The rheological and structural characteristics of gastric (MUC5AC) and duodenal (MUC2) mucin solutions, the structural basis of the adherent mucus layer in the two organs, and their interactions with polyphenols, the phytochemicals which are linked with a number of health benefits, were investigated using particle tracking microrheology and scattering techniques. We used biochemically well characterised porcine mucins as models for human mucins to characterise their viscoelasticity, structure and dynamics as a function of concentration and pH. Additionally, the mesoscopic forces that mediate the integrity of the network were investigated using reducing (dithiothreitol) and chaotropic agents (guanidinium chloride and urea). Mucins in solution were found to be flexible and three distinct viscoelastic regimes were identify ed. At neutral pH, both types of mucin were found to form flexible self-assembled semi-dilute networks above a critical concentration (c*) where the viscosity scales as c 0.53+-0.08 and c 0.53 +-0.06 for MUC5AC and MUC2 respectively. Above a second critical concentration, the entanglement concentration (Ce), the peptide backbones reptate and entangle and there is a sharp increase in viscosity, c 3.92+- 0.38 for MUC5AC and c 5.1 0+-0.08 for MUC2. At low pH, both types of mucin solution undergo a sol-gel transition, forming pH-switchable gels. The addition of tea-derived polyphenols and tea extracts to the mucin solutions has revealed the strong interaction of galloylated phenolic molecules with mucins, which eventually leads to the gelation of the solution. Cross-linking of mucins by galloylated polyphenols is thus expected to have an impact on the physicochemical environment of the stomach and small intestine; the alteration of the organisation of the mucin polymer network is expected to modulate the barrier properties of the two adherent mucus layers which will affect nutrient absorption and the viscoelastic microenvironment of intestinal bacteria.

La relation de la microflore lactique avec l'homme n'a été que très peu étudiée dans le contexte des aliments amylacés fermentés tropicaux. La plupart des recherches dans ce domaine sont réalisées par des combinaisons de tests phénotypiques (modèles cellulaires et animaux) et des essais cliniques. Cependant, la disponibilité des données génomiques permettent d'envisager de nouvelles stratégies. L'objectif de ce travail est de rechercher la présence d'une cinquantaine de gènes impliqués dans des fonctions probiotiques dans une collection de 152 bactéries lactiques isolées d'un aliment fermenté africain à base de mil, le ben-saalga, ainsi que dans le métagénome de différents aliments amylacés fermentés. Plusieurs couples d'amorces ont été dessinés par nos soins et ont permis de détecter par PCR la présence de ces gènes. Le criblage génétique est efficace pour déterminer le potentiel lié à certaines fonctions « simples » (synthèse de vitamines B et caroténoïdes, métabolisme de l'amidon, etc.), puisqu'il permet le plus souvent de réduire le nombre de tests phénotypiques à réaliser aux souches porteuses des gènes d'intérêt. Au contraire, des tests in vitro complémentaires (résistance au pH acide et aux sels biliaires, adhésion sur des modèles cellulaires, imagerie à résonnance plasmonique de surface) montrent les limites de l'approche moléculaire appliquée à la détection de fonctions plus complexes que sont l'adhésion et la survie des bactéries. Par ailleurs, les profils d'expression des gènes impliqués dans la fonction d'adhésion par PCR en temps réel sont fonction du modèle utilisé (cellules ou rats). Nous avons montré qu'un mélange des trois souches les plus prometteuses modifie le profil de protéines impliquées dans la maturation de l'épithélium intestinal de rats initialement axéniques. Nous pouvons conclure que le criblage génétique des métagénomes d'aliments amylacés fermentés tropicaux permet de mettre en évidence un potentiel probiotique et nutritionnel prometteur. / The relationship between the lactic acid bacteria composing the microbiota of tropical starchy fermented foods and humans has been poorly investigated. Most of the studies focus on a combination of phenotypical (cells models, animals) and clinical trials. However the increasing numbers of genomic data allow new strategies. The objective of this work was to screen the presence of around 50 genes involved in probiotic functions in a collection of 152 lactic acid bacteria isolated from an African fermented cereal based food called ben-saalga, and in the metagenome of various starchy fermented foods. In this study, several primers have been designed allowing the detection of genes of interest by PCR. The genetic screening is efficient for determining the potential linked to simple functions (B vitamins and carotenoids synthesis, starch metabolism, tannin degradation) as in most cases it allows to limit the number of phenotypical tests to the strain harbouring the genes of interest. On the opposite, more complex functions such as cell binding or bacterial survival, estimated in vitro (low pH, bile salts, cell models, surface plasmonic resonance imagery) revealed the limit of the approach. The expression of genes involved in cell adhesion measured by real time PCR vary depending on the model used (cells or animal).We showed that a cocktail of three potentially probiotic strains modifies the profile of proteins involved in the maturation of the intestinal epithelium of initially germ free rats. The genetic screening of the metagenomes shows that the traditional starchy fermented foods harbour a promising probiotic and nutritional potential.

