Keywords: MAIT cells, lamina propria, microbiota, riboflavin, colitis, mice Mucosal-associated invariant T (MAIT) cells IBDs: Inflammatory bowel diseases MHC: Major histocompatibility complex Glutathione disulfide (Oxidized GSH Invariant natural killer T cells EAE: Experimental autoimmune encephalomyelitis Intestinal epithelial cells AMPs: Antimicrobial peptides FGF2: Fibroblast growth factor STAT3: Signal transducer and activator Reactive oxygen species LEfSe: Linear discriminant analysis effect size Amplicon sequence variant Integrated Mouse Gut Metagenomic Catalog Ct: Threshold cycle C. difficile: Clostridioides difficile C. perfringens: Clostridium perfringens C. butyricum: Clostridum butyricum PCR: Polymerase chain reaction CFUs: Colony forming units LB: Luria broth GUT LUMEM O2 gradient PPAR-g b-oxidation nucleus O2 Butyrate Obligate anaerobes Facultative anaerobes

Mucosal associated invariant T (MAIT) cells are an evolutionary conserved T cell subset, which recognize riboflavin precursor derivatives presented by MR1. Intestinal bacteria from the Bacteroidetes and Proteobacteria phyla can produce MAIT antigens, suggesting a direct interplay between MAIT cells and the microbiota. In humans, MAIT cells have been implicated in pathologies associated with intestinal dysbiosis such as inflammatory bowel diseases, but the role of MAIT cells in these diseases remains unknown. Increased levels of oxygen and oxidative stress are hallmarks of intestinal inflammation, and riboflavin contributes to bacterial respiration and to oxidative stress resistance, suggesting increased needs for riboflavin during intestinal inflammation. Supporting this hypothesis, bacteria possessing the riboflavin biosynthesis pathway are enriched in Crohn's disease patients.

In the first part of this work, we explored the hypothesis that MAIT cells monitor a bacterial metabolic pathway associated with altered gut ecosystem. We showed that hypoxia disruption in the colon, upon antibiotic treatment or dextran sodium sulfate (DSS)-induced colitis, increased MAIT antigen production by the microbiota. We sequence the 16S rRNA genes from the cecum of antibiotic-and mice, and analyzed expression of the riboflavin pathway genes in the Helicobacter hepaticus model of colitis. Dysanaerobiosis was associated with expansion of Enterobacteriaceae upon vancomycin treatment, and Bacteroidaceae and Enterobacteriaceae in colitic mice. Riboflavin production provided a fitness advantage to Escherichia coli in the inflamed intestine. Both Bacteroidaceae and Enterobacteriaceae can produce high amounts of MAIT antigens in vitro. In the H. hepaticus model of colitis, ribD, which controls MAIT antigen production, was over-expressed by Bacteroidaceae, Clostridiaceae and Enterobacteriaceae during colitis. MAIT antigens crossed the intestinal barrier and induced T cell receptor (TCR) signalling in MAIT cells, which produced the tissue-repair mediator amphiregulin and reduced colitis severity. In the second part of this work, we characterized MAIT cells in the ileal environment, wherein high levels of oxygen have been observed at steady state. A range of bacteria that encode the riboflavin pathway populated the ileum, however ileal MAIT cells were not activated through TCR engagement and expressed distinct phenotypic profiles. We discuss the future studies that will be needed to understand MAIT-microbiota interactions in the ileum. Collectively, in the colon, MAIT cells directly sense and react to changes in bacterial metabolism associated with intestinal inflammation and provide host protection in return. This new hostmicrobiota interaction may explain MAIT cell activation in other pathologies associated with dysbiosis such as colorectal cancer. ABSTRACT Mucosal associated invariant T (MAIT) cells are an evolutionary conserved T cell subset, which recognize riboflavin precursor derivatives presented by MR1. Intestinal bacteria from the

I would like to thank my thesis committee members for all their guidance through this process; their insightful discussions, ideas, and feedbacks that have been absolutely invaluable. Namely I would like to thank Dr. Anna-Maria Lennon, Pr. Gérard Eberl, Pr.

Sophie Caillat-Zucman, and Dr. Stanislas Mondot. Special thanks go to my friends and lab colleagues in Institut Curie who have emotionally and technically supported me to overcome the hardships and complete this work.

I am greatly indebted to my amazing family for their unconditional love, prayers, care and continuous support. All my love goes to my father Naji, my mother Joceline, my sisters Diala and Maria Celesté. Also, I express my thanks to my grandparents for their support and valuable prayers. I undoubtedly could not have done this without you.

Last but not least, I want to express my utmost gratitude towards my closest friends in France and Lebanon who kept encouraging me and giving me hope. Lots of love and big thanks to all of you.

H&E: Hematoxylin and eosin

MyD88: Myeloid differentiation primary response 88 Nlrp6: NOD-like receptor family pyrin domain containing 6

List of illustrations

Introduction

Through an enduring mutualistic partnership, humans have co-evolved with the trillions of microbes that inhabit different sites of our bodies. Host-specific microbiota is a result of this continuous mutual selection between the host and the microbes [START_REF] Ley | Worlds within Worlds: Evolution of the Vertebrate Gut Microbiota[END_REF]). The highest concentration of mutualistic microorganisms is found in the gastrointestinal tract with the colon being colonized with the densest and most metabolically active community [START_REF] Rooks | Gut Microbiota, Metabolites and Host Immunity[END_REF]. Research from the past 20 years has revealed critical functions of the gut microbiota, which is often considered as a new organ system.

In the gut, the microbiota relies on the host environment and nutrients for its survival, and in return provides benefits to the host in many ways. Indeed, commensal bacteria influence host nutrient uptake, food metabolism and support host resistance to invading pathogens [START_REF] Wu | A Systematic Review of the Interaction Between Gut Microbiota and Host Health from a Symbiotic Perspective[END_REF]. In recent years, several studies have revealed that the gut microbiota provides the host with essential metabolites and components for host immune development and regulation (G. Wang et al. 2019;[START_REF] Ahern | Understanding Immune-Microbiota Interactions in the Intestine[END_REF]. There is an increasing awareness that microbial communities can modulate locally the gut immune system, which encounters more antigens than any other part of the body. Changes in the composition or function of the microbiota (dysbiosis) are associated with many pathologies, but how the intestinal immune system monitors such alterations remains poorly understood. This question is of particular importance for mucosal-associated invariant T (MAIT) cells, which are predominantly found in tissues colonized by the microbiota and recognize metabolites broadly produced by many bacteria and fungi.

In this introduction, I will first review what is currently known about MAIT cells. Second, I will describe the interplay between the gut microbiome and the host immune system at steady state and in diseases with a particular focus on the inflammatory bowel diseases (IBDs).

Chapter I: Mucosal associated invariant T cells

MAIT cells represent the most abundant αβT cells recognizing a given bacterial compound. Unlike conventional T cells, which recognize peptide antigens presented by highly polymorphic MHC Class I or MHC Class II molecules, MAIT cells recognize microbial-derived metabolites presented by the MHC-I related protein, MR1.

MR1 genomic organization and transcripts 1.

MR1 is a non-classical member of the MHC class I family of proteins that is encoded by the non-polymorphic Mr1 gene. This Mr1 gene appeared in mammals and is the most highly conserved Mhc gene between human and mouse, with ≈90% identity in coding sequences for the α1 and α2 domains that bind the ligand (S. [START_REF] Huang | MR1 Antigen Presentation to Mucosal-Associated Invariant T Cells Was Highly Conserved in Evolution[END_REF]). In addition, the similarity scores of the α1 and α2 domains of MR1 across mammals show that MR1 sequences have evolved more slowly compared with CD1 or MHC1 (Fig. 1) [START_REF] Boudinot | Restricting Nonclassical MHC Genes Coevolve with TRAV Genes Used by Innate-like T Cells in Mammals[END_REF]. This high evolutionary conservation of MR1 reflects the conserved structure of the antigen presented.

Interestingly, Mr1 has been pseudogenized or lost in carnivores, lagomorphs and the armadillo.

Very interestingly, the TRAV1 TCR gene segment used by MAIT cells (see section 2 below) is absent in all species in which a functional Mr1 gene is also lacking [START_REF] Boudinot | Restricting Nonclassical MHC Genes Coevolve with TRAV Genes Used by Innate-like T Cells in Mammals[END_REF]) (Fig. 2). This observation strongly suggests a co-evolution between Mr1 and TRAV1 and indicates that the main function of MR1 is to present antigens to TRAV1 + cells.

The Mr1 message and encoded protein are expressed in various cell types and tissues. In the steady state without antigen, the majority of MR1 resides in an incompletely folded conformation INTRODUCTION Chapter I: Mucosal associated invariant T cells [START_REF] Bourhis | Antimicrobial activity of mucosal-associated invariant T cells[END_REF] In accordance with the above-mentioned endosomal trafficking of MR1, using anti-MR1 antibodies (clones 26.5 and 12.2), which only recognize MR1 in a folded form, MR1 appears to have limited surface expression and is more readily detectable at the cell surface in the presence of ligand [START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF][START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF]. Despite considerable effort, surface expression of endogenous MR1 remains challenging. Nonetheless, MR1 is likely to be expressed on different human and mouse cells (double positive thymocytes, monocytes, dendritic cells, macrophages, and epithelial cells) that have been shown to activate MAIT cells in vitro in an MR1-dependent manner [START_REF] Gold | Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells[END_REF]Le Bourhis et al. 2010;[START_REF] Ussher | TLR Signaling in Human Antigen-Presenting Cells Regulates MR1-Dependent Activation of MAIT Cells[END_REF][START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF][START_REF] Harriff | Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium Tuberculosis Ligands to MAIT Cells[END_REF]. In Carnivora (Cat, Dog, Panda, Ferret) and Armadillo, the MR1 region is conserved but MR1 is a pseudogene (red sphere). A functional Mr1 is also missing in the rabbit and pika in which the MR1 region has been lost. Chicken and Zebrafish do not have Mr1.

Ligands for MR1 2.

The MR1 binding groove is characterized by an A' pocket and an F' pocket. The A' pocket is lined with aromatic residues that facilitate the binding of small organic metabolites derived from two vitamin B sources: metabolites derivatives from the riboflavin (vitamin B2) biosynthesis pathway that activate MAIT cells, and metabolites derivatives from folate (vitamin B9) that act as competitive inhibitors of MAIT cell activation [START_REF] Kjer-Nielsen | MR1 Presents Microbial Vitamin B Metabolites to MAIT Cells[END_REF][START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF][START_REF] Eckle | Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells[END_REF][START_REF] Kjer-Nielsen | An Overview on the Identification of MAIT Cell Antigens[END_REF]).

MR1 antigens that activate MAIT cells

The MAIT cell activating metabolite antigens are generated during the synthesis of riboflavin inside all plants and fungi and most bacteria. Almost all Bacteroidetes, Fusobacteria and Proteobacteria are able to synthesize riboflavin. However, half of the Firmicutes species and very few Actinobacteria are predicted to be riboflavin producers [START_REF] Magnúsdóttir | Systematic Genome Assessment of B-Vitamin Biosynthesis Suggests Co-Operation among Gut Microbes[END_REF]. To note, animal cells and the non-producing riboflavin microorganisms rely on external riboflavin which they take up using specialized transporter proteins [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF][START_REF] Kanehisa | The KEGG Databases at GenomeNet[END_REF]. The pathways of riboflavin synthesis are identical in eubacteria and plants but different in fungi and archaea [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF][START_REF] Garcia-Angulo | Overlapping Riboflavin Supply Pathways in Bacteria[END_REF].

INTRODUCTION

Chapter I: Mucosal associated invariant T cells [START_REF] Seeley | The role of riboflavin in the formation and disposal of hydrogen peroxide by streptococcus faecalis[END_REF] The next two reactions, the deamination produces 5-amino-6-(5' -phosphoribosylamino)uracil and the reduction produces 5-amino-6-(5' -phosphoribitylamino)uracil. Those steps are catalyzed by a bifunctional pyrimidine deaminase/ reductase enzyme, encoded by the ribD gene.The ribD product undergoes further dephosphorylation giving uracil . The mechanism of dephosphorylation remained unknown until recently. Multiple broad-spectrum hydrolases belonging to the haloacid dehalogenase (HAD) superfamily are able to mediate this dephosphorylation. 5-A-RU is the direct precursor of MAIT cell antigens. It gives rise to lumazine derivatives (Fig. 4, violet) or pyrimidine adducts (5-OP-RU and 5-OE-RU, red) which are formed from the non-enzymatically spontaneous reaction of the unstable 5-A-RU with methylglyoxal or glyoxal, respectively (Fig. 4) [START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF].

Methylygyoxal and glyoxal are produced by several metabolic pathways, in particular glycolysis and therefore can be provided by both prokaryotic and eukaryotic cells.

Next, 5-A-RU condensates with 3,4-dihydroxy-2-butanone-4-phosphate [START_REF] Serriari | Innate mucosal-associated invariant T (MAIT) cells are activated in inflammatory bowel diseases[END_REF] by lumazine synthase encoded by ribE. The condensation leads to 6,7-dimethyl-8-ribityllumazine (RL-6,7-diMe), the riboflavin immediate precursor. The 3,4-DHBP is synthesized from ribulose 5-phophate by the 3,4-DHBP synthase coded by ribB. The final step is the dismutation of 6,7dimethyl-8-ribityllumazine mediated by riboflavin synthase coded by ribC. In this step, riboflavin and 5-A-RU, the substrate of the lumazine synthase reaction, are generated.

All organisms, including animal cells, generally do not use free riboflavin but riboflavin is utilized to synthesize flavin nucleotides; flavin mononucleotide (FMN) and flavine adenine dinucleotide (FAD). FMN is produced by specific phosphorylation of riboflavin by the riboflavin kinase and is then converted to FAD by the action of FAD synthetase or FMN adenylyltransferase in the presence of ATP [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF][START_REF] Garcia-Angulo | Overlapping Riboflavin Supply Pathways in Bacteria[END_REF][START_REF] Kjer-Nielsen | An Overview on the Identification of MAIT Cell Antigens[END_REF]. (KEGG database). Riboflavin biosynthesis proceeds through different enzymatic steps starting from GTP and ribulose 5-phosphate. This pathway is conserved in many species with few variations in enzymes nomenclature. Bacillus gene names are shown in green and E. coli gene names are shown in blue. Note: In fungi and archaea, despite few variations in some reaction's mechanisms [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF], the intermediary compound 5-A-RU is still produced and used for the spontaneous generation of 5-OP-RU and 5-OE-RU.

Figure 4: Riboflavin biosynthesis pathway and formation of MAIT antigens in bacteria

Regulation of riboflavin synthesis 2.1.2.

The details of riboflavin synthesis regulation are still emerging. It occurs at the level of enzyme activity and synthesis and differs in bacteria, yeasts, and fungi. However, the physiological role of these regulations is not known.

In yeasts, different possible feedback regulations of riboflavin biosynthetic enzymes have been observed. FAD and other nucleotides containing an adenylic moiety can allosterically inhibit GTP cyclohydrolase II. In addition, riboflavin has been shown to inhibit the activity of lumazine synthase from Pichia guilliermondii [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF]. In bacteria, production of riboflavin is regulated by the FMN riboswitch (also known as RFN element). This is found upstream several rib operons and monocystronic rib genes. The binding of FMN to riboswitch element (noncoding RNA) of the nascent mRNA causes transcription termination of rib genes. In fact, the conformation of the FMN riboswitch element changes resulting in terminator hairpin formation. In the absence of FMN, the conformation of the FMN element forms an antiterminator structure that allows normal transcription of downstream genes.

By competing RNA structures, the FMN riboswitch attenuates also translation mechanisms by sequestering of the ribosomal binding site (also called the shine-Dalgarno (SD) box) in bacterial mRNA (Fig. 5) [START_REF] Pedrolli | The RibB FMN Riboswitch from Escherichia Coli Operates at the Transcriptional and Translational Level and Regulates Riboflavin Biosynthesis[END_REF][START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF][START_REF] Vitreschak | Regulation of Riboflavin Biosynthesis and Transport Genes in Bacteria by Transcriptional and Translational Attenuation[END_REF]. Thus, riboflavin gene expression levels in some eubacteria but also in few archaea and eukaryotes can be directly coupled with the intracellular availability of FMN. Except for a direct function as a redox cofactor for the Na+ -pumping NADH: quinone oxidoreductase [START_REF] Juárez | Riboflavin Is an Active Redox Cofactor in the Na+-Pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio Cholerae[END_REF]), riboflavin usually does not have direct metabolic functions in the living cell. Indeed, the biological role of riboflavin, in most cases, is connected with the functions of FMN and FAD which are the most important organic cofactors. FMN and FAD bind to proteins, producing flavoproteins (flavoenzymes). It is currently estimated that around 2% of the genes in bacterial and eukaryotic genomes code for flavin-binding proteins [START_REF] Abbas | Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers[END_REF]. Because of their three redox states (oxidized, one-electron reduced or twoelectron reduced) (Fig. 6), flavins are involved in several oxidation-reduction reactions that occur in plants, animals, and bacteria. and in oxidative stress reduction (glutathione reductase, alkyl hydroperoxide reductase). The fumarate reductase uses FAD and allows the strict anaerobe Bacteroides fragilis to grow in the presence of nanomolar concentrations of oxygen (Baughn and Malamy 2004). The glutathione reductase utilizes FAD to catalyze the reduction of glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH), which is a critical molecule in resisting oxidative stress [START_REF] Ashoori | Riboflavin (Vitamin B₂ ) and Oxidative Stress: A Review[END_REF]. The alkyl hydroperoxide reductase, which is overproduced in bacteria following H 2 O 2 treatment or oxygen exposure, requires FAD as cofactor to mediate bacterial protection against oxidative damage [START_REF] Poole | Bacterial Defenses against Oxidants: Mechanistic Features of Cysteine-Based Peroxidases and Their Flavoprotein Reductases[END_REF][START_REF] Rocha | Role of the Alkyl Hydroperoxide Reductase (AhpCF) Gene in Oxidative Stress Defense of the Obligate Anaerobe Bacteroides Fragilis[END_REF]. Taking these implications into consideration, researchers observed that strict anaerobe Clostridioides difficile

INTRODUCTION

Chapter I: Mucosal associated invariant T cells

24

In bacteria, in addition to their role as ubiquitous intracellular cofactors, flavins are involved in extracellular electron transfer mechanisms. For example, the human gut anaerobe Faecalibacterium prausnitzi employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. This mechanism allows F. prausnitzi to survive in the mucosal-luminal interface in the presence of some oxygen [START_REF] Khan | The Gut Anaerobe Faecalibacterium Prausnitzii Uses an Extracellular Electron Shuttle to Grow at Oxic-Anoxic Interphases[END_REF].

Discovery of MAIT antigens 2.1.4.

In 2009, more than 10 years after the discovery of MAIT cells, MAIT ligands were discovered using a combination of methodologies. An important step in identifying MAIT ligands was the discovery that various strains of bacteria and yeasts and not viruses can activate MAIT cells in a MR1-dependent manner [START_REF] Gold | Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells[END_REF]Le Bourhis et al. 2010). It was suggested by Lantz's team that MAIT ligand could be an extremely conserved compound among microbes (Le Bourhis et al. 2010).

Subsequent experiments based on refolded MR1 with the culture supernatant of Salmonella Typhimurium lead to the discovery of several MR1 stimulatory ligands. High resolution mass spectrometry of the ligands eluted from MR1, allowed determination of ligands from the riboflavin biosynthetic pathway: 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 6, [START_REF] Kjer-Nielsen | An Overview on the Identification of MAIT Cell Antigens[END_REF]. In accordance, it was noted that only riboflavin producing microbes can activate MAIT cells [START_REF] Gold | Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells[END_REF]Le Bourhis et al. 2010).

Further mass spectrometry experiments [START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF]) allowed the determination of the most potent organic MAIT antigens identified to date, the 5-OP-RU and 5-OE-RU which are active in the low nanomolar range. However, these metabolites are very unstable in water unless they are stabilized by the covalent bound with K43 of the MR1 groove [START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF]. They have a short half-life in water where they spontaneously cyclize to form weak agonists for MAIT cells (t 1/2 1.5 h, 37 °C) [START_REF] Kjer-Nielsen | MR1 Presents Microbial Vitamin B Metabolites to MAIT Cells[END_REF][START_REF] Mak | Stabilizing Short-Lived Schiff Base Derivatives of 5-Aminouracils That Activate Mucosal-Associated Invariant T Cells[END_REF]. A synthesis was therefore devised to prepare 5-OP-RU in dimethylsulfoxide (DMSO), where it was indefinitely stable in water [START_REF] Mak | Stabilizing Short-Lived Schiff Base Derivatives of 5-Aminouracils That Activate Mucosal-Associated Invariant T Cells[END_REF]. Compared to aqueous solution, 5-OP-RU in DMSO is a potentially useful new reagent for a better understanding of MAIT cell recognition and activation in vitro. Nevertheless, understanding the biology of microbial 5-OP-RU remains hampered by the low stability of free 5-OP-RU in vivo.

To further confirm the major role of 5-A-RU as a precursor for MAIT cell ligands, culture supernatant from Lactococcus lactis (Gram-) or E. coli (Gram+) with individual mutation in genes of riboflavin pathway either upstream or downstream of MAIT antigen production were tested for activation of MAIT cells [START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF][START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF]. Lantz group and McCluskey group showed that MAIT activation is directly dependent upon ribA and ribD/G genes expression and 5-A-RU production by bacteria.

In another series of experiments, it was found that 5-A-RU can react through nonenzymatic reaction with dihydroxyacetone (DHA), another intermediate from glycolysis pathway, to form less potent MAIT antigens [START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF]. This was explained by probably an intracellular conversion of DHA into methylglyoxal, given that DHA was able to react with 5-A-RU in the cells and not in the medium. Besides metabolites from the riboflavin pathway, synthetic small molecules can also bind the MR1 groove and react with the MAIT TCR. These chemical agonists, such as diclofenac, present a 100 000 fold lower ability to activate MAIT cells than 5-OP-RU (Keller et al. 2017).

The discovery of the potent activators of MAIT cells enabled the construction of antigen-loaded MR1 tetramers. The resulting 5-OP-RU:MR1 tetramer (available at the NIH tetramer facility) allowed, in both humans and mice, the identification and study of MAIT cells replacing less specific approaches [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF][START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF].

Interaction between the MR1-antigen complex and MAIT TCR 2.1.5.

In contrast to conventional αβ T cells, MAIT cells express semi-invariant T cell receptors (TCR) made of a non-variable TCR-α chain paired with a β-chain of limited diversity. In humans [START_REF] Xiao | Mucosal-Associated Invariant T Cells Expressing the TRAV1-TRAJ33 Chain Are Present in Pigs[END_REF], the TCR-α chain of MAIT cells is made of the evolutionarily conserved TRAV1 (Vα7.2) and TRAJ33/12/20 (Jα33/12/20) genes, preferentially joined to Vβ2 or Vβ13 (TRBV20 or TRBV6). In mice [START_REF] Tilloy | An Invariant T Cell Receptor Alpha Chain Defines a Novel TAP-Independent Major Histocompatibility Complex Class Ib-Restricted Alpha/Beta T Cell Subpopulation in Mammals[END_REF], MAIT cells express an orthologous TCR-α chain consisting of TRAV1 and TRAJ33, which pairs with TRBV13 and TRBV19 TCR-β chains.

INTRODUCTION

Chapter I: Mucosal associated invariant T cells

26

The interaction is mediated by the evolutionarily conserved CDR3α-encoded Tyrosine at position 95 (Tyr95α) (Fig. 7A and Fig. 8A-B) [START_REF] Corbett | T-Cell Activation by Transitory Neo-Antigens Derived from Distinct Microbial Pathways[END_REF][START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF]2015). Despite the varied TRAJ and TRBV usage in the MAIT TCR repertoire, Tyr95α is conserved making conserved interactions with MAIT antigens [START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF].

Interestingly, a human TCRγδ T cell subset was found recently to exhibit MR1 autoreactivity and to sense with moderate affinity any MR1 ligand able to induce surface MR1 expression.

However, these cells adopt distinct and unusual binding modes to the a3 domain of MR1 (Le [START_REF] Nours | A Class of Γδ T Cell Receptors Recognize the Underside of the Antigen-Presenting Molecule MR1[END_REF]) (Fig. 7B).

MR1 antigens that act as competitive inhibitors of MAIT cell activation

The discovery of MAIT non-stimulatory metabolites was based on interesting observation of MR1 refolded in the presence of cell culture medium RPMI 1640 [START_REF] Kjer-Nielsen | MR1 Presents Microbial Vitamin B Metabolites to MAIT Cells[END_REF]. This observation suggested that RPMI medium contained a ligand that could bind to MR1 in the absence of bacterial culture.

