New Insights into the Regulation of Intestinal Immunity by Nod1 and Nod2

by

Stephen J. Rubino

A thesis submitted in conformity with the requirements for the degree of a Doctorate of Philosophy

Laborartory Medicine and Pathobiology University of Toronto

© Copyright by Stephen Rubino 2014

ii

New Insights into the Regulation of Intestinal Immunity by Nod1 and Nod2

Stephen J. Rubino

Doctor of Philosophy

Laboratory Medicine and Pathobiology

University of Toronto

2014

Abstract

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific

moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate

immune responses. Importantly, mutations in the human NOD2 gene have been associated with

increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease.

However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear.

In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-)

exhibited delayed induction of intestinal inflammation at early timepoints after infection with

Citrobacter rodentium compared to wild-type mice, which correlated with compromised control

of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17

and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection

with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that

Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid

kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response

the innate Th17 (iTh17) response.

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The iTh17 cells exhibited an effector memory phenotype and required priming from the

enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major

histocompatibility complex (MHC) class II expression in hematopoietic cells was required for

the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-

17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a

dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa.

In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited

increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an

unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent

inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP.

Together, the data presented in this thesis expand our current understanding of the roles

of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and

the therapeutic potential of novel Nod2 agonists.

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Acknowledgments

First and foremost, I must thank Steph for all his support, guidance and, most

importantly, friendship that he provided during the course of my PhD studies. I also wish to

thank Dana for all her help over the years and essentially acting as my de facto co-supervisor. I

know I could not have completed even one iota of the work in this thesis without your

mentorship and I will forever be in your debts.

I wish to acknowledge all my labmates, past and present, for making the lab such an

enjoyable workplace. Special thanks goes to: Kaoru, for his help with many of the experiments

conducted as part of this thesis and for his advice on all matters scientific or otherwise; Joao, for

showing me the ropes of in vivo work and for his lost-in-translation “Joaoisms”; Ivan, for helping

me get started when I joined the lab; Fraser, for keeping the softball team alive; Susan, for

teaching me to never use the term “flora” when referencing the gut microbiota, since “bacteria

are not plants”; Kavi and Vinicius, for being such considerate desk neighbors and putting up with

my endless discussions over the years. I will miss the lab’s annual hockey pool (the NODHL).

I would also like to thank my Committee members, Dr. Phil Sherman and Dr. Jeremy

Mogridge, for providing excellent support and ideas over the course of my degree. Moreover, I

wish to acknowledge the invaluable work of my collaborators, including: Dr. Catherine

Streutker, Dr. Rupert Kaul, Dr. Jennifer Gommerman, Dr. Michelle Bendeck and Connie Kim. I

would also like to thank the graduate student coordinator, Dr. Harry Elsholtz, and the graduate

student administrators, Rama and Ferzeen, for their help in keeping me on track to finish my

degree.

I wish to thank my friends: Vince, Jayesh, Nick and Sarah, and especially my girlfriend

Cat for the memories that I will cherish for the rest of my life and for keeping me sane all these

years. Furthermore, I would like to acknowledge my LMP colleagues- Paul and Amy- for

making sure CLAMPS didn’t fall apart during our presidency.

Finally, I must thank my family for their love and support. Special thanks are reserved for

my mom, Mary, and my dad, Michael, who were always there when I needed it most; in

particular the much appreciated care packages that always seemed to arrive at precisely the right

moments. I also wish to thank my aunt, Pierina, for taking me in when I moved to Toronto and

for essentially being my home away from home. I also must thank Lisa, Anthony, Mikey, Josie,

Frank, Emma, Francis, Lucas, Evelina, Santi, Diane, David, Damiano, Chris, Matilda, Daniel,

Alex, Joe, Lina, Sara, Anthony P. and last but not least my grandmothers, Nonna Antoinetta and

Nonna Maria.

Research is what I'm doing when I don't know what I'm doing.

Wernher von Braun

Chance favors the prepared mind.

Louis Pasteur

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Table of Contents

Section Page

Title page………………………………………………………………………………… i

Abstract………………………………………………………………………………….. ii

Acknowledgements……………………………………………………………………… iv

Table of Contents………………………………………………………………………... v

List of Tables……………………………………………………………………………. ix

List of Figures………………………………………………………………………….... x

List of Abbreviations……………………………………………………………………. xiii

Dissemination of Work Arising from the Thesis…………………………………….…… xv

Chapter 1: General Introduction……………………………………………………… 1

1.1 Introduction to Innate Immunity…………………………………………………….. 2

1.1.1 Discovery of Pattern Recognition Receptors ………………………….... 3

1.1.2 Overview of the Nod-Like Receptor Family……………………………. 4

1.2 Nod1 and Nod2: Sensors of Bacterial Peptidoglycan………………………………. 8

1.2.1 Structural Determinants of Nod1 and Nod2 Ligands…………………… 8

1.2.2 Activation of NF-kB Signaling Pathway………………………………... 10

1.2.3 Linking Innate and Adaptive Immunity………………………………… 13

1.2.4 Mediating Host Responses to Bacterial Infections……………………… 14

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1.3 Role of Nod1 and Nod2 in the Intestine…………………………………………… 16

1.3.1 Association between the NOD2 gene and Crohn’s Disease …………… 18

1.3.2 Regulation of the Intestinal Microbiota by Nod1 and Nod2…………… 19

1.3.3 Nod1 and Nod2 in Murine Models of Colitis……………………...…… 20

1.4 Models of Enteric Pathogen-Induced Colitis……………………...…………….…. 22

1.4.1 Citrobacter rodentium-induced colitis……………………....…………. 22

1.4.2 Salmonella enteric serovar Typhimurium-induced colitis…………….. 24

1.5 IL-17 and IL-22: Mediators of Mucosal Immunity……………………....………… 26

1.5.1 Differentiation Program of Th17 Cells………………………………… 29

1.5.2 Homeostatic Regulation of Th17 Cells by Intestinal Microbiota……… 29

1.5.3 Importance of Intestinal IL-17/IL-22 Responses to Bacterial

Pathogens……………………………………………………….……… 30

1.5.4 Cellular Sources of IL-17 and IL-22 in the Gut………………….…….. 32

1.5.5 Induction of IL-17 Responses by Pattern Recognition

Receptors………………………………………………………………. 36

1.6 Thesis Overview……………………………………………………………...……. 40

Chapter 2: Identification of an Innate Th17 Response to Enteric Bacterial

Pathogens………………………………………………………………………..…….. 41

2.1 Abstract…………………………………………………………………………….. 42

2.2 Introduction………………………………………………………………………… 43

2.3 Material and Methods……………………………………………………………… 44

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2.4 Results……………………………………………………………………………… 52

2.4.1 Nod1 and Nod2 are required to control infection with the enteric

pathogen C. rodentium.…………………………………………………..…..… 52

2.4.2 Nod1 and Nod2 are required for induction of early intestinal

Th17 responses……………………………………………………………….… 58

2.4.3 Nod-dependent IL-6 induction is required for early Th17

responses……………………………………………………..………………… 68

2.4.4 Induction of early Th17 responses to bacterial pathogens

requires priming by the intestinal microbiota. …………………………….… 76

2.5 Discussion……………………………………………………………………..…… 81

Chapter 3: Constitutive Induction of Tc17 Cells does not protect against

Citrobacter rodentium infection. ……………………………………………………... 84

3.1 Abstract…………………………………………………………………………….. 85

3.2 Introduction………………………………………………………………………… 86

3.3 Material and Methods………………………………………………………………. 88

3.4 Results……………………………………………………………………………… 90

3.4.1 MHCII is necessary for early mucosal Th17 responses to

Citrobacter rodentium nfection. ………………………………………………. 90

3.4.2 Deletion of hematopoietic MHCII signaling results in the upregulation

of IL-17+ and FOXP3+ CD8+ T cells in the cecal lamina propria……………... 98

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3.4.3 Expression of MHCII on hematopoietic cells is necessary to control C.

rodentium infection..……………………………………………………………. 107

3.5 Discussion……………………………………………………..……………………. 110

Chapter 4: Identification of a Muramyl Peptide with Enhanced Nod2

Stimulatory Capacity……………………………………………………..………...…. 113

4.1 Abstract…………………………………………………………………………….. 114

4.2 Introduction………………………………………………………………………… 115

4.3 Material and Methods……………………………………………………………… 117

4.4 Results…………………………………………………………………………….... 119

4.4.1 NF-b stimulatory activity of MP derivatives…………………….…… 119

4.4.2 In vitro and in vivo analyses of MDP(D-Val1)……………….…………. 131

4.4.3 In vitro and in vivo analyses of MDP(D-Glu2)-OCH3…………….……. 134

4.5 Discussion…………………………………………………………………….……. 140

Chapter 5: General Discussion and Future Directions…………………………..…. 142

5.1 Linking Nod1 and Nod2 to mucosal Th17 responses: implications for Crohn’s

disease pathogenesis……………………………………………………………………. 143

5.2 Memory T cell responses to the enteric microbiota ……………………………….. 145

5.3 Role of Nod2 in CD103+Dendritic cell biology …………………………………... 146

5.4 Therapeutic potential of Nod1 and Nod2 agonist………………………………….. 148

References Cited………………………………………………………………………. 150

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List of Tables

4.1 List of tested muramyl dipeptide derivatives…………………………………… 117

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List of Figures

1.1 NLR Family Members…………………………………………………………... 7

1.2 Structures of Nod1 and Nod2 Ligands…………………….……………………. 9

1.3 Cellular pathways downstream of Nod1 and Nod2 activation………………….. 12

1.4 Mechanisms of Nod1 and Nod2 mediated regulation of gut homeostasis……… 17

1.5 The role of innate IL-17/IL-22 responses to enteric bacterial infections……….. 28

1.6 Innate IL-17 producing lymphocytes in the gut………………………………… 34

1.7 Dendritic cells sense infection and drive innate IL-17 and IL-22 production….. 38

2.1 Purity of MACS-sorted populations…………………………………………….. 50

2.2 Phenotypic characterization of Nod1–/–, Nod2–/– and Nod1–/–Nod2–/– mice

during infection with C. rodentium…………………………………………………… 54

2.3 Nod1 and Nod2 differentially modulate early and late inflammation during C.

rodentium colitis………………………………………………………………… 55

2.4 Phenotypic characterization of bone-marrow chimeras infected with

C. rodentium………………………………………………………….…………. 57

2.5 Early IL-17 responses during C. rodentium colitis are Nod1 and Nod2

dependent………………………………………………………….…………….. 59

2.6 Analysis of lamina propria T cell responses during C. rodentium infection…… 61

2.7 Acute IL-17 responses during S. typhimurium colitis are dependent on

hematopoietic and non-hematopoietic Nod1 and Nod2………………………… 65

2.8 CD4+ T cells from human colonic biopsies produce IL-17A and IL-22 in

response to short term S. typhimurium infection………………………………… 67

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2.9 IL-6 expression during C. rodentium and Salmonella colitis is Nod1 and Nod2

dependent………………………………………………………………………… 70

2.10 IL-6 expression during the acute phase of infectious colitis is critical for TH17

development. ……………………………………………………………………. 72

2.11 Analysis of cytokine expression in infected IL-6 knockout mice and

pathological scores in IL-6 depletion experiments.……………………………… 74

2.12 Early TH17 cells express memory surface markers and require microbiota for

activation. ……………………………………………………………………….. 78

2.13 Intestinal colonization with segmented filamentous bacteria (SFB)……………. 80

3.1 CD4+, CD8+ and MHCII+ cell characterization in MHCII-/-WT mice. ……. 93

3.2 Induction of early Lamina Propria Th17 responses after C. rodentium infection

is dependent on MHCII signaling. ……………………………………………… 94

3.3 Intracellular IL-22 expression in CD4+TCRb+ LPLs…………………………... 96

3.4. Characterization of CIITA-/-WT chimeric mice. ……………………………. 97

3.5. Enrichment of IL-17+CD8+ T cells in the lamina propria of MHCII-/-WT

mice …………………….………………………………………………………. 99

3.6 Intracellular IL-22 expression in CD8+TCRb+ LPLs………………………….. 103

3.7 Analysis of FOXP3+CD4+ and FOXP3+ CD8+ T cells in the lamina propria

of MHCII-/-WT mice………………………………………………………… 104

3.8 Induction of of IL-17+ and FOXP3+ CD8+ T cells in the intraepithelial

lymphocyte compartment of MHCII-/-WT mice. …………………………… 105

3.9 CD44 expression on IL-17+, FOXP3+ and IFNg+ CD8+ T cells in the

lamina propria of MHCII-/-WT mice. ……………………………………….. 106

3.10 MHCII-/-WT mice are more susceptible to C. rodentium infection...……….. 108

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4.1. NF-B stimulatory capacity of MDP-derivative compounds modified at the

2nd

amino acid. ………………………………………………………….……… 121

4.2 NF-B stimulatory capacity of MDP-derivative compounds modified at the

1st amino acid. ……………………………………………………………..…… 123

4.3 NF-B stimulatory capacity of MDP-derivative compounds modified at the

MurNAc carbohydrate. ………………………………………………………… 125

4.4 NF-B stimulatory capacity of MDP-derivative compounds modified at

two or more sites. ………………………………………………………………. 127

4.5. NF-B stimulatory capacity of MDP-derivative compounds with either the sugar or

an amino acid removed. ………………………………………….…………….. 129

4.6. In vitro and in vivo responses observed with MDP(D-Val1)..………….……….. 130

4.7 In vitro and in vivo responses observed with MDP(D-Glu2)-OCH3……………. 135

4.8 Head-to-head comparison of in vivo responses observed with

MDP(D-Glu2)-OCH3 and N-Glycolyl-MDP.………………………………….. 137

4.9 Muramyl dipeptide (MDP) (D-Glu2)-OCH3 induces enhanced cytokine and

chemokine production by human dendritic cells compared to MDP..…………. 138

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List of Abbreviations

AHR: Aryl hydrocarbon receptor

APC: Antigen-presenting cell

ATP: Adenosine Triphosphate

BMDM: Bone-marrow derived macrophages

CARD: Caspase-active recruitment domain

CD: Crohn’s disease

CIITA: Major histocompatibility complex II transactivator

DC: Dendritic cell

DKO: Double-knockout

DSS: Dextran sodium sulphate

IBD: Inflammatory bowel disease

DAMP: Damage-associate molecular pattern

DAP: Di-aminophilic acid

EHEC Enterohemorrhagic E. coli

EPEC: Enteropathogenic E. coli

GF: Germ-free

GWAS: Genome-wide association study

IEL: Intra-epithelial lymphocyte

IFN: Interferon

IL: Interleukin

ILC: Innate Lymphoid Cell

JNK: c-Jun N-terminal kinase

KO: Knockout

LP: Lamina propria

LPL: Lamina propria lymphocyte

LPS: Lipopolysaccharide

LRR: Leucine-rich repeat

LTi: Lymphoid-tissue inducer

MAMP: microbial-associated molecular pattern

MAPK: Mitogen-activated protein kinase

MDP: Muramyl di-peptide

MHC: Major histocompatibility complex

MP: Muramyl peptide

MyD88: Myeloid differentiation primary response protein 88

NACHT: domain in NAIP, CIITA, HET-E and TP1

NBD: Nucleotide-binding domain

NK: Natural killer

NKT: Natural killer T cell

NOD: Nucleotide-binding and oligomerization domain-containting protein

NLR: Nod-like receptor

PCR: Polymerase Chain Reaction

PMA: Phorbyl 12-myristate 13 acetate

PRR: Pathogen recognition receptor

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PYR: Pyrin

RA: Retinoic acid

RIP2: Receptor-interacting serine/threonine-protein kinase 2

SFB: Segmented filamentous bacteria

SPF: Specific-pathogen free

ssRNA: Single-stranded ribonucleic acid

TA: Acid Transactivation domain

TIR: Toll IL-1b receptor domain

Th1: T-helper type 1

Th2: T-helper type 2

Th17: T-helper type 17

TLR: Toll-like receptor

TMCH: Transmissable Murine Colonic Hyperplasia

TNBS: Trinitrobenzenesulfonic acid

TRIF: TIR-domain-containing adapter-inducing interferon-β

Treg: Regulatory T cell

UC: Ulcerative Colitis

xv

Dissemination of Work Arising from the Thesis

Chapter 1:

Stephen J. Rubino, Thirumahal Selvanantanam, Stephen E. Girardin and Dana J. Philpott. Nod-

like receptors in the control of intestinal inflammation. Current Opinion in Immunology, 2012

Aug;24 (4):398-404. (Review)

Stephen J. Rubino, Kaoru Geddes and Stephen E. Girardin. Innate IL-17/IL-22 Responses to

Enteric Pathogens. Trends in Immunology, 2012 Mar;33(3):112-8. (Review)

Susan J. Robertson, Stephen J. Rubino, Kaoru Geddes and Dana J. Philpott. Examining host-

microbial interactions through the lens of NOD: from plants to mammals. Seminars in

Immunology, 2012 Feb;24(1):9-16. (Review)

Chapter 2:

Stephen J. Rubino* and Kaoru Geddes* (*co-first author publication), Joao Gamelas

Magalhaes, Catherine Streutker, Lionel Le Bourrhis, Joon H. Cho, Susan Robertson, Connie J.

Kim, Rupert Kaul, Dana J. Philpott and Stephen E. Girardin. Identification of an innate Th17

response to intestinal bacterial pathogens. Nature Medicine, 2011 Jun 12.

Chapter 3:

Stephen J. Rubino, Kaoru Geddes, Joao Gamelas Magalhaes, Catherine Streutker, Dana J.

Philpott and Stephen E. Girardin. Induction of mucosal Tc17 cells in the absence of MHCII

signaling does not protect against infection with Citrobacter rodentium. European Journal of

Immunology. 2013 Jul 23.

Chapter 4:

Stephen J. Rubino* and Joao G. Magalhaes* (*co-first author publication), Dana Philpott,

George M. Bahr, Didier Blanot and Stephen E. Girardin. Identification of a muramyl dipeptide

derivative with enhanced Nod2 stimulatory capacity. Innate Immunity. 2013 Jan 22.

Additional publications:

Joao G. Magalhaes, Stephen J. Rubino, Travassos LH, Le Bourhis L, Duan W, Sellge G,

Geddes K, Reardon C, Lechmann M, Carneiro LA, Selvanantham T, Fritz JH, Taylor BC, Artis

D, Mak TW, Comeau MR, Croft M, Girardin SE, Philpott DJ. Nod1 and Nod2 activation in the

stromal compartment instructs dendritic cells to initiate Th2 immunity. Proc Natl Acad Sci U S

A, 2011 Sep 6;108(36):14896-901.

Kaoru Geddes, Stephen J. Rubino, Catherine Streutker, Cho JH, Magalhaes JG, Le Bourhis L,

Selvanantham T, Girardin SE, Philpott DJ. Nod1 and Nod2 regulation of inflammation in the

Salmonella colitis model. Infection and Immunity, 2010 Dec;78(12):5107-15.

xvi

Jörg H. Fritz, Olga Lucia Rojas, Nathalie Simard, Doug McCarthy, Siegfried Hapfelmeier,

Stephen Rubino, Robertson SJ, Larijani M, Gosselin J, Ivanov II, Martin A, Casellas R, Philpott

DJ, Girardin SE, McCoy KD, Macpherson AJ, Paige CJ, Gommerman JL. Acquisition of a

multifunctional TNFα/iNOS-producing IgA+ plasma cell phenotype in the gut. Nature, Dec

11;481(7380):199-203.

Susan J. Robertson, Jun Yu Zhou, Kaoru Geddes Stephen J. Rubino, Joon Ho Cho, Stephen E.

Girardin and Dana J. Philpott. Nod1 and Nod2 signaling does not alter the composition of

intestinal bacterial communities at homeostasis. Gut Microbes, 2013.

Joao G. Magalhaes, Jooeun Lee, Kaoru Geddes, Stephen Rubino, Stephen E. Girardin and Dana

J. Philpott. Essential role of Rip2 in the modulation of innate and adaptive immunity triggered

by Nod1 and Nod2 ligands. European Journal of Immunology, 2011 May;41(5):1445-55.

Ingrid Stroo, Loes M. Butter, Nike Claessen, Gwen J. Teske, Stephen J. Rubino et al.

Phenotyping of Nod1/2 double deficient mice and characterization of Nod1/2 in systemic

inflammation and associated renal disease. Biology Open. 2012 Dec 15;1(12):1239-47.

Catherine Werts, Stephen J. Rubino, Arthur Ling, Stephn E. Girardin, Dana J. Philpott. Nod-

like receptors in intestinal homeostasis, inflammation, and cancer. J Leukoc Biol, 2011

Sep;90(3):471-82. (Review)

Stephen J. Rubino, Jooeun Lee and Stephen E. Girardin. Mammalian PGRPs also mind the fort.

Cell Host and Microbe, August 2010 Aug 19;8(2):130-2. (Review)

1

Chapter 1:

General Introduction

Excerpts of section 1.3 were originally published in: Stephen J. Rubino, Thirumahal

Selvanantanam, Stephen E. Girardin and Dana J. Philpott. Nod-like receptors in the control of

intestinal inflammation. Current Opinion in Immunology, 2012 Aug;24 (4):398-404.

Excerpts of section 1.5 and Figures 1.5-1.7 were originally published in: Stephen J. Rubino,

Kaoru Geddes and Stephen E. Girardin. Innate IL-17/IL-22 Responses to Enteric Pathogens.

Trends in Immunology, 2012 Mar;33(3):112-8.

Figure 1.4 was originally published in: Susan J. Robertson, Stephen J. Rubino, Kaoru Geddes

and Dana J. Philpott. Examining host-microbial interactions through the lens of NOD: from

plants to mammals. Seminars in Immunology, 2012 Feb;24(1):9-16.

2

1.1 Introduction to Innate Immunity

Innate immunity is defined as the rapid first-line response to infectious agents such as

bacteria, viruses and fungi that precedes the onset of specific pathogen-targeted adaptive

immunity. Classically, macrophages, dendritic cells (DCs) and granulocytes (neutrophils,

basophils and eosinophils) are characterized as the innate cells that mediate the innate immune

response, which encompasses three broad arms: 1) the phagocytocis and destruction of invading

pathogens; 2) the initiation of inflammation through the secretion cytokines and chemokines; 3)

the recruitment and activation of the adaptive immune response through antigen presentation and

co-stimulation(1). However, more recent studies now suggest that all cells, including lympoid

cells, such as T, B and Natural killer (NK) cells and innate lymphoid cells (ILCs), epithelial cells

and stromal cells can also potentiate innate immune responses, particularly at mucosal surfaces

such as the gastrointestinal (GI) tract and lungs.

The induction of innate immune responses is critically dependent on the ability of the

mammalian host to discriminate between self and non-self. Non-self can be recognized by

molecularly conserved products that are expressed on invading microbes, but not in host cells,

and are termed microbial associated molecular patterns (MAMPs). Alternatively, innate immune

responses can also be initiated by recognition of “altered” self in the form damage associated

molecular patterns (DAMPs), such as adenosine triphosphate (ATP) or double-stranded DNA

(dsDNA), released from damaged or dying cells. The cellular detection of MAMPs and DAMPs

is performed by pattern recognition receptors (PRRs), which include evolutionarily conserved

families of receptors such as the Toll-like receptors (TLRs) and Nucleotide-binding and

oligomerization domain-containting protein (Nod) Nod-like receptors (NLRs)(2,3). In this

3

section, we will review the discovery of the surface-bound TLRs and then provide a detailed

summary of the cytosolic NLRs.

