The intestinal tissue is charged with a delicate immunological task. The intestinal immune system needs to be tolerant towards nutrients and microbiota present in the intestinal lumen, while simultaneously detecting and responding to dangers such as pathogens. A single-cell layer of intestinal epithelial cells (IECs) acts as a first line of defence. There is a T cell population located between the IECs that have been named intraepithelial lymphocytes (IELs). As the main lymphoid population within the intestinal barrier, IELs are thought to have an important role in intestinal homeostasis maintenance, as well as a role in intestinal inflammatory and autoimmune diseases such as inflammatory bowel disease and celiac disease. Despite extensive research on IEL biology, there are still questions remaining in terms of the development, maintenance and activation of IELs. Furthermore, IELs survive poorly in vitro, which hinders mechanistic insights. In this thesis, a co-culture system between IELs and intestinal organoids, "mini-guts", provides an in vitro model for IELs. With this IEL-organoid co-culture system, IELs associated with the organoids survive for at least 4 days. Additional findings suggest that IELs are kept in a poised state of activation due to differences in their mitochondria compared to other T cells found in spleen, lung and skin. Upon activation or intestinal inflammation, the mitochondrial mass in IELs increases. This increase correlates with effector functions such as cytokine production and proliferation. In addition, the composition of the mitochondria-specific lipid, cardiolipins, alters drastically in IELs after activation. These data support a model of mitochondria-dependent activation of IELs. The mitochondria-dependent activation in IELs appears to have at least two pathways: one T cell receptor-dependent and one microbiota-dependent. The latter pathway suggests a model in which IELs can become activated regardless of the cause of intestinal epithelial barrier damage.

This thesis reports investigations of the interactions between the intestinal epithelial barrier and the intracellular apicomplexan Eimeria spp., both in vivo and in vitro. Initially, conventional in vivo studies using genetically modified animals were used to investigate the contribution of innate lymphoid cells (ILCs) to immune protection of the intestinal barrier. Additionally, to understand complex epithelial host-pathogen interactions a novel in vitro model of small intestine organoids was developed. Data suggest that immunoprotection against Eimeria vermiformis infections is mediated by T cells. Furthermore, there is an indication that ILCs have a detrimental effect in Eimeria vermiformis-infected immunocompromised animals. However, the role for ILCs in the regulation of the immune response remains unclear. The life cycles of Eimeria vermiformis and Eimeria falciformis are highly complex, comprising multiple schizogonies followed by a gametogony. In vitro life cycle completion has not been achieved to date due to the limitations of monolayer cell line models. It is likely that for a successful parasite development the interaction of the different epithelial cell types present in intestinal organoids is required. The development of intestinal organoids by Sato and colleagues gave rise to a breakthrough in cellular studies, providing the tools to study complex interactions between host tissues and invading pathogens in vitro. I showed that small intestine-derived organoids grow exponentially after passage and that each organoid contains distinct specialised epithelial cell types, such as Paneth, Goblet or enteroendocrine cells, suggesting that the organoid model closely resembles the native intestinal epithelium and that Eimeria spp. benefit from the three-dimensional structure and physiological characteristics of the organoid model. Intestinal organoids were infected with E. vermiformis or E. falciformis sporozoites. These completed several rounds of asexual replication but did not proceed to the final gametogony. Despite the need for the development of sensitive techniques applicable to three-dimensional cell culture models, these results indicate that intestine-derived organoids are a promising model to study host-parasite interactions at the intestinal epithelial barrier at the cellular and molecular levels.

No description available.

No description available.

