Introduction: Gut microbial composition has been associated with immune-mediated diseases. Breastfeeding yields a microbiota rich in bifidobacteria and promotes colonization by lactobacilli. Bifidobacteria and lactobacilli are considered health-promoting and are used as probiotics, i.e. live microbial food supplements which when ingested in adequate amounts confer a beneficial effect on the host. During weaning the developing gut immune system is exposed to an increasing variety of antigens from both foods and gut microbiota. Aims: We aimed to determine if daily feeding of 1x108 colony-forming units (CFU) of the probiotic Lactobacillus paracasei ssp. paracasei strain F19 (LF19) to healthy term infants from 4 to 13 months of age could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, T cell function, Th1/Th2 immune balance and eczema incidence. Study design: Infants were randomized to daily intake of cereals with (n=89) or without LF19 (n=90) from 4-13 months of age. Clinical outcome measures were monitored by diaries and a questionnaire. Stool and blood samples were obtained at 4, 6½, 9, 13 and 5½, 6½, 12 and 13 months of age, respectively. Stool samples were analyzed for lactobacilli counts by conventional culture methods and the presence of LF19 was verified by randomly amplified polymerase chain reaction (RAPD-PCR). Fecal short-chain fatty acid (SCFA) pattern, a proxy for gut microbial function, was determined by gas-liquid chromatography. After polyclonal or specific activation of T cells, the cytokine mRNA expression levels [interleukin 2 (IL2), IFN-, IL4 and IL10] were determined on isolated mRNA by quantitative real time reverse transcriptase-PCR. Serum concentrations of total and specific IgE antibodies, Haemophilus influenzae type b, diphtheria and tetanus toxoid specific IgG antibodies were analyzed by enzyme immunoassay. Results: Feeding LF19 maintained high fecal lactobacilli counts during weaning. Persistent colonization with LF19 induced differences in the fecal SCFA pattern. The cumulative incidence of eczema was lower in the probiotic group, in conjunction with a higher IFN-γ/IL4 mRNA ratio in polyclonally activated T cells. Even though there was an effect by LF19 on Th1/Th2 immune balance, there was no effect on IgE sensitization. Infants in both groups increased their capacity to express both Th1 and Th2 cytokines during the second half of infancy but the expression was still lower than that of adults. Infants in the probiotic group had lower IL2 levels after polyclonal T cell activation at 13 months of age compared with infants in the placebo group. Infants fed LF19 did not have fewer infections, but had fewer days with antibiotic prescription compared with infants fed placebo. In addition, compared to placebo, persistent colonization by LF19 enhanced specific vaccine responses to protein antigens during the course of vaccination. Conclusions: We conclude that feeding LF19 was safe, based on no observed adverse effects in our study. Infants in both groups demonstrated maturation of adaptive immune responses during weaning. Adding probiotics in complementary foods during weaning reduced the risk of eczema by 50%, with a concomitant shift towards an enhanced Th1/Th2 ratio. The reduction of eczema might be explained by probiotic effects on both T cell-mediated immune responses and reinforced gut microbial function.

