Foundation, CH 1066 Epalinges, Switzerland
GORDON LANGSLEy
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France
LEWIS L. LANIER
Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143
CAROLE A. LONG
Malaria Immunology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
TATIANA M. LOPERA-MESA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
NICHOLAS J. LyNCH
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
STEVEN M. HOLLAND
SHUHUA MA
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada
contributors
LAURENCE MADERA
MARTIN OLIVIER
RICK M. MAIzELS
GABRIELA O’NEILL
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada Centre for Immunity, Infection and Evolution, and Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom
JOHN M. MANSFIELD
Department of Microbiology and Immunology, Duff Medical Building, 3775 University, McGill University, Montréal, Québec, Canada. Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
EDWARD J. PEARCE
Department of Bacteriology, Microbial Sciences Building, 1550 Linden Drive, University of Wisconsin-Madison, Madison, WI 53716
Trudeau Institute Inc., 154 Algonquin Avenue, Saranac Lake, Ny 12983
DAVID MASOPUST
Center for Vaccine Development, Howard Hughes Medical Institute and University of Maryland School of Medicine, Baltimore, MD 21201
Department of Microbiology, Center for Immunology, University of Minnesota, 2-182 Medical Biosciences Building, 2101 6th St. SE, Minneapolis, MN 55455
MARy ANN McDOWELL
The Eck Institute for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556
JOHN D. McKINNEy
Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
RAFAEL A. MEDINA
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
FRITz MELCHERS
Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany
DENNIS W. METzGER
Center for Immunology and Microbial Disease, Albany Medical College, Albany, New york 12208
STEPHEN D. MILLER
Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
NEIDA K. MITA-MENDOzA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
MARIAN R. NEUTRA
Department of Pediatrics, Harvard Medical School, and GI Cell Biology Laboratory, Children’s Hospital, Boston, MA 02115
JANKO NIKOLICH-ŽUGICH
Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, Az 85718
STEPHEN NORLEy
Robert Koch Institute, 13353 Berlin, Germany
MyAING M. NyUNT
Department of International Health, Global Disease Epidemiology and Control Program, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205
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CHRISTOPHER V. PLOWE
ANNETTE PLüDDEMANN
Department of Primary Health Care, University of Oxford Old Road Campus, Oxford, Ox3 7LF, UK
ANDREA PUHAR
Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France
BALI PULENDRAN
Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329
TROy D. QUEREC
Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329
IRENE RAMOS
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
JüRGEN A. RICHT
Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, KS 66506-5601
ELEANOR M. RILEy
Department of Infectious and Tropical Diseases, London School of Tropical Medicine and Hygiene, London, UK
RICHARD ROBINSON
Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny
KAREN ROBINSON
Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
LUIGINA ROMANI
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy
GRAHAM A. W. ROOK
Centre for International and Medical Microbiology (CIMM), Windeyer Institute for Medical Sciences, University College London (UCL), 46 Cleveland Street, GB - London W1T 4JF, United Kingdom
xii
contributors
BARRy T. ROUSE
JEANETTE G. TSE
DAVID L. SACKS
JOANNE TURNER
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996 Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.
PHILIPPE J. SANSONETTI
Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France
BERNADETTE M. SAUNDERS
Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia
WILHELM J. SCHWAEBLE
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
PHILLIP SCOTT
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
SHARVAN SEHRAWAT
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996
ALAN SHER
Laboratory of Parasitic Diseases NIAID, NIH Building 50, Room 6140, 50 South Drive, MSC-8003, Bethesda, MD 20892-8003
EMIL SKAMENE
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852 Center for Microbial Interface Biology, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University, 460 West 12th Ave., Columbus, OH 43210
PAUL VAN HELDEN
DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
SILVIA VIDAL
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
MATTHIAS VON HERRATH
Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037
CHRISTOPHER WALKER
Nationwide Children’s Hospital and the Departments of Pediatrics, Pathology, and Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43205
RUSSELL WALLIS
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
GERHARD WALzL
MARK K. SLIFKA
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Ave., Beaverton, OR 97006
DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
JOSEPH C. SUN
DAVID I. WATKINS
Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 6152 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706
EVA SzOMOLANyI-TSUDA
RAyMOND M. WELSH
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
RICK L. TARLETON
E. JOHN WHERRy
Center for Tropical & Emerging Global Diseases, Coverdell Center for Biomedical Research (Rm 310B), 500 D.W. Brooks Drive, University of Georgia, Athens, GA 30602
Department of Microbiology and Institute for Immunology, University of Pennsylvania, School of Medicine, 421 Corie Blvd., Room 312, Philadelphia, PA 19104
ANNA D. TISCHLER
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
SEAN WILTSHIRE
contributors
THOMAS A. WyNN
Immunopathogenesis Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-8003
JONATHAN W. yEWDELL
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209
ALAN yOUNG
Department of Veterinary Science, South Dakota State University, Box 2175, ARW168F, Brookings, SD 57007
xiii
FIDEL P. zAVALA
Department of Molecular Microbiology and Immunity, Bloomberg School of Public Health, Malaria Research Institute, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205
PETER F. zIPFEL
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany
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Preface
“Excellent textbooks and review volumes on immunology, virology, parasitology, medical microbiology, and infectious diseases abound. So what gap is this book aimed to fill?” This question was posed in the preface of the ASM Press book Immunology of Infectious Diseases (edited by Stefan H. E. Kaufmann, Alan Sher, and Rafi Ahmed), published in 2002. The explanation provided then still holds true today. Microbiology and immunology, despite their common roots, have matured as distinct disciplines, and infectious diseases are too often viewed from the perspective of either the microbe or the host. A more holistic approach was provided by that book and a second one published by ASM Press in 2004, The Innate Immune Response to Infection (edited by Stefan H. E. Kaufmann, Ruslan Medzhitov, and Siamon Gordon). These books now urgently need updating because the knowledge base in immunology, as well as with all types of infectious agents, has expanded dramatically. The present volume, The Immune Response to Infection, covers all aspects of innate and adaptive immune mechanisms and describes how they interact with pathogens of different types, resulting in either success or failure to control infection and clinical disease. This volume also emphasizes how our understanding of mechanistic events is leading the design and production of more effective prophylactic and therapeutic control measures for infectious agents. Most of the chapters here consider host-pathogen interactions in the context of the broad divisions of the microbial world—either viruses, bacteria, or parasites—
and do not confine their discussion to any individual pathogen. The exceptions are for the agents of the “big three” infectious diseases—HIV/AIDS, tuberculosis, and malaria—which account for almost one-third of human deaths from infections, as well as influenza, which is the focus of much media and public attention. We have also included chapters that consider the detrimental sequelae of infection that are an indirect result of the infectious process, such as chronic inflammation, cancer, and autoimmunity. Finally, all of the chapters emphasize the special attributes that make pathogens difficult to control, and they appraise the prospects of current and future prophylactic and therapeutic vaccines. We hope that this book, which comprises the rich variety of aspects of infection and immunity, helps to further promote the important relationship between immunology and medical microbiology. We express our deep appreciation to the editorial staff of ASM Press, in particular, Greg Payne and Ellie Tupper. We also want to thank our associates Mary Louise Grossman and Lisa Washington for their secretarial help and for their wonderful dedication. Most of all, we thank our colleagues for sacrificing so much of their valuable time to generously share their outstanding expertise with us and with the readers of this book. STEFAN H. E. KAUFMANN, BARRy T. ROUSE, AND DAVID L. SACKS
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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
The Immune Response to Infection: Introduction STEfAN H. E. KAUfMANN, BARRy RoUSE, AND DAvID SACKS
HISTORICAL PERSPECTIVE
immune system. from a coevolutionary perspective, these different stratagems in the struggle for life between microbes and host might seem to offer neither any clear-cut advantage. yet humans have one major advantage: our brain that can envision, design, and develop new countermeasures. In the case of infectious disease these are anti-infectives, mostly antibiotics and vaccines. from the beginning, plagues have written history (fig. 1) (Brock, 1999). However, it took until the late 19th century until we understood what causes plagues and how our body fights against them. Investigations of plagues started almost 500 years bce in ancient Greece, when Thucydides (460–396 bc) assumed contagiousness of the plague in Athens because he realized that those who recovered from disease would not fall ill again. Even though Hippocrates (460–375 bc) and his school recognized that some disease can be transmitted, their main concept viewed disease as an imbalance among the four humors: phlegm, black bile, yellow bile, and blood. Hence their contribution to our understanding of contagious diseases was minimal. Some time later, Diodorus Siculus (1st century bc) impressively described plagues at the times of Pericles and considered that gas arising from the muddy ground was responsible. Bad air is termed miasma in Greek, and a long line of investigators believed in miasma as a cause of infectious diseases. Marcus Terentius varro (116–27 bc) argued against miasma in his treatise on agriculture, where he assumed that minute animals invisible to the naked eye cause severe diseases when they are inhaled through the nose and mouth. This knowledge would have been lost during the Middle Ages were it not continued by the Islamic world. Notably, the Persian Rhazes (864–830 ad), in his treatise about smallpox and measles, precisely described differences and similarities of these two plagues. A major breakthrough, however, came with the work of Girolamo fracastoro (1478–1553 ad), from verona, Italy. In his work About the Contagious Diseases and Their Treatment, he foresaw the major characteristic features of infectious diseases, speculating that small organisms were responsible for transmission. The German Jesuit Athanasius Kircher (1602–1680 ad) was a universally educated man who was also curious about the causes of plague, and was the first to claim to have seen microbes, which cause infectious diseases. This, however, was impossible because the magnification glasses he used were insufficient. What he probably
Microbes were the first living organisms to colonize the world some 3 billion years ago. When our human ancestors entered the scene some millions of years ago, microbes had already occupied every niche on the globe. The human body was no exception to this occupation. Microbes colonized and readily exploited the outer and inner surfaces of our ancestors’ bodies; every now and then they entered into deeper tissue sites where they caused harm—something we now call infectious disease. As long as humans were nomadic and did not settle in larger communities, infectious diseases were restricted to small groups of individuals and could not spread. once humans switched to stable settlements and started to grow crops and tend animals, microbes could better spread from contaminated water, soil, and vegetable sources to animal and human, from animal to human, and from human to human. farming and animal husbandry practices began 10,000 to 20,000 years ago and provided fertile ground for insects, which served as vectors for disease transmission. In fact, we can trace several major infectious diseases of today back to these times. Currently, we witness a resurgence of plagues with the emergence of new diseases such as acquired immunodeficiency syndrome (AIDS) and new strains of influenza virus as well as the reemergence of old diseases such as tuberculosis (TB). In the struggle between microbe and human, the central strategy microbes trust on is rapid replication time paired with frequent mutation so that a few microbes surviving under catastrophic conditions can emerge and conquer a new and previously hostile niche. In contrast, humans developed along a different path based on specialization and complexity. We possess specialized organs that serve as pumps for blood circulation, the heart; as site of metabolite production and detoxification, the liver; or as site for perception and thought, the brain. The combat of infectious agents is the responsibility of a specialized organ, the Stefan H. E. Kaufmann, Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117 Berlin, Germany. Barry Rouse, Department of Pathobiology, 1414 W. Cumberland Ave, WLS Rm B408, University of Tennessee, Knoxville, TN 37996-0845. David Sacks, Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, NIAID, Bldg 4, Rm 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.
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FIGURE 1 Timeline of infectious disease research. The timeline illustrates major breakthroughs in the area of infection and immunity.
observed were some blood cells rather than microbes. The first step from concept to experiment was carried out by the textile merchant Antonius van Leeuwenhoek (1632–1723 ad) from Delft, The Netherlands, who, in his free time, crafted small lenses that allowed him to see real microbes for the first time (which he called “Animalcules”). Thus Leeuwenhoek could be named the father of experimental microbiology, although he had no particular interest in infectious diseases. The next issue to clarify was whether life was generated spontaneously or was derived from living organisms. This question was resolved by the Italian Lazzaro Spallanzani (1729–1799 ad) who showed that after heating, a closed flask containing broth remained free of living organisms, whereas broth in an open flask was soon recolonized by microbes, even after being heated. Louis Pasteur (1822–1895 ad) refined this
experiment by using a flask with an s-shaped neck, which prevented entry of microbes when stored, but allowed exchange of air. Even when left open, the heated broth in these flasks was not colonized. Pasteur also showed that fermentation and putrefaction were not just chemical processes but the function of microorganisms. Pasteur went one step further and opened the way for infectious disease research by showing that putrefaction was the underlying mechanism of diseases that were accompanied by pus formation. Moreover, he laid the foundation for rational vaccine design by attenuating microbes and using them for prevention or therapy of infectious diseases. He is best known for the development of two vaccines, one to prevent anthrax and one for treating rabies. The AustroHungarian Ignaz Semmelweis (1818–1865 ad) emphasized the importance of careful hand washing before diagnosing
The Immune Response to Infection: Introduction
healthy pregnant women. In hospital stations where doctors who had been working in pathology prior to diagnosing pregnant women there was a far higher mortality rate of women and infants at delivery. Semmelweis’s advice for disinfection prior to contact with the patient during delivery was not appreciated by the medical community at the time. The Scottish surgeon Joseph Lister (1827–1912 ad) was more successful in convincing his colleagues to recognize the value of wound disinfection, and, together with Semmelweis, established hygiene as a principle for the prevention of the spread of infectious diseases. The Englishman John Snow (1813–1858 ad) furthered the principles of public health for control of plagues and established the discipline of epidemiology. Robert Koch (1843–1910 ad), by revealing the etiology of TB, anthrax, and cholera, founded the golden age of medical microbiology in Berlin, Germany (Kaufmann & Winau, 2005). Within the next decades, dozens of pathogens were discovered and the etiologies of equally numerous infectious diseases were established. Robert Koch’s school also paved the way for immunology, which was established by his scholars, Emil von Behring (1854–1917 ad) and Paul Ehrlich (1854–1915 ad) (fig. 2). Behring developed the concept of passive vaccination, and Ehrlich built the concepts of antibody specificity and specific immunity in general. Pasteur’s Russian colleague Elie Metchnikoff (1845–1916 ad) described phagocytosis and phagocytes as cellular mediators of nonspecific immunity. While the immunologic concepts introduced by Ehrlich and Metchnikoff provided the scientific basis for an understanding of the mechanisms underlying
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vaccination, it was Edward Jenner (1749–1823 ad), an English country physician, who had developed the first active vaccine a century before. Jenner used smallpox with low virulence for humans to protect against a highly virulent related human pox. Medical microbiology paved the way for the emergence of immunology as a new discipline. At the same time, it hampered its development because of its focus on exogenous invaders as being exclusively responsible for pathogenesis. This was a necessary step to challenge the prevailing dogma, which considered disease exclusively as result of malfunctioning host cells. Ehrlich and Metchnikoff balanced this out. The host is not a helpless prey for foreign invaders. A highly sophisticated host defense system—the immune system—exists to combat predators. At the same time, not only microbes, but also the host contributes to pathogenesis. The concept of infectious disease as the result of a cross talk between pathogen and host had developed. Even though the Paris and Berlin schools of immunology hotly disputed their opposing theories, we now know how much they complement each other. Both host cells as first described by Metchnikoff and soluble mediators as described by Ehrlich comprise the two pillars of the immune system: Metchnikoff’s nonspecific innate immune system and Ehrlich’s specific, acquired immune system (Kaufmann, 2008). first signs for a synergy between specific and nonspecific immunity emerged when researchers, notably Hans Buchner (1850–1902 ad), Jules Bordet (1870–1961 ad), as well as Ehrlich himself described complement as a soluble factor of innate immunity, which was activated by antibodies.
FIGURE 2 The merger of innate and acquired immunity. The figure depicts the major events that led to the merger of innate and acquired immunity to a global view of the immune response.
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Complementarities were further emphasized by the Englishman Almroth Wright (1861–1947 ad), who demonstrated the cooperation between antibodies and phagocytes in a process termed opsonization, which allowed engulfment of microbes that resist phagocytosis by means of a capsule (fig. 2). Thus the specific antibodies of Behring and Ehrlich and the nonspecific phagocytes of Metchnikoff could be fused in a more general concept of immunology. It took another half century, however, before the specific mediators of acquired cellular immunity, T lymphocytes, were discovered. This was achieved in the 1960s when J. f. A. P. Miller described the thymusderived lymphocytes. Through the work of Jim Gowans, it was already known that such lymphocytes recirculated through the body and initiated acquired immune responses. The interaction between T lymphocytes and macrophages in defense against intracellular bacteria was described by George B. Mackaness (1924–2007 ad). Communication between T cells and macrophages was facilitated by soluble mediators now called cytokines, as revealed (independently) by Barry R. Bloom and John R. David. Toward the end of the 20th century, the central importance of the innate immune system became clear when it was shown to instruct the acquired immune system and hence play a much more decisive role than originally assumed. Even though numerous researchers contributed to these advances, one of the first who foresaw the intensive cross talk between innate and acquired immunity was Charles A. Janeway (1943–2003 ad). Today, most immunologists agree that cooperation between innate and adaptive immune mechanisms is critical to the success of the immune response. Even though at its birth, immunology was viewed as our defense mechanism against pathogens, it subsequently became the target of more academic research lines focusing on the molecular mechanisms underlying antigen specificity, cell–cell communication, and intracellular signaling processes. The ease with which lymphocytes can be obtained from peripheral blood of humans or lymphoid organs of experimental animals made them preferred targets for many investigators interested in more general biologic issues. In the mid-20th century, diagnosis of infectious diseases had become routine and major intervention measures— notably vaccination to prevent and antibiotics to treat infectious disease—had become broadly available. It is no wonder that the perceived threat of infectious disease had subsided substantially (Kaufmann, 2009). How wrong we were. In fact, globalization has increased the risk of emerging and reemerging plagues, and recent years witness heightened interest in immunity against microbial pathogens. It
soon became clear that the canonical approach to vaccine development had been exhausted. Thus far, vaccines had been developed mostly by trial and error with only minor impact from immunologic knowledge. Principally, these vaccines stimulate antibodies, which fight against pathogens or neutralize their toxic products. This approach, however, is insufficient for design of novel vaccines against agents that are not controlled by antibodies alone. These include the three major killers, malaria, HIv/AIDS, and TB, as well as vaccines against pandemic flu. Here, knowledge in immunology is required for rational vaccine design, since not only antibodies but also multiple subsets of T cells need to be stimulated. Broader immune responses against a variety of antigens are required, which are also directed not only against dominant epitopes, but also against subdominant ones. This needs novel adjuvants designed on the basis of our knowledge about pathogen recognition by the innate immune system, about antigen presentation by antigen-presenting cells through the different presentation pathways, and about stimulatory and inhibitory signalling pathways that control activation and suppression of T lymphocyte populations of distinct functions. It is against this background that we have decided to publish a book on the immunology of infectious diseases, which comprises the whole spectrum of immunology as it relates to infection. It is our goal to describe the cross talk between the different kinds of infectious agents with the different host factors involved in immunity. focus is not only laid on underlying mechanisms, but also at the different sequelae of host–pathogen interactions ranging from sterile eradication of the invader to controlled chronic infection to pathologic corollas of the host–pathogen crosstalk. We also consider the pathogenesis of certain autoimmune disorders and cancers that are induced by infectious agents but then apparently become independent of the infectious process.
REFERENCES Brock, T. D. 1999. Milestones in microbiology: 1546 to 1940. ASM Press, Washington, DC. Kaufmann, S. H. 2008. Immunology’s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat. Immunol. 9:705–712. Kaufmann, S. H. E. 2009. The new plagues: pandemics and poverty in a globalized world. Haus Publishing, London. Kaufmann, S. H. E., and F. Winau. 2005. from bacteriology to immunology: the dualism of specificity. Nat. Immunol. 6:1063–1066.
host defense: general
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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
1 Invertebrate Innate Immune defenses LAURE EL CHAMY, CHARLES HETRU, AND JULES HOFFMANN
IntrodUCtIon
an attempt to trace the origin of essential elements of the innate immune system.
All metazoans are able to mount an innate immune response. Whereas invertebrate species rely solely on this type of defense, gnathostome vertebrates have developed, in addition, a sophisticated adaptive immune system centered on lymphocytes that are absent from invertebrates. Importantly, these adaptive responses require strong stimulatory signals from cells of the innate immune system, and during infections in vertebrates, both arms of defenses cross talk in their fight against invading microorganisms. Current estimates are that 95% of all metazoan species are invertebrates. Consequently, they lack adaptive immune defenses, which are believed to have appeared in ancestral cartilaginous fish some 450 million years ago and are restricted to vertebrates. In this review, we will focus on invertebrate immune defenses and address the question of when and how they evolved in sponges and in recent forms of arthropods. For lack of space, we will restrict our analysis to invertebrate protostomes and will not consider here immune defenses of deuterostomes such as sea urchins and sea squirts. Recent reviews on these groups are found in Rast and Messier-Solek (2008) and Khalturin et al. (2004). Beyond the long-standing concepts that innate defenses depend on the presence of barrier epithelia, phagocytosis by blood cells, and a variety of circulating proteins, our understanding of the mechanisms of invertebrate immune defenses largely depends on studies performed on the model organism Drosophila melanogaster. Flies are easily induced by septic injuries to mount an immune response and are amenable to large scale genetic analysis to decipher the gene cascades required to mount this response. The overall picture that has evolved on the Drosophila host defense is still unique among invertebrates and we will first present its general outlines. With this platform of information in mind, we will then turn our attention to other arthropod groups and to other invertebrate phyla and provide short overviews of recent advances in the studies of their immune defenses. We will extend our review to currently available data from the simplest metazoan species, the Porifera and Cnidaria, in
a PrototYPICal Innate IMMUne resPonse: the host defense of DROSOPHILA
The Drosophila host defense comprises both humoral and cellular reactions. The hallmark of the humoral response is the challenge-induced synthesis, mainly by the fat-body cells (equivalent of the mammalian liver), of potent antimicrobial peptides (AMPs) and their secretion into the hemolymph (blood), (which is the systemic immune response). AMPs are also produced by epithelial barriers where they contribute to repel the infections, (which is the local immune response). When these barriers are breached, an additional layer of defense is provided by rapidly activated proteolytic cascades leading to coagulation and melanization (i.e., synthesis and deposition of melanin) at the injury site and around the invading microorganisms. The cellular reactions essentially correspond to the phagocytosis of invading microorganisms by professional phagocytes (the so-called plasmatocytes). Encapsulation of parasites by dedicated blood cells (the lamellocytes) can also contribute to the cellular host defense of larvae. Each of these aspects of the Drosophila host defense is presented in the following sections.
the systemic Immune response
Septic injury of Drosophila larvae and adults induces the appearance of an antimicrobial activity in the hemolymph. Seven families of antimicrobial peptides have been identified to date in Drosophila. Their immune-inducibility relies on the activation of transcription factors of the NF-kB family by two signaling cascades, referred to as the Toll and IMD (immune deficiency) pathways. Interestingly, the molecular characterization of these pathways has pointed to significant similarities with NF-kB activating pathways controlling mammalian innate defenses, namely the interleukin 1 receptor/Toll-like receptor (IL-1R/TLR) and tumor necrosis factor-receptor (TNFR) pathways (reviewed in Hoffmann, 2003). The Toll pathway is predominantly activated upon fungal and gram-positive bacterial infections, whereas the IMD pathway is mainly induced by gram-negative bacterial
Laure El Chamy, Charles Hetru, and Jules Hoffman, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France.
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challenge, although there are some exceptions to this rule. The JNK (Jun N-terminal kinase) and JAK (Janus kinase)/ STAT (signal transducers and activator of transcription) pathways are also involved in some aspects of the Drosophila immune response, as described below.
Immune effectors
Antimicrobial peptides are the best characterized Drosophila immune effectors. The Drosophila genome encodes at least 25 immune-inducible AMPs that belong to seven families (reviewed in Imler & Bulet, 2005). Although structurally diverse, these peptides are mostly small in size, cationic, and predominantly membrane-active. Whereas Drosomycins and Metchnikowins target mainly fungi, Defensin is active against gram-positive bacteria, and Attacins, Cecropins, Diptericins, and Drosocin predominantly affect gram-negative bacteria. Note that the activity spectra can overlap (e.g., Cecropins have been reported to act against fungi). The combined concentrations of the AMPs can reach up to 400 μM in the hemolymph of infected flies. Some AMPs are very stable and are still detected in the hemolymph several weeks after challenge (e.g., Drosomycins) whereas others (e.g., Cecropins) are rapidly degraded by proteolysis. In addition to the genes encoding AMPs, a large set of genes is up-regulated following microbial infection (reviewed in Lemaitre & Hoffmann, 2007). Many of these can be assigned immune functions such as: regulation of AMP gene expression, phagocytosis, melanization, and production of reactive oxygen species (ROS). Other induced genes are related to iron sequestration. A large fraction of the induced genes have as of yet no known functions. Among these is the Turandot family consisting of eight proteins secreted by the fat body under stress conditions including bacterial infections, heat-shock, or exposure to ultraviolet light. Another group of induced genes encodes the DIMs (Drosophila immune-induced molecules) family, with at least 17 members of generally small sized cystein-containing peptides that do not exhibit antimicrobial activities in conventional in vitro assays.
sensing and signaling
The recent years have seen significant progress in the understanding of the mechanisms underlying microbial detection and the selective activation of the Toll and IMD signaling pathways by various classes of invaders. Our current view is that bacteria are detected through the sensing of distinct forms of peptidoglycan (PGN), whereas fungi are detected through the sensing of b-glucans. Genetic studies have revealed that these tasks are assigned to two families of germ-line encoded receptors, referred to as pattern recognition receptors PRRs: the peptidoglycan recognition proteins (PGRPs) and the glucan binding proteins (GNBPs) (reviewed in Ferrandon et al., 2007). PGRPs are innate immune proteins highly conserved from insects to mammals. These proteins are characterized by a PGRP domain that is evolutionarily related to bacteriophage type II amidases and is implicated in the immune response by either interacting with or degrading microbial PGN. The Drosophila genome encodes 13 PGRP members. Some of these PGRPs have retained the amidase activity while other members have lost crucial amino acid residues that are essential for catalysis and serve as recognition proteins (reviewed in Charroux et al., 2009). A remarkable feature of recognition PGRPs is their ability to discriminate between gram-positive and gram-negative bacteria based on single amino acid substitution in the composition of their respective PGNs and to subsequently initiate distinct signaling pathways. PGN consists of long glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues that are cross-linked to each other
by peptide bridges. The majority of gram-positive bacteria are characterized by a lysine residue at the third position of the stem peptide that is replaced by a meso-diaminopimelic acid (DAP) in the PGN of gram-negative bacteria (and the gram-positive Bacillus species) (Fig. 1). To date, four members of the PGRP family have been assigned PRR functions, namely PGRP-SA and PGRP-SD which mediate Toll pathway activation upon gram-positive bacterial infections, and PGRP-LC and PGRP-LE which trigger IMD signaling upon infections with gram-negative bacteria (reviewed in Charroux et al., 2009). GNBPs, also known as b-GRPs (b-glucan recognition proteins) are related to a group of b-glucanases that are found in bacteria, fungi, plants, and invertebrates. Two domains characterize insect GNBPs: a N-terminal glucan binding domain that binds to b-(1,3)-glucans, and a C-terminal glucanase-like domain (reviewed in Royet et al., 2005). The Drosophila genome encodes three GNBP members, of which GNBP3 was shown to mediate fungal detection upstream of Toll. Unexpectedly, GNBP1 was shown to be required for the sensing of gram-positive bacteria in complex with PGRP-SA (reviewed in Ferrandon et al., 2007).
