Nod1 and Nod2 in innate immune responses, adaptive immunity and bacterial infection.
By
Lionel Le Bourhis
A thesis submitted in conformity with the requirements of the degree of
Doctor of Philosophy
Graduate Department of Immunology
University of Toronto
Nod1 and Nod2 in innate immune responses, adaptive immunity and bacterial infection.
Lionel Le Bourhis, PhD thesis, 2009, Departmenent of Immunology, University of Toronto
Abstract
The last decade has been witness to a number of seminal discoveries in the field of innate immunity. The discovery that microbial molecules and endogenous danger signals can be detected by germ-line encoded receptors has changed the way we study the immune system. Indeed, the characterization of Toll in Drosophila as a sensor of microbial products in 1997 then led to the discovery of a family of Toll Like Receptors (TLRs) in mammals. TLRs are critical for the induction of inflammatory responses and the generation of a successful adaptive immune response. The array of ligands that these transmembrane proteins recognized mediates defense against bacteria, viruses, fungus and parasites, as well as, possibly, cancerous cells.
In addition to this membrane-bound family of recognition proteins, two families of pattern recognition receptors have been recently shown to respond to microbial and chemical ligands within the cytosol. These represent the Nod Like Receptors (NLRs) and RIGI-like helicase receptor
(RLH) families. Nod1 and Nod2 are members of the NLR family of proteins, which are responsible for the recognition of components derived from the bacterial cell wall, more precisely, moieties of peptidoglycan. As such, Nod1 and Nod2 are implicated in the recognition and the defense against bacterial pathogens. Importantly, the genes encoding these two proteins have also been linked to the etiology of several inflammatory disorders such as Crohn’s disease and asthma.
In this thesis, we show that recognition of Nod1 and Nod2 ligands generates a rapid and transient inflammatory response in vivo. When co-injected with a model protein, Nod1 and Nod2 ligands exhibit adjuvant properties that lead to the generation of an antigen-specific Th2 type adaptive immune response. Surprisingly, recognition of the Nod1 ligand in non-hematopoietic cells
ii is critical for the generation of this immune response. In contrast, TLRs classically tip the balance towards a Th1 response and interestingly, co-injection of TLR and Nod ligands synergize to generate a more potent immune response characterized by the generation of Th1, Th2 and Th17 T cell respones.
To study the role of Nod1 and Nod2 in the context of a bacterial infection in vivo, we used an intestinal mouse pathogen, Salmonella enterica serovar Typhimurium . We were able to show that
Nod1-deficient mice, but not Nod2-deficient mice, are more susceptible to the strain of this bacterium, which enters the host through the active pickup in the intestinal lumen by underlying myeloid cells. This sampling mechanism is mediated by a subset of dendritic cells that populate the intestinal lamina propria. Accordingly, the defect seen in Nod1-deficient mice localizes to the mucosal barrier where these dendritic cells appear to have an impaired response towards the bacteria.
Taken together, these results increase our knowledge on the general role of Nod1 and Nod2 in immunity and might generate new avenues of research and potential therapeutic targets.
iii Acknowledgements
I would like to thank Dr Dana Philpott and Dr Stephen Girardin for their support, guidance and friendship. You gave me way more than the tools to be a good scientist.
I am also greatful for the help that I received coming to Toronto by my committee members Dr Tania Watts and Dr James Carlyle, Rejeanne Puran and the graduate student of the department of immunology. I shouldn’t forget the members of the laboratories past and present, especially Jörg and Joao for science and much more.
Je voudrais remercier mes parents, Chantal et André, pour leur confiance, une denrée dont tout scientifique à besoin. Je ne serais pas qui je suis sans mes trois frères, Gaël, Joël et Mikaël ; je les remercie pour m’avoir poussé à réussir, ils m’ont fait aimer le travail d’équipe et avoir un esprit compétitif, qualitées importantes chez un scientifique. Je voudrais remercier les amis qui m’ont accompagné, pour certains depuis longtemps. Enfin je voudrais remercier Julie pour sa patience, indispensable dans la vie d’un scientifique.
Work for this thesis have been supported by the Pasteur Institut, l’Association François Aupetit, la Fondation pour la Recherche Médicale, as well as the Canadian Institutes of Health Research (CIHR) and the Howard Hughes Medical Institute (HHMI).