27

MR1 folded in the presence of RPMI medium analyzed by mass spectrometry revealed the 6-Formylpterin (6-FP) [START_REF] Kjer-Nielsen | MR1 Presents Microbial Vitamin B Metabolites to MAIT Cells[END_REF]). The 6-FP is a photo-degradation product derived in ultraviolet light from the folates (vitamin B9) present in the culture medium. Analogs of 6-FP have been synthesized such as acetyl-6-FP and other derivatives of 6-FP [START_REF] Eckle | Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells[END_REF][START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF]. These compounds can form a covalent bond with MR1, specifically a Schiff-base bond between the formyl group and the residue K43 of MR1 [START_REF] Kjer-Nielsen | MR1 Presents Microbial Vitamin B Metabolites to MAIT Cells[END_REF][START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF]2015). Although these compounds up-regulate MR1 cell surface expression [START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF][START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF], they fail to promote MAIT TCR recognition and activation [START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF]2015). Given that the ribityl chain is crucial for MAIT activation through direct TCR contacts [START_REF] Awad | The Molecular Basis Underpinning the Potency and Specificity of MAIT Cell Antigens[END_REF], the ribityl-less 6-FP forms a single hydrogen bond with the TCR Tyr95α and fails to stimulate MAIT cells [START_REF] Eckle | Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells[END_REF]) (Fig. 8C). 6-FP and their analogs are competitive antagonists of MAIT cells [START_REF] Eckle | A Molecular Basis Underpinning the T Cell Receptor Heterogeneity of Mucosal-Associated Invariant T Cells[END_REF][START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF] and they can be used to modulate pharmacologically the MAIT cell activation.

Interestingly, folate is mainly provided in the diet and also can be made by fungi and some prokaryotes in the body. In fact, vitamin B9 biosynthesis pathway is present in nearly all Bacteroidetes genomes as well as in most Fusobacteria and Proteobacteria and in few Firmicutes and Actinobacteria (Revuelta et al. 2018;[START_REF] Magnúsdóttir | Systematic Genome Assessment of B-Vitamin Biosynthesis Suggests Co-Operation among Gut Microbes[END_REF]. However, whether folate-based metabolites are present in biological fluids remains unknown.

Finally, all structures to date have shown that the antigens fill only the A' pocket of recombinant MR1 which contains K43 residue for Schiff base formation. Thus, the F' pocket remains empty suggesting that bigger molecules might occupy both A' and F′ pockets. However, whether these ligands exist, and whether they would be of bacterial or other origins are unknown.

Thymic development of MAIT cells 3.

MAIT cells arise in the thymus from a CD4 + CD8 + double-positive (DP) precursor [START_REF] Martin | Stepwise Development of MAIT Cells in Mouse and Human[END_REF][START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF]. TRAV1 + immature thymocytes undergo positive selection by MR1 expressed at the surface of either DP thymocytes [START_REF] Seach | Double-Positive Thymocytes Select Mucosal-Associated Invariant T Cells[END_REF] or thymic epithelial cells (TECs) [START_REF] Legoux | Molecular Mechanisms of Lineage Decisions in Metabolite-Specific T Cells[END_REF]) (Fig. 9).

Positive selection on hematopoietic cells (Fig. 9A) results in the engagement of SLAM molecules and the activation of the SAP signalling pathway [START_REF] Legoux | Molecular Mechanisms of Lineage Decisions in Metabolite-Specific T Cells[END_REF], which drives expression of the master transcription factor promyelocytic leukemia zinc finger PLZF (also known as zinc finger and BTB domain-containing protein 16, encoded by Zbtb16). PLZF in turn suppresses the naive T cell program and controls the "preset" phenotype of MAIT cells. As for iNKT cells [START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF][START_REF] Savage | The Transcription Factor PLZF Directs the Effector Program of the NKT Cell Lineage[END_REF] Those cells patrol the non-lymphoid tissues for immediate immunity, contrary to mainstream T cells. Although, the development of MAIT cells strongly resembles that of iNKT cells, mature mouse MAIT cells do not display a phenotype of NKT2 cells [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF]Salou et al. 2019). This could result from weaker TCR signals during MAIT cells positive selection, possibly in relation with lower amounts of MR1 as compared to CD1d.

Positive selection on TECs (Fig. 9B) does not engage the SLAM-SAP pathway, and results in the differentiation of 5-OP-RU-specific T cells into naïve conventional-like CD4 + T cells that remain in secondary lymphoid organs [START_REF] Legoux | Molecular Mechanisms of Lineage Decisions in Metabolite-Specific T Cells[END_REF]. Following positive selection on hematopoietic cells [START_REF] Seach | Double-Positive Thymocytes Select Mucosal-Associated Invariant T Cells[END_REF], MAIT cells develop in the thymus through a three-stage process [START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF]. In mice, the stage 1 immature MAIT cells downregulates CD24 and develops into stage 2 CD24 -CD44 -MAIT cells. PLZF expression begins at stage 2 and is highest at stage 3 characterized by the high expression of CD44. The third mature stage differentiates subsequently into Tbet-or RORγt-expressing cell subsets (Salou et al. 2019;[START_REF] Legoux | Molecular Mechanisms of Lineage Decisions in Metabolite-Specific T Cells[END_REF]. The mechanisms controlling MAIT1 versus MAIT17 lineage choice remain unknown.

Ontogeny studies of human MAIT cells have identified three similar stages defined as CD27 - CD161 -, CD27 + CD161 -, and CD27 + CD161 + cells respectively [START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF].

Interestingly, analysis of SAP-deficient patients revealed normal frequency of blood MAIT cells expressing PLZF [START_REF] Martin | Stepwise Development of MAIT Cells in Mouse and Human[END_REF] suggesting the contribution of additional mechanisms in human MAIT cell development. Furthermore, in humans, stage 3 MAIT cells exit the thymus as Tbet + RORγt + cells [START_REF] Leeansyah | Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection[END_REF] whereas mouse MAIT cells express either Tbet or RORγt [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF].

Role of bacterial metabolites in the ontogeny of MAIT cells 4.

In the thymus of germ-free (GF) mice, both immature CD24 + and mature CD44 + MAIT cells are reduced [START_REF] Koay | A Three-Stage Intrathymic Development Pathway for the Mucosal-Associated Invariant T Cell Lineage[END_REF][START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]. As MAIT cells do not recirculate from the periphery to the thymus [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF], MAIT intrathymic development is likely directly controlled by the microbiota. Experiments with GF mice monocolonized with either ΔribD or ΔribE E. coli, showed that 5-A-RU production by bacteria is essential for positive selection and complete thymic maturation and proliferation of MAIT17 cells [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF] (Fig. 9A). These findings indicate that microbial riboflavin derivatives travel from the gut and reach mouse thymus with enough concentration to select MAIT cells. In agreement, results showed that 5-OP-RU applied onto intact skin or delivered by oral gavage is detected shortly after in the thymus [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]. This is due to the nature of 5-OP-RU which is a metabolite and not lipid or peptide antigen.

In the thymus, numbers of both immature and mature MAIT cells increase and reach normal levels 2 weeks after colonization of GF mice with a complex microbial flora [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]). However, these newly generated mature MAIT cells do not populate peripheral tissue when the bacterial colonization occurs in adults [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF][START_REF] Constantinides | MAIT cells are imprinted by the microbiota in early life and promote tissue repair[END_REF].

By contrast, early life 5-OP-RU administration restores MAIT cell development and enables their skin homing [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF].

Importantly, the absence of the microbiota and the colonization of GF mice with 5-OP-RUproducing E. coli had no effect on the number of MAIT1 cells, which was explained by a possible inhibition of the TCR signalling in this subset [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]. A small number of mature and functional MAIT17 cells develop in GF mice, indicating that positive selection still occurs in these mice [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]. It was suggested that MR1, which is required for each stage of MAIT development [START_REF] Legoux | Molecular Mechanisms of Lineage Decisions in Metabolite-Specific T Cells[END_REF], binds unidentified self-ligands to govern the positive selection of MAIT cells.

In humans, Vα7.2+ CD161+ T cells are found in fetal thymus [START_REF] Leeansyah | Acquisition of Innate-like Microbial Reactivity in Mucosal Tissues during Human Fetal MAIT-Cell Development[END_REF]. It is unclear whether they are selected on DP thymocytes expressing endogenous self-antigen(s) presented by MR1, or whether riboflavin metabolites produced by the microbiota of the mother influence MAIT cell development in the fetal thymus. In fact, as shown for other microbial metabolites [START_REF] Agüero | The Maternal Microbiota Drives Early Postnatal Innate Immune Development[END_REF], riboflavin metabolites produced by the microbiota of the mother might be able to reach the thymus.

In contrast to mouse MAIT cells that become antigen-experienced memory cells in the thymus, human mature MAIT cells exit the thymus as "naïve" cells and acquire a memory phenotype and expand in the periphery [START_REF] Youssef | Ontogeny of Human Mucosal-Associated Invariant T Cells and Related T Cell Subsets[END_REF]. This suggests that a final maturation occurs in the periphery likely derived by the commensal microbiota. Due to the human body size, it was suggested that the concentration of microbial metabolites reaching the thymus is not sufficient to affect MAIT cells in the thymus (Legoux, Salou, and Lantz 2020).

Tissue tropism and residency properties of MAIT cells 5.

INTRODUCTION

Chapter I: Mucosal associated invariant T cells

32

In mice, each functional program associated with RORγt versus Tbet expression seems to correlate with a specific tissue location. MAIT1 are preferentially positioned in the spleen, lymph nodes, and liver, while MAIT17 cells are present in mucosal tissues such as skin [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF]) and lung [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF]Salou et al. 2019) (Fig. 9). In the spleen, liver and lung, MAIT cells are resident. They exhibit a tissue residency transcriptomic signature and do not exchange between 5-week-long parabiotic pairs, except for MAIT1 in the lungs being mostly intravascular (Salou et al. 2019). The tissue residency signature of both MAIT1 and MAIT17 is expressed in the thymus suggesting that the tissue tropism is acquired during thymic development (Salou et al. 2019).

In C57BL/6J mice, MAIT cells represent 0.08% in the spleen, 0.6% in the liver, 0.7% in the lamina propria and 3.3 % of T cells in the lung [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF]. However, in Castaneus inbred SPF mouse strain, MAIT cells are 20 times more abundant compared to C57BL/6J strain [START_REF] Cui | Mucosal-Associated Invariant T Cell-Rich Congenic Mouse Strain Allows Functional Evaluation[END_REF]. This increase in MAIT cell frequency results from one CAST genetic factor mapped to the TCRα locus leading to higher frequency of Vα19 transcripts in the thymus and thus an increased generation of the iVα19-Jα33 rearrangements. Accordingly, B6-MAIT CAST congenic mouse strain, bearing the CAST locus associated with high numbers of MAIT cells on a B6 background, was generated by backcrossing the MAIT CAST trait to C57BL/6J mice for more than 10 generations [START_REF] Cui | Mucosal-Associated Invariant T Cell-Rich Congenic Mouse Strain Allows Functional Evaluation[END_REF]. This model facilitates phenotype analysis and functional studies of MAIT cells in mice.

In humans, as in mice, thymic MAIT cells exhibit tissue homing properties given that they express already in the thymus tissue-homing molecules such as CCR5, CCR6, and CXCR6 (Legoux, Salou, and Lantz 2020). In human thymus and in cord blood, the frequency of MAIT cells is very low (0.07% of T cells). MAIT cell frequency increases in the blood during the first 5-6 years of life, to reach 1-8% in adults (Ben [START_REF] Youssef | Ontogeny of Human Mucosal-Associated Invariant T Cells and Related T Cell Subsets[END_REF]. Accordingly in adults, human MAIT cells comprise 2%-4% of T cells in the lung, 20%-50% in the liver, and up to 60% of CD4 -T cells in the jejunum [START_REF] Dusseaux | Human MAIT Cells Are Xenobiotic-Resistant, Tissue-Targeted, CD161hi IL-17-Secreting T Cells[END_REF]Ayako Kurioka et al. 2016). In addition, MAIT cells from human liver (Salou et al. 2019) and buccal mucosa [START_REF] Sobkowiak | Tissue-Resident MAIT Cell Populations in Human Oral Mucosa Exhibit an Activated Profile and Produce IL-17[END_REF]) display tissue resident property compared with the blood (Salou et al. 2019).

However, how resident MAIT cells respond to tissue microenvironment, and how they are affected in different disease conditions are unclear.

MAIT cell response to stimulation 6.

In humans and mice, MAIT cells may be activated either through their TCR, which recognizes a ligand presented on MR1, or by inflammatory cytokines [START_REF] Franciszkiewicz | MHC Class I-Related Molecule, MR1, and Mucosal-Associated Invariant T Cells[END_REF]. MAIT cells are likely activated through TCR triggering during bacterial infection, given that most bacteria can make 5-A-RU [START_REF] Mondot | MAIT, MR1, Microbes and Riboflavin: A Paradigm for the Co-Evolution of Invariant TCRs and Restricting MHCI-like Molecules?[END_REF]. However, TCR-independent MAIT cell activation occurs during viral and non-riboflavin synthesizing bacterial infections and any pro-inflammatory conditions (van [START_REF] Wilgenburg | MAIT Cells Are Activated during Human Viral Infections[END_REF]2018;[START_REF] Paquin-Proulx | MAIT Cells Are Activated in Acute Dengue Virus Infection and after in Vitro Zika Virus Infection[END_REF][START_REF] Loh | Human Mucosal-Associated Invariant T Cells Contribute to Antiviral Influenza Immunity via IL-18-Dependent Activation[END_REF]. Once activated MAIT cells secrete various cytokines and chemokines and alter their cytotoxic granule content and transcription factor expression. However, MAIT cell effector functions vary according to the context of stimulation [START_REF] Hinks | Activation and In Vivo Evolution of the MAIT Cell Transcriptome in Mice and Humans Reveals Tissue Repair Functionality[END_REF][START_REF] Lamichhane | TCRor Cytokine-Activated CD8+ Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]. MAIT cell responses following various activation protocols, at the RNA and/or the protein levels, are developed below.

TCR-independent activation

Resting human and mouse MAIT cells harbor several interleukin-receptors such as IL-18Rα, IL-12Rβ, IL-23R and IL-7Rα and can be activated by corresponding cytokines independent of their TCR [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF][START_REF] Dusseaux | Human MAIT Cells Are Xenobiotic-Resistant, Tissue-Targeted, CD161hi IL-17-Secreting T Cells[END_REF]. Given that IL-15 may trigger IL-18 production, MAIT cells can also be activated by IL-15 (van Wilgenburg et al. 2016). Moreover, type I interferons (IFN-α or -β) and the gut-associated pro-inflammatory cytokine TNF-like protein 1A (TL1A), were found to potentiate cytokine-mediated MAIT cell activation (van [START_REF] Wilgenburg | MAIT Cells Are Activated during Human Viral Infections[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]. The TCR-independent activation of human MAIT cells induces a modest cytokine response dominated by IFN-γ production which is consistent with the INTRODUCTION Chapter I: Mucosal associated invariant T cells 34

TCR mediated activation

In human, in contrast to cytokine stimulation, MAIT TCR triggering results in a rapid, polyfunctional and proinflammatory response [START_REF] Hinks | Activation and In Vivo Evolution of the MAIT Cell Transcriptome in Mice and Humans Reveals Tissue Repair Functionality[END_REF][START_REF] Lamichhane | TCRor Cytokine-Activated CD8+ Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]) (Fig. 10B). Following TCR activation, blood MAIT cells robustly and rapidly upregulate RORγt and Tbet and the transcription of multiple inflammatory cytokines including IL-17A, IL-22, TNF-α, IFN-γ, IL-1B, IL-6, IL-21, and GMCSF [START_REF] Hinks | Activation and In Vivo Evolution of the MAIT Cell Transcriptome in Mice and Humans Reveals Tissue Repair Functionality[END_REF][START_REF] Lamichhane | TCRor Cytokine-Activated CD8+ Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]. At the protein level, TCR stimuli result in production of TNF-α and to a lesser extent IFN-γ by MAIT cells from the blood and the gut [START_REF] Lamichhane | TCRor Cytokine-Activated CD8+ Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]. Although MAIT cell phenotype strongly suggests their potential to secrete IL-17, TCR stimulation does not result in IL-17 secretion. However, in response to phorbol 12-myristate 13acetate (PMA) and ionomycin stimulation, peripheral blood MAIT cells produce IL-17 in addition to TNF-α, IFN-γ and IL-22 [START_REF] Billerbeck | Analysis of CD161 Expression on Human CD8+ T Cells Defines a Distinct Functional Subset with Tissue-Homing Properties[END_REF][START_REF] Dusseaux | Human MAIT Cells Are Xenobiotic-Resistant, Tissue-Targeted, CD161hi IL-17-Secreting T Cells[END_REF]. This suggests that MAIT TCR stimulation alone is insufficient to fully activate MAIT cells and requires costimulation. Co-stimulation may include TLR agonists (Z. [START_REF] Chen | Mucosal-Associated Invariant T-Cell Activation and Accumulation after in Vivo Infection Depends on Microbial Riboflavin Synthesis and Co-Stimulatory Signals[END_REF], which promote MAIT cell activation mainly in an indirect way through the activation of antigen presenting cells [START_REF] Hinks | MAIT Cell Activation and Functions[END_REF]. Moreover, CD28 [START_REF] Turtle | Innate Signals Overcome Acquired TCR Signaling Pathway Regulation and Govern the Fate of Human CD161(Hi) CD8α + Semi-Invariant T Cells[END_REF]) and cytokines (Z. [START_REF] Chen | Mucosal-Associated Invariant T-Cell Activation and Accumulation after in Vivo Infection Depends on Microbial Riboflavin Synthesis and Co-Stimulatory Signals[END_REF][START_REF] Tang | IL-7 Licenses Activation of Human Liver Intrasinusoidal Mucosal-Associated Invariant T Cells[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]) have been shown to maximize MAIT cell activation and to make MAIT cells potent early responders. Indeed, costimulation with IL12, IL-18 and IL-15 markedly increases TNF-α and IFN-γ secretion by MAIT cells from the blood and the gut [START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]. Likewise, increased IFN-γ and IL-17 secretion by blood and liver human MAIT cells was seen following stimulation through TCR and IL-7, as well as IL-1β and IL-23 [START_REF] Tang | IL-7 Licenses Activation of Human Liver Intrasinusoidal Mucosal-Associated Invariant T Cells[END_REF].

In mice, in accordance with the abundance of MAIT17 subset, TCR stimulation of MAIT cells from the spleen using anti-CD3/CD28-coated bead, leads to the production of large amounts of IL-17, lower levels of TNF-α and GM-CSF, but almost no IFN-γ. Notably, murine MAIT cells from the spleen also secrete IL-4 and IL-10 (Cui et al. 2015).

MAIT cells in infectious diseases

Bacterial infections

Given their antigen specificity, their ability to be directly activated by certain cytokines and their rapid polyfunctional response, MAIT cells represent the most abundant T cell subset reacting against bacteria in humans. MAIT cell activation and functions have been addressed in various bacterial infectious diseases, both in humans and mice (reviewed in [START_REF] Salou | MAIT Cells in Infectious Diseases[END_REF].

In various riboflavin-producing bacterially infected patients, the frequency of MAIT cells in the blood is decreased and seems to accumulate at the inflammatory sites [START_REF] Salou | MAIT Cells in Infectious Diseases[END_REF]. The hypothesis of migration toward infected sites is supported by the decreased number of MAIT cells and the alteration in their proliferation and homing characteristics in the blood after Salmonella typhimurium infection (Salerno-Goncalves et al.

2017) or in response to

MAIT cells in non-infectious diseases

Wound

A role for MAIT cells in cutaneous wound healing has been observed in the absence of compensatory responses by γδT cells. Indeed, skin wounds heal faster in MAIT cell-sufficient Tcrd -/-mice as compared to MAIT cell-deficient Mr1 -/-Tcrd -/-mice [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF].

Importantly, topical application of 5-OP-RU MAIT antigen to the skin of WT mice before wounding promotes wound healing [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF]). These findings are corroborated by the results of an in vitro wound-healing assay combining the intestinal epithelial cell line Caco2 with supernatants from human TCR-activated MAIT cells. Interestingly, MR1

blockade abolished the accelerating effect that these supernatants had on the closure of a scratch on Caco2 monolayer [START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF]). These findings confirm the TCR-dependent tissue repair potential of MAIT cells. However, when and under which circumstances MAIT TCR signalling is triggered in vivo is unclear.

Obesity and type 2 diabetes

Similar to several infectious diseases, in obese and/or type 2 diabetes (T2D) patients, MAIT cells dramatically decline in the blood as well as in adipose tissue [START_REF] Magalhaes | Mucosal-Associated Invariant T Cell Alterations in Obese and Type 2 Diabetic Patients[END_REF];

INTRODUCTION Chapter I: Mucosal associated invariant T cells 39
These findings in humans are in line with more recent results in obese mice [START_REF] Toubal | Mucosal-Associated Invariant T Cells Promote Inflammation and Intestinal Dysbiosis Leading to Metabolic Dysfunction during Obesity[END_REF]. Indeed, MAIT cells are decreased in the blood, adipose tissue and ileum of obese mice, associated with increased apoptosis in MAIT cells. Moreover, MAIT cells display an activated phenotype that is associated with elevated pro-inflammatory cytokines in adipose tissue and ileum, lipid and glucose metabolism deregulation and increased gut leakiness in obese mice [START_REF] Toubal | Mucosal-Associated Invariant T Cells Promote Inflammation and Intestinal Dysbiosis Leading to Metabolic Dysfunction during Obesity[END_REF].

Autoimmune diseases

As discussed above, MAIT cell characteristics including responsiveness to inflammatory cytokines, expression of chemokine receptors and their localization at mucosal surface imply that they could be involved in immune-mediated diseases. Numerous reports, mostly in human samples and sometimes in mouse models, have described alterations in the frequency, location, phenotype and/or functions of MAIT cells within various autoimmune pathologies (Fig. 11).

Multiple sclerosis

Three studies reported decreased frequency of MAIT cells in the blood of multiple sclerosis (MS) patients correlating with the clinical activity of the disease. Salou et al showed decreased frequency of blood MAIT cells in the progressive form of the disease as compared to relapsing ones and to healthy controls [START_REF] Salou | Neuropathologic, Phenotypic and Functional Analyses of Mucosal Associated Invariant T Cells in Multiple Sclerosis[END_REF]). Miyazaki et al. observed a dramatic reduction in the frequency of blood MAIT cells in MS patients with active disease, which increases in parallel with clinical recovery [START_REF] Miyazaki | Mucosal-Associated Invariant T Cells Regulate Th1 Response in Multiple Sclerosis[END_REF]. Finally, Willing et al reported a reduction of MAIT cells in the blood of MS patients compared with healthy donors, which significantly correlated with IL-18 serum levels in MS patients [START_REF] Willing | CD8 + MAIT Cells Infiltrate into the CNS and Alterations in Their Blood Frequencies Correlate with IL-18 Serum Levels in Multiple Sclerosis[END_REF]).

In addition to CCR5 expressed at steady-state, blood MAIT cells from MS patients upregulate the expression of homing molecules involved in migration across the blood-brain barrier such as VLA-4 [START_REF] Willing | CD8 + MAIT Cells Infiltrate into the CNS and Alterations in Their Blood Frequencies Correlate with IL-18 Serum Levels in Multiple Sclerosis[END_REF], and the homing receptors P-selectin glycoprotein ligand 1 and αL integrin [START_REF] Salou | Neuropathologic, Phenotypic and Functional Analyses of Mucosal Associated Invariant T Cells in Multiple Sclerosis[END_REF]. Consistent with these results, MAIT cells are found by immunohistochemistry within the inflamed central nervous system (CNS) lesions from MS patients, while they are almost absent in the cerebrospinal fluid [START_REF] Willing | CD8 + MAIT Cells Infiltrate into the CNS and Alterations in Their Blood Frequencies Correlate with IL-18 Serum Levels in Multiple Sclerosis[END_REF][START_REF] Salou | Neuropathologic, Phenotypic and Functional Analyses of Mucosal Associated Invariant T Cells in Multiple Sclerosis[END_REF]. Over-expression of MR1 transcripts as well as IL-18 and IL-23 in MS lesions, suggests that infiltrating MAIT cells could be activated in situ in CNS lesions of MS patients [START_REF] Salou | Neuropathologic, Phenotypic and Functional Analyses of Mucosal Associated Invariant T Cells in Multiple Sclerosis[END_REF]). However, the role of MAIT cells in the inflamed brain remains to be addressed. and IL-17) and increased production of IL-10 in the draining lymph nodes and spleen [START_REF] Croxford | Invariant V(Alpha)19i T Cells Regulate Autoimmune Inflammation[END_REF].

Type 1 diabetes

MAIT cell are decreased and altered in the blood of children with recent-onset type 1 diabetes (T1D) as compared to age-matched control children [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF]. Indeed, blood MAIT expression of CD25 and PD-1 and production of TNF-α, IL-4 and granzyme B are increased in children with recent onset T1D [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF].