Introduction: Intestinal bacteria influence gut morphology by affecting epithelial cell proliferation, development of the lamina propria, villus length and crypt depth [1]. Gut microbiota-derived factors have been proposed to also play a role in the development of a 30 % longer intestine, that is characteristic of PRM/Alf mice compared to other mouse strains [2, 3]. Polyamines and SCFAs produced by gut bacteria are important growth factors, which possibly influence mucosal morphology, in particular villus length and crypt depth and play a role in gut lengthening in the PRM/Alf mouse. However, experimental evidence is lacking. Aim: The objective of this work was to clarify the role of bacterially-produced polyamines on crypt depth, mucosa thickness and epithelial cell proliferation. For this purpose, C3H mice associated with a simplified human microbiota (SIHUMI) were compared with mice colonized with SIHUMI complemented by the polyamine-producing Fusobacterium varium (SIHUMI + Fv). In addition, the microbial impact on gut lengthening in PRM/Alf mice was characterized and the contribution of SCFAs and polyamines to this phenotype was examined. Results: SIHUMI + Fv mice exhibited an up to 1.7 fold higher intestinal polyamine concentration compared to SIHUMI mice, which was mainly due to increased putrescine concentrations. However, no differences were observed in crypt depth, mucosa thickness and epithelial proliferation. In PRM/Alf mice, the intestine of conventional mice was 8.5 % longer compared to germfree mice. In contrast, intestinal lengths of C3H mice were similar, independent of the colonization status. The comparison of PRM/Alf and C3H mice, both associated with SIHUMI + Fv, demonstrated that PRM/Alf mice had a 35.9 % longer intestine than C3H mice. However, intestinal SCFA and polyamine concentrations of PRM/Alf mice were similar or even lower, except N acetylcadaverine, which was 3.1-fold higher in PRM/Alf mice. When germfree PRM/Alf mice were associated with a complex PRM/Alf microbiota, the intestine was one quarter longer compared to PRM/Alf mice colonized with a C3H microbiota. This gut elongation correlated with levels of the polyamine N acetylspermine. Conclusion: The intestinal microbiota is able to influence intestinal length dependent on microbial composition and on the mouse genotype. Although SCFAs do not contribute to gut elongation, an influence of the polyamines N acetylcadaverine and N acetylspermine is conceivable. In addition, the study clearly demonstrated that bacterial putrescine does not influence gut morphology in C3H mice. / Einleitung: Die intestinale Mikrobiota beeinflusst die Morphologie des Darmes durch Beeinflussung der Epithelzellproliferation, Entwicklung der Lamina Propria, Zottenlänge und Kryptentiefe [1]. Zudem stehen bakterielle Faktoren im Verdacht, die Entwicklung eines 30 % längeren Darmes in der PRM/Alf Maus gegenüber anderen Mausstämmen zu begünstigen [2, 3]. Die von der intestinalen Mikrobiota produzierten Polyamine und kurzkettigen Fettsäuren (SCFA) stellen wichtige Wachstumsfaktoren dar, die bei der Ausbildung des Darmes sowie an der Darmverlängerung in der PRM/Alf Maus beteiligt sein könnten. Zielstellung: Ziel dieser Arbeit war, den Einfluss von bakteriell-produzierten Polyaminen auf die Kryptentiefe, Schleimhautdicke und Epithelzellproliferation zu untersuchen. Zu diesem Zweck wurden keimfreie C3H Mäuse mit einer vereinfachten menschlichen Mikrobiota (SIHUMI) assoziiert und mit C3H Mäusen, die mit einer SIHUMI plus dem polyaminproduzierendem Fusobacterium varium (SIHUMI + Fv) besiedelt worden waren, verglichen. Weiterhin sollte der mikrobielle Einfluss sowie die Rolle von SCFAs und Polyaminen bei der Ausbildung eines verlängerten Darms in der PRM/Alf Maus untersucht werden. Ergebnisse: Die SIHUMI + Fv Mäuse zeigten eine bis zu 1,7 fach höhere intestinale Polyaminkonzentration im Vergleich zu SIHUMI-Mäusen, welche vor allem auf eine Erhöhung von Putrescin zurückzuführen war. Trotz der höheren Polyaminkonzentrationen wurden keine Unterschiede in der Kryptentiefe, Schleimhautdicke und Epithelzellproliferation beobachtet. Die Untersuchung der Darmlänge in PRM/Alf Mäusen in Abhängigkeit vom Besiedlungsstatus ergab einen 8,5 % längeren Darm in konventionell besiedelten PRM/Alf Mäusen im Vergleich zu keimfreien PRM/Alf Mäusen. Im Gegensatz dazu wurden in C3H-Mäusen keine Unterschiede in der Darmlänge in Abhängigkeit von der Besiedlung beobachtet. Der Vergleich zwischen PRM/Alf und C3H Mäusen, die beide mit der SIHUMI + Fv Mikrobiota assoziiert wurden, zeigte einen 35,9 % längeren Darm in PRM/Alf Mäusen. Trotz des längeren Darmes waren die intestinalen SCFA- und Polyaminkonzentrationen vergleichbar bzw. geringer als in C3H Mäusen, mit einer Ausnahme: Die Konzentration von N Acetylcadaverin war in PRM/Alf Mäusen 3,1-fach erhöht. Wurden keimfreie PRM/Alf Mäuse mit einer komplexen PRM/Alf Mikrobiota assoziiert, so war ihr Darm ein Viertel länger als bei PRM/Alf Mäusen, die mit einer C3H Mikrobiota besiedelt wurden. Dieser längere Darm korrelierte mit der N Acetylsperminkonzentration. Schlussfolgerung: Die intestinale Mikrobiota ist in der Lage, die Darmlänge abhängig von der mikrobiellen Zusammensetzung und von dem Genotyp des Wirtes zu beeinflussen. Obwohl SCFAs die Darmlänge nicht beeinflussten, ist eine Beteiligung der Polyamine N Acetylcadaverin und N Acetylspermin denkbar. Darüber hinaus zeigte die Studie, dass Putrescin die Anatomie des Darmes in C3H Mäusen nicht beeinflusst.