1.1.1 Discovery of Toll and the Toll-like Receptor Family

Lipopolysaccharide (LPS, also referred to as endotoxin) is a glycolipid expressed on the

outer membrane of all Gram-negative bacteria. Systemic injection of LPS had been known for

decades to induce a host cytokine storm that could result in septic shock, a condition

characterized by high fever and collapse of the circulatory system, however the host sensing

system mediating LPS-induced shock remained unknown. It wasn’t until the late-1990s that

Hoffman and colleagues identified the transmembrane protein Toll in Drosophila melanogaster

as the critical mediator of LPS recognition and the antimicrobial peptide response to bacterial

infection in this organism. Subsequently, Beutler et al and Janeway et al discovered a

mammalian homologue of Toll, which they termed Toll-like receptor 4 (TLR4), and this protein

was described as the essential host receptor that is activated by LPS injection(4,5). These seminal

studies resulted in the Nobel Prize of medicine to Beutler and Hoffman in 2011, and ushered in a

wave of research into the field of innate immunity and microbial recognition.

The TLRs are all transmembrane proteins located at the plasma membrane or within

intracellular endosomes and they all share a similar domain structure: an extracellular Leucine-

rich repeat (LRR) domain that mediates MAMP recognition, a hydrophobic transmembrane

domain and an intracellular Toll IL-1b receptor (TIR) domain involved in downstream signaling.

In humans, there are ten members in the TLR family and they can all function as PRRs(2,3).

TLR1 can dimerize with TLR2 to detect triacyl lipopeptides; TLR2 homodimers detect

lipoteichoic acid; TLR3 recognizes double-stranded RNA (dsRNA); TLR4 detects LPS; TLR5

detects bacterial flagellin; TLR6 can dimerize with TLR2 to detect zymozan; TLR7 and TLR8

4

can detects single-stranded (ssRNA); TLR9 detects CpG containing DNA motifs; finally the

ligand for TLR10 remains unknown(2,3). Upon activation, the TLRs recruit the adaptors

Myeloid differentiation primary response protein 88 (MyD88) and/or TIR-domain-containing

adapter-inducing interferon-β (TRIF) to activate Nuclear Factor-B (NF-B), mitogen-activated

protein kinase (MAPK) and type 1 interferon response intracellular signaling pathways(2,3).

Induction of these signaling pathways results in numerous innate immune responses, including

increased phagocytosis and macrophage activation, cytokine and chemokine secretion and the

up-regulation of co-stimulatory molecules on antigen presenting cells.

1.1.2 The Nod-like Receptor (NLR) Family

In addition to the membrane-bound TLRs, there are also numerous intracellular PRRs

that mediate cellular anti-microbial responses, including the RIG-I/MDA-5 and AIM2-like anti-

viral pathways (reviewed in depth elsewhere(6,7)) and the NLRs. The NLRs are an evolutionary

conserved family of proteins that, while not present in protostomes (ie Drosophila,

Caenorhabditis elegans), are expanded in early deuterostomes such as sea urchins and sea

sponges(8,9). All NLRs share a similar domain structure: a LRR domain at the carboxy-

terminus; a central nucleotide binding domain (NACHT or NBD); and finally a protein-protein

interaction domain at the amino-terminus(9,10). In humans, there are 22 NLR family members

that can be divided into five subfamilies based on domain structure: NLRA, NLRB, NLRC,

NLRP and NLRX (see Fig 1.1).

The NLRA subfamily consists of the major histocompatibility class II transactivator

(CIITA), which contains a caspase recruitment domain (CARD) and acid transactivation domain

in the amino-terminus. Interestingly, CIITA does not function as a PRR, instead this NLR

functions as the critical regulator of major histocompatibility (MHC) class II expression(11).

5

The NLRB subfamily is comprised of only one member in humans, NAIP, and six

members in mice, NAIP1-6, and contain a baculoviral inhibition of apoptosis (BIR) domain at

the amino-terminus. NAIP2, NAIP5, and NAIP6 act as PRRs and can recognize bacterial

flagellin and type 3 secretion effector proteins(12-15) to induce the activation of the

inflammasome, an intracellular complex containing the adaptor ASC and Caspase-1 that, when

activated, mediates the cleavage of the cytokines interleukin (IL) IL-1, IL-18 and IL-33 into their

active forms(16).

The NLRC family consists of five members that are well conserved in most vertebrates

ranging from zebrafish to humans(17). The two most characterized members are Nucleotide-

binding and oligomerization domain-containing protein (Nod) 1 (also referred to NLRC1) and

Nod2 (NLRC2), and they contain CARD domains in their amino-terminus. Both Nod1 and Nod2

recognize structures derived from bacterial peptidoglycan to initiate inflammatory pathways(18-

21) (reviewed in greater detail in section 1.2). NLRC3 has been reported to function as a

negative regulator of T cell function and TLR responses(22,23). NLRC4 (IPAF) activates the

inflammasome in response to bacterial flagella delivered via bacterial type III or type IV

secretion systems(24-26). NLRC5 was reported to function as negative regulator of

inflammatory pathways(27,28) and has more recently been described to regulate MHC class I

expression (29-32).

The NLRP subfamily consists of 14 members that are generally characterized by the

presence of a pyrin domain at the amino-terminus. The most studied members of this group are

NLRP1 (also referred to as NALP1) and NLRP3 (also referred to as NALP3) that activate the

caspase-1-containing inflammasome(16). NLRP1 recognizes lethal toxin produced by Bacillus

anthracis(33,34), whereas NLRP3 has been reported in numerous studies to be activated by a

6

wide range of ligands, including: viral RNA, ATP, bacterial toxins, uric acid crystals, silica

crystals, beta-amyloid and low intracellular levels of potassium(35-40). NLRP6 has been also

shown to initiate inflammasome activation(41,42), and more recently NLRP6 was found regulate

intestinal homeostasis through IL-18 secretion(42). NLRP2, NLRP10 and NLRP12 were initially

reported to act as negative regulators of inflammation(43-45), however NLRP10 was recently

found to be essential for the induction of systemic adaptive responses by mediating the migration

of antigen-presenting cells (APCs)(46).

The NLRX subfamily consists of only one member, NLRX1, and is the only NLR that

contains a mitochondrial targeting sequence that mediates localization to the inner matrix of the

mitochondria(47). Functionally, NLRX1 has been found to regulate NF-KappaB and ROS

production(48,49) and has also been reported to modulate type-1 interferon responses to viral

infections(49-51), although this putative function remains controversial in light of in vitro and in

vivo findings suggesting NLRX1 does not play a role during influenza infection(52,53).

7

Figure 1.1 Human NLR Family Members. This table presents a schematic representation of

the domain organization of the five NLR subfamilies found in humans. CARD = Caspase-active

recruitment domain; TA = Acid Transactivation domain; NACHT = nucleotide-binding domain;

LRR = Leucine-rich repeat domain; PYR = Pyrin; X = Undefined domain.

8

1.2 Nod1 and Nod2: Sensors of Bacterial Peptidoglycan (PG)

Similarly to LPS, the capacity of PG, a critical component of the cell wall of Gram-

positive and Gram-negative bacteria, to induce both inflammatory and adaptive immune

responses has been studied for decades(54-57). The PG heterogeneous polymer is composed of a

sugar backbone comprised of alternating N-acetylglucosamine and N-acetylmuramic acid

(MurNAc) residues. A peptide chain is attached to the MurNAc sugar, forming a muramyl

peptide (MP), and these stem oligopeptides can be crosslinked to form the 3-dimensional lattice

structure of PG. It is only in the past decade that Nod1 and Nod2 have been identified as the

critical cytosolic PRRs that sense specific MPs in mammals(18-21). In this section, we will

review the structural determinants of Nod1/2 sensing, the cytosolic pathways activated by these

receptors, the link between Nod1/2 activation an adaptive immunity and finally the role of these

receptors in cellular responses to bacterial infection.

1.2.1 Structural Determinants of Nod1 and Nod2 Ligands

Nod1 detects meso-diaminopimelic acid-containing MurNAc-tripeptide (Mur-TriDAP)

predominantly found in Gram-negative bacteria(18,20), whereas Nod2 detects muramyl-

dipeptide (MDP) found in Gram-negative and Gram-positive bacteria(19,21) (see Fig 1.2). More

detailed studies on the minimal structural requirements of the MP ligands needed for Nod1 or

Nod2 activation revealed that the MurNAc sugar is not required for Nod1 activation, since the D-

Glu-meso-DAP dipeptide (iE-DAP) is sufficient for detection and innate immune activation by

this PRR(58,59). Nod2 on the other hand can only be activated by muramyl dipeptides that have

an intact MurNAc ring structure, and the sugar has to be attached to a dipeptide moiety (L-Ala-D-

Glu or L-Ala-D-isoGln)(59).

9

Figure 1.2 Structures of the Nod1 and Nod2 Ligands. Schematic representation of the

minimal molecular requirements of the Nod1 ligand: Muramyl-tri-DAP (M-TriDAP); and the

Nod2 ligand: Muramyl-di-peptide (MDP). For Nod1, the terminal the D-Glu-meso-DAP

dipeptide (iE-dAP) is sufficient for stimulation of NF-B signaling pathways.(59)

10

Recently, Nod2 was shown via a number of biochemical assays to bind directly to its

ligand MDP(60,61), suggesting that this protein acts as a bona fide cytosolic receptor. It was also

previously reported that an intact cellular endocytic pathway was critically required for the

activation of both Nod1 and Nod2(62,63), indicating that cytosolic internalization of ligands was

necessary for activation. The discovery of di- and tripeptide transporters located at the plasma

membrane that are needed to internalize Mur-TriDAP and MDP further reinforces the idea that

binding of MP ligands to Nod1 and Nod2 is essential for the induction of NF-B signaling by

these proteins. Specifically, it was shown that the oligopeptide transporter hPepT1 (also known

as SLC15A1) acts as a specific transporter for MDP(64), but not Nod1 ligands(65), whereas the

peptide transporter SLC15A4, which is expressed in early endosomes, is required for Nod1

stimulation by MurNAc-TriDAP and iE-DAP(62). Finally, hPepT2 (also known as SLC15A2)

was also found to be involved in MP internalization(66,67).

1.2.2 Activation of Downstream Cellular Pathways

Upon activation, Nod1 and Nod2 can stimulate a number of downstream cellular pro-

inflammatory pathways; the best characterized of which is the NF-B signaling cascade (Fig

1.3). Nod1 and Nod2 are intracellular receptors that preferentially localize to the cytosolic

surface of the plasma membrane(68,69). After detecting their cognate ligands, Nod1 and Nod2

oligomerize, a step mediated by the central NACHT domain, and recruit the adaptor protein

Receptor-interacting serine/threonine-protein kinase (RIP2)(70,71). A carboxy-terminal CARD

domain on RIP2 mediates a CARD-CARD interaction with the amino-terminal CARD in Nod1

and Nod2. After activation, RIP2 is poly-ubiquitinylated(72), which leads to the recruitment of

NEMO (IKK), a critical scaffold of the IKK complex, and the phosphorylation of

IKKPhospohorylated IKKwill then in turn phosphorylate IB, which will lead to its

11

ubiquitinylation and degradation by the proteosome(73). The destruction of IB is followed by

the release of the p50 and p65 subunits of NF-B, which are then free to translocate into the

nucleus and stimulate the transcription of NF-B-dependent pro-inflammatory genes, including

cytokines and chemokines.

In addition to NF-B, Nod1 and Nod2 activation has also been reported to induce

MAPK/MAPKK signaling (Fig 1.3). Specifically, stimulation of Nod1 and Nod2 leads to the

phosphorylation of c-Jun N-terminal kinases (JNK) and p38, which will induce activator protein-

1 (AP-1)-dependent gene transcription(74-77). The exact mechanism linking Nod1/2 MAMP

detection to JNK and p38 phosphorylation remains unclear, however one study suggested that

the adaptor protein CARD9 plays a role in mediating this response in hematopoietic cells(74).

Finally, Nod1 and Nod2 activation can also trigger the autophagy pathway (Fig. 1.3), a

highly conserved cytosolic process used by the cell to remove misfolded proteins, damaged

organelles and invading microbes(78,79). Indeed, Nod1 and Nod2 can interact directly with

ATG16L1 and recruit this adaptor to sites of bacterial entry to target bacteria to double-

membrane autophagosomes(78). ATG16L1 is located in a complex with ATG5 and ATG12 to

form the core autophagic machinery that, once activated by Nod stimulation, is responsible for

converting LC3 into LC3II, a key step in autophagosome formation. The autophagosome will

then fuse to lysosomes, where proteases will then mediate the degradation of the

autophagosome’s contents(80). Nod1/2-induced autophagy has been shown to limit intracellular

growth of the pathogens Shigella flexneri and Salmonella enterica serovar Typhimurium in

epithelial cells, fibroblasts and macrophages(78,79). Moreover, Nod2-induced autophagy was

also reported in one study to play a role in antigen presentation by MHCII in human DCs(79).

12

Figure 1.3 Cellular pathways downstream of Nod1 and Nod2 activation. Schematic

representation of the three most well characterized signaling pathways downstream of Nod1 and

Nod2. Recognition of Tri-DAP by the LRR domain of Nod1 or MDP by the LRR domain of

Nod2 leads to the recruitment and ubiquitinylation of the adaptor RIP2. RIP2 can activate the

NF-B signaling cascade (1) and/or the JNK and p38 pathway (2). Alternatively, Nod1 and

Nod2 can bind and recruit AT16L1 to target bacteria to the autophagy pathway (3).

13

1.2.3 Linking Innate and Adaptive Immunity

Adjuvants are defined as agents that enhance systemic adaptive immunity to injected

antigens and the adjuvant capacity of MAMPs, such as LPS and PG, has been studied for

decades. Indeed, one of the most commonly used adjuvants in biomedical research is Complete

Freund’s Adjuvant (CFA), an emulsion of mycobacterial cell wall fragments dissolved in a

mineral oil and is primarily composed of NLR and TLR agonists. Adjuvants such as CFA

enhance adaptive immune responses primarily by enhancing the capacity of APCs, such as DCs

and macrophages, to activate T and B-lymphocytes(54). Specifically, MAMPs stimulate the

upregulation of co-stimulatory molecules, such as B7.1, B7.2 and CD40, on the surface of APCs,

which bind to their cognate receptors on T cells to mediate T cell activation. Furthermore,

MAMP-stimulated APCs secrete cytokines, such as IL-12, IL-4 and IL-6 that are crucial for

polarizing the adaptive response to a Th1, Th2 and Th17 response, respectively(2).

Importantly, Nod1, Nod2 and RIP2 have all been reported to mediate the adjuvant ability

of MPs to potentiate antigen specific immune responses(81-84). Specifically, Nod1-/- mice

exhibited blunted Th1, Th2 and Th17 antigen-specific immune response after systemic injection

of CFA + antigen. Interestingly, injection of antigen + FK156, a synthetic Nod1 agonist, drove a

Th2-polarized response, characterized by T cells that predominantly secrete IL-4 and IL-5 and B

cells that primarily secrete IgG1, which was completely abrogated in Nod1-/- mice(81). In line

with these results, systemic injection of MDP + antigen was also shown to induce a Th2

polarized response that was dependent on Nod2(83). Subsequently, another study demonstrated

that RIP2-/- mice also exhibited ablated adaptive responses during vaccination experiments using

Nod1 or Nod2 ligands as adjuvants(82). Together, these findings indicate that Nod agonists

injected in the presence of TLR agonists will drive a mixed adaptive response, whereas injection

of Nod-specific ligands (MDP, TriDAP, etc.) will specifically induce a Th2 response. Indeed,

14

previous studies had demonstrated that Nod and TLR agonists induce a synergistic response in

epithelial and macrophage cells(85-87).

A recent study using bone-marrow chimeric mice demonstrated that the Th2-polarizing

capacity of Nod1 and Nod2-dependent agonists was controlled by signals derived from both

hematopoietic and non-hematopoietic cells(84). Indeed, FK156 or MDP could stimulate

epithelial cells to secrete thymic stromal lymphopoietin (TSLP), a cytokine that in turn activated

APCs to express the pro-Th2 co-stimulatory molecule OX40L(84). Moreover, deletion of TSLP

in the non-hematopoietic compartment or OX40L in hematopoietic cells was sufficient to

abrogate Nod-mediated adjuvanticity(84). These findings are in sharp contrast with what is

observed for TLR ligands, which stimulate APCs directly, and non-hematopoietic cells do not

contribute to their adjuvanticity. Thus, the ability of Nod ligands to initiate a stromal cell-

regulated adaptive response represents a fundamental difference in the capacity of Nod and TLR

signaling to potentiate adaptive immune responses and could explain why FK156 and MDP

induce a Th2-specific response, whereas TLR agonists induce predominantly a Th1 and Th17

response.

1.2.4 Mediating Host Responses to Bacterial Infections

Numerous studies have examined the importance of Nod1 and Nod2 signaling for host

clearance of infections with intracellular and extracellular bacterial pathogens both in vitro and

in vivo. Nod1 was first reported to function as a critical mediator of NF-B-dependent immune

responses in epithelial cell lines after infection with the intracellular pathogen Shigella

flexneri(75). Subsequently, Nod1 has been shown to mediate cellular innate immune responses

against numerous other Gram-negative pathogens, including: Helicobacter pylori(88,89),

Pseudomonas aeruginosa(90), entero-invasive Esherichia coli(91) and Chlamydia species(92).

15

In numerous in vivo studies, Nod1 has been shown to contribute to protection against infection

by H. pylori(88,89), attenuated Salmonella Typhimurium(93), Haemophilus influenzae(94) and

Listeria monocytogenes(77,95) (a Gram-positive bacterium that also expresses DAP-containing

PG). Moreover, Nod1 can trigger NF-B-dependent pro-inflammatory pathways in response to

outer membrane vesicles (OMVs) secreted by H. pylori, Pseudomonas aeruginosa and Neisseria

gonorrhoeae(96).

Nod2 has also been shown to mediate the host defense against a number of bacteria

pathogens including: Listeria monocytogenes(77), Yersinia pseudotuberculosis(97),

Mycobacterium tuberculosis(98,99), Streptococcus pneumoniae(100,101), adherent-invasive E.

coli(102), S. Typhimurium(103) and Staphylococcus aureus(104).

Since both Nod1 and Nod2 share a common downstream adaptor, it is not surprising that

there is a degree of overlap in the function of these receptors. Indeed, Nod1Nod2 double-

knockout mice (DKO) exhibited increased systemic colonization by Listeria monocytogenes

compared to Nod1-/- or Nod2-/- mice(95). Moreover, we recently found that Nod1/Nod2 double-

knockout mice exhibited increased bacterial translocation and more severe colonic pathology

during infection with the Gram-negative enteric pathogens S. Typhimurium(105) and

Citrobacter rodentium(106) (see section 1.4 and Chapter 2).

16

1.3 Role of Nod1 and Nod2 in the Intestine

Considering the roles of Nod1 and Nod2 in mediating host protection against bacterial

infections and modulation of innate and adaptive immune response to bacterial products, it is not

surprising that these PRRs have emerged as important sentinels in the gastrointestinal tract, a site

that is in intimate contact with over 1014

resident bacteria and under constant assault from

invading pathogens(107).

Since their discovery, Nod1 and Nod2 have been reported to regulate intestinal

homeostasis and immunity by a number of mechanisms (see Fig 1.4), these include: 1) mediating

Paneth cell secretion of anti-microbial peptides(108,109); 2) enhancing the barrier function of

enterocytes(110,111); 3) regulating the recruitment and function of mucosal DCs(93,112); 4)

controlling the formation of intestinal lymphoid follicles (ILFs)(111,113,114); 5) initiating

inflammatory pathways after breach of the epithelial barrier by enteric bacterial

pathogens(105,106,115).

In this section, we will review in greater detail the genetic association between Nod2 and

susceptibility to develop the inflammatory bowel disease (IBD), Crohn’s disease (CD); the link

between Nod1 and Nod2 signaling and the enteric microbiota and finally summarize the studies

that have assessed the physiological functions of Nod1 and Nod2 in murine models of colitis.

17

Figure 1.4 Mechanisms of Nod1 and Nod2 mediated regulation of gut homeostasis. (a)

NOD2 expressed in Paneth cells directs the secretion of antimicrobial peptides, such as -

defensins, which modulate the composition of the enteric microbiota. (b) Stimulation of CD103+

dendritic cells (DC) with NOD2 ligands results in IL-10 production that promotes regulatory T

cells responses. (c) NOD1-dependent CCL20 is critical for the development of intestinal

lymphoid follicles (ILFs). (d) NOD2 is important for maintaining the integrity of the epithelial

barrier, which if compromised results in increased bacterial translocation and thus more ILFs and

Peyer’s patches. Upon infection with an intestinal bacterial pathogen, (e) NOD1 and NOD2 are

important for the early production of IL-6 and the induction of an innate Th17 response, (f) while

epithelial NOD2 dependent CCL2 is needed for the early recruitment of monocyte/macrophages

to the site of infection.(9)

18

1.3.1 Association Between the NOD2 Gene and Crohn’s Disease

IBD can be broadly be classified as one of two distinct disorders: CD and ulcerative

colitis (UC). CD histopathology is characterized by punctate intramural inflammation with

neutrophil infiltration and granulomas often present throughout the entire mucosa and can affect

the entire gastrointestinal tract, although most patients with CD exhibit pathology in the terminal

ileum or ileal-cecal regions of the intestine. UC on the other hand is characterized by diffuse

inflammation and ulceration of the epithelial layer, with the terminal colon being the

predominant region of the colon affected. Early studies determined that both CD and UC are

complex genetic disorders(116,117), indicating that both genetic and environmental elements

contribute to the pathogenesis of these diseases. More recently, genome-wide association studies

(GWAS) have revealed a number of susceptibility genes for CD and UC: 71 for CD, 47 for UC,

and 28 loci that shared between both diseases(118,119). Indeed, many of the susceptibility genes

for CD and UC are implicated in innate immunity and barrier function, established and

reproduced susceptibility loci include: Nod2, ATG16L1, IL-23R, IL-22, STAT3, TLR4 among

others(118,120,121).

One of the first identified susceptibility loci for CD was a Nod2 was a frameshift

mutation in the LRR domain discovered by Ogura et al by examining gene loci that were in

linkage disequilibrium between CD patients versus controls(122). In line with these findings,

Hugot et al identified in an independent study three Nod2 variants (R702W, G908R, and

fs1007insC) associated with CD patients(123). These variants lead to loss of function mutations

within Nod2 that affect MDP sensing(124). More recently, a study examined multiple GWAS

loci that were deep-sequenced and identified five new rare variants of Nod2 (R311W, S431L,

R703C, N852S and M863V) that were present at higher frequencies in CD patients versus

19

controls(125). One of the new rare variants, the S431L mutant, demonstrated impaired MDP-

driven NFkB activation that resembles the defect of the fs1007insC mutant(125). Another newly

identified variant, N852S mutant, in the LRR region exhibited impaired NF-kB activation but

normal membrane localization of the protein(125).

1.3.2 Regulation of the Intestinal Microbiota by Nod1 and Nod2

The gastrointestinal intestinal tract requires constant interaction with a complex community

of resident microbes, dominated by the Bacteroidetes and Firmicutes phyla, in order to function

and develop properly(126). For example, the gut microbiota is essential for regulating proper

nutrient and vitamin uptake, preventing colonization by bacterial pathogens and promoting the

development of secondary lymphoid structures such as Peyer’s patches and intestinal lymphoid

follicles (ILFs)(126). Moreover, dysbiosis, defined as the alteration of the microbiota from its

normal state, is also commonly observed in patients with IBD, however it remains largely

unknown whether these changes drive or are caused by disease pathogenesis(126).