The non-obese diabetic (NOD) mouse Mr1 -/-mice allowed to directly investigate the role of MAIT cells in T1D. Importantly, Mr1 -/-NOD mice have accelerated diabetes and increased gut permeability to FITC-Dextran after gavage [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF]. Even though MAIT cells infiltrate in the pancreas and could contribute to islet destruction in diabetic stage [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF], the exacerbation of diabetes development in Mr1 -/-NOD mice suggested a protective role of MAIT cells against diabetes. Accordingly, MAIT cells were found enriched in the ileum at the prediabetic stage associated with increased level of IL-17 and IL-22 mRNA in MAIT cells, suggesting a capacity for regulating intestinal epithelium tissue integrity [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF]. Of note, a protective role of IL-17 and IL-22 has been established in several intestinal inflammation models as discussed below.

Further work is needed to determine whether impaired functions of MAIT cells triggers loss of gut integrity, which would increase MAIT cell activation in the pancreas probably due to the translocation of bacterial compounds from the gut to pancreatic lymph nodes.

INTRODUCTION

Chapter I: Mucosal associated invariant T cells 41

Inflammatory bowel diseases

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory conditions of the gastrointestinal (GI) tract. In contrast to CD, which can affect any part of the GI tract, UC is characterized by inflammation that is usually limited to the colon and/or the rectum.

The exact etiology of IBD has not yet been fully understood. The prevailing hypothesis is that IBD is a result of inappropriate immune response to antigens from the intestinal lumen in genetically susceptible individuals. In fact, IBD is associated with a breach in the intestinal barrier [START_REF] Michielan | Intestinal Permeability in Inflammatory Bowel Disease: Pathogenesis, Clinical Evaluation, and Therapy of Leaky Gut[END_REF], which results in the development of innate and adaptive immune responses targeting commensal antigens [START_REF] Shen | Enhanced CBir1-Specific Innate and Adaptive Immune Responses in Crohn's Disease[END_REF]. Furthermore, it is the dysregulated intestinal immune responses to these organisms that result in chronic intestinal inflammation. Indeed, IBD patients exhibit reduced numbers of Treg cells in the blood and gut mucosa and increased numbers of peripheral Th17 cells [START_REF] Li | Expression of Foxp3 MRNA in Peripheral Blood Monocytes of Patients with Ulcerative Colitis[END_REF][START_REF] Boden | Regulatory T Cells in Inflammatory Bowel Disease[END_REF][START_REF] Eastaff-Leung | Foxp3+ Regulatory T Cells, Th17 Effector Cells, and Cytokine Environment in Inflammatory Bowel Disease[END_REF]. Given that Treg cells are extremely important for intestinal homeostasis through mainly the release of IL-10 and TGF-β, dysfunction of Treg cells may play a crucial role in the development of IBD lesions [START_REF] Ishimaru | Development of Inflammatory Bowel Disease in Long-Evans Cinnamon Rats Based on CD4+CD25+Foxp3+ Regulatory T Cell Dysfunction[END_REF]. Antigen presenting cells and effector T cells secrete cytokines that have a complex role in the development of IBD. Elevated levels of TNF-α, IL-12, IL-18, IL-23, IL-1 and IL-6 are detected in IBD and were identified to have important role in the pathogenesis of the disease [START_REF] Neurath | Cytokines in Inflammatory Bowel Disease[END_REF]. Accordingly, targeting IL-12, IL-23 or TNF-α alleviates symptoms in IBD patients [START_REF] Sandborn | Ustekinumab Induction and Maintenance Therapy in Refractory Crohn's Disease[END_REF][START_REF] Sands | Efficacy and Safety of MEDI2070, an Antibody Against Interleukin 23, in Patients With Moderate to Severe Crohn's Disease: A Phase 2a Study[END_REF][START_REF] Lichtenstein | Comprehensive Review: Antitumor Necrosis Factor Agents in Inflammatory Bowel Disease and Factors Implicated in Treatment Response[END_REF]. Other cytokines such as IL-17 and IL-22 are also increased in IBD [START_REF] Fujino | Increased Expression of Interleukin 17 in Inflammatory Bowel Disease[END_REF][START_REF] Kobayashi | IL23 Differentially Regulates the Th1/Th17 Balance in Ulcerative Colitis and Crohn's Disease[END_REF][START_REF] Andoh | Interleukin-22, a Member of the IL-10 Subfamily, Induces Inflammatory Responses in Colonic Subepithelial Myofibroblasts[END_REF][START_REF] Brand | IL-22 Is Increased in Active Crohn's Disease and Promotes Proinflammatory Gene Expression and Intestinal Epithelial Cell Migration[END_REF].

While IL-17 is mostly a pro-inflammatory cytokine that provides a protective inflammatory response towards pathogens, it has been shown to be protective in several models of intestinal inflammation [START_REF] Eyerich | IL-17 and IL-22 in Immunity: Driving Protection and Pathology[END_REF]. Clinical trials designed to block IL-17 signalling using anti-IL-17A or anti-IL-17RA antibodies actually worsened inflammation in patients with IBD [START_REF] Hueber | Secukinumab, a Human Anti-IL-17A Monoclonal Antibody, for Moderate to Severe Crohn's Disease: Unexpected Results of a Randomised, Double-Blind Placebo-Controlled Trial[END_REF]). These results were in line with the effect of IL-17

blockade in mouse models of colitis [START_REF] O'connor | A Protective Function for Interleukin 17A in T Cell-Mediated Intestinal Inflammation[END_REF][START_REF] Ogawa | Neutralization of Interleukin-17 Aggravates Dextran Sulfate Sodium-Induced Colitis in Mice[END_REF][START_REF] Song | Growth Factor FGF2 Cooperates with Interleukin-17 to Repair Intestinal Epithelial Damage[END_REF][START_REF] Maxwell | Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation[END_REF]. Subsequent studies revealed that IL-17 preserves the intestinal epithelial

INTRODUCTION

Chapter I: Mucosal associated invariant T cells 42 barrier through the increase expression of claudin 1 and claudin 2, that are involved in the formation of tight junctions between T84 cell line [START_REF] Kinugasa | Claudins Regulate the Intestinal Barrier in Response to Immune Mediators[END_REF], and by enhancing the epithelial release of antimicrobial peptides (AMPs) [START_REF] Maxwell | Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation[END_REF] during epithelial injury.

Moreover, IL-17 cooperates with the growth factor FGF2 to induce genes in mouse primary intestinal epithelial cells to repair damaged intestinal epithelium [START_REF] Song | Growth Factor FGF2 Cooperates with Interleukin-17 to Repair Intestinal Epithelial Damage[END_REF].

Likewise, the function of IL-22 is difficult to generalize and continue to be under further investigation [START_REF] Sonnenberg | Border Patrol: Regulation of Immunity, Inflammation and Tissue Homeostasis at Barrier Surfaces by IL-22[END_REF]. Some studies have reported that IL-22 exerts proinflammatory role after Toxoplasma gondii infection and in mouse models of arthritis and psoriasis (Y. [START_REF] Zheng | Interleukin-22, a T(H)17 Cytokine, Mediates IL-23-Induced Dermal Inflammation and Acanthosis[END_REF]Ma et al. 2008, 17;[START_REF] Geboes | Proinflammatory Role of the Th17 Cytokine Interleukin-22 in Collagen-Induced Arthritis in C57BL/6 Mice[END_REF][START_REF] Muñoz | Interleukin (IL)-23 Mediates Toxoplasma Gondii-Induced Immunopathology in the Gut via Matrixmetalloproteinase-2 and IL-22 but Independent of IL-17[END_REF]).

In contrast, other studies in mouse models of IBD, have demonstrated tissue-protective properties of IL-22. Indeed, IL-22 activates STAT3 in intestinal epithelial cells (IEC) and therefore promotes IEC proliferation and migration [START_REF] Brand | IL-22 Is Increased in Active Crohn's Disease and Promotes Proinflammatory Gene Expression and Intestinal Epithelial Cell Migration[END_REF]) and the production of mucin which is one of the major components in the intestinal mucus [START_REF] Sugimoto | IL-22 Ameliorates Intestinal Inflammation in a Mouse Model of Ulcerative Colitis[END_REF]).

These findings suggest important functions of IL-22 in mucosal healing and in preserving the mucus layer to prevent bacterial invasion. Like IL-17, IL-22 induces the production of AMPs by IEC and thus limits bacterial replication and dissemination. Accordingly, IL-22 attenuates the intestinal inflammation in mice (Sugimoto et al. 2008, 22), while blockade of IL-22 signalling using anti-IL-22 antibodies or in IL-22 knockout mice worsens colonic injuries [START_REF] Cl | IL-22-Producing Neutrophils Contribute to Antimicrobial Defense and Restitution of Colonic Epithelial Integrity during Colitis[END_REF].

Interestingly, IL-17-and TNF-α-producing MAIT cells are significantly increased in the blood of CD patients accompanied by a decreased IFN-γ secretion. In addition, IL-22 producing MAIT cells trend toward increasing in UC [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF][START_REF] Haga | MAIT Cells Are Activated and Accumulated in the Inflamed Mucosa of Ulcerative Colitis[END_REF][START_REF] Tominaga | Possible Involvement of Mucosal-Associated Invariant T Cells in the Progression of Inflammatory Bowel Diseases[END_REF]). In the blood of IBD patients, MAIT cells identified as CD161hi Vα7.2+ CD3+ are significantly reduced and accumulate in the inflamed intestinal mucosa [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF][START_REF] Haga | MAIT Cells Are Activated and Accumulated in the Inflamed Mucosa of Ulcerative Colitis[END_REF][START_REF] Tominaga | Possible Involvement of Mucosal-Associated Invariant T Cells in the Progression of Inflammatory Bowel Diseases[END_REF]). These findings, together with the increased proportion of cycling MAIT cells in the blood of IBD patients [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF] and their expression of CD69, NKG2D or BTLA, suggest a chronic state of activation of MAIT cells in IBD patients [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF][START_REF] Haga | MAIT Cells Are Activated and Accumulated in the Inflamed Mucosa of Ulcerative Colitis[END_REF][START_REF] Tominaga | Possible Involvement of Mucosal-Associated Invariant T Cells in the Progression of Inflammatory Bowel Diseases[END_REF]) (Fig. 11). However, studies are required to address the question of the role of MAIT cells upon intestinal inflammation. In terms of bacterial composition, the healthy human fecal microbiome is composed of five dominant bacterial phyla. Strict anaerobic bacteria from the phyla Firmicutes and Bacteroidetes are the most relatively abundant while Actinobacteria, Proteobacteria and Verrumicrobia constitute a minority in healthy individuals [START_REF] Tap | Towards the Human Intestinal Microbiota Phylogenetic Core[END_REF]). To note, the low abundance of the facultative anaerobic Proteobacteria is indicative of a healthy gut microbiota [START_REF] Hollister | Compositional and Functional Features of the Gastrointestinal Microbiome and Their Effects on Human Health[END_REF].

As in humans, Firmicutes and Bacteroidetes are the two most abundant bacterial phyla in mice feces [START_REF] Gu | Bacterial Community Mapping of the Mouse Gastrointestinal Tract[END_REF]. Even though this general profile remains constant, clear differences can be observed between the murine and human gut microbiota when exploring deeper taxonomic classifications [START_REF] Nguyen | How Informative Is the Mouse for Human Gut Microbiota Research?[END_REF]. Despite that, mouse models remain a powerful tool for studying gut-microbiota-associated diseases [START_REF] Nguyen | How Informative Is the Mouse for Human Gut Microbiota Research?[END_REF].

Several factors influence the composition and functionality of the microbiota within the gut. The abundance of oxygen is considered to be one of the most important forces by which the host shapes the microbiota in the gut. Indeed, intestinal oxygen is regulated by the host and altered in many situations. Evidences for oxygen to shape the configuration of the microbiome are developed below.

Role of oxygen in shaping the microbiota 2.

From birth to adulthood

It is well established that the postnatal gut environment is different from the mature adult gut environment. Notably, the neonatal gut is characterized by a high abundance of oxygen while mature adult gut is almost completely hypoxic [START_REF] Sanidad | Neonatal Gut Microbiome and Immunity[END_REF]. Accordingly, immediately after birth, the neonatal gut becomes dominated by the facultative anaerobes Enterobacteriaceae and Lactobacillaceae in both human [START_REF] Chu | Maturation of the Infant Microbiome Community Structure and Function across Multiple Body Sites and in Relation to Mode of Delivery[END_REF][START_REF] Combellick | Differences in the Fecal Microbiota of Neonates Born at Home or in the Hospital[END_REF]) and mouse [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF]. The expansion of the facultative anaerobes leads to a gradual consumption of oxygen, which create a new environment suitable for the 45 colonization by the obligate anaerobes Bifidobacteriaceae and Clostridiaceae originating from the milk (Sanidad and Zeng 2020) (Fig. 11). The introduction of solid foods at weaning initiates the fermentation of indigestible dietary fibers in the gut, which leads to the production of short-chain fatty acids (SCFAs) like butyrate.

Butyrate serves as the main carbon source of colonocytes and its metabolism has a direct bearing on the bioavailability of oxygen in the gut lumen. Indeed, butyrate binds to the intracellular receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) which is synthesized at high levels in differentiated colonocytes at the tip of the villi. Butyrate-induced PPAR-γ signalling drives the energy metabolism of colonocytes towards mitochondrial fatty acids β-oxidation. In this process, high amounts of oxygen are consumed through oxidative phosphorylation, making the top of the intestinal crypts hypoxic. This ensures limited oxygen diffusion from the mucosal surface, and creates an environment suitable for obligate anaerobes (Fig. 12) (Litvak, Byndloss, and Bäumler 2018).

INTRODUCTION

Chapter II: Intestinal microbiota and interactions with the host 46 Over time, the decrease in the luminal oxygenation allows obligate anaerobic species to expand and outnumber facultative bacteria. Within 3 years after birth, infant fecal microbiota reaches a mature adult-like profile [START_REF] Yatsunenko | Human Gut Microbiome Viewed across Age and Geography[END_REF], characterized by preponderance of the strict anaerobes Lachnospiraceae and Ruminococcacea (from the phyla Firmicutes), and Bacteroidaceae, Prevotellaceae and Rikenellaceae (from the phyla Bacteroidetes), with low abundance of Actinobacteria, Proteobacteria and Verrumicrobia [START_REF] Tap | Towards the Human Intestinal Microbiota Phylogenetic Core[END_REF][START_REF] Sanidad | Neonatal Gut Microbiome and Immunity[END_REF] Zeng 2020) (Fig. 11).

Thus, the regulation of oxygen availability enables the host to control the ecological niche of the colon. The establishment of hypoxic conditions selects for anaerobic bacteria equipped with metabolic pathways for the fermentation of fibers, a feature beneficial to the host. By contrast, aerobic Proteobacteria can represent opportunistic pathogens, because they can strive in oxygen-rich host tissues in the gut and systemically.

Along the longitudinal axis of the adult gut

The small intestine is subject to pronounced fluctuations of the partial pressure of oxygen (pO2) associated with ingestion of nutrients. However, in contrast to the colonic epithelium, which favors oxidative metabolism of butyrate, small intestinal epithelial cells are more flexible and use glucose and glutamine as metabolic substrates (L. [START_REF] Zheng | Physiologic Hypoxia and Oxygen Homeostasis in the Healthy Intestine. A Review in the Theme: Cellular Responses to Hypoxia[END_REF].

Accordingly, Guanglong He et al., by Electron Paramagnetic Resonance (EPR) imaging, measured a steep oxygen gradient within the murine intestinal tract going from 32 mmHg in the duodenum to deeply anaerobic conditions (3 mmHg) in the distal colon [START_REF] He | Noninvasive Measurement of Anatomic Structure and Intraluminal Oxygenation in the Gastrointestinal Tract of Living Mice with Spatial and Spectral EPR Imaging[END_REF]). The hypoxia-inducible factor (HIF)-luciferase reporter mouse has also enabled the study of oxygen availability along the gut [START_REF] Safran | Mouse Model for Noninvasive Imaging of HIF Prolyl Hydroxylase Activity: Assessment of an Oral Agent That Stimulates Erythropoietin Production[END_REF]). In fact, the transcription factor HIF is the major cellular mechanism coordinating the transcriptional response to hypoxic conditions [START_REF] Papandreou | HIF-1 Mediates Adaptation to Hypoxia by Actively Downregulating Mitochondrial Oxygen Consumption[END_REF]). This oxygen-sensitive transcription factor is rapidly degraded in the presence of oxygen. Fusing luciferase to HIF enabled visualization of the proximal-to-distal gradient of oxygen along the length of the gut (L. Zheng, Kelly, and Colgan 2015) (Fig. 13).

48

The high oxygen availability of small intestine limits bacterial load and selects for the facultative anaerobes. Indeed, the small intestine is enriched in Proteobacteria (mainly E. coli), Streptococcus spp. and Lactobacillaceae as well as Bacteroidetes and Lachnospiraceae in human [START_REF] Zoetendal | The Human Small Intestinal Microbiota Is Driven by Rapid Uptake and Conversion of Simple Carbohydrates[END_REF][START_REF] Pereira | Microbial Nutrient Niches in the Gut[END_REF] and Lactobacillaceae, Bacteroidales and Desulfovibrionaceae in mice [START_REF] Gu | Bacterial Community Mapping of the Mouse Gastrointestinal Tract[END_REF][START_REF] Pereira | Microbial Nutrient Niches in the Gut[END_REF]. In contrast, in the large intestine, where less oxygen is available, microbiota is dominated by the strict anaerobes and is almost similar to fecal microbiota [START_REF] Gu | Bacterial Community Mapping of the Mouse Gastrointestinal Tract[END_REF][START_REF] Yasuda | Biogeography of the Intestinal Mucosal and Lumenal Microbiome in the Rhesus Macaque[END_REF]) (Fig. 14).

Along the cross-sectional axis of the adult gut

Beside the gradient of oxygen along the longitudinal axis of the gut, oxygen drops along the radial axis from the highly vascular lamina propria to the lumen (L. [START_REF] Zheng | Physiologic Hypoxia and Oxygen Homeostasis in the Healthy Intestine. A Review in the Theme: Cellular Responses to Hypoxia[END_REF]. Indeed, using the phosphorescence quenching method, the pO2 of mouse cecal tissue was found to be ∼40 mmHg, while in the cecal lumen pO2 was <1 mmHg [START_REF] Albenberg | Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of Intestinal Microbiota in Humans and Mice[END_REF]). The higher oxygen concentration in the gut tissue compared to the lumen, enables oxygen-tolerant organism's adherence to the mucosal surface. Compared to the lumen community, the mucosa is enriched in Proteobacteria in humans [START_REF] Albenberg | Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of Intestinal Microbiota in Humans and Mice[END_REF]) and macaques [START_REF] Yasuda | Biogeography of the Intestinal Mucosal and Lumenal Microbiome in the Rhesus Macaque[END_REF], and in Lactobacillus in mice [START_REF] Hill | Metagenomic Analyses Reveal Antibiotic-Induced Temporal and Spatial Changes in Intestinal Microbiota with Associated Alterations in Immune Cell Homeostasis[END_REF]) (Fig. 14). The consumption of host-derived oxygen by the facultative anaerobes close to the mucosa contributes to the steep gradient of oxygen that decreases in concentration from tissue to lumen (L. [START_REF] Zheng | Physiologic Hypoxia and Oxygen Homeostasis in the Healthy Intestine. A Review in the Theme: Cellular Responses to Hypoxia[END_REF].

At the oxic-anoxic interface near the intestinal epithelium, oxygen influx from the epithelium reacts directly with hydrogen sulfide (H 2 S) generated by luminal bacteria, producing H 2 O 2 (Imlay 2019) (Fig. 15). Accordingly, in humans, the mucosally adherent microbiota is enriched in genes required for resistance to reactive oxygen species (ROS) relative to the microbiota in the feces [START_REF] Albenberg | Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of Intestinal Microbiota in Humans and Mice[END_REF]. Moreover, the riboflavin biosynthesis pathway, which is required for the regeneration of reduced glutathione, were found enriched in the macaque mucosal community [START_REF] Yasuda | Biogeography of the Intestinal Mucosal and Lumenal Microbiome in the Rhesus Macaque[END_REF]. This was identified using LEfSe analysis [START_REF] Segata | Metagenomic Biomarker Discovery and Explanation[END_REF]) on the community metabolic pathways inferred by PICRUSt. PICRUSt for "phylogenetic investigation of communities by reconstruction of unobserved states", is a computational approach that serves to predict the metabolic potential of the microbiota communities, based on 16S rRNA microbial composition and a database of reference genomes [START_REF] Langille | Predictive Functional Profiling of Microbial Communities Using 16S RRNA Marker Gene Sequences[END_REF].

Upon antibiotic treatment

Antibiotic treatment targeting fermenting microbes such as Clostridia or Bacteroides depletes butyrate and thus silences epithelial PPAR-γ signalling. As a result, antibiotic-mediated depletion of butyrate shifts the colonocytes metabolism toward anaerobic glycolysis (fermentation of glucose). This metabolic reprogramming of colonocytes conserves oxygen within the cell, resulting in the loss of epithelial hypoxia and in increased amounts of oxygen emanating from the mucosal surface. Because oxygen can freely diffuse across biological membranes, increased oxygenation of the epithelium is predicted to increase the luminal bioavailability of oxygen, and the expansion of facultative anaerobic bacteria (Litvak, Byndloss, and Bäumler 2018). This scenario was supported by the decreased butyrate levels, increased epithelial oxygenation and increased abundance of endogenous E. coli in the colon of mice treated with streptomycin [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF]. Streptomycin is an aminoglycoside antibiotic, and causes a marked depletion of Clostridia which include abundant butyrate-producers, specifically Lachnospiraceae and Ruminococcaceae [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF].

INTRODUCTION

Chapter II: Intestinal microbiota and interactions with the host 51

The expansion of E. coli upon streptomycin treatment required the cydAB gene, which encodes for cytochrome bd oxidase and is essential for E. coli aerobic respiration. Interestingly, hypoxia restoration through tributyrin (analogue of butyrate having delayed absorption in the colon) supplementation abrogated the fitness advantage conferred by aerobic respiration in streptomycin-treated mice [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF]).

Upon intestinal inflammation

Two phenomena contribute to increased luminal oxygen concentrations during chronic intestinal inflammation. First, epithelial injury leads to mucosal bleeding and to the direct release of haemoglobin carrying oxygen within the luminal environment in the inflamed tissue (Rigottier-Gois 2013). Second, oxygen consumption by the inflamed epithelium is reduced (Litvak, Byndloss, and Bäumler 2018). In fact, the epithelial repair responses to injury drive an accumulation of dividing transit-amplifying epithelial cells, thereby causing colonic crypt hyperplasia, a common feature of ulcerative colitis. Consequently, a reduction in the density of terminally differentiated epithelial cells, such as goblet cells, and a concomitant thinning of the adherent mucus layer are observed in ulcerative colitis patients [START_REF] Strugala | Thickness and Continuity of the Adherent Colonic Mucus Barrier in Active and Quiescent Ulcerative Colitis and Crohn's Disease[END_REF]. Because PPAR-γ is only expressed in terminally differentiated colonocytes, the accumulation of undifferentiated colonocytes in ulcerative colitis patients reduces mitochondrial β-oxidation of butyrate to CO 2 . Dividing transit-amplifying cells convert glucose into lactate, a metabolism characterized by low oxygen consumption. This shift in colon epithelial metabolism reduces epithelial oxygen consumption, leading to increased amounts of oxygen emanating from the colonic surface (Litvak, Byndloss, and Bäumler 2018) (Fig. 16).

The dextran sodium sulfate (DSS)-induced colitis mouse model recapitulates several changes observed in intestinal tissues from patients with ulcerative colitis. DSS treatment triggers colitis in mice by inducing ER stress in colonocytes similar to that observed in IBD patients [START_REF] Cao | The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice[END_REF]. Chronic ER stress impairs cell homeostasis and induce apoptosis leading to epithelial injuries, crypt elongation and increased epithelial oxygenation in DSS-treated mice [START_REF] Cao | The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice[END_REF][START_REF] Chassaing | Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice[END_REF][START_REF] Cevallos | Increased Epithelial Oxygenation Links Colitis to an Expansion of Tumorigenic Bacteria[END_REF]). On the other hand, the destruction of mucosal barrier function upon DSS treatment, allows the translocation of microbes, leading to the induction of inflammatory responses [START_REF] Kitajima | Changes in Colonic Mucosal Permeability in Mouse Colitis Induced with Dextran Sulfate Sodium[END_REF].