The Toll Pathway
The Toll pathway was initially discovered for its role in the control of dorsoventral patterning in the Drosophila embryo. A genetic analysis revealed more recently that many components of this pathway are also used for the control of AMP gene expression following fungal and gram-positive bacterial infections (reviewed in Hoffmann, 2003) . Toll is the eponymous member of the conserved family of Tolls and Toll-like receptors (TLRs). Its extracellular domain is characterized by multiple leucine rich repeats (LRRs) and its intracytoplasmic domain shares significant sequence similarities with the corresponding domain of the IL-1R and is referred to as TIR (Toll/IL-1R) domain (reviewed in Leulier & Lemaitre, 2008). Toll signaling in Drosophila drives the expression of immune responsive genes through the activation of two closely related NF-kB transcription factors, DIF in adults, and DIF and/or dorsal in larvae (reviewed in Hoffmann, 2003; Lemaitre & Hoffmann, 2007). Unlike mammalian TLRs, which directly bind microbial elicitors, Drosophila Toll does not act as a prototypical PRR but is activated by an endogenous ligand, the cytokine-like spaetzle. Spaetzle is a cystine-knot polypeptide with structural and functional similarities to mammalian neutrophins (Zhu et al., 2008 and references therein). In the absence of stimuli, spaetzle is present in the hemolymph as an inactive dimeric precursor. Pattern recognition involves circulating PRRs, acting upstream of Toll, which activate a proteolytic cascade leading to the processing of spaetzle to its active Toll ligand form (Fig. 2). The data available to date indicate that recognition of gram-positive bacteria occurs in a protein complex of PGRP-SA and GNBP1; the exact role of the latter is still under investigation. Interestingly, PGRP-SD is required as a third partner in this PRR complex for sensing of some gram-positive bacterial strains. As regards fungal recognition, GNBP3 is the sole PRR that has been identified to date (reviewed in Ferrandon et al., 2007). Binding of microbial ligands to the receptors described above leads, through as yet unknown mechanisms, to the activation of a proteolytic cascade in which four zymogens, ModSP (modular serine protease), grass, spirit, and SPE (spaetzle processing enzyme), have been identified (Buchon et al., 2009 and references therein). ModSP is a modular serine protease characterized by four low-density lipoprotein-receptor class A (LDLa) domains and one complement control protein (CCP) domain at its N-terminus. Genetic analysis has revealed a role for ModSP in integrating signals from the circulating PRRs
1. Invertebrate Innate Immune defenses
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FIGURE 1 Peptidoglycans as inducers of the Drosophila Toll and IMD signaling pathways. Peptidoglycan (PGN) is a glucopeptide polymer consisting of long chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues connected to each other by short peptide bridges. The nature of the peptide bridge varies depending on the bacterial strains. Most gram-positive bacterial PGN carries a lysine residue at the third amino acid position in the peptide stems (Lys-type PGN), that is replaced by a diaminopimelic acid residue in most gram-negative bacteria (DAP-type PGN). In Drosophila, sensing of Lys-type PGN and DAP-type PGN is mediated by dedicated recognition PGRPs that activate the Toll and IMD pathways, respectively. Catalytic PGRPs have a zinc-dependant amidase activity (scissors). They reduce the immune stimulatory potency of PGN by removing the peptide bridges from the sugar backbone.
and connecting them to the downstream elements of the proteolytic cascade. Grass, spirit, and SPE are characterized by an inhibitory domain, called clip-domain that is only found in arthropods. SPE is the ultimate protease in this cascade and directly cleaves spaetzle to its Toll ligand form. In addition to the detection of microbial moieties a Toll-dependent immune response is also driven by the sensing of microbial virulence factors, notably, by secreted proteases. This reaction is centered on the clip-domain serine protease persephone, which was shown to be activated by secreted fungal subtilisins (Gottar, 2006). Persephone mediates activation of SPE and subsequently that of Toll. This reaction can be mimicked by the injection of purified bacterial proteases, hence the proposal that persephone is a sensor of danger signals (El Chamy, 2008). In agreement with this hypothesis, a recent analysis has revealed that, in the absence of any infection, tracheal melanization triggers the systemic activation of the Toll pathway in a persephone-dependent manner. The current model proposes that a host factor deriv-
ing from the melanization reaction diffuses from the tracheal system into the hemolymph where it triggers persephone activation, further driving the systemic Toll response in the fat body (Tang et al., 2008). Activation of the Toll receptor triggers an intracellular signaling cascade, which has partial similarities with the TLR and IL-1R signaling pathways (Color plate 1 a and b) (reviewed in Hoffmann, 2003; Ferrandon et al., 2007). This process involves the assembly of a multivalent complex, including death-domain-containing proteins around the intracellular TIR domain. In Drosophila, this complex comprises two adaptor molecules: a homologue of myeloid differentiation factor 88 (MyD88), which, in addition to the death domain, has a TIR domain similar to that of Toll and tube, which has a bivalent death domain. Pelle, the third partner, is a member of the IL-1R associated kinase (IRAK) family of serine-threonine kinases. The end result of Toll signaling is the dissociation of the NF-kB transcription factors from the ankyrin repeat inhibitor protein cactus, a homologue of mammalian inhibitor
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FIGURE 2 Activation of the Drosophila Toll pathway during fungal and gram-positive bacterial infections. The Toll receptor is activated by a proteolytically processed form of the cytokine-like polypeptide spaetzle (present as a dimer in the hemolymph). Microbial cell wall components (right panel) interact with circulating receptors (fungal b-glucans with GNBP3; gram-positive bacterial peptidoglycan (PGN) with a PGRP-SA/GNBP1 complex). This recognition activates, via a still unidentified mechanism, a proteolytic cascade including the zymogen modular serine protease (ModSP) and the serine protease grass, leading to the activation of the spaetzle processing enzyme (SPE), which cleaves pro-spaetzle into its active Toll-ligand form. Alternatively, microbial secreted proteases (left panel) can activate the circulating zymogen persephone, which, directly or indirectly (still unknown), activates SPE to cleave pro-spaetzle.
NF-kB (IkB). This process involves signal-dependent phosphorylation of cactus and its subsequent degradation. By analogy with mammals, it was suggested that kinases related to mitogen activated protein kinase kinase kinase (MAPKKK) and to IkB kinase (IKK) families might be involved in this process. However, such molecules have not been assigned a function in the Toll pathway in Drosophila and the identity of the cactus kinase has remained elusive so far. Liberated DIF and/or dorsal translocate to the nucleus and trigger the expression of hundreds of genes including those encoding some of the AMPs, notably the antifungal drosomycin, which is often used as a read-out of Toll pathway activation. Dorsal and DIF seem to require additional modifications for full biological activity. A protein cassette, which includes dTRAF2 (Drosophila homolog of TNFR-associated factor 2), aPKC (atypical protein
kinase C), and the scaffolding protein Ref2(P) (homologue of mammalian p62), has been suggested to participate in this process. Toll signaling shows slow kinetics, as revealed by transcriptional profiling of AMP gene expression, with maximum peaks at 24 to 48 hours following gram-positive bacterial and fungal infections, respectively. Negative regulators of this pathway have not been investigated in detail. However, following septic injury, the expression of the gene encoding cactus was shown to be up-regulated in a Toll-dependent manner, thus pointing to a negative feedback loop on Toll signaling. In addition to Toll, the Drosophila genome encodes 8 Toll receptors (18 wheeler, Toll 3 to 9). An immune function has so far only been assigned to Toll. Toll 5 and Toll 9 were also proposed to induce AMP gene expression based on
1. Invertebrate Innate Immune defenses
transfection assays in hemocyte-derived Drosophila cell lines but the in vivo significance of these results is not clear at this stage. Conversely, all Drosophila Toll members seem to be required for developmental processes as suggested by their expression patterns during embryogenesis and metamorphosis. These observations were confirmed by genetic analysis for Toll 2 and Toll 8 mutants (reviewed in Leulier & Lemaitre, 2008). This contrasts with the situation in mammals where all TLRs are devoted to immune detection (reviewed in Kawai & Akira, 2009). Further, Drosophila Tolls share sequence characteristics both in the LRR ectodomain and the intracellular TIR domain that distinguish them from mammalian TLRs. Whereas mammalian TLRs (and all identified vertebrate TLRs) contain a single cystein cluster flanking the C-terminal end of the extracellular LRR, Drosophila Tolls contain multiple cystein clusters including two or more of the characteristic C-terminal cluster in addition to a N-terminal cluster. The only exception is Toll 9, which has a single cystein cluster and is more closely related to mammalian TLRs (Fig. 3).
The IMD Pathway
The IMD pathway controls the activation of the third Drosophila NF-kB transcription factor, relish (reviewed in Lemaitre & Hoffmann, 2007). The IMD gene encodes an intracellular death-domain-containing protein with significant similarities to mammalian RIP1 (receptor interacting protein 1), which is associated with the tumor necrosis factor-receptor 1 (TNF1R). As indicated above, the IMD pathway shows striking similarities with the intracellular signaling cascade activated downstream of the TNFR in mammals (reviewed in Hoffmann, 2003; Ferrandon et al., 2007) (Color plate 2 a and b).
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The IMD pathway is activated through the direct interaction of microbial ligands with the transmembrane receptor PGRP-LC (reviewed in Ferrandon et al., 2007; Charroux et al., 2009). The PGRP-LC receptor displays three distinct isoforms, PGRP-LCa, PGRP-LCx, and PGRP-LCy, resulting from alternative splicing of its transcript. These receptors share identical intracellular domains but present different PGRP exodomains. Our current understanding is that IMD pathway activation is driven by the association of at least two PGRP molecules: PGRP-LCx homodimers mediate the recognition of polymeric DAP-type PGN and PGRP-LCa/ LCx heterodimers sense monomeric PGN. In addition, a secreted form of the PGRP-LE protein, consisting of its PGRP domain, is proposed to enhance the PGRP-LC receptor activity by binding to PGN in circulation and bringing it into close proximity of the cell surface. In the malpighian tubules, the full-length, non-secreted form of PGRP-LE can function as an intracellular receptor and trigger the IMD pathway in a PGRP-LC independent way (reviewed in Charroux et al., 2009; Ferrandon et al., 2007). Upon stimulation of PGRP-LC, the IMD protein is recruited to the intracellular domain of this receptor with which it interacts. The N-terminal domain of PGRP-LC (and that of PGRP-LE) contains a core motif named RHIM domain (for its homology to the RIP homotypic interaction motif) which is crucial for signaling, but not for PGRP-LC and IMD interaction. Thus, signaling via the RHIM domain seems to require a so far unidentified third partner of the PGRP-LC receptor and the IMD adaptor protein. A signaling complex is further established around IMD. FADD, a homologue of mammalian Fas-associated death domain, interacts with IMD via its death domain
FIGURE 3 Characteristic structural domains of Drosophila Toll and Toll9 and of mammalian TLR2.
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and with the caspase-8 DREDD via its death effector domain (DED). This complex triggers the activation of the MAP3K, TAK1 (transforming growth factor [TGF]-b activated kinase 1), which in turn activates the IKK signalosome. The latter protein complex includes kenny, a regulatory subunit homologous to IKKg/nemo and IRD5, a catalytic subunit homologous of IKKb, which phosphorylates relish on specific serine residues. The precise molecular mechanisms leading to the activation of TAK1 and of the IKK signaling complex remain unclear. However, convincing evidence points to a K63-linked polyubiquitination process involving the ubiquitin conjugating enzyme complex (including the Drosophila homologues of Uev1a and Ubc13), the RING-finger domain protein DIAP2 (Drosophila inhibitor of apoptosis protein 2) and the adaptor protein TAB2 (TAK1 binding protein 2). In contrast to dorsal and DIF, relish carries its own IkB-like inhibitory sequences in the form of several ankyrin repeats located at its C-terminal end which is cleaved, following relish phosphorylation, most probably by the caspase-8 DREDD. The N-terminal fragment bearing the Rel-homology domain translocates to the nucleus and drives the expression of hundreds of genes including diptericin. Although the IKK complex is required for relish cleavage, this process does not depend on its phosphorylation. Instead, phosphorylation is critical for the adequate transcriptional activation of relish target genes (Erturk-Hasdemir, 2009). In addition, a newly identified nuclear protein, akirin is required for the control of relish transcriptional activity. Homology search has led to unravel two akirin homologs in mammals. One of these exerts similar functions in NF-kB -dependent gene expression downstream of TNFR, TLR, and IL-1R (Goto, 2008). It is currently understood that the differential expression profiles of IMD- and Toll-immune induced genes are related to distinct sequence codes of kB-response elements in the promoter regions of the target genes responsive to the various NF-kB family members of Drosophila (for references see recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007). In contrast to Toll, IMD pathway-dependent signaling is rapid and short-lived. Maximum levels of AMP gene expression are detected around six hours following infection. Negative regulation of the IMD pathway occurs at different levels (reviewed in Aggarwal & Silverman, 2008). First, secreted catalytic members of the PGRP family, namely PGRP-SC and PGRP-LB degrade DAP-type PGN, thus reducing its immune stimulatory potency. In addition, the transmembrane PGRP-LF prevents the inappropriate activation of the IMD pathway by sequestering circulating PGN (reviewed in Charroux et al., 2009). Further, a homologue of the Fas-associating factor 1, caspar, interferes with the DREDD-dependent cleavage of relish. Finally, an intracellular protein, pirk (also designated pims and rudra), is induced in a relish-dependent manner following gram-negative bacterial infections and interferes with the activation of the IMD pathway by interacting with PGRP-LC and IMD (Aggarwal et al., 2008; Lhocine, 2008; Kleino et al., 2008).
The JNK Pathway
The JNK signaling pathway is involved in early developmental processes of the Drosophila embryo where it controls dorsal closure. It is also required for wound healing in adults. Microarray analysis of Drosophila hemocyte-derived cell lines and adult flies indicated an immune induction of the JNK pathway through the upstream components of the IMD signaling cascade (reviewed in Lemaitre & Hoffmann, 2007). Thus, similar to TNFR signaling in mammals, the IMD pathway in Drosophila bifurcates downstream of TAK1,
activating both IKK and JNK signaling. The precise role of the JNK pathway in the Drosophila host defense is not yet firmly established, as genetic analyses are hampered by the early embryonic lethality of JNK pathway null mutants. The transcriptional program triggered by this MAPK signaling cascade is enriched for genes related to cytoskeleton remodeling. These findings are consistent with the role of the JNK pathway in controlling epithelial sheet movement and tissue repair following septic injury and may also be linked to hemocyte activation (all references are included in a recent review by Lemaitre & Hoffmann, 2007). JNK signaling also promotes pro-apoptotic gene expression, an observation that correlates with early lethality of transgenic flies overexpressing IMD and developmental defects associated with the reduction of PGRP-LF expression levels (Georgel, 2001; Maillet et al., 2008). JNK signaling mediates immediate but very short-lived responses (up to 3 hours) following septic injury. Some observations point to negative feedbacks between the IMD/relish and IMD/JNK branches (Aggarwal & Silverman, 2008).
The JAK/STAT Pathway
The JAK/STAT pathway was originally identified for its role in embryonic segmentation and was later shown to control other developmental processes including larval hematopoiesis (reviewed in Agaisse & Perrimon, 2004). The four main components of this pathway in Drosophila are: the cytokine-like ligand Unpaired (Upd), the receptor Domeless (a member of the type-I cytokine receptor family), the Janus Kinase Hopscotch, and the STAT92E transcription factor. Septic injury triggers a transient activation of the JAK/STAT pathway with maximum levels reached 6 hours following infection. The induced genes encode, among others, secreted proteins such as TEP (thiol ester-containing proteins) and Turandot (Tot) family members. TEPs show significant similarities to members of the complement C3/a2macroglobulin family and could function as opsonins as was shown for Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The functional relevance of JAK/STAT pathway activation upon septic injury remains unclear, as JAK/STAT-deficient flies are resistant to bacterial infections. An interesting new finding has linked JAK/STAT pathway activation to the resistance to viral infections in Drosophila (reviewed in Kemp & Imler, 2009). Genome-wide expression profile analysis of flies infected by DCV (Drosophila C Virus) has revealed the activation of a dedicated transcriptional program, which is different from that induced following bacterial and fungal infections. Part of the induced genes, such as vir-1, were shown to be JAK/STAT dependent. In addition, Hopscotch mutant flies exhibited accelerated lethality following viral infection, which correlated with an increase in viral loads. These results suggest that the antiviral immune response in Drosophila triggers the production of a cytokine that activates the receptor Domeless and JAK/STAT-dependent induction of a set of genes to control the viral load in infected flies. In mammals, the innate antiviral defense is characterized by the production of interferons (IFN absent from the Drosophila genome), which trigger JAK/STAT pathway transcriptional activation of IFN-stimulated genes (ISGs). Thus, these observations in Drosophila point to an evolutionarily conserved role of the JAK/STAT pathway in the control of viral infections from insects to mammals. It is important to note here that, in addition to the inducible antiviral response, an intrinsic response based on RNA interference (RNAi) also plays a crucial role in the control of viral infection in Drosophila (reviewed in Kemp & Imler, 2009) (Fig. 4). This is reminiscent of the findings in plants and other invertebrates (Caenorhabditis elegans), suggesting that RNAi represents an ancestral response to
1. Invertebrate Innate Immune defenses
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FIGURE 4 The Antiviral Response in Drosophila. RNA interference and inducible gene reprograming are two major arms of the Drosophila antiviral defense. Viral nucleic acids are recognized by the RNase III enzyme dicer 2, which activates the small interfering RNA (siRNA) pathway leading to the degradation of viral RNA. In addition, dicer 2 can trigger the inducible expression of a variety of genes via a yet unidentified signaling pathway. Among the induced genes is that encoding the vago cystein-rich polypeptide, which is involved in the control of the viral load. Further, a cytokine-mediated response activated upon viral infection leads to the expression of genes via the activation of the JAK/STAT pathway in neighboring cells. The mechanism controlling this antiviral-inducible response is so far unknown.
viral infection. Viral double-stranded RNA is recognized by the RNAse III enzyme dicer 2, which drives the degradation of viral RNA through the small interfering RNA (siRNA) pathway. Intriguingly, dicer 2 (but no other mediators of the siRNA pathway) is also required for the expression of some DCV-inducible genes, such as vago, in the infected cells. Dicer 2 contains an amino-terminal DExD/H box helicase domain, which is important for the inducible vago expression. This domain is similar and is phylogenetically related to the helicase domain of mammalian RIG-I-like receptors, which mediate the sensing of viral nucleic acids in the cytosol and the induction of interferon gene expression (reviewed in Kawai & Akira, 2009). Hence, although Drosophila lacks interferon homologues and the mechanisms of antiviral responses are different between Drosophila and mammals, many similarities can be drawn on sensing (requirement of intracellular DExD/H box helicases) and signaling (cytokine production and JAK/STAT signaling) during viral infection in these evolutionarily distant organisms.
epithelial defense reactions
Epithelial barriers constitute critical interfaces between the host and microorganisms of the environment. Antimicrobial defense reactions deployed at the level of these epithelial layers provide a first tier of defense counteracting microbial invaders. In Drosophila, various spectra of AMPs have been reported in the epidermis, the reproductive system, the respiratory, and the digestive tracts. Whereas some AMPs are constitutively expressed (such as Drosomycin in the salivary glands and Cecropin in the ejaculatory duct), others are induced upon infection, and by contrast to the
systemic response, their regulation is solely dependent on the IMD pathway (including that of Drosomycin in the trachea and of Diptericin in the gut). The development of appropriate food-borne infection models in Drosophila has now uncovered the central role of locally synthesized AMPs in the gut for the survival of pathogenic infections such as Serratia marcescens or Pseudomonas entomophila. Recognition of ingested DAP-type PGN is mediated by the PGRP-LC receptor, triggering the local activation of the IMD pathway, and immune reactivity is attenuated by secreted catalytic PGRPs in the gut lumen, which degrade bacterial PGN into nonstimulatory molecules. It is proposed that this modulation of the IMD pathway protects the gut flora from excessive AMP production. Furthermore, in the absence of gut infection, a selective repression of NF-kB-dependent AMPs is provided by the homeobox gene caudal (Ryu, 2008). In addition to AMPs, ROS are rapidly produced following microbial ingestion and critically contribute to the bactericidal reactions in the gut. ROS production is mediated by dual oxidase (DUOX), a member of the conserved family of NADPH oxidases. DUOX enzymatic activity is Ca2+-dependent and is regulated by the Gaq-Phospholipase C-b (PLC-b) pathway mediating the mobilization of intracellular Ca2+. The identity of the receptor acting upstream of this pathway and the mechanism triggering this oxidative reaction remain unsolved (Ha, 2009). Excessive ROS produced following microbial infection are eliminated by a secreted antioxidant enzyme, the immune regulated catalase (IRC), thereby protecting the host from the deleterious effects of the oxidative burst. (All references for epithelial defense reactions in Drosophila are
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found in recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007.)
Melanization
Melanization is a particular feature of invertebrate immune reactions that is immediately activated at the site of cuticular wounding and around invading microorganisms. It relies on the activation of phenoloxidase (PO), a key enzyme that catalyzes the conversion of phenols to quinones, which polymerize nonenzymatically to form melanin. Melanization is generally assumed to contribute to the arthropod defense by facilitating wound healing, sequestrating microorganisms and preventing or retarding their growth, and by the production of toxic intermediates that can kill microbial intruders (for reference see Cerenius & Soderhall, 2004).
Cellular responses Drosophila Blood Cell lineages and functions
Compared to the complexity of blood cell lineages in mammals, the picture is relatively simple in Drosophila as there are no equivalents to lymphoid cells and as hemocyte lineages are restricted to three cell types: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes represent the predominant population of embryonic and larval hemocytes (95%) and are the unique blood cells found in adults. They are professional phagocytes, which are in charge of the uptake and clearance of both apoptotic cells and microorganisms. Plasmatocytes also contribute to the humoral response by secreting AMPs and, as previously mentioned, they are required for the activation of the systemic response of adults exposed to stress conditions (i.e., JAK/STAT pathway activation). Crystal cells and lamellocytes have no mammalian counterparts and are both involved in defense reactions that are restricted to invertebrates (i.e., melanization for crystal cells and encapsulation for lamellocytes) (reviewed in Meister, 2004).
Phagocytosis
Several studies have revealed the importance of the cellular arm of defense in Drosophila. To give but one example: mutant larvae devoid of hemocytes are filled with microorganisms and cannot reach the adult stage unless reared on germ-free media. Further, adult flies deprived of hemocytes by targeted cell death of hemocyte lineages, are highly susceptible to some bacterial and fungal infections, and this lethality correlates with higher bacterial loads (Charroux & Royet, 2009; Defaye et al., 2009). Phagocytosis is a central theme in innate immunity and is conserved in all metazoans. High-throughput RNAi screens in the embryonic hemocyte-derived S2 cells have deciphered the phagocytic machinery and have identified multiple components of the basic mechanisms of phagocytosis, namely regulators of vesicle transport and actin cytoskeleton remodeling, which are preserved in other animal species. A recent proteomic analysis identified approximately 600 proteins potentially associated with the Drosophila S2 cell phagosome. Of the identified proteins, many are evolutionarily conserved and 70% have mammalian orthologues. By combining several approaches, this study generated a detailed model of the phagosome, which is also valid in mammalian cells. Finally, studies performed in S2 cells and other in vivo and ex vivo analysis using different microorganisms identified diverse phagocytic receptors, some of which have mammalian homologues required for the phagocytic process. (All references are found in a recent review on phagocytosis by Stuart & Ezekowitz, 2008.) These receptors belong to three families
of evolutionarily conserved proteins: scavenger receptors, EGF-like repeat containing receptors, and immunoglobulin repeat containing proteins. Other molecules, including complement-like opsonins are proposed to promote the phagocytic uptake of microorganisms.
Scavenger Receptors
Scavenger receptors represent a large family of structurally heterogeneous receptors, which bind to and internalize a broad array of endogenous molecules and microbial ligands with polyanionic character. Three members of the Drosophila scavenger receptor family were shown to be involved in phagocytosis: croquemort and peste are homologous to mammalian scavenger receptors, and dSR-CI belongs to an insect-specific family of scavenger receptors. Croquemort is required for the phagocytosis of apoptotic cells (in the Drosophila embryo) and is also involved in the phagocytosis of bacteria. In cell culture experiments, peste was shown to be required for the entry of intracellular Mycobacteria and Listeria. Finally, dSR-CI contributes to the phagocytic uptake of both gram-positive and gram-negative bacteria in Drosophila S2 cells.
Receptors Containing Multiple EGF-like Repeats
Receptors containing multiple EGF-like repeats are found in diverse species, including humans, and are emerging as a new family of phagocytic receptors. The best characterized member in Drosophila is eater, a type I transmembrane receptor with 32 typical EGF-like repeats in its extracellular domain. This receptor has a broad reactivity, like scavenger receptors, and directly binds to microbial surfaces. Eater seems to be the major phagocytic receptor in Drosophila, as silencing of its transcript dramatically decreases the phagocytic index of Drosophila S2 cells. Furthermore, eater mutant flies are highly susceptible to infections with both gram-positive and gram-negative bacteria. Another member of this family of EGF-like repeat receptors, nimrod C1, is involved in the uptake of Staphylococcus aureus and may participate to the phagocytosis of Escherichia coli. Finally, draper contains 16 EGF-like repeats and is required for the phagocytosis of apoptotic cells. Interestingly, this receptor contains an ITAM (immunoreceptor tyrosine-based activation motif) in its intracytoplasmic domain.
Immunoglobulin-like Repeat Proteins
Another member of Drosophila phagocytic receptors, Dscam (Down syndrome cell adhesion molecule), belongs to the immunoglobulin superfamily. Dscam is a single pass transmembrane receptor characterized by 10 immunoglobulinlike domains and 6 fibronectin type III domains in its extracellular part. This receptor was identified for its essential functions in neuron wiring and is also expressed in hemocytes. The dscam gene comprises a cluster of variable exons flanked by constant exons which can theoretically generate, by alternative splicing, a large number of distinct protein products. Silencing of the dscam transcript in hemocytes decreases the phagocytic uptake of E. coli by these cells. In addition, some recombinant isoforms of Dscam directly bind to E. coli. Secreted isoforms of Dscam are detected in Drosophila hemolymph and a role of these isoforms in opsonization of pathogens has also been proposed.
Complement-like Opsonins
Other molecules proposed to mediate phagocytosis in Drosophila comprise the secreted family of TEPs. Based on their homology to the complement C3/a2-macroglobulin superfamily, these molecules were anticipated to function either as opsonins to promote phagocytosis and/or as
1. Invertebrate Innate Immune defenses
protease inhibitors. Some data so far support the opsonin function, namely from the studies performed on Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The Drosophila genome encodes four expressed TEPs (TEP I to IV) and a closely related gene, Mcr (macroglobulin complement related), which encodes a protein lacking the characteristic thiol ester motif of TEPs. Based on RNAi-mediated gene silencing in Drosophila S2 cells, TEP II and TEP III are proposed to mediate phagocytosis of E. coli and S. aureus respectively, whereas Mcr is proposed to mediate binding and internalization of the yeast Candida albicans. Confirmation of TEP functions during phagocytosis awaits further in vivo studies.
CoMParatIVe analYsIs of the Innate IMMUne sYsteM Insects other than drosophila
Most of the data obtained from the analysis of gene families related to the immune system in other insect species are compatible with the picture that we have described for Drosophila melanogaster. This is namely the case for the two dipteran species, Anopheles gambiae and Aedes aegypti, the hymenopteran Apis mellifera, the lepidopteran, Bombyx mori, and the coleopteran, Tribolium castaneum (Christophides et al., 2004; Waterhouse, 2007; Evans et al., 2006; Tanaka, 2008; Zou et al., 2007). Phylogenetic analysis of the insect immune repertoires (including those of twelve additional Drosophila species [Sackton et al., 2007]) has revealed dynamic changes of genes related to recognition and effector functions, probably as a result of the selective pressure imposed by distinct pathogens encountered by each insect species. These include PRRs (PGRPs and GNBPs) as well as phagocytic receptors (i.e., scavenger receptors) and opsonins (TEPs). With regard to the effector molecules, novelties are noted among insect orders mainly with the emergence of lineage-specific AMPs such as Drosomycin in Drosophila, Gambicin in mosquitoes, and Apisimin and others in bees (Christophides, 2002; Evans et al., 2006; Sackton et al., 2007). In contrast, intracellular signaling mediators are highly conserved in all studied insects. Notably, a nearly complete tool kit of both Toll and IMD pathways has been identified in all analyzed genomes. Some experimental data support the functional conservation of these pathways for immune defenses in the respective insect species. Mosquitoes lack the NF-kB transcription factor dif and rely on a dorsal orthologue, REL1, and on a relish orthologue, REL2. These mosquito REL factors have been reported to control the induced expression of AMPs and the resistance to bacterial infections (Shin et al., 2005; Bian, 2005; Antonova et al., 2009; Meister, 2005). In Anopheles gambiae, REL1 and REL2 are also involved in the resistance to the rodent malaria parasite Plasmodium berghei by controlling the basal expression of the major antiparasitic factors (Blandin et al., 2008). Of the five Toll receptors and the three spaetzle ligands encoded in the genome of the yellow fever mosquito, Aedes aegypti, a role for Toll5A and its putative ligand spaetzle1C is documented in the defense against the entomopathogenic fungus Beauveria bassiana. This receptor ligand complex triggers the activation of the REL1 transcription factor regulating the antifungal immune response (Shin et al., 2006; Shin et al., 2005; Bian, 2005). The Toll pathway, together with the JAK/STAT pathway, has also been linked to the transcriptional program triggered by Dengue virus infection in Aedes aegypti and a contribution of the Toll pathway in the defense against Dengue virus in this species has also been proposed (Xi et al., 2008).