Given the roles of Nod1 and Nod2 as sentinels at the mucosa, many studies in recent years

have examined how deletion of these PRRs in mice can affect the composition of the

microbiota(107). Indeed, the microbiota in the terminal ileum of Nod1-/- mice was initially

reported to be altered compared to WT control mice(114). Specifically, there was an observed

increased in bacterial density, increased percentage of Bacteroides and Enterobacteriaceae and

decreased percentage of Lactobacteriacae as measured by 16S qPCR analysis in Nod1-/-

mice(114). The authors of this study suggested that the changes in the microbiota were caused by

reduced -defensin secretion in Nod1 mice(114). Moreover, Nod1 was found to be required for the

proper generation of intestinal lymphoid follicles by directly detecting ligands from the enteric

microbiota(114). In addition to maintaining intestinal homeostasis, Nod1-activating peptidoglycan

20

ligands from the resident gut microbes are continuously released into the circulation, thereby

promoting systemic priming of the innate immune system, and in particular neutrophils(127).

Regarding Nod2, two studies showed that Nod2-/- mice also contained a microbiota that

exhibited altered bacterial density and percentages of Bacteroides and Firmicutes compared to WT

ileums(128,129). Another study suggested that CD patients harboring the Nod2 frameshift

mutation demonstrated a shift in the composition of their mucosal-attached microbiota compared

to CD patients that did have the frameshift variant(130). However, a detailed study that used

littermate controls and a large number of mice per group recently reported that there were no

appreciable differences in the composition of the enteric microbiota Nod1-/- and Nod2-/-

compared to F2 littermate controls as determined by 16 qPCR analysis(131), highlighting the

importance of properly controlling these types experiments with littermates, and putting into doubt

the results obtained in the previous studies.

1.3.3 Nod1 and Nod2 in Murine Models of Colitis

The first, and still the best characterized, Nod2 SNP that was found to be associated with

CD was the fs1007insC frameshift variant which leads to loss of function of Nod2 sensing of

MDP. Therefore, studying the impact of Nod2 deficiency on in vivo intestinal homeostasis would

provide critical insights into how this NLR is impacting CD pathogenesis. Chemical-induced

models of colitis such as Dextran Sodium Sulphate (DSS) and Trinitrobenzenesulfonic acid

(TNBS) are commonly used murine models of colitis that are characterized by severe destruction

of the intestinal epithelial layer and massive neutrophil influx(132). In both DSS and TNBS-

induced colitis, Nod2-/- mice were more susceptible and demonstrated excessive intestinal

inflammation compared to wild-type treated control mice(112,133-135). In wild-type mice,

treatment with Nod2 ligands (peptidoglycan or MDP) ameliorated TNBS and DSS-driven weight

loss in wild type animals(136). More recently, a study by Couturier-Maillard et al determined

21

that the microbiota of Nod2-/- and RIP2-/- mice predisposes these mice to developing more

severe pathology than wild type mice during DSS colitis(135). Moreover, the authors suggested

that transferring the microbiota of these mice into WT mice rendered the mice sensitive to DSS,

indicating that the sensitivity to develop colitis was communicable from mouse to mouse(135).

In enteric pathogen driven colitis, Nod2-/- mice were more susceptible to Helicobacter hepaticus

infection, which correlated with increased intestinal inflammation and increased frequency of

IFN- secreting Th1 cells in the Peyer’s patches of Nod2-/- mice(137). Finally, a mouse

harbouring the fs1007insC Nod mutant alleles (Nod2m/m) has recently been generated and,

similarly to Nod2-/- mice, the Nod2m/m strain exhibited severely impaired sensing of MDP and

increased susceptibility to the enteric pathogen Enterococcus faecalis(138).

Unlike Nod2, SNPs in Nod1 have not been robustly genetically linked with increased

susceptibility to develop IBD, however Nod1 is highly expressed intestinal epithelial cells and

likely plays an important role in regulating host responses to the normal gut microbiota and to

enteric pathogens in these cells(121). Upon challenge with DSS, Nod1-/- mice exhibited

exacerbated intestinal inflammation compared wild-type mice, which was partially attributable to

an increased intestinal permeability observed in these mice(139). Moreover, Nod1-/- mice had an

increased frequency of colonic polyps after injection with the carcinogen azoxymethane (AOM)

followed by DSS administration (AOM/DSS model of colitis-associated carcinoma (CAC))(139).

The increased susceptibility of Nod1-/- mice to CAC was dependent on signals from the

microbiota as antibiotic depletion before inducing colitis prevented this increased frequency of

polyps(139). Nod1-/- mice were recently shown to have a greater mortality rate to oral infection

with the enteric pathogen Clostridium difficile, which correlated with reduced CXCL1 expression

and neutrophil recruitment to the cecum and colon(140).

22

Nod1-/-/Nod2-/- mice provide a useful tool to address the importance of total PG

recognition by the mammalian intestinal immune system, which is especially relevant for host

sensing of gram-negative bacteria that express ligands for both Nod1 and Nod2. Similarly to Nod1

or Nod2 single knockout mice, Nod1-/-/Nod2-/- mice have increased intestinal permeability,

exhibit increased translocation of and are more susceptible to DSS-colitis(110). Moreover, Nod1-/-

/Nod2-/- mice could be partially rescued from colitis by altering their normal microbiota by

feeding the mice with the probiotic Bifidobacterium breve(110).

1.4 Models of Enteric Pathogen-Induced Colitis

There does not exist a model that can fully recapitulate the full pathological spectrum of

either CD or UC. However, over the years several infectious-colitis models have emerged as

useful tools to study the initiation, progression and resolution of physiologically relevant

inflammatory responses in the gastrointestinal tract. Of these enteric pathogen induced-models,

the Citrobacter rodentium and streptomycin-Salmonella enterica serovar Typhimurium-induced

models colitis are the most reproducible, routinely used, and well characterized and will be

reviewed in this section(141).

1.4.1 Citrobacter rodentium-induced colitis

C. rodentium is a gram-negative bacterium that was originally described in the 1960s by

Barthold and colleagues as the etiological agent of transmissible murine colonic hyperplasia

(TMCH) in mice(142,143). C. rodentium-induced TMCH colitis displays many of the hallmarks

seen during IBD, including: inflammatory cell infiltration, goblet cell depletion and epithelial

layer remodelling(144). C57Bl6 mice infected with C. rodentium demonstrate peak pathology in

the distal colon at 10-14 days post-infection (p.i.) and the mice clear the pathogen after 21-28

23

days p.i. Studies using a bioluminescent C. rodentium have demonstrated that this pathogen

initially colonizes the cecum and then moves to the distal colon at day 3-4 p.i(144).

C. rodentium is a strict murine pathogen in the same family as enteropathogenic

Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), which cause severe diarrheal

diseases in humans (ie “Hamburger Disease”). C. rodentium, EPEC and EHEC are all classified

as attaching/effacing (A/E) pathogens because they colonize the colon by first firmly attaching to

the luminal surface of enteroctyes, which is then followed by the effacement of the surrounding

microvili and distinctive pedestal formation(144). The A/E lesions are induced by bacterial

effectors secreted into the enterocyte by a type-three secretion system (TTSS) encoded on the

locus of enterocyte effacement (LEE)(144). Of the more than 41 genes located in the LEE, the

effectors intimin and Tir (translocated intimin receptor) are essential for initial A/E pedestal

formation and, therefore, colonization by this pathogen(145). Specifically, C. rodentium will

inject its own receptor (Tir) into the enteroctye via a TTSS needle that will then tightly bind to

intimin located on the outer membrane of the bacterium(145).

C. rodentium-induced TMCH has proven to be a very useful mouse model to dissect the

functions of various arms of the adaptive and innate immune system in the intestinal mucosa

during a physiologically relevant inflammatory response. The adaptive response generated by C.

rodentium is thought to be Th1- and Th17-driven, and both CD4+T cells and B cells are needed

to clear the infection at later stages(146-149) (2-3 weeks post-infection). However, mice

deficient for CD8+T cells, T cells and most classes of immunoglobulins (IgA, IgM, IgE and

IgG1) do not exhibit any defects in controlling this pathogen(146,147,149). The mucosal Th17

response induced by this pathogen is critical to clear infection and will be reviewed in greater

detail in section 1.5.

24

With regards to the innate immune recognition of C. rodentium, the TLR adaptor protein

MyD88 is critically required to prevent necrotic lesion formation in C. rodentium-infected

colons(150,151). Other studies have further characterized that mice deficient in either TLR4 or

TLR2 have altered inflammatory responses to this pathogen in vivo(152,153). Finally, we

determined that Nod1/Nod2 double-deficient mice exhibit reduced inflammation and a blunted

Th17 response during the early stages of infection (day 4 p.i.) compared to infection in wild-type

(WT) C57Bl/6 mice(106). This delayed response correlated with exacerbated pathology and

systemic spread of C. rodentium at later time points (day 14 p.i) (see Chapter 2). Moreover,

Nunez and colleagues reported a blunted onset of the inflammatory response in Nod2-/- mice

infected with C. rodentium, which was characterized by impaired recruitment inflammatory

monocytes and reduced induction of Th1 response in the mucosa(115).

1.4.2 Salmonella enterica serovar Typhimurium-induced colitis

In humans, Salmonella Typhimurium infections result in a gastrointestinal disease that

ranges from gastroenteritis to enterocolitis, with symptoms including: nausea, abdominal pain

and diarrhea. In genetically susceptible mice, S. Typhimurium infection results in systemic

bacteremia that resembles the typhoid fever induced by Salmonella typhi infections in

humans(154,155). However, S. Typhimurium can induce colitis in C57Bl/6 mice following pre-

treatment with the antibiotic streptomycin and infection with a streptomycin resistant strain of S.

Typhimurium (strain SL1344)(156). This model generates an acute and severe inflammatory

response in the cecum that occurs as early as 20 hours p.i, and is characterized by neutrophil

infiltration, edema and goblet cell depletion. Infection of C57Bl6 mice with 5X107 colony-

forming units (CFU) of this pathogen results in peak cecal pathology occurring at 72 hours p.i,

and a 100% mortality rate with the first mice dying as early as day 5 p.i(154,155).

25

S. Typhimurium is a facultative intracellular pathogen that displays two very well

characterized pathogenecity loci, SPI-1 and SPI-2 (Salmonella pathogenicity island 1 and 2,

respectively), which encode two distinct sets of TTSS virulence effectors. SPI-1 effectors are

required for S. Typhimurium invasion of fibroblasts and epithelial cells in vitro and mediate in

vivo the entry into M cells, which are specialized enterocytes located at the base of Peyers

patches.(155) SPI-2 effectors are critical for the maturation of the Salmonella-containing vacuole

(SCV) in infected cells. The SPI-2 permits the intracellular replication of this pathogen inside

epithelial cells and macrophages, and SPI-2-deficient mutants are completely avirulent in both in

vitro and in vivo infections(154,155).

S. Typhimurium infection induces the upregulation of numerous cytokines and

chemokines in the cecal mucosa very early during infection in the streptomycin pretreatment

model, and these include IFN, IL-1, KC and CXCL-2. KC and CXCL-2 are potent granulocyte

chemo-attractants and mediate the recruitment of neutrophils observed in the first hours after

infection(154,155). IFN-/- mice display a blunted induction of the inflammatory response in the

first 10 hours p.i, and increased S. Typhimurium colonization at later time points, indicating a

role for IFNin controlling pathogen burden(157,158). Moreover, the cytokines IL-17 and IL-22

are also significantly induced after infection and play a significant role in mucosal immunity

(reviewed in Section 1.5).

With regards to the innate immune system in the streptomycin-Salmonella model,

MyD88-/- mice exhibit reduced inflammation at early time points and impaired clearance of the

pathogen at later time points compared to WT counterparts(159). Similarly, RIP2-/- and

Nod1/Nod2 DKO mice also demonstrated a delayed onset of cecal inflammation at early time

points after infection(105). Nod1-/- mice were found to be more susceptible to infection with

26

SPI-1 deficient Salmonella mutant, suggesting a role for Nod1 signaling in gut resident DCs that

regulate engulf the pathogen(93). Finally, mice deficient for Caspase-1, NLRP3 and NLRC4

exhibited increased systemic spread of S. Typhimurium, as IL-1 and IL-18 are required for full

initiation of the host inflammatory response(160).

1.5 IL-17 and IL-22: Mediators of Mucosal Immunity

In humans and mice, the IL-17 cytokine family consists of six members: IL-17A (IL-17),

IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F. IL-17 and IL-17F share close sequence

homology and can both signal through the receptors IL-17RA and IL-17RC found on both

hematopoietic and non-hematopoietic cells(161-164). IL-17 was initially found to be secreted by

a subset of CD4+ T cells termed T helper type 17 (Th17) cells that also express the cytokines IL-

22, IL-17F and IL-21(161,165) Functionally, IL-17 induces neutrophil granulopoiesis by

stimulating epithelial cells to secrete G-CSF. Furthermore, IL-17A and IL-17F can directly

recruit and activate neutrophil responses at sites of inflammation(161,164). Moreover, IL-17 was

also recently described to play a critical role for B-cell class switching to IgA-producing plasma

cells in the Peyer’s patches of the small intestine(166). Often acting in concert with IL-17, IL-22

signals through the IL-22R, which is exclusively located on non-hematopoietic cells and highly

expressed on enterocytes, to induce STAT3-dependent innate epithelial defense

mechanisms(167,168). These include: stimulating the secretion of antimicrobials such as Reg

proteins, lipocalin-2 and defensins; reinforcing tight junctions between enterocytes; and

enhancing epithelial cell proliferation(167,168). Both IL-17 and IL-22 have emerged as critical

mediators of mucosal innate immunity (Fig 1.5) and in this section we will review in greater

detail the physiological regulation and importance of these cytokines for mucosal defense against

enteric pathogens

27

28

Figure 1.5 Role of innate IL-17/IL-22 responses to enteric bacterial infections. Extracellular

pathogens (such as C. rodentium) and intracellular pathogens (such as S. Typhimurium) are

detected via either direct or indirect mechanisms by dendritic cells that produce cytokines (TGF-

, IL-6, IL-23 and IL-1) that drive IL-17 and IL-22 production by innate lymphocytes. IL-22

primarily acts on epithelial cells (such as Paneth cells) to promote barrier functions such as

enhancing production of antimicrobial peptides that control bacterial growth while IL-17 acts to

promote recruitment and activation of neutrophils that prevent bacterial spread.(169)

29

1.5.1 Differentiation Program of Th17 Cells

Similarly to Th1 or Th2 cells, differentiation of naïve CD4+ T cells into Th17 cells

requires T cell receptor recognition to its cognate antigen presented on MHC class II by

professional APCs such as DCs or monocytes. Moreover, Th17 cell differentiation in vivo is

driven by the cytokines IL-6 and TGF-, whereas IL-23 and IL-1 are required for the rapid and

sustained activation of these cells(161,163,165). The transcription factors RORt and ROR

coordinate the differentiation of Th17 cells, while STAT3 (a transcription factor downstream of

IL-6R and IL-23R) and the aryl hydrocarbon receptor (AHR) control RORt expression levels

and enhance cytokine secretion(170-172). In addition to Th17 cells, a number of other RORt-

expressing cell types can secrete IL-17 and IL-22, these include: CD8+ T cells (“Tc17”

cells)(173), T cells, Lymphoid Tissue Inducer cells (LTi)(174), LTi-like ILCs(175), NKp46+

ILCs(176-178), iNKT cells(179)and mucosal-associated invariant T- cells (MAIT cells)(180).

Unlike Th17 cells, antigenic priming is not required for activation of LTi, T cells, iNKT and

NK cells and IL-23 stimulation is often sufficient for inducing IL-17 and IL-22 secretion by

these cell types(181) (reviewed in section 1.5.3).

1.5.2 Homeostatic Regulation of Th17 cells by the Intestinal Microbiota

Studies using germ-free mice have recently demonstrated that the microbiota is required

for the generation of Th17 cells in the intestinal lamina propria (LP)(106,182,183). In one study,

bacteria-derived ATP was determined to be the critical factor for stimulating DC-driven Th17

differentiation(182). Subsequently, a seminal paper by Ivanov and colleagues identified

segmented filamentous bacteria (SFB), a member of the Clostridia genus that attaches itself to

the surface of enterocytes in the gut lumen, as one of the specific microbial species that induces

homeostatic Th17 responses in the gut(184). Another group confirmed that SFB could strongly

30

induce LP Th17 responses, however in this report SFB also upregulated basal gut IFN+ Th1 and

FOXP3+ regulatory T cell responses(185). Moreover, reduction of SFB levels in the enteric

microbiota by Paneth cell-derived defensins was also shown to concomitantly decrease LP Th17

cell numbers(186). Importantly, SFB-primed homeostatic Th17 responses enhanced host

protection during infection with C. rodentium by limiting the colonic colonization of this

pathogen(184). In line with these findings, recent work from our lab demonstrated that germ-free

mice are severely impaired in their ability to mount a LP Th17 response during infection with S.

Typhimurium(106) (see Chapter 2), which indicates that the microbiota is needed to prime gut

Th17 cells so that they can respond to a bacterial pathogenic insult.

1.5.3 Importance of Intestinal IL-17/IL-22 Responses to Bacterial Pathogens

The C. rodentium model has been very useful for dissecting how various arms of the

intestinal immune system respond to a bacterial pathogen. Indeed, C. rodentium has been shown

to induce a robust adaptive IL-17 response at 10 to 14 days post-infection, while also inducing a

more modest IL-17 response in the cecum and colon at earlier timepoints, days 4 to 7 post-

infection(106,187,188). A study using IL-17A/IL-17F-/- double-knockout mice demonstrated

that both IL-17A and IL-17F are required to properly contain C. rodentium colonization and

prevent aberrant intestinal pathology, however IL-17A-/- and IL-17F-/- single knockout mice

exhibited a comparable phenotype to C57Bl6 wild-type (WT) mice, highlighting a level of

overlap in the functionality of these two cytokines(189). IL-22 expression from ILCs, Th22 and

Th17 cells in the cecum and colon is also induced very early during C. rodentium infection(190-

192), at day 4 post-infection, and IL-22-/- mice succumb to disease by 10 days post-

infection(187). Notably, IL-22 dependent RegIII secretion was found to be critical for

mediating protection against C. rodentium as exogenously added RegIIIrescued IL-22-/- mice

31

from infection induced mortality and morbidity(187). Moreover, IL-6-/- and IL-23-/- mice, two

inflammatory cytokines that induce IL-17/IL-22 responses, also fail to contain C. rodentium and

die during the course of the infection(187,193). More recently, two studies reported that IL-17C

levels in intestinal epithelial cells are maximally induced at 4 days post C. rodentium challenge

and that IL-17C signaling through IL-17RE was essential to limit systemic spread of the

pathogen and prevent mortality(188,194).

The streptomycin treated mouse model of S. Typhimurium colitis is also frequently used

to investigate severe gut inflammatory responses. In this model, IL-6, IL-1, IL-17 and IL-22 are

all robustly induced in the cecum within 24 hours post-infection(105,106,159,195,196), while

IL-23 is induced at 48 hours post-infection. S. Typhimurium infected IL-17RA-/- mice have

reduced levels of inflammatory cytokines and neutrophil recruitment and increased levels of

bacterial translocation to the spleen and mesenteric lymph nodes(197). IL-23 is essential for

early IL-17 and IL-22 expression in cecal tissue as IL-23-/- mice exhibit significantly reduced

levels of these cytokines following S. Typhimurium challenge leading to decreased neutrophil

recruitment(198). Finally, depletion of IL-6 with a monoclonal antibody results in decreased

expression of IL-17A and IL-22 expression in Th17 cells, but not any other cell type

investigated, during the early stages of S. Typhimurium infection(106). In agreement with this

finding, a blunted LP Th17 response has also been observed in IL-6-deficient mice(106) after

infection with S. Typhimurium.

Together, these studies suggest that bacterium-induced IL-23 and IL-6 act in concert to

drive IL-17A and IL-22 expression during what can be classified as the ‘innate’ stage of the

course of an infection. Moreover, rapid IL-17- and IL-22-dependent innate response

mechanisms, including neutrophil activation and epithelial cell antimicrobial peptide secretion

(Fig 1.5), appear essential for proper host defense against extracellular and intracellular bacte-

32

rial pathogens in the gut mucosa. In the next section, we review the rapidly expanding number of

studies that describe the various cell types that drive innate IL-17A and IL-22 responses to

bacterial pathogens in the gut (see Fig 1.6 for a graphical summary).

1.5.4 Cellular Sources of IL-17 and IL-22 in the Gut Mucosa

CD4+ T helper cells were the first cell population described to secrete IL-17 and IL-22

and although new IL-17 and IL-22-producing cell types are emerging, Th17 cells remain the

most abundant cellular source of these cytokines in human gut tissue. Indeed, Rafatellu and

colleagues demonstrated in a SIV infection macaque model that depletion of gut CD4+Th17

leads to an overaggressive infection and increased mortality during S. Typhimurium

challenge(197). Similarly, depletion of CD3+ T cells in the streptomycin S. Typhimurium mouse

model results in a blunted innate IL-17 response and associated decrease in mucosal

protection(199). Recent reports have also identified two novel subsets of Th17 cells, a pro-

inflammatory subset that produces GM-CSF(200,201) and a regulatory subset that produces IL-

10(202).

During S. Typhimurium infection IL-17, and to a lesser extent IL-22, expression is

strongly induced in gut T cells(106,159). T cells are predominantly found in the intestinal

epithelial lymphocyte (IEL) compartment of the intestinal mucosa and these cells can be divided

into IL-17 producing or IFN- producing subsets(181). Indeed, CD27-RORt+ T cells secrete

IL-17 when stimulated with IL-23, IL-1 or a phorbyl 12-myristate 13 acetate (PMA) and

ionomycin cocktail(203,204). Alternatively, CD27+ T cells express the transcription factor T-

bet which governs IFN- secretion(203,204).

Murine lymphoid tissue inducer cells (LTi) cells are defined based on the surface marker

phenotype: leukocyte lineage marker negative (LIN-) CD4+CCR7+THY1+(171,174). In humans

33

LIN-CD4-CD127+CD45int cells are classified as LTi(205). LTi cells can be isolated from the

spleen, lymph nodes and gut LP and are believed to play a critical role in lymphoid organ

development through the production of lymphotoxin- (LT and lymphotoxin-LT(178). In

ex vivo experiments, LTi cells can secrete IL-17 and IL-22 after stimulation with IL-23(171,174).

In vivo, LTi cells are a major source of IL-22, but not IL-17, in the gut LP and early IL-22

production by intestinal epithelial LTi cells was required to contain C. rodentium infection(191).

Notably, two recent studies demonstrated that the (LT)/LTR pathway, which was previously

shown to be critical to survive C. rodentium infection(206,207), is triggered by IL-22 producing

LTi cells located in intestinal lymphoid follicles to provide mucosal protection against this

pathogen(208,209). Moreover, a population of IL-17 producing LTi-like cells expressing THY1,

stem cell antigen 1 (SCA-1), and RORt were discovered in RAG-deficient mice infected with

Helicobacter hepaticus(175). Similarly to LTi cells, IL-23 is required to induce IL-17, IFN, and

to a lesser extent IL-22, expression by THY1+SCA1+ LTi-like ILCs(175).

A novel subset of IL-22 producing NKp46+ ILCs was recently identified by a number of

independent groups(176,177,210). In humans, NK1.1+NKp44+ cells from tonsils were

discovered to produce IL-22 when stimulated with IL-23 or the chemokine CCL20 ex vivo(176).

In mice, NKp46+ ILCs can be stimulated by IL-23 to release IL-22(177,210). NKp46+ ILCs are

also upregulated during infection with C. rodentium, however NKp46 is not required to fight off

infection with this pathogen(211). Studies have demonstrated that a fraction of both murine and

human LTi cells can express NK markers in vitro(205). Accordingly, a recent report suggested

that NKp46+, CD4+ and CD4- LTi-like cells could all derive from a common fetal liver-derived

precursor cell in vivo(212). However, a definitive answer regarding the exact lineage

relationship between various IL-22-producing ILC subsets still requires further investigation.

