Regulation of Mucosal T cell Activation and Inflammation by NLRC3 and Tritrichomonas muris

by

Nichole Kathryn Escalante

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

Department of Immunology University of Toronto

© Copyright by Nichole Kathryn Escalante 2017 Regulation of Mucosal T cell Function and Inflammation by NLRC3 and Tritrichomonas muris

Nichole Kathryn Escalante

Doctor of Philosophy

Department of Immunology University of Toronto

2017

Abstract

The mucosal immune system in the gut is specialized to protect the host from infection by pathogenic microbes while maintaining tolerance towards the antigen-rich microbiota. T cells are a major component of the mucosal immune system that help to respond to pathogenic microbial invasion while maintaining a tolerogenic environment during times of homeostasis. These T cells can be regulated by genetically inherited factors as well as factors from the environment. Since defects in T cell function can contribute to the development of inflammatory bowel diseases, it has become increasingly important to understand how these factors are regulating T cell function. In this thesis we seek to explore how an extrinsic microbe, Tritrichomonas muris and an intrinsic factor, NLRC3, separately contribute to T cell function and disease regulation in various models of colitis.

ii Acknowledgments

As advancements in biomedical research are rarely made alone, I have many friends, colleagues and family members to thank for contributing to the completion of this thesis.

First and foremost, I would like to thank Dr. Dana Philpott for her endless support and guidance throughout the ups and downs of my PhD research. Without her encouragement, insight and positivity I would not have been so successful with my studies. Thank you for your never ending generosity and for truly caring about me not just as a student but as a person. I will forever be grateful for the opportunities you gave me as a student and for your mentorship.

I would like to thank Dr. Stephen Girardin, Dr. Thierry Mallevaey, Dr. Jennifer

Gommerman and Dr. Phillipe Poussier for their scientific discussion, ideas and support as co- supervisors and committee members. I would especially like to thank Dr. Jennifer Gommerman for her support as the Graduate Coordinator.

My graduate work was funded by a CIHR Vanier Canada Scholarship, Ontario Graduate

Scholarship and Queen Elisabeth II/ Aventis Pasteur Graduate Scholarship. I’d like to thank all the relevant funding agencies and those who submitted reference letters on my behalf.

I wish to acknowledge my collaborators: Dr. Catherine Streutker for tirelessly scoring my histology samples and Dr. Arthur Mortha for his parasite advice. Furthermore, I would like to thank the many University of Toronto staff who supported my research and made my day by day work more enjoyable. These include Dione White and Joanna Warzysynska in the flow cytometry facility; Laura Kent, Lisa Scott, Leslie, plus all the other staff in the animal facility; and the wonderful past and present administrative staff for the Department of Immunology. I would especially like to thank Dionne for giving me the opportunity to be her flow cytometry minion. iii To my many, many lab mates and summer students, thanks for making the lab so much fun and the long days in the lab durable. I will miss all our discussions on Science, Life, etc. I appreciate all the help, encouragement, training and ideas you guys have provided me over the years. I couldn’t have asked for a nicer group of people to work with.

I’d like to thank all my friends and fellow graduate students in the Department of

Immunology for making graduate studies and Toronto a fun and memorable experience outside of the lab. I am grateful to all the IGSA members who organized the events and sporting activities I enjoyed participating in. Thanks to Angela Zhou for co-directing SciChat with me and for all the volunteers who helped us along the way. Special thanks to Nyrie Israelian for our soccer sideline discussions and Mayra Cruz Tleugabulova for being a wonderful friend.

Finally I would like to thank my family and significant other for their endless love and support throughout my studies. I would especially like to thank Jayesh Salvi for encouraging me to do my best and for showing me the wonders of his favorite city; my aunt, Ghislaine Pilon, and uncle, Richard Ayuen, for adopting me when I moved to Toronto; my Mom, Kathleen Escalante, for making me suitcases of delicious frozen food; my Dad, Christopher Escalante, for helping me pack those suitcases at the last minute and for reminding me to keep a work-life balance; my sister,

Alena Escalante, for her “encourage mint”; my brother, Gerard Escalante, for his computer tech support; and my baby nephew Nolan Escalante, for always giving me a reason to smile. I could not have completed this journey without you all.

iv Table of Contents

Acknowledgments...... iii

Table of Contents ...... v

List of Tables ...... vii

List of Abbreviations ...... viii

List of Figures ...... x

Chapter 1 ...... 1

Introduction ...... 1

1.1 The microbiome ...... 1

1.2 Microbial sensing and regulation in the gut ...... 2

1.3 Intestinal T cells ...... 2

1.3.1 Regulatory T cells ...... 3

1.3.2 Effector T cell subsets ...... 4

1.3.3 Intrinsic and Extrinsic Regulation of T cells by NOD-like Receptors ...... 8

1.3.4 Extrinsic regulation of T cells by the intestinal microbiota ...... 16

1.4 Inflammatory Bowel Disease ...... 22

1.4.1 T cells in IBD ...... 23

1.4.2 Models of colitis ...... 25

1.4.3 NLRs in IBD ...... 28

1.4.4 Microbiome & IBD ...... 30

1.5 Thesis Overview ...... 34

Chapter 2 ...... 35

The Common Mouse Protozoa Tritrichomonas muris alters Mucosal T cell Homeostasis and Colitis Susceptibility ...... 35

v

2.1 Abstract ...... 36

2.2 Introduction ...... 36

2.3 Materials and Methods ...... 38

2.4 Results ...... 44

2.4.1 Rip2-/-Rag1-/- mice have accelerated T cell transfer colitis pathology ...... 44

2.4.2 Rip2-/-Rag1-/- mice are not protected by regulatory cells ...... 47

2.4.3 Exacerbated Rip2-/-Rag1-/- pathology is transmissible ...... 51

2.4.4 T. muris infection exacerbates T cell transfer colitis ...... 53

2.4.5 Chronic T. muris infection alters baseline T cell homeostasis...... 55

2.5 Discussion ...... 57

Chapter 3 ...... 60

Exploring a Role for NLRC3 in T cells and Mucosal Inflammation ...... 60

3.1 Abstract ...... 61

3.2 Introduction ...... 61

3.3 Materials and Methods ...... 63

3.4 Results ...... 68

3.4.1 Preliminary evidence suggesting a role for NLRC3 in T cell function ...... 68

3.4.2 NLRC3 does not control Foxp3+ CD4 T cell development ...... 71

3.4.3 NLRC3 deficient T cells can efficiently induce colitis and NLRC3 deficient Tregs retain suppressive function in vivo ...... 75

3.4.4 T cell intrinsic NLRC3 does not regulate C. rodentium induced pathology but may govern Tc17 differentiation ...... 77

3.4.5 In vitro CD4 T cell differentiation is not regulated by NLRC3 ...... 83

3.4.6 NLRC3 does not regulate chemically induced colitis ...... 87

3.5 Discussion ...... 89

Chapter 4 Overall Discussion and Future Directions ...... 92

References ...... 102 vi

List of Tables

Table 1 - NLR Function in T cells ...... 9

Table 2 - NLRs in Experimental Colitis ...... 29

Table 3 – List of Primers ...... 67

vii

List of Abbreviations

AHR – Aryl Hydrocarbon Receptor ANOVA – Analysis of Variance AOM – Azoxymethane APC – Antigen Presenting Cell BMI – Body Mass Index CARD- Caspase activation and recruitment domain CD – Cluster of Differentiation CD – Crohn’s Disease cDNA – Complementary Deoxyribonucleic acid CFU- Colony Forming Unit DSS – Dextran Sodium Sulfate GWAS – Genome Wide Association Studies IBD – Inflammatory bowel disease IFN- Interferon Ig - Immunoglobulin IL – Interleukin ILC – Innate Lymphoid Cell iLN – Inguinal Lymph Node IMDM – Iscove’s Modified Dulbecco’s Medium LP – Lamina Propria LPS - Lipopolysaccharide LRR- Leucine Rich Repeat MHC – Major Histocompatibility Complex MLN – Mesenteric Lymph Node MNV – Murine Norovirus MyD88- Myeloid Differentiation Primary Response Protein 88 NACHT – Domain in NAIP, CIITA, HET-E and TP1 NKT- Natural Killer T cell NLR – NOD-Like Receptor NOD – Nucleotide Oligomerization Domain PAMPs- Pathogen Associated Molecular Patterns PCR – Polymerase Chain Reaction

viii

PMA – Phorbyl 12-myristate 13 acetate PMN – Polymononuclear PRR – Pattern Recognition Receptor REGIIIγ – Regenerative islet-derived protein 3 gamma RIP2 – Receptor Interacting Serine/Threonine Protein Kinase 2 RPMI - Roswell Park Memorial Institute RT-PCR – Reverse Transcription Polymerase Chain Reaction RT-qPCR – Real Time Quantitative Polymerase Chain Reaction mRNA- Messenger Ribonucleic acid SFB- Segmented Filamentous Bacteria SPF – Specific Pathogen Free TCR – T Cell Receptor TGFβ – Transforming Growth Factor Beta Th – Helper T cell TLR – Toll Like Receptor TNBS – Trinitrobenzenesulfonic acid Treg – Regulatory T cell UC – Ulcerative Colitis

ix

List of Figures

Figure 1 - CD4 T cell subsets in the intestine...... 6

Figure 2 – NLRC3 Domain Organization...... 15

Figure 3 – Sorting strategy for Tritrichomonas muris...... 43

Figure 4 - Non-littermate Rip2-/-Rag1-/- mice develop accelerated colitis...... 46

Figure 5 - Non-littermate Rip2-/-Rag1-/- are not protected from pathology by regulatory CD45RBLow T cells...... 49

Figure 6 - Rip2-/-Rag1-/- are not protected from pathology by regulatory CD45RBLow T cells .... 50

Figure 7 - Co-caged and littermate mice develop equally exacerbated T cell induced colitis ..... 52

Figure 8 - Tritrichomonas muris accelerates T cell induced colitis...... 54

Figure 9 - Tritrichomonas muris infection chronically increases cecal IFN-ɣ+ T cells ...... 57

Figure 10 – Preliminary analysis of NLRC3 deficient mice ...... 70

Figure 11 – NLRC3 does not regulate in vivo or in vitro Treg development...... 73

Figure 12 - NLRC3 deficient cells have normal peripheral T cell distribution ...... 75

Figure 13 – NLRC3 does not influence CD4 T cell function in T cell Transfer Colitis ...... 77

Figure 14 – T cell intrinsic NLRC3 does not alter C. rodentium pathology ...... 78

Figure 15 – Full body NLRC3 deficient mice do not have an altered response to C. rodentium . 81

Figure 16 – NLRC3 controls IL-17A production in CD8 T cells...... 83

Figure 17 – NLRC3 does not control in vitro T cell differentiation...... 84

Figure 18 – NLRC3 does not control Th17 or Tc17 in vitro differentiation ...... 87

x

Figure 19 – NLRC3 does not regulate the response to DSS induced colitis...... 89

Figure 20 – Wildtype mice from the NLRC3 colony display highly variable T cell responses to C. rodentium infection ...... 98

Figure 21 – NLRC3 is highly expressed in innate lymphoid cells...... 100

xi

Chapter 1

Introduction

1.1 The microbiome

Throughout evolution, mammals have adapted to living in a microbe rich world (Ley et al.,

2008b); the air, the earth, our food, are all teeming with microbes. While some of these microbes have evolved mechanisms to invade our tissues and exploit our cellular machinery, many microbes have evolved a more balanced approach where the host and microbes can live symbiotically. In particular, many microbes have found a niche in the mammalian intestine, benefiting from the nutrients we consume while also contributing to host health by metabolizing nutrients indigestible by the host, providing vitamins and protecting us from invasion of pathogenic species (Cho and

Blaser, 2012; Topping and Clifton, 2001; Venema, 2010). As a whole, these microbes and their collective genetic functions are termed the microbiome (Lederberg, 2000).

The microbiome is a multi-kingdom community consisting of bacteria, viruses, protozoa, fungi and helminths. While attempts have been made to define the microbial load in the human gut, it is well appreciated that microbial abundance and diversity varies between animals within the same species (Ley et al., 2008a; Turnbaugh et al., 2009). Many factors determine the microbial abundance and diversity in the gut, including diet, lifestyle, environment, medications, genetics and health status (Dominguez-Bello et al., 2011). Understanding these relationships and what

1 2 constitutes a healthy microbiome is an area of increasing interest as a healthy microbiome is thought to promote health of the host.

1.2 Microbial sensing and regulation in the gut

Mammals have developed a sophisticated mucosal immune system to protect themselves from invading pathogens and to retain the microbiota within their intestinal niche (Macpherson et al., 2009). Mucus, antimicrobial peptides, IgA and epithelial cells serve as a first barrier in the host. If microbes pass this barrier, they are sensed by various pattern recognition receptors such as

NOD-Like Receptors (NLRs) and Toll-like receptors (TLRs) in epithelial cells, stromal cells and immune cells (Caballero and Pamer, 2015; Hooper et al., 2012; Philpott et al., 2014). This sensing leads to a cascade of signals and host cell responses such as reinforcement of the primary barrier or the further activation of innate and adaptive immune cells. Through these varying levels of immune activation and concomitant regulatory mechanisms, the immune system is able to effectively control infections by pathogenic microbes while maintaining homeostasis with the microbiota.

1.3 Intestinal T cells

The ability of the immune system to adaptively select for a diverse range of beneficial microbes while excluding and maintaining memory for pathogenic microbes emerged in early vertebrates with the development of lymphocytes. Interestingly, this development of adaptive immunity is thought to be driven by the survival advantages conferred by maintaining a diverse microbiota (McFall-Ngai, 2007). Co-evolution has fine-tuned the symbiotic microbe-immune cell

3 relationship by reciprocally altering their repertoires to promote tolerance. The outcomes of this are highly apparent in the microbe rich gastrointestinal tract where these complex interactions frequently occur.

The gastrointestinal tract is one of the largest compartments of T cells within the body. It includes innate-like γδ T cells, CD8αα T cells and NKT cells along with conventional CD4 and

CD8αβ T cell subsets. These cells reside in the intraepithelial compartment, lamina propria (LP),

Peyer’s patches, isolated lymphoid follicles and mesenteric lymph nodes (MLN) where they are well positioned to react to invading microbes. Naïve T cells circulating in the blood can become primed in the mesenteric lymph nodes or Peyer’s patches by CD103+ dendritic cells to express

α4β7 and CCR9 that allows them to home to the intestinal tissues (Johansson-Lindbom et al., 2005;

Mora et al., 2003; Stagg et al., 2002). As such, the majority of gut resident T cells have an antigen experienced, effector or memory phenotype (Lundqvist et al., 1996; Masopust et al., 2001; Stagg et al., 2002; Zeitz et al., 1988). Interestingly, some of these T cells have microbiota specific T cell receptors (Cong et al., 2002; Hand et al., 2012; Lathrop et al., 2011; Yang et al., 2014). Although a quick response to pathogens is important, a fine balance must be maintained between effector and regulatory T cells to maintain a healthy gut. This section reviews the conventional T cell subsets found in the intestinal tract and explores some of the intrinsic and extrinsic factors that can shape their development and function.

1.3.1 Regulatory T cells

Within the gastrointestinal tract resides a large population of regulatory T cells (Tregs).

These Tregs are induced by the TGFβ rich intestinal environment and marked by the expression of the master transcriptional regulator Foxp3 (Fontenot et al., 2003; Marie et al., 2005). They help maintain tolerance to the microbiota and protect from intestinal inflammation by actively

4 suppressing innate and adaptive immune cells (Maloy et al., 2003; Read et al., 2000). Mechanisms of Treg suppression in the gut include secretion of suppressive cytokines such as TGFβ and IL-10 and contact-dependent suppression of cells through receptors such as CTLA-4 and ICOS (Li et al.,

2007; Tanoue et al., 2016). While some Tregs develop naturally within the thymus, then home to the gut, other intestinal Tregs are induced in the periphery by microbial products and food antigens

(Atarashi et al., 2011; Coombes et al., 2007; Kim et al., 2016; Round and Mazmanian, 2010; Sun et al., 2007). The transcription factor Helios has been described as a potential marker for thymically derived (Helios+) versus peripherally derived (Helios-) Tregs (Singh et al., 2015a; Thornton et al.,

2010). Indeed, both Helios- and Helios+ Tregs can be found in the intestine and both populations have been shown to be important for intestinal homeostasis (Cebula et al., 2013; Lathrop et al.,

2011). In addition to Foxp3+ regulatory T cells, a subset of Foxp3- regulatory T cells that secrete high amounts of IL-10, termed T regulatory type 1 cells, can also contribute to intestinal homeostasis (Groux et al., 1997).

1.3.2 Effector T cell subsets

1.3.2.1 CD4 T cells

Although the gut is generally a tolerogenic environment, invasion of pathogenic microbes into the intestinal tissue initiates an inflammatory response that leads to the presentation of antigen, upregulation of co-stimulatory molecules and secretion of pro-inflammatory cytokines. Depending on the type of insult, these signals will promote various types of CD4 helper T cell (Th) responses that are characterized by specific effector cytokines (Figure 1) (Mosmann et al., 1986). Th1 cells develop in response to IL-12 and are regulated by the transcription factor T-bet (Hsieh et al., 1993;

Seder et al., 1993; Szabo et al., 2000). They are characterized by IFN-γ and TNF secretion that leads to the activation of immune pathways necessary for the clearance of predominantly

5 intracellular pathogens such as bacteria and viruses. Th17 cells are induced by TGFβ with IL-6 and/or IL-21 and are regulated by RORγT (Bettelli et al., 2006; Harrington et al., 2005; Ivanov et al., 2009; Mangan et al., 2006; Park et al., 2005; Veldhoen et al., 2006). Their main effector cytokines are IL-17A, IL-17F and IL-22 and they are important for responses to mostly extracellular pathogens, such as bacteria and fungi. Th2 cells develop in response to IL-4 and IL-

25 and are regulated by the transcription factor GATA3 (Kopf et al., 1993; Seder et al., 1992;

Zheng and Flavell, 1997). They secrete IL-4, IL-5 and IL-13 that can promote humoral responses and protect against extracellular parasites. T follicular helper (TFH) cell development is promoted by IL-21 and IL-6 and regulated by the transcription factor Bcl-6 (Chtanova et al., 2004; Nurieva et al., 2008; Vogelzang et al., 2008). These cells secrete IL-21, enhancing germinal center formation and B cell class switching. Th9 are thought to develop in response to IL-4 and TGFβ and are under the control of PU.1 and IRF4 transcription factors (Chang et al., 2010; Schmitt et al., 1991; Schmitt et al., 1994; Staudt et al., 2010). They produce IL-9 and IL-10 in response to extracellular parasites (Licona-Limon et al., 2013; Veldhoen et al., 2008). Finally, Th22 cells have recently been identified as a potential new subset produced in response to IL-6 and TNFα and under the control of the Aryl Hydrocarbon Receptor (AHR) (Duhen et al., 2009; Trifari et al.,

2009). These cells produce IL-22, but no IL-17A or IFN- and are important for maintaining intestinal homeostasis (Backert et al., 2014).