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In the silkworm Bombyx mori and the beetle Tenebrio molitor, the expression of some AMPs is up-regulated following the injection of a maturated form of spaetzle orthologues (Wang, 2007; Roh et al., 2009). Due to the lack of appropriate genetic tools, functional data for immune-related genes are restrained in the lepidopteran and coleopteran species, as compared to Drosophila. However, these large insects have provided valuable biochemical tools for the investigation of pathogen recognition processes and the characterization of the proteolytic cascades subsequently activated downstream of PRRs (i.e., melanization and the processing of spaetzle orthologues) (reviewed in Iwanaga & Lee, 2005; Roh et al., 2009). Recently, the components of the extracellular pathway leading to the processing of spaetzle were purified from the hemolymph of the beetle Tenebrio molitor. Biochemical assays have allowed the in vitro reconstruction of the proteolytic cascade activated downstream of TmGNBP3, TmPGRP-SA, and TmGNBP1 (Roh et al., 2009). This work led to the identification of the apical protease, DmModSP, acting downstream of PRRs for the activation of the Toll pathway in Drosophila (Buchon et al., 2009).
horseshoe Crab
The horseshoe crab is often referred to as a “living fossil” and first appeared more than 500 million years ago. Its host defense largely relies on granular hemocytes, which constitute 99% of the circulating blood cells. These hemocytes contain secretory granules that store the major elements of the horseshoe crab’s defense arsenal. They include AMPs, serine protease zymogens, the clotting protein coagulogen and several lectins (reviewed in Iwanaga & Lee, 2005). The innate immune system of this chelicerate arthropod recognizes invading pathogens through a combinatorial system involving several lectins with distinct affinities for carbohydrates exposed on the surface of pathogens. Another recognition molecule playing a key role at the frontline of the horseshoe crab antimicrobial defense is the serine protease zymogen Factor C. This factor is mainly found in the hemocyte granules, but is also detected at the hemocyte surface. Factor C is highly sensitive to gram-negative bacterial lipopolysaccharide (LPS) and is autocatalytically activated upon stimulation by this microbial elicitor. This reaction leads to the secretion of the defense molecules from granular hemocytes by exocytosis (reviewed in Kurata et al., 2006). Of the released molecules, Factor C and another serine protease zymogen, factor G, which is responsive to b-(1-3)-glucan, trigger a two-step proteolytic cascade leading to hemolymph coagulation. This reaction consists in the conversion of coagulogen into an insoluble protein, coagulin. Hemolymph clotting is believed to contribute to the animal’s defense by preventing blood leakage and by trapping the pathogen. The immobilized microorganisms can subsequently be recognized by the large number of plasma and hemocyte-delivered lectins and killed by AMPs. Phagocytosis also contributes to the horseshoe crab defense reactions. Homologs of vertebrate complement C3 and C2/ Bf were reported from the horseshoe crab Carcinoscorpius rotundicauda and complement mediated phagocytosis of bacteria by hemocytes was observed (Zhu et al., 2005). These findings imply an early origin of core elements of the complement system. Further evidence for an archaic role of NF-kB signaling cascades in the antimicrobial defense is also provided from the study of this living fossil. Two NF-kB transcription factors, CrNF-kB and CrRelish, and an IkB homologue, CrIkB, have been cloned in C. rotundicauda. CrNF-kB belongs to the dorsal-like group of transcription factors and interacts with CrIkB, which inhibits its nuclear translocation. Bacterial
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infection causes rapid degradation of CrIkB and subsequent release of CrNF-kB, which activates the expression of immune-related genes such as Factor C (Wang, 2006). Experimental data support the conservation of the CrRelish function and point to its up-regulation following bacterial infection, suggesting an involvement of CrRelish in antibacterial defense (Fan, 2008). A TRAF homolog has also been reported from C. rotundicauda (Wang, 2006). How these signaling cascades are activated is not yet established. A Toll receptor has been identified in the Japanese horseshoe crab Tachypleus tridentatus, called tToll, and is closely related to Drosophila Toll. Its biological function has not been investigated so far and its ligand has not been defined. The expression pattern of tToll does not suggest an immune function (reviewed in Inamori et al., 2004).
Mollusks and annelids
Investigations of the immune reactions of several Annelid and Mollusk species have revealed a potent cellular arm of defense, involving phagocytosis, encapsulation, and production of lytic activities often referred to as natural killer-like (NK) activities. These cellular defenses are combined with humoral reactions involving lysozymes and AMPs (reviewed in Salzet et al., 2006; Cheng-Hua et al., 2009). These reactions are mostly inducible. At present, the mechanisms controlling these responses are still poorly defined. In Mollusks, recognition of nonself relies in particular on lectins. Of special interest is the family of fibrinogenrelated proteins (FREPs) identified from the gastropod Biomphalaria glabrata, an intermediate host of the human parasite Schistosoma mansoni. FREPs were first described as plasma proteins that are induced following trematode infection. They bind to trematode sporocysts and precipitate glucan-bearing molecules released by the parasite. FREPs consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyterminal fibrinogen domain. Interestingly, for one subfamily, FREP3, it was observed that the IgSF1 is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants were found to derive from a small set of source sequences by point mutation and recombinatorial processes. Importantly, diverse FREP3 transcripts were also produced. Loker and colleagues, who discovered this phenomenon, have hypothesized that snails have evolved a mechanism capable of diversifying molecules involved in recognition during innate defenses (Zhang et al., 2004). Further, members of the PGRP and GNBP families have also been identified in snails and are proposed to contribute to the microbial detection in these species (reviewed in Bayne, 2009). In the context of immune signaling in Mollusks, genome-based approaches identified several components of NF-kB signaling pathways in various species. In the squid Euprymna scolop, several ESTs (expressed sequence tags) encoding homologues of proteins associated with putative Toll and IMD pathways were identified. These include a Toll receptor; four PGRPs; orthologues of several intracellular signaling mediators including IRAK, TRAF, and the IKK complex; an IkB-like protein; and two NF-kB-like transcription factors (Goodson, 2005). Evidence for the presence of NF-kB signaling pathways have also been reported from other Mollusk species, including an NF-kB transcription factor from the marine gastropod Haliotis diversicolor supertexta, a Toll receptor and a MyD88 homologue in the marine bivalve Chlamys farreri, and orthologues of NF-kB and IKKb from the pacific oyster Crassostrea gigas (Jiang & Wu, 2007; Qiu et al., 2007; Escoubas, 1999; Qiu, 2007; Montagnani et al., 2004).
In Annelids, the molecular mechanisms underlying the antimicrobial defenses are, as yet, poorly understood. The analysis of genome sequence reads of the leech Helobdella robusta and the polychaete Capitella capitata have identified homologues of Toll receptors in both species (Davidson et al., 2008). Surprisingly, the family of TLRs is largely expanded in Capitella with at least 105 members inferred from this analysis. The majority of these receptors are structurally related to vertebrate TLRs (i.e., having a membrane proximal single cystein cluster in their LRR ectodomain). In contrast, of the 16 Toll homologues identified in Helobdella, most show multiple cystein-rich clusters in their LRR ectodomain, as is the case for most insect Toll receptors. Mediators of the NF-kB signaling cascades have also been identified in both species, notably, two dorsal-like transcription factors and most mediators of the Toll signaling (but not yet of the IMD) pathway.
orIgIn of the Innate IMMUne sYsteM: the Innate IMMUne rePertoIre of CnIdarIa and PorIfera
The phylum Cnidaria includes aquatic species such as sea anemones, corals, and jellyfish. These animals provide crucial insights into the early origin of metazoans. The genome sequence is now available for two Cnidarian species: the sea anemone Nematostella vectensis and the fresh water polyp Hydra magnipapillata. Amazingly, analysis of these databases uncovered a complete Toll/TLR pathway in the genome of N. vectensis, indicating that this pathway predates the divergence between Cnidarians and Bilaterians some 600 million years ago (reviewed in Hemmrich et al., 2007). The Nematostella Toll receptor is characterized by multiple cystein clusters in its LRR extracellular domain, suggesting that this form likely represents the ancestral structural organization of Toll receptors. A single NF-kB transcription factor and its IkB inhibitory protein have been identified in the genome of N. vectensis together with many intracellular signaling mediators of the NF-kB signaling pathways, including MyD88, IRAK/Pelle, TRAF, TAK1, and elements of a IKK signalosome. In contrast, and quite intriguingly, the Hydra genome apparently lacks a conventional NF-kB transcription factor and a canonical Toll receptor, although it features two genes encoding transmembrane TIR-domain containing proteins with a short ectodomain deprived of LRR. Hydra mounts an inducible expression of AMPs upon infection and it has been suggested that the TIR-domain transmembrane molecules might function in an atypical signaling complex with separate LRR-containing proteins to control these Cnidarian immune defenses (Bosch, 2009). Other potential immune molecules that have been identified in the genome of the sea anemone are absent from Hydra. These include homologs of C3, Bf, and MASP elements involved in the complement cascades. These observations suggest that the multicomponent complement system was already established in the genome of the common ancestor of Cnidaria and Bilateria (Kimura et al., 2009). The absence of these elements from Hydra likely reflects secondary gene losses during evolution. Evidence for MAPK signaling pathways (including JNK, p38, and AP1) has also been obtained from the analysis of the genomes of both Cnidarian species. Sponges, which belong to the phylum Porifera, represent an even earlier branching lineage than Cnidaria, occupying a basal position near the root of the metazoan tree of life. Investigations of cDNA libraries from the sponge Suberites domuncula and a thorough analysis of the genome of Amphimedon queenslandica have revealed the presence of an impressive armamentarium of genes encoding potential
1. Invertebrate Innate Immune defenses
recognition proteins, including scavenger receptors and several members of NF-kB signaling pathways (reviewed in Hemmrich et al., 2007). Of note, in contrast to the sea anemone NF-kB, the sponge NF-kB is structurally related to Drosophila relish, in that it carries its own inhibitory sequences. The expression pattern of AmqNF-kB during embryonic and larval stages suggests a developmental role, although these observations do not exclude a potential defense function of AmqNF-kB (Gauthier & Degnan, 2008).
ConClUsIons
As illustrated in this presentation, we now have at our disposal a wealth of information on innate defenses in invertebrates ranging from relatively simple organisms to highly evolved animals. Obviously, for many species, we still lack crucial functional data and the cellular and molecular mechanisms underlying the immune reactions still await further analysis in several large invertebrate phyla. In all frankness, we must admit that even our view of the much studied Drosophila immunity is still fragmentary and that it requires intense further investigations. With these caveats in mind, the essential message that we want to convey here is that innate immunity, as we understand it today, has appeared very early in life’s history, probably with the first multicellular organisms. There is clearly a common thread through all invertebrate phyla regarding recognition of microorganisms, activation of intracytoplasmic signaling cascades, and induction of gene programs that culminate in the synthesis of potent defense molecules, among which the antimicrobial peptides feature prominently. Recognition of microorganisms by the invertebrate immune system is a field that has significantly advanced in recent years. A variety of phagocytic receptors have been described, most of which have mammalian counterparts. They interact with an intracellular machinery of vesicular trafficking and cytoskeleton rearrangements, ultimately leading to large phagosomal structures where microorganisms can be disposed of. Again, from our recent information, it appears that most of the players in a few well-studied cases are highly similar to the molecules that govern these processes in mammals. This hardly comes as a surprise as it is largely surmised that phagocytosis evolved very early in evolution for purposes of nutrition, development, and defenses. Intriguingly, some putative phagocytic receptors, such as Dscam in Drosophila and the circulating opsonin-like FREPs in Mollusks, which feature IgSF domains, are encoded by genes that have the potential to generate multiple receptor isoforms. It will be interesting to follow future studies on the functional relevance of these arrangements. Parallels with the now well-established variable lymphocytes receptor (VLR) system of jawless fish (Agnathans) recently described by Cooper and colleagues (Pancer, 2004) appear premature at this stage. Every subfield has its own potential pitfalls. This is also the case in the Toll/TLR context. In the best documented cases, that is Drosophila and mammals, a very important difference is that the Toll receptor becomes activated by a cleaved form of the cytokine spaetzle, whereas mammalian TLRs interact directly with microbial ligands. The expression “pattern recognition receptors,” coined by Janeway (1989) thus perfectly applies to mammalian TLRs but not to that of the Drosophila Toll. This remark underlines that, pending functional studies, we are uncertain how to interpret results on dozens or more Toll/TLRs in various invertebrates. Therefore, it is certainly premature to propose that the increase in number may reflect environmental pressures by microorganisms.
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One of the beauties of innate immunity, as illustrated by the case of Drosophila, is that the number of microbial ligands that actually activate gene reprogramming is extremely limited: Firm evidence exists to date only for PGNs and b-glucans. More recently, viral RNAs have been added to the list. The receptors, as we see them today, have essentially derived from ancestral enzymes, namely amidases, glucanases, and helicases. In Drosophila at least, the number of documented receptors is less than a dozen proteins to date. Would a larger repertoire be beneficial for this organism? We believe that a larger repertoire of receptors has to be viewed in the context of lymphocyte development, clonal selection, and expansion and memory. In the absence of these features, increasing the repertoire of innate immune receptors might be of little improvement for the defense, but we are ready to reevaluate our view if clear and reproducible data will instruct us otherwise in the future. In the early days of our research on the signaling cascades that led to the expression of immune genes in Drosophila, we were extremely interested by the parallels between these cascades and those of mammalian immune defenses. This is now commonplace, but the data reviewed here strongly suggest that these cascades were in place already at the very earliest stages of evolution. Again, we lack functional data for most of the species in which NF-kB is present, but we anticipate that future studies will show that from sponges to insects, and further to mammals, similar molecular modules direct essentially similar cascades to transcribe gene programs that ultimately are beneficial to the host and deleterious to the invader. Further, as illustrated above, many of the effector molecules appear to be similar in the various phyla and are present in vertebrates as well. An interesting question pertains to the parallel evolutions of developmental regulations and immune defenses. How do we explain that Drosophila has nine Toll receptors and six spaetzle paralogs, three of which have been documented to act as neurotrophins (Zhu et al., 2008), all of which are involved in development and that just one pair, Toll-1 spaetzle, were selected to direct antifungal and some antibacterial defenses? We hope that functional studies in other phyla will help clarify our view on these dual roles in immune and developmental regulation during evolution. Finally, a word of apology to our friends in the growing field of Caenorhabditis elegans defense reactions. C. elegans lacks so many of the features described in this review (namely NF-kB) that we have decided to put this organism apart and refer to excellent recent reviews covering this special field (Schulenburg et al., 2004; Kim & Ausubel, 2005; Shivers et al., 2008). The common view in the community today is that C. elegans has lost most of the conventional traits of the innate immune system of invertebrates as they were described here. In fact, a similar situation might also prevail for the Cnidarian Hydra. Although innate immunity was discovered in invertebrates by Metchnikov nearly 130 years ago, it has long remained a relatively neglected area of research. In the 1990s, molecular genetics have given a potent stimulus to the field, initially in Drosophila and now in many phylogenetically diverse groups. The progress has been astounding over the last 15 years and hundreds of laboratories worldwide are now engaged in research on innate immunity. Moreover, in some precise areas of high significance (e.g., the discovery of antimicrobial peptides, the role of Toll receptors in innate immunity, and the role of peptidoglycan recognition proteins) invertebrate immunity has led the way. We hope that this thread will continue and that this review article has stimulated some interest among potential future students of the field.
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The authors apologize for their inability to quote many additional contributions due to space limitations. Work in the laboratory of the authors has been generously supported by the French National Research Agency (CNRS), the National Institute of Health (NIH), the European Union, and the University of Strasbourg. We thank our past and present associates in the laboratory for their contributions and for continuous helpful discussions.
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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
2 The Ontogeny of the Cells of the Innate and the Adaptive Immune System FRITZ MELCHERS
INTRODUCTION
Recognition of specific antigen by binding at sufficiently high avidity induces clonal lymphocyte proliferation and maturation to effector functions—in the case of T cells to helper, cytotoxic, or regulatory cells; in the case of B cells to antigen receptor-hypermutated, Ig class-switched cells and Ig-secreting plasma cells, which generate and maintain the Ig levels in the extracellular spaces, notably the blood, or, after transport through epithelial layers, in the lumen of the gut.
A single pluripotent hematopoietic stem cell (pHSC) can be the progenitor of all the erythrocytes, megakaryocytes, and blood platelets, of all the cells of the myeloid cell lineages, of all the natural killer (NK) cells, of all the different functional types of T lymphocytes, and of all the sublineages of B lymphocytes. Hence, the ontogeny of these different types of blood cells, the cells of the innate and the adaptive immune system, is very complex. Some of the major molecular steps and cellular stages, including a few of the interactions of the developing hematopoietic cells with the endogenous environment of cooperating cells, as well as with the exogenous environment of antigens and polyclonal activators will be described, mainly with the mouse but also with references to the development in humans. Development begins, shortly after the fertilization of the egg, with the development of embryonic stem (ES) cells; from there, the progenitors of the vascular and hematopoietic cells (called hemangioblasts) are developed; and thereafter is the generation of a pluripotent hematopoietic stem cell. From that point, it continues first in embryonic development and, thereafter, throughout life through the different stages of hematopoietic, myeloid, and lymphoid progenitors, and finally to mature cells of the myeloid cells and of NK cells (i.e., of the innate immune system) and to the development of antigen receptor-expressing lymphocytes of the adaptive immune system and their deposition and use in different parts of the immune system.
The Clonal Selection Hypothesis
The clonal selection hypothesis (Jerne, 1955; Burnet, 1957; Talmage, 1959; Nossal & Lederberg, 1958) proposes that one lymphocyte makes only one structure of an antigenbinding receptor, that this one structure on the surface of one lymphocyte binds one antigen of the wide world of structures (antigens) surrounding these lymphocytes, and that many different antigens bind to many different antigen receptors, but always one antigen to one of the wide world of lymphocytes in the system. To deal with the tolerance of the immune system to its own antigens, Lederberg (1959) extended this hypothesis by proposing that the emerging repertoires of antigen receptors on lymphocytes would first enter a phase—today recognized as immature lymphocytes—in which binding of the body’s own antigens, the autoantigens, would select their specific cells and eliminate them. The surviving lymphocytes would then further differentiate with the next stage of cellular development—now known as mature lymphocytes—in which binding of a foreign antigen would initiate a positive immune response (i.e., the proliferation and maturation of cells to effector functions that the system can use to eliminate the invading antigen). One prediction of this hypothesis was that the second allele of an antigen receptor-encoding gene would not be activated for expression in a single lymphocyte, termed allelic exclusion of the receptor loci. Another expectation was that a single lymphocyte would only rarely find a second, structurally nonidentical antigen to which it could bind to elicit a response. Furthermore, autoantigens would only negatively select and foreign antigens would only positively select cells of the system for a response.
The Adaptive Immune System of Lymphocytes
The major hallmark and specific feature of T and B lymphocytes is their capacity to produce a wide variety of antigenbinding receptors, in T cells, the a/b- or g/dT-cell receptors (TcR), and in B cells, immunoglobulin (Ig) heavy/light (H/L) chain-containing B-cell receptors (BcR), which they deposit for antigen recognition on their surface.
Fritz Melchers, Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany.
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Our current knowledge of the generation and selection of lymphocyte receptor repertoires shows that the clonal selection hypothesis, in its initial definition, is often no longer tenable. Nevertheless, the adaptive immune system manages to engage only a small part of its final, available repertoire of antigen receptors on lymphocytes for the recognition of, and response to, an antigen.
DEVELOPMENT OF HEMATOPOIETIC CELLS AND LYMPHOCYTES Embryonic and Adult Development In Vivo
Three waves of hematopoietic cell developments colonize the mouse embryo (Ling & Dzierzak, 2002; Godin & Cumano, 2002; Cumano et al., 2001). The first begins in extra embryonic tissue (i.e., in the yolk sac at day 7 to 7.5 of development). This so-called primitive hematopoiesis (which shows similarities to hematopoiesis in lower vertebrates) generates primitive erythrocytes (synthesizing “fetal” hemoglobin), megakaryocytes (with lower ploidy) and platelets, and myeloid cells (such as macrophages with a special set of enzymes) (Shivdasani et al., 1995). Lymphocytes are not generated. Although these primitive blood cell lineages are developed at extraembryonic, ventral sides of the embryo, they migrate to the embryonic, dorsal side as soon as blood circulation is established at day 8 to 9 of development with the development of vascular endothelium. At day 8.5 to 9 of mouse embryonic development, the second wave of hematopoiesis is started within the intraembryonic, anterior portion of the aorta-gonad-mesonephros region (Medvinsky & Dzierzak, 1996; Cumano et al., 1996). Undifferentiated, apparently pluripotent stem cells (Ohmura et al., 2001) then migrate through the blood and colonize the rudiments of the thymus and fetal liver. At these sites, the second, so-called definitive wave of hematopoiesis is initiated. It now generates erythrocytes producing adulttype hemoglobin, definitive megakaryocytes and platelets, and adult-type myeloid cells and lymphocytes. In the thymus, several waves of thymopoiesis are initiated. The T cells that are generated in these first, fetal waves of development from day 16 of mouse embryonic development onwards are first g/d and, thereafter, a/b TcRexpressing cells, all with little or no N-region insertions at their V(D)J junctions because of the lack of expression of the enzyme TdT, and they display a restricted repertoire of v-region combinations. They migrate to intraepithelial sites of the body where they might perform first line of defense functions. In the fetal liver of the mouse, between day 13 of embryonic development until birth at day 19, a first, apparently synchronous wave of B-lymphocytes is generated (Strasser et al., 1989). Two other sites of embryonic B-cell development, generating B cells with properties similar to those of the fetal liver, are the embryonic side of the placenta and the omentum (Kincade, 1981; Owen et al., 1976; Melchers, 1979; Gekas et al., 2005, Ottersbach & Dzierzak, 2005; Solvason & Kearney, 1992). Since most of these B cells do not express the enzyme TdT, they rearrange the V(D) J segment of the IgH and IgL chain loci without N-region diversity. The early waves of embryonic B cells preferentially home to the gut-associated lymphoid tissues, belong, in large part, to the BIa-types of B cells, and form a first line of defense and cohabitation against and with the developing intestinal bacterial flora. The third wave of hematopoiesis (i.e., the second definitive hematopoiesis) starts in bone marrow between days 17
and 19 (i.e., at around the birth of a mouse) (Kincade et al., 2002). It initiates a continuous process of hematopoietic cell generation throughout life. B cells develop to form at least three distinct lineages: B1a, B1b, and B2 cells (Melchers, 2009; Tung & Herzenberg, 2007). They are then distributed into at least two major subcompartments of mature, BcR-expressing B-lymphocytes of the mature, peripheral system. Approximately half of them are found in the follicular regions of the spleen and lymph nodes and in the recirculating blood and lymph, a majority of which are the B2-type B cells. The other half is mainly found in the gut-associated lymphoid tissues (e.g., in Peyer’s patches, either in follicular structures near the flat M-cell regions of the gut epithelia, or as single intraepithelial lymphocytes in the lamina propria of the gut). At least a majority of these B cells appear to belong to these so-called BI subpopulations. Most B1a cells develop during the prenatal period in the fetal liver from precursors that lack lg rearrangements and are distinct from those that give rise to B1b and B2 cells (Tung & Herzenberg, 2007). B1a cells persist for some time after birth even though they are derived from precursors that disappear during the neonatal period. B1a cells contribute a large part to the natural lgM antibodies in serum, respond to T cell-independent antigens, express unmutated V regions, contain very few (if any) N-region sequences in their complementarity determining region 3 (CDR3) sequences, and need the spleen to develop (Wardemann et al., 2002). T cells develop in the thymus. Progenitors from bone marrow with myeloid and lymphoid, or only lymphoid potentials migrate to the thymus to continuously generate, until sexual maturity, the different subpopulations of T cells. After sexual maturity, the thymus begins to involute and decreases T-cell production progressively. Even after adult thymectomy the mature T-cell compartments are maintained in size and function, indicating that T-cell maintenance in the system becomes extrathymic. The early stages of thymocytes are CD4 through CD8 and are either CD441CD252 (DN1), CD441CD251 (DN2), CD442CD251 (DN3) or CD442CD252 (DN4) cells in that developmental sequence. At the transition of DN2 to DN3 cells the expression of TcRs decides whether they develop either to a/bTcR- or to g/dTcR-expressing cells. The a/bTcR1 cells become CD41CD81 (DP) cells that are then selected to either the CD41 (SP) or the CD81 (SP) lineages of cells. After exit from the thymus, the a/bT cells mature to either regulatory, helper, or cytotoxic functions. The CD41 a/b T cells further differentiate into helper type 1 (Th1), helper type 2 (Th2), follicular B helper T, helper T-17 (Th17), or regulatory CD41CD251 T cells. The CD81 a/b T cells differentiate into type 1 or type 2 cytotoxic T cells (Tc1,Tc2), or NK T cells (NKT). g/dT cells also differentiate to helper or cytotoxic cells. It will be described below that the specificity of the TcRs determines lineage choice (Kyewski & Klein, 2006; Haks et al., 2005; Hayes et al., 2005; Kreslavsky et al., 2009). In human embryonic development, mature T and B lymphocytes are already detectable at 10 to 12 weeks, hence, well before birth. Therefore, human embryos have mature, antigen-reactive lymphocytes several months before birth, ready to react with a positive memory response to a foreign antigen if challenged by one. However, a more detailed description of prenatal human hematopoietic development is missing, as is a detailed assessment of the postnatal changes in these developments. It is safe to expect that immune systems of mice, as well as of humans, will be different at different times of an aging individual.
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
Development of Hematopoietic Cells from Embryonic Stem (ES) Cells In Vitro
From the 64cell blastocyst stage of the mouse embryo at day 3.5 of development—and from the comparable stage of human embryonic development—ES cell lines have been established in tissue culture that will proliferate for many generations without losing their ability to participate in the in vivo development of all cell lineages when reinjected into a recipient blastocyst and retransplanted into a foster mother, leading to a fully chimeric mouse (Martin, 1981; Evans & Kaufman, 1981). Furthermore, mouse ES cells are capable of homologous recombination (i.e., of introduction of genes) without the help of retroviral transfection at their sites in the chromosomes, thereby allowing proper transgenic changes and repair of selected gene loci (Matise et al., 2000). Furthermore, ES cells can be induced in vitro to differentiate to various cell lineages, notably also to hematopoietic cells and, more specifically, to cells of the innate and the adaptive immune system. These ES cell-differentiated hematopoietic cells, depending on their state of differentiation, can be transplanted in recipients to populate some or all of the hematopoietic compartments. It is surprising that these tissue culture conditions can be chosen in such a way that they will follow the embryonic time program in a wave of development, such that mesodermal cells appear at day 5 (equivalent to 8.5 of embryonic development), pHSC at day 8 to 9 (equivalent to 11.5 to 12.5), and T- and B-lymphocyte progenitors and precursors after day 12 (equivalent to day 15.5) (Nakano et al., 1994; Tsuneto, in preparation). Since it is now also possible to generate ES cell-like iPS (induced pluripotent stem) cells from peripheral, differentiated cells such as epithelial cells or lymphocytes, (Okita et al., 2007; Wernig et al., 2007; Hanna, 2008) and from them transplantation-tolerant hematopoietic cells, all in tissue culture, the study and use of any defined or mutable gene in its function in the development of the innate and the adaptive immune system can be studied. Such studies will help to develop protocols for genetic therapies of defective, disease-inducing gene loci in histocompatible ES-like iPS cells and their subsequent in vitro development to transplantable pHSC, with the hope to cure immunodeficiencies and, hopefully, to interfere with autoimmune diseases.
CELLULAR STAGES OF HEMATOPOIETIC DEVELOPMENT Pluripotent Hematopoietic Stem Cells
Pluripotent hematopoietic stem cells (pHSCs) generate the cells of the innate and the adaptive immune system first as the embryo develops, and thereafter continuously throughout life, not only to build up, but to maintain the numbers of cells (estimated as more than 2 3 109 in an adult mouse, and more than 2 3 1012 in an adult human, of which a large part turns over with half-lives of less than a week. These pHSCs derive from embryonal mesoderm, which also generates the environments of the hematopoietic system. These environments are the vascular endothelial cells that form blood and lymph vessels through which the blood cells travel and the microenvironmental stromal cells, forming hematopoietic “niches” in bone marrow and thymus for the developing progenitors (Jordan & Lemischka, 1990; Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). The cooperating stromal cells that help to induce these different hematopoietic lineage differentiations belong, in part, to mesenchymal stem-cell-derived osteoblasts, and, in other parts, to epithelial cells. Osteoblasts have been found
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to be stimulatory for some early hematopoietic progenitor functions, while differentiated adipocytes are either no longer stimulatory, or even inhibitory (unpublished observations). In part, this may be due to the spectrum of cytokines produced by these different cellular stages of mesenchymal cell differentiation. Thus, in contrast to a series of stromal cell lines derived from wild-type mouse bone marrow the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells. Five properties characterize pHSCs: 1. Their capacity to renew themselves upon cell division, 2. Their pluripotency to develop into the various differentiation lineages of blood cells, 3. Their chemotactic attraction to home back to specific niches in bone marrow upon transplantation, 4. Their long-term reconstituting ability to again serve as a stem cell when used from the transplanted host in secondary transplantations, and 5. Their potency to protect a transplanted host from death as consequence of lethal irradiation after (socalled bone marrow) transplantation.
self-renewal
pHSCs have the capacity to renew themselves upon cell division. In a steady state of the hematopoietic system selfrenewal will be by asymmetric divisions, generating one further differentiated cell while preserving the other as pHSC when the cell divides.
Intracellular Control Mechanisms
Several factors control the self-renewing proliferation of pHSCs, most notably the Polycomb gene family member bmi-I (Iwama et al., 2004; Park et al., 2003) and the transcription factor HOX B4, (Sauvageau et al., 1995; Helgason et al., 1996; Thorsteinsdottir et al., 1999; Kyba et al., 2002; Antonchuk et al., 2002; Buske et al., 2002; Krosl et al., 2003), both of which are controlled in their gene expression by the nuclear factor NF-Ya (Zhu et al., 2003). The Wntsignaling pathway, including b-catenin (Baba et al., 2005; Duncan et al., 2005; Reya et al., 2003) has been found to be involved in the control of pHSC proliferation, as it does in other stem cell programs. Thus, transgenic expression of NF-Ya, bmi-I, HOX B4, or b-catenin favor long-term proliferation of pHSC. In tissue culture, pHSs can now be expanded and cloned in the presence of appropriate cytokines (e.g., TPO [thrombopoietin], IL3, IL6, and stem cell factor [SCF, the ligand for the receptor tyrosine kinase c-kit]), all on a supportive layer of mesenchymally derived stromal cells. Prolonged, transgenic expression of these genes, however, may inhibit efficient hematopoietic differentiation into the different blood cell lineages (Schiedlmeier et al., 2003).