34

35

Figure 1.6 Innate IL-17 producing lymphocytes in the gut. NKp46 cells produce IL-22 in

response to IL-23 and IL-1, express the surface markers NK1.1, NKp46 and NKp44, and the

transcription factors ID2, IRF4, AHR, and RORt. Lti-like ILCs produce IL-17, IL-22 and IFN-

in response to IL-23, express the surface markers THY1, SCA-1 (stem cell antigen-1), and

CCR6, and the transcription factors T-bet and RORt. Lti-like cells produce lymphotoxin and

, IL-17, and IL-22 in response to IL-23 and TLR2 stimulants such as zymozan, express the cell

surface markers CD4, THY1, cKit, CD127, OX40L, CCR7 and CX3CR5, and the transcription

factors ID2, STAT3, AHR and RORt. T cells produce IL-17 and IL-22 in response to IL-23,

IL-1, IL-21 and stimulation with TLR agonists (Curdland and PAM3CSK that act through

TLR1/TLR2 and TLR2/Dectin-1, respectively), express the surface markers TCR, CD3, and

CCR6, and express the transcription factors RUNX1, IRF4, AHR, RORt. Innate Th17 cells

produce IL-17 and IL-22 in response to TGF- and IL-6 or IL-1 and IL-23, express CD4,

TCR ion factors RORt, ROR, AHR

and STAT3.(169)

36

1.5.5 Induction of IL-17 responses by Pattern Recognition Receptors

Stimulating APCs in ex vivo systems with LPS, detected by toll-like receptor 4 (TLR4),

or other TLR-recognized MAMPs leads to the induction of MHC antigen presentation, up

regulation of co-stimulatory molecules and secretion of a number of pro-inflammatory cytokines,

including the Th17-polarizing IL-6, IL-23 and IL-1 (Fig 1.7). Indeed, Torchensky and

colleagues demonstrated that DCs stimulated with apoptotic cells and TLR agonists drive IL-6-

dependent Th17 differentiation, whereas stimulation with apoptotic cells alone results in a TGF-

-induced regulatory T cell response(213). TLR agonists can synergize with Dectin-1, a PRR

that detects fungal -glucans, to directly stimulate T cells to produce IL-17 and IL-22 via

TLR1 and TLR2 pathways without the need for TCR recognition(214) and this response can be

amplified by addition of IL-23 or IL-1(215). More recently, flagellin, a TLR5 agonist, injected

intra-peritoneally into mice was shown to induce an innate IL-17 and IL-22 response in both the

spleen and gut by a novel population of CD3-CD127+ cells(216). Direct stimulation of TLR2

with its cognate ligand can also induce the rapid induction of IL-17 and IL-22 in vivo, and

TLR2-/- mice demonstrated a blunted IL-17/IL-22 response after infection with S.

Typhimurium(195).

Myd88 is critically required to mount a proper host response to S. Typhimurium as

Myd88-/- mice exhibit a delayed mucosal inflammatory response and blunted IL-17 and IL-22

levels at 24 hours post-infection compared to wild-type C57Bl/6 mice in the streptomycin-S.

Typhimurium model(159). Similarly, Myd88-/- mice also display a reduced innate mucosal IL-

17 response as compared to WT mice during infection with Klebsiella pneumonia in a lung

model(217). Myd88-/- mice also exhibit increased mortality, intestinal pathology and bacterial

37

systemic spread during infection with C. rodentium(150,151), however the role of Myd88 in

mediating innate IL-17 and IL-22 responses in this model have yet to be elucidated.

Acting upstream of the Th17 response, we determined that Nod1 and Nod2 were required

for induction of IL-6, but not IL-23, in LP DCs during infection with S. Typhiumurium or C.

rodentium(106) (see Chapter 2). Similarly, in a cutaneous Staphylococcus aureus infection

model, Nod2 was previously shown to mediate the early induction of IL-6 in the skin(104),

which was required for clearance of the pathogen. Moreover, a study by Van Beelen and

colleagues had demonstrated that stimulating murine and human DCs with a Nod2 agonist

induces Th17 cell differentiation ex vivo through an IL-23-dependent mechanism(218). In

agreement, injection of CFA results in a Nod1 and Nod2-dependent Th1 and Th17 polarized

immune responses(81,83) (Fig. 1.7). Furthermore, Nod2 and RIP2 controlled joint targeted Th17

responses in a mouse model of arthritis(219). Moreover, in the CFA-induced model of

experimental autoimmune encephalitis, RIP2 was shown to be required for DCs to induce a

robust myelin-specific Th17 response(220), further reinforcing that DCs are the nexus point

governing the NOD-Th17 axis (Figure 1.7).

Finally, given the association between IL-1β and Th17 cell activation, NLRP3 activation

could putatively regulate IL-17 responses. In support for this, IL-1β produced by the NLRP3-

inflammasome has recently been shown to mediate a protective Th17 response against

Bordetella pertussis in a lung infection model(221) and against E. coli heat-labile

enterotoxin(222) in another infection model. However, at this time, the role of NLRP3 or other

inflammasome-triggering NLRs, such as NLRC4 and NLRP6, in mediating colonic IL-17A and

IL-22 expression has yet to be determined.

38

39

Figure 1.7 Dendritic cells sense infection and drive innate IL-17 and IL-22 production.

Intracellular or extracellular bacterial pathogens are detected via cell surface TLRs or cytosolic

NLRs (Nod1 and Nod2) that activate signal transduction cascades, via MyD88/TRIF and RIP2,

respectively, which promotes NF-B nuclear translocation. NF-B mediates expression of

cytokines including IL-6, IL-23 and the proform of IL-1. Detection of infection via NLRs (such

as NLRC4 or NLRP3) or other inflammasome activation leads to processing of caspase-1 into its

enzymatically active form. Caspase-1 in turn cleaves pro-IL-1, releasing active IL-1 that can

work in concert with IL-23 to drive IL-17 production by innate lymphocytes (NK-22 cells, Lti-

like ILCs, Lti-like cells, T cells and iTh17 cells). TGF- production is upregulated in dendritic

cells upon detection of phosphatidylserine released by apoptotic cells and works in concert with

IL-6 to drive IL-17 and IL-22 production by iTh17 cells.(169)

40

1.6 Thesis Overview

Rationale and Hypothesis

Since their discovery over ten years ago, Nod1 and Nod2 have emerged as important

mediators of intestinal immunity during both homeostatic conditions and in response to infection

with enteric pathogens. Moreover, Nod1 and Nod2 signaling has been reported to regulate Th17

responses in vitro and in vivo, however whether these pathways are linked in the gastrointestinal

tract remains unclear. Given the roles of the NLR and Th17 pathways in intestinal immunity, we

hypothesized that Nod1 and Nod2 could regulate Th17 responses in the mucosa.

Objectives

Chapter 2)

Determine the roles of Nod1 and Nod2 in mediating an innate Th17 response (iTh17) to

the enteric bacterial pathogens C. rodentium and S. Typhimurium.

Chapter 3)

Further characterize the requirement for antigen specificity of the iTh17 response by

deleting MHC class II –dependent antigen presentation in hematopoietic cells.

Chapter 4)

Turn our focus away from gastrointestinal tract and instead assess the capacity of a

library of novel Nod2 agonists to induce NF-B responses in vitro and innate and

adaptive responses in vivo.

41

Chapter 2

Identification of an Innate Th17 Response to Enteric

Bacterial Pathogens

Stephen J. Rubino*

and Kaoru Geddes* (*: co-first author publication), Joao G.

Magalhaes, Catherine Streutker, Lionel Le Bourhis, Joon H. Cho, Susan

Robertson, Connie J. Kim, Rupert Kaul, Dana J. Philpott and Stephen E. Girardin

Nature Medicine, 2011 Jun 12.

I designed and performed all the experiments and wrote the manuscript. K.G. helped design and

perform experiments and write the manuscript. J. G. M. helped with in vivo experiments. C. S.

performed pathological scoring. L. L. B. made the Nod1-/-Nod2-/- mice. J.H.C helped with

certain in vivo experiments. S. R. did the microbiota analysis. C. J. K. and R. K. provided human

gut samples. D. J. P and S. E. G. supervised the research and helped write the manuscript.

42

2.1 Abstract

Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial

defense. Following infection, both innate and adaptive IL-17 responses have been reported,

but the nature of the cells involved in innate IL-17 induction, as well as their in vivo

importance, are poorly understood. Herein, we demonstrated that Citrobacter and

Salmonella infection triggered innate IL-17 responses, which were critical for host defense

and were mediated by CD4+ T helper cells. Enteric innate TH17 (iTH17) responses occurred

principally in the caecum, were critically dependent on the Nod-like receptors Nod1 and

Nod2, required IL-6 induction, and were associated with a decrease in mucosal CD103+

DCs. Moreover, imprinting by the intestinal microbiota was fully required for the

generation of iTH17 responses. Together, these results identify the Nod-iTH17 axis as a

central element in controlling enteric pathogens, which may implicate Nod-driven iTH17

responses in the development of inflammatory bowel diseases.

43

2.2 Introduction

The Th17 response has emerged as a critical component of mucosal immunity to bacterial

pathogens in the lung and intestine. In particular, the IL-17/IL-22 axis has been shown to

mediate protection in a number of lung infection models including Klebsiella pneumoniae,

Pseudomonas aeruginosa, Shigella flexneri and several Mycobacterium species(223-228). In the

gastrointestinal tract, IL-17/IL-22 has been shown to confer protection against Helicobacter

pylori, Citrobacter rodentium and Salmonella enterica serovar Typhimurium(189,196-198,229).

C. rodentium-induced colitis triggers a vigorous colonic Th17 response by the second week post-

infection, which is required for full protection against this pathogen(187,230). Streptomycin pre-

treated mice infected with S. typhimurium develop an acute inflammatory response in the cecum,

with IL-17A produced early (24-48 hours) by T cells and other unidentified

cells(196,198,199).

Depending on the infection model used, IL-17A production in the intestine could occur

immediately following infection (hours to days p.i.)(187,198) or at late stages (weeks

p.i.)(187,230), suggesting the involvement of distinct levels of control by the innate and adaptive

immune systems. In particular, early IL-17-dependent responses following bacterial infection

suggest the existence of regulatory pathways directly linking innate microbial detection to the

activation of IL-17-producing cells. However, neither the host sensing systems responsible for

early activation of IL-17 secretion, nor the identity of the IL-17-producing cells providing early

responses to bacterial pathogens in vivo, have been clearly identified.

In the present study, we determined that the innate immune receptors, Nod1 and Nod2,

were critical for induction of an early inflammatory response during C. rodentium- colitis.

44

Interestingly, blunted pathology was associated with a severely depleted mucosal Th17 response

at early stages of infection in the caecum (day 4 p.i.). In the S. typhimurium model, we observed

induction of a robust cecal Th17 response in wild-type mice 24 hours post infection that was

abrogated in Nod1/Nod2-deficient mice. We termed these cells innate Th17 cells (iTh17)

because of their early induction and their distinct regulation by Nod1 and Nod2 compared to

late-stage (day 10 p.i.) adaptive phase Th17 cells. Regulation of the intestinal Nod-iTh17 axis

was dependent upon the expression of IL-6 and required microbiota for induction. Taken

together, these results identify the Nod-iTh17 axis as a critical element of mucosal immunity

against bacterial pathogens.

2.3 Material and Methods

Mice C57Bl/6 (Charles River), IL-6-/- (Jackson Laboratories), Germ-free (GF) and Swiss-

Webster (Taconic Farms), Nod1-/- (Millenium Pharmaceuticals), Nod2-/- (from Jean-Pierre

Hugot) and Nod1/Nod2-/- mice were bred and housed according to specific pathogen free

conditions (SPF) in the Center for Cellular and Biomolecular Research, University of Toronto,

Canada (see details in Supplemental Methods). No gender-specific differences were observed in

the colitis models, and both female and male mice were used for experiements but within a

single experiment gender and age was matched. All animal experiments were approved by the

Animal Ethics Review Committee of the University of Toronto.

Bacterial infections Unless otherwise indicated, 1x109 colony-forming units (CFU) of an

overnight culture of naladixic-acid resistant Citrobacter rodentium strain DBS100 (provided by

Dr. Brett Finlay) were used to infect 6-10 week old mice that were fasted for 3 hours. For S.

typhimurium infections, mice were fasted for 3 hours then orally administered 20 mg of

streptomycin, 24 hours later the mice were fasted again prior to oral inoculation with 5X107 CFU

45

of an overnight culture of SL1344, a streptomycin resistant strain of S. typhimurium(156). C.

rodentium colonic and splenic colonization was determined by homogenizing fecal pellets or

spleens, respectively, in sterile PBS using a rotor homogenizer followed by serial dilution plating

on naladixic acid-containing LB plates.

Pathological scoring At sacrifice the mouse colons were collected and cleaned, then cut open

longitudinally and rolled, then immediately fixed with 10% formalin. Fixed samples were stained

with H/E at the Toronto Center of Phenogenomics using standard procedures. Pathological

scoring was performed blindly by a pathologist specializing in intestinal inflammation using

previously established scoring system to assess C. rodentium pathology(150).

Chimeras Chimeras were generated by lethally irradiating recipient mice with 900 centiGray of

ionizing radiation. A day later, these mice were reconstituted with 4x106

donor mouse bone

marrow cells. The mice were then allowed to reconstitute for at least 6 weeks prior to

experimental procedures.

Quantitative real-time PCR Cecum and colon samples for quantitative real-time PCR (qRT-

PCR) were collected and stored RNAlater (Sigma), then RNA was extracted using Qiagen

RNeasy Extraction kits. Genomic DNA was digested using Turbo DNase (Ambion) before

reverse transcription to cDNA with superscript RTIII (Invitrogen). qRT-PCR was performed

using either SYBR green (Applied Biosystems) or TaqMan probes (ABI). Values were

calculated using the Ct method and were normalized to the housekeeping gene RPL-19.

QRT-PCR primer sequences. The following primer sequences, which have been described

elsewhere, were used in the current study: Il22, forward, 5’-TCCGAGGAGTCAGTGCTAAA-

3’, reverse, 5’-AGAACGTCTTCCAGGGTGAA-3’, probe, TGAGCACCTGCTTCA

46

TCAGGTAGCA (FAM, Black Hole Quencher (BHQ));Il17a, forward, 5’-GCTCCAGAAGGC

CCTCAGA-3’, reverse, 5’-CTTTCCCTCCGCATTGACA-3’, probe, 5’-ACCTCAACCG

TTCCACGTCAC-3’ (FAM, BHQ); housekeeping gene Rpl-19, forward, 5’-GCATCCTCA

TGGAGCACAT-3’, reverse, 5’-CTGGTCAGCCAGGAGCTT-3’, probe, 5’-CTTGCGGGC

CTTGTCTGCCTT-3’ (FAM,BHQ); Il6, forward, 5’-TCCAATGCTCTCCTAACAGATAAG-

3’, reverse, 5’-CAAGATGAATTGGATGGTCTTG-3’, probe, 5’-TCCTTAGCCACTC

CTTCTGTGACTCCA-3’ (FAM, BHQ); Reg3g, forward, 5’-ATGGCTCCTATTGCTATGCC-

3’, reverse, 5’-GATGTCCTGAGGGCCTCTT-3’, probe, 5’-TGGCAGGCCATAT

CTGCATCATACC-3’ (FAM, BHQ); Il23a, forward, 5’-GGTGGCTCAGGGAAATGT-3’,

reverse, 5’-GACAGAGCAGGCAGGTACAG-3’; Lcn2, forward, 5’-ACATTTGTTCCAAG

CTCCAGGGC-3’, reverse, 5’-CATGGCGAACTGGTTGTAGTCCG-3’; Il23r, forward, 5’-

TGAAAGAGACCCTACATCCCTTGA-3’, reverse, 5’-CAGAAAATTGGAAGTTGGGATAT

GTT-3’.

Enzyme-Linked Immunosorbent Assay (ELISA). Cecums were excised, feces washed away

and then placed in ice-cold PBS. Tissue was weighed then homogenized using a rotor

homogenizer then samples were centrifuged and supernatants were collected. IL-6 levels in the

supernatants were quantified by ELISA (R&D Systems) and normalized to tissue weight.

In vivo cytokine neutralization. For in vivo cytokine neutralization, anti-IL-6 (R&D systems,

AB-406-NA), or control IgG (AB-108-C) was intraperitoneally injected at 48, 24, 4, and 0 hours

(50 µg per injection) prior to SL1344 infection and again at 4 hours post infection (75 µg).

LPL and IEL isolation. Cecal tissue was extracted, washed with ice-cold PBS, and cut into 1-2

cm segments that were washed three times (37O Celsius, 10 minutes) in stripping buffer (PBS,

1% FBS, 5mm EDTA, 1mm DTT). After each wash the buffer was filtered through a 100µm

47

cell-strainer then allowed to sediment. Intra-epithelial lymphocytes (IEL) were collected by

centrifuging the cells that did not sediment, washing twice in DMEM (20% FBS), and passing

the cells through a 40µm cell-strainer. After stripping, the tissue segments were minced, digested

in digestion buffer (DMEM, 20% FBS, 2 mg/ml Collagenase D (Roche), 20 µg/ml DNaseI

(Sigma)) for two 30 minute incubations at 37O Celsius. Digested material was passed through a

100µm cell-strainer and the cells were collected by centrifugation, washed twice in DMEM then

passed through a 40µm cell-strainer to obtain LP lymphocytes (LPL).

Flow cytometry. For intracellular cytokine staining (ICCS), LPL and IEL were incubated for

four hours with DMEM containing PMA (50 ng/ml), Ionomycin (Sigma) (1 µg/ml) and Golgi

Stop (BD bioscience). Dead cells were stained with violet live/dead fixable stain (Invitrogen)

then LPL and IEL were then stained for surface antigens (see supplementary methods for

antibody list). Cells were then fixed with 4% paraformaldehyde and permeabilized with BD

perm/wash buffer (BD bioscience) and stained for intracellular cytokines. FACS was performed

immediately following ICCS using either a Canto II or LSR II (BD bioscience) and analyzed

using FlowJo software (TreeStar).

Cell sorting. Magnetic bead-conjugated antibodies to CD4, CD11c and CD11b were used to

label cecal IEL and LPL cells and these cells were then sorted using LS columns (Miltenyi

Biotec) according to the manufacturer’s protocol (see Fig. 2.1 for FACS control staining of

sorted populations). RNA was immediately isolated from sorted cells.

Antibody list. The following anti-mouse antibodies were used in the current study: anti-CD4-

alexa730, anti-CD4-PECy7, anti-TCRβ-alexa647, anti-TCRβ-alexa780, anti-TCRγ-PE, anti-

TCRγ-alexa647, anti-IL17A-PerCP5.5, anti-IL22-PE, anti-IFNγ-PECy7, anti-GR1-FITC, anti-

F4/80-PerCP5.5, anti-CD11b-PECy7, anti-CD103-APC, anti-CD44-PE,anti-CD62L-FITC, anti-

48

CD69-PECy7, anti-CCR6-APC, anti-CD11c-alexa780, anti-B220-PE, isotype control–PE,

isotype control-PerCP5.5, isotype control-PECy7 (eBiosciences) and CD1d-tetramerAPC

(PBS57, obtained from the NIH tetramer facility). The following human specific antibodies were

used in the current study: anti-TCRγδ-FITC (BD), anti-CD4-PE (BD), anti-CD8-PETexasRed

(Invitrogen), anti-CD3-e780, anti-IL-17APercp5.5, anti-IL-22-eF660 (eBiosciences).

Bacterial 16S rRNA qPCR. DNA was extracted from the contents of wild-type and Nod1–/–

Nod2–/– ileum and caecum samples using a QIAamp DNA Stool mini kit (Qiagen). Quantitative

real-time PCR (qPCR) analysis was conducted using an AB 7300 system (Applied Biosystems,

Foster City, CA) and sequence detection software (version 1.3.1; Applied Biosystems, Foster

City, CA). Amplification reactions consisted of 5 μl Power SYBR green PCR master mix

(Applied Biosystems, Foster City, CA) mixed with 1 μl of forward and reverse primers (0.5 μM)

and 4 μl of genomic DNA (diluted to approximately 10 ng μl–1). Primer pairs were as follows:

Eubacteria, UniF340 (5’-ACTCCTACGGGAGGCAGCAGT-3’) and UniR514 (5’-

ATTACCGCGGCTGCTGGC-3’)2; Segmented Filamentous Bacteria, SFB736F (5’-

ACGCTGAGGCATGAGAGCAT-3’) and SFB844R (5’-GACGGCACGGATTGTTATTCA-

3’).Relative quantity was calculated by the ΔCt method and normalized to the amount of total

Eubacteria in the sample.

Recruitment of human participants. Two healthy male volunteers were recruited through the

Maple Leaf Clinic in Toronto, Canada. All participants provided written and informed consent,

and the Research Ethics Boards at St. Michael’s Hospital, Toronto and the University of Toronto

approved the study protocol originally designed by Dr. Rupert Kaul

Cell isolation from human sigmoid colon biopsies. Recto-sigmoid biopsies were sampled

approximately 25–30 cm from the anal verge and immediately placed into RPMI-1640 media

49

containing 100 U ml–1 penicillin, 100 μg ml–1 streptomycin, and 1x GlutaMAX-1 (Invitrogen,

Carlsbad, CA). Sigmoid mucosal mononuclear cells were isolated by two sequential Collagenase

type II digestions at 0.5 and 1.0 mg ml–1 (Clostridiopeptidase A; Sigma-Aldrich, St Louis, MI)

for 30 min at 37 °C each on a shaking heated block. Mucosal cells were passed through a 100 μm

filter and enumerated. Then, two million colonic mononuclear cells were infected ex vivo with

SL1344 at a 1:1 ratio and stimulated with phorbol 12-myristate 13-acetate (PMA; 1 ng ml–1) and

ionomycin (1 μM ml–1; Sigma), or negative control media alone for 8 h and Brefeldin A (1 μm

ml–1). The cells were then collected and analyzed by flow cytometry.

Statistical analysis. Mann-Whitney tests or student’s t-tests were performed using Graphpad

Prism and p values < 0.05 using a 95% confidence interval were considered significant.

50

51

Figure 2.1 Purity of MACS-sorted populations. Cecal LP and intraepithelial lymphocytes

from six mice were pooled together then subjected to MACS sorting as described in the methods

section. (a) Total cells present prior to sorting (pre-sort), CD4+ sorted, CD11b+CD11c+ sorted and

cells remaining after other populations were removed (unsorted) were analyzed for expression of

cell surface markers by FACS . The levels of TCRβ and CD4 (top row), TCRβ and TCRγδ

(middle row), and CD11b and CD11c (bottom row) were analyzed. Note that staining for CD4,

CD11b and CD11c in the sorted population resulted in decreased staining intensity due to

interference of staining by the antibodies that were used to MACS-purify the populations. (b)

The cells that were not TCRβ+ in the CD4+ sorted fraction were primarily B220-expressing cells.

(c) NKT cells were a negligible population in the caecum (data not shown) and there were only

insignificant levels of NKT cells in the CD4+ sorted population. Staining of CD4+ sorted spleen

cells is shown to demonstrate the efficacy of the PBS57-CD1d tetramer used to stain NKT cells.

52

2.4 Results

2.4.1 Nod1 and Nod2 are required to control infection with the enteric pathogen C.

rodentium.

Nod1 and Nod2 are intracellular sensors of bacterial peptidoglycan and play key roles in host

responses to bacteria(73), and have been implicated in cellular defense against C.rodentium

infection in vitro(231). In order to assess the in vivo importance of Nod1 and Nod2 in regulating

mucosal inflammation, we used the C.rodentium-colitis model. Infected Nod1-/- or Nod2-/-

single-knockout had no change in pathology or bacterial load when compared to WT mice (Fig.

