Sorbonne Université

Sorbonne Université Ecole doctorale 394 : Physiologie, Physiopathologie et Therapeutique INRA Jouy-en-Josas, Institut MICALIS, Equipe FInE Activation mechanism of AhR pathway in intestinal epithelial cells by commensal bacteria Par Marinelli Ludovica Thèse de Doctorat en Biologie Dirigée par le Dr Blottière M. Hervé et le Dr Lapaque Nicolas Présentée et soutenue publiquement le 30 mai 2018 Devant un jury composé de : CLEMENT Karine, Professeur des Universités, Paris 6 Président du Jury SEGAIN Jean-Pierre, Directeur de Recherche, INRA Rapporteur THEODOROU Vassilia, Professeur des Universités, Toulouse Rapporteur COUMOUL Xavier, Professeur des Universités, Paris 5 Examinateur SOKOL Harry, Professeur des Universités, Paris 6 Examinateur BLOTTIERE Hervé, Directeur de Recherche, INRA Directeur de Thèse LAPAQUE Nicolas, Chargé de Recherche, INRA Directeur de Thèse Ai miei gentitori, che mi hanno insegnato ad affrontare ogni sfida con il sorriso Acknowledgments Je remercie chacun des membres du jury de me faire l’honneur d’évaluer ce travail: - Mme le Professeur Karine Clement, merci de présider ce jury et de partager votre expertise ; - M le Docteur Jean-Pierre Segain et Mme le Professeur Vassilia Theodorou, merci d’avoir accepté d’etre rapporteur de ma thèse et d’apporter vos corrections afin d’améliorer ce travail. - M le Professeur Xavier Coumoul et M le Professeur Harry Sokol, merci d’avoir accepté d’examiner ce travaille et de partager vos competences sur AhR. - M le Docteur Hervé Blottière et M le Docteur Nicolas Lapaque, mes directeurs de thèse. Merci à Hervé de m’avoir accepté en thèse et de m’avoir proposé ce sujet. Merci a Nico de me avoir encadré a la paillasse comme au coin café, de m’avoir donné pleines des conseilles, de correctionnes et de m’avoir aussi stimulé avec ton interet scientifique. Merci a tous les deux de m’avoir fait confiance et de m’avoir permis de completer ce beau parcours de thèse. Table of Contents Acknowledgments 3 Table of Figures 7 Chapter 1. Introduction 1 The gastrointestinal tract 1 1.1.1. Stomach 3 1.1.2. Intestines 3 The immune system in the GI tract 7 The Human Gut Microbiota 11 1.3.1. Composition of the gut microbiota 11 1.3.2. Development and age-related variation of gut microbiota 15 1.3.3. The study of gut microbiota 18 The Role of Gut Microbiota in the Host-Bacteria Cross-Talk 20 1.4.1. The Barrier Homeostasis in Intestine 20 1.4.2. Mechanisms for Sensing Microbial Signals by IECs 23 1.4.3. Intestinal Microbiota and the Immune System beyond the Gut 26 1.4.4. Microbial-derived Metabolites in Host-Bacterial Cross-Talk 28 1.4.5. Dysbiosis in Human Pathology 42 Aryl Hydrocarbon Receptor: Description, Characterization and Physiological Role 48 1.5.1. Expression and Localization 49 1.5.2. Structure and Functional Domains 51 1.5.3. AHR pathway 55 1.5.4. Physiological role of AhR 78 1.5.5. Cross-talk with other signalling pathways 87 Chapter 2. Rationale and Objectives 89 Chapter 3. Results 91 Paper I: Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells. 91 Paper II: Identification of Bifidobacteria as novel activators of AhR pathway in human intestinal epithelial cells 133 Chapter 4. Discussion 163 Chapter 5. Supplementary 176 Choline-TMA catabolism by gut microbiota 176 5.1.1. Comments on Objectives and Methods 176 5.1.2. Experimental set-up 177 5.1.3. Comments on the Results and Discussion 177 Bibliography 180 Table of Figures Figure 1: Human digestive system. 1 Figure 2: Major layers and organisation of the digestive tract. 2 Figure 3: Regions and histological organisation of the stomach. 3 Figure 4: Organization of cell populations in the crypt and villi of small intestine. 4 Figure 5: Localization of Peyer's patches and M cells in small intestine. 5 Figure 6: Organization of cell populations in the crypt of large intestine. 6 Figure 7: Intestinal microenvironment and niches. 10 Figure 8: Characteristics of the normal gastrointestinal tract. 11 Figure 9: Spatial distribution of microbiota in the mucus layer of small and large intestine. 12 Figure 10: Phylogenetic differences between enterotypes. 13 Figure 11: Stratification of the microbial composition landscape of the human gut microbiome. 14 Figure 12: Human microbiota composition through life stages. 17 Figure 13: Gut Bifidobacteria composition through life stages. 18 Figure 14: The regulation of Intestinal Barrier. 22 Figure 15: IECs sense microbial signals. 25 Figure 16: Microbiota-derived SCFA shape cellular metabolism in intestine. 32 Figure 17: Modeling of butyrate into the PPAR binding pocket. 33 Figure 18: Synthetic molecular mechanism of action of indole and its metabolites. 37 Figure 19: Gut microbiota-dependent metabolism of dietary choline and atherosclerosis. 39 Figure 20: Bile acids in the intestine. 41 Figure 21: Schematic representation of the bHLH-PAS protein domains. 52 Figure 22: Schematic representation of AHR domains architecture. 53 Figure 23: Overall architecture of AHR/ARNT heterodimer in complex with XRE 53 Figure 24: AhR activation signalling. 