INTRODUCTION

Chapter II: Intestinal microbiota and interactions with the host 52 Studies in humans have shown that the gut microbiota of patients with UC or CD is characterized by a decrease in commensal bacteria diversity, an increased abundance of facultative anaerobic Proteobacteria (particularly Enterobacteriaceae), and a concomitant decreased proportion of Firmicutes compared with healthy subjects [START_REF] Morgan | Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment[END_REF]. Within the phylum Firmicutes, the highly oxygen-sensitive F. prausnitzii, which is a major butyrateproducing bacteria, is decreased or even disappears in IBD patients [START_REF] Sokol | Low Counts of Faecalibacterium Prausnitzii in Colitis Microbiota[END_REF].

Likewise, a luminal expansion of the phylum Proteobacteria (in particular, members of the Enterobacteriaceae family) is observed in the cecal microbial communities upon DSS treatment [START_REF] Hughes | Microbial Respiration and Formate Oxidation as Metabolic Signatures of Inflammation-Associated Dysbiosis[END_REF]. Consistent enrichment in facultative anaerobes including Pediococcus, Weissella, Lactococcus, Enterococcus and Streptococcus also occurs in the bacterial-induced model of colitis involving infection of mice with Helicobacter hepaticus and treatment with anti-IL10 receptor antibody (Ilott et al. 2016a). This model is highly relevant to human IBD as loss of function mutations in IL-10 and IL-10-R are associated with severe infantile IBD [START_REF] Kotlarz | Loss of Interleukin-10 Signaling and Infantile Inflammatory Bowel Disease: Implications for Diagnosis and Therapy[END_REF]. In the inflamed intestine, the disruption in anaerobiosis is suggested to induce this shift in the microbiota composition towards greater abundance of facultative anaerobes (Rigottier-Gois 2013) that are also equipped to confront ROS [START_REF] Morgan | Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment[END_REF]. Several ROS are continuously produced in bacteria as byproducts of aerobic metabolism [START_REF] Imlay | Where in the World Do Bacteria Experience Oxidative Stress?[END_REF]. Moreover, in addition to endogenous ROS, host immune responses under inflammatory conditions favor oxidative stress. For example, neutrophils transmigrate into the lumen and release ROS upon intestinal inflammation [START_REF] Chin | Neutrophil Transepithelial Migration and Epithelial Barrier Function in IBD[END_REF]. Moreover, proinflammatory cytokine signalling induces the expression of dual oxidase 2 (DUOX2), which is a predominant source of intestinal epithelial ROS along the gut [START_REF] Harper | Differential Regulation of Dual NADPH Oxidases/Peroxidases, Duox1 and Duox2, by Th1 and Th2 Cytokines in Respiratory Tract Epithelium[END_REF]. Accordingly, DUOX2 expression is elevated across all forms of IBD in association with an expansion of Proteobacteria [START_REF] Haberman | Pediatric Crohn Disease Patients Exhibit Specific Ileal Transcriptome and Microbiome Signature[END_REF]. Moreover, the antiinflammatory/antioxidant APOA1 gene, which is expressed in the unaffected ileum, is suppressed in CD patients [START_REF] Haberman | Pediatric Crohn Disease Patients Exhibit Specific Ileal Transcriptome and Microbiome Signature[END_REF].

A B

Associated with the release of ROS, genes of the riboflavin pathway are enriched (at the DNA level) in the microbiota of patients with ileal CD. Furthermore, the pentose phosphate pathway, which feeds the riboflavin pathway and produces the NADPH required for glutathione reduction, is overrepresented as well in ileal CD. Genes involved in the biosynthesis of cysteine (a precursor to glutathione) and glutathione transport genes are also increased in abundance in all forms of IBD [START_REF] Morgan | Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment[END_REF]. Remarkably, in this study, patients with ileal CD were the only ones to have a significant increase in the abundance of Enterobacteriaceae. This bacterial family can exacerbate intestinal inflammation by stimulating the production of pro-inflammatory cytokines by intestinal dendritic cells (Y.-L. [START_REF] Huang | Sialic Acid Catabolism Drives Intestinal Inflammation and Microbial Dysbiosis in Mice[END_REF]. Altogether, these findings suggest a shift in the microbiota composition towards greater abundance of microbes able to grow in the inflamed intestine.

Role of intestinal epithelial barrier in shaping the microbiota 3.

Under normal circumstances, the intestinal epithelium is composed of a single layer of This intestinal barrier allows for physical segregation of antigenic peptides from T cells.

However, there are still communication channels between the microbiota and their mammalian host, in particular for the monitoring of bacterial activity, ie through the detection of microbial metabolites. Only a few have been identified to date, but there are probably many more conserved molecular interactions between the 2 symbionts. Among the metabolites generated by microbial metabolism are SCFAs, tryptophan-derived microbial metabolites, and secondary bile acids (BAs). These metabolites are small molecules (<1500Da) that have been shown to penetrate distant host tissues [START_REF] Uchimura | Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response[END_REF], and to impact both locally and systemically host metabolism and immune functions.

Influence of microbial metabolites on the host 4.

Short-chain fatty acids

SCFAs such as butyrate, acetate and propionate, are the end-products of microbial anaerobic fermentation of dietary fibers, which is the dominant metabolism in the colon. Members of Firmicutes are the main producers of butyrate, whereas most acetate and propionate producers belong to Bacteroidetes (G. Wang et al. 2019;[START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF].

INTRODUCTION

Chapter II: Intestinal microbiota and interactions with the host 55 Among the SCFAs, butyrate has been investigated most extensively. As described earlier, butyrate is the preferred energy substrate for colonocytes and the primary determinant of physiologic hypoxia in the colon by promoting intestinal epithelial O2 consumption.

Consequently, butyrate stabilize HIF (Caleb J. [START_REF] Kelly | Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function[END_REF] which in turn induce the expression of barrier-protective HIF target genes, including genes critical for defensin expression (C. J. [START_REF] Kelly | Fundamental Role for HIF-1α in Constitutive Expression of Human β Defensin-1[END_REF]) and mucin production [START_REF] Louis | Selective Induction of Mucin-3 by Hypoxia in Intestinal Epithelia[END_REF]. By this mechanism, SCFAs fortify IEC barrier function.

Additionally, butyrate and to a lesser extent propionate function as inhibitors of histone deacetylases (HDACs) and therefore act as an epigenetic regulator of host physiology [START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF]. Via HDACs inhibition, butyrate and propionate make the immune system hyporesponsive to commensal bacteria. Indeed, butyrate and propionate block the differentiation of dendritic cells (DC) from bone marrow stem cells by suppressing the transcription factors necessary for DC development [START_REF] Singh | Blockade of Dendritic Cell Development by Bacterial Fermentation Products Butyrate and Propionate through a Transporter (Slc5a8)-Dependent Inhibition of Histone Deacetylases[END_REF]. Moreover, butyrate promotes tolerance towards beneficial microorganisms by inducing functional colonic Treg cells among CD4 + T cell subsets, through acetylation at the FoxP3 gene locus [START_REF] Furusawa | Commensal Microbe-Derived Butyrate Induces the Differentiation of Colonic Regulatory T Cells[END_REF].

Beside their ability to inhibit HDACs, SCFAs can signal through the G-protein-coupled-receptors (GPR41, GPR43 and GPR109A) which are expressed on the surface of numerous cell types, including IECs and immune cells [START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF]. Butyrate inducing Gpr109A signalling promotes anti-inflammatory properties in colonic antigen-presenting cells, which in turn induce differentiation of naïve T cells into Treg cells and IL-10-producing T cells [START_REF] Singh | Activation of Gpr109a, Receptor for Niacin and the Commensal Metabolite Butyrate, Suppresses Colonic Inflammation and Carcinogenesis[END_REF].

Accordingly, GPR109A deficient mice harbor significantly less frequency and number of Treg cells in the colon and show enhanced susceptibility to colitis [START_REF] Singh | Activation of Gpr109a, Receptor for Niacin and the Commensal Metabolite Butyrate, Suppresses Colonic Inflammation and Carcinogenesis[END_REF].

Interestingly, metabolic studies have demonstrated a reduction in fecal SCFAs levels in IBD patients (Aonghus Lavelle and Sokol 2020; [START_REF] Marchesi | Rapid and Noninvasive Metabonomic Characterization of Inflammatory Bowel Disease[END_REF], which is consistent with the loss of butyrate-producing bacteria and impaired butyrate oxidation in the colonic mucosa of UC patients [START_REF] Roediger | THE COLONIC EPITHELIUM IN ULCERATIVE COLITIS: AN ENERGY-DEFICIENCY DISEASE?[END_REF]. Given their role in maintaining homeostasis, SCFAs are a promising therapeutic target in IBD. Indeed, SCFAs administration attenuates colonic inflammation in mice (P. M. [START_REF] Smith | The Microbial Metabolites, Short-Chain Fatty Acids, Regulate Colonic Treg Cell Homeostasis[END_REF][START_REF] Furusawa | Commensal Microbe-Derived Butyrate Induces the Differentiation of Colonic Regulatory T Cells[END_REF][START_REF] Singh | Activation of Gpr109a, Receptor for Niacin and the Commensal Metabolite Butyrate, Suppresses Colonic Inflammation and Carcinogenesis[END_REF], and butyrate enemas reduce clinical symptoms in IBD patients [START_REF] Scheppach | Effect of Butyrate Enemas on the Colonic Mucosa in Distal Ulcerative Colitis[END_REF][START_REF] Sabatino | Oral Butyrate for Mildly to Moderately Active Crohn's Disease[END_REF].

INTRODUCTION

Chapter II: Intestinal microbiota and interactions with the host 56 To note, metabolically beneficial effects of SCFAs (mostly acetate and possibly propionate) extend beyond the gut to the adipose tissue, brain, and liver [START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF]).

Tryptophan-derived microbial metabolites

Tryptophan is an essential amino acid that can only be acquired through dietary proteins.

Intestinal bacteria can catabolize dietary tryptophan as a precursor for synthesis of immunologically important metabolites, with indole and indole derivatives being the main tryptophan-derived microbial metabolites in the gut. These metabolites modulate the activity of aryl hydrocarbon receptor (Ahr), which is a ligand-dependent transcription factor expressed by epithelial cells and immune cells (G. Wang et al. 2019;[START_REF] Rooks | Gut Microbiota, Metabolites and Host Immunity[END_REF].

In an Ahr-dependent manner, indole derivatives improve the barrier functions of epithelial cells in vitro through up-regulation of genes encoding for actin cytoskeleton, tight junction proteins, mucin production, and IL-10R [START_REF] Bansal | The Bacterial Signal Indole Increases Epithelial-Cell Tight-Junction Resistance and Attenuates Indicators of Inflammation[END_REF][START_REF] Alexeev | Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor[END_REF]. Such activity is associated with protection in colitis models. Moreover, via Ahr stimulation, indole derivatives promotes intestinal homeostasis through IL-22 production by type 3 innate lymphoid cells (ILC3s) in the colon [START_REF] Zelante | Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22[END_REF].

Importantly, tryptophan-indole derivative metabolites were found decreased in both serum and colons of colitic mice, and serum samples from UC patients as compared to controls [START_REF] Alexeev | Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor[END_REF]. Administration of indole derivatives to DSS-treated mice reduced the severity of colitis [START_REF] Zelante | Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22[END_REF][START_REF] Alexeev | Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor[END_REF], highlighting the crucial role of the microbial metabolism of dietary tryptophan for achieving and maintaining homeostasis.

Secondary bile acids

Primary BAs are the end products of cholesterol metabolism in the liver and are further secreted Microbially transformed bile acids have also been potentially linked with IBD. Indeed, altered BA transformation in the gut lumen has been reported in IBD, involving increased fecal primary BAs and decreased serum and fecal secondary BAs [START_REF] Duboc | Connecting Dysbiosis, Bile-Acid Dysmetabolism and Gut Inflammation in Inflammatory Bowel Diseases[END_REF]. In this study, the imbalance in BAs towards primary BAs has been proposed as a mediator of the pathogenesis of human

IBDs by erasing the anti-inflammatory effects of secondary BA on gut epithelial cells. Indeed, secondary and not primary BAs inhibited anti-inflammatory effect of IL-1β on Caco-2 intestinal epithelial cells [START_REF] Duboc | Connecting Dysbiosis, Bile-Acid Dysmetabolism and Gut Inflammation in Inflammatory Bowel Diseases[END_REF]. Several studies showed activation of MAIT cells in inflammatory bowel diseases [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF][START_REF] Haga | MAIT Cells Are Activated and Accumulated in the Inflamed Mucosa of Ulcerative Colitis[END_REF][START_REF] Tominaga | Possible Involvement of Mucosal-Associated Invariant T Cells in the Progression of Inflammatory Bowel Diseases[END_REF]) in which high oxygen levels and oxidative stress have been reported in the gut (Rigottier-Gois 2013). Riboflavin contributes to bacterial respiration and oxidative stress resistance, and bacteria harboring the riboflavin biosynthetic pathway are enriched in the microbiota of IBD patients [START_REF] Morgan | Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment[END_REF]. Accordingly, we suggest that MAIT cell activation in IBD patients results from the overproduction of riboflavin precursor derivatives by the microbiota.

Aims of the thesis

In this thesis, we explored the hypothesis that dysbiosis drives increased concentrations of riboflavin precursor derivatives which are sensed by MAIT cells.

The overall objective of my thesis studies was to decipher MAIT cell interactions with the microbiota which helps understanding why the mammalian immune system has evolved recognition of riboflavin-derived metabolites.

The study has the two following specific aims:

I.

Identify the mechanisms of MAIT antigen production by the microbiota.

II.

Define the function of MAIT cells in the gut.

Results

Part I: MAIT cell surveillance of colitis-associated bacterial metabolites

Summary

Changes in the composition and metabolic activity of the gut microbiota are associated with many pathological processes, but how the host monitors such changes remains poorly understood. Mucosal associated invariant T (MAIT) cells recognize metabolites from the microbial riboflavin biosynthesis pathway presented by the MHC-related molecule MR1 1,2 .

Riboflavin contributes to bacterial respiration and oxidative stress resistance, but why T cells recognize riboflavin precursor derivatives is unclear. Here we show that hypoxia disruption in the colon, upon antibiotic treatment or colitis is associated with increased production of MAIT antigen by the microbiota and triggers a protective MAIT cell response. The bacterial genes ribA, ribB and ribD, which control MAIT antigen production, were over-expressed during colitis.

Riboflavin production provided a fitness advantage to Escherichia coli in the inflamed intestine.

Riboflavin precursor adducts rapidly crossed the intestinal barrier and induced T cell receptor (TCR) signalling in MAIT cells, which produced the tissue-repair mediator amphiregulin and reduced colitis severity. Thus, MAIT cells directly monitor a bacterial metabolism associated with colitis and provide host protection in return. The study helps understand the role of T cell recognition of riboflavin precursor derivatives. Manipulation of this new host-microbiota interaction may provide clinical benefits in inflammatory bowel diseases.

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Main text

MAIT cells are evolutionarily conserved T cells that recognize metabolic adducts derived from the riboflavin biosynthetic pathway. MAIT cells preferentially localise in barrier tissues and have been implicated in pathologies associated with intestinal dysbiosis such as inflammatory bowel diseases 3,4 and colorectal cancer 5-7 . Intestinal bacteria from the Bacteroidetes and Proteobacteria phyla can produce large amounts of riboflavin pathway-derived MAIT antigens 8 , suggesting a direct interaction between MAIT cells and the microbiota. Bacteria harbouring the riboflavin biosynthetic pathway are enriched in the microbiota of patients with inflammatory bowel diseases 9-11 , raising the possibility that MAIT cell activation in these patients results from the overproduction of riboflavin adduct antigens by the microbiota. The amounts of MAIT antigens produced in the gut by the microbiota, and the mechanisms controlling this process remain unknown. The role of MAIT cells during colitis is also unknown.

MAIT cells represented 1-10% of T cells in the colonic lamina propria of B6-MAIT Cast mice 12 (Fig. 1a). Parabiosis experiments indicated that the population of colonic MAIT cells, like invariant natural killer T (iNKT) cells and unlike conventional T cells, was not replaced by blood-derived cells during a 5-week time period (Fig. 1b), indicating residency in the colon. Colonic MAIT cells expressed the activation marker CD25 at high levels as compared to iNKT cells (Fig. 1c). Given the shared functional programs but distinct specificities of MAIT and iNKT cells 13 , CD25

expression by MAIT cells suggested MAIT antigen availability in the colon. To assess TCR signalling in MAIT cells, we used the Nr4a1-GFP reporter mouse 14 , in which TCR but not cytokine stimulation drives expression of GFP in MAIT cells (Fig. S1a). iNKT cells expressed GFP in the mesenteric lymph nodes (mLN), but not in the colon, consistent with iNKT cell priming in the mLN 15 . By contrast, MAIT cells expressed high levels of GFP in the colon (Fig. 1d). Lower but significant levels of GFP were also detected in MAIT cells from the mLN and the liver, which drain intestinal lymph and blood, respectively, further suggesting MAIT antigen production in the colon.

To quantify MAIT antigens in the large intestine, we used an in vitro biological assay using MAIT ligand-specific reporter T cells and MR1-expressing antigen-presenting cells 16 loaded with intestinal contents (Fig. 1e). The assay enables the detection of the canonical MAIT ligand 5-(2oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) at a sensitivity of 0.1 nM (Fig. S1b).

The reporter T cells were activated in the presence of cecum, colon and feces contents, revealing the presence of MAIT antigens throughout the large intestine (Fig. 1e andS1c).

RESULTS

Part I: MAIT cell surveillance of colitis-associated bacterial metabolites 61

Both fungi and bacteria can produce riboflavin-derived MAIT antigens 1,17 . MAIT antigens were depleted from the intestinal contents when mice were treated with broad-spectrum antibiotics (Fig. 1e), but not with the anti-fungi drug fluconazole (Fig. S1d), indicating that MAIT antigens were mostly produced by the commensal bacteria. The colon presents ecological niches populated by microbial communities with distinct metabolic requirements 17 , which could affect MAIT antigen production. In particular, the oxygen concentration is higher in the mucosa as compared to the lumen and shapes the composition of bacterial communities 18 . Mucosal scraps contained fewer bacteria (Fig. S1e), but were more potent than luminal content at activating reporter T cells (Fig. 1f), indicating higher MAIT antigen concentration in the mucus layer than in the lumen. Thus, colonic bacteria residing close to the epithelium produce MAIT antigens, which are sensed by MAIT cells in the underlying lamina propria.

To explore the mechanisms underlying increased MAIT antigen production in the mucosa compared to the lumen, we sequenced the 16S rRNA genes from the two compartments. In both cecum and colon, the mucosal niche was enriched in Proteobacteria (Fig. 2a and S2a), in particular in the facultative anaerobes Enterobacteriaceae (Fig. 2b and S2b), consistent with higher oxygen availability in the mucus as compared to the lumen 18 . The higher production of MAIT antigens by the microbiota of the mucus may be a consequence of higher oxygen availability in that location, as riboflavin contributes to oxidative stress reduction in bacteria. For example, riboflavin accumulates and reduces hydrogen peroxide formation in the gut bacterium

Enterococcus faecalis under aerobic conditions 19 . Riboflavin is also used by the anaerobe Faecalibacterium prausnitzii to reduce oxygen and grow in contact with air 20 . Consistent with a role for riboflavin in the oxidative stress response, the ribD and ribH genes of the riboflavin biosynthesis operon were expressed upon oxidative stress in Clostridioides difficile (Fig 2c andS2c), consistent with a previous report 21 . Clostridium perfringens and Clostridium butyricum also expressed riboflavin biosynthesis genes upon air exposure (Fig. 2c andS2c), suggesting that riboflavin production is a conserved oxidative stress response in Clostridia. Escherichia coli and

Bacteroides thetaiotaomicron constantly produce high amounts of riboflavin as compared to

Clostridia 8 , which may explain that ribD or ribH were not overexpressed in these bacteria upon exposure to oxygen (Fig. 2c andS2c).

RESULTS

Part I: MAIT cell surveillance of colitis-associated bacterial metabolites 62

Intestinal hypoxia is established at weaning and is accompanied with a reduction in the abundance of Proteobacteria 22,23 . Compatible with a link between intestinal oxygen levels and MAIT antigen production, the colonic microbiota produced more MAIT antigens before weaning than in adults (Fig. S2d). Colonic hypoxia in adults is maintained by epithelial cells through the β-oxidation of microbial fermentation products such as the short-chain fatty acid butyrate, and can be disrupted upon antibiotic-mediated depletion of butyrate producers 24,25 . To increase oxygen availability in the gut, we treated mice with antibiotics and quantified short-chain fatty acids. Ampicillin and vancomycin, but not neomycin treatment, depleted short-chain fatty acids (Fig. S2e) and concomitantly induced the production of MAIT antigens in the large intestine (Fig. 2d). The loss of hypoxia was evidenced by the expansion of facultative anaerobes such as Enterobacteriaceae (Fig. 2f) upon vancomycin treatment, and by the loss of the exogenous hypoxic marker pimonidazole 26 at the surface of colonic epithelial cells (fig 2g and S2f).

Additional treatment with fluconazole had no effect on MAIT antigen concentrations in the cecum (not shown), which indicates that bacteria, not fungi, produced most MAIT antigens in this setting. Thus, increased oxygen availability in the colon is associated with overproduction of MAIT antigen by the microbiota.

At steady-state, the intestinal barrier separates microbes from the host immune system, preventing T cell responses to commensal bacteria antigens 27 . We used fluorescently labelled dextran administered by oral gavage to assess the integrity of the epithelial barrier upon vancomycin treatment. By contrast with dextran sodium sulfate (DSS) treatment, which increased gut permeability, the intestinal barrier was not affected by vancomycin treatment (Fig. S2g). Since MAIT antigens can rapidly cross the mouse skin 28 , we asked whether intact intestinal epithelium was permeable to 5-OP-RU. A mixture of antigenic peptide and 5-OP-RU metabolite was deposited into the lumen of intestinal explants and incubated for 10 minutes on top of antigen-presenting cells (Fig. 2h). Reporter T cells detected the presence of 5-OP-RU antigen, but not peptide, in the supernatant (Fig. 2h and S2h), which indicated efficient transfer of 5-OP-RU across the intestinal barrier ex vivo. Given the hydrophilic nature of 5-OP-RU, this result suggests the presence of a transporter expressed by intestinal epithelial cells.

In vivo, vancomycin treatment resulted in increased Nr4a1-GFP expression in colonic MAIT cells (Fig. 2i), indicating that overproduction of MAIT antigens by the microbiota was efficiently detected by MAIT cells through the TCR. MAIT cells also received more TCR signals in the mLN, liver and spleen upon vancomycin treatment (Fig. 2i), consistent with a transfer of MAIT

RESULTS

Part I: MAIT cell surveillance of colitis-associated bacterial metabolites 63 antigens through the lymph and the blood to these tissues. By contrast, vancomycin treatment had no effect on Nr4a1-GFP expression in iNKT cells (Fig. S2i). Collectively, MAIT cells detect microbial MAIT antigens produced upon antibiotic-mediated dysbiosis.

Loss of intestinal hypoxia is a hallmark of gut inflammation 29 and bacteria having the riboflavin biosynthesis pathway are enriched in patients with inflammatory bowel diseases 9-11 . To investigate the potential role of MAIT cells during intestinal inflammation, we analyzed expression of the riboflavin pathway genes in the Helicobacter hepaticus model of colitis using a published metatranscriptomic dataset 31 . Principal component analysis (PCA) of riboflavin biosynthesis gene expression readily distinguished mice before and after colitis induction (Fig 3a), which indicates that the riboflavin biosynthesis pathway can be used as a biomarker of colitis. All the genes controlling production of the MAIT ligand precursor 5-amino-ribityl uracil (5-A-RU), including ribD coding for the 5-A-RU-producing protein RibD, were over-expressed during colitis (Fig. 3b). The bacteria driving ribD over-expression belonged to the Bacteroidaceae, Clostridiaceae and Enterobacteriaceae families (Fig. 3c). In particular, Bacteroidaceae contributed up to 70% of the expressed ribD upon colitis induction. Total gene expression by Bacteroidaceae was also increased upon colitis (Fig. S3a), suggesting their expansion in the inflamed gut.

We then assessed whether changes in microbiota composition would be associated with MAIT antigen production in another colitis model. In the DSS model of intestinal inflammation (Fig. S3b), colitis was associated with reduced hypoxia (Fig. 3d) and with a bloom of cecal Bacteroidaceae and Enterobacteriaceae from the Escherichia/Shigella genera (Fig. 3e andS3c). The dysbiotic expansion of Escherichia coli relies on microaerobic respiration 32 . To assess the role of riboflavin production in this process, wild-type (WT) or isogenic ∆ribC E. coli, which lack riboflavin production 16 , were administered to mock-or DSS-treated mice by oral gavage and were quantified in the cecum a day later. ∆ribC bacteria were recovered in smaller numbers than WT bacteria (Fig. 3f), indicating that riboflavin production contributes to the fitness advantage of E. coli during intestinal inflammation. The mechanisms underlying Bacteroidaceae expansion during colitis are unclear, but may also involve respiration given the ability of some representatives from the Bacteroidaceae family such as B. thetaiotaomicron, which expanded during colitis (Fig. 3g), to respire under nanomolar concentrations of oxygen 33 . Consistent with the strong MAIT stimulatory capacities of Bacteroides species 8 , B. thetaiotaomicron secreted large amounts of MAIT antigens in vitro (Fig. 3h).