2.2a,b) indicating that these receptors are somewhat redundant in function. However, we found

that Nod1/Nod2 double knockout (DKO) mice had significantly lower pathological scores with

less visible colonic inflammation at 7 days post-infection (p.i.), as well as reduced inflammation-

induced crypt-elongation (Fig. 2.3a,b), although no differences in colonic colonization were

observed between both groups (Fig. 2.2c). This initially blunted colonic pathology observed in

DKO mice was followed by exacerbated crypt hyperplasia and ten-fold increased translocation

of C.rodentium to the spleen at 14 days p.i. compared to what was observed in WT mice (Fig.

2.3b), thus indicating that DKO mice could not effectively control the infection. Moreover,

C.rodentium-induced inflammation frequently extended into the proximal colon of DKO mice,

while WT mice were affected only in the medial-distal region (Fig. 2.3a). Finally, while WT and

DKO mice did not succumb to the infection with 109 CFU of C.rodentium, DKO mice were

more susceptible and lost more weight than WT mice when naladixic acid-treated mice were

infected with 1010

CFU of C.rodentium (Fig. 2.2d).

Bone-marrow chimeras generated by reconstituting WT mice with DKO bone marrow

(DKOWT), WTDKO and the control DKODKO mice all had increased splenic CFU and

53

pathology 12 days after C.rodentium infection than the WTWT mice (Fig. 2.3c), thus showing

that Nod-dependent signaling in both radio-resistant and radio-sensitive compartments is

required for the full control of the infection. Nevertheless, Nod-dependent signaling in the radio-

resistant compartment played a more prominent role in the control of C.rodentium infection,

since DKO-recipient animals, regardless of the origin of the donor bone marrow, had increased

bacterial translocation (Fig. 2.3c) and frequently developed ulcerations that were rarely observed

in WT-recipients (Fig. 2.4).

Together, these results establish the importance of Nod1 and Nod2 for initiating an early

host inflammatory response to effectively contain C.rodentium infection.

54

Figure 2.2. Phenotypic characterization of Nod1–/–, Nod2–/– and Nod1–/–Nod2–/– mice

during infection with C. rodentium. (a) The crypt lengths in the proximal, medial and distal

regions of the colon of wild-type, Nod1–/– and Nod2–/– mice infected with 109 colony forming

units (CFU) of C. rodentium. (b) The levels of C. rodentium found in the spleen at 7 and 14 d

post infection. (c) Bar graph depicts CFU of C. rodentium isolated from the fecal pellets of wild-

type and Nod1–/–Nod2–/– mice at 7 and 14 d postinfection. (d) Survival (top) and weight change

(bottom) of wild-type and Nod1–/–Nod2–/– mice pre-treated with 100 g nalidixic acid (Nx) and

infected with either 109 or 10

10 CFU C. rodentium. A log-rank test was used to assess differences

in survival and a Mann-Whitney test was used for the weight change graph. (Error bars represent

± SEM. * = P < 0.05, NS = not significant).

55

56

Figure 2.3 Nod1 and Nod2 differentially modulate early and late inflammation during C.

rodentium colitis. (a) The levels of colonic histopathology, crypt lengths and bacterial splenic

translocation assessed in wild-type and Nod1–/–

Nod2–/–

mice at 7 and 14 d post-infection. (b)

Representative images (20 magnification) of H&E stained colon sections of wild-type and

Nod1–/–

Nod2–/–

uninfected mice and C. rodentium infected mice at 7 and 14 d post-infection; :

depict areas of goblet cell depletion and sub-mucosal edema; *: depict proximal regions of colon.

(c) Lethally irradiated wild-type mice were reconstituted with either wild-type (WTWT) or

Nod1–/–

Nod2–/–

bone marrow (DKOWT) and Nod1–/–

Nod2–/–

mice were reconstituted with

either wild-type (WTDKO) or Nod1–/–

Nod2–/–

(DKODKO) bone marrow. The levels of

colonic histopathology, crypt length and splenic translocation were assessed in these chimeras at

12 d post-infection. (Error bars represent SEM. * = P < 0.05, ** = P < 0.01, *** = P < 0.001,

NS = not significant).

57

Figure 2.4 Phenotypic characterization of bone-marrow chimeras infected with C.

rodentium. (a) Macroscopic visualization of colons and (b) microscopic visualization of H&E

stained colon sections of ulcerative lesions (black arrows) in wildtype"Nod1-/-Nod2-/–

(WT"DKO), DKO"DKO, WT"WT, DKO"WT chimeric mice at 12 d post infection. (One

representative of n = 2 shown, three mice were pooled for each group).

58

2.4.2 Nod1 and Nod2 are required for induction of early intestinal Th17 responses.

C.rodentium-induced colitis triggers a strong enteric Th17 response that modulates

inflammation and bacterial colonization(189); therefore we investigated the role of Nod1 and

Nod2 in Th17 development in this infection model. At the peak of infection (10 days p.i.),

similar IL-17A expression was detected in the colons of DKO and WT mice, indicating that Nod

proteins do not modulate the adaptive phase of Th17 responses to C. rodentium (Fig. 2.5a).

However, IL-17A expression was significantly reduced in cecal tissue from DKO mice during

the very early stages of infection (4 days p.i) (Fig. 2.5a). Surprisingly, we observed significantly

fewer IL-17A+CD4+TCR+ cecal LPLs from DKO mice than in WT LPLs at 4 days p.i. (Fig.

2.5b,c), but not at 10 days p.i., where roughly 30% of the LPLs were IL-17A+ in both WT and

DKO mice (Fig. 2.6a). Of note, the differences in IL-17A+ cells were not evident in Nod1-/- or

Nod2-/- single knockout mice (Fig 2.6b). At 4 days p.i., there were no significant difference in

IL-17A production between DKO and WTT-cell LPLs (Fig. 2.6c). We also determined that

the number of interferon--producing CD4+TCR+ LPLs during the early phase of C.rodentium

infection was similar between WT and DKO mice, suggesting that Nod proteins can selectively

induce Th17 and not Th1 responses during the early stages of intestinal inflammation (Fig. 2.6c).

In support of a blunted early Th17 response, the expression of the Th17-associated cytokine IL-

22 in CD4+LPL T-cells by flow cytometric analysis or quantitative real-time PCR analysis in

cecal tissue was also significantly reduced in DKO mice at 4 days p.i. compared to WT mice

(Fig 2.5b,d). Moreover, C.rodentium-infected DKO cecal tissue exhibited lower levels of RegIII

and Lipocalin-2 mRNA, two anti-microbial proteins that are important mediators of IL-22-

dependent mucosal defence against enteric bacterial pathogens (Fig. 2.5d)(187,196,232).

59

60

Figure 2.5 Early IL-17 responses during C. rodentium colitis are Nod1 and Nod2

dependent. (a) Il17a expression in C. rodentium infected wild-type and Nod1–/–

Nod2–/–

mice

was quantified by qRT-PCR from the caeca at 4 d (top) and colons at 10 d (bottom) post-

infection. (b) Intracellular cytokine staining (ICCS) was performed on cecal LPL from wild-type

and Nod1–/–

Nod2–/–

mice (uninfected or 4 d) and IL-17A and IL-22 was analyzed by flow

cytometry on either all LPLs (left column) or CD4+TCR

+ LPLs (right column). (c) The relative

number of IL-17A+CD4

+TCR

+ (TH17) cecal LPLs from wild-type and Nod1

–/–Nod2

–/– mice

(uninfected or 4 d, n = 5, three mice per group). (d) Il22, Lcn2, and Reg3g expression in C.

rodentium infected wild-type and Nod1–/–

Nod2–/–

mice was quantified by qRT-PCR from caeca

at 4 d post-infection. For qRT-PCR the average fold change in expression over PBS-treated wild-

type mice is shown (n = 2, ten mice per group). (Error bars represent SEM. * = P < 0.05)

61

62

Figure 2.6 Analysis of lamina propria T cell responses during C. rodentium infection. (a)

Dot plots show the relative frequency of IL-17A+ and IL22+ cells in TCR+CD4+ LPLs

isolated from the colons of wild-type and Nod1–/–Nod2–/– mice infected with C. rodentium for

10 d (one representative of n = 2, three mice pooled per group). (b) Dot plots show the relative

frequency of IL-17A+ and IL22+ cells in TCR+CD4+ LPLs isolated from ceaca of wild-type,

Nod1–/– and Nod2–/– mice infected with C. rodentium for 4 d (one representative of n = 2, three

mice pooled per experiment). (c) The percentage of IL-17A+TCR+ (top) and IFN-

+TCR+CD4+ (bottom) LPLs from wild-type and Nod1–/–Nod2–/– mice infected for 4 d with

C. rodentium. (d) qRT-PCR analysis of the relative mRNA levels of Il17a and Il22 in the

caecum of wild-typeNod1–/–Nod2–/– (WTDKO), WTWT, DKOWT, and DKODKO

chimeric mice infected with C. rodentium for 4 d. (e) Dot plots show the relative frequency of

IL-17A+ and IL22+ cells in TCR+CD4+ LPLs from C. rodentium-infected chimeric mice. (Bar

graphs depict the average percentage from n = 3–5, three mice were pooled per experiment.

Error bars represent mean ± SEM).

63

To determine whether Nod1/2-dependent early induction of Th17 responses was

restricted to the C.rodentium model or if it was a general host response to enteric infections, we

assessed the role of Nod1 and Nod2 in the acute model of colitis induced by Salmonella in

streptomycin-treated mice, for which we also observed delayed intestinal pathology in DKO

animals(105). Strikingly, by just 24 hours p.i. with SL1344 (a streptomycin resistant strain of

S.typhimurium), DKO mice had much lower cecal expression of IL-17A, IL-22 and Lipocalin-2

than WT mice (Fig. 2.7a). Similar to the C. rodentium model, there was a lower percentage and

fewer overall IL-17A+CD4+TCR+ LPLs recovered from infected DKO mice compared to WT

mice at this early stage of infection (Fig. 2.7b,c). However, in contrast to what was observed

with C. rodentium, T-cell specific IL-17 production in DKO LPLs was blunted compared to

WT LPLs following infection with SL1344 (Fig. 2.7b,c). The discrepancy between models with

regards to the intestinal T-cell response probably results from the difference in timing, with

SL1344 inducing more severe and acute inflammation at an earlier time-point.

To confirm that IL-17A and IL-22 was primarily being produced by CD4+ LPLs, we

compared IL-17A and IL-22 expression in MACS-sorted CD4+, CD11b+CD11c+ and unsorted

ceacal lymphocytes (Fig. 2.1). Indeed, the WT CD4+ cecal population expressed the highest

levels of IL-17A and IL-22, and DKO CD4+ cells produced much less of these cytokines after

infection (Fig 2.7d). We also wanted to assess whether early Th17 responses can occur in the

human intestinal mucosa and determined that isolated human intestinal lymphocytes exhibited a

trend for increased levels of IL-17A and IL-22 following a 8 hour SL1344 challenge ex vivo

(Fig. 2.8). In bone marrow chimeric mice, we observed less IL-17A+ CD4+TCR+ LPLs from

either SL1344-infected (Fig. 2.7e) or C.rodentium-infected DKODKO mice compared to

WTWT mice. From our analysis using both C.rodentium and SL1344 colitis models, we

64

concluded that Nod1 and Nod2 play a critical role in inducing early Th17 responses to bacterial

infections in vivo.

65

66

Figure 2.7 Acute IL-17 responses during S. typhimurium colitis are dependent on

hematopoietic and non-hematopoietic Nod1 and Nod2. (a) qRT-PCR analysis of Il17a, Il22

and Lcn2 in the caecum of wild-type and Nod1–/–

Nod2–/–

mice (uninfected or SL1344 infected

for 24 h). (b) Bar graphs show average fold change over uninfected controls (n = 3, six mice per

group). ICCS analysis of IL-17A and IL-22 in total LPL (top row), TCR+CD4

+ (middle row) or

TCR+ cells (bottom row) in cecal LPL from wild-type and Nod1

–/–Nod2

–/– mice (uninfected or

24 h). (c) The bar graphs depict the average relative frequency of all IL-17A+, TH17 or

TCR+IL-17A

+ cells in wild-type and Nod1

–/–Nod2

–/– mice (uninfected or 24 h, n = 6, three

mice per group). (d) qRT-PCR analysis for Il17a and Il22 on total cells (pre-sort), CD4+ cells,

CD11b+CD11c

+ cells, and cells remaining after MACS purification (unsorted). The bar graphs

show fold change in expression over unsorted cells from uninfected mice (one representative of n

= 2 is shown, 6 mice pooled per group) and the numbers above the bars represent the fold change

between wild-type and Nod1–/–

Nod2–/–

for each population of cells. (e) ICCS analysis of IL-17A

and IL-22 in TCR+CD4

+ cecal LPLs from chimeric mice (24 h post-infection, one

representative of n = 3 is shown, three mice pooled per group.) (Error bars represent SEM. * =

P < 0.05, ** = P < 0.01)

67

Figure 2.8 CD4+ T cells from human colonic biopsies produce IL-17A and IL-22 in

response to short term S. typhimurium infection. Colonic biopsies obtained from two healthy

volunteers were digested and the resulting cell suspensions were infected with SL1344 for 8 h.

(a) ICCS was performed to analyze IL-17A and IL-22 expression in TCRb+ CD4+ cells from

uninfected or SL1344 infected biopsies and representative dot plots are shown. (b)The average

relative frequency of IL-17A+ (left) or IL-22+ (right) TCR b+CD4+ cells from SL1344 infected

or uninfected samples from two individuals is shown. Error bars represent mean ± SEM.

68

2.4.3 Nod-dependent IL-6 induction is required for early Th17 responses.

Th17-cell development and activation are dependent on the inflammatory cytokines IL-6

and IL-23. Furthermore, the homeostatic Th17 response to enteric microbiota(170,183) and the

inflammatory Th17 response to C.rodentium(187,189) both necessitate functional IL-6 in vivo.

Importantly, we determined that in both C.rodentium (Fig. 2.9a) and Salmonella colitis (Fig.

2.9a, b), early induction of IL-6 in cecal tissue, but not of IL-23 or the IL-23 receptor, was

dependent on Nod1/Nod2 signaling. Moreover, analysis of IL-6 levels in chimeras indicated that

Nod1/Nod2 signaling from both hematopoietic and non-hematopoietic cells regulate IL-6

production (Fig. 2.9c). Interestingly, IL-6 mRNA was expressed in CD4+-sorted,

CD11b+CD11c+-sorted and unsorted cecal cells (Fig. 2.9d), reaffirming the diverse cellular

origins of this cytokine. However, only in the CD11b+CD11c+ DC fraction was there a dramatic

decrease in IL-6 mRNA in infected DKO mice compared to WT mice, indicating Nod1 and

Nod2 are needed for full induction of IL-6 in cecal DCs.

We further investigated how Nod1/2 signaling modulated CD11b+CD11c+ DC

dynamics. Recent studies have demonstrated that the intestinal mucosa harbors distinct DC

subsets that differentially express the surface marker CD103, correlating with either tolerogenic

(CD103+) or pro-inflammatory (CD103-) properties(233,234). Hence, we assessed how Nod1/2

signaling in the intestinal mucosa affects the balance of CD103+DCs during infection with

SL1344 (24 hours) and C.rodentium (4 days). In IELs, the numbers of CD103+CD11b+CD11c+

DCs were significantly reduced in WT but not DKO mice following bacterial challenge (Fig.

2.9e), which correlated with the blunted inflammatory profile of the DKO mice after enteric

infection. These results support the notion that Nod-dependent modulation of intestinal mucosal

DC subsets would help regulate early Th17 responses in infected mice.

69

At 24 hours p.i. with SL1344, WT mice treated with a neutralizing anti-IL-6 antibody,

but not with control IgG, failed to generate a robust Th17 response in cecal LPLs (Fig. 2.10a,b);

however IL-17A production by T-cells was not significantly affected by anti-IL-6 treatment

(Fig. 2.10b). In addition, we also determined that only the Th17 response to SL1344, but not

global induction of IL-17A transcript levels or pathology (Fig. 2.11), was blunted in IL-6

knockout mice (Fig. 2.10c). Furthermore, lack of IL-6 production by hematopoietic cells in IL-6

KOWT chimeric mice was sufficient to decrease the numbers of Th17 cells at 24 hours post-

SL1344 infection and 4 days after C. rodentium infection (Fig. 2.10d). These results suggest that

an acute requirement for IL-6 likely drives the observed Nod1/2-dependent defects in the early

Th17 responses.

Taken together, we determined that Nod1/2-driven expression of the Th17-inducing

cytokine IL-6 is a critical factor controlling the early induction of cecal Th17 responses

following enteric infection.

70

71

Figure 2.9 IL-6 expression during C. rodentium and Salmonella colitis is Nod1 and Nod2

dependent. (a) Expression of Il6, Il23a and Il23r in the caecum of wild-type and DKO mice; C.

rodentium 4 d post-infection, (top); SL1344 24 h post-infection (bottom). Average fold change

over uninfected controls is shown (n = 3, six mice per group). (b) IL-6 levels in SL1344 infected

wild-type and Nod1–/–

Nod2–/–

mice (24, 48, and 72 h) were measured by ELISA. (c) IL-6 levels

in SL1344-infected chimeric mice were measured by ELISA (n = 3, six mice per group). (d)

qRT-PCR analysis for Il6 on total cells (pre-sort), CD4+ cells, CD11b

+CD11c

+ cells, and cells

remaining after MACS purification (unsorted). The bar graphs show fold change in expression

over unsorted cells from uninfected mice (one representative of n = 2 is shown, six mice pooled

per group) and the numbers above the bars represent the fold change between wild-type and

Nod1–/–

Nod2–/–

for each population of cells. (e) Histograms represent the expression of CD103

on either CD11b–CD11c

+ cells or CD11b

+CD11c

+ cecal IEL cells from wild-type (red) and

Nod1–/–

Nod2–/–

(blue) mice (uninfected, C. rodentium 4 d, SL1344 24 h). (Representative data of

n = 3 is shown, three mice pooled per group). (Error bars represent SEM. * = P < 0.05, ** = P

< 0.01, *** = P < 0.001, NS = not significant)

72

73

Figure 2.10 IL-6 expression during the acute phase of infectious colitis is critical for TH17

development. (a) ICCS analysis of IL-17A and IL-22 on total cecal LPL (top row), TCR+CD4

+

(middle row) or TCR+ cells (bottom row) from SL1344 infected wild-type mice (uninfected or

24 h) treated with either control IgG or IL-6 neutralizing antibody. (b) Average relative

frequency of all IL-17A+, TH17 or TCR

+IL-17A

+ cells from control IgG or IL-6 neutralizing

antibody-treated SL1344 infected wild-type, (c) and SL1344 infected wild-type and IL-6

knockout mice (24 h post-infection, n = 3, 3 mice per group). (d) ICCS analysis for IL-17A and

IL-22 expression in TCR+CD4

+ cecal LPLs from C. rodentium (4 d) and SL1344 (24 h)

infected chimeric mice that were generated by reconstituting irradiated wild-type mice with

either wild-type (WTWT) or Il6–/–

(Il6–/–WT) bone-marrow. (Dot plots depict one

representative of n = 3, two mice per group). (Error bars represent SEM. * = P < 0.05, ** = P

< 0.01, NS = not significant)

74

75

Figure 2.11 Analysis of cytokine expression in infected IL-6 knockout mice and

pathological scores in IL-6 depletion experiments. (a) Cecum samples stained with H&E and

analyzed for pathological changes. The pathological scores for edema, neutrophil recruitment

(PMN), goblet cell depletion and epithelial erosion are shown for uninfected, or SL1344 infected

(24 h) wild-type mice treated with control IgG or IL-6 neutralizing antibody. The scatter plots

show the total cumulative pathological scores for individual SL1344 infected mice with the

horizontal bar indicating the average. (b) The cecal tissue mRNA levels of Il17a were assessed

24 h after infection of wild-type and Il6–/– knockout mice. The average fold change in

expression over uninfected wild-type controls is shown. (six mice per group, one representative

of n = 2 is shown, error bars represent mean ± SEM. * = P < 0.05)

76

2.4.4 Induction of early Th17 responses to bacterial pathogens requires priming by the

intestinal microbiota.

To further characterize the Th17 cells that are induced at early time-points after bacterial

infection we determined that gated IL-17A+CD4+TCR+ LPLs exhibited a CD44+CD62L-

CD69hiCCR6hi phenotype compared to IL-17A-negative CD4+T-cells in the caecum (Fig

2.12a). Interestingly, homeostatic IL-17A+CD4+TCRb+ LPLs were also CD44+CD62L-,

however SL1344 infection induced the upregulation of CD69 and CCR6 on these cells (Fig

2.12b,c). The CD44+CD62L-CD69hi profile is associated with an effector memory T-cell

phenotype(235).

Bacteria in the normal intestinal microbiota such as Segmented Filamentous Bacteria

(SFB) have been shown to influence the development of Th17 responses(183,184). It is

important to note that our mouse colony harbors SFB but we found, just as others have

reported(184), that Nod1/Nod2 did not influence colonization by this bacteria and that SFB

preferentially colonized the ileum and is virtually absent from the caecum (Fig. 2.13). We

postulated that Th17 cells in the LP might be conditioned by the intestinal microbiota to induce a

state that can rapidly respond to subsequent bacterial infections. To address the importance of the

microbiota in the generation of early Th17 responses, we compared the mucosal IL-17A/IL-22

responses in germ-free (GF) or specific pathogen free (SPF) mice. We determined that, as

previously reported(183), uninfected GF mice have fewer Th17 LPLs than SPF mice (Fig

2.12d,e). However, the global numbers of IL-17A+LPLs were not significantly reduced, as there

was a large increase in the number of IL-17A+T-cells in GF mice (Fig 2.12d,e). This

observation indicates that mucosal IL-17A/IL-22 responses have some degree of plasticity in the

caecum, where other cell types can compensate for the loss of IL-17 expression by Th17 cells.

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Strikingly, 24 hours post-SL1344 infection there were significantly less Th17 cells in GF mice

compared to SPF mice (Fig. 2.12d,e). The numbers of IL-17A+CD4+TCR+cells actually

decreased after infection in GF ceacal LPLs compared to uninfected mice. These results

demonstrate the requirement of a normal bacterial microbiota to mount an early Th17 response to

an enteric pathogen.

78

79

Figure 2.12 Early TH17 cells express memory surface markers and require microbiota for

activation. (a) Expression of CD44, CD62L, CD69 and CCR6 on either all TCR+CD4

+ or TH17

cells in cecal LPL from SL1344 infected mice (top row) and compared expression of these cell

surface markers on TH17 from the LPL of uninfected and SL1344 infected mice (24 h post-

infection) (bottom row). (b) Mean fluorescence intensity (MFI) for CD69 expression on LPL

from uninfected or SL1344 infected mice (n = 3, three mice per group). (c) ICCS analysis of IL-

17A and IL-22 in total cecal LPL (top row), TCR+CD4

+ (middle row) or TCR

+ cells (bottom

row) from SL1344 infected specific pathogen free (SPF) and germ-free mice (uninfected or 24

h). (d) Average relative frequency of all IL-17A+, TH17 or TCR

+IL-17A

+ cells from SL1344

infected SPF and germ-free mice (uninfected or 24 h). (Uninfected n = 2, Infected n = 3, three

mice per group). (Error bars represent SEM. * = P < 0.05, ** = P < 0.01)

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Figure 2.13 Intestinal colonization with segmented filamentous bacteria (SFB). (a) Levels of

SFB DNA encoding 16s rRNA were measured by qPCR in both the ileum and caecum of

uninfected wild-type and Nod1–/–Nod2–/– mice. (b) Numbers of SFB were also quantified in

uninfected Ripk2–/– and Ripk2+/+ littermate mice. Bar graphs depict four–eight mice per group.

(NS = not significant, error bars represent mean ± SEM.)

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2.5 Discussion

Elucidating the role of Nod proteins in intestinal barrier defense is of fundamental

importance for our understanding of the etiology of inflammatory bowel disease (IBD). In this

study, we investigated the role of Nod1 and Nod2 in C.rodentium and S.typhimurium colitis.

