CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O epitÃlio intestinal à formado e mantido por uma populaÃÃo de cÃlulas-tronco capaz de gerar diferentes linhagens celulares, mantendo a pluripotÃncia e a capacidade de auto-renovaÃÃo. A glutamina à um aminoÃcido essencial condicional importante para a manutenÃÃo do epitÃlio do intestino. No entanto, poucos estudos tÃm explorado o papel da glutamina na regulaÃÃo fina do turnover celular da cripta intestinal. Com o propÃsito de avaliar o papel da glutamina n o turnover de cÃlulas da cripta, foi utilizado um modelo in vitro de enteroide, onde as cÃlulas-tronco sÃo capazes de gerar um epitÃlio contendo as principais linhagens de cÃlulas secretoras intestinais (cÃlula de Paneth, cÃlula caliciforme e cÃlula enteroendÃcrina), alÃm dos enterÃcitos absortivos, com a formaÃÃo de uma estrutura tipo vilosidade-cripta. Este modelo foi usado para testar o efeito de 24 horas de privaÃÃo de glutamina (meio padrÃo com glutamina a 2 mM vs meio livre de glutamina) no turnover epitelial atravÃs da contagem de cÃlulas marcadas por Edu/nÃmero total por secÃÃo e ainda na apoptose celular, atravÃs da contagem de cÃlulas marcadas para caspase 3-clivada/nÃmero total por secÃÃo. A fim de avaliar a funÃÃo secretora das criptas, as razÃes das cÃlulas de Paneth e caliciformes por cripta (cÃlula alvo/nÃmero total de cÃlulas secretoras por cripta) foram medidas. O nÃmero de cÃlulas de Paneth e caliciformes foi obtido com o auxÃlio da microscopia confocal e imunomarcaÃÃo para lisozima e mucina-2. AlÃm disso, os transcritos dos produtos das cÃlulas de Paneth e caliciformes (lisozima e mucina, respectivamente) foram analisados utilizando o mÃtodo de PCR quantitativo em tempo real. Com intuito de avaliar o potencial papel imunomodulador da glutamina, transcritos de elementos da imunidade inata, receptor de tipo Toll e sua proteÃna acessÃria MD-2, e citocinas, a saber: TNF-α, IL-1β e CXCL-1, foram medidos. A privaÃÃo de glutamina reduziu o nÃmero de cÃlulas Edu positivas, em comparaÃÃo com o enteroide sob meio padrÃo (p=0,006). NÃo houve diferenÃa significativa em relaÃÃo Ãs razÃes das cÃlulas de Paneth e cÃlulas caliciformes entre os grupos apÃs privaÃÃo de glutamina. A privaÃÃo da glutamina diminuiu significativamente os transcritos de lisozima, em comparaÃÃo com o enteroide sob meio padrÃo (p = 0,007), mas nÃo para mucina-2, transcriÃÃo relacionada com a funÃÃo das cÃlulas caliciformes. Uma transcriÃÃo reduzida para TNF- α e MD-2 (p=0,005 e p=0,016, respectivamente) foi observada apÃs a privaÃÃo de glutamina. Ao todo, nossos achados reforÃam o papel positivo da glutamina sobre o turnover do epitÃlio intestinal e, alÃm disso, sugerem um importante efeito regulador da glutamina sobre as cÃlulas de Paneth e resposta imune inata. O modelo enteroide fornece uma ferramenta importante para dissecar os mecanismos de proteÃÃo pela glutamina e guiar estudos futuros. / The intestinal epithelium is formed and sustained by a population of stem cells capable of generating different cell lines while maintaining pluripotent and self-renewal capacity. Glutamine is a conditional essential amino acid important for the maintenance of the intestinal epithelium. However, few studies to date have explored the role of glutamine in the fine regulation of the intestinal crypt cell turnover. In order to evaluate the role of glutamine in the crypt cell turnover, an in vitro enteroid model was used, where stem cells are capable of generating an epithelium containing the main intestinal secretory cell lines (Paneth, goblet, and enteroendocrine cells) and absorptive enterocytes as well, while forming a villus-crypt like structure. This model was used to test the effect of 24h of glutamine deprivation (standard media with 2mM glutamine vs glutamine-free media) on epithelial turnover by counting EdU- labeled cells/total number per section and cell apoptosis by counting cleavage-caspase 3- labeled cells/total number per section. In order to assess crypt secretory function, Paneth and goblet crypt cell ratios (target secretory cell/total cell number per crypt) were measured. The number of Paneth and goblet cells was measured with the aid of confocal microscopy and lysozyme and mucin-2 immunostainning. In addition, Paneth and globet cell product transcripts (lysozyme and mucin, respectively) were measured using quantitative Real-time PCR. In order to assess the potential immunomodulatory role of glutamine, innate immune element transcripts, Toll like receptor and their accessory protein MD-2, and cytokines, as follows: TNF-α, IL-1β and CXCL-1, were measured. Glutamine deprivation reduced the number of EdU positive cell ratio as compared with the enteroid under the standard media (p=0.006). No significant differences regarding Paneth and goblet cell ratios were seen between groups following glutamine deprivation. Glutamine deprivation significantly decreased lysozyme transcripts as compared with the enteroid under the standard media (p=0.007), but not for mucin-2 transcripts, related to goblet cell function. Decreased TNF-α and MD-2 transcription (p=0.005 and p=0.016, respectively) were found following glutamine deprivation. Altogether, our findings reinforce the glutamine positive role on the intestinal epithelial turnover and furthermore suggest an important glutamine regulatory effect over Paneth cells and the innate immune system. The enteroid model provides an important tool the dissect the mechanisms of glutamine protection and shed light for future studies.