RESULTS

Part I: MAIT cell surveillance of colitis-associated bacterial metabolites 64

As a whole, the microbiota produced high concentrations of MAIT antigens during DSS-induced colitis (Fig. 3i), in agreement with the increased expression of riboflavin pathway genes in the H. hepaticus model of intestinal inflammation (Fig. 3b). MAIT cells from the colon, but also from the mLN, liver, inguinal and brachial (I+B) LN and spleen up-regulated Nr4a1-GFP upon DSS treatment (Fig. 3j), indicating TCR-mediated activation. By contrast, intestinal inflammation had no effect on Nr4a1-GFP expression in iNKT cells (Fig. S3d). Thus, colitis drives expression of the riboflavin pathway, resulting in MAIT antigen production and in MAIT cell activation.

To assess the effect of TCR signalling on intestinal MAIT cells without affecting the microbiota, we generated a conditional MR1 transgenic mouse (LSL-MR1) for Cre-mediated overexpression of MR1 (Fig. 4a). The LSL-MR1 mouse was crossed to the Villin-CreER mouse for inducible expression in intestinal epithelial cells 34 . Transgenic MR1 expression in intestinal epithelial cells elicited strong TCR signalling in MAIT cells from the colon, but not spleen and liver, as expected (Fig. 4b). Robust TCR signalling in MAIT cells from the colon induced the production of the epidermal growth factor amphiregulin (Areg) (Fig. 4c), suggesting that MAIT cells may promote epithelial function during colitis.

MAIT cells are activated and recruited to inflammatory lesions in patients with ulcerative colitis and Crohn's disease 3,4 , but whether MAIT cells worsen or ameliorate intestinal pathology is unclear. We used a chronic DSS model (Fig. S4a) to investigate the role of MAIT cells in colitis.

The proportion of colonic MAIT cells expressing RORγt increased during colitis, while the proportion of MAIT cells co-expressing RORγt and Tbet was reduced (Fig. 4d). Consistent with an activated phenotype, MAIT cells from colitic mice spontaneously produced interferon-gamma (IFN-γ), interleukin (IL)-17, IL-22 and Areg in the colon (Fig. 4e). IFN-γ promotes mucus secretion by goblet cells 35 , while IL-17 and IL-22 promote the secretion of anti-microbial peptides and the expression of tight-junction proteins in epithelial cells 36 , thus reinforcing the epithelial barrier. Accordingly, B6 Cast Mr1 -/-mice, which lack MAIT cells 12 , presented higher pathophysiology than Mr1 + counterparts upon chronic colitis (Fig. 4f andS4b). The presence of MAIT cells resulted in reduced epithelial damage, inflammatory cell infiltrates, splenomegaly and weight loss (Fig. 4f andS4b). Thus, MAIT cells protect against chronic intestinal inflammation. El Morr et al, Figure 1 67.9 86.9 84.8

RESULTS

Part

Methods

Mice

B6 and CD45.1 mice were purchased from Charles River Laboratories. iVα19 and iVα19 Vβ8

TCR transgenic Cα -/-B6 mice, B6-MAIT Cast mice have been described 12,38 . Nr4a1-GFP mice 14 were imported from the MMRRC (UC Davis). OTII mice were maintained on a RAG -/- background. All mice were bred and maintained on a standard chow diet in SPF conditions at Institut Curie. Prior to inclusion in experimental groups, mice from different litters were cohoused, or cage contents were mixed for at least two weeks in order to normalize microbiota. All experiments were performed according to national and international guidelines and were approved by the Institut Curie Ethic committee (APAFIS#25203-2020042318109119, #24245-2020021921558370 and #IOI68-2017060909448377).

To generate Lox-stop-Lox-MR1 (LSL-MR1) transgenic mouse, a DNA segment containing a stop cassette flanked with loxP sites upstream of the murine Mr1 gene was prepared by digestion of the pOD2G-LS CAGGS (7279 bp) plasmid 39 (a gift from J. J. Moon, Massachusetts General Hospital) with the enzymes HindIII and SpeI. Efficient induction of MR1 protein expression under the control of the chicken actin promoter was validated in HEK293 cells cotransfected with a Cre expression vector (data not shown). The construct was then injected into pronuclei of fertilized oocytes. Founders were crossed to transgenic mice bearing a tamoxifendependent Cre recombinase expressed under the control of the villin promoter 35 .

Antibiotic treatments, colitis induction, and tamoxifen administration

Female mice aged 6-10 weeks at the start of the experiments were used throughout the study.

For microbiota depletion, mice were given four antibiotics (vancomycin (0.5 g/l, Mylan), ampicillin (10 g/l, Coophavet), metronidazole (1 g/l, B Braun) and neomycin (1g/l, Coophavet) in the drinking water supplemented with 2% sucrose for 7 days. Single antibiotics were also provided for 7 days or for the indicated period of time. Fresh antibiotic solutions were provided after 3 days of treatment. For fungi depletion, fluconazol (0.5 mg/ml, Sigma-Aldrich) was provided in the drinking water for 7 days.

Acute colitis was induced in mice by adding 2% (wt/vol) DSS (molecular weight: 36,000-50,000 Da; MP Biomedicals) to the drinking water for 7 days or until mice had lost 15% of initial weight. For bacterial gene amplification and sequencing, DSS-treated mice were switched to normal drinking water 1 day prior to the end of the experiment to reduce DSS concentration from samples and avoid PCR inhibition. For modeling chronic colitis, mice received 3 to 5 5-day treatments with DSS. Each DSS treatment was followed by 4 days with water only. Control groups (Mock) received only water.

For inducible MR1 over-expression, transgenic mice were injected intraperitoneally with 2 mg tamoxifene (T5648, Sigma-Aldrich) in a corn oil-ethanol mixture once per day for 3 consecutive days. Tissues were collected 24 h after the last injection.

Parabiosis

Parabiosis surgery was performed as described previously 40 . Briefly, age-and weight-matched CD45.1/2 and CD45.2/2 B6-MAIT Cast female mice were surgically joined through the abdominal skin, olecranon and knee joints. Parabiotic pairs were euthanized 5 weeks later.

Cell isolation

Spleens were mashed over 40 μm cell strainer to create single cell suspensions followed by red blood cell lysis. Livers were perfused with PBS through the portal vein, mashed over 100 μm cell strainer, and centrifuged over Histopaque 1077 (Sigma) layers to isolate the lymphocytecontaining fraction. Lymph nodes were incubated with collagenase D (1 mg/ml; Roche) and DNAse I (100 ug/ml; Roche) for 30 min at 37°C, and mashed over 30 μm filters (Miltenyi).

Colons were opened longitudinally and cut into approximately 0.5 cm pieces. Dissociation of epithelial cells was performed by incubation with constant stirring in HBSS w/o Ca/Mg (Life Technologies) containing 5 mM EDTA (5mM; Thermo Fisher Scientific), DTT (1mM;

Euromedex), and 5% FCS twice for 20 min at 37 °C. After each step, samples were vortexed and the epithelial fraction discarded. Tissue fragments were then washed in HBSS and enzymatic digestion was performed in CO 2 -independent medium (Life Technologies) containing collagenase D (1 mg/ml; Roche), liberase TM (0.17 U/ml; Roche) and DNase I (100 ug/ml; Roche) on a shaker for 30 min at 37 °C. Colon fragments were then dissociated with a gentleMACS dissociator according to the manufacturer's instructions (Miltenyi). Lymphocytes were collected at the interface of a 40%/80% Percoll gradient (GE Healthcare).

Tetramers and flow cytometry

CD1d tetramers conjugated to BV421, and MR1 tetramers loaded with

76

Tetramer staining was performed at room temperature for 30 min. Surface staining was performed at 4 °C with fluorescently labeled antibodies and fixable viability dye eFluor780 (eBioscience, Hatfield, UK).

Antibodies were purchased from Biolegend, eBioscience, BD, Invitrogen or R&D Systems. The following antibodies were used for surface staining: anti-CD19 (1D3), -B220 (RA3-6B2), -CD11c (N418), -CD5 (53-7.3), -TCRβ (H57-597), -CD24 (M1/69), -CD44 (IM7), -CD45.1 (A20), -CD45.2

(104), -CD4 (RM4-5), -CD8 (53-6.7), -CD69 (H1.2F3), -CD25 (PC61), -Vα2 (B20.1).

Fixation and permeabilization were performed for intranuclear staining (FoxP3 staining kit; eBioscience) and intra-cytoplasmic staining (Cytofix/Cytoperm kit; BD Biosciences) according to the manufacturer's instructions. For intranuclear staining, cells were stained with anti-RORγt (Q31-378) and -Tbet (4B10) for 20 min at 4 °C. For intra-cytoplasmic staining, cells were cultured for 3 h at 37 °C in complete RPMI in the presence of GolgiPlug (1:1000; BD). Cultured cells were subsequently stained for surface antigens as above. Cells were fixed and permeabilized, washed, and incubated for 30 min at 4 °C with anti-IL17 (TC11-18H), -IL22

(IL22JOP) and -IFN-γ (XMG1.2). Cells were then stained overnight with anti-amphiregulin-biotin (BAF989) followed by FITC-or BV605-conjugated streptavidin.

In some experiments, non-fixed dead cells were stained with the DAPI staining solution and directly analyzed by flow cytometry. Cells were acquired either on Cytoflex (Beckman), LSRII (BD Biosciences) or Fortessa (Becton Dickinson), and the results were analyzed with FlowJo V10.2 (Treestar).

5-OP-RU preparation

5-A-RU was synthesized as described 16 . For preparation of 5-OP-RU, 5-A-RU was incubated with equimolar amounts of methylglyoxal (Sigma) for 10 min at room temperature.

In vitro validation of the Nr4a1-GFP mouse

Splenic T cells from a transgenic Nr4a1-GFP mouse were isolated by negative cell enrichment using the Magnisort mouse T cell enrichment kit (Thermo Fisher Scientific). Isolated cells were cultured at a density of 20x10 6 /ml in complete RPMI and stimulated with either recombinant mouse IL-12 (10 ng/ml) and IL-18 (12.5 ng/ml) or with 10 nM 5-OP-RU in the presence of murine MR1-overexpressing WT3 (WT3-MR1 cells, 5x10 6 /ml) 41 for 16h at 37°C. GolgiPlug was added during the final 6h of culture. MAIT cells were then analysed for Nr4a1-GFP expression and IFN-γ production.

In vitro detection of MAIT antigens

To detect intestinal MAIT antigens, 50 mg or 100 mg of luminal contents were collected from the cecum or colon. Mucosal scrapings were sampled by scraping biomass from intestinal walls with a sterile scalpel. Feces were collected from cage floors immediately after defecation.

Samples were weighted and dissolved in complete RPMI medium (RMPI without folic acid supplemented with 10% FCS, penicillin and streptomycin), and kept on ice. Tubes were vortexed to homogenize the mixture and centrifuged at maximum speed for 1 min at 4°C. Supernatants were filtered through a 0.22-μm-pore-size filter and loaded at different dilutions on WT3-MR1 cells for 2h30 min at 37°C.

To detect MAIT antigens in bacterial culture supernatants, Bacteroides thetaiotaomicron, Escherichia coli and Clostridium perfringens grown to late exponential phase in an anaerobic chamber were centrifuged at 14000 g for 30 s. The supernatant of each sample was collected and flash-frozen in liquid nitrogen. Supernatants were thawed and incubated with WT3-MR1 cells at 37°C for 2h 30 min.

WT3-MR1 cells were then washed and co-cultured overnight at a 1:1 ratio with splenocytes from a Vα19 TCR Tg Cα -/-or Vα19Vβ8 TCR Tg Cα -/-mouse. Cells were then harvested and stained for CD25 and CD69 expression on 5-OP-RU:MR1-specific T cells. MAIT antigen concentrations were estimated using a standard curve prepared with serial dilutions of synthetic 5-OP-RU.

Intestinal permeability to 5-OP-RU

75 µl of PBS containing the indicated amounts of mixed OVA 323-339 peptide (Invivogen) and 5-OP-RU were added into the lumen of freshly isolated colons from healthy mice. Colon segments were then immersed in complete RPMI medium at 37°C in a well on top of mixed MutuDC and WT3-MR1 cells, for OVA and 5-OP-RU presentation, respectively. As positive control, antigen solutions were also added directly on top of mixed antigen presenting cells.

Colon segments were removed from the wells after 10 min of incubation. For the measurement of antigen-presentation, splenocytes from a Vα19 TCR Tg Cα -/-mouse and from a OTII-Rag1 -/- mouse were cultured overnight with antigen presenting cells at respectively 1:1 and 1:10 ratios.

Reporter splenocytes were then harvested and stained for activation markers.

Intestinal sample collection for nucleic acids extraction

After sacrifice, the intestinal tract was quickly removed and splayed open through a longitudinal incision. Luminal contents and mucosal scrapings were collected from the cecum and the colon.

78

Intestinal contents were resuspended in ATL lysis buffer (Qiagen) and bacteria were lysed using a Fast Prep Bead beater (MP Biomedicals) for 3 times 1 min at 6.5 m/s. Genomic DNA was purified using the Qiamp DNA Mini Kit (Qiagen).

16S rRNA gene PCR amplification and sequencing

16S rRNA gene sequencing was performed in-house or at the University of Minnesota Genomics center with similar results. In-house library preparation was performed as described 42 . Briefly, a single PCR step was used for amplification of the V3-V4 region of the 16S rRNA gene and for dual-index labelling of each sample. PCR amplification was performed using the Accuprime Pfx DNA polymerase (ThermoFisher). PCR products were purified (AMPure XP beads, Beckman Coulter) and sequenced on MiSeq (Illumina, USA) to generate paired-end 300-bp reads.

Bioinformatic analysis of 16S rRNA sequencing data

Adapter sequences were trimmed from the raw fastq files using Cutadapt v 2.10 43 . Subsequent analyses were performed using a modified DADA2 microbiome pipeline v 1.12.11 44 (https://benjjneb.github.io/dada2). In brief, raw sequence reads were first filtered using DADA2 default parameters. Filtered reads were then dereplicated and denoised using DADA2 default parameters. The DADA2 pipeline produced the non-chimeric ASV table containing the frequency with which each ASV was present in each sample. Taxonomic assignment of selected ASVs was performed against the SILVA reference database (v 132 -https://www.arbsilva.de) 45 .

Downstream analyses, statistical tests, and visualization were performed using different packages in the R environment v 4.0.4 (R Core Team 2019). To search for differentially abundant taxa and differentially expressed genes among different treatments, locations, and layers, DESeq2 (v 1.24.0) 46 was used by performing the Wald significance test.

Metatranscriptomic data analysis

Metatranscriptomic datasets 32 stored under the accession number E-MTAB-4082 were downloaded from EBI ENA. Paired end reads were quality checked and trimmed using TrimGalore v0.6.6 (https://github.com/FelixKrueger/TrimGalore). Host reads were removed by aligning metatranscriptomes against mouse genome (http://ftp.ensembl.org/pub/release-102/fasta/mus_musculus/dna/ Mus_musculus.GRCm38.dna.primary_assembly.fa.gz) using STAR v2.7.7 47 . Ribosomal reads were removed from metatranscriptomic datasets using sortmerna v4.2.0 48 . Finally, remaining non ribosomal reads were mapped against the iMGMC mouse gene catalogue 49 using bwa v0.7.17-r1188 with default parameters. Read counts aligned to the iMGMC gene catalogue were obtained from bam files processed with featureCounts v2.0.1 50 . Read counts table were parsed using in-house custom scripts to extract rib gene expression data used as input for DESeq2 analysis. Heatmaps were generated using the pheatmap R package v 1.0.12.

Estimation of bacterial number in intestinal samples

Total bacterial numbers from intestinal samples were estimated by TaqMan qPCR quantification of bacterial 16S rRNA (LightCycler 480 Instrument II, Roche). The universal 16S rRNA gene primers and probe sequences used in this study are listed in table S1. A standard curve was built using Escherichia coli DNA to convert the threshold cycle (Ct) values into colony-forming unit (CFU) equivalents.

Bacterial cultures

All bacterial strains were first grown anaerobically (5% H 2 , 5% CO for the other strains, cultures were distributed into 24-well plates, 1 mL per well and placed in aerobiosis at 37°C. Bacteria were harvested after 0, 30 or 60 min air-exposure and fast frozen in liquid nitrogen. Bacterial RNAs were extracted, cDNAs synthesised and real-time quantitative PCRs performed as previously described 55,56 using the primers listed in table S1. The relative change in gene expression was given by the [∆C T ] method 57 with cDNA quantity of the rib genes normalised to the gyrA gene.

In vivo expansion of E. coli during DSS treatment

Six days after the start of DSS treatment (3%), B6 mice were intragastrically inoculated with 10 9

CFUs of E. coli wild-type strain carrying an ampicillin-resistance plasmid, or with the isogenic ΔribC strain resistant to chloramphenicol. The mice were euthanized after one day.

Cecum contents were collected in sterile phosphate buffered saline for bacterial counts. The bacterial loads of E. coli were determined by plating serial 10-fold dilutions on LB plates containing the appropriate antibiotic and supplemented with 20 µg/mL of riboflavin.

Short-chain fatty acid (SCFA) analysis

SCFA analysis was carried out as described previously 58 , with slight modifications. Samples were water-extracted and proteins were precipitated with phosphotungstic acid. A volume of 0.3 µl of the supernatant fraction was injected onto a gas-liquid chromatograph (Agilent 6890; Les Ulis, France) equipped with a split-splitless injector, a flame-ionization detector and a capillary polyethylene glycol column (15 m x 0.53 mm, 0.5µm). Carrier gas (H 2 ) flow rate was 10 ml/min and inlet, column and detector temperatures were 200°C, 100°C and 240°C, respectively.

Between each sample injection, the column was cleaned by increasing the temperature from 100°C to 180°C (20°C/min) followed by holding at 180°C for 2 min before returning to 100°C for the next analysis. 2-Ethylbutyrate was used as an internal standard. Samples were analyzed in duplicate. Data were collected and peaks integrated with the OpenLab CDS Chemstation Edition software (Agilent, Les Ulis, France).

Detection of intestinal hypoxia

Pimonidazole (PMDZ) (Sigma-Aldrich) was administered at 60 mg/kg by intraperitoneal injection 1h prior to sacrifice. Colon samples were fixed in formalin solution 10% neutral buffer (Sigma-Aldrich). Paraffin-embedded sections were blocked with protein block (Dako, X0909) and treated with avidin/biotin blocking reagents (Dako, X0590) followed by overnight incubation at 4°C with mouse monoclonal anti-pimonidazole IgG1 (Hypoxyprobe TM -1) at 1.2 μg/ml of concentration according to the manufacturer's instructions. Negative control sections were treated identically except that primary antibody was substituted with control antibody (BD) for overnight incubation. All sections were then incubated for 30 min with biotinylated antimouse IgG secondary antibody (Vectastain) and then stained for 30 min with Alexa546conjugated Streptavidin (ThermoFisher). Tissues were counterstained with DAPI. PMDZ staining was quantified using Fiji and plotted as fluorescence intensity per 400 μm 2 .

FITC-Dextran permeability assay

Mice were given an oral gavage of FITC-dextran (4 kDa; Sigma-Aldrich FD4) solution at 60 mg/100 g body weight. FITC fluorescence preand 4h post-gavage was measured in plasma, and FITC dextran concentrations were calculated with the help of a standard curve prepared with serial dilutions of FITC-dextran in PBS.

Assessment of intestinal pathophysiology

In chronic DSS experiments, the spleen and colon were dissected from each mouse. Individual spleen tissues were weighed. The entire colon was fixed in formalin solution 10% neutral buffer, embedded in paraffin and stained with hematoxylin and eosin (H&E). H&E-stained sections of the colon were analysed for colitis severity in an investigator-blinded manner. Epithelial damage (architectural lesion), inflammatory cell infiltration and villus length were scored using the histopathological scoring system described previously 59 .

Statistical analyses

Statistical analyses and graphical representations related to the microbiome pipeline were conducted in R (v 4.0.4) (R Core Team 2019) with the R package ggplot2 v 3.2.0. Inkscape 0.92 was used to finalize the figures. All other statistical analyses were performed with Prism software (GraphPad). Two-tailed P values were determined using Wilcoxon's and Mann-Whitney's tests for paired and non-paired samples, as appropriate.

Table S1:

Primers and probe for real-time PCR used in this study. 117-125 (2004).

Primers and probe

Supplementary Information is available for this paper.

Correspondence and material requests should be addressed to: F. L. (francois.legoux@curie.fr) and OL (Olivier.lantz@curie.fr).

RESULTS

Part II: Characterization of MAIT cells in the ileal environment 88

Part II: Characterization of MAIT cells in the ileal environment

The intestinal tract is composed of different ecological niches shaped by dynamic parameters that determine bacterial colonisation and metabolism. Examples of variations from the proximal to the distal end of the gut, include decreasing concentrations of nutrients, bile acids, antimicrobials and oxygen, and increasing mucus density (Donaldson, Lee, and Mazmanian 2016). Using the exogenous hypoxia marker pimonidazole, we analysed ileal and colon sections from healthy mice for pimonidazole adduct formation. In contrast to the apical surface of colonocytes, which is hypoxic, ileal epithelial cells did not sustain pimonidazole adduct formation, indicating a relatively high oxygen concentration in that location (Fig. 1). These results are consistent with the proposed oxygen gradient along the intestinal tract. The contribution of riboflavin to bacterial respiration and oxidative stress resistance, suggested increased needs for riboflavin in the ileum. In view of this, the production of riboflavin metabolites may be higher in the ileum as compared to the colon, potentially affecting MAIT cell function in each location. To detect MAIT antigens along the gut of healthy mice, we used the in vitro assay using 5-OP-RU:MR1-specific reporter T cells and MR1-expressing antigen-presenting cells loaded with luminal contents from the ileum, cecum, colon, or feces. Reporter T cells were not activated with contents from the ileum, but up-regulated the activation markers CD25 and CD69 in the presence of cecum, colon contents and feces extracts (Fig. 2). These findings suggested that bacteria colonizing the ileum do not produce sufficient levels of metabolites that are required for the activation of reporter cells in vitro. We then characterized MAIT cells in the ileum and compared them to colon MAIT cells. In the ileum, MAIT cells were present in the lamina propria at lower frequencies than in the colon. By contrast, iNKT cells, a subset with similar transcriptional program but distinct specificity (Salou et al. 2019), were not enriched in the colon (Fig. 3a). Just as in the colon, ileal MAIT cells do not circulate between 5-weeks parabiotic mice, indicating residency properties of MAIT cells within the gut (Fig. 3b). In contrast to the thymus, spleen and lung where RORγt and Tbet expression by MAIT cells appears to be mutually exclusive [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF], in the ileum and the colon, MAIT cells coexpressed RORγt and Tbet (Fig. 3c). However, the proportion of RORγt + Tbet -was higher in the ileum, and localization in the colon was associated with reduced RORγt and increased Tbet expression (Fig. 3c,d), suggesting different effector programs within the gut.

We then examined the activation state of MAIT cells along the gut. MAIT cell expression of the activation marker CD25 was lower in the ileum compared to the colon, but higher than the iNKT expression of CD25 (Fig. 3e). These data suggested that MAIT cells are less activated in the ileum where MAIT antigens were not detected. We further assessed TCR signalling in MAIT cells, using the Nr4a1-GFP reporter mouse. In the ileum, MAIT cells expressed the lowest levels of GFP compared to MAIT cells from other tissues such as spleen (Fig. 3f), and to conventional T cells and iNKT cells from the ileum (Fig. 3g). These weak TCR signals in MAIT cells exclusively in the ileum suggested a mechanism controlling ileal MAIT cell activation at steady state. MAIT TCR stimulation in the ileum may be muted either by low production of riboflavin pathway-derived antigens by the microbiota and/or by low expression of MR1 in the lamina propria. We sequenced the 16S rRNA gene to characterize bacterial communities along the gut of healthy B6 mice. As compared to the cecum and colon samples, the ileal lumen of healthy mice had ~10 times lower numbers of CFU equivalents per gram (Fig. 4a). At the phylum level, Bacteroidetes and Firmicutes dominated the different regions along the mice intestinal tract (Fig. 4b). Bacteroidetes, Firmicutes and other phyla such as Proteobacteria were similarly abundant in all three anatomical regions (Fig. 4b).