Human pluripotent stem cells (hPSCs) have the ability to self-renew and differentiate into all specialized body cells, providing material suitable for studying basic biology, modeling disease, and for regenerative medicine. The differentiation of hPSCs into functional cell types has been further enhanced by the production of organoids, miniature 3D organ-like structures that mimic the architecture and function of their in vivo counterparts, representing more physiologically relevant models of native tissues than monolayer cultures. Our initial aim was to differentiate hPSCs into lung epithelial organoids in vitro, and we hypothesized that applying knowledge of signaling cues during embryonic development to the dish would produce lineage-specific tissue. Using a multi-stage differentiation strategy, we derived organoids sharing properties with the developing lung as well as intestine. From this work, we learned the importance of purification, selection, and using singularized precursor cells to produce populations of bona fide lineage-restricted organoids. Upon developing a type of intestinal organoid technology from hPSCs not reported before, we shifted our focus to the intestine. We generated cystic intestinal epithelial organoids called enterospheres (hEnS) in vitro from hPSCs, which mimic structural and cell type properties of the native small intestinal epithelium. hEnS growth, differentiation, and long-term culture can be controlled by modulating media conditions. Importantly, hEnS are functional in that they elicit an innate immune response upon treatment with enteric pathogens. We established hEnS as an attractive in vitro model system for studying human gastrointestinal biology. We then developed an automated hEnS imaging assay to measure responses to growth factors, bacterial products, and enteric bacteria themselves. In doing so, we demonstrated the utility of hEnS as a germ-free system for studying host-microbe interactions and intestinal maturation. Finally, we investigated the expression of protein markers of intestinal maturation in tissue sections of primary human intestine spanning gestation, and made observations that are different from those reported in mice. Overall, our work provides new and important insights into hPSC differentiation, organoid technologies, and intestinal development in humans. / Thesis / Doctor of Philosophy (PhD)

Les cellules entéroendocrines sécrétrices d’hormones représentent 1% de l’épithélium intestinal mais sont des régulateurs essentiels du métabolisme énergétique et l’altération de leur différenciation provoque de graves pathologies métaboliques. Leur différenciation est régie par une cascade de régulations transcriptionnelles qui est encore peu décrite, particulièrement chez l’homme. L’objectif de ce projet de thèse était d’évaluer l’implication de plusieurs facteurs de transcription préalablement identifiés chez la souris (NGN3, RFX6, ARX, PAX4) dans la différenciation entéroendocrine humaine. Pour ce faire, ces gènes ont été inactivés par la technique CRISPR/Cas9 dans des cellules souches pluripotentes induites humaines (hiPSCs), qui ont ensuite été différenciées in vitro en organoïdes intestinaux (HIOs). Les analyses des HIOs déficients pour NGN3 ne permettent pas de conclure quant à un rôle dans la différenciation entéroendocrine mais indiquent une altération de la régionalisation du tissu formé chez les mutants. RFX6 semble important pour la différenciation et/ou la fonction des cellules entéroendocrines, bien que sa fonction précise n’ait pas pu être déterminée. / Hormone-producing enteroendocrine cells represent 1% of the intestinal epithelium but are key regulators of the energetic metabolism and alteration of their differentiation is associated with severe metabolic disorders. Enteroendocrine differentiation is governed by a transcriptional regulatory cascade that is poorly described, especially in humans. This thesis project aimed to evaluate the implication of several transcription factors, previously identified in mice (NGN3, RFX6, ARX, PAX4), in human enteroendocrine differentiation. To do so, these genes were disrupted with the CRISPR/Cas9 system in human inducible pluripotent stem cells (hiPSCs), which were then differentiated in intestinal organoids (HIOs). Preliminary analysis of NGN3-deficient HIOs did not allow a firm conclusion regarding NGN3 implication in enteroendocrine differentiation but showed a tissue regionalization alteration. RFX6 seems important for the differentiation/function of enteroendocrine cells, although its precise function is still to be determined.