Les N-acyl homosérine lactones sont des molécules du quorum sensing impliquées dans la communication interbactérienne mais elles sont également capables d'intéragir avec les cellules eucaryotes. Rechercher ces molécules dans le contexte des maladies inflammatoires chroniques intestinlaes (MICI) et plus particulièrement dans le cadre de l'étude des conséquences de la dysbiose sur les voies de l'inflammation intestinale est séduisant. En utilisant la spectrométrie de mase, nous avons mis en évidence pour la première fois des AHLs dans l'écosystème intestinal humain, et plus particulièrement une nouvelle AHL, 3-oxo-C12 :2, qui est prédominante. Cette AHL est corrélée à la normobiose, est perdue au cours des MICI et exerce un effet protecteur sur les cellules épithéliales intestinales. En effet, la 3-oxo-C12 :2 exerce un effet anti-inflammatoire in vitro sur les cellules Caco-2 sans augmenter la perméabilité paracellulaire. De plus, les premiers résultats in vivo montrent que la 3-oxo-C12 est également capable d'influencer la composition du microbiote intestinal des souris. Ces résultats ouvrent de nombreuses perspectives notamment dans la recherche de traitements écologiques au cours des MICI. / Quorum sensing molecules N-acyl-homoserine lactones (AHLs) involved in bacterial communication network are also able to interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel diseases (IBD) and more precisely when studying consequences of dysbiosis on gut inflammation pathways is appealing. Using mass spectrometry, we identified for the first time AHLs in human intestinal ecosystem, and among them a new AHL, 3-oxo-C12:2 which is prominent. This AHL correlates with normobiosis, is lost IBD and exerts protective effect on gut epithelial cells. In fact, 3-oxo-C12:2 exerts anti-inflammatory effect in vitro on Caco-2 cells without increased paracellular permeability. Furthermore, first results from in vivo experiments show that 3-oxo-C12:2 is also able to influence mice gut microbiota composition. These results open multiple perspectives especially on new ecological treatments in IBD.