6

Figure 1 - CD4 T cell subsets in the intestine.

In response to various stimuli, antigen presenting cells (APC) produce numerous cytokines which promote the differentiation of intestinal CD4 T cells into T helper (Th) subsets (Treg, Th9, Th2, Th17, Th22, Th1, Tfh). These T cell subsets produce specific cytokines which help protect against bacteria, viruses, parasites, helminths and fungi. Tregs are involved in the suppression of T cells and Tfh contribute to B cell responses.

7

Although CD4 T cells were initially thought to commit to and maintain one helper subset, there is evidence for plasticity between subsets (DuPage and Bluestone, 2016; Hori, 2014). In addition, examples of cells with multiple phenotypes, such as inflammation driven IFN-γ- and IL-

17A- double producing T cells have also been described (Lee et al., 2009). Certainly, the classification of T cells into distinct subsets is oversimplified as many more cytokines and transcription factors have been shown to regulate the development and function of CD4 helper T cells than described here.

1.3.2.2 CD8 T cells

CD8 T cells were thought to mainly function as cytolytic effectors that secrete perforin and granzyme to kill infected and malignant cells (Butz and Bevan, 1998; Moskophidis and Kioussis,

1998). However, it is now appreciated that they too can take on various helper phenotypes.

Although less studied, their development into Tc1, Tc2, Tc9, Tc17 and Foxp3+ cells have been described to mirror their CD4 counterparts in terms of stimulating cytokines, transcriptional regulators and effector cytokines. These CD8 T cell subsets have been observed in various diseases such as psoriasis, asthma, allergy, graft versus host disease, multiple sclerosis, contact hypersensitivity and Citrobacter rodentium infection but their contribution to intestinal homeostasis needs to be further defined (He et al., 2006; Kryczek et al., 2008; Robb et al., 2012;

Rubino et al., 2013; Schedel et al., 2016; Tang et al., 2012; Tzartos et al., 2008).

1.3.2.3 Regulation of T cells

The level of T cell activation during homeostasis or infection is regulated by many T cell intrinsic and extrinsic factors. T cell intrinsic factors are the result of genetic modifications within

T cells that alter their ability to sense and respond to external cues; alter their ability to become polarized and maintain their effector subsets; or alter their ability to survive. Intrinsic factors can

8 include transcriptional regulators, pattern recognition receptors, metabolic sensors, enzymes and signaling molecules. T cell extrinsic factors, on the other hand, originate from environmental factors. These can include microbial products, nutrients, drugs, cytokines, chemokines, cell surface receptors or antigen presented on MHC class I or II. It is important to understand how T cells are regulated by both intrinsic and extrinsic factors as the consequences of dysregulated T cell activation can range from immunodeficiency to autoimmunity. The following section will touch on some T cell modulating factors that are relevant to this thesis.

1.3.3 Intrinsic and Extrinsic Regulation of T cells by NOD-like Receptors

1.3.3.1 NOD-Like Receptors

The NOD-Like Receptor (NLR) family is a broad family consisting of receptors with shared features but varied functions. Most NLRs contain a ligand sensing c-terminal leucine rich repeat domain, a central nucleotide-binding domain for oligomerization and a varied n-terminal protein binding domain which dictates their downstream function (Jones et al., 2016). Some NLRs have been attributed with microbial sensing capabilities, however, other NLRs do not directly sense microbes but may still contribute to host responses to pathogens by regulating essential immune related pathways. T cells express many NLRs such as NOD1, NOD2, NLRP3, NLRC5,

NLRX1 and NLRC3 (Table 1), however, their function in T cells is largely understudied and published reports are often conflicting. This section will review what is currently known about

NLRs in T cells.

9

Table 1 - NLR Function in T cells

NOD-Like Function in T cell Implications for Disease Citations Receptor

NOD2 Protects Human Tregs from No role in T cell Transfer (Rahman et al., 2010; FAS-mediated cell death. colitis Zanello et al., 2013)

NOD1 Synergizes with TLR-2 in Promotes DSS colitis and (Mercier et al., 2012; CD8 T cells to promote IFN- anti-tumor activity Zhan et al., 2016) γ response

NLRP3 Produces IL-1β in response to Protects from LCMV (Arbore et al., 2016; nigericin & intracellular C5. infection, Graft versus host Bruchard et al., 2015; Promotes Th2 differentiation disease and T cell transfer Doitsh et al., 2014) colitis. Promotes asthma and melanoma growth.

NLRX1 Suppresses Th1 & Th17 Protects from DSS colitis, T (Leber et al., 2017) differentiation. Controls cell transfer colitis and C. metabolism rodentium infection

CIITA Upregulates MHC class II. Enhances HIV replication. (Gourley et al., 1999; Promotes Antigen Promotes oxazolone-induced Kim et al., 2006; presentation and cell colitis. Protects from Lanzavecchia et al., proliferation. Alters Th1/Th2 autoimmune 1988; Odum et al., balance encephalomyelitis. 1991; Park et al., 2004; Saifuddin et al., 2000)

NLRC5 Upregulates MHC class I. Protects from LCMV (Ludigs et al., 2016) Protects from NK cell killing infection

NLRC3 Suppresses IL-2 & CD25 Unknown (Conti et al., 2005) mRNA. Suppresses NFκB, NFAT, AP1

10

1.3.3.2 NOD1 & NOD2

NOD1 and NOD2 are cytosolic pattern recognition receptors that sense different moieties of peptidoglycan from bacterial cell walls (Chamaillard et al., 2003; Girardin et al., 2003a;

Girardin et al., 2003b; Inohara et al., 2003). NOD1 detects meso-diaminopimelic acid found mostly in Gram-negative bacteria while NOD2 detects muramyl-dipeptide found in Gram-negative and Gram-positive bacteria. Upon ligand binding, both NOD receptors self-oligomerize, and associate with their downstream adaptor molecule Receptor-interacting serine/threonine-protein kinase 2 (RIP2) (Girardin et al., 2001; Inohara et al., 2000; Park et al., 2007). This interaction occurs through the caspase recruitment domains (CARDs) on both NOD1/NOD2 and RIP2 and promotes RIP2 ubiquitination followed by recruitment and ubiquitination of NEMO (Abbott et al.,

2004; Yang et al., 2007). This then leads to the phosphorylation of IKKγ, degradation of IkB and release of NFkB subunits that translocate into the nucleus to initiate transcription of NFkB regulated genes such as pro-inflammatory cytokines. In addition, NOD1 and NOD2 can also stimulate pro-inflammatory cytokine production through the MAPK signaling cascade, though the mechanisms are less clear (Girardin et al., 2001; Hsu et al., 2007). Independent of RIP2, NOD1 and NOD2 activation can also enhance bacterial targeting by the autophagosome (Travassos et al.,

2010). This occurs through the direct recruitment of ATG16L1 by NOD1 and NOD2 to the site of bacterial entry and subsequent recruitment of autophagy machinery.

Since Crohn’s Disease (CD) is characterized by aberrant T cell responses within the intestinal wall and NOD2 is a known CD risk allele, the potential for a T cell intrinsic role for

NOD2 was explored by multiple groups. In human T cells, NOD2 was found to be fully functional and was shown to protect Tregs from Fas-mediated cell death (Rahman et al., 2010). Similarly, in mouse CD4 T cells, Zanello et al found that NOD2 is functionally responsive to its ligand muramyl

11 dipeptide (Zanello et al., 2013). Contrary to the human study though, NOD2 deficiency did not alter the development and function of mouse regulatory T cells or T cell mediated intestinal disease. An early study reported Th1 defects in NOD2 deficient T cells in response to Toxoplasma gondii and in the T cell transfer model of colitis (Shaw et al., 2009), however, this report has since been challenged (Caetano et al., 2011). Furthermore, no role for NOD2 in CD8 T cell immunity was reported during viral infection (Lin et al., 2013).

RIP2 expression is upregulated in T cells following TCR activation (Chin et al., 2002; Hall et al., 2008). Early studies described a central role for RIP2 in T cell receptor signaling, proliferation, survival and T cell differentiation into the Th1/Th2 subsets (Chin et al., 2002;

Kobayashi et al., 2002; Ruefli-Brasse et al., 2004). Unfortunately, many of these reports were contradictory and have since been disproven in fully backcrossed mice (Hall et al., 2008; Nembrini et al., 2008).

NOD1 and NOD2 share a similar signaling pathway through RIP2, however, in addition to differing ligands, they also have different cellular expression levels (Philpott et al., 2014).

Interestingly, NOD1 is more highly expressed in CD8 T cells than NOD2 and was reported to synergize with TLR2 to enhance CD8 T cell activation after TCR stimulation (Mercier et al.,

2012). It was also recently reported that NOD1 stimulation during TCR activation could enhance

IFN- production in CD8 T cells, enhancing their anti-tumor response (Zhan et al., 2016).

While a T cell intrinsic role for NOD1 & NOD2 remains mostly enigmatic, many T cell extrinsic roles for NOD1 & NOD2, in the regulation of gastrointestinal immunity, have been described. These include stimulating antimicrobial peptide secretion from Paneth cells, regulating the formation of isolated lymphoid follicles, controlling the function of dendritic cells, maintaining intestinal integrity and inducing inflammatory responses after an intestinal infection (Barreau et

12 al., 2007; Bouskra et al., 2008; Geddes et al., 2010; Geddes et al., 2011; Lala et al., 2003; Le

Bourhis et al., 2009; Magalhaes et al., 2011a; Natividad et al., 2012; Ogura et al., 2003). In relation to T cells, extrinsic NOD2 controls T cell numbers and cytokine production within Peyer’s patches,

(Barreau et al., 2010; Barreau et al., 2007) and the development of CD103+ DCs, which can stimulate intestinal Treg development (Macho Fernandez et al., 2011). Both NOD1 and NOD2 are also important for the generation of an early Th17 response to Citrobacter or Salmonella infection

(Geddes et al., 2011) and can also mediate Th2 responses (Fritz et al., 2007; Magalhaes et al.,

2008; Magalhaes et al., 2011a; Magalhaes et al., 2011b). How NOD1 and NOD2 contribute to intestinal pathogenesis is described in section 1.4.

1.3.3.3 Inflammasome associated NLRs

A subgroup of NLRs form multiprotein complexes, called inflammasomes, after sensing of various microbial and danger signals in the cytosol (Martinon et al., 2002). Canonical inflammasomes can activate caspase-1 through the adaptor molecule ASC, leading to the processing of pro-IL-1β and pro-IL-18 into their bioactive forms or the induction of cell death by pyroptosis (Broz and Dixit, 2016). The most well described canonical inflammasomes are NLRP1,

NLRP3 and NLRC4 but other less defined NLRs such as NLRP6, NLRP7 and NLRP12 may also contribute to caspase-1 activation. Epithelial cells, macrophages and dendritic cells express many of these NLR containing inflammasomes and processing of IL-1β and IL-18 from these cell types has been shown to alter T cell activation and differentiation in several models.

Evidence for T cell intrinsic inflammasome function was first reported in CCR5 expressing human memory CD4 T cells that activate IL-1β through the NLRP3 inflammasome in response to nigericin (Doitsh et al., 2014). Following this, a study of ASC deficient T cells found ASC to be important for T cell autocrine production of IL-1β, which enhanced Th17 cell survival and

13 promoted disease development during Experimental Autoimmune Encephalomyelitis (EAE)

(Martin et al., 2016). Recently, the NLRP3 inflammasome was reported to be activated in human

T cells by intracellular components of the complement cascade (Arbore et al., 2016). TCR and

CD46 induced expression of complement C5 reportedly leads to autocrine production of IL-1β, which supports Th1 differentiation. Contrary to these studies, Bruchard et al. previously reported no role for the NLRP3 inflammasome in development of Th1 or Th17 subsets (Bruchard et al.,

2015). Instead, they reported an inflammasome independent role for NLRP3 in T cells whereby

NLRP3 transcriptionally regulated Th2 cell differentiation by facilitating the binding of the transcription factor IRF4 to Th2 associated genes.

Overall, further studies are needed to clarify the role of NLRP3 in T cells. Thus far, no T cell intrinsic role has been ascribed to other inflammasome-associated NLRs.

1.3.3.4 NLRX1

NLRX1 is a mitochondrial associated NLR that has been shown to be involved in various cellular functions such as the formation of reactive oxygen species, the function of the mitochondrial respiratory chain, TRAF6 signaling and cell death (Allen et al., 2011; Singh et al.,

2015b; Soares et al., 2014; Tattoli et al., 2008). Consequently, NLRX1 is important for controlling inflammation in the brain, lungs and colon and can act as a tumor suppressor (Eitas et al., 2014;

Kang et al., 2015; Soares et al., 2014; Tattoli et al., 2016).

NLRX1 was recently shown to intrinsically regulate T cell effector and metabolic function

(Leber et al., 2017). In various intestinal models, NLRX1 deficient T cells were more prone to develop into IFN-γ and IL-17A secreting effector cells. Furthermore, these T cells had altered

14 expression of several genes involved in the metabolic uptake and utilization of glucose and fatty acids.

Evidence of T cell extrinsic modulation by NLRX1 is demonstrated by increased central nervous system tissue damage when wildtype myelin-specific T cells are transferred into NLRX1 deficient hosts compared to wildtype hosts (Eitas et al., 2014). This was attributed to increased

MHC class II and pro-inflammatory cytokine expression in NLRX1 deficient microglia and spinal cord antigen presenting cells.

1.3.3.5 NLRs as Transcriptional Regulators

Some NLRs have been shown to be transcriptional regulators, rather than direct pathogen sensors. For example, CIITA and NLRC5 have been shown to regulate MHC class II and MHC class I expression, respectively (Beresford and Boss, 2001; Masternak et al., 2000; Meissner et al.,

2010) Since presentation of antigen to CD4 or CD8 T cells through MHC class I or MHC class II is an essential step for T cell activation during infection and disease, there are many studies demonstrating deficient T cell responses in CIITA and NLRC5 deficient hosts (Abrahimi et al.,

2016; Rubino et al., 2013; Staehli et al., 2012; Thelemann et al., 2014; Yao et al., 2012).

As for a T cell intrinsic role, CIITA can upregulate surface MHC class II in human T cells after

TCR stimulation. In addition to conferring antigen-presenting abilities to activated T cells, MHC class II can signal through its cytoplasmic tail, enhancing T cell proliferation (Lanzavecchia et al.,

1988; Odum et al., 1991). Overexpression of CIITA in mouse T cells has resulted in conflicting results where CIITA was reported to suppress Th1 while promoting Th2 differentiation in one report and the opposite in another (Gourley et al., 1999; Park et al., 2004). Interestingly, T cell intrinsic NLRC5 was shown to be important for the upregulation of MHC class I in T cells. This

15 led to subsequent protection of T cells from NK cell mediated killing (Ludigs et al., 2016). The role for other NLRs as transcriptional regulators, especially in T cells, needs to be further studied.

1.3.3.6 NLRC3

NLRC3 also known as NOD3 or Caterpillar 16.2, is an NLR that is highly expressed in both mouse and human lymphocytes (Conti et al., 2005). It contains the canonical tripartite NLR structure with a NACHT domain similar to NOD1, NOD2, NLRC5 and CIITA and an LRR domain resembling that of NLRX1 (Figure 2) (Benko et al., 2010). Unlike NOD1 & 2, NLRC3 does not have a bona fide N-terminal CARD domain but has a CARD-like domain of unknown function.

NLRC3 is reported to be expressed in the cytoplasm, however, its ligands and functions have not been fully explored (Conti et al., 2005).

Figure 2 – NLRC3 Domain Organization.

NLRC3 contains 14 c-terminal Leucine Rich Repeat (LRR) domains, a central NACHT domain for oligomerization and an n-terminal undefined CARD-like domain (X).

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Initial studies in a human CD4 T cell line suggest an inhibitory role for NLRC3 in T cells.

In support of this, NLRC3 mRNA expression is rapidly decreased following TCR stimulation

(Conti et al., 2005). Furthermore, overexpression of NLRC3 suppresses the induction of IL-2 and

CD25 mRNA after TCR stimulation. This was attributed to the suppression of NFκB, NFAT and

AP-1 signaling pathways, however, these results were based on luciferase reporter assays and should be considered with caution since NLRC3 was shown to non-specifically inhibit luciferase assays at the post-translational level (Ling et al., 2012). Overall, these initial findings lead to the theory that NLRC3 may act as a negative regulator of T cell activation.

Follow up studies on NLRC3 have been focused on its regulatory role in macrophages and epithelial cells. In macrophages, NLRC3 was reported to inhibit TLR mediated activation of NFκB

(Schneider et al., 2012). This occurred through the interaction of NLRC3 with TRAF6 and subsequent TRAF6 degradation. In a second report, NLRC3 was reported to negatively regulate macrophage STING in response to challenges with viral DNA (Zhang et al., 2014). In this study,

NLRC3 bound to STING, impeding its interaction with TBK1, thus inhibiting type-1 interferon production. More recently, NLRC3 was shown to regulate PI3K-mTOR signaling in epithelial cells (Karki et al., 2016). Here, NLRC3 bound PI3K, inhibiting its activation of AKT, leading to downstream suppression of mTOR and to the suppression of colon cancer. While these studies highlight a role for NLRC3 in inhibiting key inflammatory pathways in macrophages and epithelial cells, it is still unknown why NLRC3 is highly expressed in lymphocytes and how it may be regulating lymphocyte function.

1.3.4 Extrinsic regulation of T cells by the intestinal microbiota

It is now appreciated that the microbiome is integral for the development of the intestinal immune system (Lathrop et al., 2011) (Bouskra et al., 2008; Hooper et al., 2012; Mazmanian et

17 al., 2005). This is evidenced by studies in germ-free mice; germ-free mice have immature lymphoid tissues, impaired epithelial function and lack lymphoid cells in their intestinal lamina propria (Hamada et al., 2002; Hooper et al., 2012; Pabst et al., 2006). Microbial antigens, along with food antigens, help prime and maintain T cells within the intestinal tissue (Atarashi et al.,

2013; Geuking et al., 2011; Kim et al., 2016). In general, microbial signals activate epithelial cells, stromal cells or antigen presenting cells, which results in antigen presentation and increased expression of T cell modulating cytokines and receptors. As such, the composition of the gut microbiome can alter the type and level of immune responses within the gut. This section will review the regulation of intestinal T cells by intestinal microbes.