2 rights make 1 wrong…
iv Table of Content
Abstract 2
Acknowledgement 3
Table of content 4
List of Publications 8
List of Figures and Tables 9
List of Abbreviations 11
Chapter I: Introduction 14
1. Innate immune recognition 15
A. TLRs 15
1) Structure and signaling. 15
2) Ligands specificity 18
B. NLRs 21
1) Structure and signaling. 21
2) Ligands specificity 25
C. Other sensors 27
2. Role of the NLR in the immune response 28
A. Barrier and antimicrobial functions 29
B. Role in inflammation and cellular activation 30
C. Impact on the adaptive response 31
D. Gut mucosal immunity 33
3. Innate immune recognition and bacterial infection 38
A. NLRs and bacterial infections 38
i. Nod1 38
v ii. Nod2 39
B. Salmonella enterica Typhimurium 39
i. Overview 40
ii. Innate host factors 41
iii. Implication of the immune response 44
Chapter II: Research 46
1. Innate immune recognition by Nod1 in vivo 47
a. Introduction 47
b. Results 49
c. Discussion 55
2. Nod1 signaling induces adaptive immune response 56
a. Introduction 56
b. Results 58
Nod1 Is Required for Optimal Generation of T Cell Responses
Optimal Antibody Responses Require Nod1 Triggering
Induction of Adaptive Immunity by Nod1-Specific Stimulation
TLRs and Nod1 Synergize to Elicit Adaptive Immune Responses
Stimulation of Nod1 in Nonhematopoietic Cells Is Required for Priming Antigen-Specific Immunity
c. Discussion 73
3. Nod2 signaling induces adaptive response 77
a. Introduction 77
b. Results 79
Induction of Th2 immunity by Nod2-specific stimulation
Nod2 is required for optimal elicitation of T and B cell responses in a model of CFA immunization
vi Nod2 cooperates with TLRs for the increased production of Th1-polarizing mediators
c.Discussion 93
4. Nod1 and Nod2 in Salmonella enterica Typhimurium infection 97
a. Introduction 97
b. Results 99
Nod1 deficiency impairs intestinal LPMC homeostasis
LPMC-dependent entry of Salmonella uncovers a role for Nod1 in host defense
Nod1 expression in LPMCs controls host colonization
Nod1 deficiency impairs the response of specific LPMC subsets to Salmonella infection.
c. Discussion 114
5. Unpublished observation and work in progress 118
a. Detection of Salmonella enterica Typhimurium in epithelial cells 118
b. Adaptive response against S. Typhimurium attenuated strain 121
Conclusion and Future directions 125
Experimental procedures 128
Mice and Immunizations. 128
Bone marrow chimeric mice and adoptive transfer 128
Limulus amebocyte assay. 129
Expression plasmids and transient transfections. 129
Mouse peritoneal macrophages. 130
Peripheral blood mononuclear cell isolation. 130
Reagents. 130
Reporter Assay for NF-κB Activation. 131
Cytokine Dosage. 132
vii Flow Cytometry. 132
Analysis of T Cell Responses. 132
Analysis of B Cell Responses. 133
Bone Marrow-Derived DCs and Macrophages. 134
Lymphocyte Proliferation and RT-PCR Analysis. 134
Infections. 135
Analysis of immune cells from lamina propria. 136
References 138
viii List of Publications
Le Bourhis L , Magalhaes JG, Selvanantham T, Travassos LH, Geddes K, Fritz JH, Viala J, Tedin K, Girardin SE, Philpott DJ. (2009) Nod1 controls Salmonella infection through regulation of intestinal lamina propria dendritic cells. (submitted) Magalhaes JG, Fritz JH, Le Bourhis L , Sellge G, Travassos LH, Selvanantham T, Girardin SE, Gommerman JL, Philpott DJ (2008) Nod2-dependent th2 polarization of antigen-specific immunity. J Immunol 181(11): 7925-7935 Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Le Bourhis L , Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB (2008) Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis. Proc Natl Acad Sci U S A 105(26): 9017-9022 Fritz JH, Le Bourhis L , Magalhaes JG, Philpott DJ. Innate immune recognition at the epithelial barrier drives adaptive immunity: APCs take the back seat. Trends Immunol. 