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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
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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
83
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.
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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
92
(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.
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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.
107
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.
116
# 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.
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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.
135
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.
137
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.
140
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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