Extracellular Controls
Mesenchyme-derived as well as epithelial stromal cells provide several pHSC-cooperative, stimulatory interactions, among them SCF, interacting with c-kit on pHSCs (Gilliland & Griffin, 2002); angiopoietin, interacting with Tie-2 on pHSC (Arai et al., 2004; Zhang et al., 2006); and bone morphogenic protein 4 (BMP-4), interacting with its receptor on pHSC (Maeno et al., 1996; Hogan, 1996; Graff, 1997). Also, they chemotactically attract pHSC by the production of CXCL-12 (SDF-1), interacting with its receptor CXCR4 on pHSC (Nagasawa et al., 1996; Egawa et al., 2001; Ma et al., 1999; Ma et al., 1998; Kawabata et al., 1999; Melchers et al., 1999) and Ca-2, which is the Ca11-receptor
24
host defense: general
protein expressed on pHSC, probably sensing Ca11 near the marrow of bones (Adams et al., 2006). Activation of these stromal cells, especially the osteoblasts among them, in hematopoietic cell niches (Wilson & Trumpp, 2006) result in subsequent activation or inhibition of pHSC activities. Furthermore, nor-adrenaline produced by sympathetic nerves in collaboration with actions of the stem-cell-mobilizing cytokine G-CSF induce apoptosis of stromal cells, thereby mobilizing pHSC to leave the marrow and enter blood circulation (Osmond et al., 1985; Rico-Vargas et al., 1995; Medina et al., 1993; Medina et al., 2001; Katayama et al., 2006). Since early progenitors of B lymphocyte development in mice and humans have been shown to decrease in absolute numbers in bone marrow during life (Rolink et al., 1993), it is reasonable to expect that absolute numbers of earlier progenitors and of pHSC also decrease with age, even though they never disappear completely.
Pluripotency
A single pHSC can repopulate all cell lineages of blood and fill their compartments in the body with normal numbers of cells (Jordan & Lemischka, 1990 Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). pHSCs give rise to erythrocytes, megakaryocytes, and platelets that are not considered to be parts of the immune system. pHSCs also generate the cells of the innate immune system (i.e., myeloid cells such as monocytes, macrophages, various forms of dendritic cells, granulocytes such as basophils, neutrophils and eosinophils, and osteoclasts). Finally, pHSCs are the origin of lymphoid cells (i.e., the various forms of NK cells and cells of the adaptive immune system, T and B lymphocytes). Pluripotency is extracellularly controlled by the environment of cell contacts, chemokines and cytokines provided by stromal cells, and is intracellularly regulated by hematopoietic cell–autonomous genetic programs (i.e., receptor-mediated signal transductions and regulations of gene expressions).
MYELOID-LYMPHOID PROGENITORS Generation by Environmental Influences
When pHSCs are exposed to the action of FLT3L, the ligand of the membrane-bound tyrosine kinase receptor flt3 they lose their long-term repopulating capacity (Adolfsson et al., 2001). FLT3L is provided by “niches” of stromal cells in the bone marrow. FLT3L induces an apparently irreversible change to short-term repopulating stem cells (ST-pHSC), also called multipotent progenitors (MPP). In tissue cultures on OP9 stromal cells these MPPs can be grown in the presence of FLT3L and SCF. When regrown in tissue cultures with TPO, IL3, IL6, and SCF, they do not regain pHSC potential. Upon transplantation, these cells give rise to one wave of myeloid and lymphoid cell differentiation which is first seen in bone marrow (for myeloid, progenitor lymphoid and B-lymphoid progenitors) and in the thymus (for T-lymphoid progenitors) at 3 to 4 weeks, then as mature myeloid, and T- and B-lymphoid cells in the periphery, such as the spleen, 6 to 8 weeks after transplantation. While myeloid cells disappear again due to their short half-life, long-lived T and B cells remain for more than 4 months in the periphery (e.g., spleen, peritoneum, blood) of the transplanted host.
Induction by Intracellular Controls
GATA-1 controls erythroid development (Pan et al., 2005) while Icaros and PU-I induce myeloid-lymphoid cell development (Klemsz et al., 1990; Hromas et al., 1993; Singh & Pongubala, 2006; Dakic et al., 2005; Nutt et al., 2005).
THE CHOICE BETWEEN MYELOID AND LYMPHOID DEVELOPMENT Environmental Influences
While stromal cell lines derived from wild-type mouse bone marrow most often do not support the development of lymphoid, but do support myeloid cells, it was found that the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells, to proliferate pHSC with TPO, IL3, IL6 and SCF, and MPP with FLT3L and SCF. When M-CSF is added to such proliferating MPPs, they develop to myeloid cells that cease to proliferate after 1 week. On the other hand, when IL7 is added to the proliferating MPPs they become lymphoid progenitors, with the properties of common lymphoid progenitors (CLP) that can be induced to become either B lymphoid, T lymphoid, or NK cells. Hence, progenitors with the dual capacity to develop to myeloid and to lymphoid cells are induced on OP9 stromal cells, by M-CSF to myeloid, or by IL7 to lymphoid development. It appears that the constitutive production of M-CSF by wild-type stromal cells favors the development of myeloid progenitors, thereby disfavoring lymphoid progenitors. Once MPPs have chosen one of the two ways, the “other” cytokine no longer influences these cells in their continued differentiation (e.g., M-CSF is no longer active on lymphoid, and IL7 no longer on myeloid development). When MPPs or the IL7-induced CLPs are cultured on transgenic NOTCH-ligand DELTA-I expressing OP9 stromal cells, in vitro differentiation and proliferation of thymocytes to the CD4/CD8-double negative state is induced (Schmitt et al., 2004). Finally, osteoclast development is induced on stromal cells expressing transgenic TRANCE (Tsuneto et al., 2005).
Intracellular Controls
High levels of PU.1 induce myeloid and low levels lymphoid cell development (Nutt et al., 2005). Mef2 is required for proper expression of key lymphoid regulators, among them the IL7 receptor. It is in agreement with the expectation that external recognition of IL7 induces lymphoid lineage choice (Stehling-Sun et al., 2009), while at the same time, it represses myeloid development through the downregulation of C/EBPa. It appears to act in concert with PU.1 to modulate the intracellular decisions between myeloid and lymphoid lineage development, maybe differently at different concentrations of complexes containing PU.1 and Mef2c.
EXCLUSIVE B- VERSUS T-LYMPHOID LINEAGE DECISIONS
At least a part of the CD19-negative, CD42/CD8-double negative (DN), IL7-receptor-expressing, receptor-tyrosine kinases c-kit and flk-2-double positive CLP progenitor cells in bone marrow have yet to make the decision to enter either the T- or B-lymphoid lineage pathway of differentiation. Some of these early lymphoid progenitors in bone marrow express both the T-lymphoid lineage specific preTa gene as well as the surrogate L chain-encoding VpreB and 5 genes. Similarly, cellular functions control the proliferative expansion of DN2 to DN3 and 4 and large DP (CD41, CD81) thymocytes once the precursor have migrated to the thymus, and SL chain controls proliferative expansion of pre-BI to large pre-BII cells in the bone marrow. Hence, while these common lymphoid progenitors already express
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
both surrogate chains as well as the rearrangement machinery (i.e., RAG1, RAG2 and TdT), they still have to commit themselves to the expression of only one of the two—either pre-Ta, express CD3 and enter D- to J-rearrangements at the TcRd or b loci, or SL chain, Iga and b and enter D- to J-rearrangements at the IgH locus.
B-Lineage Commitment environmental Influences
We do not know which microenvironmental influences positively stimulate the development of sIgM1 immature B cells from B-lineage-committed progenitors and precursors, in contrast to the other lineages described above in bone marrow. B-progenitors of wild-type mice differentiate spontaneously—without addition of cytokines or exposure to stromal cells—to sIgM1 B-lymphocytes (Rolink et al., 1991). Contrary to the lineage-inducing capacities of other cytokines, IL7 locks DHJH-rearranged pre-BI cells into their early stage of B-lineage differentiation. In the added presence of stromal cells, which provide, for example, stem cell factors (SCF) for interaction with the receptor tyrosine kinase c-kit, these pre-BI cells proliferate but, again, do not differentiate to later stages of B-cell development (Rolink et al., 1995). Removal of IL7, rather than the addition of a different set of cytokines, induces the development of IgM1 B cells.
transcription Controls
Three transcription factors—E2A, EBF, and PAX5—control the initiation and maintenance of B-lymphoid development (Bain et al., 1994; Zhuang et al., 1994; Lin et al., 1995; Sigvardsson et al., 1997; Kee & Murre, 1998; Roessler & Grosschedl, 2006; Busslinger, 2004). Among the three, E2A dominates this control by not only initiating, but also maintaining the expression of EBF1, PAX5, and, thus, the B-cell program. E2A or EBF-deficient mice are arrested in B-cell development, just as PAX5-deficient mice are. However, E2Adeficient progenitor B cells carry no DHJH-rearrangements (Ikawa et al., 2004). By contrast, PAX5-deficiency still allows DH to JH-rearrangements on the IgH chain loci to the same extent (i.e., on both alleles) as PAX5-proficient, wild-type progenitor B cells (Rolink et al., 1999b). Transgenic expression of E2A (Singh & Pongubala, 2006), but not of EBF or PAX-5, or expression of important lineagespecific genes controlled by these early regulators (e.g., the IL7-receptor) (Choi et al., 1996) induce autonomous, environment-independent, B-lineage specific differentiation. Transfection, and subsequent transgenic expression of the E47 component of E2A in nonlymphoid fibroblasts, activates the terminal deoxynucleotidyl transferase (TdT) gene and the IgH chain locus (Choi et al., 1996). When transgenic expression of the rearrangement machinery (i.e., RAG1 and RAG2) is also provided in an embryonic kidney cell line, DH to JH rearrangements are induced at the endogenous IgH chain loci (Romanow et al., 2000). Endogenous rearrangements at the L chain loci in the same nonlymphoid cells require more selective actions of transcriptions factors; in the RAG1/RAG2-transgenic cells, E2A allows for endogenous VK to JK rearrangements, and EBF for V to J rearrangements (Romanow et al., 2000). Finally, myeloid cell differentiation can be autonomously induced by the transgenic expression of high levels of PU-I, or of the gene encoding the receptor for GM-CSF (Singh, 1996) as lymphoid differentiation can be directed by the transgenic expression of the receptor for IL7 (Singh & Pongubala, 2006). Both E2A-deficient, as well as PAX5-deficient progenitor B cells can be grown—like wild-type pre-BI cells—for
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long periods and cloned on stromal cells in the presence of IL7. In the case of PAX5-deficient pre-B cells, they are cell clones that are genetically identifiable by individual sets of DHJH rearrangements at both IgH chain alleles, just as wildtype, DHJH-DHJH rearranged pre-BI cells are (Rolink et al., 1991; Schaniel et al., 2002a, 2002b). The same is true for RAG-deficient as well as SL-chain-deficient, CD191 pro/ pre-B cells, the former without any DHJH rearranged IgH chain loci, the latter with normally DHJH/DHJH rearranged loci. Hence, the cellular differentiation program of the expression of the tyrosine kinases flk2 and ckit, and of CD19, accompanied by reactivity to IL7 and to stromal cell interactions that provide the ligands for flk2 and ckit and the cytokine IL7, can become disconnected from the DH to JH rearrangement program in progenitor B cells. The PAX5-deficient progenitors can be induced in vitro to (DHJH/DHJH rearranged) macrophages by M-CSF, to (DHJH/DHJH rearranged) dendritic cells by GM-CSF and M-CSF, to (DHJH/DHJH rearranged) granulocytes by GMCSF and G-CSF, to (DHJH/DHJH rearranged) NK cells by IL-2, to (DHJH/DHJH rearranged) osteoclasts by TRANCE expressed on mesenchyme-derived stromal cells, and to (DHJH/DHJH rearranged) thymocytes by NOTCH-ligand DELTA I, again expressed on stromal cells (Rolink et al., 2002a; Radtke et al., 2004; Höflinger et al., 2004). Transplantation of pre-BI cells, grown on stromal cells in the presence of IL7 from day 18 fetal liver, populate exclusively the B1a compartment in spleen, peritoneum, and blood, and do not populate the bone marrow, and only rarely and transiently populate the thymus. In contrast, pre-BI cells derived from bone marrow in SCF/FLT3L/IL7-stimulated cultures on stromal cells populate transiently (i.e., 2 to 4 weeks bone marrow with B-cell progenitors and thymus with T-cell progenitors and, thereafter, spleen and blood with T and B cells, and peritoneum with B cells resembling B1b cells (Vegh et al., 2010). On the other hand, transplantation of PAX5-deficient pre-BI-like cells, grown from bone marrow on stromal cells in the presence of IL7, populate all the myeloid, NK cell, and T cell compartments (with the exception of B-cell compartments in bone marrow), the thymus, and the peripheral lymphoid organs in a long-term repopulating fashion. Hence, such PAX5-deficient pre-BI-like cells can be isolated, regrown, and retransplanted several times into secondary, tertiary, and onward recipients, always repopulating all the different myeloid and lymphoid compartments except the B-lineage compartments (Schaniel et al, 2002).
T-Lineage Commitment Commitment by Internal Controls
Expression of NOTCH-1 accompanies development to the T lineage (Rolink et al., 2002a). Ectopic expression of “active” NOTCH-1 allows early thymocyte development in the absence of a thymus and, at the same time, inhibits B-cell development (Pui et al., 1999). Ectopic expression of Id2 induces differentiation to the NK cell lineage (Yokota et al., 1999; Ikawa et al., 2001; Engel & Murre, 2001) and, again, inhibits B-cell development, possibly by forming heterodimeric complexes with E2A (Engel & Murre, 2001). On the other hand, “inactive” NOTCH-1 inhibits T-cell development at the earliest, DNI stage of T-cell development, but promotes B-cell development even in the thymus (Radtke et al., 1999; Wilson et al., 2001). NOTCH-1 can also be inactivated by Lunatic Fringe (Koch et al., 2001) and by Deltex (Izon, 2002). Again, such activations lead to arrest of T-cell development and stimulation of B-cell development.
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host defense: general
Whenever activated, NOTCH-1 releases its intracellular domain, which acts on the transcription factor RBPJ to convert it from a repressor to an activator (Tanigaki & Honjo, 2007). Direct targets of the activator are Ptcra, CD25, and Hes-1.Thereafter and throughout a/b TcR T-cell differentiation from DN1 to CD41CD81DP thymocytes, activities of the transcription factors RUNX-1-CFBb, MYB, GATA-3, Icaros, PU.1, ETS-1, ETS-2, SPIB, E2A, HEB, and LEF1 all participate in controls at different developmental points, as described in detail in Rothenberg et al. (2008).
Commitment by external Controls
When CLPs are cultured in vitro not on OP9 stromal cells, but on OP9 stromal cells that express the ligand for NOTCH1 and DELTEX 1 constitutively after transgenic transfection, they develop in the presence of FLT3L and IL7 to the four stages of CD4/CD8-double negative (i.e., DN1, DN2, DN3, and DN4 cells, as well as to CD4/CD8double positive thymocytes) (Schmitt & Zuniga-Pflücket, 2002). In prolonged cultures, cells with a DN2/DN3 phenotype expand. The chemokine CCL25 attracts progenitors expressing its receptor, CCR9, within the thymus.
REVERSIBLE DECISIONS OF EXCLUSIVE B-CELL DIFFERENTIATIONS – THE FLEXIBILITY OF PROGENITOR CELLS
The hierarchical models of hematopoietic cell differentiation propose that commitment to a given cell lineage, with concomitant loss of the respective pluripotency or multipotency, is achieved in irreversible steps. This appears to be the case when FLT3L commits pHSCs to myeloid or lymphoid (i.e., differentiation to MPPs) and also when M-CSF commits MPPs to myeloid (i.e., to CML), and IL7 to lymphoid (i.e., CLP differentiation). By contrast, this appears not to be the case with PAX5deficient pre-BI-like cells. While they are blocked in further B-cell development, they differentiate to all the possible hematopoietic cell lineages (i.e., erythroid, myeloid, NK, and T-lymphoid cells), depending on the external environment of cytokines and niche cell contacts. However, they do so with rates that are too slow to allow protection of the transplanted host from irradiation-induced death. Hence, while they appear to be pluripotent, they are not pHSCs, although they home back to their original sites in bone marrow, retaining the original phenotype of ckit1flk-21CD192 IL7Ra1sca-11 cells (Rolink et al., 1999a; Schaniel et al., 2002b). The same multipotency has also been seen with E2A-deficient pro-B cells (Ikawa et al., 2004). By contrast, wild-type pre-BI cells do not home back to the bone marrow but rather, upon transplantation, give rise to a single wave of B-cell development, mainly to BI type (Rolink et al., 1991). B2-type B cells are developed when CD41 helper T cells, together with CD41CD251 regulatory T cells, are cotransplanted. Hence, PAX5-deficient progenitors are fully functional, pluripotent, long-term reconstituting, and flexible stem cells, but they are not pHSCs since they do not differentiate to all the hematopoietic cell lineages at appropriate rates to allow protection of the host from death by lethal irradiation. The expression of PAX5 can be seen as the final, decisive step to commit hematopoietic progenitors to the B-lineage pathway of development. The importance of PAX5 in this commitment process is further documented by experiments, in which lox-flanked PAX5 has been “knocked into” the PAX5 locus, rendering the locus conditionally inactive. When pre-BI cells of such conditionally PAX5-deficient pre-BI cells are treated with CRE-recombinase, thus deleting the PAX5-locus, these wild-type pre-BI cells revert
back to the PAX5-deficient pro/pre-B-cell phenotype. They become inducible to enter erythroid, myeloid, NK-, and Tlymphoid development (Mikkola et al., 2002). T-lymphoid cells in the thymus derived from the PAX5-inactivated and transplanted pre-BI B cells retain the DHJH/DHJH rearrangements of the original pre-B cells. When mature, sIgM1 B cells of conditionally PAX5deficient mice are rendered PAX-5 deficient by treatment with CRE-recombinase, the cells revert back to a pre-B-like stage (Schebesta et al., 2002). In vitro, such reverted cells regain their IL7-receptor expression and regain the capacity to respond to IL7 by proliferation. Since these cells have already expressed a set of productively VHDHJH/VLJL rearranged IgH/IgL-chain loci (i.e., sIgMs with μH- and kLor L-chains), and since PAX5 deficiency leads to lack of SLP65 expression (Schebesta et al., 2002), the resulting pre-B cells express a polyclonal spectrum of VH-domains on μH chains, and reexpress the SL chain (i.e., reexpress pre-BcRs but cannot signal downregulation of SL chain expression). Hence, they continue to proliferate, stimulated by IL7. Interestingly, the induced PAX5-deficiency turns off the expression of the productively VLJL rearranged Lchain loci (i.e., of L chains), suggesting a tight regulation of L chain expression by PAX5. In vivo transplantation of the CRE-induced, PAX5-deficient pre-B cells leads to repopulation by polyclonal μH chain-expressing thymocytes in the thymus, again without expression of the VLJL rearranged Lchain loci. It remains to be determined whether the CREinduced PAX5 inactivation dedifferentiates these cells to a large pre-BII cell-like stage or to a pro/pre-BI-like stage of B-cell differentiation.
THE GENERATION OF THE ANTIGEN RECEPTOR-EXPRESSING LYMPHOCYTE REPERTOIRES
In order to generate lymphocytes expressing antigen receptors, CLPs have to enter the appropriate primary lymphoid organs and begin to differentiate and rearrange antigen receptor loci. From the moment a lymphocyte precursor expresses the first chain of its antigen-specific receptor, that receptor becomes the dominating control element of all further steps of proliferation or death, and survival and location in the immune system. T- and B-cell antigen receptors (i.e., TCR, BCR) are generated by joining of V, D, and J segments at the TcR and Ig loci in a process ordered in time and cell differentiation stages. First, D to J rearrangements at the TcRb, TcRd, and IgH loci are initiated. For B-lineage cells this happens first during embryonic development in omentum and fetal liver, and later in adult life, in bone marrow, while for the T-lineage this is initiated in the fetal, and then in the adult thymus. Fetal cells appear to originate from progenitors that are distinct from those used after birth and all throughout life. In the B-lineage, D to J-rearrangements are begun in CD19-FLT31B2201c-kit1 pro/pre-B cells and are complete in CD191FLT3-B2201c-kit1pre-BI cells. T-lineage cells begin their D to J rearrangements at the transition from DN1 to DN2 cells, and have completed them in DN2 cells.
V to DJ Rearrangements and Synthesis, and Use of the First Chain of the Heterodimeric Antigen Receptors as Pre-Lymphocyte Receptors
The first functional Ig TcR chains are made when V to DJ rearrangements are initiated that can generate productively rearranged IgH, respectively, TcRb or TcRd loci. In B-lineage cells, V to DJ rearrangements occur in the
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
CD191B2201c-kit-CD251 large pre-BII stage, in the a/b TcR T-lineage at the DN3 stage (Kyewski & Klein, 2006; Melchers, 2005). All T-cell lineages with the possible exception of fetal TcRg/d expressing T cells, are derived from the same intrathymic precursors. All B cells, with the exception of the fetal wave of development in omentum and fetal liver generating B1a cells are derived from the same precursor in bone marrow. Productive TcRg and TcRd rearrangements result in expression of a g/d TcR, which commit cells mostly to the gd lineage (Kyewski & Klein, 2006). Since they do not form a TcRd chain-containing pre-lymphocyte receptor, these T-lineage thymocytes do not expand by proliferation in the thymus as TcRb chain-containing T-lineage cells do. Hence, they make up only 5% of all thymocytes and, consequently, generate correspondingly lower numbers of T cells for the peripheral immune system, at least in the spleen, lymph nodes, and blood. a/b TcR T-cell development and B-cell development show striking similarities. Productive IgH gene and TcRb gene rearrangements generate Igμ-H chains and TcRbchains which are assembled with SL chains—composed of the Vpre-B and 5 proteins, respectively—with pre-Ta chains to form pre-lymphocyte receptors (pre-BcR, pre-TcR) that are deposited on the cell surface of large pre-BII cells, such as DN3 thymocytes. In this way, the Vpre-B part of the SL chain in pre-BcRs (but not pre-TcRs, which lack such Vprobing Vpre-T parts) probe the newly generated IgH chains for their ability to form structurally intact heterodimers, first with the surrogate light chain, and later, in development with IgL chains (Melchers, 2005). Between 50% and 80% of the newly formed IgH chains do not pair well enough to form a pre-BcR on the cell surface, and hence, are excluded from the positive selection for further use in the B-lineage that follows. Initially, all TcRb chains may form a pre-TcR. The newly formed pre-BCRs and TcRb-pre-TcRs induce proliferation of large pre-BII cells, respectively, DN3 thymocytes. In the case of pre-BcRs, the number of induced cell cycles (between 2 and 7) depend on the fitness of the IgH chain/SL chain pairing, while fitness between TcRb chains and pre-Ta chains appear not to influence a larger number of induced cell cycles (7 or more). Mutations that abolish the proper structure of the preBcR (i.e., B-lineage progenitors deficient in VpreB1 and VpreB2 or 5, or VpreB1, VpreB2, and 5), or that interfere with pre-BcR signaling (Melchers, 2005; Melchers et al., 2000), abolish the proliferative expansion of large pre-BII cells, as do mutations that interfere with or abolish pre-TcR functions. Pre-BcRs and pre-TcRs may not even use the complementarity determining regions (CDRs) of the V-domains to bind ligands that could induce proliferation. The newly generated VH-domain repertoire of pre-BcRs is screened for fitness to pair (i.e., to be prepared for the eventual combination with conventional L chains). Thereby unfit H chains are excluded that may have other unwanted properties, such as the formation of self-aggregating immune complexes that might bear the danger of glomerulonephritis and vasculitis (Melchers, 2005). It appears that the non-Ig-portion of the 5-subunit of an SL chain is mandatory for the capacity of pre-BcRs to stimulate pre-BII cell proliferation (Ohnishi & Melchers, 2003). Crosslinking of pre-BCRs via positively charged arginine residues in the non-Ig portion of 5, possibly mediated by repetitive negative charges on molecules such as negatively charged nucleic acids and other molecules, either on the same pre-BII cells or on environmental stromal cells (Ohnishi & Melchers, 2003; Bradl et al., 2003), initiates pre-BCR signaling. Some CDR3 regions of newly generated
27
VH-domains contain argine residences that are apparently capable of mediated pre-BcR signaling functions even in the absence of an SL chain (Doyle et al., 2006). The pre-BCR also induces the downregulation of SL, thereby limiting proliferative expansion of IgH chain-expressing cells through exhaustion of pre-BcR formation. Furthermore, the pre-BcR down regulates recombinase activating gene (RAG1 and 2) expression, thereby preventing VDJ rearrangements on the second allele, possibly because the locus is rendered inaccessible for the rearrangement machinery. This should secure allelic exclusion at the IgH chain locus, so that one B cell produces only one type of H chain. Membrane-bound IgH chains signal the cell to turn off the rearrangement machinery and make the second, DHJH rearranged IgH chain allele inaccessible for further rearrangements. However, it appears that the classical pre-BcR with SL chain is not involved in mediating this allelic exclusion, since SL-triple-deficient (Vpre-B12/2 /vpreB22/252/2) B cells still are allelically excluded (Shimizu et al., 2002; Melchers, 2005). In pre-BII cells where pre-BCRs cannot be formed, these nonproliferating cells nevertheless proceed in differentiation (Grawunder et al., 1993; Rolink et al., 1996; Grawunder et al., 1995). However, their contribution to the developing B-cell compartment is, at best, 20- to 40-fold lower than that of their pre-BCR-expressing counterparts (Rolink et al., 1993b). Deletion of the gene encoding the adaptor protein SLP-65 directly by targeted mutagenesis (Flemming et al., 2003; Jumaa et al., 2003) or indirectly by targeted inactivation of PAX5 controlling SLP-65 expression (Koch et al., 2001) abolishes the capacity of large pre-BII cells to downregulate surrogate L-chain expression. This leads to continued pre-BcR formation in dividing large pre-BII cells so that, in fact, SLP-65-defective mice have a hyperplastic large pre-BII cell compartments. This chronic pre-BII cell proliferation is a breeding ground for secondary mutation so that, in a period of weeks, pre-B cell lymphomas (lymphocytic leukemias) develop in these mice. Do some autoantigens, nevertheless, influence—either positively or negatively—the pre-BcR repertoire development? It has been observed that a subset of autoreactive and polyreactive pre-BCRs expressed in pre-BII-like cells induced a proliferative boost in tissue culture cells, suggesting a positive rather than a negative selection by polyreactive pre-BCRs (Köhler et al., 2008) while other experiments indicated that autoantigens suppress pre-BcR-dependent pre-B-cell development (Keenan et al., 2008). Still other experiments indicate that pre-BCR cross-linking in fetal liver organ culture did not boost pre-B-cell proliferation, nor did it inhibit it (Ceredig et al., 1998). Pre-BCR formation leads to a shift in the originally generated heavy chain repertoire (ten Boekel et. al., 1997) and this shift is delayed in SL chain-deficient bone marrow. It facilitates a loss of positively charged and aromatic amino acid residue-containing polyreactive μH-chains, and thus may represent negative selection of μH-chain repertoires by the pre-BCR (Keenan et al., 2008). It is not clear how much of this negative selection of pre-BCRs is mediated by autoantigens binding to CDRs of μH-chain chains and how much is due to lack of pairing with SL. Productive TcRb rearrangements generate pre-TcRs, that induce proliferation of DN3 thymocytes in a cell autonomous fashion without any apparent requirement for a ligand. As for the B lineage in pre-BII cells, formation of a pre-TcR on the surface of DN3 thymocytes leads to the termination of V to DJ rearrangements on the second TcRb allele. More than 99% of thymocytes and T cells are allelically excluded at their TcRb locus. It is unknown whether
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TcRb loci can replace their Vb -regions in secondary replacements. Polar residues in the extracellular domain of the pre-TcRa chain, as well as its cytoplasmic tail are likely to contribute to this autonomous signalling (Yamasaki et al, 2006; Aifantis et al., 2002). However, the only conserved portion of the pre-TCRa chain is the transmembrane region and not its polar residues, so that other structural features might contribute to this autonomous signaling. Pre-TcRs terminate further rearrangements at the TcRb locus, but, unlike the pre-BcRs, do not select cells expressing particular TcRb chains by pairing, because pre-Ta does not have a Vpre-B-like structure that would allow such pairing with V regions of TcRb chains. Hence, the distribution of TcRb V regions in unselected and pre-TCR-selected thymocytes is the same (von Boehmer et al., 2003). All pre-TcR-expressing cells commit to the a/b TcR T-lineage with the help of Notch signaling, and they subsequently all undergo TcRa rearrangements.