2008 Jan;29(1):41-9. Le Bourhis L , Benko S, Girardin SE (2007) Nod1 and Nod2 in innate immunity and human inflammatory disorders. Biochem Soc Trans 35(Pt 6): 1479-1484 Werts C, Le Bourhis L , Liu J, Magalhaes JG, Carneiro LA, Fritz JH, Stockinger S, Balloy V, Chignard M, Decker T, Philpott DJ, Ma X, Girardin SE (2007) Nod1 and Nod2 induce CCL5/RANTES through the NF-kappaB pathway. Eur J Immunol 37(9): 2499-2508 Fritz JH, Le Bourhis L , Sellge G, Magalhaes JG, Fsihi H, Kufer TA, Collins C, Viala J, Ferrero RL, Girardin SE, Philpott DJ (2007) Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 26(4): 445-459 Le Bourhis L , Werts C (2007) Role of Nods in bacterial infection. Microbes Infect 9(5): 629-636 Tien MT, Girardin SE, Regnault B, Le Bourhis L , Dillies MA, Coppee JY, Bourdet-Sicard R, Sansonetti PJ, Pedron T (2006) Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol 176(2): 1228-1237 Ferwerda G, Girardin SE, Kullberg BJ, Le Bourhis L , de Jong DJ, Langenberg DM, van Crevel R, Adema GJ, Ottenhoff TH, Van der Meer JW, Netea MG (2005) NOD2 and toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis. PLoS Pathog 1(3): 279-285 Magalhaes JG, Philpott DJ, Nahori MA, Jehanno M, Fritz J, Le Bourhis L , Viala J, Hugot JP, Giovannini M, Bertin J, Lepoivre M, Mengin-Lecreulx D, Sansonetti PJ, Girardin SE (2005) Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin. EMBO Rep 6(12): 1201-1207 Leonardo H. Travassos, Leticia A. M. Carneiro, Séamus Hussey, Mahendrasingh Ramjeet, Linda Yuan, Joao G. Magalhaes, Lionel Le Bourhis , Ivo G. Boneca, Abdelmounaaim Allaoui, Nicola L. Jones, Stephen E. Girardin and Dana J. Philpott. Essential role of Nod1 and Nod2 in bacterial induced autophagy. 2009 (submitted)
ix List of Figures and Tables
Figure 1: TLR structure and signaling. Table 1: TLR ligands Figure 2: Nod-Like Receptors (NLR) Family Table 2: NLR ligands Figure 3: NLR triggers and signaling pathways Figure 4B: Response of lamina propria myeloid cells to microbial trigger in the gut. Figure 4A: Role of lamina propria myeloid cells in gut immune homeostasis. Figure 5: Salmonella enterica Typhimurium model of entry. Figure 6: Chemical composition of Nod1 and Nod2 ligands Figure 7: Human Nod1 detects diaminopimelic acid-type muramyl tripeptides in both epithelial cells and immune cells. Figure 8: Murine Nod1 preferentially detects diaminopimelic acid-type muramyl tetrapeptides. Figure 9: Nod1 agonists induce cytokine and nitrite responses in mouse peritoneal macrophages. Figure 10: Murine Nod1 agonist FK156 induces innate immune responses in vivo in mice. Figure 11: Altered Antigen-Specific T Cell Responses in Nod1-Deficient Mice Immunized with CFA. Figure 12: Altered Antigen-Specific Immunoglobulin Production in Nod1-Deficient Mice upon Immunization with CFA and Bacterial Infection. Figure 13: Impaired Antigen-Specific T and B Cell Immunity in Nod1-Deficient Mice Immunized with FK156. Figure 15: Analysis of Nod1 Expression and Nod1-Deficient T and B Lymphocytes Function Figure 16: TLRs and Nod1 Synergize for Dendritic Cell Cytokine Production and NF-κB Signaling Figure 17: The Nod1 Agonist FK156 Synergizes with TLR Agonists for Priming of Antigen- Specific Immunity Figure 18: Impaired FK156-Mediated Chemokine Production in Nod1-Deficient Mice Figure 19: Stimulation of Nod1 in Nonhematopoietic Cells Is Required for Priming Antigen- Specific Immunity