We note that intestinal bacteria from the Bacteroidetes and Proteobacteria phyla can produce large amounts of riboflavin pathway-derived MAIT antigens [START_REF] Tastan | Tuning of Human MAIT Cell Activation by Commensal Bacteria Species and MR1 Dependent T Cell Presentation[END_REF], suggesting that MAIT-stimulating bacteria can be found in the ileum. We analysed expression of the riboflavin

RESULTS

Part II: Characterization of MAIT cells in the ileal environment 92 ribD gene in the feces of mice, using a published metatranscriptomic dataset (Ilott et al. 2016b).

Except for Prevotellaceae and Deferribacteraceae, all the families contributing to ribD expression in the feces including strong ribD contributors such as Muribaculaceae, Lachnospiraceae, Lactobacillaceae, Desulfovibrionaceae, Bacteroidaceae and Clostridiaceae (Fig. 4c), were as abundant in the ileum as in the feces (Fig. 4d). The high MAIT TCRstimulators belonging to Enterobacteriaceae were enriched in the ileum (Fig. 4d). Although the total number of bacteria is lower in the ileum (Fig. 4a), Tastan et al. observed no significant correlation between the bacterial counts and the level of MAIT antigens they secreted in vitro [START_REF] Tastan | Tuning of Human MAIT Cell Activation by Commensal Bacteria Species and MR1 Dependent T Cell Presentation[END_REF]. Thus, the ileum may be populated by strong MAIT-TCR stimulators.

However, further experiments are needed to directly assess bacterial transcriptional activity in the ileum.

93 To test this hypothesis, we compared the ability of total cells from the thymus (positive control), small intestine and colon lamina propria to capture and present synthetic 5-OP-RU in vitro. In contrast to thymic and colon lamina propria cells harvested from Mr1 +/+ mice, small intestine lamina propria cells were not efficient at activating reporter cells in vitro (Fig. 5a).

These results suggested low MR1 expression in the small intestine, which correlates with the low MAIT TCR stimulations in the ileum (Fig. 3f,g). We further investigated whether MR1 expression could impact on MAIT cell activation in the ileum. We used the transgenic LSL-MR1

x Villin-CreER mouse that we generated to increase MR1 expression in gut epithelial cells. LSL-MR1 x Villin-CreER mice were crossed to the Nr4a1-GFP reporter mouse to assess TCR signalling in MAIT cells after MR1 induction with tamoxifen. Inducible MR1 expression elicited TCR signalling in MAIT cells from the ileum, and increased TCR signalling in colon MAIT cells (Fig. 5b). Although no MAIT antigens were detected in the ileum contents using the in vitro assay (Fig. 2), these data suggested that some MAIT ligands were produced in the ileum, possibly at concentrations below the detection limits of our in vitro assay. Robust TCR signalling in MAIT cells from the ileum resulted in reduced RORγt and increased Tbet expression (Fig. 5c). These results indicated that Tbet expression in intestinal MAIT cells is a consequence of TCR triggering by riboflavin-derived metabolites. These results are consistent with previous data showing increased Tbet expression in lung MAIT cells after infection with bacteria that encode the riboflavin pathway (H. Wang et al. 2019). In the colon, MAIT cells were constantly activated through TCR, which resulted in the basal expression of Tbet. Further increase in MR1 expression did not change MAIT cell effector programs in the colon (Fig. 5c).

RESULTS

Part II: Characterization of MAIT cells in the ileal environment 95 Finally, we asked whether additional factors drive phenotypic differences in ileal MAIT cells. The adaptor MyD88 is required for TLR and IL1R family signalling and contributes to the maintenance of intestinal homeostasis [START_REF] Frantz | Targeted Deletion of MyD88 in Intestinal Epithelial Cells Results in Compromised Antibacterial Immunity Associated with Downregulation of Polymeric Immunoglobulin Receptor, Mucin-2, and Antibacterial Peptides[END_REF]. We examined whether MAIT cell functional program was altered in the gut of MyD88 -/-mice. In the ileum, MyD88 -/-mice had a marked reduction in the proportion of RORγt + MAIT cells, and increased Tbet + MAIT cells compared with WT mice (Fig. 6a). In contrast, in the colon, there was no difference in MAIT cell expression of RORγt and Tbet in WT versus MyD88 -/-mice (Fig. 6b). These experiments need to be repeated, and may suggest a role of MyD88 for the maintenance of RORγt expression by MAIT cells in the ileum. NOD-like receptor (NLR) family member, Nlrp6, is essential for the production of mature IL-18 [START_REF] Elinav | NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis[END_REF], and plays a critical role in maintaining gut homeostasis (G. Y. [START_REF] Chen | A Functional Role for Nlrp6 in Intestinal Inflammation and Tumorigenesis[END_REF]. By contrast with MyD88, NLRP6 did not affect MAIT cell functional program in the ileum and the colon (Fig. 6c-d). Collectively, the ileum is characterized by high levels of oxygen at steady state and is colonized by a range of bacteria including bacteria producing high concentrations of MAIT antigens in vitro. However, MAIT cells are not activated through TCR engagement and express distinct phenotypic profiles in the ileum. MAIT TCR stimulation may be muted by low levels of MR1 expression in the ileum. Alternatively, dietary MAIT antagonists such as folic acid-derived metabolites may be more abundant in the ileum than in the colon and may compete with microbial antigens. Further experiments will be necessary to define how MAIT cells are inhibited in the ileum.

Potential of oxygen to induce riboflavin over-production 1.

The in vitro MAIT-TCR activation assay we used enabled us to quantify MAIT antigens in the gut. Although mucosal scraps contained fewer bacteria compared to the lumen, we found significantly increased amounts of riboflavin pathway-derived MAIT antigens in the mucosa.

Consistent with our observation, the riboflavin biosynthesis pathway was enriched in the macaque mucosal community as compared to the lumen [START_REF] Yasuda | Biogeography of the Intestinal Mucosal and Lumenal Microbiome in the Rhesus Macaque[END_REF]. Riboflavin derivatives such as FMN and FAD contribute to bacterial respiration and oxidative stress resistance, thus bacterial production of riboflavin at the oxic-anoxic interface near the intestinal epithelium may reflect a mechanism by which the gut microbiome responds to oxygen and oxidative stress, or a selection of the bacteria able to survive in these environmental conditions. This notion was supported by the data showing over-expression of the enzymes responsible for riboflavin production upon air exposure in vitro and upon hypoxia disruption in vivo. Thus, we propose that riboflavin over-production by intestinal bacteria is a consequence of increased oxygen in the gut environment. However, we acknowledge that more experiments are necessary to get a better insight. As described earlier, butyrate is the preferred energy substrate for colonocytes and the primary determinant of physiologic hypoxia in the colon by promoting intestinal epithelial O2 consumption. Antibiotic treatment and colitis are associated with a depletion of butyrate-producing bacteria [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF][START_REF] Sokol | Low Counts of Faecalibacterium Prausnitzii in Colitis Microbiota[END_REF], which results in the loss of epithelial hypoxia and in increased amounts of oxygen emanating from the mucosal surface [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF]Litvak, Byndloss, and Bäumler 2018). Even though butyrate supplementation was able to restore hypoxia [START_REF] Byndloss | Microbiota-Activated PPARγ-Signaling Inhibits Dysbiotic Enterobacteriaceae Expansion[END_REF], it did not abrogate the increase in MAIT antigens following antibiotic treatment (data not shown). To get a better insight, staining for hypoxia and metagenomic and metatranscriptomic profiling of the gut microbiome after butyrate supplementation would be necessary in future studies.

We and others [START_REF] Neumann-Schaal | Tracking Gene Expression and Oxidative Damage of O2-Stressed Clostridioides Difficile by a Multi-Omics Approach[END_REF] have provided evidence for the potential of oxygen to induce riboflavin biosynthesis gene expression in the strict anaerobes C. difficile. The induction of riboflavin seems to be a conserved response in Clostridia under microaerobiosis, as C. butyricum and C. perfingens overexpressed also riboflavin genes upon air exposure.

Although expression of ribD in Streptococcus pneumoniae isolates correlated with MAIT cell response [START_REF] Hartmann | Riboflavin Metabolism Variation among Clinical Isolates of Streptococcus Pneumoniae Results in Differential Activation of Mucosal-Associated Invariant T Cells[END_REF]), suggesting differences in the production of MAIT antigens, we did not detect higher frequencies of MAIT antigens in the supernatants of C. difficile and C. perfingens after ribD over-expression (data not shown). One possible explanation is that these bacteria do not produce or release sufficient amounts of metabolites that are required for the activation of reporter cells in vitro. Mass spectrometry analysis of recombinant MR1 protein refolded in the presence of bacterial supernatant could be an alternative way to detect MAIT antigens in the supernatants. The enzymes responsible for riboflavin metabolism (RibD, RibH, RibB, and RibE) are encoded on a single operon in Clostridia. Thus, it is also plausible that the absence of increased riboflavin derived metabolites in bacterial supernatants could result from over-expression of ribB or RibE, which encodes the enzyme that consumes 5-A-RU. Accordingly, over-expression of ribB by a strain of Salmonella typhimurium (strain ST313 lineage 2) results in lower production of MAIT antigens, and has been proposed as an escape mechanism (Preciado-Llanes et al. 2020).

Modifications in bacterial community structure and MAIT cell 2. activation in human diseases

An important finding in colitic mice was the bloom of Bacteroidaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) which we and others [START_REF] Tastan | Tuning of Human MAIT Cell Activation by Commensal Bacteria Species and MR1 Dependent T Cell Presentation[END_REF]) have found to produce high levels of MAIT antigens in vitro, as compared to bacterial species from Firmicutes phylum. It is also worth noting that in contrast to Firmicutes, almost all Bacteroidetes and Proteobacteria encode the riboflavin pathway [START_REF] Magnúsdóttir | Systematic Genome Assessment of B-Vitamin Biosynthesis Suggests Co-Operation among Gut Microbes[END_REF]. Interestingly, the Bacteroidetes to Firmicutes ratio is perturbed in patients with Crohn's disease who exhibit an increase in the Bacteroidetes to Firmicutes ratio compared with healthy controls, as well as an increase in Enterobacteriaceae [START_REF] Wright | Recent Advances in Characterizing the Gastrointestinal Microbiome in Crohn's Disease: A Systematic Review[END_REF]. Colorectal adenoma, the precursor for the majority of colorectal cancer (CRC), and CRC cancer are other examples of such a shift in gut microbiota [START_REF] Lu | Mucosal Adherent Bacterial Dysbiosis in Patients with Colorectal Adenomas[END_REF]T. Wang et al. 2012). Although COVID-19 is primarily a respiratory illness, gut microbiota composition of patients with COVID-19 during hospitalization and after recovery is significantly altered. Particularly, members of the Bacteroidetes were enriched in patients with COVID-19 [START_REF] Yeoh | Gut Microbiota Composition Reflects Disease Severity and Dysfunctional Immune Responses in Patients with COVID-19[END_REF]). Thus, we speculate that the increase in ratio of high-stimulator to low-stimulators of MAIT cells could be associated with increased concentrations of MAIT antigens and could be one of the reasons for the activation of MAIT cells in IBDs [START_REF] Serriari | Innate Mucosal-Associated Invariant T (MAIT) Cells Are Activated in Inflammatory Bowel Diseases[END_REF][START_REF] Haga | MAIT Cells Are Activated and Accumulated in the Inflamed Mucosa of Ulcerative Colitis[END_REF][START_REF] Tominaga | Possible Involvement of Mucosal-Associated Invariant T Cells in the Progression of Inflammatory Bowel Diseases[END_REF], CRC [START_REF] Berzins | A Role for MAIT Cells in Colorectal Cancer[END_REF], and patients with COVID-19 [START_REF] Flament | Outcome of SARS-CoV-2 Infection Is Linked to MAIT Cell Activation and Cytotoxicity[END_REF], although the levels of IL-12 and IL-18 are elevated and likely activate MAIT cells (van [START_REF] Wilgenburg | MAIT Cells Are Activated during Human Viral Infections[END_REF]).

On the other hand, numerous studies have investigated gut microbial communities in obese individuals, but the results have not always been consistent. In a majority of studies, obesity is associated with a decrease in Bacteroidetes while Firmicutes increase in both humans and animal models (reviewed in [START_REF] Abenavoli | Gut Microbiota and Obesity: A Role for Probiotics[END_REF]). Only one in three studies reported greater abundance of Enterobacteriaceae in obese participants [START_REF] Peters | A Taxonomic Signature of Obesity in a Large Study of American Adults[END_REF][START_REF] Turnbaugh | An Obesity-Associated Gut Microbiome with Increased Capacity for Energy Harvest[END_REF][START_REF] Zhang | Human Gut Microbiota in Obesity and after Gastric Bypass[END_REF]. The increase in the ratio of Firmicutes to Bacteroidetes could explain the low abundance of genes of riboflavin biosynthesis enzymes in obese mice [START_REF] Toubal | Mucosal-Associated Invariant T Cells Promote Inflammation and Intestinal Dysbiosis Leading to Metabolic Dysfunction during Obesity[END_REF]). Post-gastric-bypass individuals had an increase of Proteobacteria particularly Enterobacteriaceae [START_REF] Zhang | Human Gut Microbiota in Obesity and after Gastric Bypass[END_REF]) and increase circulating MAIT cell frequency, which was associated with amelioration in patient's metabolic and inflammatory status [START_REF] Magalhaes | Mucosal-Associated Invariant T Cell Alterations in Obese and Type 2 Diabetic Patients[END_REF].

An important question is still open as to whether increased MAIT antigen production upon colitis results from upregulation of rib genes or from the expansion of strong producers of MAIT antigens. The two possibilities are not mutually exclusive. To address this question, combining metagenomic and metatranscriptomic profiling of the gut microbiome would be valuable in future studies to assess modifications to both bacterial community structure and transcriptional activity.

Intestinal permeability to MAIT antigens 3.

A striking finding from our study was the rapid and efficient transfer of 5-OP-RU MAIT antigen across the colonocyte lipid bilayer membrane and the whole intestinal barrier. A previous study from the lab showed that 5-OP-RU delivered by oral gavage is detected shortly after in the thymus [START_REF] Legoux | Microbial Metabolites Control the Thymic Development of Mucosal-Associated Invariant T Cells[END_REF]. These findings are consistent with known properties of other microbiota-derived metabolites, such as microbial SCFAs which can reach distant host tissues [START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF]. It is important to note that 5-OP-RU is hydrophilic and may require a transporter given that passive diffusion through the lipid bilayer membrane is low for hydrophilic molecules. Nr4a1-GFP expression by MAIT cells in the mLN and liver, suggests a scenario where riboflavin precursor adducts cross the intestinal barrier to reach lymph circulation and portal blood before reaching the general blood circulation. This pathway may lead to a progressive dilution of MAIT antigen concentration and could explain the low TCR signalling in MAIT cells from the spleen at steady state. Nonetheless, upon antibiotic treatment and colitis, where the microbiota produced high concentrations of MAIT antigens, MAIT cells received strong TCR signals in the colon and distant tissues such as spleen.

MR1-dependent and -independent activation of MAIT cells upon 4. colitis

This is the first demonstration that the activation of MAIT cells upon colitis involves cognate interaction with their restricting molecule, MR1. In line with published data showing MAIT cell expression of genes linked with the tissue-repair program upon TCR stimulation [START_REF] Hinks | Activation and In Vivo Evolution of the MAIT Cell Transcriptome in Mice and Humans Reveals Tissue Repair Functionality[END_REF][START_REF] Lamichhane | TCRor Cytokine-Activated CD8+ Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses[END_REF][START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF], we demonstrated that MAIT TCR activation induced the production of Areg, an important mediator of tissue repair at mucosal sites [START_REF] Zaiss | Emerging Functions of Amphiregulin in Orchestrating Immunity, Inflammation and Tissue Repair[END_REF]. However, given that Areg is involved in epithelial malignancies [START_REF] Chayangsu | The Correlations between Serum Amphiregulin and Other Clinicopathological Factors in Colorectal Cancer[END_REF][START_REF] Normanno | Amphiregulin Anti-Sense Oligodeoxynucleotides Inhibit Growth and Transformation of a Human Colon Carcinoma Cell Line[END_REF], chronic MAIT TCR stimulation could be deleterious. This raises the possibility that MAIT cells could be helpful or harmful, depending on the microenvironment they are in, or depending on the duration of TCR stimulation.

From the data obtained, we would argue that both MR1-dependent and -independent activation [START_REF] Ghilardi | Compromised Humoral and Delayed-Type Hypersensitivity Responses in IL-23-Deficient Mice[END_REF]) have been imported in our animal facility and will be studied.

Role of MAIT cells in the inflamed gut 5.

Histopathological analysis showed that MAIT cells attenuate DSS-induced inflammation. Owing to the known tissue-protective properties of effector molecules they produce, MAIT cells likely interact with intestinal epithelial cells and preserve the intestinal barrier. This suggestion is corroborated by the loss of small intestine integrity in mice lacking MAIT cells [START_REF] Rouxel | Dual Role of Mucosal-Associated Invariant T Cells in Type 1 Diabetes[END_REF] and by the in vitro [START_REF] Leng | TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions[END_REF] and in vivo [START_REF] Constantinides | MAIT Cells Are Imprinted by the Microbiota in Early Life and Promote Tissue Repair[END_REF]) results demonstrating a role of MAIT cells in wound healing.

Whether MAIT cells interact with other immune cells in the inflamed gut, which in return maintain homeostasis, has not been determined. Previous study has shown that γδT17 cells, through IL-17, are important in the recruitment of immunosuppressive Gr-1 + CD11b + myeloid cells to the inflamed gut lesions [START_REF] Sun | Innate ΓδT17 Cells Play a Protective Role in DSS-Induced Colitis via Recruitment of Gr-1+CD11b+ Myeloid Suppressor Cells[END_REF]). Thus, it is possible that MAIT cells promote inflammation resolution through a new immune pathway involving myeloid cells recruitment to the site of colitis inflammation.

On the other hand, we speculate that MAIT cells can reciprocally regulate the microbiota through different mechanisms. For example, MAIT-derived IFNγ, IL-17 and IL-22 can influence the compartmentalization of the intestinal microbiota by inducing expression of mucins and by promoting the secretion of AMPs. Indeed, mice lacking the large gel-forming mucin Muc2 or the antibacterial lectin RegIIIγ, lack spatial segregation of commensal bacteria [START_REF] Johansson | The Inner of the Two Muc2 Mucin-Dependent Mucus Layers in Colon Is Devoid of Bacteria[END_REF][START_REF] Vaishnava | The Antibacterial Lectin RegIIIgamma Promotes the Spatial Segregation of Microbiota and Host in the Intestine[END_REF]. It is also possible that MAIT cells inhibit the expansion of colitogenic bacteria, such as E. coli (Y.-L. [START_REF] Huang | Sialic Acid Catabolism Drives Intestinal Inflammation and Microbial Dysbiosis in Mice[END_REF], by preserving the integrity of the intestinal epithelial barrier and thus by promoting oxygen consumption by colonocytes and hypoxia. Analysis of commensal microbiota composition in colitic mice deficient or not in MAIT cells would be valuable in future studies to address these possibilities. Only few studies are available that have characterized gut microbiota composition in mice deficient in MAIT cells.

Smith et al. showed a greater proportion of particularly Bacteroidaceae and Lactobacilliaceae in

Mr1 -/-mice as compared to WT mice (A. D. [START_REF] Smith | Microbiota of MR1 Deficient Mice Confer Resistance against Clostridium Difficile Infection[END_REF]. However, in that study, microbiota differences were abrogated upon co-housing of WT and Mr1 -/-mice, suggesting that the differences observed were weak and due to original breeding location. It is also worth noting that we did not find any differences in MAIT ligand concentration in the gut between WT and Mr1 -/-mice (data not shown).

MAIT cells in the ileal environment 6.

A striking observation from our study was the low TCR signals in MAIT cells from the ileum.

Because ileal and colon samples were processed similarly at the same time, the observed difference is probably not an experimental artefact. One possible explanation for the low TCR signals in MAIT cells from the ileum as compared to the colon, was the low expression of MR1.

However, further microscopy and in vitro assay experiments are needed to investigate MR1 expression along the gut and to identify the antigen presenting cells. Although inducible MR1 expression in intestinal epithelial cells elicited TCR signalling in MAIT cells from the ileum, MAIT TCR signals in the ileum were lower than in the colon. These data and MAIT cell activation in bioassays suggest low production of MAIT antigens in the ileum. We envision that genes controlling production of the MAIT ligand precursor in some bacteria in the ileum are attenuated by riboflavin originating from food. It is worth noting that some bacteria express riboflavin transporter proteins [START_REF] Gutiérrez-Preciado | Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species[END_REF] and could bypass the need for de novo synthesis of riboflavin which is regulated by FMN riboswitch [START_REF] Pedrolli | The RibB FMN Riboswitch from Escherichia Coli Operates at the Transcriptional and Translational Level and Regulates Riboflavin Biosynthesis[END_REF][START_REF] Vitreschak | Regulation of Riboflavin Biosynthesis and Transport Genes in Bacteria by Transcriptional and Translational Attenuation[END_REF]. Analysis of bacterial transcriptional activity along the gut of mice fed a normal or riboflavin-deficient diet would be valuable in future studies to address this possibility. Riboflavin transporters are observed in Firmicutes but not in Bacteroidetes [START_REF] Gutiérrez-Preciado | Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species[END_REF] which could explain the high and constant production of riboflavin by species belonging to Bacteroidetes. It is also plausible that food-derived antagonists such as folate-derived metabolites, compete with the microbial antigens for binding to MR1 in the small intestine.

Contact of intestinal epithelial cells (IECs) with MAIT cells in the 7. intestinal mucosa

While MAIT cells from the lamina propria exist below the basement membrane, in this study we showed that transgenic MR1 expression in intestinal epithelial cells elicited strong TCR signalling in MAIT cells from the lamina propria. This could be explained by IECs contact with MAIT cells in the underlying lamina propria via basolateral projections through the semi-porous basement membrane. In this regard, evidence from a number of studies show that IECs can transfer information from luminal microorganisms and antigens by directly processing and presenting them in highly polarized fashion, to lymphocytes in the underlying lamina propria [START_REF] Dahan | Epithelia: Lymphocyte Interactions in the Gut[END_REF][START_REF] Hershberg | Antigen Processing and Presentation by Intestinal Epithelial Cells -Polarity and Complexity[END_REF].

Tbet and RORγt coexpression in the lamina propria 8.

Surprisingly, we found that many MAIT cells co-expressed Tbet and RORγt in the lamina propria, which has not been reported yet in mice at steady-state [START_REF] Rahimpour | Identification of Phenotypically and Functionally Heterogeneous Mouse Mucosal-Associated Invariant T Cells Using MR1 Tetramers[END_REF]Salou et al. 2019), but found in human MAIT cells from the thymus and periphery [START_REF] Leeansyah | Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection[END_REF]. This suggests a form of plasticity within MAIT17 lineage that occurs post-thymically and in a tissue-specific manner. The importance of Tbet in MAIT17 cells is currently unknown.

However, the co-expression of Tbet suggests that MAIT cell may acquire type 1 transcriptional and functional traits which may give the cell an advantage for fighting infections. Regulatory cues governing Tbet expression in MAIT17 are to be defined. The current study shows that expression of Tbet was independent of MyD88 but depends on TCR signalling. Further experiments using floxed MR1 mouse that we recently generated are needed to better investigate such an aspect. Previous studies have provided evidence that STAT5B signalling favors Tbet-expressing γδT17 cells in the lamina propria [START_REF] Kadekar | The Neonatal Microenvironment Programs Conventional and Intestinal Tbet+ ΓδT17 Cells through the Transcription Factor STAT5[END_REF], raising the possibility that the gut-specific cytokine milieu that activate STAT5 may also regulate Tbet expression in MAIT cells.

Conclusion and therapeutic prospects

In summary, we show that intestinal inflammation drives riboflavin production and the accumulation of metabolite antigens captured by MR1 and presented to MAIT cells in the lamina propria, which provide tissue-repair mediators in return. Overproduction of riboflavin by the microbiota may result from increased oxygen concentration in the colon. The study helps understand how the host immune system monitors and responds to altered microbial metabolism during intestinal inflammation. Our findings establish a new molecular interaction between the microbiota and mammalian hosts. The identification of the riboflavin pathway as a functional marker of colitis may explains why MAIT cells recognize riboflavin metabolites.

The demonstration that MAIT cells protect against colitis may facilitate the development of novel therapeutic approaches in inflammatory bowel diseases. Conditional MR1 transgenic mice (LSL-MR1) crossed to the Villin-Cre mice will be very useful in future experiments to assess the contribution of MAIT TCR activation to protection from colitis. The preparation of MAIT antigens in DMSO was found to improve its stability [START_REF] Mak | Stabilizing Short-Lived Schiff Base Derivatives of 5-Aminouracils That Activate Mucosal-Associated Invariant T Cells[END_REF]) and allows their utilization in preclinical and clinical settings. Therefore, we will assess the therapeutic potential of activating MAIT cells with a stimulating ligand and the effect of blocking MAIT cells function with a nonactivating ligand. It is worth noting that in contrast to T cells which are often difficult to exploit therapeutically because the diversity of the TCR repertoire, MAIT cells are an attractive target because they express a semi-invariant TCR that binds to well characterized antigens.