V to J Rearrangements and Synthesis and Use of the Second Chains of the Heterodimeric Antigen Receptors as TcRs and BcRs in Immature T- and B-Cell Compartments
V to J rearrangements at the TcRa loci occur last in DN4 and CD41/CD81 DP cells, as do rearrangements at the Igk and Igl loci in CD191 B2201c-kit-CD251small preBII and in IgM1 immature B cells. Furthermore, except for most of the embryonically generated V(D)J-joints, N-region sequences of differing lengths are inserted at most joining sites, although this is more evident in human, and less so in murine B-cell development. Most cells with productive TcRd and TcRg rearrangements develop to the gd lineage without allelic exclusion of either TcRd or TcRg chains (Rothenberg et al., 2008). It is in fact not the isotype of the TcR but the signaling strength (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) that commits cells to either the d/g or a/b lineage. Some 20% g/d TcR-expressing cells that undergo weak signaling can still enter the a/b lineage by silencing TcRg expression and deleting the TcRd locus by TcRa rearrangement (Nossal & Lederberg, 1958). Little is known about tolerance mechanisms in g/d T cells except that TcR ligands appear to have a role in committing these cells to the g/d lineage (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) rather than in causing apoptosis. In fact, TcR ligation results in the generation of a subset of g/d T cells that assume the characteristics of innate lymphocytes in that they express the PLZF transcription factor, which is also is expressed in NKT cells (Schebesta et al., 2002).
Secondary Rearrangements at the IgL and TcRa Loci
A first rearrangement on the Ig L-chain loci can be followed by replacements by secondary VL to JL rearrangements on an already VLJL rearranged L-chain locus (Yamagami et al., 1999a, 1999b). TcRa can enter secondary Va to Ja rearrangements, and they can be extensive since more than 50 Ja segments are available in the mouse for such secondary, tertiary, and other rearrangements (Nossal & Lederberg, 1958). Secondary rearrangements can be made in immature lymphocytes that lack IgL chain, respectively, TcRa chain protein expression as heterodimeric antigen receptors on the cell surface, either as a result of a nonproductive V to J rearrangement, or because the chains do not fit their corresponding partner chain in the heterodimer. They can also be the result of an inappropriately affine binding of a BcR to an autoantigen, in the case of an a/b TcR to MHC-self-peptide. Changing the Ig L chain, respectively TcR a chain in this way has been termed editing. Continued rearrangements at
the IgL chain loci of autoreactive BcR-expressing, immature B cells increase the chance save this cell from apoptosis by a change of the specificity away from autoantigen recognition. By contrast, continued rearrangements at the TcRa locus increase the chance to salvage an immature thymocyte by changing the TcR specificity towards self-MHC/self-peptide recognition. During the process of L-chain editing, evolutionarily selected editor Ig light chains that have low isoelectric points (due, for example, to the presence of aspartate residues) appear to neutralize the DNA binding properties of certain IgH chains; this may facilitate avoidance of diseases mediated by antibody DNA complexes such as SLE (Li et al., 2001). Editing can also lead to the expression of two or even more different L chains (and possibly also two different H chains) in a single immature B cell (Gerdes & Wabl, 2004; Khan et al., 2008; Doyle et al., 2006). If editing results in the expression of one autoreactive and one nonautoreactive BCR, immature B cells can become unreactive to autoantigen due to dilution of the autoreactive with the nonautoreactive BCR. These dual-expressor B cells can enter the mature B cell pool while remaining potentially autoreactive. L chain editing can also lead to polyreactivity. Interestingly, some human B cells coexpress V pre-B and conventional L chains together with H-chains containing CDR3 regions enriched in positively charged and/or aromatic amino acids. Two-thirds of these cells are autoreactive, hence, they appear to have escaped central tolerance. Although human Vpre-B, which has an isoelectric point of 5.67, may act as an “editor” by neutralizing positively charged CDR3 regions, mouse Vpre-B proteins have an isoelectric point of 9.37 and thus may not be able to perform this function.
THE SELECTION OF IMMATURE LYMPHOCYTES BY THEIR ANTIGEN SPECIFICITIES Selection For and Against Autoantigen Reactivities
From the random generation of the antigen-binding regions, especially of the third complementarity region (CDR3) of the V regions of TcRs and BcRs, it is predictable that some of these V regions expressed on BcRs and TcRs of immature T and B cells should be fully autoreactive. In fact, it has been estimated that more than half of all newly generated BCRs are capable of binding autoantigens (Nemazee, 1995). The reactivity or responsiveness of B cells to antigens and autoantigens remains imprecisely defined; however, as the correlation between the binding affinity of an antigen to a BCR and the functional response of the B cell expressing this BCR cannot be given as a quantitative term. Furthermore, such antigen-sensing mechanisms are not necessarily the same in developmentally distinct subsets of lymphocytes. The same limitations in the judgement of quantitations of avidities exist for the interactions of TcRs with MHC-self-peptide or MHC-foreign peptide interactions. There is no reason to expect that the diversities of the originally generated repertoires of TcRb and TcRd chains and, thereafter, of the TcRa and TcRg chains are different from those of the IgH and L chain, since all loci are rearranged by the same rearrangement machinery, including the insertion of N regions by TdT. The originally generated repertoires of TcRs have not been tested for binding to the same collection of autoantigens and foreign antigens as BcRs have. It is, therefore, reasonable to expect that these original TcR repertoires are similarily polyreactive and autoreactive, and many of them should be heterodimer-unfit
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
to pair. The more extensive pre-TcR-driven expansion of TcRb chains may just simply increase the chances to find a pairing TcRa chain later. However, neither polyreactive nor autoreactive a/b TcR matter, because all of them, in contrast to BcRs, are selected to fit only one selected set of autoreactivities, namely recognizing MHCI or MHCII-self-peptide complexes (see below). Only those will be allowed to mature to single-positive (SP) CD41 or CD81 thymocytes, and to leave the thymus and mature in the periphery to effector cytotoxic or helper T cells. It is surprising—and also not in agreement with the hypothesis of clonal selection—that a large part of the originally generated repertoire of IgM1 B cells are most likely are polyreactive (i.e., bind a large collection of structurally seemingly unrelated antigens).
Autoantigen-Presenting Cells in Primary Lymphoid Organs for t Cells
To begin T-cell development 10 to 100 CLP migrate through the blood from the adult bone marrow into the cortical regions of the thymus (Kyewski & Klein, 2006). After commitment to the T lineage they proliferate as CD4-CD8-DN1 and -DN2 thymocytes and, thereby, expand to 5 3 107 cells in 15 to 20 divisions within nearly 2 weeks. In these proliferating cells, D to J rearrangements are initiated and completed. V to DJ rearrangements follow in DN3 and 4 thymocytes. The pre-TcR of the a/b T lineage allows another expansion by more than seven divisions, so that the ratio of a/b versus g/d TcR thymocytes after V to J rearrangements settles at 95% to 5%. However, most of these newly generated thymocytes never leave the thymus; only 1 to 2 3 10106 cells do. Productive TcRa rearrangements in TcRb-expressing cells lead to expression of an a/b TcR on the cell surface if the two chains can form a proper heterodimer. In marked contrast, TcRa rearrangements are only halted when the TcR can bind to MHC I or II self-peptide complexes with sufficient avidities (Borgulya et al., 1992; Casanova et al., 1991). In fact, all mature a/b TcR T cells contain both TcRa alleles in VJ-rearranged forms, and 30% of these thymocytes and T cells express two productive TcRa rearrangements (Padovan et al., 1993). Therefore, the TcRa loci appear to be kept open for secondary rearrangements as long as no MHC-self-peptide complex fits, thereby increasing the chances to positively edit the a/b TcR specificity for self-MHC/self-peptide recognition. However, not all the 30% of T cells express two TcRs on the cell surface (Saito et al., 1989) because pairing preferences exist for some TcRb chains in pairing with certain TcRa chains (Heath et al., 1995). Hence, only 10% of T cells express two different TcRa chains on the cell surface. In summary, a/b TcR T cells are frequently not allelically excluded. The specificity of the a/b TcRs determines whether cells are neglected and die, or are positively or negatively selected (von Boehmer, 2004). a/b TcRs on CD41CD81 thymocytes that recognize MHC class I self-peptide complexes on antigen-presenting cells in the thymus bring these MHCI molecules on the antigen-presenting cells in contact with CD8, which induces their differentiation to naïve CD81 cytotoxic T cells (Tc), on which CD4 has been downregulated. Conversely, other a/b TcRs on other thymocytes that recognize MHC class II self on antigen-presenting cells allow the contact of MHCII with CD4 on thymocytes and induce the differentiation to CD41CD8− helper T cells (Th), or to CD41 CD251 regulatory T cells (Treg) (von Boehmer, 2008). The induction to cytotoxic and helper
29
T-cells differentiation—also called positive selection—is dependent on the avidity of interaction between MHC selfpeptide complexes and a/b TcRs. If these avidities are too high, the thymocytes are driven into programmed cell death (apoptosis), also called negative selection. High avidity interactions can, however, also result in a diversion of developing abTCR1 cells into unconventional lineages involved in innate immunity, such as NK T cells. For the selection of such NK T cells, the selecting ligand is expressed on lymphoid cells (Bendelac et al., 2007).
Generation and Selection of Treg Cells in the Thymus
High affinity TcR ligands can also induce the generation of Treg cells that express the transcription factor Foxp3 and have an essential role in preventing autoimmune disease (Yamagata et al., 2004; Guy-Grand & Vassalli, 2004). Lineage fate of such cells is instructed by TcR binding to agonist ligands expressed on stromal cells within the thymus (Guy-Grand & Vassalli, 2004; Jordan et al., 2001). The Treg cell lineage differentiation process might begin either with CD41CD81 double positive, FOXP3-expressing cells in the thymic cortex (Apostolou et al., 2002), or with CD418− single positive cells in the medulla. It is evident that in developing T cells, the TcR determines lineage fate. The developing thymocytes are confronted with different types of autoantigen-presenting cells: (i) epithelial cells in the subcapsular regions, cortical epithelial cells, and epithelial cells in the medulla (mTEC [medullary thymic epithelial cells]); (ii) hematopoietically derived dendritic cells (DC); and (iii) hematopoietically derived B lymphocytes. All of them can take up, process, and present autoantigens (proteins) as self-peptides in MHC I and II molecules. mTECs and DCs express the highest levels of MHC. Hence, these three types of cells are prime sites for thymocytes probing the specificities of their a/b TcRs on MHC-self-peptide complexes (Kyewski & Klein, 2006). High-avidity interactions lead to negative selection by apoptosis, or to anergy, and short time survival. The ignorance of TcRs with no avidity for MHC-self-peptide complexes does not allow such thymocytes to further differentiate to a state where they could leave the thymus. These thymocytes remain short-lived and die by neglect. Low-avidity interactions lead to positive selection to more mature, CD41 or CD81 cells that are allowed to leave the thymus. Hence, the emerging T-cell repertoire in the peripheral immune system is restricted in the recognition of peptides of foreign antigens by the MHC of the antigenpresenting cells that they have encountered in the thymus. mTECs have the most unusual capacity to transcribe and translate a large variety of autoantigen-encoding gene loci, a capacity that appears to be in part controlled by the AIRE gene product (Peterson et al., 2008). Some thymic DCs also express AIRE and may, therefore, have the same expanded autoantigen-presenting capacity. Therefore, positive and negative selection of helper CD41, cytotoxic CD81, and regulatory CD41CD251 thymocytes does not only include the autoantigens expressed in the normal cells of the thymus; thereby, central tolerance can be generated for all autoantigens.
for B Cells
In immature B cells, surface membrane expression of IgM appears stop further Ig rearrangements. At least two-thirds of such immature B cells are capable of doing so, because two-thirds of the immature, as well as the peripheral, splenic mature B1 and B2 compartments have one of the two IgL chain alleles in germline configuration. They are allelically excluded, since
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less than 0.5% of all immature or mature B cell express more than one IgH chain allele as BcR on the surface. Quite comparably to T cells, the specificity of the Ig receptor influences the selection into the different B cell lineages, shown in experiments where the transgenic change of a B2-derived Ig receptor to a B1a-derived one changes the selection of the Ig-transgenic cells from B2 to B1a. It is remarkable that almost nothing is known of the autoantigen-presenting modes that select B-cell repertoires and establish central B-cell tolerance in the different B-cell compartments. Genetic deficiencies in the complement components C1q, C4, serum amyloid protein, complement receptor 2, or secreted natural serum IgM lead to systemic autoimmune disease characterized by production of autoantibodies against DNA, and other nuclear antigens suggest that these gene loci may contribute to central B-cell tolerance (Melchers & Rolink, 2006). Two models have been proposed to explain the emergence of autoantigen-reactive B-cell repertoires in these knockout animals. In one model (Carroll, 2004), macrophages expressing the appropriate complement receptors (e.g., C1qR, CR1) efficiently remove apoptotic cells that are bound by natural IgM, C1q, and C4b, thereby preventing accumulation of these cells and subsequent activation of mature B cells. The other model (Melchers & Rolink, 2006), suggests that autoantigens, including those released from apoptotic cells are presented to immature B cells by immune complexes containing C1q, C4b, and IgM on as yet unidentified cells expressing the appropriate receptors (e.g., C1qR, CR1, and possibly FcRμ). In this model, the result of the autoantigen presentation depends on the avidity of interaction of the immune complex-bound autoantigens with the BcR expressed on an immature B cell. If the avidity is very high, it can result in negative selection (i.e., induction of apoptosis and/or anergy in the immature B cell). If the avidity is low, the corresponding immature B cell can be positively selected, maybe into the B1 compartments, if this low autoantigen recognition is maintained in the B1 populated sites. If the BcR on an immature B cell does not bind any autoantigen with even a low avidity this cell is ignored and is allowed to enter (maybe into the B2) conventional B cell compartments. It is notable that, in this view, ignored B cells are allowed to enter the mature, peripheral compartments while T cells must be MHC-self-peptide-selected. A better understanding of the modes of central B-cell tolerance induced by apoptosis, editing, anergy, and ignorance requires identification of the relevant antigen-presenting cell populations. At this first checkpoint of B-cell repertoire development, the contribution of polyreactive BcRs to the total repertoire drops from 60% to 6%. In contrast, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients with these autoreactive and polyreactive BcRs are not lost at this checkpoint (Carroll, 2004), providing strong evidence of the role of this checkpoint in preventing autoimmune disease.
POSITIVE SELECTION OF B CELLS
Autoreactive, in fact, often polyreactive B cells (Leslie et al., 2001) can be positively selected and appear to accumulate in the BI compartments of the gut-associated lymphoid tissues (i.e., in the follicles below the M cells of the flat epithelium of the gut). Some of them are also found as single, intraepithelial cells. Many positively selected B cells accumulate in the gutassociated lymphoid tissues (GALT), including the lamina propria. They produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epi-
thelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high crossreactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. B cells within the marginal zone of the spleen also appear to be positively selected. However, it remains to be seen whether this major traffic intersection, in fact, is a selection site for newly generated (as well as possibly of recirculating) B cells, potentially, these B cells could be in contact with cellular debris of dying blood cells and of killed bacteria, which are found at the same sites and which are about to be removed from the circulation. Many of them have a BI phenotype (Hayakawa et al., 1999; Stall et al., 1992). BcRs of BI B cells are often found to cross-react with a variety of auto-antigens and bacterial antigens at low avidities. B1 cells appear mildly activated (i.e., “tickled”) do not proliferate in their GALTs but appear to be long lived. Transplantation of BI cells into secondary hosts, in contrast to the transplantation of conventional B cells is easy and repeatable in subsequent hosts. Even low numbers of BI cells will fully replenish the GALTs. This suggests that the low avidity–autoreactivity, combined with a polyreactivity to several antigens, could be the stimulatory influence for BI cells to replenish their lymphoid sites after transplantation and to attain longevity (Craig & Cebra, 1971; Förster & Rajewsky, 1990). Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side, it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. At least in parts this IgA production by gut-associated B cells appears to be quite distinct from the Ig-production elicited in a helper T-cell-dependent, inflammatory response of B cells in germinal centers of peripheral lymph nodes and the spleen. First, while the follicular B cell response beneath the M cells may be helper T-cell-dependent; hence, germinal center-derived, the submucosal, intraepithelial response in the lamina propria can be helper T-cell-independent. Onethird of the IgA might derive from such T-cell-independent B cell responses in these T-cell-independent responses (Rolink et al., 1991). The expression of the Ig class-switchinducing AID (activation-induced cytidine deaminase) gene (Muramatsu et al., 2000; Revy et al., 2000; Shinkura et al., 2004) is normally induced by a CD40 ligand-CD40 interaction. However, the cooperating cell providing the CD40 ligand in the GALT, especially in the intraepithelial spaces is not known, and must not be a helper T cell. Furthermore, these B cell responses could be costimulated by other cell–cell contacts and cytokines that might not be typical for a T-cell-dependent, germinal center response of B cells. Hypermutation of the V regions of the IgM, and later IgA, expressed and secreted by the lamina propria of the intestine BI cells could well occur in the AID-expressing, IgA class switching cells. However, the lack of the selection
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
of better fitting v region mutants by bacterial antigens and the lack of germinal center formation (i.e., exclusive B-cell proliferation) may decrease the probability to generate and select better fitting antibacterial antibodies—in as much as that also should decrease the probability of generating high affinity autoreactive antibodies.
IGNORANCE OF B CELLS
Emerging B cells with no apparent autoreactivity pass the two checkpoints in bone marrow and the spleen as long as they express a surface membrane-deposited BcR. A variety of Ig-transgenic mouse models support this view (Fields & Erikson, 2003). The ignored B cells enter the spleen via the terminal branches of the central arterioles and populate the follicular regions as conventional, B2-type B cells. Initially they are short-lived, with half-lives of 2 to 4 days. They then mature to longer-lived B cells, with half-lives longer than 6 weeks. Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. Conventional, B2-type B cells are most often triggered into responses by helper T-cell-dependent antigens. These foreign antigens also stimulate helper T cells to cooperate with the follicular B cells in a response, which takes place mainly in germinal centers within the follicular regions of secondary lymphoid organs. This induces B cells in a CD40 (B)-CD40 ligand (T) cell cytokine (e.g., IL4- or TGF-b-)dependent fashion to switch to IgG, IgE and IgA, and to preferentially hypermutate the V regions of the rearranged IgH- and L-chain genes, leading to affinity maturation of B cells. The switched, hypermutated BcR-expressing B cells have the choice either to mature to Ig-secreting plasma cells or BcR-expressing memory cells. Both of them gain longevity in such a T-cell-dependent response with half-lives changed from a few days to weeks and months of survival in the immune system. The memory B cells and the long-lived plasma cells appear to leave the germinal centers to lodge in special niches in the bone marrow until they are recalled by a secondary challenge of the same antigen. While BI B cells are apparently unable to be stimulated to longevity by BAFF, a member of the TNF family of cytokines, conventional B cells express BAFF-receptor, a member of the TNF-receptor family, and respond to BAFF by polyclonal maturation to long-lived B cells (Rolink & Melchers, 2002). The TNF family ligands BAFF and APRIL and their receptors BCMA, TACI, and BAFF-R control the selection of short-lived immature B cells with no apparent positively (or negatively) selecting specificities for autoantigens to long-lived mature B cells. Experiments of the in vivo administration of soluble BAFF-R ligands and of soluble decoy receptors, the analysis of BAFF-transgenic, BAFF-deficient and BAFF-receptor (BAFF-R)-deficient mice, as well as the in vitro responses of immature and mature B cells of BAFF (all reviewed in Rolink & Melchers, 2002) have shown that immature B cells from bone marrow and the spleen (initially
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immature as well as transitional B cells) and mature B cells respond to BAFF by polyclonal maturation to long-lived B cells without proliferation. BAFF and BAFF-R deficiencies arrest B-cell development at the transition from immature to transitional B cells. Although the action of BAFF in vitro is polyclonal and independent of BCR occupancy, it remains to be seen whether ligand selection through BCR occupancy plays a role in this selection of the conventional “virgin” antigen-reactive mature B cells. B2 cells can also respond, as B1 cells do, to T-cellindependent antigens (i.e., in the absence of an activation of helper T cells and their cooperative actions on the B cells). In that situation the Ig-secreting cells, which are generated to mainly produce IgM, remain short-lived and do not generate memory B cells. It remains also possible that gut-associated B1 cells can be flexible enough to respond in a T-cell-dependent fashion, form germinal centers, and generate long-lived, Ig class-switched plasma cells and memory B cells. In such cases they are, or become, conventional, B2-type B cells. Germinal center-like lymphoid aggregations have been seen to form near the flat epithelium M cells.
PERIPHERAL B CELLS WITHOUT Ig
In normal B cell development, B-lineage cells that cannot express Ig molecules on their surface are restricted to the primary lymphoid organs and will die there, usually with half-lives of 2 to 4 days. Experimentally induced ablation of surface Ig expression in peripheral mature B cells induces their rapid death (Lam et al., 1997). Therefore, neither immature, mature, memory B cells, nor plasma cells without Ig expression can ever be detected in the peripheral lymphoid organs, and even most B-cell tumors appear to be selected to express Ig. However, several observations, most of them made in vitro, suggest that B-lineage cells from as early as the Blineage committed progenitor B cells and pre-BI cells can differentiate all the way to memory-phenotype and plasma cell-like stages without ever expressing Ig. In kL-chainrearrangements-deficient bone marrow, over 90% of the small pre-BII cells have the proper phenotype, but no Lchain rearrangements, since rearrangements at the L chain loci are so slow that they can only be detected in the remaining 10% of the small pre-BII cells (Yamagami et al., 1999a, 1999b). Most striking is the development of RAG-deficient pre-BI cells (hence with IgH and L-chain loci in nonrearranged, germline configurations) in vitro in the stimulatory presence of CD40-specific monoclonal antibody and IL4 to sμ-sg-switched cells with a mature phenotype (Rolink et al., 1996). Hence, the differentiation of B-lineage cells is a stepwise process driven by cell–cell contacts and cytokines that can occur without the synthesis or surface deposition of Ig. The roles of the pre-BcR and BcR also become apparent from these experiments. They control, positively and negatively, proliferation, anergy, and apoptosis of B-lineage cells and, thereby, ascertain the production of appropriate numbers of cells with selected specificities. Viral infection of B cells by EBV (Epstein-Barr virus) may be another way, by which the requirement of surface Ig deposition on B cells for their survival and use in the peripheral lymphoid organs may be circumvented. The EBV encodes latent membrane proteins (LMP), of which LMP 2A, a multispanning transmembrane protein, expressed in B lymphocytes, can associate with Iga and Igb and share with these two anchor proteins an ITAM motif (Gross et al., 2005; Fruehilng & Longnecker, 1997; Caldwell et al., 1998). In fact, the phosphorylated form of the ITAM
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motif of LAMP 2A recruits tyrosine kinases and adaptor proteins, which are also used by the BcR complex for signaling. In this way, LMP 2A acts as a BcR analog, supporting the selection into the peripheral mature B-cell compartments of B cells lacking BcRs. High-level transgenic expression of LMP 2A selectively recruits cells into the BI compartments (e.g., into the gut and intestine) while low level expression promotes development of conventional, B2-type, and marginal zone B cells (Casola et al., 2004). Neither of the high-level or low-level LMP2A expressing BcR-negative B-cell populations can be stimulated by T-cell-dependent antigens to develop germinal centers in peripheral lymphoid organs, indicating that BcRs on the surface of B cells are needed for peripheral germinal center responses. By contrast, the low-level, but not the high-level (i.e., the conventional) B2 (and not the B1) cell-directed LMP 2A expression allows germinal centers to be formed in the gut. Hence, the role of BcRs in germinal center formation in the gut might be replaced by an antigen-unspecific mechanism of antigen uptake, which allows processing and presentation by MHC class II molecules to helper T cells. Alternatively, these gut-associated BcR-deficient B cells might be stimulated by bystander helper T cells activated with bacteria of the gut or by bacteria-derived polyclonal activators, such as LPS and lipoprotein (Kawai & Akira, 2009), in the latter case via TLR2, TLR4, and other receptors (Akira & Takeda, 2004), or by other T-lineage cells or NK cells not restricted by the recognition of MHC class/ peptide complexes. In summary, T-cell (or NK-cell) dependent, BcR-independent (and perhaps MHC class II-independent) activation of B cells in germinal centers can lead to GC responses in which the activated B cells (as well as T or NK cells) can be expected to secrete pro-inflammatory cytokines such as IFN-g, IL-1, IL-4, IL-5, IL-6, and TNFa as essential mediators of immune responses. Furthermore, it has been observed in pre-BI cell-transplanted RAG-deficient mice, which contained only pre-BI cell-derived BI cells but no T cells, that one-third of the normal levels of IgA can be produced—apparently T-cellindependently—mainly by cells in the intestinal lamina propria without the formation of germinal centers. It is likely that these cells are also present in BcR-deficient, high-level LMP 2A-expressing lamina propria BI cells. Obviously they cannot produce IgA, but, again, they can be expected to respond to bacteria by the secretion of cytokines that might be of pro- or anti-inflammatory types. In conclusion, the GALT-associated B-cell compartments can develop even when the B cells do not express BcRs, or even MHC class II (i.e., can function in cellular responses of proliferation and differentiation to effector functions in an antigen-independent, and perhaps bacteriadependent way, leading to pro-inflammatory (and maybe also anti-inflammatory) responses. From all of these results, it becomes evident that peripheral mature B cells have important functions beyond their capacity to produce, secrete, class switch, and hypermutate antibodies in the regulation of immune reactions.
LINKING THE DEFENSIVE POWER OF THE INNATE IMMUNE SYSTEM WITH ANTIGEN SPECIFICITY OF THE ADAPTIVE IMMUNE SYSTEM
Since B lymphocytes can be stimulated to develop into cells that no longer deposit their BcRs (Ig) as antigen-specific receptors into the surface membrane but rather secrete this Ig as so-called plasma cells into the blood stream, antigen specificity of a B cell can be transferred to the innate
immune system in three ways. (i) Ig molecules of different IgH chain classes can find receptors for the constant regions of their H chains (so-called Fc receptors) and, thereby, can transfer this antigen specificity (by so-called opsonization) to antigen-nonspecific cells (e.g., cells of the innate immune system, such as macrophages or dendritic cells). (ii) The antigen-unspecific cells of the system can bind antigen–antibody (Ig)-complement immune complexes via complement receptors, again gaining antigen specificity by opsonization. (iii) As mature B cells displaying Ig on their surface, they can attract “their” specific protein antigens with high avidity, take it up, process it, and present it as peptides complexed with MHC class II molecules to helper T cells (i.e., they can act as antigen-specific antigen-presenting cells).