1.3.4.1 Bacteria

With advances in sequencing technology, development of new culture methods and increased availability of germ free mice, much focus has been placed on elucidating the immune modifying capabilities of various symbiotic gut bacteria (Hooper et al., 2012). As such, specific bacteria are now implicated in influencing the level of specific T cell subsets. A primary example is the colonization of the small intestine with Segmented Filamentous Bacteria (SFB), which induces the development of SFB-specific Th17 cells (Gaboriau-Routhiau et al., 2009; Ivanov et al., 2009; Yang et al., 2014). These Th17 cells require SFB-induced Serum Ameloid A proteins

1/2 for robust production of IL-17A and can contribute to pro-inflammatory responses (Sano et al., 2015). In another case, certain Clostridial strains were found to promote regulatory T cell accumulation in the colonic lamina propria (Atarashi et al., 2013). This occurs through the production of short chain fatty acids. Specifically, bacterial-derived butyrate and propionate can stimulate epithelial cells to produce TGFβ, suppress dendritic cell activation and directly promote thymic and peripheral Treg development (Arpaia et al., 2013; Furusawa et al., 2013). As a final

18 example, Polysaccharide A (PSA) expressing Bacteroides fragilis promotes an anti-inflammatory response by inducing intestinal T cells to secrete IL-10 (Round and Mazmanian, 2010). This occurs through direct sensing of PSA by TLR2 in FoxP3+ T cells (Round et al., 2011). Further research into the T cell modulating capacities of specific bacterial strains opens the door to the development of novel treatments that could shift the activation profile of intestinal T cells.

1.3.4.2 Viruses

The identification and study of the enteric virome has been limited due to a lack of affordable technology and techniques to analyze the viral community as a whole (Virgin, 2014).

Recent advances, studying specific known viruses, however, have described a role for some viruses as immune modulating, symbiotic members of the human and mouse microbiome (Pfeiffer and Virgin, 2016). Intriguingly, the intestines of healthy individuals carry many viruses such as non-pathogenic Anelloviridae and Circoviridae viruses, persistent pathogenic Herpesveridae and

Caliciviridae viruses along with many unidentified families of viruses (Popgeorgiev et al., 2013).

Accordingly, how persistent viruses contribute to T cell homeostasis and host fitness is of increasing interest.

Chronic infection of mucosal tissues requires constant immune monitoring, even in asymptomatic conditions, and can cause T cell exhaustion, skew the T cell repertoire and/or contribute to T cell aging (Virgin et al., 2009). On the other hand, some chronic viral infections can prime cross-reactive T cells that can protect from subsequent infections by other microbes or exacerbate disease (Virgin et al., 2009; Welsh et al., 2010). Additionally, the chronic virus HIV can directly target and kill T cells, leading to immunodeficiency (Deeks et al., 2015). While informative, these studies mostly focus on the role of persistent pathogenic viruses in T cell modulation and may not reflect non-pathogenic persistent viruses.

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Murine norovirus (MNV), is a positive strand RNA virus of the Caliciviridae family that is highly prevalent in animal facilities (Henderson, 2008). Although initially discovered in ailing immunodeficient mice, certain strains of MNV can establish persistent intestinal infections in immunocompetent hosts without causing obvious disease (Karst et al., 2003). This makes MNV infection a suitable model for studying the contribution of the non-pathogenic virome on host mucosal immunity. Thus far, one study has reported that MNV infection of germ-free mice is sufficient to stimulate innate and adaptive mucosal immune cell reconstitution in the gut

(Kernbauer et al., 2014). Indeed, both CD4 and CD8 T cell numbers increase in the lamina propria and MLN of MNV positive germ-free mice. Although interesting, T cell numbers in MNV infected specific pathogen free (SPF) mice were unchanged, suggesting MNV may play a redundant role in mucosal T cell recruitment and maintenance when other microbes are present. In addition to stimulating T cell numbers, MNV infection supported a protective Th1 response in antibiotic treated mice subsequently infected with Citrobacter rodentium (Kernbauer et al., 2014). Whether or not this is relevant in non-antibiotic treated mice remains unknown as MNV positive mice did not develop an increased Th1 response to Salmonella typhimurium in a separate report (Higgins et al., 2011).

In addition to viruses that infect eukaryotic cells, the virome is proposed to consist of viruses that infect bacteria or archaea (bacteriophages) and of virus-derived genetic elements such as prophages, endogenous retroviruses and endogenous viral elements (Virgin, 2014). In one study, it was found that the human commensal Enterococcus faecalis strain V583 produced a composite prophage (ɸV1/7) (Duerkop et al., 2012). This prophage conferred a survival advantage for E. faecalis strain V583 over other strains of E. faecalis, but how these phage-induced changes alter host immunity remains to be explored. Overall, the contribution of viral elements to T cell function and intestinal homeostasis need to be further studied.

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1.3.4.3 Helminths

Helminths are complex multicellular organisms that are becoming steadily less prevalent in the human microbiome with improvements to sanitation (Elliott and Weinstock, 2012).

Helminths generally induce Th2 responses that lead to eosinophilia, mastocytosis and IgE production (Finkelman et al., 1991; Jarrett and Bazin, 1974). Concurrent with this Th2 shift, Th1 and Th17 cells and cytokines in the intestine are reduced (Elliott et al., 2008; Houlden et al., 2015).

This is due to dendritic cells and macrophages that develop a regulatory or pro-Th2 phenotype in response to helminth products, antigens or induced changes to the intestinal environment (Hang et al., 2010; Johnston et al., 2010; Schnoeller et al., 2008). Interestingly, this Th2 bias is sufficient to hamper Th1 responses to subsequent infection with vaccinia virus or HIV-1 but conversely, provides colonization resistance to pro-colitogenic species of Bacteroides (Actor et al., 1993;

Ramanan et al., 2016). Moreover, helminths can also induce Treg development and enhance secretion of IL-10 and TGFβ, further suppressing pro-inflammatory responses (Elliott et al., 2004;

Grainger et al., 2010; Setiawan et al., 2007). Based on the current evidence, helminths and helminth-derived products are attractive candidates for T cell modulating therapies.

1.3.4.4 Fungi

The mammalian gastrointestinal tract houses a diverse community of fungi termed the mycobiota (Hoffmann et al., 2013; Iliev et al., 2012; Scupham et al., 2006). These fungal populations are less stable than bacterial populations as they undergo episodic variation without experimental perturbation and are very sensitive to alterations in the diet (Dollive et al., 2013;

Hoffmann et al., 2013). C-type lectin receptors, TLRs and scavenger receptors on dendritic cells are important for detection of intestinal fungi and the initiation of protective Th17 and Th1 responses during fungal infection (De Luca et al., 2010; Drummond et al., 2016; LeibundGut-

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Landmann et al., 2007; Saijo et al., 2010). Interestingly, disruption of the natural gut fungal community with anti-fungal agents led to increased Th1 and Th17 cells during dextran sodium sulfate and T cell transfer colitis (Wheeler et al., 2016). Little is known about the role of the long- term, resident mycobiota in regulating the development and function of the mucosal T cell compartment during homeostasis.

1.3.4.5 Protozoa

The human intestinal tract can be colonized with a variety of protozoa, which have classically been deemed pathogenic parasites (Lukeš et al., 2015). Indeed, intestinal infections by pathogenic species (Entamoeba Histolytica, Giardia Lamblia, Toxoplasma gondii,

Cryptosporidium, etc.) are a major health burden worldwide (Fletcher et al., 2012). Many protozoan infections, however, can remain asymptomatic in a substantial portion of those infected and can even persist for years (Audebert et al., 2016; Fletcher et al., 2012; Parfrey et al., 2014).

Host genetics, environmental factors and the microbiota have all been described as determining factors for protozoan virulence and survival (Abd-Alla et al., 2006). As such, there have been many conflicting opinions on the pathogenicity of several enteric protozoa (Blastocytosis, Dientamoeba fragilis) and their potential as symbiotic members of the microbiota (Audebert et al., 2016; Barratt et al., 2011; Lukeš et al., 2015; Stark et al., 2016).

Similarly, laboratory mice can be infected with a range of protozoa – those considered pathogenic (Cryptosporidium spp., Spironucleus spp., Giardia spp., etc) have generally been eradicated from housing facilities, while those thought harmless (Entamoeba spp., Tritrichomonas spp., Hexamastix spp., etc.) often pass unmonitored (Baker, 2008). Most wild mice, however, carry various enteric protozoa without obvious disease.

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Trichomonads are flagellated Parabasilids from the Trichomonadida order (Lindsay et al.,

2008). Both parasitic and symbiotic species of Trichomonads are found in mammals (Baker,

2008). The human Trichomonads Pentatrichomonas hominis and Dientamoeba fragilis have been described, albeit with controversy, as gastrointestinal symbionts (Lukeš et al., 2015; Maritz et al.,

2014; Stark et al., 2016). Likewise, Tritrichomonas muris (T. muris) and the related

Tritrichomonas musculus (T. musculus) have been described as mouse symbionts which are highly prevalent in animal facilities (Baker, 2008).

A recent study by Chudnovskiy et al. sought to describe the commensal relationship between T. musculus and it murine host (Chudnovskiy et al., 2016). Acute infection with T. musculus induced epithelial derived IL-18, which promoted the expansion of Th1 and Th17 responses in the colon. Interestingly, this amplified T cell response conferred protection from

Salmonella typhimurium infection but consequently increased host susceptibility to colitis and colon cancer. On the contrary, a recent study by Howitt et al. described a Th2 type immune response to T. muris, characterized by tuft-cell secretion of IL-25 and expansion of IL-13 secreting

ILC2s (Howitt et al., 2016). While these studies begin to define host-protozoan interactions by non-pathogenic species, more work is needed to understand the role of protozoa in the development and long-term homeostasis of the mucosal T cell compartment.

1.4 Inflammatory Bowel Disease

A fine balance between inflammatory and tolerogenic responses needs to be maintained within the gastrointestinal tract in order to protect from pathogens while sustaining gut integrity during homeostasis. Failure of regulatory mechanisms to maintain T cell homeostasis can lead to

23 the development of uncontrolled chronic inflammation and Inflammatory Bowel Diseases (IBD), namely Crohn’s Disease (CD) and Ulcerative colitis (UC). This section will review the role of classical T cells, NLRs and the microbiota in IBD while touching on some of the current models used to study their etiology.

1.4.1 T cells in IBD

CD and UC are chronic gastrointestinal diseases with increasing prevalence throughout the world (Molodecky et al., 2012). A combination of genetic, microbial and environmental factors have been identified as contributors to immune dysregulation in IBD (de Souza and Fiocchi, 2016).

There is substantial evidence implicating defects in T cell regulation in the initiation and perpetuation of IBD. Evidently a large portion of tissue-infiltrating immune cells in IBD are activated T cells. CD was initially characterized as a Th1 disease but is now known to also include aberrant Th17 responses (Breese et al., 1993; Fujino et al., 2003; Fuss et al., 1996). UC was initially described as a Th2 disease but high levels of IFN-γ and IL-17A have also been found in some UC patients and biopsies, muddying that depiction (Fujino et al., 2003; Fuss et al., 1996; Kobayashi et al., 2008; Rovedatti et al., 2009). Furthermore, a role for Th9 responses in UC was recently defined

(Gerlach et al., 2014). Interestingly, IL-23 and IL-21, which support Th17 and potentially Th1 maintenance, have also emerged as important cytokines in IBD (Kobayashi et al., 2008;

Monteleone et al., 2005).

In addition to excessive expansion of inflammatory T cells during IBD, Tregs are also expanded in the lamina propria (Makita et al., 2004; Maul et al., 2005; Saruta et al., 2007).

Curiously, these Tregs are not sufficient to quell the aberrant inflammatory response, even though they maintain suppressive capacities in vitro (Kelsen et al., 2005). This may be due to the high levels of inflammatory cytokines, such as TNFα, which can inhibit Treg suppressive abilities

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(Valencia et al., 2006). Another theory argues that effector T cells and immune cells may be resistant to Treg suppression in IBD (Fantini et al., 2009; Monteleone et al., 2008). This was attributed to the upregulation of SMAD7, an inhibitor of TGFβ signaling, in effector T cells.

Notably, a phase II clinical trial with a SMAD7 inhibitor showed favorable rates of remission in

CD (Monteleone et al., 2015).

Several of the described IBD susceptibility loci are involved in altering T cell responses

(IL-2RA, IL-23R, IL-10, IL-12B, IFN etc.) (Anderson et al., 2011; Beaudoin et al., 2013; Duerr et al., 2006; Jostins et al., 2012; Momozawa et al., 2011; Neurath et al., 1995; Rivas et al., 2011).

Correspondingly, mice with altered expression of these gene products or of their ligands display dysregulated T cell activation and differentiation in various models of colitis (Aggarwal et al.,

2003; Okada et al., 2005; Rennick et al., 1997). Nevertheless, a defect in one of these genes alone is often insufficient for spontaneous intestinal inflammation, suggesting that an accumulation of various defects and contributions from the environment and the microbiota are required to disrupt intestinal homeostasis.

While many microbiota-specific antibodies have been found in human and experimental colitis, limited evidence has been found for microbiota specific T cells (de Souza and Fiocchi,

2016). An early study found T cells reactive to microbial antigens in inflamed CD tissue but not healthy tissue (Pirzer et al., 1991). Following this, flagellin specific CD4+ Th1 T cells were found in IBD patients and in mice exhibiting spontaneous colitis (Cong et al., 1998; Lodes et al., 2004);

Transfer of these T cells was sufficient to induce colitis in a murine model. These studies suggest that there may be a breach in microbial tolerance in IBD, but whether this is a cause, promoter or consequence of IBD isn’t fully understood.

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Although many T cell cytokines are upregulated in the intestines of human IBD patients, their roles in disease pathogenesis remain unclear since clinical attempts to disrupt these signaling pathways have produced mixed results (Neurath, 2017). Anti- IFN-γ and anti-IL-13 treatment were ineffective for human CD and UC respectively and anti-IL-17A treatment worsened CD disease symptoms (Hueber et al., 2012; Reinisch et al., 2006; Reinisch et al., 2015). Anti-IL-6 and anti-

IL-12/IL-23p40, however, have both shown promising results in clinical trials (Ito et al., 2004;

Mannon et al., 2004; Sandborn et al., 2008; Sandborn et al., 2012). Furthermore, anti-TNFα remains a leading therapeutic for IBD (Neurath, 2017). Nevertheless, therapeutics aimed at controlling T cell survival, activation, and migration are still actively being investigated with the hopes of developing more specific and efficacious treatments for IBD.

1.4.2 Models of colitis

Identification of microbial and genetic contributors to human IBD have largely depended on sequencing of patients and their microbiomes. These studies are largely correlative and require experimental manipulation of identified factors to uncover causal relationships. For this purpose, many murine models of IBD have been developed. These include genetically, bacterially, chemically and T cell induced models. This section will review the colitis models pertinent to this thesis.

1.4.2.1 T cell transfer colitis

The T cell transfer model of colitis is useful for investigating intrinsic and extrinsic factors that regulate T cell mediated colitis. Undeniably, seminal work on IBD from the Powrie lab and others have stemmed from this model. In short, the transfer of naïve T cells, identified as CD4+

CD45RBhi, into T cell deficient SCID or Rag1/2 knockout (KO) mice induces inflammation along

26 the intestine after 5-8 weeks (Powrie et al., 1993). This inflammation requires the homeostatic expansion and activation of T cells plus the presence of microbial antigens, as IL-7-/-Rag-/- and reduced flora SCID hosts have attenuated disease (Aranda et al., 1997; Totsuka et al., 2007). In this model, lamina propria infiltrating T cells are predominantly Th1 (Powrie et al., 1993). Th17 cells also develop and contribute to pathogenesis by becoming IFN- and IL-17A double- producing T cells or by promoting Th1 development (Harbour et al., 2015). Conveniently, co- transfer of regulatory cells, identified as CD4+ CD45RBlow, can prevent CD45RBhi T cell mediated colitis (Powrie et al., 1993). Protection is attributed to the Foxp3+ Tregs found in the CD45RBlow population (Uhlig et al., 2006). Notably, many of the genes upregulated in human CD, such as

IFN- and TNF, are similarly regulated in the colons of mice during T cell transfer colitis (te

Velde et al., 2007). Despite the similarities to IBD and usefulness of the model, one should keep in mind that the lymphopenic expansion of T cells during T cell transfer colitis may not be reminiscent of true IBD.

1.4.2.2 Dextran-sodium sulfate induced colitis

An intact epithelial barrier is essential for maintaining a tolerogenic and non-inflamed state in the intestine (Hermiston and Gordon, 1995). Undoubtedly, disruption of the epithelial barrier leads to translocation of bacteria into the lamina propria and activation of an immune response.

The dextran sodium sulfate (DSS) model of colitis exploits this process to mimic inflammation seen in IBD (Cooper et al., 1993; Okayasu et al., 1990). DSS is a water soluble, sulfated polysaccharide that can be administered to mice through their water. It is thought to be toxic for colonic epithelial cells, causing erosion, loss of epithelial tight junction proteins and decreased expression of protective molecules (Hernandez-Chirlaque et al., 2016). Primary epithelial damage occurs in the absence of the microbiota, however, subsequent inflammation in the tissue requires

27 microbial antigens (Hernandez-Chirlaque et al., 2016). The initiated immune response is largely characterized by infiltration of granulocytes, monocytes and macrophages, which control the influx of microbes while promoting repair of the epithelium (Bernasconi et al., 2010; Cooper et al., 1993; Okayasu et al., 1990).

DSS colitis occurs in the absence of adaptive immune cells and is typically viewed as an innate model of intestinal injury (Dieleman et al., 1994; Krieglstein et al., 2002). However, in immunocompetent hosts, Th1 and Th2 cells accumulate and may contribute to chronic DSS pathogenesis (Dieleman et al., 1998). In addition, expansion of Tregs has recently been shown to protect from DSS colitis by promoting IgA production from B cells (Chen et al., 2011b; Wang et al., 2015). Overall, the lack of T cell driven inflammation and artificial nature of disease onset makes the classification of DSS as a model of IBD debatable. Nevertheless, DSS is still a suitable model for studying factors involved in maintaining and repairing the epithelial barrier.