Environmental factors such as diet contribute to the pathogenesis of inflammatory bowel disease (IBD). Epidemiological evidence suggests a robust linkage between IBD and the Western diet, which is often characterized by a high intake of food additives. These additives, including synthetic colourants, are widely used, leading to significant human exposure. Allura Red AC (AR) is one of the most popular synthetic colourants, yet little is known about its impact on human health and the role of AR in the pathogenesis of colitis remains elusive. Serotonin (5-hydroxytryptamine; 5-HT), which regulates various gut physiological processes, has been shown to modulate the gut microbiota and enhance susceptibility to colitis. In this thesis, it was discovered that chronic exposure to AR, at a dose found in commonly consumed dietary products, exacerbated dextran sulfate sodium (DSS)-induced colitis and triggered early onset of disease in the CD4+CD45RBhigh T cell-induced colitis model. AR also induced low grade colonic inflammation in naïve C57BL/6 mice. Exposure to AR was associated with increased colonic 5-HT levels and impaired intestinal barrier function via activation of the myosin light chain kinase (MLCK) pathway. However, AR did not promote colitis in mice lacking tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme responsible for colonic 5-HT synthesis. Further, AR increased colonic 5-HT levels in germ-free (GF) mice and perturbed the gut microbiota composition in specific pathogen-free (SPF) mice. Transfer of this altered microbiota from the dye-exposed SPF mice to GF mice conferred enhanced susceptibility to DSS-induced colitis. Mechanistically, AR induced reactive oxygen species (ROS) generation and promoted 5-HT secretion via the NF-κB pathway in BON cells. Data in this thesis indicate that the widely used synthetic colourant, AR, promotes colitis via colonic 5-HT in microbiota-dependent and -independent pathways. Collectively, these findings provide important information on enhancing public awareness of its detrimental effects on human health. / Thesis / Candidate in Philosophy / Epidemiological and experimental studies suggest a potential link between inflammatory bowel disease (IBD) and diet. The Western diet, often characterized by a high intake of processed foods, is associated with the growing incidence of IBD. Allura Red AC (AR) is a popular artificial food dye found in highly common processed foods, yet little is known about its impact on human health and disease. Serotonin, a key molecule in the gut, has been implicated in large bowel inflammation. Herein, the potential role of AR in the development of colitis was examined. Across multiple models, AR exposure heightened vulnerability to colitis in mice, an effect attenuated by reduced serotonin production in the gut. The effect of AR in enhancing colitis vulnerability occurred via gut microbiota-dependent and -independent pathways. These studies have identified how AR promotes colitis, findings that may advance public health awareness and impact the health of patients with IBD.

Les maladies inflammatoires de l’intestin (MII), incluant la maladie de Crohn et la colite ulcéreuse, sont des maladies chroniques qui résultent d’un dérèglement de la réponse immunitaire aux microorganismes de la lumière intestinale. Des études de séquençages réalisées par le laboratoire du Dr. Rioux, avec ses collègues du International IBD Genetics Consortium ont identifié quatre variants rares, indépendants et causals pour les MII, dans le gène IFIH1. La protéine d’IFIH1 (MDA5) interagit avec certains virus à ARN, afin de déclencher une réponse antivirale de l’immunité innée. Nous avions émis l’hypothèse que ces variants dans IFIH1 diminuaient la réponse antivirale de l’épithélium intestinal, suite à une infection. Nous avons d’abord travaillé avec des lignées cellulaires lymphoblastoïdes (LCLs) obtenues à partir d’individus atteints de MII et qui sont homozygotes ou hétérozygotes composés pour ces variants, ainsi qu’à partir d’individus contrôles (IFIH1 wt). Ces LCLs ont été reprogrammées en cellules souches pluripotentes induites humaines, avant d’être différenciées en cultures épithéliales intestinales. Nos résultats ont d’abord confirmé l’impact des variants sur la structure génique et protéique (IFIH1/MDA5), dans ces modèles cellulaires. Puis, la réponse antivirale a été induite, grâce à la stimulation avec des agents (moléculaire et viral) connus pour stimulés la protéine MDA5. Nous avons démontré que ces variants dans IFIH1 induisaient effectivement une moins grande réponse antivirale, caractérisée par une plus faible expression d’IFNs, comparativement aux contrôles. Finalement, la modulation des IFNs constituerait une avenue potentiellement intéressante pour le traitement des patients atteints des MII et porteurs des variants causals. / Inflammatory Bowel Disease (IBD), including Cronh’s disease and ulcerative colitis, are chronic inflammatory diseases of the gastro-intestinal tract. IBD is associated with a disturbance of the immune response to the microorganisms of the intestinal lumen. Sequencing studies conducted by the laboratory of Dr. John Rioux, in collaboration with his colleagues of the International IBD Genetics Consortium, identified four rare and independent variants in IFIH1, associated to IBD. The protein of IFIH1 (MDA5) interacts with certain RNA viruses to trigger the innate mechanism of antiviral defense. Our hypothesis was that these IFIH1 variants decreased the intestinal epithelial antiviral response, following an infection. We first worked with lymphoblastoid cell lines (LCLs) obtained from IBD patients who are homozygotes or compound heterozygotes for the different variants, as well as from control individuals (IFIH1 wt). These LCLs were reprogrammed into human induced Pluripotent Stem Cells (hiPSCs), before being differentiated into intestinal epithelial cultures. Our results first confirmed the impact the variants on the genetic and protein structure for these models. Then, the antiviral response was triggered by the stimulation of LCLs and intestinal epithelial cells, with agents (molecular and viral) known to stimulate MDA5. We have demonstrated that these IFIH1 variants did indeed induce a lower antiviral response, characterized by lower IFNs expression, compared to control cell lines. Finally, modulation of IFNs could be an interesting avenue for the treatment of IBD patients with the causal variants.