Our findings pave the way to further elucidate the interaction between the gut microbiota and MAIT cells in other pathologies associated with dysbiosis such as colorectal cancer. Previous studies have shown that MAIT cells are present in tumors, and that they are capable of cytokine release in response to in vitro stimulation [START_REF] Berzins | A Role for MAIT Cells in Colorectal Cancer[END_REF]. There is an ongoing debate about whether (1) MAIT cell activation in tumors occurs through direct recognition of microbial antigens or via TCR-independent activation, (2) MAIT cell activation promotes malignancy or inhibit cancer progression. MAIT cells from CRC patients can release IFNγ [START_REF] Ling | Circulating and Tumor-Infiltrating Mucosal Associated Invariant T (MAIT) Cells in Colorectal Cancer Patients[END_REF] which has been shown to control cancer progression by inducing senescence in cancers [START_REF] Braumüller | T-Helper-1-Cell Cytokines Drive Cancer into Senescence[END_REF].

On the other hand, MAIT cells can also express IL-17 [START_REF] Ling | Circulating and Tumor-Infiltrating Mucosal Associated Invariant T (MAIT) Cells in Colorectal Cancer Patients[END_REF]) which can promote cancer progression [START_REF] Grivennikov | Adenoma-Linked Barrier Defects and Microbial Products Drive IL-23/IL-17-Mediated Tumour Growth[END_REF]. Our study showed that MAIT cells can produce Areg when stimulated in a TCR-dependent manner. While Areg is important in tissue repair, it can also promote tumor growth [START_REF] Chayangsu | The Correlations between Serum Amphiregulin and Other Clinicopathological Factors in Colorectal Cancer[END_REF]. Therefore, studies are required to address the question of the role of MAIT cells in CRC and the possible correlations between their activation and the course of the disease. Depending on whether MAIT cells are beneficial or deleterious in CRC, they can be activated or inhibited in therapeutic settings.

Les lymphocytes MAIT et la riboflavine dans les interactions hôte-microbiote

Figure 1 :

 1 Figure 1: Neighbor-joining tree based on similarity scores of the α1-α2 domains of MR1, CD1 and classical MHC proteins in mammals ...................................................................................17

Figure 2 : 18 Figure 3 : 18 Figure 4 : 21 Figure 5 : 22 Figure 6 : 23 Figure 7 : 26 Figure 8 : 28 Figure 9 : 29 Figure 10 :

 21831842152262372682892910 Figure 2: Inactivation or loss of Mr1 in several groups of mammals .........................................18 Figure 3: The Intracellular Pathway of MR1 .............................................................................18Figure 4: Riboflavin biosynthesis pathway and formation of MAIT antigens in bacteria ............21 Figure 5: The FMN riboswitch regulates riboflavin genes in bacteria ........................................22 Figure 6:The three redox states of flavin mononucleotide ........................................................23 Figure 7: TCR recognition of MR1-5-OP-RU. ..........................................................................26 Figure 8: MAIT TCR recognition of MR1-binding ligands derived from riboflavin ......................28 Figure 9: Intra-thymic development of MAIT cells .....................................................................29 Figure 10: Responses of human MAIT cells to TCR and cytokine stimulation. .........................35

Figure 11 :

 11 Figure 11: MAIT cells in autoimmune and immunological diseases ..........................................43

Figure 12 : 45 Figure 13 :

 124513 Figure 12: Human fecal microbiota composition from early life to adulthood ............................45 Figure 13: Microbiota-derived butyrate metabolism maintains anaerobiosis in a virtuous circle 46

Figure 14 : 47 Figure 15 : 49 Figure 16 : 50 Figure 17 :

 14471549165017 Figure 14: Oxygen gradient along the length of the gut ............................................................47 Figure 15: Spatial heterogeneity of the gut microbiota in the intestinal tract .............................49 Figure 16: Plausible source of H2O2 in the oxic-anoxic interfaces near the intestinal epithelium .................................................................................................................................................50 Figure 17: Increased epithelial oxygenation in inflamed tissues ...............................................52

Figure 1 :

 1 Figure 1: Neighbor-joining tree based on similarity scores of the α1-α2 domains of MR1, CD1 and classical MHC proteins in mammals. Boudinot et al showed a strong conservation of MR1 sequences evolving more slowly than other MCH1-like proteins (Boudinot et al. 2016).

Figure 2 :

 2 Figure 2: Inactivation or loss of Mr1 in several groups of mammals.In Carnivora (Cat, Dog, Panda, Ferret) and Armadillo, the MR1 region is conserved but MR1 is a pseudogene (red sphere). A functional Mr1 is also missing in the rabbit and pika in which the MR1 region has been lost. Chicken and Zebrafish do not have Mr1.

Figure 3 :

 3 Figure 3: The Intracellular Pathway of MR1. MR1 surface expression and antigen presentation are almost strictly dependent on antigen availability within the ER (McWilliam and Villadangos 2017).

Figure 5 :

 5 Figure 5: The FMN riboswitch regulates riboflavin genes in bacteria. (A) Transcription attenuation; (B) translation attenuation[START_REF] Vitreschak | Regulation of Riboflavin Biosynthesis and Transport Genes in Bacteria by Transcriptional and Translational Attenuation[END_REF]

Figure 6 :

 6 Figure 6:The three redox states of flavin mononucleotide.

Figure 7 :

 7 Figure 7: TCR recognition of MR1-5-OP-RU (Le Nours et al. 2019). (A) MAIT αβTCR and (B) γδTCR footprint on surface MR1.

Figure 8 :

 8 Figure 8: MAIT TCR recognition of MR1-binding ligands derived from riboflavin. Two-dimensional diagram interaction summary of the crystal structure of the MAIT TCR-MR1-5-OP-RU complex. MR1 and TCR residues and their hydrogen bonds are colored in orange and maroon, respectively (Awad et al. 2020). Structural characterization of MAIT TCR recognition of MR1-binding (B) 5-OP-RU, (C) 6-FP (Eckle et al. 2015).

  , PLZF induces in MAIT cells the acquisition of effector programs associated with the transcription factors Tbet or RORγt which define the INTRODUCTION Chapter I: Mucosal associated invariant T cells 29

Figure 9 :

 9 Figure 9: Intra-thymic development of MAIT cells (Legoux, Salou, and Lantz 2020). During thymic development, both (A) TECs and (B) DP thymocytes can mediate positive selection of 5-OP-RU-specific thymocytes, but with distinct outcomes.

INTRODUCTION 35 Figure 10 :

 3510 Figure 10: Responses of human MAIT cells to TCR and cytokine stimulation (Figure adapted from (Lamichhane et al. 2019)). (A) TCR-independent activation results in a slow and modest cytokine response dominated by IFN-γ under the control of Tbet. (B) TCRdependent activation induces a rapid and stronger response. TCR stimuli produces broad pro-inflammatory cytokines and chemokines dominated by TNF-α under the control of Rorγt and Tbet. TCR and not cytokine triggered MAIT cells results in the expression of tissue repair signature.

INTRODUCTION

  Chapter I: Mucosal associated invariant T cells40 In experimental autoimmune encephalomyelitis (EAE), the mouse model of MS, Vα19 + cells seem to protect mice from clinical EAE. In fact, adoptive transfer of invariant Vα19 transgenic cells into C57BL/6 mice with EAE reduces the disease severity. In addition, similar regulatory effect was observed in Va19 transgenic mice compared to MR1-/-mice. The suppression of EAE was associated with reduced expression of proinflammatory cytokines (IFN-γ, TNF, IL-2

INTRODUCTION

  Chapter I: Mucosal associated invariant T cells 43 MS, T1D and IBDs are associated with important alterations in the composition and function of the gut microbiota (Honda and Littman 2016), which may impact on the production or availability of riboflavin metabolite antigens for MAIT cells. Because of the strong link between microbial groups imbalance and health impairment or diseases, the following section will discuss the current understanding of the host-microbiota interactions.

Figure 11 : 44 Chapter

 1144 Figure 11: MAIT cells in autoimmune and immunological diseases. MAIT cells are reduced in the blood and accumulate in inflamed tissues, including central nervous system, pancreatic islets and intestine. The protective roles of MAIT cells were demonstrated in animal models of multiple sclerosis and type 1 diabetes. The possible contribution of MAIT cells to pancreatitis was demonstrated in T1D. (H= Human; m= mouse).

Figure 12 :

 12 Figure 12: Human fecal microbiota composition from early life to adulthood. Facultative anaerobes Enterobacteriaceae and Lactobacillaceae are the first colonizers of the gut after birth. During the following 6 months, obligate anaerobes, including Bifidobacteriaceae and Clostridiaceae, begin to accumulate in the gut. Butyrate-producing Clostridia decrease epithelial oxygenation in the colon and allow for the colonization of Bacteroidaceae, Porphyromondaceae, Lachnospiraceae, and Ruminococcacea in the adult gut (Sanidad and Zeng 2020).

Figure 13 :

 13 Figure13: Microbiota-derived butyrate metabolism maintains anaerobiosis in a virtuous circle. Butyrate, which is produced from fermentation of undigested carbohydrates and protein, enters the differentiated colonocytes via the monocarboxylate transporter 1 (MCT1). Butyrate induces PPAR-γ-dependent activation of mitochondrial βoxidation. By analogy with the metabolic programming of M2 macrophages, coloncytes having increased fatty acid oxidation are in a C2-metabolic state. C2-colonocytes consume oxygen and therefore maintains anaerobiosis and the dominance of obligate anaerobic bacteria in the microbial community. SC, stem cell; TA, undifferentiated transitamplifying cell; C2, terminally differentiated C2-skewed colonocyte; GC, goblet cells(Litvak, Byndloss, and Bäumler 2018).

Figure 14 :

 14 Figure 14: Oxygen gradient along the length of the gut. Intestine from hypoxia-inducible factor-luciferase reporter mouse (L. Zheng, Kelly, and Colgan 2015).

INTRODUCTION 50 Figure 16 :

 5016 Figure 16: Plausible source of H2O2 in the oxic-anoxic interfaces near the intestinal epithelium (Imlay 2019).

Figure 17 :

 17 Figure 17: Increased epithelial oxygenation in inflamed tissues. To repair colon epithelial injury, pericryptal myofibroblasts release R-spondin 2, which stimulates division of undifferentiated transitamplifying (TA) cells. Accumulation of dividing TA cells leads to crypt elongation, increased epithelial oxygenation, and expansion of facultative anaerobic bacteria (Litvak, Byndloss, and Bäumler 2018).

  absorptive epithelial cells that are interspersed by specialized cells called Paneth cells and goblet cells. Paneth cells are enriched in the bases of the small intestinal crypts and secrete an array of AMPs including defensins and C-type lectins, whereas goblet cells are highly abundant in the large intestine and continuously produce the gel-forming mucin to maintain a functional mucus layer. The mucus layer in the large intestine is composed of two distinct layers: an inner INTRODUCTION Chapter II: Intestinal microbiota and interactions with the host 54 mucus layer that contains large concentrations of antimicrobial factors, and an unattached outer mucus layer. However, the small intestine harbors a thinner and discontinuous mucus layer.The production of AMPs is induced by the activation of pattern recognition receptors (TLRs and NLRs) by microbial products, and in response to cytokines secreted by immune cells in the surrounding milieu[START_REF] Levy | Dysbiosis and the Immune System[END_REF]. The release of mucin by goblet cells is triggered by immune mediators such as IFN-γ(Klose et al. 2013a). Through the secretion of antimicrobial peptides and mucus the host maintains bacteria away from the epithelium (De Weirdt and Van de Wiele 2015;[START_REF] Levy | Dysbiosis and the Immune System[END_REF]. Indeed, the mucus layer is characterized by low bacterial density compared to the lumen, and is restricted to bacterial species that can survive host antimicrobial strategies and can resist to ROS produced by aerobic metabolism.Moreover, as a result of exposure to intestinal microbes, B cells residing in the intestinal mucosa induce secretory IgA antibody which decreases microbial motility, accelerates in vivo intestinal transit, and limits exposure of the mucosa to microbial products[START_REF] Uchimura | Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response[END_REF]).

  into the small intestine to facilitate absorption of lipids or fat-soluble vitamins. In the colon, BAs that escape active transport in the distal ileum are transformed by bacteria to secondary BAs (G. Wang et al. 2019). Song et al. observed that mice fed with minimal-diet had lower levels of fecal primary and secondary BAs with lower number of colonic RORγt + Treg than did rich-diet INTRODUCTION Chapter II: Intestinal microbiota and interactions with the host 57 SPF mice. A combination of secondary BAs restored the number of colonic RORγt + Treg cells in minimal-diet mice, indicating that certain BAs biotransformed by bacteria are essential for the maintenance of colonic RORγt + Treg cells (Song et al. 2020).Another study revealed additional anti-inflammatory roles of secondary BA metabolites by inhibiting Th17 and enhancing Treg cell differentiation, through respectively, a direct binding and inhibition of RORγt protein, and by increasing the expression of FOXP3[START_REF] Hang | Bile Acid Metabolites Control T H 17 and T Reg Cell Differentiation[END_REF].Thus gut-residing bacteria may modulate the balance of Th17 and Treg cells through BA metabolites. Secondary BA supplementation alleviated colitis in minimal-diet mice by increasing the number of RORγt + Treg cells[START_REF] Song | Microbial Bile Acid Metabolites Modulate Gut RORγ + Regulatory T Cell Homeostasis[END_REF]).

  MAIT cells preferentially localize in barrier tissues and recognize microbial metabolites derived from the riboflavin biosynthesis pathway. Intestinal bacteria can produce riboflavin pathwayderived MAIT antigens[START_REF] Tastan | Tuning of Human MAIT Cell Activation by Commensal Bacteria Species and MR1 Dependent T Cell Presentation[END_REF], suggesting a direct interaction between MAIT cells and the microbiota. Nevertheless, the concentration of MAIT antigens produced in the gut, and the mechanisms controlling MAIT antigen production by the microbiota have not been determined. Moreover, how MAIT cells monitor disturbances of the composition and function of the microbiota remains unknown.

Figure 1 :

 1 Figure 1: MAIT cells are resident in the colon and sense mucosal bacterial antigens. a. Left: CD1d:aGC tetramer (iNKT) and MR1:5-OP-RU tetramer staining of B6-MAIT Cast colon lamina propria T cells. Right: Pooled data from five independent experiments. b. Parabiotic CD45.1/2 and CD45.2/2 congenic B6-MAIT Cast mice were examined 5 weeks after establishment. Left: Example of CD45.1 and CD45.2 staining of the indicated T cell subsets in the colon of a CD45.1/2 parabiont. Right: Chimerism of the indicated T cell subsets from n = 4 mice corresponding to two pairs. c. Left: CD25 expression by iNKT and MAIT cells in the colon lamina propria. Right: Pooled data from 4 independent experiments. d. Left: Nr4a1-GFP expression in the indicated cell subsets and tissues; representative of at least three independent experiments. I+B LN: Inguinal and brachial lymph nodes. mLN: mesenteric lymph nodes. Right: Mean fluorescence intensity (MFI) in the indicated cell subsets and tissues (n= 4 mice; representative of at least three independent experiments). e. Left: Scheme of the in vitro assay used to quantify MAIT antigens in intestinal contents. Middle: Activation of MR1:5-OP-RU-specific reporter T cells after overnight incubation with WT3-MR1 cells pulsed with 100 mg of luminal contents from the indicated segment of the large intestine of control mice (vehicle) or of mice treated with an antibiotic (ATB) cocktail for 7d. Right: Relative expression of CD25 and CD69 by reporter T cells in the indicated conditions (n = 3-5 mice per group; representative of two independent experiments). f. Left: Activation of reporter T cells after overnight incubation with WT3-MR1 cells left untreated or pulsed with 50 mg of lumen or mucosal cecal contents. Right: Relative expression of CD25 and CD69 by reporter T cells in the indicated conditions (means SEMs, n = 10 mice; data pooled from two independent experiments), (UD: undiluted). *p<0.05, ** p<0.01 and ****p<0.0001 by Student's t (b, c, e and f) and by Mann-Whitney U test (d).

Figure S1 :Figure 2 :Figure S2 :

 S12S2 Figure S1: MAIT cells are resident in the colon and sense mucosal bacterial antigens. a. Interferon (IFN)-g and Nr4a1-GFP expression in splenic MAIT cells untreated or incubated overnight with interleukin (IL)-12 and -18 or with 5-OP-RU-pulsed WT3-MR1 cells. b. Left: Activation of MR1:5-OP-RU-specific reporter T cells after overnight incubation with WT3-MR1 cells pulsed with 5-OP-RU (100 nM). Right: Relative expression of CD25 and CD69 by reporter T cells in the indicated conditions (data pooled from three independent experiments). c. Staining of reporter T cells after overnight incubation with WT3-MR1 cells pulsed with luminal contents at the indicated dilution. d. Left: Activation of reporter T cells after overnight incubation with WT3-MR1 cells pulsed with 100 mg of luminal contents from the cecum of control (vehicle) or of mice treated for 7d with fluconazole. Right: Relative expression of CD25 and CD69 by reporter T cells in the indicated conditions (means SEMs, n = 3 mice per group). e. Quantification of bacterial numbers in the cecal lumen and mucosa by qPCR amplification of the 16S rRNA gene. Colony-forming unit (CFU) equivalents were estimated using a standard curve with Escherichia coli (data pooled from 2 independent experiments). ** p<0.01 by Mann-Whitney U test.

Figure 3 :IL

 3 Figure 3: Up-regulation of the riboflavin biosynthetic pathway during colitis a. Principal component analysis (PCA) of the riboflavin biosynthesis pathway genes expressed in the feces before (steady state, blue) or after helicobacter-induced colitis (red) (n= 8 mice); in a-c, original data are from ref 31. b. Top: Heatmap showing the genes of the riboflavin biosynthetic pathway found up-regulated in the feces upon colitis induction (p<0.01 by Mann-Whitney U test). Bottom: Scheme of the riboflavin biosynthesis pathway. The genes found up-regulated during colitis are in red. c. Proportion of ribD expression contributed by the indicated bacterial families during colitis as compared with steady-state. Only families contributing 2% or more of total ribD expression are shown. d. Left: Representative images showing PMDZ adduct (red) and DNA (blue) staining of the colon from control (Mock) and DSS-treated mice. Right: Quantification of PMDZ adduct staining at the epithelium surface of the colon (each dot represents one mouse). e. Abundance of the Bacteroidaceae and Enterobacteriaceae families in the cecum of the indicated mice as determined by 16S rRNA gene sequencing. f. Left: Experimental strategy. Right: Numeration of the indicated isogenic WT or DribC E. coli strains after administration to control (Mock) or DSS-treated mice (n= 6 mice per group; data pooled from two independent experiments). g. Abundance of B. thetaiotaomicron in the cecum of the indicated mice, as determined by 16S rRNA gene sequencing. h. Left: Activation of MR1:5-OP-RU-specific reporter T cells after overnight incubation with WT3-MR1 cells pulsed with supernatant from the indicated bacterial cultures. Right: MAIT antigen concentration in the supernatant of the indicated bacterial cultures as estimated using 5-OP-RU (data from 4 biological replicates). i. Left: Activation of reporter T cells after incubation with WT3-MR1 cells pulsed with 50 mg of cecal contents from control (Mock) or DSS-treated mice. Right: Pooled data from two independent experiments (means SEMs, n= 6 mice per group). j. Left: Nr4a1-GFP expression in MAIT cells from the indicated tissues. Right: Data pooled from three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 by Mann-Whitney U (c, d, e, f, g, h, and j) or by Student's t tests (i).

Figure 4 :

 4 Figure 4: MAIT cells protect against colitis. a. Scheme of the lox-stop-lox-MR1 transgenic construct. b. Left: Nr4a1-GFP expression by MAIT cells from the indicated tissues. Both LSL-MR1 + and LSL-MR1 -mice received tamoxifen injections. Right: Pooled data from four independent experiments. c. Left: Areg expression by colonic MAIT cells from the indicated mice. MAIT cells were stained for Areg without in vitro re-stimulation. Right: Pooled data from two independent experiments (n= 7 mice per group). d and e. Left: Representative staining for the indicated proteins of colonic MAIT cells from control (Mock) mice and from mice with chronic colitis. Right: Percentages of colonic MAIT cells expressing the indicated transcription factors (d, n=6 mice per group; representative of two independent experiment) and cytokines (e, n=9-11 mice per group; pooled data from two independent experiments) without in vitro re-stimulation. f. Top: Hematoxylin and eosin staining of the colon of the indicated control (Mock) mice and mice with chronic colitis. Representative 2X and 10X magnifications are shown. Bottom: Summary of the investigator-blinded inflammatory and epithelial lesion scores assessments in the indicated conditions. Summary of villi length measured in the indicated conditions (n=5-7 mice per group; data representative of three independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by Mann-Whitney U (b) or by Student's t tests (c, d, e and f).

Figure S4 :

 S4 Figure S4: MAIT cells protect against colitis. a. Scheme of the chronic DSS-induced colitis model. b. Weight of the indicated mice following mock or DSS treatment. c. Summary of spleen weight in Mr1 +/+ and Mr1 -/-mice in the indicated conditions; in b and c, n=12-13 mice per group; data are representative of three independent experiments. *p<0.05, ns p>0.05, ****p<0.0001 by Student's t tests.

Figure 1 :

 1 Figure 1: Immunohistochemical visualization of pimonidazole adducts. Right: Representative images showing pimonidazole (PMDZ) adduct (red) and DNA (blue) staining of the ileum and colon of healthy mice. Left: Quantification of PMDZ adducts staining at the epithelium surface of the ileum and colon, each dot represents a mouse.

Figure 2 :

 2 Figure 2: Gradient of MAIT antigens in the gut of healthy mice. Left: Representative flow cytometry of MR1:5-OP-RU-specific reporter T cells after overnight incubation with WT3-MR1 cells pulsed with 100 mg of feces or luminal contents from the indicated segments of healthy mice. Right: Summary of the relative expression of CD25 and CD69 on reporter T cells. ***p<0.001 and ****p<0.0001 by Student's t test.

  Characterization of MAIT cells in the ileal environment 90

91 Figure 3 :

 913 Figure 3: Distinct MAIT phenotypic profiles in the ileum. a. Left: Representative flow cytometry of B6-MAIT Cast lamina propria T cells in the ileum and the colon. Right: Summary of CD1d:αGC tetramer+ (iNKT) and MR1:5-OP-RU tetramer+ (MAIT) cell frequencies in the ileum and colon lamina propria. b. CD45.1/2 and CD45.2/2 congenic B6-MAIT Cast mice joined in parabiotic pairs were examined 5 weeks later. Left: Representative flow cytometry of the indicated T cell subsets in the ileum. Right: Summary of chimerism of the indicated T cells subsets. c. Top: Representative flow cytometry of MAIT cell expression of RORγt and Tbet in the ileum and colon of healthy mice. Bottom: Summary of the percentages of MAIT cells expressing the indicated transcription factors in the ileum and the colon. d. Left: Representative flow cytometry of MAIT cells in the ileum and the colon. Right: Summary of RORγt-GFP mean fluorescence intensity (MFI) in the indicated tissues. e. Left: Representative flow cytometry of iNKT and MAIT cells in the ileum and the colon. Right: Summary of CD25+ cell frequencies in the indicated cell subsets and tissues. f. Left: Representative flow cytometry of MAIT cells in the indicated tissues. I+B LN: Inguinal and brachial lymph nodes. mLN: mesenteric lymph nodes. Right: Summary of Nr4a1-GFP MFI in MAIT cells from the indicated tissues. g. Left: Representative flow cytometry of memory (CD44+) CD8+ T cells, iNKT and MAIT cells in the ileum. Right: Summary of Nr4a1-GFP MFI in the indicated cell subsets from the ileum. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by Wilcoxon (a), Student's t (b, c, d and e) and by Mann-Whitney U (f and g) test.

Figure 4 :

 4 Figure 4: The gut microbiota composition along the gut of healthy mice. a. Summary of colony-forming unit (CFU) equivalents per gram in the indicated tissues, determined by qPCR quantification of the 16S rRNA gene. b. Relative abundance of the indicated bacterial phyla in the indicated regions determined by 16S rRNA gene sequencing. c. Heatmap showing ribD expression in the indicated families in fecal samples. Data are expressed as percentage of total ribD expression. Columns indicate individual mice. d. Relative abundance of Muribaculaceae, Prevotellaceae, Lachnospiraceae, Lactobacillaceae, Desulfovibrionaceae, Deferribacteraceae, Bacteroidaceae, Clostridiaceae and Enterobacteriaceae in the indicated regions as determined by 16S rRNA gene sequencing. **p<0.01 by Mann-Whitney U test.