Thesis (Master of Science) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 147-160.

The intestinal mucosa is made up of complex secreted mucus layer consist of mainly mucin 2 (MUC2) and antimicrobial components that defend the underlining cellular barrier from intrusion by luminal microbiota and toxins. In inflammatory bowel diseases (IBD), the mucosal integrity is compromised. This can result from a combination of altered host genetics, gut immune responses and environment factors. However, it is the presence of intestinal bacteria that is central to the pathogenesis of IBD. As part of the dynamic gut microbial flora, mucolytic bacteria produce a wide range of glycosidases that are able to remove the outer oligosaccharide chains of MUC2, which allow other luminal bacteria to further degrade the mucin. We hypothesised that increased mucolytic bacteria will cause excessive degradation of the mucus layer, which in turn, allow more luminal bacteria to be in close proximity to the underlining epithelial cells resulting in inflammation. Consistent with our group’s previous semi-quantitative bacterial 16S rRNA gene clone library analysis, we found increased Ruminococcus gnavus in non-inflamed ulcerative colitis (UC) mucosa. R. gnavus was previously isolated by others based on its mucolytic property. In this study, we quantify total mucosa-associated bacteria and mucolytic bacteria, namely, R. gnavus, R. torques, Akkermansia muciniphila and bifidobacteria. We were able to show quantitatively that total mucosa-associated bacteria were increased in IBD. There was also a population shift in the mucosa-associated mucolytic bacteria, which were increased overall. There was significantly more R. gnavus in non-inflamed IBD biopsies. For the first time, we were also able to demonstrate that R. gnavus can degrade human MUC2 in vitro. To examine whether the numerical association of R. gnavus in IBD does have functional influence on intestinal inflammation and Paneth cell antimicrobial peptide gene expression, we fed mice with R. gnavus. Interestingly, R. gnavus feeding did not result in histological or molecular evidence of gut inflammation; however, it was able to specifically induce Paneth cell cryptdins and lysozyme P genes expression in 3 week old, antibiotic pre-treated C57BL/6 mice. This demonstrated that R. gnavus is not a pathogenic bacterium, which will directly cause colitis. However, the increased Paneth cell response suggested the need for host innate defence when R. gnavus is increased. Other than bacterial degradation, altered host genetics will also influence the mucus barrier. There is evidence to suggest that the MUC2 gene is highly unstable and is susceptible to gene copy number variation (CNV). Therefore, we hypothesised that MUC2 CNV is present, which may result in altered oligomerisation of the MUC2 glycoprotein causing endoplasmic reticulum stress of the goblet cells that appears to be characteristic of UC. Currently, our data partly support the presence of MUC2 CNV. However, further investigation is required to verify the MUC2 CNV identity. Only then can a high throughput methodology be designed to screen a large population for any association with IBD.

(En) Paneth cells are one of the major player in the maintenance of the homeostatic relationship between intestinal microbiota and the immune system. This function is largely achieved by their production of bactericidal enteric α-defensins (ED) and other antimicrobials. Disruption of Paneth cell functions is associated with severe human disorders such as Crohn's disease (CD) and Autoimmune Polyendocrinopathy- Candidiasis-Ectodermal Dystrophy (APECED). However, there is only a very limited information regarding the interactions and regulatory circuits operating between Paneth cells and intestinal immune system in either health or under pathological conditions. The previous study conducted in our laboratory described a new mechanism for the initiation and maintenance of Paneth cells targeted autoimmunity. The suggested model was that ED-specific T cells escape the selection in the thymus, infiltrate the intestine and diminish Paneth cell numbers through autoimmune destruction. This process also lead to the accumulation of inflammation- inducing bacteria, which were implied to exacerbate the inflammatory autoimmunity. Since this model of intestinal autoimmunity is of correlative nature, its intrinsic mechanism and functional relationships between immune system, Paneth cells and microbiota are largely...