1.4.2.3 Citrobacter rodentium

Citrobacter rodentium (C. rodentium) is an attaching-effacing Gram-negative murine pathogen similar to human enterohaemorrhagic E. coli and enteropathogenic E. coli (Collins et al.,

2014). Upon oral gavage with in vitro cultures, C. rodentium initially colonizes the cecum then progresses to the distal colon and rectum where it induces a mild colitis (Wiles et al., 2004; Wiles et al., 2006). The resulting inflammatory response is characterized by an influx of neutrophils, macrophages and dendritic cells, followed by activation of early and late Th1, Th17 and B cell responses (Geddes et al., 2011; Gibson et al., 2008; Higgins et al., 1999; Lebeis et al., 2007;

Mangan et al., 2006; Simmons et al., 2003; Vallance et al., 2002). Recently, group 3 innate lymphoid cells (ILC3) have also been described as important for full defense against infection

(Rankin et al., 2016; Song et al., 2015). In immunocompetent hosts, bacterial load and epithelial

28 damage peak around 10-14 days and are resolved in 3-4 weeks. However, mice deficient for key immunoregulatory molecules (IL-22, IL-17A, IFN, etc.) or cell types (ILC3, CD4 T cells, B cells, etc.) succumb to or develop persistent infection (Collins et al., 2014).

C. rodentium pathogenesis has a close relationship to the microbiota. C. rodentium colonization and its accompanying inflammation alter the abundance and diversity of the microbiota (Lupp et al., 2007). Furthermore, susceptibility to C. rodentium infection is highly influenced by the composition of the microbiota (Ghosh et al., 2011; Ivanov et al., 2009; Willing et al., 2011). For example, colonization with SFB can induce a protective Th17 intestinal response

(Ivanov et al., 2009). Correspondingly, there is evidence for increased abundance of diverse strains of E. coli in some CD patients, however, it is still unclear where E. coli fit into disease pathogenesis

(Martinez-Medina and Garcia-Gil, 2014). Overall, C. rodentium infection has many of the hallmarks of IBD including immune cell infiltration, epithelial damage and microbial dysbiosis making it a useful model for studying intestinal pathogenesis (Higgins et al., 1999).

1.4.3 NLRs in IBD

Considering the importance of NLRs in regulating T cell homeostasis, it is not surprising that NLRs can contribute to or protect from intestinal pathogenesis. Indeed, NLRX1, NLRP3,

NLRP6, NLRC4, NOD1 and NOD2 protect from intestinal damage in various models of colitis

(Table 2) (Rubino et al., 2012). Despite this evidence, only mutations to NOD2 have been strongly linked to IBD susceptibility (Rivas et al., 2011). This association is found in CD patients from

Jewish or European ancestry but not in Japanese or Chinese populations (Inoue et al., 2002; Leong et al., 2003). In addition, not all carriers of CD-associated NOD2 variants in susceptible populations develop CD, highlighting the multifaceted nature of the disease.

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Table 2 - NLRs in Experimental Colitis

NOD-Like Contribution to experimental colitis Citations Receptor

NOD2 Protects from DSS, TNBS, C. rodentium, (Barreau et al., 2007; Biswas et al., S. typhimurim (with NOD1), H. 2010; Couturier-Maillard et al., 2013; hepaticus and E. faecalis induced colitis Geddes et al., 2010; Geddes et al., 2011; Kim et al., 2011a; Kim et al., 2011b; Macho Fernandez et al., 2011; Natividad et al., 2012; Watanabe et al., 2008)

NOD1 Protects from AOM/DSS and C. difficile. (Chen et al., 2008; Geddes et al., 2010; Protects from C. rodentium & S. Geddes et al., 2011; Hasegawa et al., typhimurium (with NOD2). 2011; Zhan et al., 2016)

NLRP3 Protects from DSS, TNBS, AOM/DSS & (Allen et al., 2010; Hirota et al., 2011; C. rodentium. Liu et al., 2012; Zaki et al., 2010)

NLRP6 Protects from C. rodentium infection, (Chen et al., 2011a; Elinav et al., 2011; DSS and AOM/DSS Levy et al., 2015; Normand et al., 2011; Seregin et al., 2017; Wlodarska et al., 2014)

NLRX1 Protects from DSS colitis, T cell transfer (Koblansky et al., 2016; Leber et al., colitis, AOM/DSS and C. rodentium. 2017; Soares et al., 2014; Tattoli et al., 2016)

CIITA Promotes oxazolone-induced colitis (Kim et al., 2006)

NLRC4 Protects from DSS, AOM/DSS, C. (Broz et al., 2010; Carvalho et al., 2012; rodentium & S. typhimurium. Franchi et al., 2012; Hu et al., 2010; Liu et al., 2012)

NLRC3 Protects from AOM/DSS (Karki et al., 2016)

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Interestingly, the majority of the disease-predisposing NOD2 variants have loss of function mutations within the peptidoglycan sensing LRR domain (Lesage et al., 2002). This suggests that a deficiency in microbial sensing may be a contributor to CD and that NOD2 deficient mice could be an effective tool to develop mechanistic insights into the disease. In accordance with CD being a complex disease, NOD2 deficient mice do not develop spontaneous colitis but develop more severe colitis after intestinal assault with DSS (Barreau et al., 2007; Couturier-Maillard et al.,

2013; Magalhaes et al., 2008; Natividad et al., 2012). This increased colitis susceptibility was recently ascribed to a transferrable, colitis-inducing microbiota in NOD2 or RIP2 deficient mice

(Couturier-Maillard et al., 2013). However, it is unclear whether this pro-colitogenic microbiota is a result of genetics or an artifact of long-term breeding of isolated colonies (Al Nabhani et al.,

2016; Robertson et al., 2013b). Interestingly though, injection of NOD2 ligands or colonization with probiotic MDP-expressing bacteria, could protect wildtype mice from some symptoms of chemically induced colitis (Macho Fernandez et al., 2011; Natividad et al., 2012; Watanabe et al.,

2008). Notably, the induction of colitis by transfer of NOD2 deficient T cells into T cell deficient hosts is no different than with transferred wildtype T cells (Zanello et al., 2013). This suggests that

NOD2 deficiency in non-T cells may play a more prominent role in colitis susceptibility. More research is required to further understand the NOD2 regulated pathways that mediate CD susceptibility and the potential role for other NLRs in IBD.

1.4.4 Microbiome & IBD

Enteric bacteria, viruses, helminths, fungi and protozoa can play major immunomodulatory roles in murine models of IBD. Indeed, many models of colitis depend on microbial colonization for full pathology (Bamias et al., 2007; Hernandez-Chirlaque et al., 2016; Niess et al., 2008; Sellon

31 et al., 1998). Whether or not these microbes can modulate human IBD onset and progression is a growing area of research that could potentially lead to novel therapeutic development.

Bacterial dysbiosis (altered bacterial composition) is found in a subset of CD and UC patients. In CD, some patients have decreased bacterial diversity and outgrowths of bacteria not usually dominant in healthy humans (Hansen et al., 2012; Man et al., 2011; Martinez-Medina and

Garcia-Gil, 2014). However, it is uncertain whether these bacterial shifts arise spontaneously in genetically susceptible hosts, were acquired from the environment or are a result of intestinal inflammation. In mouse models, specific bacteria such as Helicobacter hepaticus, Camplylobacter jejuni, Mycobacterium avium paratuberculosis and adherent-invasive Escherichia coli can contribute to intestinal pathogenesis while others such as Bacteroides fragilis, Bifidobacterium breve and Lactobacillus salivarius can protect (Macho Fernandez et al., 2011; Man et al., 2011;

Mazmanian et al., 2008; Natividad et al., 2012). Whether or not these and other pro/anti- colitogenic bacteria are relevant to human IBD is uncertain, but a better understanding of their immunomodulatory potential opens the door to treatment with antibiotics or pre-/probiotics.

Viruses are known to cause acute intestinal diseases such as diarrhea, however their role in

IBD is less clear. Some viruses have been associated with IBD or IBD severity but whether or not they cause disease or are bystanders of the disease is unknown (Powell et al., 1961; Sun et al.,

2011). For example, a recent study surveying the intestinal virome found an expansion and diversification of the virome in IBD patients (Norman et al., 2015). In particular, they found an expansion of Caudovirales bacteriophages - viruses that have the potential to alter bacterial communities – identifying them as potential instigators of disease. More causative roles for viruses in IBD were recently described in mice with defects in CD susceptibility genes (ATG16L1 or IL-

10). ATG16L1 deficient mice have abnormal Paneth cell function only in the presence of MNV

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(Cadwell et al., 2010). Moreover, this MNV-driven Paneth cell defect sensitizes these mice to DSS colitis. In another example, IL-10 deficient mice develop epithelial barrier disruption and inflammation after MNV infection whereas wildtype mice are unaffected (Basic et al., 2014).

These intriguing findings highlight a potential role for viruses in IBD, especially in the context of a genetically susceptible host, but specific mechanisms of pathogenesis need to be defined.

Increased fungal abundance and diversity are found in both UC and CD (Odds et al., 2006;

Ott et al., 2008; Sokol et al., 2016; Standaert-Vitse et al., 2006). Interestingly, CARD9, a signaling molecule downstream of fungi sensing lectin receptors, is a known IBD susceptibility gene

(Beaudoin et al., 2013; Rivas et al., 2011). CARD9 and Dectin-1 deficient mice have defective fungi sensing and are more susceptible to DSS colitis and Citrobacter rodentium induced colitis

(Iliev et al., 2012; Sokol et al., 2013). Increased intestinal pathology was attributed to blunted Th17 and IL-22 responses and to defective regeneration of the epithelium. Fungal regulation of bacterial populations was also shown to be a potential driver of intestinal inflammation as mice treated with anti-fungal agents developed an altered bacterial and fungal community and more severe acute colitis (Qiu et al., 2015; Wheeler et al., 2016). How sensing, regulation and outgrowth of fungi fit into the etiology of IBD remains to be explored.

Remarkably, Helminths can protect from T cell transfer colitis, DSS colitis, TNBS colitis and spontaneous colitis in IL-10 deficient mice (Elliott et al., 2003; Elliott et al., 2004; Hang et al.,

2010; Smith et al., 2007). Many of these protective mechanisms stem from the regulation of immune cell populations such as T cells (described in section 1.3.4). Indeed many human studies suggest helminths may be an effective therapy for some IBD patients (Broadhurst et al., 2010;

Buning et al., 2008; Croese et al., 2006; Summers et al., 2003; Summers et al., 2005). More

33 research is needed to explore which helminth products are protective and the safety of using helminths as human therapeutics.

The protozoa T. musculus was recently described to exacerbate inflammation in a mouse model of colitis, however, no definitive link for protozoa in the onset and progression of human

IBD has been found so far (Chudnovskiy et al., 2016). There are conflicting results on the frequency of protozoa in IBD patients compared to healthy controls and both populations seem to be equally colonized (Cekin et al., 2012; Nagler et al., 1993; Petersen et al., 2013). One report surveying Dientamoeba fragilis and Blastocytosis infections in IBD patients found a correlation between active inflammation and the lack of protozoa, however, it is unclear if protozoa protect from inflammation or are reduced beyond detection during an active inflammatory response

(Petersen et al., 2013). Overall, the role of protozoans in IBD remains severely understudied.

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1.5 Thesis Overview

Rational and Hypothesis

The intestinal immune system is a complex network that simultaneously integrates host genomics and environmental cues to protect from invading species while maintaining tolerance to innocuous antigens. T cells are important drivers of these responses and require close regulation since a failure to react or an over-reaction are equally detrimental to the host. Given the sensitivity of the immune system to changes in the intestinal environment and the growing list of IBD-associated loci, we hypothesize that a microbial factor, Tritrichomonas muris, and a genetic factor of little known function, Nlrc3, can independently regulate intestinal T cell activation and function, leading to the development of exacerbated intestinal pathology.

Specific Aims

1) Determine if T. muris alters the onset and severity of T cell transfer colitis and describe its

influence on intestinal T cell activation, independent of disease. (Chapter 2)

2) Explore a T cell intrinsic role for NLRC3 in T cell differentiation and function during

homeostasis and intestinal disease. (Chapter 3)

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Chapter 2

The Common Mouse Protozoa Tritrichomonas muris alters Mucosal T cell Homeostasis and Colitis Susceptibility

Nichole K Escalante, Paul Lemire, Mayra Cruz Tleugabulova, David Prescott, Arthur Mortha

Catherine J Streutker, Stephen E Girardin, Dana J Philpott*, Thierry Mallevaey*

Journal of Experimental Medicine, November 11th 2016 Volume 213 No. 13 pp2841-2850

(* Co-senior authors)

Author Contributions:

Conceptualization, N.K.E., D.J.P, T.M. and S.E.G; Methodology, N.K.E., A.M., D.J.P., T.M. and

S.E.G.; Formal Analysis, N.K.E.; Investigation, N.K.E., P.L., M.C.T., D.P. and C.J.S.; Writing –

Original Draft, N.K.E.; Writing – Review & Editing, D.J.P. and S.E.G.; Visualization, N.K.E.;

Supervision, D.J.P., T.M. and S.E.G.; Project Administration, N.K.E., D.J.P. and T.M.; Funding

Acquisition, D.J.P. and T.M.

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2.1 Abstract

The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths and viruses. Through co-evolution, mammals and these microbes have developed a symbiosis that is sustained through the host’s continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing

T-cell driven colitis in non-littermate mouse strains, we serendipitously identified that a non- genetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naïve wild-type mice and accelerated colitis in Rag1-/- mice after T cell transfer. Overall, we describe a novel cross- kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research.

2.2 Introduction

Microorganisms within the mammalian gastrointestinal tract have developed complex relationships with the host, such that the host provides a nutrient-rich microbial niche while the microbes provide vitamins, metabolites and other nutrients otherwise inaccessible to the host

(Hooper et al., 2012). In order to maintain this symbiosis, the host has evolved many microbial sensors, such as Toll-like receptors and Nod-like receptors, which stimulate the immune system to secrete factors such as mucous, antimicrobial peptides and IgA, to keep microbes in check

(Caballero and Pamer, 2015; Philpott et al., 2014). In turn, these microbial signals are necessary

37 for the proper development of the mucosal immune system (Honda and Littman, 2012; Hooper et al., 2012).

While it is clear that the presence of certain bacteria (Atarashi et al., 2013; Ivanov et al.,

2009), viruses (Pfeiffer and Virgin, 2016) and helminths (Elliott and Weinstock, 2012) within the microbiota can impact intestinal homeostasis, the potential immune shaping role of other kingdoms, such as Protista, has been less well studied. Protozoa are unicellular eukaryotes known to cause human diseases such as malaria, giardiasis and trichomoniasis (Lindsay et al., 2008). In wild and laboratory mice, several protozoa, have been documented to be disease-causing whereas others, such as Entamoeba muris and Tritrichomonas muris (T. muris), are considered non- pathogenic members of the murine microbiome (Baker, 2008). Recent studies suggest, however, that T. muris and a related organism, Tritrichomonas musculis, have the potential to be pathogenic since the presence of these protists in the gut microbiota promote type 2 immune responses (Howitt et al., 2016) and Th1 inflammation (Chudnovskiy et al., 2016), respectively. These observations suggest that even though murine health is not overtly altered, protozoa colonization has the potential to modify the immune response in disease models.

While analyzing colitis induced using a T cell transfer model in two non-littermate mouse lines, we unexpectedly observed that the exacerbated disease susceptibility of one of the lines was transmissible by co-housing and was dominant in littermates. We further identified the protozoan parasite T. muris to be the pro-colitogenic confounding factor in this T cell transfer model of colitis.

Indeed, mice infected with T. muris developed an increased IFN-ɣ+ CD4 T cell response and accelerated epithelial damage within the colon. We have also found that colonizing mice chronically with isolated T. muris altered the baseline number of Th1 T cells in the mouse intestine.

This work reveals an unexpected critical role of a murine intestinal protozoan parasite in

38 exacerbating colitis. This also highlights the need for monitoring parasite infections in SPF animal facilities and argues for the standard use of littermate controls in all murine research.

2.3 Materials and Methods

Animals

All animals were housed in specific-pathogen-free conditions at the University of Toronto,

Division of Comparative Medicine. Rag1-/- and C57BL/6 mice were purchased from Jackson

Laboratory and subsequently bred in house. Rip2-/- mice were obtained from Dr. Richard A. Flavell

(Yale University School of Medicine) (Kobayashi et al., 2002). Rip2-/-Rag1-/- mice were generated by crossing Rag1-/- mice with in house Rip2-/- mice. All animal studies were approved by the animal care committee, University of Toronto.

T cell transfer colitis

CD4+CD45RBHigh and CD4+CD45RBLow T cells were sorted from T. muris negative

C57BL/6 spleens. 0.5x106 CD4+CD45RBHigh with or without 0.25x106 CD4+CD45RBLow T cells were injected intraperitoneally into 8 week old Rag1-/- or Rip2-/-Rag1-/- mice. Mice were weighed twice a week to obtain an average weekly weight. Mice were sacrificed at 4 weeks post T cell transfer and colons were fixed with 10% formalin and hematoxylin and eosin stained for pathological scoring or used for lamina propria preps and flow cytometry. Colon pathology was scored by a blinded pathologist as previously described (Ostanin et al., 2009).

Tissue preparations

Colon lamina propria cells were isolated as previously described (Goodyear et al., 2014).

Briefly, colons were washed in pre-stripping buffer (HBSS with 5mM DTT, Penicillin,

39

Streptomycin, and FBS) to remove mucous and fecal contents before incubation in epithelial stripping buffer (HBSS with 5mM EDTA, Penicillin, Streptomycin and FBS). Colons were then washed in HBSS + 10mM HBSS, minced and digested in digestion buffer (HBSS with 10mM

HEPES, Penicillin, Streptomycin, 20ug/mL Dnase and 0.2U/mL Liberase TM). Colon pieces were titurated with an 18GA needle, filtered and washed. Spleen and mesenteric lymph nodes were mashed through a filter and cells isolated by centrifugation. Red blood cells were lysed using ammonium chloride.