THE TRANSFER OF IMMATURE LYMPHOCYTES FROM PRIMARY LYMPHOID ORGANS TO MATURE COMPARTMENTS IN THE PERIPHERY
Although VH-repertoires expressed in immature B cells in the bone marrow and in immature and mature B cells in the spleen are not significantly different, almost 90% of the newly formed immature B cells never leave the bone marrow (Rolink et al., 1999a). Therefore, they should be subject to additional deletion mechanisms. In humans, there is a further reduction of the percentage of autoreactive cells (40% to 20%) across this transition, while the percent of polyreactive cells remains at the low level (6%) already achieved during the transition from pre-B to immature B cells (Wardemann & Nussenzweig, 2007). Again, SLE and RA patients fail to establish this repertoire change at this second checkpoint. Indicating that several mechanisms for establishing recessive tolerance may contribute to these diseases (Yurasov et al., 2005). Once arrived in the spleen, hardly any cells are lost at the transition from immature, including transitional T1 and T2 cells, to mature B cells. Hence, the mature B1 and B2 cells emerge only after passing through at least two checkpoints, suggesting that several mechanisms may operate to establish tolerance. Both of the checkpoints are not functioning in SLE and RA patients and, thus, may contribute to these diseases. The BcR complex with Iga and Igb is mandatory for transition to the peripheral sIgM1B cell pools—no sIg2 B cells survive normally. The transition from short-lived to long-lived, from AA4.11 to AA4.12, CD212/CD232 to CD211/CD231 cells is controlled by a series of genes, including OBF, btk, and CD40. Thus, btk/CD402 or btk2/ OBF− double deficient mice have a strong defect at the transition from the immature, transitional T1- and T2-type B cells to mature B cells (Rolink et al., 1999a). In addition, cross-linking of surface IgM by anti-IgM-antibodies (possibly a polyclonal example of a cross-linking auto-antigen) induces immature B cells of bone marrow as well as spleen (T1 and T2) to apoptosis. This apoptosis can be prevented by the transgenic expression of the anti-apoptotic transgene bcl-2, and is circumvented by polyclonal activation with LPS and with CD40-specific antibodies or CD40 ligand. Deficiency in the expression of the TNF-family member BAFF (also called B-lys), or its receptor, BAFF-R, blocks the maturation of immature B cells into conventional, B2-type B cells, but not to B1-type B cells (Rolink et al., 2002b; Rolink & Melchers, 2002; Ng et al., 2005; Moore et al., 1999; Schiemann et al., 2001; Gross et al., 2001). In vitro BAFF induces polyclonal maturation of immature B cells from bone marrow and from the spleen (T1 or T2) without proliferation. BAFF has been found in the sera of some SLE
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
patients, as well as in the sera of NZBxNZW SLE autoimmune disease prone mice. In these mice, administration of BAFF-specific antibodies prevents, or at least delays the development of SLE-disease. Therefore, excessive production of BAFF, administered at the sites of negative selection of immature B cells, could “rescue” autoreactive B cells from deletion, leading to autoimmune reactivities in the peripheral mature conventional B-cell repertoires. One major site of BAFF production has been found to be dendritic cells. Hence, in order to interfere with the deletion of autoreactive immature B cells such dendritic cells would have to be localized near the sites of B-cell deletion in bone marrow or the spleen, and would have to be strongly activated to BAFF production to effect rapid maturation of autoreactive immature B cells before they are subjected to negative selection. Such a scenario still needs to be investigated, and the observed partial rescue of B-cell tolerance by T-cell independent antigens may be one experimental way to probe the molecular and cellular requirements for such competition with clonal deletion. In T-cell development, once a/b TcR-expressing thymocytes have been probed as CD41CD81 DP cells for their appropriate fitness to MHC-self-peptide complexes, their first choice, either to become a Th or a Tc cell, has been made. Their further fate, to polarize either into Th1 or Th2 or follicular B helper T or Th17 cells or to Tc1 or Tc2 cells, appears to made as a consequence of a response to MHC-foreign peptide complexes created by introduction or infection of a foreign protein antigen in the secondary lymphoid organs. While Treg cell induction begins in the thymus and appears to be guided by MHCII-self-peptide complex recognition, the rules of their initial selection and their further maturation need to be worked out in detail. The generated repertoires of lymphocytes with BcRs and TcRs, at any time in the life of an individual, are limited by the number of cells generated per day (i.e., 0.1% of the total B- and T-cell pools in a newborn and may be less than 0.001% in an adult), possibly decreasing with age as the frequencies of CLPs and their DJ-rearranged descendants (i.e., FLT3L/IL7/stromal cell-clonal precursors have been seen, at least for the B lineage, to decrease 20- to 100-fold within a few months in mice and from birth to 10 years of age in humans (Ghia et al., 1998, 2000). Since the thymus involutes after sexual maturity, this should also be the case for T-lineage progenitors. This means that in newborn mice with around 5 3 107 B cells as well as T cells, 5 3 104 cells each may be generated per day, while in adult mice, with around 5 3 108 B cells and T cells each, around 5 3 103 B cells and T cells may be generated. Humans, which show striking similarities in the generation of B cells and T cells in bone marrow and the thymus, have approximately 1,000-fold higher numbers of B and T cells and should, therefore, also generate 1,000-fold more cells with newly formed BcRs or TcRs.
FAILURES OF CENTRAL TOLERANCE B Cells
Prolongation of the short half-life of B cells brought about either by a decrease in expression and/or activity of proapoptotic genes or by an increase in pro-survival gene expression (e.g., B lymphocyte-activating factor, BAFF) favors B-cell driven autoimmunity that manifests as SLE-like syndromes (Mackay et al., 1999; Rolink et al., 2002b). Elevated expression of the genes encoding Toll-like receptors (Deane et al., 2007; Shlomchik, 2008) can promote similar autoimmune manifestations.
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Signaling defects can also impair central tolerance induction in immature B cells. For example, the NZM2410/NZW mouse strain-derived z alleles of Sle1 and its sublocus, Sle1b, as well as the orthologous human locus impair B-cell anergy, receptor editing, and deletion (Liu et al., 2007; Kumar et al., 2006). Members of the SLAM family of costimulatory molecules are candidate Sle1b gene products, among which the Ly1081 isoform of the Ly108 gene is highly expressed in immature B cells (Xie et al., 2007). The Ly108.2 allele, but not the SLE-associated Ly1081 allele, sensitizes immature B cells to deletion and RAG reexpression. Hence, Ly108 is a potential regulator of tolerance checkpoints, as it may censor self-reactive B cells by protecting from autoimmunity. When the z allele of Sle1 is combined with the Fas lpr mutation, lymphoproliferative autoimmunity induced by the phosphatidylinositol-3-kinase-AKT-mTOR pathway is induced. As the signaling pathways in immature B cells will become known in greater detail, more genes that interfere with the proper establishment of B-cell central tolerance are likely to be identified.
T Cells
Lowered expression of pro-apoptotic genes and enhanced expression of pro-survival factors (Takahama et al., 1992; Buch et al., 2002; Bouillet et al., 2002) contribute to a shift towards inclusion of self-peptide/self-MHC-reactive T cells in the mature compartments. The mode of antigen presentation appears to play a major role in human T-cell-dependent autoimmune diseases. Juvenile diabetes contributing susceptibility factors include those that affect the amount of insulin expressed intrathymically (McCaughtry et al., 2008). Consequently, thymic overexpresssion of insulin in diabetes-prone NOD mice reduced the incidence of diabetes (Baldwin et al., 2005). Like in the B-cell lineage, mutations in signal transducing molecules, such as ZAP-70, result in autoimmune disease (Smith et al., 1989). It appears that the ZAP-70 mutations alter the balance of positive and negative selection, allowing certain T cells to escape negative selection and then to be activated by their self-ligands in peripheral lymphoid tissues. It seems possible that these mutations could also disturb the balance of positive selection and Treg cell generation in the CD41 T-cell compartments. In analogy to B cells expressing more than one BCR, it was found that expression of a second TCR in addition to an autoreactive TCR can “dilute” the signaling capacity of the autoreactive TCR such that these cells can escape negative selection (Wack et al., 1996).
CONCLUSIONS
By the time newly generated lymphocytes have been deposited in their peripheral, mature compartments of all the T cells and a large part of the B1 cells have been auto-antigenligand selected (hence their repertoire purged) even before a foreign antigen can select “its” specific lymphocytes for a response. This leaves “holes” in the diversity of these repertoires (i.e., their capacities to recognize a foreign antigen). By contrast, those conventional B cells that are ignored by negative and positive selection should be more diversely reactive, although their short half-life in the system limits them. If these conventional B cells could not enter a helper T-cell-dependent response of Ig class switching and v-region hypermutation, the capacity of most of them to recognize the wide world of foreign antigens might well be much too low to elicit a clonally selected, high-avidity immune response with defensive power.
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The author has recently written two review articles (Melchers, 2009; von Boehmer & Melchers, 2010), which have covered parts of the contents of this article. He is the recipient of a Reinhard Kosellek grant of the DFG (ME 2467/1-1).
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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
3 The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates JIM KAUFMAN
INTRODUCTION
Thus, the essence of the adaptive immune system has been said to be wide diversity, exquisite specificity, and memory. In contrast, the so-called innate immune system described in mammals lacks the capacity to generate completely new receptors within an individual and lacks memory. There is a division of labor among lymphocytes (discussed in chapter 2 and elsewhere in this book). B cells produce antibodies (Ab), which recognize shapes as the primary defense of the adaptive immune system to extracellular pathogens. Some T cells, those that carry T-cell receptors (TcRs) composed of gamma and delta chains ( TcR), also recognize shapes although these shapes are generally found on a (cell) surface. Other T cells, which carry TcRs composed of alpha and beta chains (ab TcR), recognize only major histocompatibility complex (MHC) molecules carrying molecular pieces (peptides or lipids) from within the cell, thus revealing pathogens that have escaped from antibodies to the inside of a cell. Peptides from pathogens such as viruses replicating in the cytoplasm (or contiguous spaces such as the nucleus) are presented by classical class I molecules to T cells bearing CD8 molecules, whereas pathogens such as bacteria or parasites in intracellular vesicles (such as the endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes) as well as the extracellular space, which is topologically equivalent, give rise to peptides that are presented by classical class II molecules to T cells bearing CD4 molecules. Since the CD4 cells recognize antigens from the extracellular space, they also have a role in regulating B cells that are major effectors of the adaptive immune system dealing with extracellular antigens. Nonclassical class I molecules called CD1 molecules carry lipids from intracellular vesicles to be recognized by a special group of CD8 bearing T cells. The MHC molecules are loaded with their antigens by sophisticated pathways composed of various kinds of molecules, some of which are encoded by genes located in the MHC (discussed in detail in many other chapters of this book). Classical class I molecules bind peptides, which are produced in the cytoplasm by the proteasome (under certain conditions augmented by various inducible components), and pumped into the lumen of the endoplasmic reticulum by the transporter associated with antigen presentation (TAP, a member of the ABC transporter family). The class
The pathogens and tumors confronting an individual are so many, so various, so unrelenting, and so lethal, it is a wonder that any of us survive to discuss the issue. The fact we are here demonstrates that overall and in the long run, the defense systems do work. To do so, they are incredibly complex, with a complicated interplay of many different cell types interacting in many specific locations of the body, utilizing hundreds of cell surface and soluble ligands to bind hundreds of different receptors that respond through various signaling systems to thousands of target genes, all levels of which are controlled by many layers of regulation. This extraordinary complexity is presumably the result of the accumulation of many changes appearing step by step over millions of years, each step arising by accident and, in general, being selected by the need to defend the individual organism from the particular pathogens present at the time. Here we will consider the evolution of only one part of the immune response. The so-called adaptive immune response, examined in great detail for humans (Homo sapiens) and mice (Mus musculus), is conceptually simple. The essential cell is the lymphocyte with a cell surface glycoprotein that functions as an antigen receptor, with each antigen receptor having a binding site with a different protein sequence, which is constructed by somatic changes in the genes for the antigen receptor in the lymphocyte (mediated in part by the recombination-activating genes, RAG1 and RAG2). Each lymphocyte (or clone of lymphocytes) bears a different antigen receptor that can specifically recognize a small group of complementary shapes; but, altogether, antigen receptors of the whole population of lymphocytes can bind an extraordinarily large diversity of shapes. Upon recognition of a molecule with a particular shape, only those clones of lymphocytes that recognize that shape will be stimulated to be activated and to proliferate (so-called clonal selection). Upon a second encounter with the same shape, there will be a faster, greater, and qualitatively different immune response.
Jim Kaufman, University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom.
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I molecule and TAP are part of a peptide-loading complex (PLC), which includes the dedicated chaperone tapasin (or TAP-binding protein, TAP-BP) in association with ERp57 (a member of the protein disulfide isomerase, PDI, family), the lectin chaperone calnexin and/or calreticulin, and perhaps other molecules (such as endoplasmic reticulum associated aminopeptidase, ERAAP, and PDI itself). Classical class II molecules bind to a trimer of invariant chain (Ii) in the endoplasmic reticulum and are transported (primarily via the cell surface) to a specialized endosomal compartment called MIIC, in which Ii is degraded and then antigenic peptides produced by endosomal proteases are loaded. The class II molecules are stabilized by nonclassical class II molecules that act as dedicated chaperones, DM (as well as DO in some cells) until high affinity peptides are bound. Different CD1 molecules are known to traffic to different endosomal compartments based on signals in their cytoplasmic tails and to be loaded with lipids generated by lipid hydrolases and other degradative enzymes. Of the accessory molecules in these pathways, the genes for some of the inducible components of the proteasome (LMP2 and LMP7), both TAP subunits (TAP1 and TAP2), tapasin, and both chains of DM (DMA and DMB) and DO (DZA and DOB), are located in the MHC. For all immune responses, there is an evolutionary arms race between pathogens and the host (discussed in detail in many other chapters of this book). Pathogens are under selective pressure to change the protective peptides that bind to classical MHC molecules and then stimulate effective T-cell responses, so most pathogens have many variants. In return, and more slowly, the hosts are selected to have multiple MHC molecules, both by having a multigene family and multiple alleles. The most efficient method would appear to be a large multigene family, but each additional MHC molecule increases the possibility that any given T cell will recognize a self-peptide leading to autoimmunity, which is countered (at least in part) by deletion of that T-cell clone in the thymus. Thus, if there are too many MHC molecules, the repertoire of T-cell specificities may be reduced so much that the T-cell response becomes ineffective. In addition, the total number of MHC gene loci may be limited by the number of MHC molecules that can be accommodated on the cell surface. However, there is no obvious reason why the number of MHC alleles should be limited, and, indeed, MHC molecules are among the most polymorphic known, with the human class I locus HLA-B having over 1,000 alleles worldwide. In contrast, the human Ab and TcR loci have large multigene families and further generate variability by gene rearrangement (and other processes) in somatic cells, so they have little allelic polymorphism. Also, the genes involved in processing and loading the peptides (e.g., LMPs, TAPs, tapasin, DM, DO, Ii) are relatively nonpolymorphic in typical mammals, but this becomes a crucial point in the discussion that follows. Although this all sounds quite complicated, the actual adaptive immune system elucidated in typical mammals is, in fact, significantly more complex. Even if we could, it would be beyond the scope of this review to try to explain the emergence of lymphocytes, natural killer (NK) cells, dendritic cells, regulatory T cells, follicular dendritic cells, and the like, with their many interactions, receptors, and signaling cascades. It is far beyond this review to explain the emergence of the various lymphoid tissues, including the thymus and the critical processes of self-tolerance, although some innovative work in this area has been reported recently (Boehm & Bleul, 2007; Bajoghli et al., 2009). It is also beyond our scope to consider all of the various evolutionary novelties and adaptations that have emerged
independently in each of the vertebrate taxa. In this review, we will focus on the question of how the essential components of the adaptive immune system—Ab, TcR, and MHC molecules—arose and began to interact.
THE TIME FRAME: ADAPTIVE IMMUNE SYSTEMS IN JAWED VERTEBRATES AND INVERTEBRATES
There is a long history of speculation about the origins of the adaptive immune system so extensively studied in mammals. There was certainly the possibility of a very ancient origin, given that multicellular organisms have existed for billions of years and presumably always needed protection from pathogens. Moreover, the elements of the adaptive immune system might have evolved originally for other purposes, for instance, development or the nervous system. It was also speculated that the allorecognition system of soft-bodied invertebrates was a primitive form of MHC recognition, although it is now clear that the genes involved in tunicate allorecognition are not closely related to MHC genes (Scofield et al., 1982; De Tomasso et al., 2005). A more recent suggestion is that MHC molecules arose for selection of olfactory specificity in mate recognition (Boehm & Zufall, 2006). Whatever the original function, it appears that the adaptive immune system so extensively studied in mammals arose at the base of the jawed vertebrates over 500 million years ago (Flajnik & Kasahara, 2001, 2010; Litman et al., 2005). In all jawed vertebrates examined thus far, the essential elements of the adaptive immune system are present (Fig. 1). In particular, sharks (as representatives of cartilaginous fish, the most phylogenetically primitive jawed vertebrates) have RAG1 and RAG2, IgM Ab, ab and TcR, and an MHC containing class I and class II genes, as well as genes for processing molecules such as TAPs, tapasin, and proteasome components (Bernstein et al., 1994; Rast et al., 1997; Ohta et al., 2002; Terado et al., 2003). No such genes (with two possible exceptions discussed below) have been found in more phylogenetically primitive organisms, such as the jawless fish (the two kinds of cyclostomes, the hagfish Eptatretus stoutii and the sea lamprey Petromyzon marinus, among several species examined). With the exception of apparent RAG genes in the sea urchin (Fugmann et al., 2006), no such genes were identified in the genomes of other deuterostomes (two chordates, the cephalochordate amphioxus or lancet Branchiostoma floridae and the urochordate tunicate Ciona intestinalis, and an echinoderm, the purple sea urchin, Strongylocentrotus purpuratus), nor in the genomes of any other invertebrate (Azumi et al., 2003; Rast et al., 2006; Putnam et al., 2008; Holland et al., 2008). These data intensified the contrast between vertebrate and invertebrate immune responses, and led to the concept of a “Big Bang,” during which an adaptive immune system suddenly appeared in jawed vertebrates (Schluter et al., 1999). Despite years of functional experiments with invertebrates pointing to recognition of allo- and xenoantigens, none of the essential genetic components of the adaptive immune system in jawed vertebrates were found in the first complete genomic sequences of invertebrates (C. elegans Sequencing Consortium, 1998; Medzhitov & Janeway, 2002): an insect (the fruit fly Drosophila melanogaster) and a nematode worm (Caenorhabditis elegans). Therefore, the notion that invertebrates lack an adaptive immune system was strengthened, and the question became how invertebrates managed to survive without a system that is essential to jawed vertebrates. Early thoughts included the ideas that short lifespans and/or large numbers of offspring made an adaptive immune system
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
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FIGURE 1 An idealized tree, showing the relationships of the animals (and their defense systems). Phyla are indicated above the tree with thick lines; common names of species are indicated above the tree with thin lines; defense systems are indicated.
unnecessary, although many invertebrates do not fit this profile, with examples ranging from cockroaches, which live for 3 years like a mouse to some sea urchins, which live for hundreds of years. Another possibility is that invertebrates expand their innate immunity enough to counter their pathogens, which might be (part of) the answer for the purple sea urchin and amphioxus, whose genomes contain hundreds of Toll-like receptor (TLR) and other pattern recognition genes (Fig. 1) (Rast et al., 2006; Huang et al., 2008). A key concept that has relevance no matter what the defense system might be is that pathogens that overcome all of their hosts may face a bleak evolutionary future, and so, in general, the virulence of pathogens is constrained and guided by the particular defenses that their host(s) evolve. In other words, pathogens will not be selected to overcome an adaptive immune system that is not present in their host(s), and therefore, pathogens of invertebrates will not be virulent in the same way as those facing the adaptive immune system of jawed vertebrates. The idea that pathogens evolve with their host means that invertebrate immune systems are not “worse” than vertebrate immune systems, just different, with different benefits and costs. Indeed, jawed vertebrates now have many pathogens, which evade the adaptive immune system, and, on top of that, have autoimmunity that is a consequence of the adaptive immune system, prompting the characterization of “one
moment of ecstasy followed by 400 million years of agony” (Hedrick, 2004). In fact, at least some groups of invertebrates can have adaptive immune systems based on molecules other than Ab, TcR, and MHC (Fig. 1), as first described in some molecular detail for the fibronectin-related proteins (FREPs) from a mollusk, the snail Biomphalaria glabrata. The snail FREP system consists of a multigene family, whose members are composed of immunoglobulin (Ig) V (variable)-like domains and a fibronectin-like domain, apparently undergo somatic mutation within an individual, and have expression correlated with responses to parasites such as schistosomes (Adema et al., 1997; Zhang et al., 2004). In arthropods (such as the insects, the fruit fly Drosophila melanogaster and the mosquito Anopheles gambia, and two species of the crustaceans water flea, Daphnia), a gene related to the human gene Down’s syndrome cell associated molecule (Dscam) is involved in axon guidance in neurons during development, but then is expressed in adult fat body and hemocytes, the latter involved in various kinds of immune response in insects. Alternative splicing, mainly among the extracellular Ig-like domains, gives rise to a large number of variants, and transient silencing of the Dscam gene in mosquitoes has been reported to lead to decreased resistance to the bacterial pathogens and insect malaria (Watson et al., 2005; Dong et al., 2006; Brites et al., 2008).
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In jawless fish, like lampreys and hagfish, two variable lymphocyte receptor (VLR) genes, composed mainly of leucine-rich repeats (LRRs) give rise to many variants by gene conversion from nearby LRR pseudogenes. These variants are clonally expressed in lamprey and hagfish lymphocytes. Moreover, there is evidence that the VLR-A gene product is secreted by lymphocytes with some properties in common with vertebrate B cells, in response to and then binding to specific antigens. In contrast, the VLR-B gene product is expressed on the surface of other lymphocytes with some properties in common with T cells. Thus, the adaptive immune system of the jawless fish is founded on a different set of antigen-specific receptor molecules, but otherwise has many features in common with the adaptive immune system of jawed vertebrates, suggesting that they may both have descended from a common ancestor (Pancer et al., 2004b; Cooper & Alder, 2006; Guo et al., 2009). Overall, it seems likely that many, if not most, invertebrates will have some form of adaptive immunity based on different molecular systems, which may allow us at last to understand which features of adaptive immunity are essential in a way that was impossible with just a single example. The postulated “Big Bang,” the apparently sudden appearance of all the essential components of our familiar adaptive immune system, could be explained by another, still somewhat controversial phenomenon, the 2R hypothesis. On the basis of fragmentary data on genome sizes, Susumu Ohno (1970) postulated the following scenario: two rounds of genome-wide duplication at the base of the vertebrates, which, for each gene, would have led to one old gene for the previously essential function and three new
genes (so-called paralogs) available for new functions (socalled neofunctionalization). Such a large number of new genes could allow a burst of innovation, leading to new features (e.g., notochord and vertebrae). Support for this idea came from the analysis of Hox gene clusters, for which there is one in the invertebrate amphioxus and four paralogous regions in vertebrates such as mammals (Garcia-Fernandez & Holland, 1994). Analysis of gene families found in the human MHC on chromosome 6 led to the identification of MHC paralogous regions on chromosomes 1, 9, and 19 (Fig. 2), each one of which has a subset of genes thought to have been present in the primordial cluster (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). For instance, class I genes are found in the MHC while the genes for homologs CD1 and neonatal FcR are found on chromosome 1 and 19, respectively. Similarly, the genes for inducible proteasome components LMP2 and LMP7 are located in the MHC, while the gene for MECL1 in the MHC-paralogous region is on chromosome 9. The complement component genes for C4 are found in the class III region of the human MHC on chromosome 6, and the homologs C5 and C3 are found in the MHC paralogous regions on chromosomes 9 and 19. Many other genes, which are not necessarily involved in the immune system, are also found in these MHC-paralogous regions, including the developmental factors Notch1 on chromosome 9, Notch2 on chromosome 1, Notch3 on chromosome 19, and Notch4 on chromosome 6. Thus, the apparently sudden appearance of the adaptive immune system at the base of the vertebrates could be explained by the sudden appearance of many homologous
FIGURE 2 A model for the evolution of the adaptive immune system of jawed vertebrates, from the primordial MHC (left) through two rounds of genome-wide duplication (middle) and subsequent silencing, deletion, and break up to give MHC paralogous regions (right). Black boxes indicate genes based on data from chickens; open boxes indicate genes based on data from other species. Gga and Hsa, chromosomes from chickens and humans, respectively.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
genes, which could adapt to new functions. However, the “Big Bang” might have taken as much as 70 million years, based on the apparent divergence times of jawless fish and the armored fish that led to the jawed vertebrates. Fossils of extinct intermediate forms spanning this time show no sudden jump in the body plan, but instead show a stepwise acquisition of new features (Donoghue & Purnell, 2005). Although there is little scope to observe the evolution of the immune system through fossils, it seems likely that the adaptive immune system of jawed vertebrates also evolved in steps, none of which are separated between those organisms still living. However, these steps might still be inferred by the molecular equivalent of fossil traces.
FIRST ANTIGEN-SPECIFIC RECEPTOR WITH IMMUNOGLOBIN VARIABLE REGIONS: THE T-CELL RECEPTOR
The presence of antigen-specific receptors with highly variable regions (Ab and TcR) is the major difference between the adaptive immune system in jawed vertebrates and the
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many different kinds of innate immunity. Antigen-specific receptors in innate immunity (e.g., TLRs) can have great specificity for particular antigens, which are found in pathogens and not in the host, but in the vertebrates examined thus far they lack both the enormous diversity and the memory of adaptive immunity. The single most important event in the appearance of the adaptive immune system of vertebrates was undoubtedly the appearance of split Ig V domains with the potential of the separated genomic segments to be rejoined in an imprecise manner (Fig. 3). From the first discoveries of split V genes, it was postulated that insertion(s) by a transposable element was responsible, based on the specific recognition by RAG proteins of recombination signal sequences (RSS) flanking the initial DNA cleavage site (Sakano et al., 1979). More recently, the RAG proteins have been shown to have transposase activity, and a class of transposons have been described whose integrases and recognition sequences are closely related to RAG and RSS sequences (Kapitonov & Jurka, 2005). A (relative of a) potential target gene has been described in the sea lamprey, which encodes a molecule related to TcR
FIGURE 3 A model for the evolution of split variable region-containing antigen specific receptors by insertion of transposons (top), with cartoons indicating the location of the CDRs in a V domain (lower right) and the footprint of the CDRs on an MHC molecule (lower left). In the upper left, open circles indicate domains (Ig V type and C type), lines indicate transmembrane and cytoplasmic regions, filled circles indicate D and J segments of the protein, grey bars indicate membranes; one chain is gray to indicate that the molecule may or may not have been a dimer. In the upper right, boxes indicate genes or gene segments, and dotted lines indicate regions introduced by transposon insertion with the heavy black lines indicating RSS. In the cartoons, cylinders indicate a-helices, arrows indicate b-strands, thin lines indicate other conformations of the protein, the thick line indicates peptide bound to the MHC molecule, and the dotted ovals indicate the footprint of the CDRs from the a-chain and the b-chain of a TcR on a class I molecule.
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but with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail like some NK cell receptors (Pancer et al., 2004a; Yu et al., 2009). Another potential relative of the target gene is the variable region-containing chitin-binding protein (VCBP), found as large diversified families in amphioxus and tunicate genomes (Dishaw et al., 2008). These examples are two of several gene families interpreted to have mixtures of the properties of innate and adaptive immunity, leading to the view of an intimate intertwining of the evolution of innate and adaptive immunity (Kasahara et al., 2004; Flajnik & Du Pasquier, 2004; Litman et al., 2005, 2007; Cannon et al., 2008). A single event that gave rise to all split V genes in jawed vertebrates would mean that one kind of variable antigenspecific receptor came first: either Abs (which recognize three-dimensional shapes and have a high enough affinity to bind soluble antigens), TcRs (which recognize shapes located on cell surfaces), or ab TcRs (which recognize peptide or other antigens on MHC molecules located on cell surfaces). Although in principle, Ab seem like the simplest system to evolve, the fact that joining of the segments of split V genes only generates diversity at complementarity determining region 3 (CDR3) strongly indicates that ab TcRs were the original variable antigen-specific receptor in jawed vertebrates. It was long a mystery why diversity is found in three (or four) loops of variable antigen-specific receptors, but this diversity is generated by joining split V gene segments (J and D, and even multiple D) only at CDR3 (Fig. 3). As first suggested on the basis of a model (Davis & Bjorkman, 1988), many three-dimensional structures of ab TcR bound to peptide-MHC complexes show beyond a doubt that CDR1 and CDR2 contact mainly the MHC molecule, whereas CDR3 contacts mainly the peptide. The original model envisaged a scenario, in which the primordial TcR-like molecule recognized a collection of peptide antigens bound to a single monomorphic MHC class I molecule, so that the TcR needed only to generate diversity at CDR3 in order to recognize different peptide antigens. Later acquisition of MHC polymorphism and polygenism drove the acquisition of diversity in CDR1, CDR2, and elsewhere, primarily by polygenism of ab TcR. After that, TcR evolved from ab TcR by losing the requirement for recognizing antigens in the context of an MHC molecule, and then Ab evolved from ab TcR by losing the requirement to stay bound to a cell membrane. However, an argument that Ab or TcR came first might still be made, based on the fact that CDR3 is in the center of the antigenbinding site and thus might have been particularly important for binding molecular shapes. There are other steps in the emergence of the first variable antigen-specific receptors which remain mysteries, the sequence of events perhaps obscured by further evolution (Flajnik, 2002; Eason et al., 2004; Hsu et al., 2006). TcR locus organization seems relatively stable, perhaps due to the coevolution with the MHC. However, in cartilaginous and some bony fish, certain TcR loci are organized in clusters of V-D-D-J-C, and there are many germline-joined TcR genes expressed early in ontogeny. In contrast, Ab locus organization varies wildly throughout the vertebrates, with various heavy chain and light chain isotypes found in different vertebrate groups. There are even single-chain Ab described in cartilaginous fish (new antigen receptors, NARs) and camels, which are generally thought to be derived rather than an ancestral form. One mystery is that TcR and nearly all Ab are heterodimers of two chains with different numbers of joining segments. The establishment of this pattern must have been very early, because this feature is found in all three classes
of receptor (Fig. 3): one chain (a and chains of TcR, light chains of Ab) only requiring joining of the V segment with a J segment, and the other chains (b and chains of TcR, heavy chains of Ab) requiring joining of a J segment to D segment(s) before joining with the V segment. One possibility is that multiple V segments followed by multiple J segments gives the possibility of sequential rearrangements, whereas once V-D-J joining is complete, all J segments are deleted and rearrangement generally halts. Another mystery is the curious arrangement of TcR loci in most jawed vertebrates: two separate loci for b and chains, but a combined locus for a and , with V, D and J segments as well as C exons upstream of the Ja segments and Ca exons, so that V-Ja joining would delete the D and J segments. The Ab heavy chain loci of (certain) bony fish have a somewhat similar pattern, with the IgM locus embedded in the IgZ locus (although both have J and D segments). An underlying reason for this organization (as well as for germlinejoined TcR expressed early in ontogeny) has been suggested (Danilova & Amemiya, 2009): very young animals have few lymphocytes, and these loci are organized and regulated to restrict diversity to recognition of the most dangerous pathogens during early ontogeny. A third mystery is how and why the RSS are distributed, with the heptamer-nonamer spacing of 12 and 23 nucleotides (roughly the distance of one and two turns of the DNA helix, respectively) flanking different segments in the different Ab and TcR loci. In mammals, these distributions restrict the heavy chains of Ab to one D segment, but allow TcR multiple D segments.