Mice deficient for both Nod1 and Nod2 could not efficiently generate early Th17 responses in

the caecum in both colitis models. We propose the term innate Th17 (iTh17) for these cells due

to their dependency on Nod1 and Nod2 signaling for activation at early time points after

bacterial infection (1-4 days p.i). Importantly, the lack of a protective iTh17 response correlated

with delayed pathology and increased disease burden in DKO mice infected with C.rodentium.

These results represent the first identification of a role for innate immune sensors in the control

of Th17 induction in the intestine, and suggest that Nod proteins contribute to the regulation of

the balance between signals from the intestinal microflora and from enteric pathogens. This

illustrates the expanding functional complexity of intestinal Th17 responses in conditions of

homeostasis or pathogenic infection.

The kinetics of mucosal iTh17 induction following Citrobacter and Salmonella infection

are not compatible with the kinetics of a prototypic adaptive immune response. Previously, Lti-

like cells were identified as an innate source of IL-17A and IL-22 in RAG-1 knockout

mice(175,191). However, our study is the first to identify LP iTh17 cells as an innate source of

IL-17A/ IL-22 in normal mice using in vivo infection models. This observation was unexpected

and suggests that Th17 cells may exhibit innate–like properties. The iTh17 response does not

appear to be a non-specific activation of LP Th17 cells since iTh17 cells failed to develop in the

absence of microbiota even though Th17 cells were present in germ-free mice prior to infection.

Although iTh17 express effector memory associated cell surface markers, it is not clear whether

82

bacteria-specific antigens or other microbiota derived products are required for priming the

response. Further analysis is warranted to investigate the role of the microbiota in the

development of iTh17 responses. Together our results suggest that Nod-iTh17 responses

represent a rapid bacteria-specific protective response in the intestine that bridges the gap until

adaptive Th1, Th2 and/or Th17 responses are fully developed.

In contrast to previous studies showing that IL-23 regulates IL-17 from innate sources

such as LTi and T-cells, we show that IL-6 is essential for the development of iTh17

responses. In our models IL-6 was the only factor implicated in Th17 development whose

expression was significantly influenced by Nod1 and Nod2, strongly suggesting that Nod1/Nod2

regulate iTh17 responses through modulation of IL-6 expression. Nod1/Nod2 have been

previously shown to be important for development of adaptive Th17 responses in mouse(81) and

human cells(218) in an IL-23 dependent manner. Although we found that IL-23 was expressed at

high baseline levels its expression was not dependent on Nod1/Nod2 and was not significantly

induced at very early times of infection. Thus, the discrepancy in the requirement of IL-23 or IL-

6 in these studies is likely due to the difference in kinetics and the different model systems

involved. It is important to note that although IL-23 does not appear to be directly modulated by

Nod1 and Nod2 we believe it likely works in concert with IL-6 to drive the iTh17 responses

since early IL-17 responses during both Salmonella and Citrobacter colitis have been shown to

be IL-23-dependent(187,198).

A growing body of evidence suggests that defective homeostatic immune control of the

intestinal microbiota, or impaired responses to microbial pathogens, might play important roles

in the development of intestinal inflammation. Interestingly, several studies in IBD patients have

identified risk-associated single nucleotide polymorphisms in Nod2 and in loci associated with

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Th17 responses including IL-23R, IL-22 and STAT3(120), providing independent evidence that

NLRs and pathways controlling IL-17 expression both play key roles in intestinal immune

homeostasis. Thus, the intestinal Nod-iTh17 axis that we have identified in this report provides

new mechanistic insights into the pathophysiology of the IBD conditions Crohn’s disease and

ulcerative colitis.

84

Chapter 3

Constitutive induction of intestinal Tc17 cells in the absence of

hematopoietic cell-specific MHC class II expression

Stephen J. Rubino, Kaoru Geddes, Joao G. Magalhaes, Catherine Streutker, Dana

J. Philpott and Stephen E. Girardin

European Journal of Immunology. 2013 Jul 23.

I designed and performed all the experiments and wrote the manuscript. K.G. and J. G. M.

helped with in vivo experiments. C. S. performed pathological scoring. D. J. P and S. E. G.

supervised the research and helped write the manuscript.

85

3.1 Abstract

The enteric pathogen Citrobacter rodentium induces a mucosal IL-17 response in CD4+T

helper (Th17) cells that is dependent on the Nod-like receptors Nod1 and Nod2. Here, we

sought to determine whether this early Th17 response required antigen presentation by

major histocompatibility complex (MHC) class II for full induction. At early phases of C.

rodentium infection, we observed that the intestinal mucosal Th17 response was fully

blunted in irradiated mice reconstituted with MHCII-deficient (MHCII-/-WT)

hematopoietic cells. Surprisingly, we also observed a substantial increase in the number of

IL-17+CD8+CD4-TCR+ cells (Tc17 cells) and FOXP3+CD8+CD4-TCR+ cells in the LP

and intraepithelial lymphocyte compartment of MHCII-/-WT mice compared to

WTWT counterparts. Moreover, MHCII-/-WT mice displayed increased

susceptibility, increased bacterial translocation to deeper organs and more severe colonic

histopathology after infection with C. rodentium. Finally, a similar phenotype was observed

in mice deficient for CIITA, a transcriptional regulator of MHCII expression. Together,

these results indicate that MHCII is required to mount early mucosal Th17 responses to an

enteric pathogen, and that MHCII regulates the induction of atypical CD8+ T cell subsets,

such as Tc17 cells and FOXP3+CD8+ cells, in vivo.

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3.2 Introduction

IL-17 can be secreted by a number of cell populations, including Th17 cells, T cells,

ILCs and NKT cells(161,174,175,215,236,237). In humans, Th17 cells appear to represent the

major cell population that produces IL-17 in the intestine under normal conditions, whereas in

mice IL-17 is mainly produced by Th17 and T cells. Th17 cell differentiation is mediated by

the cytokines IL-6 and TGF, whereas IL-23 and IL-1 contribute to the full activation of these

cells(193,236,238).

In addition to the cell populations that produce IL-17 in normal conditions and described

above, several recent studies have identified the existence of CD8+ T cells expressing IL-17

(Tc17 cells)(173,239-241). Interestingly, Tc17 cells were recently shown to be upregulated in a

number of pathological conditions, such as cancer(242-244) and acquired immuno-

deficiency(245,246), suggesting the existence of a dynamic regulatory balance between CD4+

and CD8+ T cells for the production of IL-17. However, the exact role and importance of Tc17

cells in non-pathological conditions remains uncertain, and it is unclear if these cells could have

similar functions to Th17 cells in vivo.

The nature of the signals that drive IL-17 secretion in the intestine in response to enteric

bacterial pathogens remains poorly characterized. In a recent study (Chapter 2), we demonstrated

that the Nod-like receptor (NLR) proteins Nod1 and Nod2 were critical for inducing cecal

production of IL-17 from Th17 cells in the early phase of infection with S. Typhymurium and C.

rodentium, in part through the control of IL-6 secretion in the intestinal mucosa(106). We termed

this response the “innate” Th17 (iTh17) response because of the rapid onset of this T cell

response and the dependency on innate immune signaling for induction. Moreover, mice

87

deficient for MyD88(159), a critical adaptor protein for Toll-like receptor and IL-1R signaling,

and mice deficient for TLR2(195) both displayed a blunted capacity to mount IL-17-dependent

host responses to Salmonella infection starting at 1 day post-infection. Together, these studies

suggest that pattern-recognition molecules of the innate immune system play key roles in driving

iTh17 response to bacterial challenge; however, a question arises regarding the nature of the

signals that are required to prime Th17 cells in the gut following infection. In particular, it is

unclear if specific pathogen-derived and/or microbiota-derived antigens are required to prime

Th17 cells. In vitro studies suggest that the induction of a specific network of cytokines is

sufficient for the activation of these cells(247); however, our previous study identified a crucial

role for the intestinal microbiota in promoting innate Th17 responses in the cecum of

Salmonella-infected mice(106). This suggests a key role for specific antigens in iTh17 induction

in the intestine following bacterial infection.

In the present study, we aimed to investigate if iTh17 responses in the intestinal mucosa

depended on antigen presentation by hematopoietic cells despite the fact that activation occurred

prior to the prototypical kinetics of adaptive immune response onset. To do so, we used mice

deficient in the major histocompatibility complex II (MHCII), or mice that are knocked-out for

the gene encoding CIITA, a master regulator of MHCII expression. We highlight here the

requirement for MHCII to control C. rodentium in vivo and to induce early Th17 responses in the

mucosa. Unexpectedly, we also provide evidence that the absence of MHCII expression resulted

in a massive upregulation of IL-17 production by mucosal Tc17 CD8+ T cells. Importantly, our

results also reveal that constitutive induction of Tc17 cells in the LP of MHCII-deficient mice

was not sufficient to provide protection against C. rodentium infection.

88

3.3 Materials and Methods

Mice. C57BL/6 (Charles River), MHCII–/–

(strain B6.129S2-H2dlAb1-Ea

/J, Jackson Laboratories)

and CIITA–/–

(strain B6.129S2-Ciitatm1Cum

/J, Jackson Laboratories) mice were bred and housed

under specific pathogen free conditions at the CCBR, University of Toronto. The University of

Toronto Animal Ethics Review Committee approved all animal experiments. Sex and age

matched mice, 6-10 weeks of age, were used for experiments.

Bacterial infections. Both C. rodentium and S. Typhimurium infections we carried out as

described previously(105,106).

CFU counting. Mesenteric lymph nodes (MLNs) and spleens were homogenized in sterile PBS

using a rotor homogenizer. C. rodentium colony forming units were counted by serial dilution

analysis using nalidixic acid–containing luria broth agar plates.

Histology and pathological scoring. Mouse colons were cut, rolled and then fixed in a 10%

formalin solution. H&E stained slides were scored with an established scoring system for C.

rodentium histopathology(150) by a pathologist who was blinded to the experiments.

Chimeras. Chimeras were generated as previously described(84). Briefly, recipient mice were

irradiated with 900cGy of ionizing radiation and reconstituted with 4 x 106 donor mouse bone

marrow cells one day later and were left for 6 weeks before being used for experiments.

Quantitative real-time PCR. Colonic tissue was excised, stored in RNAlater (Sigma) and then

mRNA was isolated using Qiagen RNeasy Extraction kits. cDNA was generated with

SuperScript RTIII (Invitrogen) and then qRT-PCR was performed with SYBR Green (Applied

Biosystems) using the following primer sequences(187): il17a, forward, 5-

89

GCTCCAGAAGGCCCTCAGA-3, reverse, 5-CTTTCCCTCCGCATTGACA-3; ifn, forward,

5-TCAGCAACAGCAAGGCGAAAAAG-3, reverse, 5-ACCCCGAATCAGCAGCGACTC-3;

rpl-19, forward, 5-GCATCCTCATGGAGCACAT-3, reverse, 5-CTGGTCAGCCAGGAGCTT-

3. Values were calculated using the Ct method and were normalized to the housekeeping gene

rpl19.

LPL isolation. LP lymphocytes (LPLs) were isolated as previously described(106). Briefly,

cecal tissue was extracted, stripped of the epithlial cells and digested with Collagenase D

(Roche) containing buffer. The cells were then passed through strainers and used for flow

cytometry.

Flow cytometry. LPLs were stimulated for 4 h with phorbol-12-myristate-13-acetate (50 ng/ml),

ionomycin (Sigma) (1g/ml) and Golgi Stop (BD bioscience) in DMEM buffer. The cells were

then collected, stained with live/dead fixable stain (Invitrogen) and then stained for surface

antigens. The next day the cells were stained for intracellular cytokines using the EBiosciences

FOXP3 staining kit, according to the manufacturer’s instructions. FACS plots were generated

using FlowJo (TreeStar).

Antibody list. The following antibodies for flow cytometry were used: anti-CD8-FITC, anti-

CD8-alexa700, anti-CD4-PECy7, anti-TCR-alexa780, anti-IL17A-PerCP5.5, anti-IL22-PE,

anti-IFNg-alexa450, anti-FOXP3-alexa647, anti-CD44-PE, isotype control–PE, isotype control-

PerCP5.5, isotype control- alexa450 (eBiosciences).

Statistical analyses. Two-tailed Student’s t tests were used in Graphpad (Prism), and P values <

0.05 using a 95% confidence interval were considered significant.

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3.4 Results

3.4.1 MHCII is necessary for early mucosal Th17 responses to Citrobacter rodentium

infection.

We first determined that MHCII-/-

mice (B6.129S2-H2dlAb1-Ea

) housed in our animal

facility, and in Specific Pathogen Free (SPF) conditions, have very few CD4+T cells in the

intestinal LP (Figure 3.1A), in agreement with a previous report that analyzed lymphocyte

numbers in the spleen and lymph nodes in mice of the same genotype(248). This impairment is

likely the consequence of a lack of thymic selection in these mice, which results in an

imbalanced CD8+/CD4+ T cell ratio. In order to circumvent the problem that the CD4+ T cells

deficiency in MHCII-/-

mice could represent a confounding factor in our analysis, we

reconstituted irradiated C57Bl/6 recipient mice with MHCII-/-

hematopoietic cells, thus

generating MHCII-/-WT chimeric mice. These chimeras displayed ratios of CD8+/CD4+ T

cells in the LP similar to those found in WTWT mice (Figure 3.1A-B) and the expression of

MHCII was completely ablated in hematopoietic cells isolated from the spleen and LP (Figure

3.1C) of these MHCII-/-WT chimeric mice.

C. rodentium infection induces a robust Th17 response that can be detected as early as 4

days post-infection (p.i.), preceding the onset of a classical adaptive response, which occurs from

7 to 14 days p.i., and has proven to be a very useful model for studying the physiological

regulation of intestinal Th17-dependent immunity. Using flow cytometry, we found that there

was a significant increase in the percentage of IL-17+CD4+TCR+ LP lymphocytes (LPL) in the

cecum at 4 and 7 days post-challenge with 109 colony forming units (CFU)/ml of C. rodentium

in WTWT mice, whereas this response was completely ablated in MHCII-/-WT

91

mice at the same time points (Figure 3.2A-B). This result was confirmed by real-time PCR, as

we observed an increase in IL-17a mRNA transcripts at 7d. p.i. in WTWT ceca, which was

fully blunted in MHCII-/-WT mice (Figure 3.2C).

For C. rodentium colitis, the cytokine IL-22 is indispensible for mice to survive infection

(187) and Th22 cells are also induced very early during infection(190). We found that the

induction of Th22 cells was blunted in the cecal LP of MHCII-/-WT mice infected with C.

rodentium for four days (Figure 3.3A). Moreover, we found a lower percentage of IL-17+CD4+

and IL-22+CD4+ T cells at day one p.i. with S. Typhimurium (Figure 3.3B), indicating that

expression of MHCII is more broadly required for the induction of innate IL-17 and IL-22 T-cell

responses to bacterial pathogens.

CIITA is a NLR that acts as a master regulator of MHCII transcription and CIITA-/-

strain

(B6.129S2-Ciitatm1Cum

/J)WT hematopoietic cells lacked MHCII expression (Figure 3.4A-B).

To confirm the results obtained with the MHCII-/-WT mice, we next used CIITA-deficient

mice. Interestingly, the induction of a LP Th17 response at 7 days p.i with C. rodentium was also

severely reduced in CIITA-/-WT mice as compared to WTWT mice (Figure 3.2D).

Therefore, the results obtained with MHCII- as well as CIITA-deficient chimeric mice clearly

indicate that hematopoietic MHCII expression is required to initiate an early mucosal Th17

response to C. rodentium.

In addition to the up-regulation of IL-17+CD4+TCR+ LPLs, we also observed a trend

for an increase in the percentage of IFN+CD4+TCR+ LPLs isolated from cecal tissue isolated

from uninfected MHCII-/-WT mice (Figure 3.2A,B). Accordingly, higher levels of IFN

mRNA were found in MHCII-/-WT uninfected intestinal tissue as compared to WTWT mice

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(Figure 3.2C), which is in line with previously published results that demonstrated increased

IFNlevels in intestinal tissue from uninfected MHCII-/-WT mice(249). However, At 7d. p.i

with C. rodentium, we observed a significant increase in the amount of IFN mRNA transcripts

over uninfected mice in cecal tissue of WTWT but not in MHCII-/-WT mice (Figure 3.2C),

which could be caused by differences in the number of IFN-producing cells in the mucosa. IFN

has been shown to inhibit Th17 cell polarization in vitro and the high basal levels of this

cytokine in MHCII-/-WT could be contributing to the blunted induction of Th17 responses in

the mucosa.

In sum, expression of MHCII on hematopoietic cells appears to play a key role in both

the homeostatic control of IL-17 and IFN production by CD4+TCR+ LPLs in the intestinal

mucosa, and the rapid capacity of these cells to differentiate into Th17 and Th22 cells in the

early stages of an enteric infection with a bacterial pathogen.

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Figure 3.1 CD4+, CD8+ and MHCII+ cell characterization in MHCII-/-WT mice. A-B)

Gated on live TCR+ lymphocytes, dot plots depict the number of CD4+ and CD8+ T cells in

the LP of WT and MHCII-/- mice (A) or WTWT and MHCII-/-WT chimeric mice (B). C)

Expression of MHC class II by flow cytometry on live lymphocytes in the spleen and LP of

WTWT and MHCII-/-WT chimeric mice. Dot plots represent one representative of 3 to 5

independent experiments.

94

95

Figure 3.2 Induction of early LP Th17 responses after C. rodentium infection is dependent

on MHCII signaling. A) Gated on live CD4+TCR+ lymphocytes, dot plots depict the

percentage of IL-17+ and IFN+ CD4+T cells in the LP of uninfected, d4 and d7 p.i WT WT

and MHCII-/-WT mice. B) The bar graphs depict the average relative frequency of all IL-

17A+ and IFN+ CD4+ T cells in WTWT and MHCII-/-WT mice (3 mice pooled per group,

average of 3 to 5 experiments). C) Quantitative RT-PCR (qRT-PCR) analysis of IL-17a mRNA

isolated from proximal colon of WTWT and MHCII-/-WT mice (uninfected or C.

rodentium-infected for 7 days). Bar graphs show relative expression units normalized to the

housekeeping gene rpl19 (6-8 mice per group). D) Gated on live CD4+TCR+ lymphocytes, dot

plots depict the percentage of IL-17+ and IFN+ CD4+T cells in the LP of uninfected and d7

post-infected WTWT and CIITA-/-WT mice (Dot plots represent one representative of two

independent experiments, 3-6 mice per group). qRT-PCR analysis of IFN mRNA isolated from

proximal colon of WTWT and MHCII-/-WT mice (uninfected or C. rodentium-infected for

7 days). Error bars represent SEM. Statistical analysis was performed using a two-tailed

student’s t-test where: * = p < 0.05, **= p < 0.01, NS= not significant.

96

Figure 3.3 Intracellular IL-22 expression in CD4+TCRb+ LPLs. A-B) Gated on live

CD4+TCRb+ lymphocytes, dot plots depict the percentage of IL-17+ and IL-22+ CD4+T cells in

the LP of WT WT and MHCII-/-WT mice infected with C. rodentium for 4 days (A) or

infected with Salmonella enterica serovar Typhimurium for 1 day (B). One representative of two

independent experiments.

97

Figure 3.4. Characerization of CIITA-/-WT chimeric mice. A) Domain organization of the

Nod-like receptor CIITA: CARD= Caspase recruitment domain, TA= transcriptional activator

domain, NBD= nucleotide binding domain and LRR=leucine-rich domain. B) Expression of

MHC class II by flow cytometry on live lymphocytes in the LP of WTWT and CIITA-/-WT

chimeric mice. C) Survival curve of WTWT and CIITA-/-WT mice infected by oral gavage

with 1x109

CFU of C. rodentium (4 to 6 mice per group).

98

3.4.2 Deletion of hematopoietic MHCII signaling results in the upregulation of IL-17+ and

FOXP3+ CD8+ T cells in the cecal LP.

We next questioned whether the lack of MHCII expression on hematopoietic cells could

have an indirect effect on the function of CD8+TCR+ T cells. Strikingly, a massive

upregulation of the percentage of IL-17+CD8+TCR+ (Tc17 cells) and IFN+CD8+TCR+

cells was observed in the ceca of uninfected MHCII-/-WT mice as compared to WTWT mice

(Fig. 3.5A). Quantification of the average relative frequency of cells revealed there was over a

tenfold increase in the number of IFN+CD8+TCR+ LPLs and approximately a hundredfold

increase in the number of IL-17+CD8+TCR+ LPLs in MHCII-/-WT mice compared to their

WTWT counterparts (Fig. 3.5B). Interestingly, IL-22 was not expressed in CD8+ T cells

isolated from the LP of MHCII-/-WT mice (Fig. 3.6). The induction of a Tc17 response in

MHCII-/-WT mice was much stronger in the cecal LP than in the spleen (Fig. 3.5C), which

reflects what is normally observed for Th17 cells, with the intestine being the major site of

regulation of these cells. However, the percentage of Tc17 cells in the LP of MHCII-/-WT

mice did not change after infection with C. rodentium (Fig. 3.5C). In addition, we observed a

higher percentage of IL-17+CD8+TCR+ and IFN+CD8+TCR+ cells in the cecal LP of

MHCII-/-

(Fig. 3.5D) and CIITA-/-WT mice (Fig. 3.5E), providing confirmation that the

phenotype observed in MHCII-/-WT mice was not an artifact of the bone-marrow

reconstitution process.

99

100

Figure 3.5. Enrichment of IL-17+CD8+ T cells in the LP of MHCII-/-WT mice. A) Gated

on live CD8+TCR+ lymphocytes, dot plots depict the percentage of IL-17+ and IFN+ CD8+T

cells in the LP of uninfected WTWT and MHCII-/-WT mice. B) The bar graphs depict the

average relative frequency of all IL-17A+ and IFN+ CD8+ T cells in WTWT and MHCII-/-

WT mice (3 mice pooled per group, average of 3 to 5 experiments). C) Gated on live

CD8+TCR+ lymphocytes, dot plots depict the percentage of IL-17+ and IFN+ CD8+T cells in

the LP or spleen of uninfected and d7 post-C. rodentium infected MHCII-/-WT mice. Dot

plots represent one representative of 3 to 5 independent experiments. D-E) Gated on live

CD8+TCR+ lymphocytes, dot plots depict the percentage of IL-17+ and IFN+ CD8+T cells in

the LP of uninfected WT and MHCII-/- mice (D) or WTWT and CIITA-/-WT mice (3) (one

representative of two independent experiments, 3 mice pooled per group). Error bars represent

SEM. Statistical analysis was performed using a two-tailed student’s t-test where: * = p < 0.05.

101

Next, we sought to determine if the numbers of other atypically differentiated CD8+ T

cells would also be amplified in the LP of MHCII-/-WT mice in addition to the induction of

Tc17 cells. We observed a significantly increased percentage of FOXP3+CD8+T cells in the LP

of MHCII-/-WT mice (Fig. 3.7A,C). Moreover, and in line with previous results, the

percentage of FOXP3+CD4+T cells, or regulatory T cells (Tregs), was reduced in MHCII-/-

WT mice (Fig. 3.7B,D). Together, these results indicate that hematopoietic deletion of MHCII

results in broad dysregulation of CD4+ and CD8+ T cell dynamics that is not limited to only

Th17 and Tc17 subsets.

We then analyzed the CD8+ T cell population in the intraepithelial lymphocyte (IEL)

compartment, a mucosal site rich in CD8+ and T cells. Similarly to what was observed in the

LP, there was a robust induction of both Tc17 and FOXP3+CD8+ T cells (Fig. 3.8) in the IELs

isolated from MHCII-/-WT ceca compared to WTWT counterparts, suggesting that these

atypical CD8 cells are upregulated throughout the entire mucosa.