Les cellules à mucus intestinales sécrètent des mucines, principalement MUC2, qui forment un gel protecteur. Malgré la place importante des acides gras à chaîne longue (AGCL) dans l'alimentation et leurs liens avec des pathologies où les cellules à mucus sont altérées il n'existe pas d'étude sur leurs impacts sur ces cellules. Mes travaux ont consisté à mettre en lumière ces interactions et à étudier leurs conséquences. In vitro, nous montrons que les AGCL saturés augmentent l'expression de MUC2 et son relarguage alors que les AGCL insaturés les inhibent et que l'acide palmitique favorise la différenciation cellulaire. In vivo, chez des ratons gavés avec de l'huile de palme, la production de MUC2 est augmentée et la perméabilité paracellulaire colique diminuée. Les huiles de colza ou de tournesol ne modifient pas la production de MUC2 et la perméabilité est identique aux contrôles malgré l'augmentation de l'expression de l'occludine. Dans un modèle de syndrome de l'intestin irritable, nous montrons que le gavage avec l'huile de palme, malgré la diminution de l'occludine, restaure la perméabilité et augmente le nombre de cellules à mucus. L'huile de colza et l'huile de tournesol ne corrigent pas le défaut de perméabilité. Parallèlement, deux études chez la souris ont mis en évidence l'impact de certains régimes sur le nombre des cellules à mucus. Ce travail a permis d'identifier les AGCL comme des nutriments capables de moduler les cellules à mucus, de faire émerger une nouvelle piste thérapeutique pour restaurer les populations de cellules à mucus et la perméabilité et d'ouvrir la réflexion sur les impacts de l'augmentation des populations de cellules à mucus chez l'individu sain / Intestinal goblet cells secrete mucins, mainly MUC2, which form a protective mucus gel. Despite the important place of long chain fatty acids (LCFA) in the diet and their links with pathologies where goblet cells are altered, there is no study dealing with their impact on goblet cells. The aim of my work was to highlight these interactions and to study their consequences. In vitro, we show that saturated LCFA increase MUC2 expression and release whereas unsaturated LCFA inhibit these process and that palmitic acid promotes cellular differentiation. In vivo, rat pups receiving oral administration of palm oil show a decrease of colonic paracellular permeability and an increase of MUC2 production. On the opposite, rapeseed and sunflower oils do not change MUC2 production and, intestinal permeability is the same as controls despite the increase of occludin expression. In an animal model of irritable bowel syndrome, palm oil effects are found again. When subjected rat pups to a maternal deprivation stress a few days after birth, they develop an intestinal hyperpermeability and a goblet cell depletion. We show that oral administration of palm oil concomitantly to the stress is able to, despite a decrease of occludin expression, restore the permeability at the level of non-stressed animals and to increase goblet cell number. Rapeseed oil and sunflower oil are not able to correct the increase of intestinal permeability. In parallel, studies in mice show that some types of diets can be associated with an increase of the number of proximal and distal goblet cells. To conclude, this work (i) helped to identify LCFA as nutrients able to modulate intestinal goblet cell number and physiology, (ii) put forward a new therapeutic track, via palmitic acid use, to restore goblet cell populations and intestinal permeability in pathologies associated with these kind of alterations and finally (iii) offered new tracks of reasoning about physiological and pathophysiological impacts of the increase of intestinal goblet cell number in a healthy subject

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.

Les mucines sont les molécules structurales de base du gel de mucus qui assure la protection et la lubrification de la muqueuse gastro-intestinale. Notre but était de déterminer l'effet de la leptine sur la sécrétion et l'expression des mucines gastro-intestinales. Cette étude a été réalisée in vivo chez le rat et in vitro à l'aide des lignées cellulaires de rat DHE et humaine HT29-MTX. Nous avons trouvé que ces 2 lignées expriment les récepteurs (Ob-R) de la leptine. La perfusion luminale de la leptine dans le côlon de rat ainsi que la stimulation des cellules DHE par la leptine a entraîné une augmentation de la sécrétion de mucines et du niveau des ARNm codant pour rMuc2, rMuc3 et rMuc4. Cet effet de la leptine était dose dépendant. Testée dans les HT29-MTX, la leptine a provoqué une augmentation de la sécrétion de mucines ainsi qu'une augmentation des transcrits de MUC2, MUC4 et MUC5AC via la voie de la PKC, la PI3K et les MAPK. Ces résultats montrent que la leptine pourrait jouer un rôle essentiel dans la protection de la muqueuse colique.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.