THE PRIMORDIAL MHC MOLECULE: SIMILAR TO A CLASS II MOLECULE
The inference that the first discernible recognition by variable antigen-specific receptors was the recognition of MHClike molecules by TcR suggests that intracellular pathogens were the first major problem tackled by the emerging adaptive immune system. In fact, there are a wide range of MHC-like molecules described in well-studied mammals, ranging from the highly polymorphic peptide-binding classical class I and class II molecules located in the MHC to a host of nonclassical molecules with a variety of polymorphism levels, genomic locations, and functions, some immunological and some not. There are many examples of MHC-like molecules with “closed grooves,” which do not bind peptide or any other ligand (Wilson & Bjorkman, 1998), and some of these molecules function as indicators of stress or infection recognized by T cells (for instance, MIC-A and MIC-B recognition by TcR) (Li et al., 1999). So, it is easy to imagine the beginning of MHC molecules as such a monomorphic MHC-like molecule appearing on the surface of a cell to signal infection through recognition by a nonvariable TcR-like molecule. The first point at which we have some direct evidence, however slight, is the recognition of peptides on an MHC-like molecule by a TcR molecule with variability in CDR3 generated by genomic joining, and that narrows the list of examples to classical class I, classical class II, and CD1 molecules. Based on structure as well as function, these molecules have all descended from a common ancestor, so which one was first? The case for class I molecules coming first has been made repeatedly (Flajnik et al., 1991; Flajnik & Kasahara, 2001), based on the hypothesis that property of peptide binding evolved first in chaperones such as heat shock protein 70 (HSP70, an inducible gene which is located in the MHC of mammals), and that the exon encoding the chaperone domain was then shuffled to the beginning of a gene encoding a transmembrane protein with an extracellular Ig domain. Other arguments that class I molecules came before
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
class II molecules have included the nearly ubiquitous tissue distribution of class I molecules compared to the relatively restricted expression of class II molecules (at least in mammals), and the direct function of class I molecules in resisting viruses compared to the indirect function of class II molecules in regulating antibody production. A major argument against this scenario is that the structures of chaperones such as HSP70 have now been solved, and they have no obvious relationship to MHC peptide binding domains (Zhu et al., 1996). Moreover, the argument that class II molecules are now specialized in tissue distribution and function is not necessarily true for the original class II molecules. CD1 molecules have a structure like class I molecules but pick up their antigen lipids from deep endosomal compartments like class II molecules, so they are certainly candidates which could give rise to both. Thus far, CD1 genes have been identified in mammals and birds, but not yet in other vertebrates, and the evidence has been interpreted both for and against an ancient origin of CD1 molecules (Miller et al., 2005; Salomonsen et al., 2005). The hypothesis for class II molecules coming first was made initially on the basis of the domain organization and later on the basis of crystallographic structures and gene organizations (Kaufman et al., 1984; Kaufman et al., 1990). This scenario (Fig. 4) envisages a single gene, which encodes a homodimeric molecule with the domain structure of a class II b chain, and then this gene duplicates and diverges to encode a heterodimeric molecule with the domain structure of a class II molecule. The fact that most class II genes are arranged in a/b gene pairs in opposite transcriptional orientation allows an inversion around a central point but
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with asymmetric breakpoints, which leads in one step to a class I heavy chain gene and a gene encoding the end of a class II a chain, that is the Ig domain with a transmembrane region and a cytoplasmic tail. Finally, a stop codon or splice site mutation leads directly to a b2-microglobulin (b2m) gene. One argument against this simple molecular scenario is that the conformations of the Ig domains in the heterodimers is not the same; the class II a2 domain and b2m are oriented differently from the class II b2 domain and the class I a3 domain, which then requires some explanation that is so far not forthcoming. The class II first scenario assumes that the initial tissue distribution and function of the class II molecule was the same as the class I molecule, except that class II molecules sampled antigen from intracellular vesicles rather than the cytoplasm. Since the extracellular space is topologically equivalent to intracellular vesicles, it was a natural extension of class II function to regulate Ab production by B cells, once they appeared. Regardless of whether it was structurally like class I or class II, the origin of the first MHC-like molecule is of interest. Thus far, no molecule with distinctive feature of MHC molecules, variously described as the “MHC fold,” “peptidebinding domain,” or “open-faced sandwich” has been found in invertebrates. Even though proto-MHC genomic regions have been proposed in organisms as distant from vertebrates as protochordates, nematodes, and fruit flies (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004), no gene with the appropriate features has been identified nearby. Perhaps part of the difficulty might be the fact that there seems to be little or no sequence identity required for generating the MHC fold, as illustrated
FIGURE 4 A model for the evolution of MHC molecules, from a class II b-chain-like homodimer to a class II heterodimer by duplication and divergence, and then to a class I chain with b2m by inversion. On the left, circles indicate domains (open for class II a-chain, gray-filled for class II b-chain, with SS for intrachain disulphide bond), lines indicate transmembrane and cytoplasmic regions, gray bars indicate membranes. On the right, boxes indicate exons (open for coding regions of class II a-chain, gray-filled for coding regions of class II b-chain, black-filled for 3 untranslated region), P indicates promoter region with arrow showing direction of transcription, curved arrow around axis, and lightning bolts indicating an inversion around a central point with asymmetric breakpoints. Adapted from Kaufman, 1988.
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by the fact that such homologous domains in class I and CD1 share only 10% sequence identity, so perhaps the gene is present but just as yet unrecognized.
COEVOLUTION OF GENES LED TO THE PRIMORDIAL MHC
The mammalian MHC is a large complex region (MHC Sequencing Consortium, 1999; Kelley et al., 2005) filled with hundreds of genes, pseudogenes, and repetitive elements, typically divided by recombination into regions including a class I region with class I genes and certain other “framework genes,” a class II region with class II genes as well as proteasome and TAP genes, and in between, a class III region with complement component genes, TNF cytokine genes, and many other kinds of genes. The outer boundaries of the MHC can be extended, so that the tapasin gene is found in the “extended class II region,” while TRIM genes and olfactory receptors might be included in the “extended class I region.” However, there is much variation in the boundaries and detailed organization of the MHC between different mammals with the gene organization of the class II and class III regions most similar but with class I genes varying wildly. For instance (Fig. 5), the human classical class I genes (HLA-A, B, and C) are located in the class I region, two mouse classical class I genes (H-2D and L) are located in the class I region but one (H-2K) is located in between the class II region and the extended class II region, while in the rat all classical class I genes (RT1-A, A1, and A2) are located in between the class II region and the extended class II region. This example is mentioned because it has functional implications discussed below, but more detailed descriptions
and the concepts of the framework in the mammalian class I regions are discussed elsewhere (Amadou, 1999). Outside of mammals, the organization of the MHC is reported to be even more various (Kelley et al., 2005). The MHC of the frog Xenopus tropicalis appears to be much the same size as the human MHC, but the MHC of chickens (Gallus gallus) and many other birds seems particularly small and compact, while some passerine birds are thought to have a much larger MHC. Of particular note is the arrangement among bony fish and at least one frog (Xenopus ruwensoriensis), in which the MHC appears to have fragmented (Du Pasquier & Blomberg, 1982; Sammut et al., 2002; Stet et al., 2003). For instance, in zebrafish (Danio rerio) the class I region includes the proteasome and TAP genes and is completely separated (on different chromosomes) from the class II genes found in at least two loci and from class III region genes, which appear to be scattered throughout the genome (Bingulac-Popovic et al., 1997; Sambrook et al., 2005). In the phylogenetically most primitive jawed vertebrates, the cartilaginous fish (as exemplified by the sharks Triakis scyllium and Ginglymostoma cirratum), the class I, TAP, inducible proteasome, class II and class III region (complement components C4 and factor B) genes are found together in a single MHC locus (Ohta et al., 2000, 2003; Terado et al., 2003), so the organization in X. ruwensoriensis and the bony fish appears to be derived. A simple explanation in both cases would be genome-wide duplication followed by silencing and deletion, as discussed in the next section. Amid all of this variability of the MHC in different vertebrates, at least one general rule has emerged about the relationship of genomic organization and function: the coevolution of polymorphic interacting genes that are closely linked in the
FIGURE 5 A cartoon showing the relationship between coevolution of interacting genes and (recombinational) distance in the genome, as illustrated for the class I and TAP genes of the MHC. (Left) The TAP heterodimer and class I molecule(s) are embedded in a membrane. The number of well-expressed class I molecules ranges from three in humans to one in chickens. Similarly, the specificity of interaction with peptide (as indicated by the number and depth of indentations) increases for TAPs and may decrease for class I molecules from humans to chickens. (Right) Different regions of the MHC labelled in bold are separated by thin vertical lines, genes labeled in normal script are indicated by thick vertical lines, and distance between the TAP genes and the most distant class I gene is indicated by the horizontal arrow. Adapted from Kaufman, 1999; B. Walker et al., submitted.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
genome. This idea was first suggested (Germain et al., 1985) to explain a curious situation in mouse class II molecules: both the Ab and Aa genes are polymorphic, but the Eb gene is also polymorphic while the Ea gene is not. A hotspot of recombination separates the A region with the Ab, Aa, and the 5 end of the Eb gene (including the exon encoding polymorphic peptide-binding domain) from the E region containing the nonpolymorphic 3 end of the Eb gene and the Ea gene (as well as other genes). The Ab and Aa chains within one haplotype (but rarely from different haplotypes) will interact to make a class II heterodimer, whereas the nonpolymorphic Ea chain will interact with Eb chains from all other haplotypes. The fact that the Ab and Aa genes are rarely separated by recombination means that optimal combinations can coevolve and remain together, whereas the frequent separation of Eb and Ea genes means that one (in this case, the Ea gene) evolves to be an “average best fit” to interact with all of the alleles of the other (in this case, the Eb gene). Once this concept is recognized, examples of it can be seen elsewhere. For instance, of the human class II genes, the DQA and DQB genes are rarely separated by recombination and coevolve as polymorphic partners in haplotype-specific manner; the same is true of DPA and DPB, but DRA is nonpolymorphic and the DRA protein interacts with the proteins encoded by multiple DRB alleles and loci. Human b2m interacts with all classical class I alleles and loci, as well as many different MHC-like proteins including those encoded by CD1 loci gene; the b2m gene is monomorphic and located on a different chromosome from the MHC and class I-like genes. Similarly CD4, CD8, and TcR loci are all nonpolymorphic and located on different chromosomes from the MHC. However, other pressures can cause interacting proteins located on different chromosomes to become polymorphic, a situation which can lead to serious problems of interaction. For instance, NK cell receptors encoded by the killer inhibitory receptor (KIR) complex are individually polymorphic as well as the multigene family expanding and contracting to give “haplotype polymorphism,” all of which are presumably selected by interactions with pathogens. Some combinations of KIR and MHC haplotypes confer poor responses to infectious pathogens, susceptibility to autoimmunity, and reproductive difficulties (Parham, 2005; Trowsdale & Moffett, 2008). In other words, in order for two interacting genes to both become polymorphic without difficulty, in general they must be genetically closely linked so that they can be selected to coevolve. The same concept applies to the genes within the MHC, most clearly shown for the presentation system of class I molecules (Fig. 5). In typical (placental) mammals, the various components involved in peptide processing and loading are located far away from the classical class I genes and are not polymorphic. For instance, in humans, the genes for two inducible components of the proteasome and TAPs are located in the class II region, tapasin is located in the extended class II region, the third inducible proteasome component, ERp57, calnexin and calreticulin are located on different chromosomes from the MHC, and the few variants described for these genes are not reported to result in functional differences. In mice, there is one classical class I gene located in the H-2K region between the class II and the extended class II region, but there is at least one other classical class I gene far away in the class I region (the H-2D region), and the TAP, tapasin, and proteasome genes are nonpolymorphic. However, in laboratory rats (Rattus norvegicus), the only classical class I genes are located in the RT1A region between the class II and the extended class II region, and there is some haplotype-specific coevolution between the class I gene(s) and the TAP2 gene (Momberg et al., 1994; Joly et al., 1998; Hurt et
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al., 2004). There are two lineages of TAP2, with the TAP2A alleles allowing transport of peptides with any amino acid at the C-terminus, but the TAP2B alleles allowing transport of peptides with a hydrophobic amino acid at the C-terminus. This specificity is generally mirrored in the closely linked class I gene(s), and mismatch of class I and TAP2 specificity found in a natural recombinant led to changes in class I maturation and presentation of peptides to cytotoxic T cells (CTLs). The Syrian hamster (Mesocricetus auratus) may also have this structural and functional oligomorphism (Lobigs et al., 1999). In chickens, the classical MHC (the BF-BL region) has two classical class I genes, but only one is expressed at a high level, apparently due to coevolution with the TAP1, TAP2 and tapasin genes (Kaufman et al., 1995, 1999a, 1999b). Recombination across the chicken MHC is very rare, with no recombinants recovered from thousands of deliberate matings and only one “natural recombinant” known among experimental chicken lines (although there is some evidence for gene conversion or double reciprocal recombination of short stretches within the MHC). No proteasome genes have been found (yet), but the TAP1, TAP2, and tapasin genes are located very close to the two classical class I genes and are highly polymorphic and moderately diverse in sequence, with each haplotype examined (except the natural recombinant) having a unique allele differing in roughly 1% of amino acid residues (Walker et al., submitted; van Hateren et al., submitted). Moreover, they are polymorphic in function, working with a dominantly expressed class I gene in a haplotype-specific manner. Peptide translocation assays show TAP specificity at least three peptide positions, which matches or even exceeds the peptide-binding specificity of the single dominantly expressed class I molecule (Fig. 5). Although such interactions have not yet been shown directly for tapasin, the natural recombinant has been used to show that class I maturation is haplotypedependent through class I residues known in mammals to be important for interaction with tapasin. Most importantly, the coevolution between TAPs, tapasin, and the class I genes leads to a single dominantly expressed class I molecule, which, in turn, has functional consequences (Kaufman et al., 1999a, 1999b; Wallney et al., 2006; Walker et al., submitted; van Hateren et al., submitted). As an example (Fig. 6), peptides isolated from cells of
FIGURE 6 A cartoon showing that coevolution between the peptide-translocation specificity of TAP and the peptide-binding specificity of class I molecules leads to a single dominantly expressed class I molecule, as illustrated with the chicken MHC haplotype B4. Thin lines indicate peptides, a minus sign within a circle indicates negatively charged residue, a plus sign within a circle indicates positively charged residue, a circle indicates a hydrophobic residue. Adapted from Kaufman et al., 1999b.
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HOST DEFENSE: GENERAL
the B4 haplotype overwhelmingly have negative charges at anchor residues (aspartic acid and/or glutamic acid at positions 2 and 5 and glutamic acid at position 8), and models of the dominantly expressed class I molecule reveal positively charged residues in the peptide-binding domain well positioned to bind such peptides, which are totally lacking in the poorly expressed molecule. The TAP1 in B4 has positive charges in three positions, which are negatively charged in other haplotypes, and overwhelmingly, peptides bearing negative charges at positions 2 and 5 and glutamic acid at position 8 are translocated in B4 cells. In general, the peptide-binding specificity of the dominantly expressed class I molecule converges (or is exceeded by) the peptidetranslocation specificity of the TAP, which means that class I molecules encoded by other loci or haplotypes will receive few, if any, peptides, and thus, will not be utilized much by the adaptive immune system. The important functional consequence of a single dominantly expressed class I molecule is that an MHC haplotype may not be able to respond effectively to a particular pathogen if the class I molecule fails to bind a peptide that confers protection, as compared with a multigene family of MHC molecules, one of which is likely to find a protective peptide (Kaufman et al., 1995; Kaufman, 2008). In fact, the chicken MHC is known for strong associations with resistance to infectious pathogens as well as responses to vaccines, whereas the human MHC is known for strong associations with autoimmune diseases, with the few associations with infectious diseases being relatively weak, and in some cases known to involve interactions with NK cells rather than peptide presentation to T cells. Although a wide range of critical tests are required to establish that this apparent difference is real, it is at least clear for a few examples that peptide presentation by the single dominantly expressed class I molecule can determine whether a chicken lives or dies. Although the data is still patchy, many, if not most, nonmammalian vertebrates examined have the salient features for the class I system first discovered for the chicken MHC: one class I gene expressed at a high level, with polymorphic TAP and tapasin genes next to the class I gene(s), and for one fish, strong genetic associations with resistance to infectious pathogens (Kaufman, 1999). For example, the duck (Anas platyrhynchos) has five class I genes, of which only one is well expressed, adjacent to the polymorphic TAP genes (Mesa et al., 2004; Moon et al., 2005). Similarly, a single classical class I gene is located next to the TAP genes in the frogs Xenopus laevis and X. tropicalis (Ohta et al., 2006). A bony fish, Atlantic salmon (Salmo salar) has a single classical class I gene, with inducible proteasome components, tapasin, and (at least one) TAP genes close by (Grimholt et al., 2002; Lukacs et al., 2007), and cartilaginous fish as exemplified by sharks (Triakis scyllium and Ginglymostoma cirratum) have one (and in some haplotypes, two) class I genes with the proteasome components and TAP genes closely linked (Ohta et al., 2002). The situation for class II genes is less clear, although many birds have elements of the simple class II system of chickens (Jacob et al., 2000; Salomonsen et al., 2003). Thus far, there is little relevant functional data available for nonmammalian vertebrates, but for X. laevis there is an association of the MHC with resistance to a bacterial pathogen (Barribeau et al., 2008), and for Atlantic salmon there are strong genetic associations of the class I region with resistance to a virus and the class II region with resistance to a bacterial pathogen (Grimholt et al., 2003). Taking this data together, there appear to be at least two strategies of MHC genomic organization leading to differences in function. The salient features of the chicken MHC (and those of most nonmammalian vertebrates) are
most likely to represent the ancestral organization, with the typical mammalian (and some nonmammalian vertebrate) MHC organizations being derived. In this view, a large inversion occurred in the lineage leading to mammals, bringing the class III region in between what we now call the mammalian class I and class II regions, but separating the TAP, tapasin, and proteasome genes from the class I genes in the process. In fact, it appears that this event took place in the lineage leading to placental mammals, since at least one marsupial, the American opossum (Monodelphis domestica), has the MHC organization typical of nonmammalian vertebrates (Belov et al., 2006). Independent events leading to the breakup of the ancestral organization of class I, TAP, tapasin, and proteasome genes may have occurred several times. For instance, another marsupial, the Tamar wallaby (Macropus eugenii), has TAP genes in the MHC, but the classical class I genes located at the ends of several other chromosomes (Deakin et al., 2007; Siddle et al., 2009). Some passerine birds have been shown to have a multigene family of expressed classical class I genes, for which individual alleles in warblers and sparrows (great reed warbler Acrocephalus arundinaceus and house sparrow Passer domesticus) confer resistance to infectious pathogens (Westerdahl et al., 2000, 2005; Bonneaud et al., 2006). The genomic sequence of another passerine bird, the zebra finch (Taeniopygia guttata), appears to have classical class I and TAP genes on different chromosomes. The class II B genes of a bony fish, the threespined stickleback (Gasterosteus aculeatus), form a multigene family with expansion and contraction, with evidence that intermediate numbers of loci/alleles are important in mating and pathogen resistance (Wegner et al., 2003). Finally, there may be yet other strategies for MHC organization and function, of which there are two examples that express a large number of classical class I genes at a low level (Sammut et al., 1999; Persson et al., 1999; Miller et al., 2002), with only one expressed class II gene in a salamander, the axolotl (Ambystoma mexicanum), and no obviously expressed class II gene in a bony fish, the Atlantic cod (Gadus morhua). What could be the reason for the presence of a single dominantly expressed MHC molecule in so many nonmammalian vertebrates when a multigene family of MHC molecules allows a much broader response to pathogens (albeit potentially at the expense of greater autoimmunity)? The answer may be that this is the way in which the class I part of the primordial MHC first got started (Kaufman et al., 2008). The primordial class I, TAP, tapasin, and proteasome genes presumably all had functions other than the ones they now fulfill, and then evolved to interact. The simplest and most efficient way for such coevolution to take place would be for the genes to be closely linked, in much the same way that the class I, TAP, and tapasin genes have coevolved as haplotypes in the chicken MHC. Conceptually, this is the same idea proposed for the evolution of metabolic pathways at the beginning of life. The natural recombinant in the chicken MHC may be one example of catching such a coevolution in the act (van Hateren et al., submitted). The Scandinavian B19 haplotype (also known as B19var1) is a recombinant of the B12 and B15 haplotypes, with the site of recombination in the middle of the TAP2 gene. Thus far, all the genes sequenced from BG1 to the middle of TAP2 are identical between B19 and B12, while the end of TAP2 and the dominantly expressed class I gene BF2 are nearly identical between B19 and B15. Transfection of BF2 cDNAs from B15 and B19 into B15 cells showed that BF2*15 matured more rapidly and more completely than BF2*19. Given that the class I peptide motif and the translocation specificity in B15 and B19 cells have the same anchor residues, the only likely
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
mismatch is with tapasin: BF2*15 interacts well with the tapasin in B15 cells, but BF2*19 expects B12 tapasin. There are only eight differences between BF2*15 and BF2*19, of which five are changes back to the residues found in the BF2*12. Some of these residues are implicated in human class I molecules with tapasin interaction, and swapping these residues between the two molecules shows that they are enough to increase or decrease maturation. Thus, small changes can allow coevolution between genes in a newly formed haplotype, and it is precisely such small changes that are envisaged to allow the primordial class I, TAP, and tapasin genes to coevolve in order to work together as a team. Does this scenario of coevolution fit with the speculations outlined above that the first variable antigen-specific receptor was a TcR recognizing a class II-like molecule? If a class II-like gene came first, then the principles are likely to be the same, although there may have been much repurposing from a molecule sampling the intracellular vesicles for class II-dependent killer cells to a molecule primarily (although not exclusively) involved in regulation of antibody responses. Consistent with coevolution leading to a single dominantly expressed class II molecule in chickens, there are strong associations of the MHC with responses to inactivated vaccines, a single nonpolymorphic class II A gene located outside of the MHC, and two classical class II B genes, only one of which is well expressed, and polymorphic DM genes nearby with which to coevolve in a haplotype-specific manner (Jacob et al., 2000; Salomonsen et al., 2003; Kaufman et al., 2008). Overall, much remains to be clarified about class II genes throughout the rest of the vertebrates, but consistent with the coevolution model, the class II-dependent genetic associations with antibody responses in mammals (immune response or Ir genes) are relatively weak, while associations of the class II region in Atlantic salmon with responses to bacteria are very strong (Benacerraf & Germain, 1978; Grimholt et al., 2003). Whether TcR was the first variable antigen-specific receptor may also involve coevolution within the primordial MHC, as explored in the next section.
THE PRIMORDIAL MHC: THE BIRTHPLACE OF THE ADAPTIVE IMMUNE SYSTEM OF JAWED VERTEBRATES
What did the primordial MHC look like? In various invertebrates, many of the genes in and around the mammalian MHC, including the complement components of the class III region, are found close together in regions dubbed the “proto-MHC” (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004). However, with the possible exceptions of a TcR-like and perhaps a TAP-like gene (Uinuk-ool et al., 2002, 2003; Pancer et al., 2004a), no invertebrate genes have been identified as obvious direct ancestors of the essential components of the adaptive immune system. Nevertheless, genomes in vertebrates provide clues, which suggest that the genes for these essential components were originally all in the same region, allowing them to coevolve into a functioning system. As described above (Fig. 2), MHC paralogous regions have been identified in mammals and are presumed to have arisen from two rounds of genome-wide duplication at the base of the vertebrates (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). They are far from perfect copies of each other, with much evidence of regions breaking up, pieces breaking off, genes deleted, and families expanded, and most importantly, members of gene families with new functions. In humans, the classical class I (and some nonclassical) class I genes are located in the MHC on chromosome 6, the nonclassical class I
51
genes for CD1 molecules are located in the MHC-paralogous region on chromosome 1, and the nonclassical class I gene encoding an Fc receptor is located in the MHC-paralogous region on chromosome 19, but no class I-like gene is located in the MHC-paralogous region on chromosome 9. Also, homologs of certain complement component genes are found in the MHC-paralogous regions (C3, C4, and C5 on chromosome 9, 6, and 19), but the last homolog (the protease inhibitor a2-macroglobulin) is found in the NK receptor cluster (NKC) on chromosome 12, rather than in the MHCparalogous region on chromosome 1. These are just a few of the many examples taken to illustrate regions breaking up and/or pieces breaking off (a2-macroglobulin in the NKC), genes deleted (class I-like genes on chromosome 1), families expanded (many class I genes on chromosome 6, several CD1 genes on chromosome 1), and genes with new functions (Fc receptor gene on chromosome 19). Examination of the chicken MHC shows some of the same genes (Fig. 2), but many expected genes are located elsewhere in the genome (or deleted from the genome entirely) and other unexpected genes are present, overall giving an alternative view of the dynamics of the MHC paralogous regions and allowing some insight into the primordial MHC (Kaufman et al., 1999a; Salomonsen et al., 2005; Rogers et al., 2005, 2008). For instance, a pair of lectin-like genes were identified in the original sequence of the chicken MHC (the BF-BL region) and, on the basis of genomic organization and expression patterns, shown to correspond to pairs of genes in the NKC (particularly the NK receptor gene NKR-P1 and its ligand LLT1 in humans, or Clr in mouse and rat). In contrast, only two lectin-like genes were identified in the chicken region corresponding to the NKC. Later, two genes encoding chicken CD1 were located immediately adjacent to the sequenced portion of the chicken MHC. The simplest explanation for the presence of both NK receptors and CD1 in the chicken MHC is that they were both present in the primordial MHC before the two rounds of genome-wide duplication, and were silenced in different genomic regions in the lineage leading to birds versus the lineage leading to mammals. With this idea in mind, traces of other gene families present in the MHC become understandable. An example recognized early on (Kasahara et al., 1997) is the presence of lectin-like genes CD23 and DC-SIGN in the MHC-paralogous region on human chromosome 19 and many other lectin-like genes, expressed in NK/T cells and myeloid cells, located in the NKC on human chromosome 12. Similarly, the lymphocyte receptor region (LRC) on human chromosome 19 contains many homologous genes, including the KIR genes expressed primarily on NK cells, and a variety of homologs (including an Fc receptor) expressed on cells of the myeloid lineage (Martin et al., 2002). A homolog called NKp30, a member of the natural cytotoxicity receptor (NCR) genes, is located in the human MHC (Moretta et al., 2001). Conversely, two class I-like Mill genes are found just outside the LRC in mice (Kasahara et al., 2002). This fits well with the suggestion that the regions syntenic to the MHC, the NKC and the KIR genes were originally next to each other based on the arrangement in the tunicate Ciona intestinalis (Olinski et al., 2006; Zucchetti et al., 2009). A most intriguing example is a gene related to TcR genes found on the edge of the MHC of the frog X. tropicalis (Ohta et al., 2006). Such examples suggest that all of these gene families were located together in the primordial MHC before the two rounds of genome-wide duplication. The two concepts of paralogous regions and coevolution taken together make a powerful prediction (Kaufman et al., 2008). The presence of MHC, NK receptor, and TcR gene
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families in the same genomic locations suggests that genes for receptors and ligands originated in the same genomic region (Fig. 2), presumably to allow coevolution so that they would function together, much in the same way as was outlined above for coevolution of disparate genes to form the class I presentation pathway. In this view, the adaptive immune system of jawed vertebrates arose in a single genomic region, the primordial MHC, which has been breaking apart ever since.
CONCLUSIONS
Some 50 years of research have shown that the adaptive immune system of jawed vertebrates appeared once at least 500 million years ago. Many important events involve drastic changes in the genome, including transposon insertion to create split V genes, horizontal transfer of recombination activating genes (RAGs), inversions and translocations of DNA to create MHC genes, genome-wide duplications to create genes for new functions, and further reorganizations to alter MHC function. Throughout the evolution of this adaptive immune system, coevolution of linked genes was important for different genes to begin to work together, a process still evident in the coevolution of antigen presentation systems in most nonmammalian vertebrates. The best guess is that the birthplace of this adaptive immune system was the primordial MHC, which has been falling apart ever since. Thanks to John Chattaway, Aimee Parker, Andrew Chan, and Hannah Siddle for reading the manuscript, and to the Wellcome Trust (089305/Z/09/Z) for support.
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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
4 Host Defense (Antimicrobial) Peptides and Proteins LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK
DEFENSINS
INTRODUCTION
Defensins are a family of small cationic peptides ranging from 3 to 5 kDa in size and possessing a -sheet structure containing three intramolecular disulfide bonds between six conserved cysteine residues (Froy, 2005). Beyond this pattern, peptides within the defensin family are highly variable. In mammals, over 50 defensin members have been identified (Yang et al., 2001). Defensins members are categorized into three families based on their sizes and patterns of disulfide bonding: the -, -, and (among the higher primates only) -defensins (Brown et al., 2007). Humans only possess the - and -defensin subfamilies (Brown et al., 2007). To date, six -defensins, HNP1–4 and HD5–6, and six -defensins, hBD1–6, have been identified in humans (Yamasaki & Gallo, 2008; Brown et al., 2007).