En période néonatale, la muqueuse intestinale est immature et particulièrement perméable, notamment aux facteurs environnementaux comme les toxiques, les infections mais aussi les événements stressants. De la bonne maturation de la barrière intestinale va dépendre la mise en place de l'homéostasie intestinale et donc la tolérance vis-à-vis des antigènes luminaux. La survenu d’évènements stressants durant la petite enfance est associée au déclenchement et/ou l’entretien de pathologies gastro-intestinales fonctionnelles comme le Syndrome de l’Intestin Irritable (SII) mais aussi organiques comme les Maladies Inflammatoires Chroniques de l’Intestin (MICI). De plus, ces pathologies malgré leurs différences dans la sévérité et les signes cliniques, partagent un certain nombre de caractéristiques physiopathologiques communes, comme la rupture de l’intégrité de la barrière intestinale associée à une rupture de tolérance vis-à-vis des antigènes luminaux et plus particulièrement contre le microbiote intestinal. L’objectif de ce travail a été d’évaluer les effets d’un Stress de Séparation Maternelle (SSM) sur l’homéostasie intestinale et la réponse immunitaire vis-à-vis du microbiote commensal chez la souris jeune adulte. Nos résultats ont mis en évidence un dimorphisme sexuel dans ce modèle. Chez les souris mâles jeunes adultes, le SSM diminue les fonctions de la barrière intestinale associé à une altération de la réponse humorale et cellulaire au niveau systémique vis-à-vis du microbiote commensal, ainsi qu’à un défaut des cellules présentatrices d’antigènes, conduisant à une inflammation de bas grade malgré un profil proinflammatoire des lymphocytes T. Chez les souris femelles jeunes adultes, le SSM altère la fonctionnalité des cellules de Paneth associée à surpopulation bactérienne intestinale, responsable de la sensibilité viscérale en réponse à une distension colorectale et une réponse humorale systémique dirigée contre le microbiote commensal. Nous avons mis en évidence une empreinte du SSM chez le jeune adulte, capable d'induire des modifications profondes de l’homéostasie intestinale ainsi que de la réponse immunitaire systémique contre le microbiote intestinal. Le SSM altère l'homéostasie intestinale et reproduit des caractéristiques communes aux pathologies digestives à savoir une rupture de barrière associée à une réponse immunitaire contre le microbiote sans symptômes majeurs. Ce travail a permis d'identifier la survenue d'événements stressants pendant la petite enfance comme un facteur environnemental important capable d'altérer l'homéostasie intestinale chez des individus sains et qui pourrait contribuer au déclenchement de pathologies intestinales chez des individus génétiquement prédisposés. / Perinatal period is characterized by an immature intestinal barrier particularly permeable to luminal antigens and as such highly vulnerable to environmental factors like toxins, infections or stressful events. The appropriate maturation of intestinal barrier leads to intestinal homeostasis and tolerance toward luminal contents. Early life stressful events are associated with the development and/or maintenance of functional gastrointestinal disorders like Irritable Bowel Syndrome (IBS) or organic one like Inflammatory Bowel Diseases (IBD). These pathologies are highly different in term of etiology and clinical severity however they share common features like alteration in intestinal barrier associated with an abnormal immune response toward luminal contents especially commensal microbiota. Our aim was to evaluate the consequences of maternal separation (MS) on intestinal homeostasis, host-microbiota relationship and the humoral and cellular response at adulthood. Due to sexual dimorphism in this model, the results are presented separately for male and female. In young adult male mice, MS decreases intestinal barrier functions associated with an alteration of systemic humoral and cellular response toward commensal microbiota. Moreover, a defect of antigen presenting cells in spleen leads to systemic low grade inflammation despite a pro-inflammatory profile of T cell. In young adult female mice, MS alters the functionality of Paneth cells associated with an intestinal bacterial overgrowth, leading to visceral sensitivity and systemic humoral response toward commensal microbiota. We highlighted that MS has long lasting adverse effects on intestinal homeostasis and systemic immune response toward commensal microbiota in young adult. MS impairs intestinal homeostasis in healthy individuals and might contribute to trigger intestinal pathologies in susceptible persons.