Notre équipe a précédemment rapporté que l'utilisation d'un traitement d'induction lympho-déplétant [les globulines anti-lymphocytaires polyclonales (GALP)], peut entrainer une lymphopénie T CD4 persistante chez certains transplantés rénaux. Celle-ci est associée à certaines anomalies biologiques telles que la présence d'un syndrome inflammatoire chronique. Ce travail de thèse s'intéresse aux facteurs impliqués dans l'induction de ces anomalies puisqu'elles sont associées à une augmentation de l'incidence d'infections, de maladies cardiovasculaires et de décès, habituellement retrouvés chez les personnes âgées. Nous avons montré que les GALP entrainent une diminution de la fonction thymique et une expansion de lymphocytes T à un stade avancé de différenciation pouvant traduire une immunosénescence prématurée. Par ailleurs, une rupture de la barrière intestinale semble favoriser la translocation bactérienne digestive et l'activation d'une inflammation chronique observée en transplantation, via les lipopolysaccharides libérés dans la circulation périphérique. La composition du rnicrobiote intestinal pourrait jouer un rôle dans l'initiation, le maintien et la sévérité de l'inflammation. Nous avons observé des modifications du microbiote après transplantation et après l' antibioprophylaxie utilisée en transplantation. Les mécanismes à l'origine de toutes ces observations restent à élucider mais ce travail permet d'améliorer la compréhension des facteurs impliqués dans le développement de l'immunosénescence et de l'inflammation chronique en transplantation rénale, ouvrant potentiellement la voie à d'intéressantes perspectives thérapeutiques. / We bave previously described that polyclonal anti-lymphocytic globulins (GALP) may contribute to prolonged CD4 T­cell lymphopenia in some renal transplant recipients, associated with some biological abnormalities, such as a chronic inflammatory syndrome. Our work focuses on the factors involved in the induction of tbese abnormalities and susceptible to increase the incidence of infections, cardiovascular diseases and deatb, comparable to the incidence observed in the elderly. We showed that GALP are implicated in the decrease in thymie output and the expansion ofT cells at an advanced stage of differentiation. Both are hallmarks of premature immune senescence. Moreover, the dysfunction of the gut epithelial barrier is responsible for gut bacterial translocation (GBT) and the activation of chronic inflammation observed in chronic kidney disease as well as in renal transplant recipients. The composition of the intestinal microbiota may play a role in the initiation, maintenance and severity of GBT and systernic inflammation. We reported the existence of a dysbiosis a.fier transplantation. The mechanisms involved remain to be elucidated, yet, this work contributes to the understanding of the potential factors involved. in the progression of immune senescence and the persistence of chronic inflammation after kidney transplantation paving the way to new fields of therapeutic research in transplantation

Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

Il a été montré que la consommation de Lactobacillus pouvait avoir des conséquences sur le poids de l’hôte, et ce, en fonction de la souche inoculée. Nous avons voulu comprendre le mécanisme par lequel ces microorganismes prolifèrent dans le tube digestif et induisent un changement de poids. Nous avons exploré la production de bactériocines permettant aux souches de Lactobacillus de croître au détriment des autres micro-organismes. Nous avons constitué une base de données de bactériocines nommée BUR et recherché les bactériocines dans les génomes de bactéries du tube digestif humain. Nous avons observé une abondance de bactériocines dans les génomes des bactéries du tube digestif, majoritairement dans les Firmicutes. Aussi, nous avons approfondi l’étude du profil métabolique des souches de Lactobacillus par une approche génomique et dans une analyse in vitro. Nous avons constaté que les souches de Lactobacillus dont la consommation induit une perte de poids possèdent des enzymes qui empêchent l'obésité en réduisant les niveaux de glucose dans le sang, les niveaux de triacylglycérols sériques, la masse corporelle et l'accumulation de graisse, tandis que les génomes des souches dont la consommation induit un gain de poids codent pour plusieurs enzymes favorisant la production de fructose, et mobilisent l'énergie et le carbone stocké dans les acides gras. De ce fait, nous pouvons dire que les espèces de Lactobacillus jouent un rôle important dans la digestion des nutriments, avec un effet direct sur le changement de poids. / It has been shown that Lactobacillus consumption could affect the weight of the host, and led consequently to significant weight gain or loss, depending on the strain. We wanted to understand the mechanism by which these microorganisms induce weight change. We explored the various known mechanisms, including the production of bacteriocins, allowing Lactobacillus strains to grow at the expense of other microorganisms. We established a bacteriocin database, named BUR, and search for bacteriocins in the genomes of human gut bacteria. We observed an abundance of bacteriocins in the genomes of bacteria in the digestive tract, predominantly in Firmicutes. We also have deepened the study of the metabolic profile of Lactobacillus strains, both by a genomic approach and in an in vitro assay, to better understand the pathophysiology of obesity. We have found that Lactobacillus strains whose consumption induces weight loss possess enzymes which prevent obesity by reducing glucose levels in blood, serum triacylglycerols levels, body weight and the accumulation of fat, whereas the genomes of strains whose consumption leads to weight gain encode several enzymes promoting the production of fructose and mobilize energy and the carbon stored in the fatty acids. Therefore, we can say that Lactobacillus species are important in the digestion of nutrients and can have a great influence on carbohydrate and lipid metabolism, with direct effect on weight change.