Das Protozoon Giardia duodenalis ist eine der Hauptursachen für infektiöse Magen-Darm-Erkrankungen. Die zugrundeliegenden Pathomechanismen sind jedoch nach wie vor unklar. Um die Pathogenität G. duodenalis‘ untersuchen zu können, wird ein Modellsystem benötigt, dass die Komplexität des Darmepithels widerspiegelt. Diese Arbeit zeigt die Etablierung eines Zellkultursystems auf der Basis von organoid-abgeleiteten Epithelien unter Verwendung von filter-basierten Zellkultureinsätzen. Wir haben Protokolle für die Etablierung von organoid-basierten Zellkulturen (ODMs) vier verschiedener Wirte zoonotischer Protozoen unter Verwendung eines einzigen Protokolls erstellt. Die Charakterisierung zeigte, dass das Modellsystem erfolgreich die Polarisierung des Darmepithels nachahmt, aus mehreren Zelltypen besteht und eine Infektion ermöglicht. Der Schwerpunkt der Arbeit lag auf der Analyse der durch G. duodenalis induzierten Barrierestörung in ODMs auf Transkriptions-, Protein- und Funktionsebene. Die Infektion von humanen duodenalen Zellen führte zu einem Verlust der epithelialen Barrierefunktion. Mit Hilfe des transepithelialen elektrischen Widerstandes und Dextran Flux wurde eine Erhöhung der Barrieredurchlässigkeit beobachtet. Die Hemmung von zuvor in immortalisierten Zellmodellen beschriebenen Reaktionswegen konnte die Barrierefunktion nicht wiederherstellen. Stattdessen konnten Veränderungen der Ionenhomöostase sowie den Zusammenbruch der zonula occludens nachgewiesen werden. Der beobachtete Phänotyp konnte auf die Aktivierung des cAMP/PKA/CREB-Signalwegs, als einen von mehreren kausalen Faktoren, zurückgeführt werden. Hier zeigen wir die Etablierung eines aus Organoiden abgeleiteten Modells, das die Untersuchung von G. duodenalis Infektionen in vitro ermöglicht. Mit unserem Modell konnten wir eine neue Reihenfolge von Ereignissen entschlüsseln, die einen der Faktoren während symptomatischer Giardiasis darstellt. / The protozoan Giardia duodenalis is a one of the major causes of gastrointestinal illness. Underlying pathomechanisms remain unclear. An in vitro model system that also mimics the complexity of intestinal epithelium is needed to allow pathogenicity studies. This thesis shows the establishment of a cell culture system based on organoid-derived epithelia using permeable cell culture inserts. We have provided guidelines on the establishment of organoid-derived monolayers (ODMs) of four different hosts of zoonotic protozoa using a single protocol. Characterization showed that the model system successfully mimics intestinal polarization, is composed of multiple cell types and allows for infection with multiple protozoan parasites. As the main focus of the thesis, analysis of G. duodenalis-induced barrier breakdown in ODMs was performed on transcriptional, protein and functional level. Infection of human duodenal, organoid-derived monolayers resulted in a time- and dose-dependent breakdown of epithelial barrier function. Barrier permeability increases were observed ranging from ions to macromolecules as measured by transepithelial electrical resistance and Dextran flux. Inhibition of previously proposed key pathogen-induced pathways observed in immortalized cell models did not rescue barrier dysfunction. We could instead show changes in ion homeostasis, and tight junctional breakdown. While none of the previously proposed effector pathways appeared to be responsible, we could pin-point the observed phenotype to activation of the cAMP/PKA/CREB signaling pathway, as one of the factors of the multifactorial barrier breakdown. The establishment of an organoid-derived infection model is shown, allowing the study of in vitro Giardia duodenalis infections. Using this model, we could decipher a new series of events that may be one of the factors causing the intestinal barrier breakdown observed in symptomatic Giardiasis.