A previous study reported that deletion of MHCII could result in the accumulation of

CD44+CD8+ memory T cells(250); therefore we were interested in determining whether the LP

Tc17 cells found in MHCII-/-WT mice were CD44 positive. Using flow cytometry, we found

that LP IL-17+CD8+TCR+, FOXP3+CD8+TCR+ and IFN+CD8+TCR+ isolated from

MHCII-/-WT mice were all positive for CD44, indicating these cells have a memory cell

surface marker phenotype (Fig. 3.9). These results highlight certain similarities between mucosal

Tc17 and FOXP3+CD8+ T cells and their mucosal CD4+ T cell counterparts, Th17 and Tregs

cells, which are also CD44+ activated cells.

102

Together, our results provide the unexpected finding that MHCII signaling is required to

prevent the induction of CD44+ Tc17 and FOXP3+CD8+ T cells in the LPL and IEL

compartments, thus demonstrating the existence of tightly controlled regulatory feedback loops,

dependent on MHCII expression on hematopoietic cells, which orchestrate the dynamic interplay

between CD4+- and CD8+-dependent differentiation programs in the intestinal mucosa.

103

Figure 3.6 Intracellular IL-22 expression in CD8+TCRb+ LPLs. Gated on live CD8+TCRb+

lymphocytes, dot plots depict the percentage of IL-17+ and IL-22+ CD8+T cells in the LP of

WT WT and MHCII-/-WT mice. One representative of two independent experiments.

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Figure 3.7 Analysis of FOXP3+CD4+ and FOXP3+ CD8+ T cells in the LP of MHCII-/-

WT mice. A-B) Gated on live CD8+TCR+ (A) or CD4+TCR+ (B) lymphocytes, dot plots

depict the percentage of FOXP3+ T cells in the LP of uninfected WTWT and MHCII-/-WT

mice. C-D) The bar graphs depict the average relative frequency of all FOXP3+CD8+ T cells (C)

or FOXP3+CD4+ T cells (D) in WTWT and MHCII-/-WT mice (3 mice pooled per group,

average of 3 to 5 experiments). Error bars represent SEM. Statistical analysis was performed

using a two-tailed student’s t-test where: * = p< 0.05, ** = p < 0.01.

105

Figure 3.8 Induction of of IL-17+ and FOXP3+ CD8+ T cells in the intraepithelial

lymphocyte compartment of MHCII-/-WT mice. Gated on live CD8+TCR+ lymphocytes,

dot plots depict the percentage of IL-17+, IFNg+ and FOXP3+ T cells in the IEL compartment of

uninfected WTWT and MHCII-/-WT mice. One representative of two independent

experiments, 3 mice pooled per group.

106

Figure 3.9 CD44 expression on IL-17+, FOXP3+ and IFNg+ CD8+ T cells in the LP of

MHCII-/-WT mice. Dot plots depict the expression CD44, IL-17 (left), FOXP3 (middle) and

IFNg (right) on gated CD8+TCRb+ LPLs isolated from uninfected MHCII-/-WT mice. One

representative of 3 independent experiments.

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3.4.3 Expression of MHCII on hematopoietic cells is necessary to control C. rodentium

infection.

We next aimed to determine if MHCII expression on hematopoietic cells was required to

control C. rodentium in vivo. WTWT mice exhibited a 100% survival rate after inoculation

with 109 CFU of C. rodentium (Fig. 3.10A), in line with the reported resistance of wild type

C57Bl/6 mice to an oral challenge with C. rodentium at this inoculum. In sharp contrast, MHCII-

/-WT mice exhibited a hundred percent mortality rate after oral challenge with C. rodentium,

and the mice started to die as early as 3 days p.i. (Fig. 3.10A). MHCII-/- mice all succame to

infection although with slightly delayed kinetics compared to the chimeras (Fig. 3.10B),

suggesting the bone-marrow reconstitution process can slightly increase susceptibility to C.

rodentium. Histologically, the colons of MHCII-/-WT mice exhibited significantly increased

pathology compared to their WTWT counterparts at 7 days p.i. (Fig. 3.10C). Moreover, over

10-fold more CFUs of C. rodentium were isolated from the spleens (Fig. 3.10D) and mesenteric

lymph nodes (Fig. 3.10E) of MHCII-/-WT mice than WTWT mice at 7 days p.i. CIITA

-/-

WT also exhibited increased mortality after infection with C. rodentium (Fig. 3.4C). Finally,

many regions of very severe ulceration could be observed in the colonic sections from MHCII-/-

WT mice (Fig. 3.10F, arrow), which were not observed in WTWT mice. In line with a

previous report [27], uninfected MHCII-/-WT colons had higher pathological scores than

WTWT colons(249), suggesting that homeostatic antigen presentation via MHCII is likely

needed to prevent the occurrence of low level of inflammation in the intestine. The basal colitic

phenotype observed in the MHCII-/-WT mice could be caused in part by the decreased

percentage of Tregs found in the mucosa of these mice. Together, these results demonstrate that

MHCII signaling is critically required to properly control C. rodentium infection.

108

109

Figure 3.10 MHCII-/-WT mice are more susceptible to C. rodentium infection. A)

Survival curve of WTWT and MHCII-/-WT mice infected by oral gavage with 1x109

CFU

of C. rodentium (8 mice per group). B-C) The levels of C. rodentium translocation to the

mesenteric lymph nodes (MLN) (B) or spleen (C) were measured at 7 days post-infection in

WTWT and MHCII-/-WT mice. Each data point represents an individual mouse,

representative experiment of two independent experiments. D) The levels of colonic

histopathology were assessed in uninfected and 7 days post-infected WTWT and MHCII-/-

WT mice (3-6 mice per group, one representative of two independent experiments). E)

Representative images (10X magnification) of H&E stained colon sections of WTWT and

MHCII-/-WT mice infected with C. rodentium infected for 7 days; depicts severe

ulceration. Error bars represent SEM. Statistical analysis was performed using a two-tailed

student’s t-test where: * = p < 0.05, **= p < 0.01.

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3.5 Discussion

In this study we demonstrated that MHCII was essential for the generation of early Th17

responses to C. rodentium challenge. These results expand our understanding of the role of iTh17

cells in early responses to C. rodentium (4 days p.i.) and S. Typhimurium (1 day p.i.)(106). The

kinetics at which MHCII-/-WT mice succumbed to the infection, with the first animal dying as

early as 3 days p.i, further suggests that CD4+ T cells play a critical protective role in the early

phases of infection with C. rodentium in the chimeras.

We previously found that the intestinal microbiota was essential for the generation of an

iTh17 response to an enteric bacterial pathogen, as this response was completely blunted in

germ-free mice infected with S. Typhimurium(106). Here, we now provide evidence that MHCII

is also required for the induction of early LP Th17 responses to bacterial infection, which

strongly suggests that specific antigens are needed to either prime or drive this response. The

antigens required for iTh17 induction could either be pathogen-derived, or alternatively antigens

from the microbiota could translocate to the intestinal mucosa following injury caused by an

enteric pathogen, thereby triggering a pool of mucosal T cells with specificity against

microbiota-derived antigens. We currently favor the second scenario, first because our previous

results have demonstrated that these iTh17 cells have a memory phenotype(106), and second

because efficient induction of IL-17 and IL-22 by these iTh17 cells occurs upon first exposure to

either C. rodentium or S. Typhimurium. Future work should delineate the nature of the antigens

specifically required to trigger iTh17 induction in a MHCII-dependent manner in the intestinal

mucosa.

Recently, a study by Eberl and colleagues demonstrated that T cell receptor (TCR)

transgenic Marilyn-Rag2-/- and TCR7-Rag2-/- TCR mice, which express a single TCR specific

for either a self male antigen or an antigen from hen egg lysozyme, respectively, had similar

111

levels of intestinal Th17 cells compared to wild-type mice in homeostatic conditions(251). These

findings suggest that antigen specificity is not required for the differentiation of LP Th17 cells in

uninfected conditions. This conclusion is in line with the results of the present study, since we

observed that in uninfected conditions there were similar numbers of Th17 cells between MHCII-

/-WT and WT

-/-WT mice in the intestinal LP (Fig 3.1A-B). However, in the aforementioned

study the authors did not explore if antigen specificity was needed for Th17 responses after

infection, which our study now suggests is needed.

Our study represents the first report that describes a key role for both MHCII and CIITA

in mediating early host defense against enteric bacterial pathogens, and is in line with previous

studies showing that CD4-deficient and RAG1-deficient mice both exhibit increased

susceptibility to infection with C. rodentium(146). Previously, depletion of CD8+T cells was

shown to have no effect on susceptibility to C. rodentium infection(146), which is in agreement

with our observation that the compensatory Tc17 response observed in MHCII-/-WT mice does

not contribute to protection. Our observations that uninfected MHCII-/-WT mice exhibited

basal intestinal inflammation are supported by a previous study, which suggested that elevated

IFN levels contributed to the basal colitis in chimeric MHCII-/-WT mice(249). However this

early study had not examined how deletion of MHCII in the hematopoietic compartment affected

mucosal IL-17 production by either CD4+ or CD8+ T cells, nor did it investigate the response to

an enteric bacterial pathogen. Finally, it must be noted that more recent studies by Irla et al(252)

and Darasse-Jeze et al(253) demonstrated that MHCII expression specifically on CD11c+ DCs

was needed to promote effective peripheral Treg function, and that loss of DC-restricted MHCII

expression resulted in exacerbated T-cell mediated autoimmunity.

One of the most interesting findings in the present study is the discovery of an in vivo

112

regulatory mechanism of Tc17 cell generation in the mucosa. Specifically, we found that Tc17

cells are constitutively inhibited by MHCII in vivo. This finding could make MHCII-/-WT

mice an interesting model to study further the physiological regulation of the Tc17 cell subset,

which to date has mainly been investigated in ex vivo settings. The induction of Tc17 cells

occurred in a LP microenvironment with elevated levels of IFN, which has been reported to

inhibit Th17 cell differentiation and could be contributing to the blunted percentage of mucosal

Th17 cells observed in our MHCII-/-WT mice. Moreover, we also surprisingly observed higher

levels of FOXP3+CD8+T cells in the LP of MHCII-/-WT mice, which now more broadly

suggests that MHCII-dependent antigen presentation limits the induction of atypical CD8+ T cell

subsets in vivo. Little is currently known about FOXP3+CD8+ T cells, however recent studies

have suggested that these cells retain regulatory function and can express surface markers

generally associated with CD4+ Tregs such as CTLA-4(254,255). Studies aiming at further

understanding the transcriptional program that regulates the differentiation of IL-17+ and

FOXP3+ CD8+ T cells both in vitro and in vivo would therefore be of interest.

Importantly, Tc17 cells were found to be upregulated in many types of tumors(242-244),

and these cells could potentially hinder anti-tumor immunity since Tc17 cells appear to exert less

cytotoxic capacity compared to normal CD8+ T cells(256). A recent study described that

CD4+CD25- T cells were required to prevent the differentiation of naïve CD8+ T cells into Tc17

cells in Th17-promoting culture conditions in vitro(257). From these findings we can now

speculate, based on the additional evidence we provide in this study, that MHCII is critical for

activating a subset of CD4+ T cells that inhibit Tc17 cell differentiation in vivo.

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Chapter 4

Identification of a synthetic muramyl peptide derivative with

enhanced Nod2 stimulatory capacity

Stephen J. Rubino* and Joao G. Magalhaes* (*: co-first author publication),

Dana Philpott, George M. Bahr, Didier Blanot and Stephen E. Girardin

Innate Immunity. 2013 Jan 22.

I designed and performed experiments, analysed the data and wrote the manuscript. J. G. M.

contributed the experiments that used murine macrophages and human DCs, and helped with in

vivo experiments. G. M. B. and D. B. provided key reagents. S.E.G. performed the initial screen

of MDP derivatives. D. J. P and S. E. G. supervised the research and helped write the

manuscript.

114

4.1 Abstract

Muramyl peptides (MPs) represent the building blocks of bacterial peptidoglycan, a critical

component of bacterial cell walls. MPs are well characterized for their immunomodulatory

properties, and numerous studies have delineated the role of MPs or synthetic MP analogs

in host defense, adjuvanticity, and inflammation. More recently, Nod1 and Nod2 have been

identified as the host sensors for specific MPs, and in particular Nod2 was shown to detect

muramyl dipeptide (MDP), an MP found in both Gram-positive and Gram-negative

bacterial cell walls. Because mutations in Nod2 are associated with the etiology of Crohn’s

disease, there is a need to identify synthetic MP analogs that could potentiate Nod2-

dependent immunity. Here, we analyzed the Nod2-activating property of 36 MP analogs

that had been previously tested for their adjuvanticity and anti-infectious activity. Using a

luciferase-based screen, we demonstrate that addition of a methyl group to the second

amino acid of MDP generates an MDP derivative with enhanced Nod2-activating capacity.

We further validated these results in murine macrophages and in vivo. These results offer a

basis for the rationale development of synthetic MPs that could be used in the treatment of

inflammatory disorders that have been associated with Nod2 dysfuntion, such as Crohn’s

disease.

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4.2 Introduction

Early studies have reported that the stimulatory capacity of MPs can be considerably

enhanced by the addition of acyl chains of various lengths to the core of what is now known as

the Nod1- and Nod2-activating ligands (i.e., iE-DAP or MDP, respectively), and this property

likely correlates with the enhanced capacity of acylated MPs to enter host cells by

endocytosis(55,258,259). In addition, earlier studies (mainly from the 1970’s and 1980’s)

provided detailed characterization of the structure/function relation for MPs, and in particular

MPs derived from MDP(56,57,260-263). However, since the identification of Nod2 as the

cellular MDP sensor, little effort was made to revisit these earlier studies and test the compounds

from the synthetic MP libraries directly for their Nod2 stimulatory capacity.

Here, we initially screened the Nod2-dependent NF-B activation capacity of a library of

36 chemical derivatives of MDP and identified MurNAc-L-Ala-D-Glu-OCH3 [MDP(D-Glu2)-

OCH3], a synthetic MDP-derivative in which the terminal D-isoglutamine was replaced by the -

methyl ester of D-glutamic acid, as a novel compound that exhibited a lower activation threshold

than MDP. Accordingly, this compound also induced more robust Nod2-dependent innate

inflammatory responses both in vitro and in vivo. Together, we found MDP analogs with either

lower or higher activation thresholds that could be used as starting points for the development of

new therapeutics with immunomodulatory properties.

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# STRUCTURE COMPOUND

NAME ADJUVANT

ANTI-

INFECTIOUS REF

1 MurNAc-L-Ala-D-isoGln-L-Lys MDP-L-Lys ++ ++ (262,264)

2 MurNAc-L-Ala-D-Gln-OCH3 MDP(D-Gln2)-OCH3 ++ ++ (262,264)

3 MurNAc-L-Ala-D-Glu-OCH3 MDP(D-Glu2)-OCH3 ++ ++ (262,264)

4 MurNAc-L-Ala-D-Glu MDP(D-Glu2) ++ ++ (262,264)

5 MurNAc-L-Ala-L-isoGln MDP(L-isoGln2) - - (262,264)

6 MurNAc-L-Ala-D-isoGln-OCH3 MDP-OCH3 ++ ++ (262,264)

7 MurNAc-L-Ala-D-isoGln-D-Ala MDP-D-Ala - -/+ (262,264)

8 MurNAc-L-Ala-D-Gln-NH2 MDP(D-Gln2)-NH2 -/+ - (262,264)

9 MurNAc-L-Ala-D-Glu(OCH3)2 Muradimetide ++ ++ (262,264)

10 MurNAc-D-Ala-D-isoGln-D-Ala MDP(D-Ala1)-D-Ala - - (262,264)

11 MurNAc-L-Ala-D-Gln-OnC4H9 Murabutide ++ ++ (262,264)

12 MurNAc-L-Ala-D-isoGln MDP ++ ++ (262,264)

13 MurNAc-N-methyl-L-Ala-D-isoGln MDP(N-Me-L-Ala1) +/- - (262,264)

14 MurNAc-D-Val-D-isoGln MDP(D-Val1) + - (262,264)

15 MurNAc-D-Ser-D-isoGln MDP(D-Ser1) + - (262,264)

16 MurNAc-L-Ser-D-isoGln MDP(L-Ser1) + - (262,264)

17 MurNAc-Gly-D-isoGln MDP(Gly1) -/+ - (262,264)

18 MurNAc-D-Ala-D-isoGln MDP(D-Ala1) - - (262,264)

19 MurNAc-L-Val-D-isoGln MDP(L-Val1) + -/+ (262,264)

20 MurNAc-L-Pro-D-isoGln MDP(L-Pro1) + - (262,264)

21 MurNAc-N-methyl-D-Ala-D-isoGln MDP(N-Me-D-Ala1) - - (262,264)

22 MurNAc-L-Thr-D-isoGln MDP(L-Thr1) ++ -/+ (262,264)

23 1-0--methyl-glycoside-MDP - - *

24 NorMurNAc-L-Ala-D-isoGln Nor-MDP ++ + (265,266)

25 1-0--methyl-glycoside-MDP - - *

26 MDP(D-Ser1)-A--L - - *

27 6-0-Suc-MDP(D-Gln1)-OnC4H9-A--La ++ ++ (261)

28 MDP(D-Val1)-A--L - - *

29 6-0-Suc-MDP(D-Gln1)-OnC4H9 ++ ++ (265)

30 MDP-A--L ++ ++ (261)

31 MDP(D-Ala1)-A--L -/+ - (261)

32 4-0-Ac-6-0-Suc-MDP-OCH3 ++ ++ (265)

33 L-Ala-D-isoGln Dipeptide - - (266)

34 MurNAc Sugar - - (266)

35 D-Ala-D-Gln-A--L - - *

36 L-Ala-D-isoGln-A--L - - (261)

Table 4.1. List of tested muramyl dipeptide derivatives. Structure information and working

names are provided. Adjuvant refers to reported adjuvant capacity of the tested compounds,

whereas anti-infectious refers to previously reported ability to enhance host immunity against the

pathogen Klebsiella pneumoniae.

*Pierre Lefrancier and George Bahr, personal observations.

aSuc, succinyl; A- -L, multi-poly(DL-alanine)- -poly(L-lysine) carrier

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4.3 Material and Methods

Reagents. The 36 MDP-derivative compounds used in this study (Table 4.1) were collected from

MP libraries previously synthesized and evaluated for their biological activity (adjuvanticity and

anti-infectious activity)(261,263,267,268). Larger quantities of MurNAc-L-Ala-D-isoGln (MDP),

MurNAc-L-Ala-D-Glu-OCH3 [MDP(D-Glu2)-OCH3] and MurNAc-D-Val-D-isoGln [MDP(D-

Val1)] that were used for in vitro and in vivo analyses were synthesized according to previously

reported procedures(267,268).

Luciferase assays. NF-B activation luciferase assays in cells over-expressing Nod2 were

performed as described previously(19). Briefly, HEK293T cells were transfected overnight with

30 ng of Nod2 and 75 ng of Ig luciferase reporter plasmid. The cells were simultaneously

treated with 1, 10, 100 or 1000 ng of the indicated muramyl peptide analog and the NF-B-

dependent luciferase activation was measured 24 h later on a luminometer.

Mice. C57BL/6 (Charles River) and Nod2–/–

mice were bred and housed under specific pathogen

free conditions at the Center for Cellular and Biomolecular Research, University of Toronto. The

University of Toronto Animal Ethics Review Committee approved all animal experiments. Sex-

matched mice that were 6-10 weeks old were used for experiments.

In vivo cytokine response. C57BL/6 (Charles River) and Nod2–/–

mice were injected

intraperitoneally (i.p.) with the indicated dose of the MDP-derivatives and serum was collected 2

h later. The concentration of the chemokine KC and the cytokine IL-6 in the serum was

measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the

manufacturer’s protocol.

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OVA-specific antibody response. WT and Nod2–/–

mice were immunized i.p. with a mixture of

OVA (100 µg/mouse) and MDP, MDP(D-Val1) or MDP(D-Glu

2)-OCH3 at the indicated

concentrations (Prime). The mice were re-injected with OVA 26 days later (boost). Blood from

tail veins was collected 16 days later (42 days after the initial immunization) and sera of

individual mice were analyzed. The levels of IgG1 in the serum were measured by sandwich

ELISA comparing serially diluted serum samples with an assay-intrinsic isotype-specific

standard as described previously(83).

Bone marrow-derived macrophages (BMDM) preparation. BMDMs were isolated from WT

and Nod2–/–

mice and cultured as described previously(269). Briefly, total bone marrow cells

were seeded at 5 106 cells in 10-cm dishes in 10 ml of complete culture medium (DMEM

supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids (50

mM each) and 10% FCS supplemented with 30% of L cell-derived medium containing CSF

activity). On day 3, 10 ml of fresh medium was added to the cell culture. After 7 days of

incubation, the non-adherent cells were removed and the remaining adherent cells were

harvested by short incubation with ice-cold 1 PBS. For experiments, BMDMs were seeded in

complete culture medium in 24-well plates at 2 106 cells/ml, pre-treated with cytochalasin D (1

M) for 30 min and then stimulated with 50 g/ml of MDP, MDP(D-Val1) or MDP(D-Glu

2)-

OCH325

. 24 h later, concentrations of KC and MIP2 were measured in the cell supernatants by

ELISA.

Statistical analyses. Student’s t tests were performed using Graphpad (Prism), and P values <

0.05 using a two-tailed 95% confidence interval were considered significant.

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4.4 Results

4.4.1 NF-b stimulatory activity of MP derivatives

In order to characterize the Nod2-dependent responses to synthetic MDP analogs, we used a

library of 36 MDP derivative compounds (which includes MDP, compound #12) that had been

tested previously for their adjuvanticity and anti-infectious activity, as summarized in Table 4.1.

Interestingly, many of the tested compounds that had a modification at the 2nd

amino acid of

MDP, such as MurNAc-L-Ala-D-Glu-OCH3 [MDP(D-Glu2)-OCH3], Murabutide and

Muradimetide, had been shown to exhibit similar levels of enhanced protection against the

bacterial pathogen Klebsiella pneumoniae and adjuvant capacity when compared to MDP (Table

4.1, compounds #1-12). This is in contrast to what was observed with MDP-derivatives modified

at the 1st amino acid, such as MDP(D/L-Val

1), MDP(D/L-Ser

1) and MDP(L-Thr

1), (Table 4.1, #13-

22) which did not exhibit a protective effect against K. pneumoniae infection in vivo. However,

many of these compounds were still reported to be functional adjuvants for the generation of

specific antibody responses to antigens injected either by the intra-venous (i.v.) or sub-cutaneous

(s.c.) routes, although they demonstrated less effective adjuvanticity than MDP (Table 4.1).

We now know that Nod2 is the PRR mediating host sensing of MDP, thus we were

interested in assessing the capacity of the 36 MDP-derivatives to activate Nod2 at the cellular

level. To do this, we used the now well-established Nod2-Ig luciferase reporter assay in

HEK293T cells, which were stimulated with our MDP-derivative library. We determined that the

same 2nd

amino acid-modified MDP-derivatives that were shown to be good anti-bacterial

compounds and adjuvants (Table 4.1) were also strong inducers of Nod2-dependent NF-B

activation in vitro (Fig. 4.1). Interestingly, MDP(D-Glu2)-OCH3 (compound #3), induced approx.

120

5-fold more NF-B activation than MDP when stimulated at the lowest dose of 1 ng/ml,

indicating that MDP(D-Glu2)-OCH3 has a lower activation threshold than MDP. Next, we

determined that MDP-derivatives with the first amino acid of the D-configuration, such as

MDP(D-Val1) (compound #14), were compounds that could not induce Nod2-dependent NF-B

activation (Fig. 4.2). MDP-derivatives modified at the anomeric function or D-lactoyl group of

MurNAc (Table 4.1, #23-25) exhibited reduced Nod2 stimulatory capacity compared to MDP

(Fig. 4.3), while MDP compounds that were modified at 2 or more sites (Table 4.1, #26-32)

generally stimulated Nod2 less efficiently at lower doses (1, 10 and 100 ng) than MDP (Fig. 4.4).