Les défauts d’odeurs, liés à l’accumulation d’androsténone et de scatol dans le tissu adipeux, sont le frein majeur à l’élevage des porcs mâles entiers. L’androsténone est produite par les testicules et le scatol dans le colon. En dehors de la génétique et de l’alimentation, les facteurs de variation ont été peu étudiés.La thèse aborde le problème des odeurs sexuelles sous quatre angles : 1) estimer l’héritabilité des composés odorants du tissu gras et leurs corrélations génétiques avec des indicateurs de santé, du développement sexuel et de l’agressivité ; 2) étudier l’effet de la dominance sur ces composés ; 3) déterminer l’impact d’un état inflammatoire chronique modéré sur le développement sexuel et la concentration en androsténone ; 4) rechercher les effets de conditions d’hygiène contrastées sur la teneur en scatol et analyser l’implication du microbiote intestinalNos résultats confirment l’héritabilité forte des teneurs en composés odorants mais les corrélations génétiques avec les paramètres de santé sont faibles. Une sélection pour réduire la concentration plasmatique en œstradiol et testostérone permettrait de réduire à la fois les teneurs en composés odorants et l’agressivité des porcs. Les porcs de rang de dominance élevé ont une teneur en androsténone supérieure dans le gras. Un état inflammatoire chronique modéré ne semble pas affecter la teneur en androsténone. Enfin, la dégradation de l’hygiène du logement augmente fortement la concentration en scatol, probablement via des changements de composition du microbiote intestinal. / Boar taint, due to the accumulation of androstenone and skatole in the fat tissue, is the major constraint to the production of entire male pigs. Androstenone is produced by the testes. Its synthesis increases during pubertal development. Skatole is produced in the hind gut. Its hepatic catabolism is inhibited by sexual steroids. Apart from genetics and feeding, the factors affecting boar taint have been little studied.The current thesis approaches four aspects of the boar taint problem: 1) the heritability of the fat content of odorous compounds in the fat tissue and their genetic correlations with indicators of health, sexual development and aggressiveness; 2) to study the effect of dominance on the fat content of boar taint compounds; 3) to determine the influence of a moderate chronic inflammatory status on the sexual development and the fat concentration in androstenone; 4) to evaluate the effect of contrasting hygiene conditions on the fat concentration in skatole and, to analyOur results confirm the high heritability of the fat concentrations of boar taint compounds but their genetic correlations with health indicators are low. A selection to decrease the plasma concentrations in estradiol and testosterone should decrease both boar taint compound concentrations and aggressiveness of boars. Boars with a high dominance rank have a higher fat concentration in androstenone. Lastly, the degradation of the housing hygiene conditions strongly increases the concentration in skatole, probably through modifications in gut microbiota composition

Les maladies psychiatriques présentent de fortes comorbidités avec des désordres gastrointestinaux, ce qui suggère l’existence de bases physiopathologiques communes. Une littérature abondante démontre que l’adversité précoce (infection, stress) augmente la vulnérabilité aux désordres psychiatriques à l’âge adulte. Chez le rongeur, le modèle de séparation maternelle induit chez la descendance adulte des comportements hyperanxieux associés à une hypersensibilité au stress, ainsi que des dysfonctionnements de la sphère gastrointestinale. De plus, des études récentes rapportent une hyperperméabilité de la barrière intestinale chez les ratons soumis au stress de séparation, un effet conduisant potentiellement à une dysbiose et une perturbation de la communication intestin-cerveau. Le but de ma thèse était donc d’étudier le rôle de l’axe intestin-cerveau dans la mise en place des effets à long terme du stress précoce. Nos travaux récents ont montré que certains effets à long-terme de la séparation maternelle peuvent être atténués par l’exposition des mères à un régime hyperlipidique. Dans un premier temps, nous avons testé les effets du régime hyperlipidique maternel sur le cerveau et l’intestin de ratons soumis à la séparation maternelle. Nos résultats montrent que le régime maternel hyperlipidique protège de l’augmentation de la permeabilité intestinale induite par le stress. Nous avons ensuite testé le rôle causal de la perméabilité intestinale sur les comportements émotionnels à travers une approche pharmacologique et une approche génétique. Nous rapportons 1) que la restauration de la fonction barrière de l’intestin atténue certains effets de la séparation maternelle et 