Figure 5 :

 5 Figure 5: MR1 is a limiting factor controlling MAIT cell activation. a. Activation (CD25 and CD69 expression measured by flow cytometry) of C+ Vβ8+ Ca-/-MR1-/-reporter T cells after overnight incubation with total Mr1 +/+ or Mr1 -/-cells from the indicated tissues in the presence or absence of 5-OP-RU. b. Left: Representative flow cytometry of MAIT cells from the indicated tissues. Both LSL-MR1+ and LSL-MR1-mice received tamoxifen injections. Right: Nr4a1-GFP expression in MAIT cells from the indicated tissues. c. Left: Representative flow cytometry of MAIT cell expression of RORγt and Tbet in the ileum and colon of LSL-MR1+ and LSL-MR1mice treated both with tamoxifen. Right: Summary of the percentages of MAIT cells expressing RORγt and Tbet in the ileum and the colon of the indicated mice. ns p>0.5, **p<0.01 and ****p<0.0001 by Mann-Whitney U (b) or by Student's t (c) tests.

Figure 6 :

 6 Figure 6: MyD88 signalling controls MAIT cell functional profile in the ileum. a-d. Left: Representative flow cytometry of MAIT cell expression of RORγt and Tbet in the ileum (a,c) and colon (b,d) of indicated mice. Right: Summary of the percentages of MAIT cells expressing of RORγt and Tbet in the ileum (a,c) and the colon (b,d) of indicated mice. Prior to inclusion in experimental groups, WT (B6) and MyD88 -/-or Nlpr6 -/-mice were co-housed, or cage contents were mixed for at least two weeks in order to normalize microbiota.

  may be involved to trigger robust MAIT cell responses in colitis. Indeed, MAIT cells from colitic mice shift towards RORγt, and show enhanced production of IFNγ, IL-17, IL-22 and Areg in the colon. However, except for Areg, increased expression of RORγt, and cytokine secretion were not observed by colon MAIT cells upon transgenic MR1 expression in intestinal epithelial cells (data not shown), suggesting that stimuli other than the TCR are responsible for IL-17, IFNγ and IL-22 production by MAIT cells during colitis. Previous study has reported that IL-23 induced human Tbet-expressing group 1 innate lymphoid cells (ILCs) differentiation into RORγtexpressing group 3 ILCs, and enhanced IL-22 production[START_REF] Bernink | Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria[END_REF]. In addition, an important role for IL-23 in MAIT cell maintenance of RORgt expression was observed in the lung in a mouse model of infection (H.Wang et al. 2019). These findings and the demonstration that IL-23 is up-regulated in inflamed mucosa[START_REF] Schmidt | Expression of Interleukin-12-Related Cytokine Transcripts in Inflammatory Bowel Disease: Elevated Interleukin-23p19 and Interleukin-27p28 in Crohn's Disease but Not in Ulcerative Colitis[END_REF], suggests that the local inflammatory milieu may contribute to MAIT cell phenotype and function in the gut. To further define the role of IL-23, mice deficient in

  Les interactions constantes et mutuelles entre hôte et microbiote jouent un rôle clé dans le maintien de la santé et du bien-être de l'hôte. Dans l'organisme, il existe différents microbiotes, dont le microbiote intestinal surtout dans la partie distale de l'intestin (le côlon) est le plus important d'entre eux. Le microbiote se met en place dès la naissance et sa composition est le résultat d'un processus complexe de sélection par l'hôte. Les cellules épithéliales jouent un rôle clé dans ce processus. Par exemple, la consommation du dioxygène par le métabolisme des cellules épithéliales coliques permet de maintenir l'anaérobie dans la lumière du côlon et donc empêche la croissance de certains micro-organismes. Ainsi, le microbiote intestinal des adultes est dominé par des espèces bactériennes anaérobies strictes appartenant à l'embranchement des Bacteroidetes et des Firmicutes. Cependant, des bactéries anaérobies facultatives comme les Enterobactéries appartenant à l'embrachement des Protéobacteria, sont trouvées mais en nombre relativement faible dans le microbiote adulte.On sait désormais que le microbiote intestinal joue un rôle important dans la maturation et l'homéostasie du système immunitaire, notamment en relarguant des métabolites microbiens.En conséquence, la dysbiose, c'est-à-dire l'altération qualitative et fonctionnelle de la communauté microbienne intestinale, est associée à de différentes pathologies notamment aux maladies inflammatoires chroniques de l'intestin (MICI). Cette dysbiose coïncide avec des modifications des molécules bactériennes produites, mais la façon dont le système immunitaire module ces modifications n'est pas bien connue. Cette question est particulièrement importante pour les cellules T invariantes associées aux muqueuses (MAIT), qui se trouvent principalement dans les tissus colonisés par le microbiote et reconnaissent des métabolites microbiens dérivés de la voie de synthèse de la riboflavine.La riboflavine, ou la vitamine B 2 , est une vitamine synthétisée par la flore intestinale de manière naturelle et peut être également absorbée par le biais de l'alimentation quotidienne. Sa plus importante fonction biologique est de participer à des reactions rédox par l'intermediaire de FAD et FMN. Par exemple, le FAD est un cofacteur nécessaire à l'activité du glutathion reductase qui sert à régénérer le glutathion, un puissant anti-oxydant, ce qui fait que la riboflavine participe aux défenses antioxydantes. Presque tous les Bacteroidetes et Proteobacteria codent pour la voie de synthèse de la riboflavine et des bactéries intestinales de l'embranchement des Bacteroidetes et Protéobacteria peuvent produire le ligand des MAIT, suggérant une interaction entre les cellules MAIT et le microbiote. Les cellules MAIT constituent une population de lymphocytes T non conventionnels et ont la particularité d'exprimer un récepteur des cellules T (TCR) semi-invariant. Ce TCR reconnaît spécifiquement des antigènes (5-OP-RU et 5-OE-RU) derivant d'un intermédiaire du métabolisme de la riboflavine et présentées aux lymphocytes MAIT par la molécule non polymorphe MR1 (MHC-related protein 1) apparentée au CMH de classe I. Ces cellules sont activées soit d'une façon MR1-indépendante, via certaines cytokines inflammatoires, notamment l'IL-12 et l'IL-18, soit par une reconnaissance d'antigènes microbiens par l'intermédiaire de MR1. Les cellules MAIT ont un rôle dans les maladies infectieuses et métaboliques, la réparation tissulaire et sont alterées chez les patients MICI. Cependant le rôle des cellules MAIT dans les MICI reste inconnu. La dysbiose au cours de MICI se caractérise par une augmentation des Entérobactéries et une diminution des Firmicutes (en particulier Faecalibacterium prausnitzii). Beaucoup d'arguments scientifiques comme l'expansion des bactéries anaerobies facultatives au cours de MICI, suggèrent une perte de l'hypoxie dans le côlon des patients MICI. Les MICI sont souvent associées à une augmentation du stress oxydant et une diminution des capacités antioxydantes. La participation de la riboflavine aux défenses antioxydantes suggère une augmentation des besoins en riboflavine pendant l'inflammation intestinale. En accord avec cette hypothèse, les bactéries prototrophes pour la riboflavine sont enrichies dans la maladie de Crohn. L'objectif de cette thèse était de comprendre l'interaction entre les MAIT et le microbiote et d'identifier le rôle des MAIT dans l'intestin. Dans une première partie, nous avons exploré l'hypothèse que les cellules MAIT détectent des alterations du métabolisme bactérien associées aux dysbioses dans le colon. Nous avons montré que la dysanaérobiose, lors d'un traitement antibiotique ou d'une colite induite par le dextran sulfate de sodium (DSS), augmente la production du ligand des MAIT dans l'intestin. Cette notion était étayée par les données montrant une concentration plus elevée du ligand des MAIT au niveau de l'interface oxique-anoxique près de l'épithélium intestinal, ainsi qu'une surexpression des enzymes responsables de la production de riboflavine après une exposition à l'air in vitro. Pour identifier les bactéries impliquées, nous avons séquencé les gènes d'ARNr 16S dans l'intestin de souris traitées aux antibiotiques et au DSS, et analysé, en utilisant des données publiées, l'expression des gènes de la voie de la riboflavine en cas de colite induite par l'infection par Helicobacter hepaticus. La dysanaérobiose était associée à l'expansion des Enterobacteriaceae lors du traitement à la vancomycine, et des Bacteroidaceae et Enterobacteriaceae en cas de colite. La production de riboflavine était bénéfique pour l'expansion d'Escherichia coli dans l'intestin enflammé. Les Bacteroidaceae et les Enterobacteriacea peuvent produire de grandes quantités de ligands des MAIT in vitro. Le gène ribD, qui contrôle la production des ligands des MAIT, était surexprimé par les Bacteroidaceae, les Clostridiaceae et les Enterobacteriaceae en cas de colite induite par H. hepaticus. Les ligands des MAIT traversaient la barrière intestinale et augmentaient la stimulation TCR des MAIT lors de traitements à la vancomycine et au DSS. La création d'une nouvelle souris transgénique a permis de montrer que la stimulation TCR des MAIT du côlon entraîne la production du facteur de croissance épidermique amphiréguline (Areg). Lors de l'inflammation intestinale chronique, les MAIT du côlon produisaient spontanément les médiateurs protecteurs Areg et l'interféron-gamma (IFNy), l'interleukine (IL) -17 et l'IL-22. En conséquence, les souris B6 Cast Mr1 -/-, dépourvues de MAIT, étaient plus sensibles à la colite que les souris Mr1 + , ce qui indique que les MAIT protègent contre la colite. En résumé de cette première partie, les MAIT détectent et répondent directement aux changements du métabolisme bactérien associés à l'inflammation du colon et assurent en retour une protection de l'hôte. La surproduction de riboflavine par le microbiote peut résulter d'une augmentation de la concentration d'oxygène dans le côlon ; toutefois des expériences ultérieures sont nécessaires sur ce point. Nos résultats établissent une nouvelle interaction moléculaire entre le microbiote intestinal et son hôte. La démonstration que les cellules MAIT protègent contre la colite peut faciliter le développement de nouvelles approches thérapeutiques dans les MICI. Il est à noter que contrairement aux cellules T qui sont souvent difficiles à exploiter thérapeutiquement en raison de la diversité du répertoire TCR, les cellules MAIT sont une cible attractive car elles expriment un TCR semi-invariant qui se lie à des antigènes bien caractérisés. Les souris transgéniques LSL-MR1 croisées avec les souris Villin-Cre seront très utiles dans de futures expériences pour évaluer la contribution de l'activation des cellules MAIT par leur TCR à la protection contre la colite. D'autre part, nous évaluerons le potentiel thérapeutique de l'activation des cellules MAIT avec un ligand d'activation et l'effet du blocage de la fonction des cellules MAIT avec un antagoniste. Nos résultats ouvrent la voie à une meilleure élucidation de l'interaction entre le microbiote intestinal et les cellules MAIT dans d'autres pathologies associées à la dysbiose comme le cancer colorectal. Des études antérieures ont montré que les cellules MAIT sont présentes dans les tumeurs et qu'elles sont capables de libérer des cytokines en réponse à une stimulation in vitro. Cependant le rôle des cellules MAIT dans le cancer colorectal reste inconnu et nécessite d'être evalué. Dans une seconde partie, nous avons caractérisé les MAIT dans l'iléon, dans lequel des niveaux élevés d'oxygène ont été observés à l'état basal. Plusieurs bactéries codant pour la voie de la riboflavine colonisaient l'iléon, mais les MAIT n'étaient pas activés par leur TCR et exprimaient un phénotype distinct dans l'iléon. Une explication possible était la faible expression de MR1 dans l'iléon. Bien que l'expression inductible de MR1 dans les cellules épithéliales de l'intestin ait déclenché une signalisation TCR dans les cellules MAIT de l'iléon, les signaux MAIT TCR dans l'iléon étaient plus faibles que dans le côlon. Nos données suggèrent une faible production d'antigènes MAIT dans l'iléon. Nous envisageons que les gènes contrôlant la production du précurseur du ligand MAIT dans certaines bactéries de l'iléon soient atténués par la riboflavine provenant de l'alimentation. Il est également plausible que les antagonistes d'origine alimentaire tels que les métabolites dérivés du folate, entrent en compétition avec les antigènes microbiens pour se lier à MR1 dans l'intestin grêle. L'analyse de l'activité transcriptionnelle bactérienne le long de l'intestin de souris nourries au régime déficient en riboflavine ou pauvre en folates serait utile pour aborder ces possibilités.

7.1.2. Viral infections

	INTRODUCTION INTRODUCTION	Chapter I: Mucosal associated invariant T cells Chapter I: Mucosal associated invariant T cells
	The most convincing data supporting the antibacterial function of MAIT cells have been Murine experimental studies support the role of MAIT cells in viral infections. In mice infected
	obtained in experimental infection mouse models. Briefly, mice deficient for MR1 and thus with influenza virus, MAIT cells are activated with upregulation of CD25, CD69 and Granzyme B
	devoid of mature MAIT cells show defective or delayed bacterial clearance. For example, and accumulate in the lungs. Mr1 -/-mice are more susceptible to the virus as compared to wild
	Mr1 -/-mice are more susceptible to E. coli (Georgel et al. 2011), Mycobacterium abscessus (Le type mice, suggesting a protective role of MAIT cells against influenza virus. (van Wilgenburg et
	Bourhis et al. 2010), Legionella longbeachae (H. Wang et al. 2018) and Francisella tularensis al. 2018). Because viruses do not produce 5-A-RU, MAIT cell activation during viral infections
	(A. Meierovics, Yankelevich, and Cowley 2013; A. I. Meierovics and Cowley 2016) as compared depends on IL-18 in synergy with IL-12, IL-15, and IFN-α/β. When co-cultured with influenza-
	to their MR1 + counterparts. Depending on the type of infection, MAIT cells produce large infected cells, MAIT cells produce IFN-γ, TNF and granzyme B that likely inhibit viral replication
	amounts of cytokines. Interestingly, during legionella infection, lung MAIT cells acquire a (Loh et al. 2016; van Wilgenburg et al. 2016).
	particular differentiation program. They express the tissue repair signature (Hinks et al. 2019)
	and both RORγt and Tbet, contrasting to the profile observed at steady state (RORγt + Tbet -).
	Altogether, these findings highlight the protective role of MAIT cells against various pathogenic
	bacteria in humans and mice.
	Shigella vaccine (Le Bourhis et al. 2013). Similarly, decreased blood MAIT cells function as innate sensors of viral infection via cytokines activation. In the blood of
	MAIT cells has been observed in patients with Mycobacterium tuberculosis infection (Harriff et patients infected with influenza, dengue, hepatitis C viruses (HCV), MAIT cells are selectively
	al. 2014; Jiang et al. 2014; 2016; Saeidi et al. 2015; Yang et al. 2015), Pseudomonas decreased compared to healthy controls. Residual MAIT cells are activated with high expression
	aeruginosa pulmonary infection (D. J. Smith et al. 2014), children with Vibrio cholerae infection of CD38, CD69 and PD-1 (van Wilgenburg et al. 2016; Bolte et al. 2017; Beudeker et al. 2018;
	(Leung et al. 2014) as well as septic patients with severe bacterial infections (Grimaldi et al. Loh et al. 2016; Hengst et al. 2016). Studies showed an inverse correlation between MAIT cell
	2014). Interestingly, septic patients who exhibit higher frequencies of circulating MAIT cells have frequencies and severity of disease in patients infected with influenza or HCV (van Wilgenburg
	better health outcomes. et al. 2016; Loh et al. 2016). Likewise, circulating MAIT cells are decreased in patients with
	human T lymphotropic virus type 1 infection (Paquin-Proulx et al. 2017) and human
	Functional in vitro studies highlighted the ability of MAIT cells to kill APCs infected with E. coli immunodeficiency virus (HIV) infection (Beudeker et al. 2018; Cosgrove et al. 2013; Fernandez
	(Le Bourhis et al. 2013; A. Kurioka et al. 2015; Lamichhane et al. 2019), Shigella flexneri (Le et al. 2015; Leeansyah et al. 2013). Interestingly, two studies reported a marked reduction of
	Bourhis et al. 2013), M. tuberculosis (Gold et al. 2010; Le Bourhis et al. 2010), P. aeruginosa MAIT cells in the blood of patients with SARS-CoV-2 infection (Parrot et al. 2020; Flament et al.
	(Jo et al. 2014; Tang et al. 2013) in a TCR-and cytokine dependent manner through the release
	of cytokines and cytotoxic effector molecules. Moreover, human MAIT cells were shown to react
	against Enterococcus faecalis, a bacteria that does not make 5-A-RU. In this case, IL-12 and IL-
		37 38

  Yara El Morr 1 , Alexey Vorobev 1 , Katarzyna Franciszkiewicz 1 , Claire Morvan 2 , Anastasia du Halgouet 1 , Marion Salou 1 , Bakhos Jneid 1 , Virginie Premel 1 , Stanislas Mondot 3 , Aurélie Darbois 1 , Mathieu Maurin 1 , Catherine Philippe 3 , Olivier Lantz 1 * and François Legoux 1 *. 1) INSERM U932, PSL University, Institut Curie, Paris, France. 2) Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, Paris, France. 3) Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

*These authors contributed equally to this work

  Saujet, L., Monot, M., Dupuy, B., Soutourina, O. & Martin-Verstraete, I. The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile. J.Bacteriol. 193, 3186-3196 (2011). 56. Soutourina, O. A. et al. Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS Genet. 9, e1003493 (2013). 57. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods San Diego Calif 25, 402-408 (2001). 58. Lan, A. et al. Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats. Br. J. Nutr. 97, 714-724 (2007). 59. Laroui, H. et al. Dextran sodium sulfate (DSS) induces colitis in mice by forming nanolipocomplexes with medium-chain-length fatty acids in the colon. PloS One 7, e32084 (2012). 60. Nonnenmacher, C., Dalpke, A., Mutters, R. & Heeg, K. Quantitative detection of periodontopathogens by real-time PCR. J. Microbiol. Methods 59,

	55.
		Sequence (5'-3')
	Universal 16S rRNA 60
	Forward	TGGAGCATGTGGTTTAATTCGA
	Reverse	TGCGGGACTTAACCCAACA
	Probe	CACGAGCTGACGACA(AG)CCATGCA
	Clostridioides difficile
	ribD Forward	GGAAAACGCCTCCTTGTGTA
	ribD Reverse	TGCCTCATTTAAATCTCTTGCTT
	ribH Forward	TTGGGTACCTGGAGCTTTTG
	ribH Reverse	TTCCAGCTTTTGTACCTGCTC
	gyrA Forward	TTTTGTTGTGTCTATGAACCTTTGT
	gyrA Reverse	TCCTTTACCAGCTCTTATTTGACTT
	Clostridium perfringens
	ribD Forward	TTGGAGGAAATCATGCTGAA
	ribD Reverse	GGATTTTGGTCTAAGGTTCCA
	ribH Forward	CTTGGGTTCCAGGAGCTTTT
	ribH Reverse	CAGCTTTAGTTCCGGCTCTTT
	gyrA Forward	CCAAACTTTGATGGTGAGGAA
	gyrA Reverse	CCTGCTGTAGGGAAATCTGG
	Clostridium butyricum
	ribD Forward	GCATGCTCATGCTGAAAGAA
	ribD Reverse	CTGCCACCAAAGGATTAGGA
	ribH Forward	TATAGCGTGGGTTCCAGGAG
	ribH Reverse	TACGCTTGCAATTCCCTTTG
	gyrA Forward	CATCGGGAATAGCTGTTGGT
	gyrA Reverse	CACCTGTTGGGAAATCTGGT
	Bacteroides
	thetaiotaomicron
	ribD Forward	CGGCAATCCCGTAGTACTGT
	ribD Reverse	GGTGGGTACTTCTCCGTCAA
	ribH Forward	TACCGGAGCCTTATTGAACG
	ribH Reverse	TCCTTGAGTCGCTCCCATAC
	gyrA Forward	GTACCATCCGCATGGAGACT
	gyrA Reverse	AGGTCATCCATCATCGCTTC
	Escherichia coli
	ribD Forward	AGGACGTTTTACCACGCATC
	ribD Reverse	ATGGCTACAGGGTTCGAGTG
	ribE Forward	GGGTACGGTTATTCGTGGTG
	ribE Reverse	GGCCTTGATGGCTTTCAATA
	gyrA Forward	ATGAACTGATGGCCGATCTC
	gyrA Reverse	ATGTGTTCCATCAGCCCTTC
		87

Acknowledgments I would like to acknowledge the jury members of this thesis who have agreed to evaluate my work. Namely I would like to thank Dr. Olivier Lantz, Pr. Harry Sokol, Dr. Julie Dechanet-Merville, Dr. Christelle Harly, Pr. Gérard Eberl, Dr. François Legoux and Dr. Stanislas Mondot. Many thanks to all the people who contributed in the success of this project. I want first and foremost express my sincere gratitude to Dr. Olivier Lantz and Dr. François Legoux for supervising this project, sharing their immense knowledge, plentiful experience and providing me invaluable guidance throughout this thesis. Everything I learned from them has been precious for my scientific and personal enrichment. I have been extremely lucky to work under your supervision. I would like to thank also the following people who contributed in this project: Alexey Vorobev, Katarzyna Franciskiewicz, Claire Morvan, Anastasia du Halgouet, Marion Salou, Bakhos Jneid, Virginie Premel, Stanislas Mondot, Aurélie Darbois, Mathieu Maurin and Sylvie Rabot. I am very grateful to all of you. Acknowledgments We thank V. Dangles-Marie, M. Garcia, the mouse facility technicians, and the flow cytometry core at Institut Curie. We thank F. El Marjou and C. Jouhanneau for the generation of the LSL-MR1 transgenic mouse and S. Robine for the Villin-CreER mouse. We are grateful to the INRAE MIGALE bioinformatics facility for providing help, computing and storage resources. We thank D. Halpern (INRAE, Jouy-en-Josas, France) for his expertise and technical help with B. thetaiotaomicron. We thank J.J. Moon (Massachusetts General Hospital) for providing the LSL-MR1 backbone. We thank the NIH tetramer core facility (Emory University) for providing CD1d and MR1 tetramers. The MR1:5-OP-RU tetramer technology was developed jointly by J. McCluskey, J. Rossjohn, and D. Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Institut Curie, Agence Nationale de la Recherche (ANR) [JCJC (MAIT) and Blanc (neoMAIT, MAIT, diabMAIT, MAITrepair) and Labex DCBIOL] and European Research Council (ERC-2019-AdG-885435).

Data Availability

16S rRNA amplicon sequencing data generated in this study are available on the EBI ENA under the accession number PRJEB44022.

49 Oxygen availability is a major determinant of microbiota composition. The longitudinal gradient of oxygen limits bacterial load and diversity in the small intestine. In contrast to the large intestine, the small intestine is characterized by a greater relative abundance of facultative anaerobic bacteria. The cross-section of the colon shows the mucosa, which is colonized by facultative anaerobes, and the lumen dominated by obligates anaerobes (

Discussion and perspectives

The microbiota has a strong influence on the host immune system and correspondingly, the immune system shapes the composition and localization of the microbiota. Yet, how the immune system monitors alterations in the composition and function of the microbiota remains poorly understood. This question is of particular importance for MAIT cells, which are predominantly found in tissues colonized by the microbiota and recognize microbial metabolites derived from a microbial riboflavin precursor. MAIT cells have been implicated in pathologies associated with intestinal dysbiosis such as inflammatory bowel diseases, but their exact role in the pathology remains unknown. This work aimed to understand the interaction between MAIT cells and the microbiota and to decipher the role of MAIT cells in the gut.

Our results show that MAIT cells directly monitor a bacterial metabolism associated with colitis and provide host protection. We establish that riboflavin over-production by intestinal bacteria is a hallmark of intestinal inflammation, and results in the production of antigens for MAIT cells.

The gene ribD, which controls MAIT antigen production, was over-expressed by Bacteroidaceae, Clostridiaceae and Enterobacteriacea during colitis. Riboflavin precursor adducts crossed the intestinal barrier and induced TCR signalling in MAIT cells, which produced the tissue-repair mediator amphiregulin and reduced colitis severity. We propose that riboflavin over-production by intestinal bacteria is a consequence of increased oxygen in the gut environment during colitis. Our study explains for the first time why the immune system monitors the activity of the riboflavin biosynthetic pathway. More generally the study describes a new molecular interaction between the microbiota and mammalian immune system. In the second part of this work, we characterized MAIT cells in the ileal environment wherein high levels of oxygen have been observed at steady state. A range of bacteria that encode the riboflavin pathway populated the ileum, however ileal MAIT cells were not activated through TCR engagement and expressed distinct phenotypic profiles.

Here, I will discuss these results and integrate future studies that will be needed to understand MAIT-microbiota interactions in the gut.