x Figure 20: Impaired MDP-mediated chemokine and cytokine production in Nod2-deficient animals. Figure 21: Specific stimulation of Nod2 elicits Ag-specific T cell immunity. Figure 22: Specific stimulation of Nod2 elicits Ag specific B cell immunity. Figure 23: Comparison of Lymphocyte Populations of Wild-Type and Nod2-Deficient Mice. Figure 24: Analysis Nod2-Deficient T and B Lymphocytes Function. Figure 25: Altered Ag-specific T cell responses in Nod2-deficient mice immunized with CFA. Figure 26: Nod2 cooperates with TLR2 and TLR4 for the increased production of Th1- polarizing mediators. Figure 27: Activation of macrophages and DCs independent of Nod2. Figure 28: Critical role of Nod2 for the production of Th1-polarizing mediators upon detection of peptidoglycan. Figure 29: Normal population in MLN, PP and LP of Nod1-/- compared to WT mice. Figure 30: Myeloid populations of WT and Nod1-/- deficient intestinal lamina propria. Figure 31: Nod1 deficiency increases the susceptibility to Spi1-deficient but not to wild type S. Typhimurium . Figure 32: Lack of contribution of Nod1 after S. Typhimurium intraperitoneal infection. Figure 33: Nod2 deficiency doesn’t confer increase susceptibility to S. Typhimurium infection neither in WT or Nod1 deficient background. Figure 34: Nod1 in the hematopoietic compartment is crucial for the response to Spi1-deficient bacteria Figure 35: Higher infection of LPMCs of Nod1 deficient mice. Figure 36: Defect of Nod1-deficient macrophages and dendritic cells to S. Typhimurium infection. Figure 37: Hematopoietic defect in the lamina propria. Figure 38: Impaired cellular response of Nod1-/- deficient LMPCs after oral infection. Figure 39: Nod1 detects invasive S. Typhimurium in HEK293 cells. Figure 40: Nod1 and Nod2 deficiency in the adaptive response towards S. Typhimurium ∆AroA Figure 41: Nod1 deficiency in the adaptive response towards S. Typhimurium ∆AroA after i.p. injection. Figure 42: Nod1 deficiency in the non hematopoietic compartment and the adaptive response towards S. Typhimurium ∆AroA.
xi List of Abbreviations
AIM2 Absent In Melanoma 2
ASC Apoptosis-associated speck-like protein containing a caspase recruitment domain
BMDC Bone Marrow derived Dendritic Cell
BMM Bone Marrow derived Macrophage
BrdU Bromodeoxyuridine
CARD Caspase Activating and Recruitment Domain
CD Crohn’s Disease
CFA Complete Freund’s Adjuvant
DAI DNA-dependent activator of IFN-regulatory factors
DAMP Danger Associated Molecular Pattern
DAP Di-AminoPimelic acid
DC Dendritic Cell
EC Epithelial Cells
ELISA Enzyme Linked Immuno Sorbant Assay
IBD Inflammatory Bowel Disease
IFA Incomplete Freund’s Adjuvant
IFN Interferon
IKK I kappa Kinase
IL- Interleukine
ILF Inducible Lymphoid Follicles
IPAF ICE protease activating factor
IPS1 interferon-β promoter stimulator 1
IRAK IL-1 Receptor Associated Kinase
KC Keratinocyte derived Chemokine
xii LP Lamina propria
LPMC Lamina Propria Myeloid Cells
LPS Lipo Poly Saccharide
LRR Leucine Rich Repeat
MAMP Microbe Associated Molecular Patterns
MAPK Mitogen-Activated Protein Kinase
MCP Monocyte chemotactic protein
MDA5 Melanoma Differentiation Associated gene 5
MDP Muramyl DiPeptide
MHCII Major Histocompatibility Complex class II
MIP Macrophage Inflammatory Protein
MLN Mesenteric Lymph Node
MyD88 Myeloid Determinant 88
NACHT domain present in NAIP, CIITA, HET-E, TP-1
NAIP Neuronal Apoptosis Inhibitor Protein
NALP NACHT-LRR-PYD-containing protein
NF-kB Nuclear Factor kappa B
NLR Nod Like Receptor
NLRC Nod Like Receptor CARD
NLRP Nod Like Receptor Pyrin
NO Nitric Oxyde
Nod Nucleotid Oligomerization Domain
NRAMP Natural Resistance Associated Macrophage Protein
OVA OVAlbumin
PGE Prostaglandine
PGN Peptidoglycan
xiii PGRP Peptidoglycan Recognition Protein
PP Peyer’s patches
PRM Pattern Recognition Molecule
RIG-I Retinoic-acid Inducible Gene I
SNP Single Nucleotide Polymorphism
Spi Salmonella pathogenicity island
TCT Tracheal CytoToxin
TGF Transforming Growth Factor
TIR TLR IL-1 Receptor domain
TIRAP TLR IL-1 Receptor Associated Protein
TLR Toll Like Receptor
TNF Tumor Necrosis Factor
TRAF TNF Receptor Associated Factor
TRAM TLR Receptor Associated Molecule
TREM Triggering Receptor Expressed on Myeloid cells
TRIF TIR domain-containing adaptor Inducing IFN-ß
WT Wild Type
xiv