2) qu’une hyperperméabilité intestinale chez des souris transgéniques non soumises à un stress produit des effets similaires à ceux de la séparation maternelle. Enfin, nous avons examiné les effets d’une adversité précoce multifactorielle sur le cerveau et l’intestin (perméabilité et microbiote) chez la descendance adulte mâle et femelle dans un modèle combinant infection prénatale et séparation maternelle. Nos résultats mettent en évidence un effet sexe très marqué sur les phénotypes comportements et intestinaux. D’autres études sont nécessaires pour identifier les mécanismes sous-tendant les effets de la perméabilité et la dysbiose intestinale sur la vulnérabilité émotionnelle associée au stress précoce. / Early-life adversity is a main risk factor for psychiatric disorders at adulthood; however the mechanisms underlying the programming effect of stress during development are still unknown. In rodents, chronic maternal separation has long lasting effects in adult offspring, including hyper-anxiety and hyper-responsiveness to a novel stress, along with gastrointestinal dysfunctions. Moreover, recent studies report gut barrier hyper-permeability in rat pups submitted to maternal separation, an effect that could potentially lead to dysbiosis and altered gut-brain communication. Therefore, the aim of my PhD was to unravel the role of the gut-brain axis in the neurobehavioral effects of early-life stress. We recently reported that some neural, behavioral and endocrine alterations associated with maternal separation in rats could be prevented by maternal exposure to a high-fat diet. We first addressed the effects of maternal high-fat diet on brain and gut during development in the maternal separation model. We show that maternal high-fat diet prevents the stress-induced decrease in spine density and altered dendritic morphology in the medial prefrontal cortex. Moreover, maternal high-fat diet also attenuates the exacerbated intestinal permeability associated with maternal separation. To explore a potential causal impact of gut leakiness on brain functions, we then examined the impact of pharmacological and genetic manipulations of intestinal permeability on brain and behavior. We report 1) that restoration of gut barrier function attenuates some of the behavioral alterations associated with maternal separation and 2) that chronic gut leakiness in naive adult transgenic mice recapitulates the effects of maternal separation. Finally, we examined the effects of multifactorial early-life adversity on behavior, gut function and microbiota composition in males and females using a combination of prenatal inflammation and maternal separation in mice. At adulthood, offspring exposed to early adversity displayed sex-specific behavioral (social behavior deficits in males and increased anxiety in females) and intestinal phenotypes. In conclusion, our work demonstrates an impact of gut dysfunctions, in particular gut leakiness, on the emergence of emotional alterations. Further studies are needed to unravel the role of the gut dysbiosis in the expression of the behavioral phenotypes associated with early-life adversity.

Ce travail de thèse a pour but de modéliser la capacité de dégradation des polysaccharides non digestibles par le microbiote intestinal humain. Nous exploitons pour cela des données métagénomiques. Il s'agit de données d'abondances de séquences de nucléotides dans 1408 échantillons dont les fonctions métaboliques sont assignées par annotation contre une base de données. Les séquences sont annotées par des marqueurs fonctionnels. Après une étape de sélection manuelle de 86 marqueurs fonctionnels pertinents à l'activité de métabolisation des polysaccharides, nous étudions leurs variations d'abondances parmi les échantillons métagénomiques.Nous proposons une approche de modélisation écologique du microbiote intestinal humain et considérons principalement la sélection fonctionnelle intense de cet écosystème pour faire l'hypothèse que des regroupements identiques de fonctions métaboliques sont présents en proportions différentes dans tous les microbiotes intestinaux humains. Nous proposons le terme d'assemblage fonctionnel qui rend compte de la co-occurrence spatiale et temporelle d'un groupement de fonctions. Ces assemblages sont en pratiques déterminés par leur composition en marqueurs fonctionnels, et peuvent s'interpréter comme une combinaison de traits fonctionnels agrégés au niveau des microorganismes composant l'assemblage.Les assemblages fonctionnels sont inférés par le biais d'une factorisation en matrice positive aussi nommée NMF de l'anglais Non-Negative Matrix Factorisation. Cette méthode permet de déterminer les assemblages fonctionnels, à la fois concernant leur composition et à la fois concernant leur abondance dans chacun des 1408 