Flow cytometry

For intracellular cytokine staining, cells were restimulated for 18hrs on plates coated with anti-CD3 and anti-CD28 antibody or for 4hrs with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Brefeldin A and Monensin (Protein transport inhibitor cocktail, eBioscience) were added during the last 4hrs of restimulation. Dead cells were marked using LIVE/DEAD Fixable

Aqua Dead Cell Stain Kit (Molecular Probes) and Fc receptors were blocked using mouse

CD16/CD32 antibody (eBioscience). Surface staining was performed with anti-mouse CD4

(GK1.5), CD45RB (C363-16A) and TCRβ (H57-597) from eBioscience. Cells were fixed and permeabilized using Foxp3/Transcription Factor staining buffer set (eBioscience) prior to staining with anti-mouse IFN-ɣ (XMG1.2), TNFα (MP6-XT22), IL-17A (eBio17B7) and Foxp3 (FJK-16s) from eBioscience. Samples were analyzed on a BD FACSCanto II or BD LSR Fortessa X20.

Colon Explants, ELISAs and Bio-Plex Assay

Proximal colon explants were weighed, rinsed in RPMI and cultured in 500uL complete

RPMI for 18-24 hours as previously described (Hue et al., 2006). Supernatants were centrifuged and used for cytokine measurement by IL-12/IL-23p40 (eBioscience, sensitivity 2pg/mL), IFN-γ

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(eBioscience, sensitivity 15pg/mL) and IL-18 (MBL, sensitivity 12.5pg/mL) ELISA or Bio-Plex

Mouse Cytokine 23-plex Assay (Bio-Rad) according to the manufacturer’s instructions. Results were normalized to tissue weight.

Immunofluorescence staining

Small intestines were collected, formalin fixed and paraffin embedded. Paraffin sections

(5 μm) were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieval was performed in 10 mM Sodium Citrate Buffer, pH 6.0 at 95°C for 20 minutes. Sections were stained with rabbit polyclonal anti-DCLK1 (Abcam: 1:1000) overnight at 4°C followed by staining with

AlexaFluor 647-conjugated anti-rabbit secondary antibody (Molecular Probes, 1:4000), and counterstaining with DAPI (Sigma-Aldrich, 1:10,000). Images were acquired with the Zeiss Axio

Scan Z.1 Slide Scanner. Quantification was performed blind by counting total number of tuft cells per crypt-villus with the Zen Blue 2 software.

Murine Norovirus Infection

MNV CR6 and CW3 stocks and plasmids were generously provided by Dr. Herbert W.

Virgin (Washington University of Medicine). MNV was propagated, quantified and detected by qPCR as previously described (Hwang et al., 2014). Mice were mock infected or infected with 1 x 106 PFU MNV and confirmed to be positive by qPCR (IDEXX Bioresearch or in house as previously described).

Tritrichomonas muris isolation and infection

Cecal contents of Rip2-/-Rag1-/- mice were resuspended in PBS and filtered twice through a 70um cell strainer. Flow through was centrifuged, resuspended and re-filtered twice. Protozoa

41 were double sorted based on size, granularity and violet autofluorescence on a BD Influx (Figure

3a) and visualized under a microscope to confirm protozoa isolation. Eubacteria 16s rDNA levels in sort samples were determined by qPCR as previously described (Robertson et al., 2013a) to measure the reduction of bacterial contamination (Figure 3b). 1x106 protozoa in PBS or protozoa free PBS was orally gavaged into Rag1-/- or C57BL/6 mice. Two weeks post infection mice were confirmed to be T. muris positive and used for T cell transfer colitis or as breeders. T. muris testing was performed by IDEXX Bioresearch, cecal content wet smear and/or by qPCR for the 28s rRNA as previously described (Howitt et al., 2016). For qPCR, fecal DNA was isolated using a

NucleoSpin Soil kit (Macherey-Nagel) and amplified using Power SYBR green mastermix

(Invitrogen). For microscopic visualization, T. muris were fixed with paraformaldehyde, adhered to slides by cytospin and stained with Giemsa.

Statistical analysis

Unpaired Student’s T-tests were performed when two groups were compared. A one-way

ANOVA was used for comparison of more than two groups, followed by a post-hoc Tukey’s multiple comparisons test if needed. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001.

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Figure 3 – Sorting strategy for Tritrichomonas muris.

(A) Filtered and washed protozoa were double FACs sorted based on size, granularity and violet autofluorescence. SSC= sidescatter, FSC= Forward Scatter, TPW = Trigger Pulse Width. T. muris negative, unsorted T. muris positive, post-sort 1 and post-sort 2 samples were visualized for purity by flow cytometry. (B) Sort sample Eubacteria 16s rDNA relative to water was quantified by qPCR. Data are one representative experiment.

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2.4 Results

2.4.1 Rip2-/-Rag1-/- mice have accelerated T cell transfer colitis pathology

With the aim of studying the role of NOD1 and NOD2 signalling in regulating non-T/B cells during colitis we generated a Rip2-/-Rag1-/- mouse line for use in the T cell transfer model of colitis. Indeed, RIP2 is an essential signaling adapter molecule downstream of both NOD1 and

NOD2 (Magalhaes et al., 2011a). After transfer of wildtype CD4+CD45RBHigh naïve T cells, Rip2-

/-Rag1-/- mice rapidly lost weight, compared to non-littermate Rag1-/- mice and were euthanized at week 4 due to their poor health (Figure 4a). Upon examination, Rip2-/-Rag1-/- mice had larger spleens and short, thick colons, consistent with an increase in cellularity and significant colon pathology (Figure 4b-e). Preparation of colon lamina propria cells revealed a decreased frequency but a significant absolute increase in the number of IFN-ɣ+CD4+ T cells (Figure 4f-g). There was also an increased number of TNFα+CD4+ T cells but no increase in IL-17A+CD4+ T cells (Figure

4f-g). Analysis of colon explant cultures revealed an elevated protein level of IFN-ɣ and IL-12/IL-

23p40 (Figure 4h). These results demonstrated that, in comparison to non-littermate Rag1-/- mice,

Rip2-/-Rag1-/- mice had an increased pro-inflammatory response and exacerbated colon pathology after naïve T cell transfer.

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Figure 4 - Non-littermate Rip2-/-Rag1-/- mice develop accelerated colitis.

Non-littermate Rip2-/-Rag1-/- and Rag1-/- mice were injected i.p. with 0.5x106 CD45RBHigh CD4 T cells.

(A) Mice were weighed weekly and the percentage of initial body weight calculated. Data from 4 experiments were pooled, n=15 mice per group. Mean ± SEM is shown with ***p≤ 0.001, ****p≤ 0.0001 using an unpaired student’s T-test. (B) Mice were sacrificed at week 4. Colons were fixed, H & E stained and scored for colitis severity. Data from 3 experiments were pooled. Mean ± SEM is shown with **p≤ 0.01 using an unpaired student’s T-test. (C) Representative spleens and colons were imaged. (D-E) Spleen, colon lamina propria (LP) and mesenteric lymph nodes (MLN) were harvested at week 4 and isolated cells were quantified by flow cytometry. (F-G) Colon cells were restimulated with plate bound anti-CD3/anti-CD28 antibody and analysed by flow cytometry to determine the (F) total and (G) frequency of cytokine positive cells. Data from 3 experiments were pooled with 5-15 mice per group. Mean ± SEM is shown with *p≤ 0.05, **p≤ 0.01 using an unpaired student’s T-test. (H) Proximal colon explants were cultured for 18hrs and supernatants were analysed by ELISA for cytokine production. Data from 2 experiments were pooled with 4-7 samples per group. Mean ± SEM is shown with *p≤ 0.05, ***p≤ 0.001 using an unpaired student’s T-test.

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2.4.2 Rip2-/-Rag1-/- mice are not protected by regulatory cells

Co-transfer of regulatory CD4+CD45RBLow T cells in Rag1-/- mice has been shown to protect mice from CD4+CD45RBHigh mediated colon pathology (Ostanin et al., 2009). Co-transfer of CD4+CD45RBLow T cells into Rip2-/-Rag1-/- mice, however, did not protect the mice from colitis

(Figure 5a). These mice still developed increased cellularity in the spleen and increased CD4+ T cell infiltration of the colon lamina propria and mesenteric lymph node (Figure 6 a-b). Increased numbers of IFN-ɣ+CD4+ T cells and TNFα+CD4+ T cells were also still observed in the colon along with an increase in colon IFN-ɣ and IL-12/IL-23p40 protein (Figure 5b-d and Figure 6 c-d).

Various other cytokines, such as IL-1, IL-6, IL-10, IL-12p70, IL-13, KC, MIP-1 & MIP-2 were also increased in Rip2-/-Rag1-/- colons, regardless of the T cells transferred (Figure 5e). This lack of protection was not due to a lack of CD4+Foxp3+ regulatory T cells (Tregs) as there was a similar ratio of CD4+Foxp3+ to CD4+Foxp3- T cells in both Rag1-/- and Rip2-/-Rag1-/- mice when

CD4+CD45RBLow T cells were co-transferred (Figure 5c). These results suggest that the early increase in pro-inflammatory mediators in the Rip2-/-Rag1-/- mice may be blocking the ability of the CD4+CD45RBLow T cells to suppress inflammation.

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Figure 5 - Non-littermate Rip2-/-Rag1-/- are not protected from pathology by regulatory CD45RBLow T cells. Non-littermate Rip2-/-Rag1-/- and Rag1-/- mice were injected i.p. with 0.5x106 CD45RBHigh with or without 0.25x106 CD45RBLow CD4 T cells and sacrificed at week 4. (A) Colons were fixed, H & E stained and scored for colitis severity. Data from 3 experiments were pooled. Mean ± SEM is shown with ***p≤ 0.001 using a one-way ANOVA and Tukey’s post-hoc analysis. (B-C) Spleen, colon lamina propria (LP) and mesenteric lymph nodes (MLN) were harvested and isolated cells were counted, restimulated with plate bount anti-CD3/anti-CD28 antibody and analysed by flow cytometry to determine (C) tissue Foxp3+ and (B) colon IFN-ɣ+ T cell numbers. Data from 2 experiments were pooled with 4-12 mice per group. Mean ± SEM is shown with **p≤ 0.01, ***p≤ 0.001 using a one- way ANOVA and Tukey’s post-hoc analysis. (D) Proximal colon explants were cultured for 18hrs and supernatants were analysed by ELISA for cytokine production. Data from 2 experiments were pooled with 3-7 samples per group. Mean ± SEM is shown with *p≤ 0.05, **p≤ 0.01 using a one-way ANOVA and Tukey’s post-hoc analysis. (E) Proximal colon explants were cultured for 18hrs and supernatants were analysed by Bio-Plex assay for cytokine production. Data is one representative of 3 experiments with 2 samples per group. *samples below minimum level of detection, # samples above maximal level of detection.

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Figure 6 - Rip2-/-Rag1-/- are not protected from pathology by regulatory CD45RBLow T cells

Non-littermate Rip2-/-Rag1-/- and Rag1-/- mice were injected i.p. with 0.5x106 CD45RBHigh with or without 0.25x106 CD45RBLow CD4 T cells and sacrificed at week 4.

(A-B) Spleen, colon lamina propria (LP) and mesenteric lymph nodes (MLN) were harvested and isolated cells were quantified by flow cytometry. (C-D) Colon LP cells were restimulated with plate bound anti- CD3/anti-CD28 antibody prior to cytokine analysis by flow cytometry. Data from 2 experiments were pooled with 4-12 mice per group. Mean ± SEM is shown with **p≤ 0.01, ***p≤ 0.001 using a one-way ANOVA and Tukey’s post-hoc analysis.

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2.4.3 Exacerbated Rip2-/-Rag1-/- pathology is transmissible

To determine whether the accelerated colitis seen in Rip2-/-Rag1-/- mice was driven by

Rip2 deficiency or was caused by other factors such as a dysbiotic microbiota, Rip2-/-Rag1-/- and

Rag1-/- mice were co-caged at weaning and 5 weeks later injected with CD4+CD45RBHigh T cells. Strikingly, 4 weeks post-transfer, co-caged Rag1-/- mice developed equally accelerated pathology as the Rip2-/-Rag1-/- mice (Figure 7a), suggesting that the disease-inducing factor could be horizontally transferred. Littermate mice were then generated and the resulting Rip2+/+Rag1-/- mice also developed accelerated colitis and had similar pro-inflammatory cytokine levels as their

Rip2-/-Rag1-/- littermates (Figure 7b-c). These results indicate that microbial factors, rather than

Rip2 genotype, was likely driving the exacerbated colitis in Rip2-/-Rag1-/- mice.

To determine the potential pro-colitogenic microbial factors, Rip2-/-Rag1-/- and Rag1-/- non- littermate and littermate mice were screened for viral infections. Interestingly, Rip2-/-Rag1-/- non- littermate and all littermates, regardless of genotype, were positive for MNV infection (IDEXX testing by qPCR), while non-littermate Rag1-/- mice were negative. MNV is commonly found in the gastrointestinal tract of laboratory mice and Rag1-/- mice have been reported to be persistently infected after exposure (Karst et al., 2003). Since previous studies had shown that MNV infection may exacerbate both chemical- and bacterial-induced colitis in mice (Cadwell et al., 2010;

Lencioni et al., 2008), we investigated the hypothesis that MNV may exacerbate colitis in Rag1-/- mice during T cell transfer colitis. To test this, we infected Rag1-/- mice with MNV CW3 or MNV

CR6 and two weeks later compared them to their non-infected littermates during T cell transfer colitis. Although MNV levels were still detectable by qPCR 4 weeks post CD4+CD45RBHigh T cell transfer, no differences were observed in pathology or weight loss, suggesting that MNV infection is not a contributing factor to T cell transfer colitis (Figure 7 d-f).

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Figure 7 - Co-caged and littermate mice develop equally exacerbated T cell induced colitis (A) Co-caged and (B) littermate Rip2-/-Rag1-/- and Rip2+/+Rag1-/- mice were injected i.p. with 0.5x106 CD45RBHigh CD4 T cells and sacrificed at week 4. Colons were fixed, H & E stained and scored for colitis severity. Data from 3 experiments were pooled. Mean ± SEM is shown and an unpaired student’s T-test was performed. (C) Proximal colon explants from week 4 post transfer littermate mice were cultured for 18hrs and supernatants were analysed by ELISA for cytokine production. Data from 2 experiments were pooled with 5-6 samples per group. Mean ± SEM is shown and a Student’s T-test was performed. (D-F) Rag1-/- mice were orally gavaged with 1x106 PFU murine norovirus (MNV) CR6 or MNV CW3 and two weeks later injected i.p. with 0.5x106 CD45RBHigh CD4 T cells. (D) Body weight was monitored and mice were sacrificed at week 4. (E) Colons were fixed, H & E stained and scored for colitis severity. (F) Fecal pellets were confirmed positive by qPCR. Data is from 1 experiment with 3-4 mice per group. Mean ± SEM is shown and a one-way ANOVA was performed.

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2.4.4 T. muris infection exacerbates T cell transfer colitis

Rip2-/-Rag1-/- and Rag1-/- non-littermate mice were also screened for parasitic infection. All mice were found to be negative for Entamoeba muris, Giardia muris and Spironucleus muris.

Notably, however, the original Rip2-/-Rag1-/- mouse line and littermate mice were all found to be positive for the protozoa T. muris while Rag1-/- non-littermates were not. Indeed large numbers of these protozoa could be observed by microscope in the cecal contents of Rip2-/-Rag1-/- but not

Rag1-/- non-littermate mice (Figure 8a). Geimsa staining revealed the characteristic three anterior flagella of T. muris (Figure 8b). qPCR screening of our colony revealed that 18 out of 24 separate mouse strains were T. muris colonized. T. muris is transferred between mice by ingestion of infected fecal pellets containing pseudocysts but transmission between cages is limited when sterile technique is strictly followed (Baker, 2008). Co-caging of mice led to transmission of the parasitic infection by two weeks (data not shown). We next tested the hypothesis that T. muris infection could be the confounding factor that accelerated colitis in our T cell transfer experiments.

T. muris was isolated from the cecal contents of Rip2-/-Rag1-/- mice (Figure 3a) and orally gavaged into Rag1-/- mice. Two weeks later, CD4+CD45RBHigh T cells were transferred into the T. muris infected Rag1-/- mice and their non-infected littermate controls. By 4 weeks post T cell transfer, the T. muris infected mice lost significantly more weight than their mock-challenged siblings (Figure 8c). The T. muris infected mice had significantly increased loss of crypt structure, epithelial damage (p=0.0545) and lymphocyte infiltration (chronic inflammation score) (Figure

8d-f). IFN-ɣ and IL-12/IL-23p40 protein levels were also elevated in the colons of the infected mice while IL-18 was not significantly increased (Figure 8g). Together, these data show that T. muris infection contributed to an elevated Th1 pro-inflammatory environment in the colon that resulted in increased colon pathology during T cell transfer colitis.

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Figure 8 - Tritrichomonas muris accelerates T cell induced colitis. (A) Fresh cecal contents were diluted in phosphate buffered saline and visualized under a microscope at 40x magnification, scale bar 100 µM. (B) T. muris from the cecal contents of Rip2-/-Rag1-/- mice were stained with Giemsa and visualized at 63x magnification. Scale bar 10µM.

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(C-G) T. muris was isolated from the cecum of Rip2-/-Rag1-/- mice and 1x106 protozoans were orally gavaged into Rag1-/- mice. Two weeks later, mice were injected i.p. with 0.5x106 CD45RBHigh CD4 T cells. (C) Body weight was measured and mice were sacrificed at week 4. (D-F) Colons were fixed, H & E stained and scored for colitis severity. Data from 3 experiments were pooled with 12 mice per group. Mean ± SEM is shown with *p≤ 0.05, **p≤ 0.01 using an unpaired Student’s T-test. (G) Proximal colon explants from week 4 post T cell transfer T. muris infected and control mice were cultured for 18hrs and supernatants were analysed by ELISA for cytokine production. Data from 2 experiments were pooled with 9-10 samples per group. Mean ± SEM is shown with **p≤ 0.01 using an unpaired Student’s T-test.

2.4.5 Chronic T. muris infection alters baseline T cell homeostasis

To determine if T. muris could be altering the steady state level of immune activation in the gut of immunocompetent mice during natural infection, we created a line of chronically infected C57BL/6 mice by infecting a pair of C57BL/6 breeders. Baseline T cell numbers and cytokine production in the offspring of these infected mice were compared to pups from non- infected breeders (littermates to the infected breeders). Interestingly, infected mice had increased frequencies and a tendency for numbers (p=0.0617) of IFN-ɣ+ CD4 T cells in their cecal lamina propria (Figure 9a). In contrast, frequencies of IL-17A+ CD4 T cells were decreased in the colon lamina propria (Figure 9b). Rort-Foxp3+ Treg frequencies and numbers were similar between infected and non-infected mice but Rort+Foxp3+ Treg frequencies were decreased (Figure 9a-b).