58 Figure 25: Chemical structure of three synthetic AhR ligands. 61 Figure 26: Structures and origins of some natural AhR ligands. 65 Figure 27: Active site of Cytochrome P450 67 Figure 28: Model structure of cytochrome P450. 68 Figure 29: Domain structure of mouse AHR and AHRR. 71 Figure 30: Models of AhRR transcriptional repression mechanism. 72 Figure 31: Schematic representation of the AhR regulation through CYP450 and AHRR. 72 Figure 32: Proposed model of repression of AHR signalling by TiPARP. 73 Figure 33: AhR serves as a ligand-dependent ubiquitin ligase, as well as as a transcription factor. 74 Figure 34: Docking orientation of TCDD into mouse and human AhR-LBD binding pocket. 76 Figure 35: In silico modelling of AHR ligand binding domain. 76 Figure 36: The role of AhR in the gut. 83 Figure 37:Mechanism of interaction between commensal bacteria and ILCs via AhR. 86 Figure 38: Butyrate incubated in presence and absence of the Sp-1 inhibitor. 164 Figure 39: Butyrate incubated in presence and absence of a NFκB inhibitors. 165 Figure 40: Schematic representation of the proposed mechanism for AhR activation by butyrate 169 Figure 41: Schematic representation of the proposed mechanism for AhR activation by Bifidobacteria 16973 Introduction – The gastrointestinal tract Chapter 1. Introduction The gastrointestinal tract The gastrointestinal tract (GI) has extensively been studied for its digestive functions. Together with accessory digestive organs, the GI tract constitutes the digestive system, whose main function consists in digestion, absorption and subsequent distribution of nutrients through the body. As a result, the GI tract is divided into functional regions, each of which is characterized by its own physico-chemical conditions (Figure 1). The ingested food is mechanically broken down in the oral cavity and mixed to the first digestive enzymes (amylases and lipases). Through the oesophagus, the food reaches the stomach where other digestive enzymes (pepsins) are released along with hydrochloride acid, causing the pH to drop (pH 1-2). The food content passes then through the different sections of the small intestine (duodenum, jejunum and ileum) where the pH increases as bicarbonate is releases (pH5.7-7.3). During this passage Figure 1: Human digestive system. Functional regions with main digestive enzymes and pH (adapted from other digestive enzymes are released Sobotta's Atlas and Text-book of Human Anatomy, 1906) (pancreatic enzymes, mucosal enzymes and bile salts), resulting in the cleavage of carbohydrates, proteins, and lipids and the absorption of low molecular amino acids, simple sugars and fatty acids. From the ileum, non-degradable food components land in the large intestine, traversing the cecum and the colon (ascending, transverse and descending; pH 5.7-6.8) where water and salts are absorbed and the fermentation of undigested food take place. Then, from the sigmoid colon, digestive wastes move through the rectum and are egested at the anus. From the histological point of view the general structure of the GI tract (Figure 2), from outer layer towards the lumen, is composed of: 1 Introduction – The gastrointestinal tract - Serosa - Loose connective tissue with elastic and collagen fibers, nerves and vessels, covered by a single layer of flat mesothelial cells. Where there is no mesothelial cover, the outermost layer is called adventitia. - Muscularis - Composed of smooth muscle cells that are Figure 2: Major layers and organisation of the digestive spirally oriented and divided tract (adapted from Janqueira's Basic Histology, 2009). into inner and outer sublayers. - Submucosa - Thick layer of denser connective tissue with numerous blood and lymphatic vessels. - Mucosa (the main object of this manuscript) - The innermost layer that come in contact with the gastrointestinal content, further divided in different layers (from the outer to the inner layer): muscularis mucosae, a thin layer of smooth muscle separating the mucosa from the submucosa; lamina propria (LP) of loose connective tissue rich in blood and lymphatic vessels, lymphocytes and smooth muscle cells and the inner epithelial layer. Despite this common histological organization, each intestinal segment is characterized by distinct histological characteristics, especially concerning the mucosa, that ensure the diverse functions performed along the GI tract. 2 Introduction – The gastrointestinal tract 1.1.1. Stomach The stomach is a direct continuation of the oesophagus and can be divided in distinct regions: the most proximal part is the cardia, followed by the fundus, corpus, antrum and pylorus (Figure 3). The fundus and corpus constitute about 80% of the stomach and differs from the antrum and pylorus both functionally and histologically. At the gastro-esophageal junction, the mucosa abruptly changes from stratified squamous epithelium to simple cuboidal. The mucosa in the stomach is thick and lined with simple columnar epithelium.

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