Die Epithelzellen des Dünndarms bilden die Hauptinfektionsroute für viele Protozoen wie zum Beispiel Toxoplasma gondii und Giardia duodenalis. Jedoch sind die Mechanismen dieser Infektionswege unbekannt, da geeignete Modelle fehlen, welche das Darmepithel nachbilden. In der folgenden Studie wurde eine in-vitro Plattform mit Darmorganoiden (organoid derived monolayers oder ODMs) etabliert, welche man für vergleichende Studien zu Parasit-Wirt-Interaktionen anwenden kann. Das ODM-System wurde angewendet, um die Anfangsphase einer T. gondii-Infektion zu modellieren, wobei der Schwerpunkt auf die Rolle von Interferon gamma (IFNγ) und immunitätsbezogenen GTPasen (Irgs) lag. Es wurde gezeigt, dass sich die Irg-abhängige Kontrolle virulenter Toxoplasma-Stämme zwischen dem Labormausmodell und anderen wildlebenden Nagetierarten unterscheidet. Aus diesem Grund wurden Vergleiche mit Organoiden verschiedener Labormausstämme und der Rötelmaus Myodes glareolus durchgeführt. Myodes glareolus ist eine Nagetierart, von der angenommen wird, dass sie eine höhere Resistenz gegen T. gondii aufweist. Basierend auf die Resultate der quantitativen Immunofluoreszentests und qPCR dieser These, führt die Stimulation mit IFNγ zu einer tendenziell verringerten Replikation der Parasitenstämme RH und Prugniaus in M. glareolus ODMs im Vergleich zu Mus ODMs. In dieser Studie, wurde zum ersten Mal die Rolle von Irgs bei intestinalen T. gondii-Infektionen identifiziert. Zu diesem Zweck wurden Organoide von M. glareolus mit einem fluoreszierend markierten Irgb10-Protein transfiziert, wodurch gezeigt werden konnte, dass Irgb10 T. gondii-Vakuolen dekoriert, was auf eine Beteiligung des Irg-Systems hindeutet. Schließlich wurde ein Koinfektionsmodell für T. gondii und/oder G. Duodenalis in Maus-ODMs etabliert. In diesem Modell wurde gezeigt, dass T. gondii weder die Induktion der Barrierestörung durch G. duodenalis noch die Replikation von T. gondii durch G. duodenalis beeinflusst. / The small intestinal epithelium is the primary route of infection for many protozoan parasites such as Toxoplasma gondii and Giardia duodenalis. Understanding the mechanisms of infection with such parasites, has been hindered due to the lack of appropriate models mimicking the complexity of the intestinal epithelium. Here, an in vitro platform was established, using intestinal organoids (organoid derived monolayers or ODMs) for comparative studies on parasite-host interactions. The ODM system was used to model the events during the early phase of a T. gondii infection, focusing on the role of Interferon gamma (IFNγ) and Immunity Related GTPases (Irgs). Irg dependent control of virulent Toxoplasma strains has been shown to differ between the laboratory mouse model and other wild-derived rodent strains. How these responses occur in rodent species that do not belong to the murine family, is yet to be determined. For this reason, comparisons were made with organoids from different laboratory mice strains and the bank vole Myodes glareolus, a non-muridae rodent species assumed to be more resistant to T. gondii. Based on this thesis, stimulation with IFNγ leads to a trend of reduced replication in M. glareolus ODMs compared to Mus ODMs for both Type I parasite strain RH and for Type II strain Prugniaud, based on quantitative immunofluorescence assays and qPCR. Analysis of the role of Irgs in intestinal T. gondii infections was performed, by transfecting organoids from M. glareolus with a fluorescently labelled Irgb10 protein, showing that Irgb10 decorates T. gondii vacuoles, suggesting Irg-system involvement. Finally, a co-infection model in murine ODMs was established for T. gondii and/or G. Duodenalis. Here, it could be observed that T. gondii did not influence G. duodenalis induction of barrier breakdown nor did G. duodenalis influence T. gondii replication.