IFN-ɣ protein levels were below the assay level of detection in cultured colon explant supernatants and IL-18 was not significantly different but interestingly IL-12/IL-23p40 protein was elevated in

T. muris colonized mice (Figure 9c). This baseline increase in IL-12/IL-23p40 protein and IFN-ɣ producing CD4 T cells, accompanied by the decrease in Tregs, indicated a shift to a more pro- inflammatory rather than tolerant mucosal environment in the presence of T. muris.

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Figure 9 - Tritrichomonas muris infection chronically increases cecal IFN-ɣ+ T cells (A-D) C57BL/6 breeders were orally gavaged with 1x106 isolated protozoans. (A) Cecums and (B) colons of infected pups and control pups from non-infected breeders were harvested. Cells were isolated and restimulated for 4hrs with PMA & Ionomycin, in the presence of protein transport inhibitor cocktail, prior to flow cytometry analysis. Data from 2 experiments were pooled with 3-6 mice per group. Mean ± SEM is shown with *p≤ 0.05 using an unpaired Student’s T-test. (C) Proximal colon explants were cultured for 24hrs and supernatants were analysed by ELISA for IL- 12/IL-23p40 and IL-18 protein. Data from 2 experiments were pooled with 6-7 samples per group. Mean ± SEM is shown and an unpaired Student’s T-test was performed. (D) Small intestines were fixed, stained for tufts cells (DCLK1+DAPI+) and quantified. Data from 2 experiments were pooled with 6 mice per group. Mean ± SEM is shown with *p≤ 0.05 using an unpaired Student’s T-test.

2.5 Discussion

While we focused on a Th1 immune response in the large intestine the wide array of cytokines found to be increased in the Rip2-/-Rag1-/- colons during T cell transfer colitis suggested that T. muris may be acting as a general stimulator of the mucosal immune system and not specifically favoring a Th1 response. This would agree with the study from Howitt et al that outlined the induction of a Type 2 response in the small intestine upon T. muris colonization

(Howitt et al., 2016). Interestingly, we also observed a significant increase in Type 2 inducing tuft cells in the small intestines of our chronically infected wildtype mice suggesting T. muris responses may differ in distinct gut regions (Figure 9d). Recently, Chudnovskiy et al reported that acute infection of mice with a related protozoan Tritrichomonas musculis, induced IL-18 driven Th1 and

Th17 responses that altered the outcome of bacterial induced colitis, T cell driven colitis and colorectal tumor formation (Chudnovskiy et al., 2016). While our study also demonstrates increased Th1 responses after protozoa infection, IL-18 and IL-17A levels were not significantly

58 increased. This difference may be the result of chronic versus acute infection or the result of different protozoa. Indeed, the 28s rRNA primers used in our study cannot differentiate between

Tritrichomonas muris and Tritrichomonas musculis. Taken together, these reports are further evidence that T. muris and related intestinal parasites could alter disease outcomes in many other mouse models.

Due to the presence of T. muris in our Rip2-/-Rag1-/- mice we were unable to determine if

NOD1 & 2 signaling could alter colitis onset in the T cell transfer model of colitis. Previous reports have described a pro-colitogenic bacterial composition in Nod2-/- and Rip2-/- mice, however, no details about the colonization of these mice with protozoa were given (Couturier-Maillard et al.,

2013). Although the relationship between T. muris and bacteria within the gut is unknown, the large number of protozoa present in the cecal contents of T. muris colonized mice suggests a likely impact on bacterial composition. Thus, along with searching for potential T. muris specific antigens, the potential of a T. muris induced bacterial dysbiosis could also be explored.

T. muris screening is not standard in animal facilities therefore the current prevalence of T. muris in laboratory mice is unknown. Treatment with metronidazole has been reported to rid mice of T. muris (Howitt et al., 2016; Roach et al., 1988), however, antibiotic treatment also alters bacterial composition, which could have other confounding effects on a given phenotype. Our experience suggests that cross-fostering newborn pups with T. muris free dams is a potential option. While T. muris may now be flagged as a potential confounding factor in intestinal disease models, whether it impacts systemic disease remains to be determined. Moreover, there still remains many other unidentified protozoans or microbes that may also play immunomodulatory roles in mouse models underscoring the importance of proper littermate controlled experiments.

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Pentatrichomonas hominis and Dientamoeba fragilis are two species of Trichomonads known to sporadically colonize the human gastrointestinal tract (Maritz et al., 2014). While these

Trichomonads were originally described to be commensal protists, the correlation between the presence of these organisms in patients presenting gastrointestinal symptoms and the work presented here calls for a re-evaluation of their pathogenicity (Meloni et al., 2011; Stark et al.,

2016). Overall, our findings highlight the need for a better understanding of cross-kingdom interactions between host and protozoa within the gastrointestinal tract and emphasizes the importance of controlling for microbial factors in mouse models of disease using littermate controls.

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Chapter 3

Exploring a Role for NLRC3 in T cells and Mucosal Inflammation

Nichole K Escalante and Dana Philpott

Unpublished Results

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3.1 Abstract

NLRC3 is an understudied member of the NLR family that is highly expressed in mouse and human lymphocytes. Initial in vitro studies on NLRC3 support a negative regulatory role for

NLRC3 in activation of the Jurkat T cell line. However, the contribution of NLRC3 to the function of primary mouse T cells and disease remains unclear. In this chapter NLRC3 deficient mice and mice with a T cell specific deletion of NLRC3 were used to explore a role for NLRC3 in regulating

T cell differentiation and intestinal pathology in several models of colitis. Overall, no regulatory role for NLRC3 was found in CD4 T cells or in chemical, T cell or bacteria induced colitis.

Interestingly though, a T cell intrinsic role for NLRC3 in IL-17A producing CD8 T cells was discovered. This suggests that further studies using CD8 T cell dependent models may help elucidate the function of NLRC3 in T cells.

3.2 Introduction

NLRC3 was initially described as a cytoplasmic NLR, with a typical NBD-LRR structure and high expression in T cells (Conti et al., 2005). Analysis of human and mouse microarray data, supported by in vitro studies with the human CD4 Jurkat T cell line, found that NLRC3 mRNA was rapidly decreased in T cells after anti-CD3 and anti-CD28 stimulation. In line with a negative regulatory role for NLRC3, overexpression of NLRC3 during TCR stimulation led to suppression of NF-kB, NFAT and AP-1 signaling, along with suppression of IL-2 and CD25 mRNA. However, the luciferase assay-based evidence for NF-kB, NFAT and AP-1 suppression must be carefully considered as a subsequent study found NLCR3 overexpression could suppress luciferase assays

62 non-specifically (Ling et al., 2012). Nevertheless, the general expression pattern of NLRC3 in resting and activated T cells supports a role for NLRC3 in negatively regulating T cell activation.

Subsequent studies using mice lacking NLRC3 have not reported any T cell intrinsic defects. Instead, NLRC3 was found to negatively regulate TLR signaling and viral DNA sensing in macrophages through interactions with TRAF6 and STING, respectively (Zhang et al., 2014).

NLRC3 was also recently found to interact with PI3K in intestinal epithelial cells, leading to the suppression of mTOR and regulation of colon cancer (Karki et al., 2016). In this study, mice with epithelial cell specific deletion of NLRC3 developed more severe intestinal damage and an increased pro-inflammatory cytokine response by day 14 of azoxymethane (AOM) and dextran sodium sulfate (DSS) treatment. Interestingly, these studies all report a negative regulatory role for NLRC3 in cellular immunity and intestinal homeostasis.

The gastrointestinal tract houses a large variety T cells that are equipped to suppress unwarranted immune responses or to promote protective responses to invasive pathogens. These responses are tightly controlled by many genetic factors as an imbalanced T cell response may lead to intestinal damage, as evidenced during inflammatory bowel diseases. T cell function in colitis can be probed in many mouse models. Specifically, Th17, Th1 and Foxp3+ CD4 T cells have all been described as essential contributors to intestinal homeostasis and are important for regulating the onset and severity of C. rodentium and T cell-induced colitis (Harbour et al., 2015;

Powrie et al., 1993; Uhlig et al., 2006) (Geddes et al., 2011; Gibson et al., 2008; Higgins et al.,

1999; Lebeis et al., 2007; Mangan et al., 2006; Simmons et al., 2003; Vallance et al., 2002).

Conversely, Tc17, Tc1 and Foxp3+ CD8 T cells, while present, are not essential for disease progression or control in these models (Rubino et al., 2013). As for the DSS model, neither CD4 nor CD8 T cells are necessary for the development or resolution of colitis even though antigen

63 specific effector T cells develop in the colon (Dieleman et al., 1998; Dieleman et al., 1994;

Krieglstein et al., 2002; Morgan et al., 2013). Expansion of Tregs, however, can protect against

DSS colitis by promoting B cell development into IgA producing plasma cells (Chen et al., 2011b;

Wang et al., 2015). In addition, DSS colitis remains a good model for evaluating the contribution of innate factors to maintaining intestinal integrity.

In this study, in vitro and in vivo models of T cell function were utilized to interrogate the function of NLRC3 in T cells and in maintenance of intestinal integrity. Preliminary results suggested a role for NLRC3 in blunting T cell activation and intestinal pathogenesis during C. rodentium infection but further experimentation with properly back-crossed and littermate mice found no conclusive evidence for this hypothesis. Interestingly, however, these studies reveal a potential role for NLRC3 in CD8 T cells.

3.3 Materials and Methods

Animals

Rag1-/- , CD4-CRE, CD45.1 and C57BL/6 mice were purchased from Jackson Laboratory and subsequently bred in house. Foxp3-GFP mice were obtained from Dr. Mohamed Oukka

(University of Washington) and crossed to Nlrc3 -/- mice. Nlrc3 -/- and Nlrc3 Floxed/Floxed mice were obtained from Dr. Philip Rosentiel, backcrossed 10 times to C57BL/6 and confirmed by single nucleotide polymorphism analysis to be more than 95% and 98% C57BL/6, respectively. Nlrc3-/- mice were bred as heterozygotes and 8-9 week old littermates were used for all experiments.

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All animals were housed in specific-pathogen-free conditions at the University of Toronto,

Division of Comparative Medicine and all animal studies were approved by the animal care committee, University of Toronto.

Antibiotic Treatment

Pregnant females were treated for 3 weeks with 1g/L ampicillin, 1g/L metronidazole and

1g/L neomycin in their drinking water. Heterozygote F1 pups were subsequently bred to generate

F2 littermates for experimental use.

T cell Transfer Colitis

T cell transfer colitis was induced as described in section 2.3 using Nlrc3-/- or Nlrc3 +/+ donors and Rag1-/- recipients.

Citrobacter rodentium Infections

Citrobacter rodentium, strain DBS 100, was cultured in LB medium overnight at 37°C.

Bacterial CFUs was calculated post-inoculation by plating on MacConkey agar. Mice were inoculated with 1x109 CFU bacteria in 200uL volume by oral gavage. Fecal pellets were collected at various time points to monitor level of infection. Colon pathology was scored based on the degree of submucosal Edema (0 = no change; 1 = mild; 2 = moderate; 3 = profound), Goblet cell depletion (scored based on numbers of goblet cells per high-power field averaged from five fields at 400x magnification, where 0 ≥50; 1 = 25–50;2 = 10–25; 3 ≤10), Epithelial hyperplasia (scored based on percentage above the height of the control, where 0 = no change; 1 = 1–50%; 2 = 51–

100%; 3 ≥100%), and Epithelial integrity (0 = no change; 1 ≤10 epithelial cells shedding per lesion;

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2 = 11–20 epithelial cells shedding per lesion; 3 = epithelial ulceration; 4 = epithelial ulceration with severe crypt destruction), as previously described (Gibson et al., 2008).

Dextran Sodium Sulfate Colitis

Mice were given 2.5% Dextran Sodium Sulfate in their drinking water ab libitum for 5 days then returned to regular drinking water until day 7 or 14. Mice were weighed daily and fresh

DSS was given every two days. DSS colon pathology was scored based on degree of acute inflammation (0- none, 1 - mild, 2 - moderate, 3 - severe), inflammatory cell depth of infiltration

(1 - mucosa, 2 - submucosa, 3 – transmural) and tissue damage (1 - minor architectural changes/small erosions, 2 - moderate damage, 3 - severe/extensive ulceration and loss of mucosa).

Total scores are reported. Polymononuclear cells per field were also counted and averaged.

Tissue Preparations

Cecum lamina propria, colon lamina propria, spleen, mesenteric lymph node and thymic immune cells were isolated following the protocols described in section 2.3

Colon Swiss rolls colons were fixed with 10% formalin and stained with hematoxylin and eosin for pathological scoring. Colon pathology was scored by a blinded pathologist.

In Vitro T cell Differentiation

T cells were enriched from spleen and lymph node preparations by magnetic bead isolation

(EasySep Mouse T cell Isolation Kit, STEMCELL Technologies) and naïve (CD62LHi CD44Low)

CD4 and CD8 T cells were sorted by flow cytometry on a BD FACS Aria. Cells were stimulated by 2.5ug/mL plate-bound anti-CD3 and anti-CD28 antibody in RPMI containing 10% heat inactivated fetal bovine serum, Penicillin/Streptomycin, L-glutamine, Beta-mercaptoethanol,

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Essential amino acids, Non-essential amino acids and Sodium pyruvate. For differentiation, the following was added to each well: Treg- TGFβ; Th1- IL-12 & anti-IL-4; Th2- IL-4 and anti-IFN-

γ; Th17- TGFβ, IL-6, anti-IFN-γ, anti-IL-4, sometimes including IL-23, IL-21, IL-2 or anti-IL-2.

After 5 days culture, cells were either restimulated with PMA and Ionomycin for analysis by flow cytometry or rested for 2 days. Rested cells were restimulated for an addional 2 days in varying cytokine conditions then restimulated with PMA and Ionomycin for flow cytometry analysis.

Flow Cytometry

For intracellular cytokine staining, cells were restimulated for 5hrs with phorbol 12- myristate 13-acetate (PMA) and ionomycin (Protein Stimulation Cocktail, eBioscience). Brefeldin

A and Monensin (Protein transport inhibitor cocktail, eBioscience) were added during the last 4hrs of restimulation. Dead cells were marked using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit

(Molecular Probes) and Fc receptors were blocked using mouse CD16/CD32 antibody

(eBioscience). Surface staining was performed with anti-mouse CD4 (GK1.5), CD8a (53-6.7),

CD45RB (C363-16A), CD44 (IM7), CD62L (MEL-14) and TCRβ (H57-597) from eBioscience.

Cells were fixed and permeabilized using Foxp3/Transcription Factor staining buffer set

(eBioscience) prior to staining with anti-mouse IFN-ɣ (XMG1.2), IL-17A (eBio17B7), IL-22

(IL22JOP) and Foxp3 (FJK-16s) from eBioscience. Samples were analyzed on a BD FACSCanto

II or BD LSR Fortessa X20.

Colon Explants and ELISAs

Proximal colon explants were performed as described in section 2.3 Supernatants were centrifuged and used for cytokine measurement by IL-22 ELISA according to the manufacturer’s instructions (Sensitivity, 8pg/mL; eBioscience). Results were normalized to tissue weight.

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Real Time Quantitative PCR

Colon samples were flash frozen in liquid nitrogen and stored at -80°C until processing.

Tissues were homogenized in Trizol and isolated RNA was treated with TURBO DNASE and reverse transcribed using SuperScript III First-Strand Synthesis System for RT-PCR (Life

Technologies). cDNA samples were analysed using PowerUp SYBR Green Master Mix (Applied

BioSystems) using a CFX384 Real Time PCR detection system (BioRad). Primers are listed in

Table 3. Results were normalized to RPL19 expression.

Table 3 – List of Primers

Primer Forward Sequence (5’-3’) Reverse Sequence (5’-3’)

IL-17A CTC CAG AAG GCC CTC AGA CTA C AGC TTT CCC TCC GCA TTG ACA CAG

IFN-γ TCA AGT GGC ATA GAT GTG GAA GAA TGG CTC TGC AGG ATT TTC ATG

IL-22 CAC AGA TGT CCG GCT CAT CG CCT GCA TGT AGG GCT GGA AC

IL-22BP TCA GCA GCA AAG ACA GAA GAA AC GTG TCT CCA GCC CAA CTC TCA

KC AGA CCA TGG CTG GGA TTC AC AGT GTG GCT ATG ACT TCG GT

IL-6 TCC AAT GCT CTC CTA ACA GAT AAG CAA GAT GAA TTG GAT GGT CTT G

IL-1β CAA CCA ACA AGT GAT ATT CTC CAT GAT CCA CAC TCT CCA GCT GCA G

REGIIIγ GCT CCT ATT GCT ATG CCT TGT TTA G CAT GGA GGA CAG GAA GGA AGC

RPL19 GCA TCC TCA TGG AGC ACA T CTG GTC AGC CAG GAG CTT

Statistical Analysis

Unpaired Student’s T-tests were performed when two groups were compared except for mixed bone marrow chimera experiments where paired Student’s T-tests were performed. A one-

68 way ANOVA was used for comparison of more than two groups, followed by a post-hoc Tukey’s multiple comparisons test if needed.

3.4 Results

3.4.1 Preliminary evidence suggesting a role for NLRC3 in T cell function

Initial characterization of Nlrc3-/- mice revealed no spontaneous pathology or altered immune cell development in the primary and secondary lymphoid tissues (Motta, unpublished).

These mice had normal CD4/CD8 T cell ratios in the thymus, spleen and mesenteric lymph node, regular frequencies of myeloid cells and developed normal IgM and IgG responses. Contrary to previously published results, Nlrc3-/- macrophages did not have altered cytokine responses to LPS and/or IFN-γ (Zhou, 2014). In fact, full length NLRC3 mRNA and protein was not detected in wildtype bone marrow derived or peritoneal macrophages.

NLRC3 was confirmed to be highly expressed in naïve and memory CD4 and CD8 mouse

T cells (Zhou, 2014). Strikingly, NLRC3 protein was found in both the cytoplasm and nucleus. In line with previously published studies in Jurkat T cell lines, NLRC3 mRNA was rapidly decreased in mouse T cells following activation with anti-CD3/anti-CD28 or Phorbol 12-myristate 13- acetate/Ionomycin. Reduced NLRC3 was also observed at the protein level and the reduction was mediated specifically by stimulation of the T cell receptor, intracellular calcium influx or antagonization of mTOR signaling. This reduction in NLRC3 was maintained for the duration of stimulation and NLRC3 mRNA returned within 1 day of stimulation withdrawal. Although

NLRC3 overexpression reportedly suppressed IL-2 mRNA expression in Jurkat cells, ex-vivo

69 stimulated Nlrc3 -/- CD4 T cells secreted normal amounts of IL-2. Furthermore, ex-vivo stimulated

Nlrc3 -/- mouse CD4 T cells proliferated normally in response to stimulation and developed equivalent levels of activation induced cell death (Motta, unpublished).