The innate immune response is an ancient defense system against pathogens that is evolutionarily conserved among plants, invertebrates, and vertebrate animals. It is characterized by a rapid, highly effective response against foreign microbial invaders. The effectiveness of innate immunity stems from many multifaceted processes working in concert to rapidly eliminate a threat. The detection of pathogens by sentry cells immediately leads to the triggering of inflammation, the effector arm of innate immunity. Inflammation leads to increased trafficking of immune cells, including phagocytes, antigen-presenting cells, and mast cells, toward the site of infection where they initiate microbial killing or further potentiate the inflammatory response, reinforcing cell recruitment. Pathogen clearance is mediated by a complex set of strategies, ranging from the ingestion of microbes by phagocytes to the production of antimicrobial molecules, including reactive chemical species or lytic compounds. Another strategy is the production of cationic host defence (antimicrobial) proteins and peptides, compounds that play an important role in innate immunity, not only as antimicrobial agents, but also as immune regulators.
Localization and Regulation
Defensins are found widely within the body depending on the family and type of defensin involved. In general, defensins are found in the granules of leukocytes and in the epithelia of the human body (Menendez & Finlay, 2007). HNP1–4 -defensins are prominent in the granules of neutrophils, from which they were first isolated (Yang et al., 2001), and also exist in the granules of other leukocytes including monocytes, lymphocytes and natural killer (NK) cells, and in intestinal Paneth cell granules, where they are secreted into intestinal crypts (Froy, 2005). The enteric -defensins HD5 and HD6 are found in the granules of epithelial cells, including Paneth cells and the cells of the urogenital tract. In contrast, -defensins are expressed in epithelial cells that line the esophagus, intestines, skin, kidney, pancreas, and gut (Brown et al., 2007), whereas hBD5 and hBD6 are found in the epididymis and airway epithelia (Froy, 2005). Unlike -defensins, epithelial -defensins are not stored in cytoplasmic granules (Brown et al., 2007). The production of defensins by the body is variable and depends on many factors, including the defensin class, cell type, and environmental stimuli. -Defensins are constitutively expressed in cell granules (Menendez & Finlay, 2007). In contrast, many -defensins are inducible by a variety of environmental factors, including microbial components and/or host inflammatory mediators, indicating that -defensins might play a role in the host antimicrobial or inflammatory response (see Table 1).
CATIONIC HOST DEFENSE PEPTIDES
Cationic host defense peptides (HDPs) encompass a diverse family of peptides that play a role in the antimicrobial innate immune response of many species. They are generally defined as peptides 12 to 50 amino acids long, containing up to 50% hydrophobic residues and 2 to 9 positively charged arginine or lysine residues (Brown & Hancock, 2006). The two major classes of HDPs found in mammals are the defensins and cathelicidins, which are produced by immune cells, including leukocytes and epithelial cells. Originally thought to play a direct antimicrobial role against pathogens, it has become evident that cationic peptides also modulate the many functions of innate immunity (Fig. 1). Laurence Madera, Shuhua Ma, and Robert E. W. Hancock, Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
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FIGURE 1 The multiple roles of host defense peptides in the immune response.
Numerous studies have shown -defensin induction in epithelial cells in response to bacterial infections (Froy, 2005; Menendez & Finlay, 2007). Specific bacterial components, including lipopolysaccharide (LPS) of gram negative bacteria and peptidoglycan of bacterial cell walls, lead to production of -defensins (Menendez & Finlay, 2007). In addition, host factors produced in response to infections play a large role in -defensin induction, especially cytokines that are cell-to-cell signalling mediators essential in the coordination of the immune response. Interleukin 1 (IL-1) and tumor necrosis factor (TNF-), cytokines that promote acute inflammation, are strong inducers of -defensin expression (Yamasaki & Gallo, 2008). Supporting the link between -defensin induction and infection is the finding that regulation of -defensin expression is dependent on signaling pathways activated in an immune response. In epithelial cells, hBD2 induction is dependent on the transcription factors NF-B and AP-1, both essential for the transcription of many inflammatory mediators (Froy, 2005). This is supported by the finding that hBD2 expression in an atopic dermatitis model is downregulated by IL-10, a suppressor of NFB activity (Menendez & Finlay, 2007). The induction of hBD3 is also dependent on the activation of the mitogen activated protein kinase (MAPK) signalling pathways involving p38 kinase and ERK-1/2, central mediators of many cellular processes including inflammation (Menendez & Finlay, 2007). -Defensins can be induced by TLRs, which are a major subset of pathogen recognition receptors that detect microbes. For example, in the lung, activation of TLR2, TLR3, TLR4, and TLR9 can lead to hBD2 and hBD3 expression, whereas in the intestine,
activation of TLR4 and the complex of TLR2 and TLR6 leads to hBD2 induction (Froy, 2005). There is a great deal of evidence that -defensin regulation is linked to the detection and progression of a microbial infection. However, the variety of roles that defensins play in the immune response is far less understood.
Antimicrobial Activity
Defensins have generated a large amount of interest due to their modest in vitro antimicrobial activity against a wide range of microorganisms, including enveloped viruses, fungi, and bacteria (Brown et al., 2007). This direct antimicrobial is likely very significant under circumstances where defensins are found in high concentrations, such as the crypts of the intestine and the granules of neutrophils where milligram per milliliter concentrations are found. Defensins also possess antiviral properties, such as against adenovirus, papilloma virus, herpes simplex virus (HSV), and human immunodeficiency virus (HIV) (Yamasaki & Gallo, 2008), and have modest antimicrobial activity against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (Chen et al., 2006). hBD3 is more potent and has a broader spectrum of antimicrobial activity against organisms including fungi and gram-positive bacteria, such as Staphylococcus aureus, possibly due to its higher net charge relative to hBD1 and hBD2 (Brown et al., 2007). Although the mechanism of antimicrobial activity by defensins has not been fully elucidated, it is believed that the positive charge of defensins allows them to bind to negatively charged molecules on microbial membranes, such as LPS and lipoteichoic acid (LTA) in gram-negative
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TABLE 1 Human cationic host defense peptides and their regulation and functions in the immune response Host defense peptide
Regulation
Immune functions
-Defensins
Constitutive
Binds and neutralizes bacterial toxins
-Defensins
Inducible in epithelia by bacterial infection: S. aureus, Campylobacter jejuni, H. pylori, and Mycobacterium bovis Inducible by LPS and peptidoglycan Inducible by inflammatory cytokines IL-17, IL-1, and TNF- Suppressed by anti-inflammatory cytokine IL-10
Chemotactic for T-lymphocytes, immature dendritic cells, mast cells, and monocytes Promotes chemokine production and mast cell degranulation Promotes production of inflammatory cytokines Promotes T-cell proliferation Promotes wound healing and regulation of the extracellular matrix
hCAP18/LL-37
Constitutive in leukocyte granules; Inducible in epithelia by bacterial infection H. pylori Inducible in epithelia by inflammatory cytokines IL-1 and IL-6 Suppressed by Shigella.
Chemotactic for monocytes, neutrophils, and T-lymphocytes Promotes chemokine production and induces mast cell degranulation Inhibits neutrophil apoptosis Suppresses of LPS-induced inflammation Regulates dendritic cell maturation and TH polarization of adaptive immunity; Promotes wound healing and angiogenesis
and gram-positive species respectively. This defensin interaction with the membrane leads to translocation across the outer membrane and access to this membrane where either the disruption of membrane integrity results in increased membrane permeability or action on other targets leads to death. The importance of defensin-mediated bacterial killing during an immune response is a point of contention, except under the previously mentioned circumstances when defensin concentrations are very high, since it has been demonstrated that in vitro defensin antimicrobial activity is dramatically reduced in a physiological environment. For example, the addition of 100 mM NaCl, approximately equivalent to the concentration found in airway fluid and blood, reduces hBD2 activity 5-fold to 500-fold and completely abolishes hBD1 activity against gram-negative bacteria (Bowdish et al., 2006). In addition, defensins are sensitive to the presence of divalent cations. It has been shown that -defensin activity is compromised by Mg21 and Ca21 at concentrations as low as 0.5 mM (Bowdish et al., 2006) even though many bodily fluids (including airway fluids, plasma, and sputum) contain Mg21 and Ca21 at concentrations of around 1 mM. Under many circumstances the physiological concentrations of defensins are too low to manifest any significant antimicrobial activity. hBD1 displays antimicrobial activity at concentrations around 1 μg/ml only under low ionic strength conditions that do not exist in vivo; however, the concentrations of hBD1 found in bronchoalveolar fluid is 10-fold lower, at 0.1 μg/ml (Bowdish et al., 2005). Thus, it is likely that defensins play a direct antimicrobial role in a limited number of environments, such as in the phagocytic granules of neutrophils and intestinal crypts where defensin concentrations can reach up to milligrams per milliliter, overcoming any ionic antagonism, or under circumstances where the ionic strength is relatively low (Froy, 2005). The antiviral effects of human defensins are far less understood. Defensins have been postulated to bind to viral
surface molecules, potentially inhibiting their entry and invasion mechanisms. Human -defensins have been shown to bind to viral glycoproteins, molecules integral to the viral invasion process, such as HSV glycoprotein B and HIV gp120 (Yamasaki & Gallo, 2008; Klotman & Chang, 2006). However the antiviral activity of defensins also varies with the physiological environment. For example, HNP-1 is able to directly neutralize HSV and HIV virions in a serum-free environment, an effect that is abolished with the addition of serum (Klotman & Chang, 2006). Interestingly, in the presence of serum, HNP-1 acts on the host cell, inhibiting HIV viral growth following the invasion and reverse transcription stages. Indeed, defensin antiviral activity can be attributed to many cell-mediated effects, including the regulation of host surface receptors used by viruses for invasion, or modulating cellular signaling pathways required for viral growth (Jenssen et al., 2006; Klotman & Chang, 2006). Although in physiological conditions and peptide concentrations the ability to act directly against pathogens is limited, recent studies show that defensins are still able to coordinate many aspects of the host immune response.
Immunomodulation
The pathogenesis of a microbe can be divided into a series of stages, which include the initial invasion by the microbe, the inflammatory innate immune response, the transition into the adaptive immune response, and the resolution of infection (or the establishment of chronic infections, or death of the host). It is becoming evident that defensins play a major role in coordinating each of these processes (see Table 1). Defensins have potent neutralizing functions against certain bacterial toxins, limiting pathogenesis at the early stages of infection. For example, HNP1–3 are able to bind and sequester the lethal factor (LF) of Bacillus anthracis from host cells, thus preventing potential cell death, and can also competitively inhibit diphtheria toxin and P. aeruginosa exotoxin A, preventing the ADP-ribosylation of host elongation factor-2 (Brown et al.,
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2007). HNP1-3 is also able to inhibit the binding of S. aureus staphylokinase to host plasminogen, preventing fibrinolysis and the spread of infection (Brown et al., 2007). Defensins also play a role in cell recruitment, influencing the ability of immune effector cells to migrate to the site of infection, which is a mainstay of the innate immune system and impacts all subsequent responses. The -defensins are directly chemotactic for naïve CD4 and CD8 T-lymphocytes as well as immature dendritic cells and monocytes (Yang et al., 2001). hBD1 and hBD2 are also chemotactic for immature dendritic cells and memory T-lymphocytes, while hBD3 is capable of attracting immature dendritic cells, mast cells, and monocytes (Menendez & Finlay, 2007; Yang at al., 2001). Similarly, hBD4 can directly chemoattract mast cells and monocytes (Froy, 2005). As defensins are secreted (e.g., through phagocyte degranulation) or induced at infectious sites, it is likely that defensins enhance the ability to chemoattract immune effector cells to the site of infection. In addition to directly promoting cellular mobilization, defensins induce the production of cytokines and chemokines, which potentiate cell recruitment. For example, -defensins can induce the production of neutrophil chemokine IL-8 in alveolar macrophages and lung epithelial cells as well as cause the degranulation of mast cells and histamine release, promoting permeabilization of blood vessels and further cell recruitment (Yang et al., 2001). hBD3 and hBD4 can also cause degranulation of mast cells (Menendez & Finlay, 2007). Defensins also induce the production of inflammatory cytokines by immune cells. For example, -defensins induce the production of proinflammatory cytokines IL-6 and IFN (gamma interferon) by T lymphocytes and IL-1 and TNF- by human monocytes (Froy, 2005). The -defensins hBD1 and hBD4 induce keratinocytes to produce IL-18, a cytokine that promotes the recruitment of neutrophils and the activation of cell-mediated immunity (Yamasaki & Gallo, 2008). Defensins can also promote the production of regulatory cytokines to balance the inflammatory response. In particular, -defensins stimulate the production of the immunosuppressive cytokine IL-10 in T lymphocytes (Froy, 2005). Defensins also contribute to the adaptive immune response by influencing lymphocyte activity. For example, in murine models, HNPs enhance the proliferation of CD4 T-lymphocytes stimulated with CD3 activating antibodies (Lillard et al., 1999). Correlated in part with this finding, HNPs enhance antigen-specific humoral and cellular responses in mice treated with antigen, indicating an adjuvant role for defensins (Bowdish et al., 2006). Defensins also contribute to wound healing; in particular, hBD2–4 promote the migration of keratinocytes and enhance their proliferation, suggesting that -defensins promote reepithelialization of a wound area, while HNP1 promotes extracellular matrix formation by increasing levels of pro-alpha collagen and decreasing levels of matrix metalloproteinase-1, an enzyme that cleaves the extracellular matrix (Yamasaki & Gallo, 2008). Thus it is known that defensins extensively regulate many immunological processes, but the overall importance of defensins in immunity during an infection or inflammation is still inadequately understood.
Defensins in Human Disease
Attempts to determine the exact roles of each defensin in the overall immune response by knockout studies have not been successful due to the functional redundancy by other defensins and immune mechanisms. Indeed, mice for example contain at least 54 -defensins and 44 -defensins (Amid et al., 2009). However, a substantial number of immunological disorders are correlated with defensin dysregulation, providing tantalizing clues as to the roles of defensins in immunity.
Defensins are upregulated in inflammatory diseases of the bowel, lung, and skin. For example, hBD2 is significantly upregulated in cases of psoriasis, an autoimmune disease of the skin (Yamasaki & Gallo, 2008). Although no causal relationship has been established between defensin dysregulation and inflammatory disease, either defensins or their regulation may be essential for inflammatory homeostasis. Interestingly, HD5–6 downregulation correlates with ileal Crohn’s disease, an inflammatory disorder of the gastrointestinal tract, while a genetic polymorphism leading to low copy number of hBD2 associates with colonic Crohn’s disease. It is hypothesized that the enteric defensins are involved in maintaining control over the normal flora and thus establishing homeostasis in the gut (Menendez & Finlay, 2007), although it is equally possible that their immunomodulatory role is influential. Altered defensin levels are also correlated with differential susceptibility to infections. Psoriatic patients with elevated defensin levels have decreased susceptibility to infection relative to atopic dermatitis patients with reduced defensin levels (Yamasaki & Gallo, 2008). Animal studies also support the role of defensins in the clearance of pathogens. (i) In a mouse model of peritoneal Klebsiella pneumoniae infection, very small doses of HNP1 (4 ng to 4 μg) caused protection that was more likely due to an increase in leukocyte accumulation than to direct antimicrobial activity. Similar results were observed in S. aureus thigh infections (Welling et al., 1998). (ii) Administration of porcine -defensin BD-1 in piglets protected them against a Bordetella pertussis challenge (Menendez & Finlay, 2007). (iii) Overexpression of rat -defensin BD-2 in the lungs increased protection against P. aeruginosa induced pneumonia (Menendez & Finlay, 2007). (iv) BD-1 knockout mice showed delayed clearance of Haemophilus influenzae in the lungs and increased levels of Staphylococcus sp. in the bladder (Menendez & Finlay, 2007).
CATHELICIDINS
Cathelicidins are a family of peptides produced from a precursor polypeptide containing a conserved N-terminal signal and propeptide sequence encompassing approximately 100 amino acids (termed the cathelin domain), as well as a highly variable C-terminal peptide domain (Zanetti, 2004). The N-terminal signal sequence allows for targeting of the polypeptide to the endoplasmic reticulum for intracellular storage (Zanetti, 2004). The cathelin domain is essential for the storage of the polypeptide, preventing its degradation by proteases and inhibiting the biological activity of the Cterminal peptide domain (Zanetti, 2004). During excretion of the precursor polypeptide from cells, the C-terminal peptide domain is cleaved, resulting in a mature cathelicidin of 12 to 80 amino acids in length (Zanetti, 2004). In mammals, more than 50 cathelicidin members have been identified according to UniProt, a comprehensive database of protein sequence and function (Uniprot Consortium, 2008). Among the different members of the cathelicidins family, there is very little structural homology in the mature cathelicidin peptide domain. The mature peptide can fold into a variety of secondary structures, including -helices, -hairpins, and looped structures (Brown et al., 2007). In humans, only one cathelicidin has been found, an 18-kDa precursor human cationic antimicrobial protein, or hCAP18, which is processed into a variety of molecules, including prominently the 37-amino-acid peptide LL-37 (Zanetti, 2004).
Localization and Regulation
In the human body, hCAP18 is found in a range of cellular environments. Although originally isolated and identified in the granules of neutrophils, it can also be found in the
4. Host Defense (Antimicrobial) Peptides and Proteins
granules of many leukocytes including NK cells, monocytes, mast cells, B-lymphocytes, and T-lymphocytes (Dürr et al., 2006). It is also constitutively expressed in epithelial cells, including those of the airways, mouth, esophagus, and intestine, and is inducible in keratinocytes (Zanetti, 2004). In addition, LL-37 is a component of most secreted bodily fluids, including sweat, gastric juices, airway fluids, seminal plasma, and breast milk (Dürr et al., 2006). Although hCAP18 is normally expressed in many intracellular environments, the levels of hCAP18 can vary and are regulated during inflammation and infection. Regulation of hCAP18 can occur at different stages, including the expression and production of the precursor polypeptide and the cleavage of the mature C-terminal peptide domain. Increased expression of hCAP18 is regularly observed during inflammation (its production during infection may indeed be related to this); hCAP18 can be modestly induced in response to the presence of host inflammatory cytokines, IL-1, and IL-6. Also, LL-37 is substantially induced in myeloid cells by the hormonal form of vitamin D3 [1,25(OH)2D3] and, interestingly, critically ill septic patients had significantly lower plasma 25(OH)D concentrations compared to healthy controls (Jeng et al., 2009). Processing of hCAP18 into the active peptide LL-37 can also be regulated. In humans, the enzymes responsible for hCAP18 processing vary between cellular environments. In neutrophil granules, hCAP18 is processed by the serine protease proteinase 3, whereas in seminal fluid and under acidic concentrations such as those found in vaginal fluid, gastricsin (Pepsinogen C) is responsible for cathelicidin processing (Zanetti, 2004). On the skin surface, the stratum corneum tryptic enzyme SCTE processes hCAP18 into LL-37 (Yamasaki & Gallo, 2008). In vitro studies using bovine neutrophil cathelicidins Bac5 and Bac7 demonstrate that the addition of small amounts of elastase results in the complete processing of their pro-forms within minutes (Zanetti, 2004). The addition of elastase inhibitors prevents the maturation and activation of these cathelicidins (Zanetti, 2004). This suggests that the maturation of cathelicidin precursors is intricately linked to the regulation of their cognate processing enzyme. Interestingly, cathelicidin levels can also be directly influenced by microbial pathogens. Cleavage of hCAP18 into LL-37 can be triggered by elastase-producing pathogens such as P. aeruginosa (Zanetti, 2004). In contrast, gut epithelial biopsies of Shigellainfected patients show low expression of hCAP18, suggesting a possible Shigella virulence strategy (Zanetti, 2004). In vitro studies support this finding by demonstrating that hCAP18 expression is downregulated in monocytes and epithelial cell lines infected with Shigella (Zanetti, 2004). It is evident that microbial infections directly regulate hCAP18/LL-37 levels via many complex avenues. In turn, hCAP18/LL-37 plays a multifunctional role in the immune response against pathogens.
Antimicrobial Activity
In a dilute medium such as phosphate buffer, the mature human cathelicidin peptide LL-37 exhibits broad-spectrum in vitro antimicrobial activity against many pathogens, although it is arguable as to whether this activity is relevant in vivo. LL-37 is antimicrobial against gram-positive bacteria, including Streptococcus sp., Listeria monocytogenes, and S. aureus, and gram-negative bacteria, including E. coli, P. aeruginosa, and Salmonella enterica serovar Typhimurium (Dürr et al., 2006), and the fungus Candida albicans (Dürr et al., 2006). LL-37 also displays moderate antiviral properties, being able to slow the growth of vaccinia virus, but having only a mild effect on HSV infection (Dürr et al., 2006). However, as
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with the defensins, LL-37 activity is sensitive to the presence of monovalent and divalent cations and its direct antimicrobial effects are severely abrogated under physiological conditions. In the presence of 100 mM sodium, LL-37 antimicrobial activity is reduced by twofold to eightfold, whereas in cell culture medium containing physiological concentrations of salts, 1 mM Mg21 and Ca21 and 150 mM NaCl, LL-37 at concentrations of up to 100 μg/ml showed no effect against S. aureus or serovar Typhimurium (Bowdish et al., 2006). To put this in context, LL-37 is normally secreted in the bronchoalveolar fluid of infants at approximately 5 μg/ml, or 10 to 15 μg/ml in children with severe inflammatory disorders (Bowdish et al., 2005). Other host factors can also interfere with the antimicrobial activity of LL-37. Apolipoprotein 1, a human serum protein, has been shown to inhibit the antimicrobial actions of LL-37 (Bowdish et al., 2006). Glycosaminoglycans like heparin strongly inhibit activity. Indeed, the direct antimicrobial role of LL-37 is likely limited to conditions where its concentration is sufficient to overcome the previous limitations, for example, in seminal fluid or in neutrophil granules (Zanetti, 2004). Although many physiological environments do not harbor the optimal in vitro conditions for LL-37 antimicrobial activity, under these discussed conditions, LL-37 is a multifunctional modulator of the host immune response.
Immunomodulation
LL-37 plays multiple roles during an immune response (see Table 1). It is a potent enhancer of immune cell trafficking. LL-37 is directly chemotactic for monocytes, neutrophils, and T-lymphocytes by engaging the G-protein coupled receptor, FPRL-1 (Yamasaki & Gallo, 2008). LL-37 also enhances immune cell recruitment by other routes (e.g., mast cells are directly chemoattracted through a separate Gprotein coupled receptor). LL-37 also induces chemokine production. Murine models have demonstrated that the administration of LL-37 increases levels of the monocyte chemoattractant protein 1 (MCP-1) (Dürr et al., 2006). In vitro studies show increased production of chemokines such as CXCL-1, MCP-1, MCP-3, and IL-8 (among others) in LL-37-stimulated human peripheral blood mononuclear cells (Bowdish et al., 2006; Mookherjee et al., 2009a). LL-37 can also play a balancing role in the immune response, having both pro- and anti-inflammatory functions. LL-37 can promote inflammation and cell-mediated immunity by inducing IL-18 in human keratinocytes, as well as by enhancing the processing of IL-1 in LPS-stimulated human monocytes (Yamasaki & Gallo, 2008). The presence of LL-37 also leads to mast cell degranulation resulting in the release of chemoattractants, prostaglandins, and histamine release (Dürr et al., 2006). LL-37 can also work in tandem with host mediators to selectively modulate inflammation and cell trafficking. Synergy between LL-37 and IL-1 in human peripheral blood cells has been observed, leading to a significant increase of cytokine production, including IL-6 and IL-10, and production of chemokines MCP-1 and MCP-3 (Yu et al., 2007). LL-37 also potentiates the production of proinflammatory cytokines TNF- and IL-6 in peripheral blood cells stimulated by unmethylated CpG DNA, a DNA motif frequently found in the bacterial genome and an activator of human TLR9 (Yu et al., 2007). In contrast, LL-37 is a strong suppressor of LPS-induced inflammation, preventing host damage from a systemic inflammatory response. It inhibits LPS-mediated induction of inflammatory mediators, including TNF- and reactive oxygen species in macrophages, and IL1-, TNF-, IL-6, and IL-8 in human peripheral blood cells (Mookherjee et al., 2006). Consequently, LL-37 is able
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to increase the survival of rats in LPS-induced sepsis models (Dürr et al., 2006). Although the LPS-suppressing effects of LL-37 were originally attributed to its capacity to bind LPS, various studies have shown that LL-37 inflammatory regulation is much more complex and likely cell mediated, especially since it can act 30 to 60 min after LPS treatment of cells. Indeed, LL-37 can also limit the production of IL-1, TNF-, IL-6, and IL-8 in TLR2-stimulated human peripheral blood cells (Mookherjee, 2006). These observations suggest that LL-37 might play an important role in coordinating the inflammatory reaction in response to different pathogens. LL-37 also plays a role at the transition between the innate and adaptive immune system and has been demonstrated to have adjuvant activity. It can increase coreceptor CD86 expression and endocytic activity (Brown & Hancock, 2006). LL-37 promotes the polarization of human monocyte-derived dendritic cells to Th-1 type responses by enhancing the secretion of the Th-1 cytokines IL-12 and IL-6 and decreasing the levels of the Th-2 cytokine IL-4, as well as proinflammatory cytokines, in LPS-stimulated dendritic cells (Brown & Hancock, 2006). In addition to its immune regulatory effects, LL-37 is a promoter of wound healing. Epithelial wounds typically display increased levels of local LL-37, which is inducible by various growth factors, leading to increased attraction of keratinocytes (Dürr et al., 2006). Indeed, LL-37-specific antibodies inhibited epithelial healing in a human skin wound model (Yamasaki & Gallo, 2008). LL-37 also promotes angiogenesis in epithelia, which may play a role in reepithelialization (Yamasaki & Gallo, 2008), while its ability to modulate inflammatory activity may also assist in wound healing.
hCAP18/LL-37 in Human Disease
Efforts to characterize the overall role of LL-37 in the immune system have been based on in vivo models and correlational studies linking LL-37 dysregulation to immune disorders. Some immune disorders suggest LL-37 is required for the proper clearance of pathogens, although it is uncertain whether these studies reflect direct antimicrobial activity, as several authors assert, or immunomodulatory activities. As with hBD2, lower expression of hCAP18 is found in atopic dermatitis patients relative to psoriatic patients, which may explain their differential susceptibilities to infection. Patients with morbus Kostmann, a neutropenic disorder, are treated with cytokine G-CSF to restore circulating neutrophils with normal oxidative responses. However, these neutrophils are deficient in LL-37 and this may correlate with the chronic periodontal disease and excessive oral inflammation observed in these patients (Brown et al., 2007). Murine models show that knocking out CRAMP, a murine cathelicidin structurally related to LL-37, increases the susceptibility to skin infections by group A Streptococcus (Zanetti, 2004).
MECHANISMS OF ANTIMICROBIAL KILLING
HDPs possess in vitro antimicrobial activity, ranging from modest (many defensins and LL-37) to potent (e.g., pig protegrin), against an extensive range of microbes including bacterial, viral, and fungal pathogens. However, the mechanisms utilized by HDPs to elicit their direct and broad-spectrum actions are quite complex and likely involve multiple targets. For bacteria, it is known that HDPs interact with negatively charged components on microbial membranes, such as LPS or LTA, and then translocate to the cytoplasmic membrane. Although it is known that HDP interaction with the membrane is a prerequisite for killing, there are many possible results leading to bacterial death, namely the disruption of membrane integrity, interference with essential processes
dependent on membrane-bound enzymes (cell division, cell wall synthesis), or translocation across the membrane to access cytosolic targets. Four models have been proposed to address peptide-membrane interactions, as reviewed by Jenssen et al. in 2006: (i) the toroidal pore, (ii) barrel-stave, (iii) carpet, and (iv) aggregate models. In the toroidal pore model, peptides are thought to intimately associate with the head groups of lipids and reaggregate into the bacterial membrane in a perpendicular orientation, resulting in the formation of a peptide-lipid pore, thus increasing membrane permeability. Certain peptides that form -helical structures in a membrane setting, such as LL-37, have been proposed to act in this fashion. In the barrel-stave model, peptides cluster on the membrane, also in a perpendicular fashion. In this case, the hydrophobic regions of the peptides interact with fatty acyl chains of membrane lipids, and their hydrophilic regions face one another, forming a pore in the microbial membrane. The carpet model, unlike the previous two, suggests that peptides remain aggregated on the membrane surface in a parallel fashion, resulting in localized peptide clusters. At high peptide concentrations the aggregates form micelles with areas of the membrane, leading to a disruption of membrane integrity. None of these models, however, really considers peptide translocation, which is more common than previously supposed, having been experimentally observed with many HDPs. One model to explain both membrane disruption and translocation is the aggregate model. In this model, peptides aggregate on the membrane surface and at a critical concentration form complexes (mixed micelles) with the membrane lipids, permitting increased membrane permeability (which for some peptides might cause death) and allowing for the translocation of peptides across the bilaye