Finally, as previously described, MDP derivatives that lacked the MurNAc sugar or the dipeptide

moiety were unable to stimulate Nod2 in vitro (Fig. 4.5), which reflected the inability of these

compounds to act as anti-bacterials or adjuvants in vivo (Table 4.1, # 33-36).

121

122

Figure 4.1. NF-B stimulatory capacity of MDP-derivative compounds modified at the 2nd

amino acid. A) Structures of compounds 1 to 11 (see Table 1 for working names), which

represent MDP derivatives modified at the 2nd

amino acid. Modifications with respect to MDP

structure (compound 12) are written in red. B) HEK293T cells that were transfected overnight

with Nod2 plasmid and Ig luciferase reporter plasmid were stimulated with 1, 10, 100 or 1000

ng of compounds 1 to 12, with 12 being MDP and acting as a positive control. The bar graphs

represent the fold NF-kB activation over unstimulated cells. Error bars depict SEM.

123

124

Figure 4.2 NF-B stimulatory capacity of MDP-derivative compounds modified at the 1st

amino acid. A) Structures of compounds 13 to 22 (see Table 1 for working names), which

represent MDP derivatives modified at the 1st amino acid. Modifications with respect to MDP

structure are written in red. B) HEK293T cells that were transfected overnight with Nod2

plasmid and Ig luciferase reporter plasmid were stimulated with 1, 10, 100 or 1000 ng of

compounds 12 to 22, with 12 being MDP and acting as a positive control. The bar graphs

represent the fold NF-kB activation over unstimulated cells. Error bars depict SEM.

125

126

Figure 4.3 NF-B stimulatory capacity of MDP-derivative compounds modified at the

MurNAc carbohydrate. A) Structures of compounds 23, 24 and 25 (see Table 1 for working

names), which represent MDP derivatives modified at the MurNAc carbohydrate. Modifications

with respect to MDP structure are written in red. B) HEK293T cells that were transfected

overnight with Nod2 plasmid and Ig luciferase reporter plasmid were stimulated with 1, 10, 100

or 1000 ng of compounds 12, 23, 24 and 25, with 12 being MDP and acting as a positive control.

The bar graphs represent the fold NF-kB activation over unstimulated cells. Error bars depict

SEM.

127

128

Figure 4.4 NF-B stimulatory capacity of MDP-derivative compounds modified at two or

more sites. A) Structures of compounds 26 to 32 (see Table 1 for working names), which

represent MDP derivatives modified at two or more sites. Modifications with respect to MDP

structure are written in red. B) HEK293T cells that were transfected overnight with Nod2

plasmid and Ig luciferase reporter plasmid were stimulated with 1, 10, 100 or 1000 ng of

compounds 12 and 26 to 32, with 12 being MDP and acting as a positive control. The bar graphs

represent the fold NF-kB activation over unstimulated cells. Error bars depict SEM.

129

130

Figure 4.5. NF-B stimulatory capacity of MDP-derivative compounds with either the

sugar or an amino acid removed. A) Structures of compounds 33 to 36 (see Table 1 for

working names), which represent MDP derivatives modified at the 2nd

amino acid. Modifications

with respect to MDP structure are written in red. B) HEK293T cells that were transfected

overnight with Nod2 plasmid and Ig luciferase reporter plasmid were stimulated with 1, 10, 100

or 1000 ng of compounds 12 and 33 to 36, with 12 being MDP and acting as a positive control.

The bar graphs represent the fold NF-kB activation over unstimulated cells. Error bars depict

SEM.

131

4.4.2 In vitro and in vivo analyses of MDP(D-Val1)

Given the previously reported capacity of MDP(D-Val1) (compound #14) to act as an

adjuvant (Table 1) and its ablated ability to induce Nod2 activation (Fig 4.2), we were interested

in further testing whether this compound could decouple the inflammation-inducing capacity of

MDP from the ability to induce adaptive immune responses. We observed that MDP(D-Val1) was

unable to stimulate KC or MIP-2 production from BMDMs when compared to MDP in vitro

(Fig. 4.6A). Moreover, MDP(D-Val1) that was injected i.p. did not induce an innate chemokine

and cytokine response in vivo, as evidenced by the lack of KC and IL-6 induction in the serum at

2 h post-injection (Fig. 4.6B). In contrast to previously reported results, we determined that

MDP(D-Val1) could not function as adjuvant as evidenced by the lack of detected OVA-specific

serum IgG1 antibodies after a prime-boost immunization regimen with OVA+ MDP(D-Val1) (Fig

4.6C). Together, our results indicate that MDP(D-Val1) cannot induce innate and adaptive

immune responses in vivo, suggesting that MDP-derivatives that cannot induce Nod2-dependent

NF-B activation result in host unresponsive compounds.

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133

Figure 4.6. In vitro and in vivo responses observed with MDP(D-Val1). A) Concentration in

pg/ml of KC (left panel) and MIP2 (right panel) measured in the supernatants of BMDMs

stimulated with PBS, MDP or MDP(D-Val1). B) Levels of KC (pg/ml) measured in the serum at

2 h post-challenge of C57Bl6 wild-type or Nod2-/-

mice injected with either PBS, 50 g/mouse of

MDP or 50 g/mouse of MDP(D-Val1) i.p. C) Bar graph represents the concentrations of OVA-

specific IgG1 in the serum of C57Bl6 wild-type or Nod2-/-

mice 42 days after prime-boost as

measured by ELISA after initial immunization with 50 g of OVA in sterile PBS, 50 g OVA

with 50 g/mouse of MDP or 50 g OVA with 50 g/mouse of MDP(D-Val1). One

representative experiment of two independent experiment. Error bars depict SEM. * represents P

< 0.05.

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4.4.3 In vitro and in vivo analyses of MDP(D-Glu2)-OCH3

Since we observed that MDP(D-Glu2)-OCH3 could activate Nod2 at lower concentrations

than MDP in the luciferase assays, we were interested in further testing this compound in more

physiological conditions. Similarly to the in vitro results, MDP(D-Glu2)-OCH3 appeared to be a

better inducer of KC and MIP-2 secretion than MDP when used to stimulate BMDMs (Fig

4.7A). Moreover, MDP(D-Glu2)-OCH3 at low concentrations (5 g/mouse) induced higher levels

of KC and IL-6 in the serum at 2 h post-injection in vivo compared to MDP at the same dose,

reinforcing the idea that MDP(D-Glu2)-OCH3 has a lower activation threshold than MDP (Fig

4.7B). At the higher dose of 50 g/mouse, MDP(D-Glu2)-OCH3 injection resulted in increased

levels of KC and a trend for increased IL-6 in the serum at the same time point (Fig 4.7B). The

increased potency of MDP(D-Glu2)-OCH3 for inducing a rapid KC response in vivo was similar

to that of N-glycolyl-MDP (Fig. 4.8), a compound that has previously been shown to exhibit

increased Nod2-dependent innate immune responses than MDP(270). Importantly, using Nod2

knockout (KO) mice, we provided evidence that the host response to both MDP and MDP(D-

Glu2)-OCH3 were fully dependent on Nod2 (Fig. 7B).

Next, we determined that at the lower dose of 5 mg/mouse, MDP(D-Glu2)-OCH3

exhibited a trend for more potent adjuvanticity compared to MDP for the generation of OVA-

specific serum IgG1 antibodies (Fig. 4.7C). In contrast, at the dose of 50 mg/mouse there was no

observable difference in potency between MDP and MDP(D-Glu2)-OCH3, as the immunization

response likely reached a plateau (Fig. 4.7C). Finally, we found that MDP(D-Glu2)-OCH3 was a

much better agonist than MDP for activating human DC ex vivo as measured by the production

of cytokine IL-6 and the chemokines MIP1a and MCP-1 (Fig. 4.9), suggesting that this

compound might be more sensitive for human cells compared to murine cells.

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136

Figure 4.7 In vitro and in vivo responses observed with MDP(D-Glu2)-OCH3. A)

Concentration in pg/ml of KC (left panel) and MIP2 (right panel) measured in the supernatants

of BMDMs stimulated with PBS, MDP or MDP(D-Glu2)-OCH3. B) Levels of KC and IL-6

(pg/ml) measured in the serum at 2 h post-challenge of C57Bl6 wild-type or Nod2-/-

mice injected

with either PBS, 5 or 50 g of MDP and 5 or 50 g of MDP(D-Glu2)-OCH3 i.p. One

representative experiment of two independent experiements. Error bars depict SEM. * represents

P < 0.05.

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Figure 4.8 Head-to-head comparison of in vivo responses observed with MDP(D-Glu2)-

OCH3 and N-Glycolyl-MDP. Levels of KC (pg/ml) measured in the serum at 2 h post-

challenge from C57Bl6 wild-type injected i.p. with either PBS, 5 mg/mouse or MDP (compound

12), 5 mg/mouse of MDP(D-Glu2)-OCH3 (compound 3) or 5 mg/mouse of N-Glycolyl-MDP.

Each data point represents one mouse, grouped from two independent experiments. Error bars

depict SEM. * represents P < 0.05, N.S.= not significant.

138

139

Figure 4.9 Muramyl dipeptide (MDP) (D-Glu2)-OCH3 induces enhanced cytokine and

chemokine production by human DCs compared to MDP. Bar graphs depict the levels of IL-

6 (A), MIP1a (B) or MCP-1 (C) as measured by ELISA in the supernatants of purified human

DCs stimulated with 0.5, 5 and 50 mg/ml of MDP or MDP(D-Glu2)-OCH3 for 16 h. Grouped

data from one or two independent experiments, error bars depict SEM.

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4.5 Discussion

In this study, we found that many MDP-derivative compounds modified at the 1st or 2

nd

amino acid can stimulate Nod2 equally well as MDP, while one in particular, MDP(D-Glu2)-

OCH3, was surprisingly shown to be a more potent immune activator than MDP in the in vitro

and in vivo stimulation assays we tested. In contrast, we also determined that MDP(D-Val1), a

compound that did not stimulate any Nod2-dependent NF-B activation in vitro, was also unable

to induce an inflammatory response in BMDMs or when injected into mice, and did not exhibit

any capacity to function as an adjuvant in OVA-specific antibody responses.

We demonstrated that none of the MDP-derivatives that had the 1st amino acid of the D-

configuration were capable of stimulating Nod2 in the luciferase assay, and given the results

observed in vivo with MDP(D-Val1) administration, we can speculate that no MDP-derivative

that harbors a D-amino acid at the first position will stimulate either innate or adaptive immune

responses. The discrepancies between our results and those obtained in the older studies could be

due to imprecision in the way the antigen-specific antibody response was previously measured.

Based on our results, an interesting avenue of investigation for future studies aiming at

decoupling the inflammation and adjuvant effects of MDP would be to use MDP(L-Val1)

(compound #19) or MDP(L-Ser1) (compound #16). Indeed, we found that these compounds still

activated Nod2 (Fig. 4.2), albeit less so than MDP, yet were still reported to exhibit adjuvanticity

and a lack of anti-infectious properties (Table 1). It would be interesting to revisit whether

MDP(L-Val1) or MDP(L-Ser

1), which have higher thresholds for Nod2 activation, retain a

significant ability to function as adjuvants.

141

Previously, it was reported that the conversion of the N-acetyl group on the MurNAc

sugar to an N-glycolyl group, which occurs naturally in mycobacterial species by the enzyme N-

acetylmuramic acid hydroxylase(270), results in an MP with a greater Nod2-stimulating activity.

In the present study, we identify what appears to be the first chemical modification to the core

amino acids of MDP that results in an analog with increased stimulatory capacity on Nod2. With

this proof-of-principle observation, we can now speculate that the relatively modest increase in

stimulatory capacity identified here (approx. 5-fold) provided by the replacement of the terminal

amide of MDP by a methyl ester could be rationally optimized by targeting the -carboxyl group

of D-Glu, and larger substitutions, such as ethyl, butyl or larger groups, would be worth testing.

Moreover, it is noteworthy that such chemical modifications of the MDP core structure could be

combined with the addition of acyl chains in order to generate MP derivatives that would be both

more active and more bioavailable than MDP.

142

Chapter 5

General Discussion and Future Directions

143

5.1 Linking Nod1/2 and Mucosal Th17 responses: Implications for Crohn’s

Disease Pathogenesis.

In the first chapter of this thesis, I demonstrated that Nod1 and Nod2 were required to

mount an early inflammatory response against the pathogen C. rodentium and this correlated

with an inability of Nod DKO mice to properly contain the infection. I then demonstrated that

Nod1 and Nod2 mediated the induction of a Th17 response that occurs very early during

infection with C. rodentium and S. typhimurium, which was termed the innate Th17 (iTh17)

response. This finding expands upon previous studies that had determined that Nod agonists

could stimulate DCs to drive Th17 differention in vitro, and that Nod agonists could synergize

with TLR agonists to potentiate systemic Th17 responses when injected in vivo. Both the Nod2

and Th17 pathways have been associated with risk to develop CD; therefore the discovery that

Nod1 and Nod2 regulate intestinal Th17 responses in two models of colitis adds to our current

understanding of the initiation of colonic inflammation.

An important question that arises from this work is how does the Nod-iTh17 pathway we

discovered in a mouse model of colitis translate to human disease? In humans, intestinal Th17

responses have generally been considered to be contributing to worse pathology in IBD since

Th17 cell-produced cytokines such as IL-17 and IL-22 are upregulated in inflamed intestinal

tissue compared to adjacent non-inflamed tissue from CD patients. However, this method

understand the pathogenesis of the diseaseis inherently flawed to since measuring cytokines and

chemokines in inflamed tissue from patients is merely giving a glimpse at the endpoint of the

pathology and does not yield any information about the early events that led to that endpoint.

Indeed, our discovery of a Nod-iTh17 axis that regulates inflammatory responses in bacterial-

induced colitis suggests the opposite is true; that proper induction of Th17 responses in the gut is

144

likely beneficial for the host in the initial stages of the pathogenesis of CD, before pathology is

observed. Furthermore, I can speculate that genetically susceptible individuals that accumulate

repeated challenges with environmental agents that cause breach of the epithelial barrier, such as

pathogens, irritants, etc, over time could result in the development of chronic inflammation.

The multi-hit mechanism of IBD induction is supported by studies that demonstrated

specific environmental triggers induce colitis only in the context of associated susceptibility

genes. Specifically, a recent study reported that certain strains of Norovirus induced colitis only

in mice that harbored defective ATG16L1 signaling in Paneth cells(271). Moreover, another

study demonstrated that specific Bacteroides species could induce colitis only in mice deficient

for IL-10R2 and defective T cell TGFR2 signaling(272). Additionally, strains of attaching-

invading E. coli and mycobacterium species have also been associated with CD. Elucidating the

nature of the environmental triggers, microbial-derived or otherwise, that could directly induce

colitis in the context of defective Nod1 and Nod2 signaling would be interest to further our

understanding of CD pathogenesis.

Another interesting finding in my study was that the iTh17 response occurred

predominantly in the cecum, a pouch that connects the terminal ileum to the colon. Indeed, the

cecum is the site in the body where the transition from under 105 bacteria/ml in the small

intestine to over 109 bacteria in the large intestine occurs. This is of relevance for CD because

patients harboring NOD2 frameshift variant typically present with inflammation in the ileo-colic

region of the GI tract(120). Whether increased density of bacteria and bacterial products or

specific bacterial species found in the cecum contribute the Nod-iTh17 responses remains to be

determined.

145

5.2 Memory T cell Responses to the Enteric Microbiota

In Chapter 2, I determined that enteric microbiota was critical for the induction of iTh17

responses; while in Chapter 3 I expanded on these findings and demonstrated that hematopoietic

MHCII is also required for Th17 responses after infection. Although I termed these cells “innate”

Th17 cells, we determined that iTh17 cells exhibited an effector memory T cell phenotype.

Indeed, I believe that the iTh17 cells are in fact memory cells that are activated by a recall

response during the infection with either C. rodentium of S. Typhimurium.

The nature of the antigens to that the iTh17 cells are responding to remains unclear.

However, considering the requirement of the microbiota and the need for antigen presentation by

MHCII to mediate iTh17 responses, I speculate that the antigens driving the memory recall

iTh17 responses are derived from the microbiota. Multiple studies have recently elucidated the

presence of commensal-bacteria specific memory T cell responses in the gut mucosa(273-276).

Specifically, Hsieh et al demonstrated that LP Tregs exhibit a restricted TCR repertoire

compared to systemic Tregs that was dependent on antigens from the microbiota(275).

Moreover, recognition of microbiota-derived antigens was shown to be required for the induction

of colitis in a TCR transgenic mouse model(276). Finally, a recent paper by Belkaid’s group

demonstrated that infection with T. gondii results in a robust generation of memory T cell

response to antigens found in the microbiota and these microbiota-specific memory T cells could

be reactivated in a recall response to a second challenge by an unrelated pathogen(274).

Together, these studies illustrate the importance of gut microbiota antigens in shaping mucosal

memory T responses. Future experiments aimed at elucidating the TCR repertoire LP Th17 cells,

and determining how changes in the microbiota shape the associated mucosal memory Th17

responses would be of interest.

146

Moreover, I also demonstrated (in Chapter 3) that in the absence of MHCII signaling,

Tc17 cells are strongly upregulated in the intestinal LP. Tc17 cells are emerging as important

mediators of pathology in a number of tumors and autoimmune disorders, and future studies

aimed at delineating their function and antigen-specificity would provide valuable insight into

this novel T cell subset.

In recent years, multiple studies have highlighted the importance of IL-17-dependent

immune responses to prevent mucosal damage during acquired immunodeficiency. Indeed, it has

been reported that during the acute-phase of CD4+ cell loss during Simian Immunodeficiency

Virus (SIV) infection in macaques there was a transient induction in the levels of mucosal Tc17

cells, which was followed by a depletion of these cells in the late stages of infection(246,277-

279). In light of my studies, it would be interesting to determine if other CD8+ subsets are

induced, such as FOXP3+CD8+ T cells, and how these subsets contribute to pathogenesis and/or

protection.

Finally, the results obtained using the CIITA-/-WT mice suggest that induction of Tc17

cells could have clinical implications for patients with Bare Lymphocyte Syndrome II, a primary

immunodeficiency often caused by mutations in gene encoding CIITA(11,280,281), in which

aberrant accumulation of non-CD4 IL-17-producing cells has not been specifically investigated

so far. Thus, an important question that arises from our studies is to determine if the

accumulation of Tc17 cells in immuno-deficient hosts is deleterious or beneficial for immune

regulation.

5.3 Role of Nod1 and Nod2 in CD103+ DCs

In addition to modulating iTh17 responses after infection with C. rodentium and S.

Typhimurium, I also demonstrated in Chapter 2 that the percentage of CD103+ DCs in Nod

147

DKO mice did not change after infection, whereas these cells were decreased in percentage in

the LP of WT mice after infection (Fig 2.9). This finding suggests that Nod1 and Nod2 play a

role in the dynamics of CD103+DC migration to and from the gut mucosa, which could have

important implications for intestinal homeostasis.

CD103 is an integrin that can bind to E-cadherin expressed on the surface of intestinal

epithelial cells, and this protein was originally identified as a marker of IELs(282,283). The

exact function of CD103 remains unclear, however it has been postulated that this surface

receptor aids in maintaining DCs and T cells to locate to the intestinal mucosa(282). The

function of LP CD103+ DCs have been extensively studied and these cells have been reported to

be capable of capturing antigens in the gut, migrating to lymph nodes, and presenting to T cells

to induce gut-homing T and B cell responses(284). This is in contrast with the other resident gut

DC population, the CX3CR1+ DCs, which cannot travel out of the LP and are thought to

mediate luminal antigen sampling by extending dendrites in between adjacent enterocytes(285).

Importantly, in co-culture experiments LP CD103+DCs preferentially polarize naïve T

cells to differentiate into FOXP3+ Tregs by secreting retinoic acid (RA) and TGF-, suggesting

that CD103+DC are important inducers of tolerance(286). Accordingly, LP CD103+DCs were

critical for Treg-mediated suppresion of T cell-driven colitis in vivo(286).

Recently, Lactobacillus PG stimulation of CD103+DCs was found to potentiate their

capacity to induce Foxp3+T cell differentiation in vitro(112). Moreover, i.p injection of

Lactobacillus PG increases the numbers of CD103+DCs in mesenteric lymph nodes and IL-10

expression in the colon(112). In line with these results, we recently determined that systemic

injection of MDP induces a dramatic recruitment of CD103+DCs to the spleen, which was

completely dependent on Nod2 signaling (Stephen Rubino and Dave Prescott, unpublished data).

148

However, whether MDP can modulate the migration of CD103+DCs in the intestinal mucosa

remains to be determined. Moreover, Nod2 was highly expressed, both at the RNA and protein

levels, in CD103+DCs compared to other myeloid and lymphoid cells (Kaoru Geddes,

unpublished data). Together, these observations suggest that Nod2 plays a pivotal role in

regulating CD103+DC responses, and opens up the possibility that CD103+DCs are the

predominant cell type bridging Nod2 signaling and adaptive immune responses.

Further studies exploring whether Nod1 agonists can also regulate CD103+DC in vivo

dynamics should be explored. Moreover, more experimentation is required to assess whether

MDP is stimulating migration of CD103+DCs directly or indirectly via epithelial or stromal-

derived signals. Indeed, epithelial-derived TSLP, which can be induced by MDP, has been

previously shown to drive CD103+DC’s ability to induce both murine and human Treg

differentiation(287-289).

5.4 Therapeutic Potential of Nod Agonists

In Chapter 4, I focused on the capacity of novel synthetic Nod2 agonists to induce NF-B

responses in vitro and innate and adaptive responses in vivo. The purposes of screening for novel

Nod2 agonists was twofold: 1) generating ligands with enhanced immunomodulatory properties

that could potentially be used to treat autoimmune disorders, such as CD; 2) identifying Nod2

ligands that would serve as more targeted adjuvants.

Many of the mutations in Nod2 associated with increased risk for CD(122,123) result in

Nod2 proteins that are either completely unresponsive to MDP, such as the 3020insC frameshift

mutant(124), or have reduced signaling capacity, such as the newly identified S431L and N852S

variants(125). Therefore, it would be interesting to determine if administration of MDP analogs

that exhibit increased Nod2 stimulatory activity, such as MDP(D-Glu2)-OCH3 identified in this

149

Chapter 4, could restore normal levels of NF-B activation in cells harboring certain CD-

associated Nod2 mutations. Such a strategy would provide the basis for a targeted therapy, using

MDP(D-Glu2)-OCH3 or other second generation MP derivatives related to MDP(D-Glu

2)-OCH3,

for CD patients with specific Nod2 mutations.

A major issue that limits the use of NLR and TLR ligands as adjuvants in a clinical

setting is the pyrogenic effects of these compounds. Therefore, identifying non-pyrogenic, or

inflammation-decoupled, novel Nod2 ligands would represent a significant advance in the

development of new adjuvants for clinical use. Moreover, identifying novel Nod1 and Nod2

agonists that could preferentially induce one arm of the adaptive response over another would be

of interest for the development of targeted adjuvants. For example, screening Nod ligands for the

specific ability to stimulate mucosal IgA responses would be of interest in the development of

mucosal vaccine, such as novel HIV vaccine where colonic and vaginal IgA mediates prevention

from infection.

Additionally, it would be very interesting to assess the ability of MDP and other novel

Nod agonists to modulate tolerogenic Treg responses in vivo. Indeed, as described in the Section

5.3, Nod2 appears to play an important functional role in CD103+DCs. Therefore, one could

screen a library of Nod2 agonists for their ability to recruit and activate CD103+DCs in vivo, or

alternatively stimulate isolate CD103+DCs ex vivo and assess the production of RA, TGF- and

IL-10.

150

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