Preliminary analysis of T cells within the cecal lamina propria of Nlrc3 -/- mice found elevated levels of CD4+ Foxp3+ regulatory T cells (Figure 10a) (Motta, unpublished). This was specific to the cecum as no differences in Treg cell frequency were noted in the spleen. At baseline, no differences in IL-17A+ or IFN-γ+ CD4, CD8 or γδ T cells were observed in the spleen or cecum lamina propria, however, preliminary results showed blunted IL-17A and IFN-γ T cell responses in the cecum lamina propria after infection with Citrobacter rodentium (Figure 10b). Moreover,

NLRC3 deficient mice were partially protected from C. rodentium induced intestinal pathology

(Figure 10c). These early results prompted further investigation into the regulatory role of NLRC3 in the gastrointestinal tract and lead to the hypothesis that NLRC3 may be regulating the differentiation of T cells into specific effector and regulatory subsets.

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Figure 10 – Preliminary analysis of NLRC3 deficient mice NLRC3 deficient (KO) or wildtype (WT) mice were left untreated (naïve) or infected with C. rodentium for 13 days (infected) prior to T cell analysis. (A)Cecum and Spleen T cells were isolated and analysed by flow cytometry for FOXP3. (B) Cecum lamina propria T cells were isolated, restimulated with PMA and ionomycin for 5 hours and then analyzed by flow cytometry for IFN-γ and IL-17A expression in CD4 and CD8 cells. (C) Colon swiss rolls were fixed and scored for pathology by a blinded pathologist. Results are a combination of 2-3 experiments. Unpaired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown with *p≤ 0.05, ***p≤ 0.001

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3.4.2 NLRC3 does not control Foxp3+ CD4 T cell development

To investigate whether or not NLRC3 deficient mice had increased intestinal Tregs, colon and cecum lamina propria lymphocytes were isolated from naïve Nlrc3 -/- and Nlrc3 +/+ fully back- crossed littermates. Upon comparison, no significant differences in Foxp3+ CD4 T cell frequencies were observed between genotypes in either compartment (Figure 11a). To examine Treg origin and phenotype, the frequency of Tregs expressing Helios and RORγT were compared but also found to be similar (Figure 11b). In support of a normal contribution of thymus derived Tregs to the intestinal compartment, no difference in Foxp3+ Tregs was found in the thymus of Nlrc3 -/- mice

(Figure 11c). Furthermore, differences in Treg frequencies were not masked by differences in cell staining protocols as Nlrc3 -/- mice expressing GFP under the control of the Foxp3 promotor also had normal levels of Tregs (Figure 11d)

Since Treg frequencies within the intestines are sensitive to environmental cues, such as microbial antigens, and these experiments were performed more than a year after the initial preliminary results, mice with an altered microbiota were generated with antibiotics and subjected to Treg analysis. In short, pregnant females were treated for two weeks with an antibiotic cocktail, non-treated F1 pups were inter-bred and the subsequent F2 generation were used for analysis.

Consistent with the above findings, no differences in Treg frequencies were observed in the cecum

(Figure 11e).

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Figure 11 – NLRC3 does not regulate in vivo or in vitro Treg development.

(A-B) Lamina propria lymphocytes from cecum and colon tissues were isolated from untreated littermate mice and CD4 T cells were analysed for Foxp3, Helios and RORγT expression by intracellular staining and flow cytometry. Each dot is one mouse. Thymus cells (C) and cecum lamina propria lymphocytes from Foxp3-GFP mice (D) or F2 littermates from an antibiotic treated line of mice (E) were isolated and analysed for Foxp3 expressing CD4 T cells. (F) Naïve CD4 T cells were isolated and stimulated with plate bound anti-CD3/CD28 for 3 days before Foxp3 analysis. Cells were either stimulated in 2.5ng/mL TGFβ with increasing concentrations of anti-CD3 or stimulated with 2ug/mL anti-CD3 with increasing concentrations of TGFβ. Results are the combination of 1-4 experiments. Unpaired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown

To further evaluate Treg development independent of the intestinal environment, naïve

Nlrc3 -/-or Nlrc3 +/+ CD4 T cells were isolated and differentiated in vitro under Treg polarizing conditions. Foxp3 was induced in an anti-CD3 or TGFβ dose dependent manner, however, no genotype specific differences were observed (Figure 11f). Finally, in an attempt to compare Treg differentiation in a more physiological setting and matched environment, mixed bone marrow chimeras were generated. Briefly, Rag1-/- mice were lethally irradiated and reconstituted with an equal ratio of congenically marked Nlrc3 +/+ (CD45.1) and Nlrc3 -/- (CD45.2) bone marrow. Six weeks post-reconstitution, CD4 or CD8 Foxp3+ T cell frequencies were analyzed in the blood, colon, MLN, spleen and inguinal lymph node. There was a non-significant trend towards increased colonic CD4 Tregs originating from Nlrc3 -/- cells (p=0.1739) but otherwise, equal frequencies of

Tregs developed from Nlrc3 -/- and Nlrc3 +/+ bone marrow in all other compartments (Figure 12a).

No differences in memory or naïve T cell subsets were observed (Figure 12). Overall, no definitive evidence was found to support a T cell intrinsic role for NLRC3 in Foxp3+ CD4 T cell development.

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Figure 12 - NLRC3 deficient cells have normal peripheral T cell distribution

Rag1-/- mice were lethally irradiated and reconstituted with an equal ratio of Nlrc3-/- (CD45.2) and Nlrc3+/+ (CD45.1) bone marrow. 6-8 weeks later, chimera blood, colon, mesenteric lymph nodes (MLN), spleen and inguinal lymph nodes (iLN) were analysed for (A) Foxp3+, (B) naïve, (C) effector memory or (D) central memory CD4 and CD8 T cells. Results are the combination of 2 experiments and each dot represents one mouse. Paired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown.

3.4.3 NLRC3 deficient T cells can efficiently induce colitis and NLRC3 deficient Tregs retain suppressive function in vivo

The T cell transfer model of colitis was used to determine whether NLRC3 deficient T cells could mount an effective, colitis inducing effector response and to test the ability of NLRC3 deficient Tregs to suppress in vivo T cell activation. CD4+ CD45RBHi and CD45RBLow T cells from Nlrc3 -/- or Nlrc3 +/+ mice were isolated and transferred into Rag1-/- mice. Mice were monitored for weight loss and sacrificed at week 8. No differences in weight loss or colon pathology were observed between mice injected with CD45RBHi cells from Nlrc3 -/- or Nlrc3 +/+ donors (Figure 13a-b). As expected, mice co-transferred with CD45RBHi and CD45RBLow T cells developed less weight loss and colon pathology than those with CD45RBHi only T cells, however, there were no genotype specific differences. CD4 T cells and Tregs expanded equally in these mice and analysis of the cecum lamina propria revealed no genotype specific differences in frequencies of IL-17A+, IFN-γ+ or IL-17A+IFN-γ+ CD4 T cells (Figure 13c-e). Overall, Nlrc3 -/- and Nlrc3 +/+ naïve T cells can equivalently induce colitis that can be equally suppressed by Nlrc3 -/- or Nlrc3 +/+

Tregs, respectively.

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Figure 13 – NLRC3 does not influence CD4 T cell function in T cell Transfer Colitis

Nlrc3-/- and Nlrc3+/+ 0.5x 106 CD45RBHi and/or 0.25x 106 CD45RBLow T cells were injected i.p. into Rag1- /- hosts. (A) Body weight was monitored and mice were sacrificed at week 8. (B) Colon pathology was scored by a blinded pathologist. Cecum lamina propria lymphocytes were isolated, restimulated for 5 hour with PMA and ionomycin in the presence of Brefeldin A and monesin and then analysed for the expression of (C) CD4, (D) Foxp3 and (E) cytokines. Results represent 1 experiment and each dot represents one mouse. A one-way ANOVA was performed to determine statistical significance. Mean ± SEM is shown.

3.4.4 T cell intrinsic NLRC3 does not regulate C. rodentium induced pathology but may govern Tc17 differentiation

To investigate a potentially T cell intrinsic role for NLRC3 during C. rodentium infection,

NLRC3 was selectively deleted from CD4 expressing cells, the majority of which are T cells, by crossing Nlrc3 flox/flox mice to mice expressing CRE under the CD4 promoter. CRE+ and CRE – littermates were infected with C. rodentium, fecal colony forming units (CFUs) were monitored and mice were sacrificed at Day 13 to survey disease severity and T cell activation. No differences in fecal pellet CFUs or distal colon pathology were observed (Figure 14a-b). In agreement, no differences in CD4 or CD8 T cell IL-17A or IFN-γ responses were found (Figure 14c). This suggests that T cell intrinsic NLRC3 does not regulate the immune response to C. rodentium.

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Figure 14 – T cell intrinsic NLRC3 does not alter C. rodentium pathology

Nlrc3floxed/floxed x CD4-Cre+ (CRE+) and Nlrc3floxed/floxed x CD4-Cre- (CRE-) mice were infected orally with 2x 109 CFU Citrobacter rodentium. (A) Fecal pellets were collected on specified days and plated on MacConkey agar to determine CFUs. (B) Mice were sacrificed at day 13 and distal colon pathology was assessed by a blinded pathologist. (C) Cecum lamina propria lymphocytes were isolated, restimulated for 5hrs with PMA and ionomycin and then analysed for IL-17A and IFN-γ production by CD4 and CD8 T cells. Results are the combination of 2-3 experiments and each dot represents one mouse. Unpaired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown.

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To broaden the search for NLRC3 regulated cells that could extrinsically regulate T cell responses to C. rodentium, mice deficient for NLRC3 in all cells (Nlrc3 -/-) were re-evaluated. After

13 days of infection, NLRC3 littermates had similar fecal CFUs and intestinal inflammation as measured by Lipocalin 2 (Figure 15a-b). Moreover, their CD4 T cells developed comparable IFN-

γ and IL-17A responses in the cecum (Figure 15c). These results show that under the current microbial environment, NLRC3 does not regulate the response to C. rodentium.

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Figure 15 – Full body NLRC3 deficient mice do not have an altered response to C. rodentium

Nlrc3 -/- and Nlrc3 +/+ were infected orally with 2x 109 CFU Citrobacter rodentium. (A) Fecal pellets were collected on specified days and plated on MacConkey agar to determine bacterial CFUs. (B) Lipocalin2 (LCN2) was measured in the fecal pellet supernatants. (C) On day 13, cecum lamina propria lymphocytes were isolated, restimulated for 5hrs with PMA and ionomycin and then analysed for IL-17A and IFN-γ production by CD4 T cells. (D) F2 mice derived from an antibiotic treated female were similarly analysed for CD4 T cell IL-17A and IFN-γ at day 13 post infection. Results are the combination of 2-3 experiments and each dot represents one mouse. Unpaired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown.

To further explore a T cell intrinsic role for NLRC3 in a comparable intestinal environment, mixed bone marrow chimeras were generated as previously described and then infected with C. rodentium (Figure 16a). No significant differences in T cell chimerism were observed between genotypes (Figure 16b). Cecum and colon CD4 T cells from donors of either genotype developed similar IL-17A and IFN-γ responses (Figure 16c). The same was observed for IFN-γ+ CD8 T cells.

Interestingly though, CD8 T cells from NLRC3 deficient donors had reduced IL-17A+ and IL-

17A+IFN-γ+ fractions in both organs (Figure 16d). These results highlight a CD8 T cell intrinsic role for NLRC3 in promoting Tc17 responses.

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Figure 16 – NLRC3 controls IL-17A production in CD8 T cells.

(A) Rag1-/- mice were lethally irradiated and reconstituted with an equal ratio of Nlrc3-/- (CD45.2) and Nlrc3+/+ (CD45.1) bone marrow. 6-8 weeks later, chimeric mice were infected with 2 x 109 CFU C. rodentium. On day 13, mice were sacrificed and lymphocytes were isolated from the spleen, cecum lamina propria and colon lamina propria. (A) CD4 and CD8 T cell frequencies were analysed. Colon and cecum (C) CD4 and (D) CD8 T cells were restimulated with PMA and ionomycin for 5hours in the presence of Brefeldin a and monesin, prior to cytokine analysis by flow cytometry. Results are the combination of 2 experiments and each dot represents one mouse. Paired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown.

3.4.5 In vitro CD4 T cell differentiation is not regulated by NLRC3

In an attempt to determine specific T cell signaling pathways regulated by NLRC3, naïve

T cells were isolated from Nlrc3 -/- and Nlrc3 +/+ donors and differentiated under various polarizing conditions in vitro. Under Th17 and Th1 conditions, NLRC3 deficient CD4 and CD8 T cells developed equal frequencies of IL-17A and IFN-γ cells (Figure 17a-b). To test the stability of the

Th17/Tc17 cells, Th17 & Tc17 cells were rested for 2 days then restimulated in the presence of

TGFβ and IL-6, IL-23 or no cytokines. IL-17A secretion was evaluated 2 days later, however,

NLRC3 deficient T cells still maintained similar IL-17A levels as wildtype cells (Figure 17c-d).

IL-1β and IL-21 promote commitment to the Th17/Tc17 lineage while IL-2 can suppress their development. Addition of IL-1β and IL-21 or neutralization of IL-2, however, did not lead to any differences in Th17/Tc17 differentiation in Nlrc3 -/- cells compared to wildtype (Figure 18a-b).

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Figure 17 – NLRC3 does not control in vitro T cell differentiation.

Naïve (CD62LHiCD44Low) T cells were isolated from Nlrc3+/+ and Nlrc3+/+ mice. (A) CD4 and (B) CD8 stimulated for 3 days under polarizing conditions, restimulated with PMA and iononmycin and analysed by intracellular flow cytometry for IL-17A and IFN-γ production. (C) CD4 and (D) CD8 T cells were stimulating under polarizing conditions, rested for 2 days, then restimulated in IL-23, no cytokines (none) or TGFβ + IL-6 for 3 days. T cells were then restimulated with PMA and ionomycin with Brefeldin A and Monensin, followed by intracellular cytokine analysis. Results are (A-B) one experiment representative of 3 repeats or (C-D) a combination of 3 experiments. Unpaired Student’s T tests were performed to determine statistical significance. Mean ± SEM is shown.

Finally, Tc17 cells were differentiated in Iscove's modified Dulbecco's medium (IMDM) instead of the commonly used Roswell Park Memorial Institute (RPMI) medium. The high levels of natural aryl hydrocarbon receptor agonists present in IMDM have previously been described to

85 enhance Th17 differentiation (Veldhoen et al., 2009). Although Tc17 differentiation was enhanced in IMDM, it was not altered by the loss of NLRC3, suggesting NLRC3 does not regulate AHR signaling (Figure 18c). Overall, these results show that under typical in vitro Th1 and Th17 conditions, NLRC3 deficient CD4 and CD8 T cells are equally capable of differentiation into IFN-

γ or IL-17A producing cells.

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Figure 18 – NLRC3 does not control Th17 or Tc17 in vitro differentiation

Naïve (CD62LHiCD44Low) T cells were isolated from Nlrc3+/+ and Nlrc3+/+ mice. (A) CD4 and (B) CD8 were stimulated for 3 days in RPMI with the indicated polarizing conditions, restimulated with PMA and iononmycin and analysed by intracellular flow cytometry for IL-17A and IFN-γ production. (C) CD8 T cells were stimulated as described above in IMDM with TGFβ and IL-6 and analysed by flow cytometry. Results are representative of 3 biological replicates.

3.4.6 NLRC3 does not regulate chemically induced colitis

A recent study showed a significant role for NLRC3 in intestinal inflammation in the DSS model of colitis (Karki et al., 2016). We therefore next examined whether NLRC3 played a role in the regulation of intestinal pathology in our animals in this model. Nlrc3 -/- or Nlrc3 +/+ littermate mice were given 2.5% DSS for 5 days in their drinking water, switched to regular water and then evaluated for disease severity and cytokines responses at the peak of inflammation (Day 7) or during the recovery phase (Day 14). No differences in weight loss or intestinal pathology were observed between genotypes (Figure 19a-b & h-i). Furthermore, cytokine levels and polymorphonuclear (PMN) infiltration were not altered by the lack of NLRC3 (Figure 19c-g & j- n). Overall, these results failed to find an immunomodulatory role for NLRC3 in intestinal pathogenesis contrary to the findings of Karki et al.

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Figure 19 – NLRC3 does not regulate the response to DSS induced colitis.

Nlrc3+/+ and Nlrc3-/- were treated ab libitum with 2.5% DSS in their drink water for 5 days then placed on regular water until (A-G) day 7 or (H-N) day 14. (A & H) Mouse weight was monitored. (B & I) Colon weight to length ratios and (C & J) pathology were measured. (D-E, K-L) Colons mRNA was isolated and analysed for various cytokine mRNA expresssions. (F & M) Proximal colon explants were cultured for 24hrs in complete RPMI and supernatant was analysed for IL-22 by ELISA. (G & N) Polymononuclear cells per field were counted and averaged. Results are a combination of 3 experiments with 4-6 mice per group. Unpaired Student’s T tests were performed to determine statistical significance with *p≤ 0.05. Mean ± SEM is shown.

3.5 Discussion

Unlike NOD2, NLRC3 has not been identified in GWAS as an IBD susceptibility gene. In agreement, this work shows that NLRC3 does not play an essential role in the progression of T cell-, bacterial- or chemically-induced models of colitis. These results are surprisingly contrary to what would be expected when taking into consideration the described roles for NLRC3 in limiting

TLR induced inflammation and T cell activation (Conti et al., 2005; Schneider et al., 2012). Both of these studies identified NFκB signaling - an upregulated pro-inflammatory pathway during IBD and experimental colitis - as the target of NLRC3 mediated suppression. Perhaps NLRC3 plays a more subtle role in the in vivo regulation of the NFκB inflammatory response than previously described in vitro. Indeed, since it is an essential pro-inflammatory pathway, NFκB signaling is tightly regulated by many different factors (Atreya et al., 2008).

It was previously reported that Nlrc3 -/- mice develop a heightened pro-inflammatory response and display increased intestinal damage in response to acute AOM/DSS treatment (Karki

90 et al., 2016). This was attributed to NLRC3’s ability to inhibit PI3K, downstream of mTOR signaling and resulting epithelial cell proliferation. Separately, our study found no differences in pro-inflammatory cytokines or intestinal pathogenesis in Nlrc3 -/- mice after acute DSS treatment.

Although DSS and AOM/DSS both initiate intestinal epithelial damage and inflammation, the two models cannot be directly compared as addition of the carcinogen AOM leads to genotoxic changes and the development of colorectal cancer, while acute DSS alone induces resolvable inflammation. In agreement, factors that promote epithelial growth and survival can often promote

AOM/DSS induced colorectal cancer but oppositely could help resolve inflammation driven by

DSS only. Either way, NLRC3’s described role as an epithelial cell intrinsic regulator of the PI3K- mTOR pathway doesn’t seem to contribute to the development or resolution of acute DSS colitis as seen in our study.

In this study, no T cell intrinsic role for NLRC3 was found in the in vivo or in vitro differentiation of CD4 T cells. This is consistent with no described T cell defects in other reports of NLRC3 deficient mice. Preliminary differences in intestinal Treg frequencies and susceptibility to C. rodentium were not reproduced in littermate mice and may have been the result of genotype- independent environmental factors. Although microbial factors were taken into consideration in this study, it remains possible that experimental conditions, in vivo or in vitro, were not optimal for parsing out the role of NLRC3 in CD4 Treg and effector cell differentiation.

Most interestingly, NLRC3 seems to be regulating the in vivo development of IL-17A producing CD8 T cells in response to C. rodentium. Since initial studies describing a role for

NLRC3 in T cells were performed using the human CD4+ Jurkat T cell line, our studies primarily focused on mouse CD4 T cells. Alas, all three of our colitis models are CD8 T cell independent.

As such, a role for NLRC3 in regulating CD8 T cells was unequally assessed and nearly missed.

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IL-17A producing CD8 T cells (Tc17) have only recently been defined as a RORγT controlled subset with similarities to Th17 cells (Happel et al., 2003; Kondo et al., 2009). They can be found at low frequencies, compared to Th17 cells, in human psoriasis or multiple sclerosis and in mouse models of inflammation such as contact hypersensitivity and C. rodentium infection (He et al.,

2006; Kryczek et al., 2008; Rubino et al., 2013; Tzartos et al., 2008). Although Tc17 are induced during C. rodentium infection, they are not sufficient to control the infection in the absence of

Th17 cells and may simply develop as bystanders in the TGFβ and Il-6 rich environment (Rubino et al., 2013). Still, understanding factors that regulate Tc17 differentiation may help further our understanding of Th17 responses and CD8 T cell biology as a whole. Moreover, it is possible that

CD4 T cells are regulated by NLRC3 in a similar way to CD8 T cells but differences in their physiologies amplifies NLRC3’s role in CD8 T cells. Further studies using CD8 T cell dependent models, such as Listeria or viral infections, may provide more insight into the T cell intrinsic role of NLRC3.

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Chapter 4 Overall Discussion and Future Directions

Overall, the development of genetically modified mice has provided researchers with many useful models to study mammalian biology and disease. When using mouse models to study mucosal immunity, however, many factors have to be considered during study design to ensure the reliability and reproducibility of experiments (Macpherson and McCoy, 2015; Stappenbeck and Virgin, 2016). These include but are not limited to housing conditions such as temperature, light cycle and noise levels; food and water sources; mouse age and sex; breeding strategies; and the presence of microbes. All these factors may directly or indirectly alter mouse health and immune responses, independent of genotype. Therefore, it is only after considering these factors that we can develop meaningful conclusions on host-genome-microbiome interactions.

Using littermate mice derived from heterozygote by heterozygote breeding pairs helps to control for many factors such as microbial differences, spontaneous genetic mutations or cage effects that occur in the maintenance of isolated mouse lines. This is supported by evidence showing maternally inherited microbiota or environmental stressors, such as temperature, can alter host phenotype, independent of genotype (Karp, 2012; Moon et al., 2015). Due to the cost and time associated with breeding littermates, co-caging has been proposed as an alternative to littermates, however, it is unclear at what age and for how long mice need to be co-housed to obtain a normalized microbiota. Furthermore, it is uncertain whether this is sufficient to control for early events which occur during the maturation of the microbiota and immune system. For example, early colonization with microbes during a critical window of development was found to have long lasting consequences for systemic iNKT cell and Treg colonization, B cell production of IgE, and intestinal epithelial cell sensitivity to bacterial ligands (Cahenzli et al., 2013; Chassin et al., 2010;

Gollwitzer et al., 2014; Olszak et al., 2012; Scharschmidt et al., 2015). None of these altered

93 immune states were rescued by microbial colonization of adult mice and while some were caused by a debatably unnatural absence of all microbes, more relevantly, others were driven by differences in bacterial diversity. Interestingly, even the microbial colonization status of pregnant dams was found to be important, as it can alter pup innate immune development independent of the maternal transfer of microbes (Gomez de Agüero et al., 2016). Further studies are required to determine whether co-housing is an appropriate control for mouse studies but either way, littermates remain a gold standard.

While it is highly important to normalize the aforementioned variables, it is equally important to be aware of the microbial factors present within the shared microbiota of littermate animals. The reason being that it is possible for genetic differences to be masked by the polarizing effects of certain microbial members. For example, the strong induction of a Th1 response and early onset of colitis in the presence of T. muris makes it difficult to determine whether or not RIP2 plays a role in regulating T cell transfer colitis (Escalante et al., 2016). Similarly, it could be speculated that the immunomodulatory role of other microbes, such as the protective roles of

Segmented Filamentous Bacteria in C. rodentium infection (Ivanov et al., 2009) or Tritrichomonas musculus in Salmonella infection (Chudnovskiy et al., 2016) could also hamper studies in these models. On the other hand, the presence of specific microbes may expose genetic phenotypes, as is the case with ATG16L1 deficient mice that develop Paneth cell defects only when infected with

MNV (Cadwell et al., 2010). This supports the need for selective inclusion of microbial factors in the study of host genetics.

Overall, being aware of the microbial factors present in mouse colonies and how they alter disease models, and controlling for their presence, are critical for the proper interpretation and reproducibility of experimental results. This not only applies to colitis models but also to non-

94 gastrointestinal models, as evidence for systemic effects of the microbiota have also been described. For example, microbial products are required for the maintenance of microglia in the central nervous system and perturbation of the microbiota can alter host susceptibility to diet- induced obesity, autoimmune diabetes and allergy (Cox et al., 2014; Erny et al., 2015; Sun et al.,

2015; Trompette et al., 2014).

This thesis highlights the importance of planning carefully controlled animal experiments and provides further evidence for the need to consider microbial factors in study design. Studies on both RIP2 and NLRC3 were initially hampered by results that were unreproducible when performed with proper littermate controls. In the case of the RIP2 studies, this was resolved by the discovery of the immune modulating protozoa T. muris (Escalante et al., 2016). As for the NLRC3 project, it is unclear if the preliminary results in non-littermate mice, showing increased cecal

Tregs and blunted T cell responses after C. rodentium infection, were solely the result of microbial factors or influenced by mouse genotype. Nevertheless, these studies have challenged us to re- think our experimental approaches and has led to the development of improved study standards for all mouse experiments.

Future directions for the study of T. muris aim at determining how T. muris is directly sensed by the immune system and how T. muris maintains a long lasting relationship with the host.

T. muris can be found closely associated with intestinal epithelial cells or free-floating in the lumen and it is unknown whether T. muris sheds common PAMPS and which pattern recognition receptors would be involved in sensing them (Chudnovskiy et al., 2016; Howitt et al., 2016). T. muris is a flagellated protozoa with no cell wall (Lindsay et al., 2008). As such, it is possible that

T. muris is sensed by the flagellin specific receptors TLR-5 and the NLRC4 inflammasome but less likely that it is sensed by receptors specific for cell wall components such as NOD1 or NOD2.

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Chudnovskiy et al. reported that infection of TLR-5 deficient mice with the related protozoa T. musculus did not result in decreased Th1 or Th17 levels compared to wildtype mice. Similarly, they found no decrease in T cell responses in mice lacking MyD88 specifically in intestinal epithelial cells or macrophages. While these findings are a start, they do not exclude the possibility that non-T cell immune responses to T. musculus may be mediated by TLR signaling. Furthermore,

TLR signaling through MyD88 could occur through other cell types such as dendritic cells or stromal cells.

In addition to cellular components shed from microbes, it is possible that T. muris is sensed through virulence factors or microbial metabolites. For example, the NAIP-NLRC4 and NLRP6 inflammasomes have been described to sense and produce bioactive IL-18 in response to components of the Salmonella type III secretion system and the microbial metabolite taurine, respectively (Levy et al., 2015; Vance, 2015). Pathogenic relatives of T. muris, such as T. vaginalis, require virulence factors for epithelial cell attachment and invasion (Menezes and Tasca,

2016). It is unknown whether T. muris would require similar virulence factors since T. muris displays a more symbiotic relationship with the host and there are no reports of active invasion into the tissue. The close association of T. muris to the epithelial layer, however, could lead to induced alterations in epithelial cell gene expression, similar to that seen with SFB adherence

(Ivanov et al., 2009). In addition, the proximity of T. muris to the epithelial layer would facilitate the rapid diffusion of potentially immune altering T. muris derived metabolites to the epithelial cells and underlying immune cells.

Intriguingly, microbial recognition through taste-chemosensory tuft cells in the gut is a newly described form of intestinal immune regulation (Gerbe et al., 2016; Howitt et al., 2016; von

Moltke et al., 2016). Howitt et al. recently showed that tuft-cell sensing of T. muris could induce

96 a type-2 immune response in the small intestine. It would be interesting to determine which T. muris products specifically mediate this tuft cell response and which tuft cell receptors initiate the taste-chemosensory response. In the future, growth of pure cultures of T. muris would simplify the detection of T. muris produced PAMPs and metabolites, providing further insight into the biology and immunomodulatory potential of T. muris. Furthermore, readouts of other types of inflammatory cytokines or cells, in conjunction with mice deficient in various receptors would help to determine the relevant pathways involved in sensing intestinal Trichomonads.

While our studies found a significant increase in IL-12/IL-23p40 in the colon of T. muris infected mice, Chudnovskiy et al. reported increased IL-18 as the driver for elevated intestinal Th1 responses to T. musculus. In our study, neutralization of IL-12/IL-23p40 and/or infection of IL-

12p40 deficient mice would be necessary to determine whether IL-12/IL-23p40 is indeed a driving factor or simply a result of other upstream factors. In addition, IL-12/IL-23p40 is a subunit of both

IL-12 and IL-23 so it would be interesting to dissect which cytokine is directing the T cell response.

T. musculus infected IL-18 deficient mice did not mount Th1 or Th17 responses, implicating inflammasomes as potential protozoan sensors (Chudnovskiy et al., 2016). While this is possible, one must remember that active IL-18 is regulated at two levels: mRNA transcription and proteolytic cleavage of the pro-form by the inflammasome. Potentially, IL-18 levels in protozoa infected mice are being regulated at the mRNA level rather than by increased activity of the inflammasome. For example, IL-22 regulates IL-18 mRNA and proIL-18 levels in epithelial cells during homeostasis or in response to T. gondii and C. rodentium infection (Muñoz et al., 2015).

Further work is required to parse out the contribution of various immune sensors and pro- inflammatory cytokines in the response to commensal protozoans. It is likely that T. muris, like many other microbes, activate multiple inflammatory pathways that can influence T cell phenotypes.

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T. muris sensing by the host initiates a strong, long-lasting immune response, yet it is not sufficient to clear the infection. How T. muris maintains its niche within the intestinal lumen without inducing pathology is an area worth exploring. It is possible that T. muris’ lack of tissue invasion and contentment living in the intestinal lumen protect it from elimination by the host.

Indeed, many intestinal pathogens, such as C. rodentium, cannot chronically survive in the intestinal lumen in the presence of other microbes and actively invade the tissue to find a more optimal niche (Kamada et al., 2012). The contribution of antimicrobial peptides, mucus and IgA towards the containment of T. muris remain to be explored.

Regulatory T cells are often thought to help promote tolerance to the intestinal microbiota but interestingly the frequency of Tregs is reduced after T. muris infection. In this study we did not test whether co-transfer of CD45RBLow regulatory T cells into T. muris positive Rag1-/- mice could protect from colitis onset. T. muris positive Rip2-/-Rag1-/- were not protected by the regulatory cell population but whether this lack of protection is a consequence of the T. muris infection or the RIP2 deficiency is currently unclear. Experiments with T. muris negative Rip2-/-

Rag1-/- mice would also help answer this question. Importantly, understanding genetic and microbial factors that control the regulatory ability of Tregs during colitis would shed light on why intestinal Tregs, while present in large numbers, are insufficient for the control of inflammation in

IBD patients.

Future directions for the NLRC3 project include examining protozoa free NLRC3 deficient mice. It is unclear when T. muris was introduced to the NLRC3 colony and we don’t know if it could mask genetic differences in non-manipulated mice or during C. rodentium infection and DSS colitis. Preliminary work in our lab suggests that T. muris may interfere with C. rodentium infections. Furthermore, high variability of immune responses to C. rodentium amongst wildtype

98 mice in the NLRC3 colony suggests a confounding microbial factor present in some but not all cages of NLRC3 littermates (Figure 20a-b). Despite this possibility of interference by T. muris, the investigation of T cell intrinsic NLRC3 in the T cell transfer colitis model and mixed bone marrow chimeras were independent of T. muris as the Rag1-/- recipients were T. muris free.

Figure 20 – Wildtype mice from the NLRC3 colony display highly variable T cell responses to C. rodentium infection

Nlrc3 +/+ mice were infected orally with 2x 109 CFU Citrobacter rodentium. On day 13, cecum lamina propria lymphocytes were isolated, restimulated for 5hrs with PMA and ionomycin and then analysed for IL-17A and IFN-γ production by (A) CD4 and (B) CD8 T cells. Results are the combination of 7 experiments. Each dot represents one mouse and each color is one experiment. Mean ± SEM is shown.

No differences in the development and distribution of T cells in steady-state NLRC3 deficient mice were found. This suggests that NLRC3 is not actively maintaining T cell homeostasis in unchallenged mice. Primary activation of T cells in vitro or in vivo was also unperturbed by the absence of NLRC3, suggesting NLRC3 does not set the tone for the immediate events that occur after TCR stimulation or the conditions of the assays were not sensitive enough to detect them. Considering the kinetics of NLRC3 mRNA and protein expression, it is possible

99 that NLRC3 may play a more prominent role in regulating secondary activation of T cells or the resolution of inflammation. Indeed, NLRC3 is rapidly downregulated after TCR stimulation, rendering wildtype T cells indistinguishable from NLRC3 deficient T cells in terms of their

NLRC3 expression. Perhaps NLRC3’s role occurs after TCR engagement is abrogated and NLRC3 expression is returned. Therefore, querying the “off” phase after TCR stimulation or the subsequent reactivation of T cells may be more informative. This could be done by evaluating the contraction of T cell responses in the later phases of C. rodentium infection or by performing secondary infections. Similarly, the duration of cytokine responses and downregulation of activation markers after cessation of T cell stimulation could be evaluated in vitro.

Further investigation into the stability and plasticity of the various T cell subsets would also be worthwhile. In vitro T cell differentiation was not altered in the absence of NLRC3 but maybe T cells would be more sensitive to reactivation in different polarizing conditions in the absence of NLRC3, such as Th1 cells restimulated in Th17 conditions. To begin, determining the expression level of NLRC3 in the different T cell subsets might provide clues to which subsets would depend on NLRC3 for their function or stability.

Expanding the inquiry of NLRC3 to other cell types may also provide further insight into its function. In addition to T cells, NLRC3 is highly expressed in innate lymphoid cells but its function in these cells types is unknown (Figure 21). Due to its low expression, a role for NLRC3 in non-lymphocytes was not highly examined in this thesis. Recent studies, however, have described roles for NLRC3 in both epithelial cells and macrophages (Karki et al., 2016; Schneider et al., 2012; Zhang et al., 2014). Although we could not reproduce some of the macrophage results

(Zhou, 2014), these studies show that low levels of NLRC3 may still be biologically relevant and worth studying.

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Figure 21 – NLRC3 is highly expressed in innate lymphoid cells.

Spleen and lymph node T cells (CD3+) and CD90+CD45.1+CD19-CD3- innate lymphoid cell subsets (CD4+, NKp46+ & NKp46-) were sorted by flow cytometry. Relative mRNA expression of NLRC3 was analysed and graphed in relation to CD3+ cell NLRC3. Data represents 3 biological replicates. Mean ± SEM is shown.

In another direction, exploring the function of NLRC3 binding partners in T cells could help identify novel NLRC3 regulated pathways. Excitingly, NLRC3 was found to bind a wide range of cytoplasmic and nuclear proteins - some whose functions have already been described in other systems (Zhou, 2014). Adding or removing NLRC3 to these known signaling pathways in vitro could reveal a new regulatory role for NLRC3 that could later be applied to a relevant model in vivo. Alternatively, the potential for NLRC3 to have an overlapping role with other NLRs could be explored.

How NLRC3 contributes to human health remains to be discovered. A recent genome wide association study identified NLRC3 as a body mass index (BMI) associated gene. NLRC3 deficient mice, however, do not have altered length, weight gain, food intake, adiposity or insulin resistance after 16 weeks on a high or low fat diet (Escalante, unpublished). That being said, we

101 do not know how the BMI-associated single nucleotide polymorphism alters NLRC3 function.

Perhaps transgenic mice with NLRC3 overexpression might fare differently in the feeding study and in other studies performed in this thesis. In another analysis of human health, investigation of human colorectal cancer samples found that NLRC3 expression was reduced in tumors and its reduction correlated with cancer progression. This parallels the recent study showing increased colorectal cancer progression in NLRC3 deficient mice. NLRC3 has yet to be associated with the progression of intestinal diseases other than colorectal cancer but that does not preclude its potential to control factors that are important for regulating IBD susceptibility.

Overall, the intestinal immune system is a complex environment and it is hard to isolate and study one part without considering the intestinal community as a whole. While studies of the microbiota have flourished in the last decade, understanding how to control and integrate microbial factors into the study of the immune system still remains a challenge. Basic controls are available to help differentiate between microbial and genotype specific effects but researchers, reviewers and editors need to take responsibility for ensuring their implementation. Developing a clear understanding of the individual contributions of specific microbial and genetic factors to intestinal immunity are not only important for the development of targeted therapeutics but also for the further study of their combined contributions to human health.

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