échantillons. Nous exploitons par ailleurs une information métabolique provenant de 190 génomes microbiens et de la bibliographie qui permet de préciser la composition de ces assemblages fonctionnels. Cette information se traduit sous forme d'une contrainte.Nous trouvons 4 assemblages en considérant un consensus entre différents critères. L'utilisation de l'information métabolique nous permet d'interpréter biologiquement ces assemblages. Les métadonnées associées aux 1408 échantillons nous permettent d'observer un comportement différent pour les échantillons provenant d'individus atteints de la maladie de Crohn. Nous validons cette observation sur des données extérieures.Nous avons proposé une approche réductionniste permettant de représenter un processus métabolique important à l'échelle du microbiote. Nous trouvons un nombre réduit de 4 assemblages fonctionnels qui sont biologiquement vraisemblables et permettent de bien approcher les 1408 échantillons métagénomiques. / The purpose of this work of thesis is to model the capacity of degradation of non-digestible polysaccharides by the human intestinal microbiote. To this end we exploit metagenomic data. We use abundances of nucleotide sequences in 1408 samples whose metabolic function are assigned by annotation against a database. The sequences are annotated with functional markers. Upon manual selection of 86 functional markers relevant to the activity of metabolisation of polysaccharides, we their abundances variation among the metagenomic samples are studied.We propose an ecological approach in modeling the human intestinal microbiote. We consider the intense functional selection of this ecosystem and assume that identical cluster of metabolic functions can be found in different proportions in every human gut microbiota. We propose the term of functional assembly as to account for spacial and temporal co-occurence of functional cluster. In practice, theses assemblies are determined by their composition and can be interpreted as combinations of functional traits aggregated at the levels of the cluster of microorganisms composing each assembly. Functional assemblies are inferred by the means of Non-Negative Matrix Factorization (NMF). This method allows to determine the composition of functional assemblies and their abundance in each of the 1408 metagenomic sample.Furthermore, we exploit metabolic information from bibliographic resources and 190 microbial genomes in order to specify the composition of these functional assemblies. This information is translated in the form of a constraint.We find 4 assemblies by considering a consensus between various criteria. The use of metabolic information allow to interpret theses assemblies biologically. By exploiting the metadata of the 1408 samples, we observe a different behaviour for the samples coming from individuals suffering from Crohn disease. We validate this observation on external data.We proposed a reductionistic approach allowing to represent an important metabolic process at the level of the microbiota. We find a small number of 4 functional assemblies which are biologically likely and approach well the 1408 metagenomic samples.

<p>Vitamin E is a natural lipophilic antioxidant contains eight structurally related forms, i.e., α-, β-, γ-, δ-tocopherols (αT, βT, γT, and δT) and corresponding tocotrienols. Recent research indicates that vitamin E forms are differentially metabolized to various carboxychromanols. Some these vitamin E metabolites have been shown to exhibit strong anti-inflammatory and anticancer effects, yet little is known about their bioavailability. Without this knowledge, it is impossible to assess the role of vitamin E metabolism in biological functions of vitamin E forms and their protective effects on chronic diseases. While αT and γT appear to improved gut health, the underlying mechanisms are not well understood. Furthermore, specific forms of vitamin E such as γT have been reported to have cancer-preventing effects, but their anticancer efficacy is relatively modest. For these reasons, this dissertation focused on the characterization of the pharmacokinetic formation of vitamin E metabolites after supplementation, and the investigation of the underlying mechanisms of the protective effect of vitamin E forms, αT and γT, on gut health, as well as anticancer efficacy of the combination of aspirin and γT on carcinogen-induced colon tumorigenesis.</p><p><br></p><p>The first project focuses on characterizing the pharmacokinetic formation of vitamin E metabolites after single dose supplementation of γ-tocopherol-rich mixed tocopherol (γTmT) and δ-tocotrienol (δTE). With our recently developed LC/MS/MS assay for quantifying vitamin E metabolites, we can simultaneously quantify the level of short-chain, long-chain, and sulfated carboxychromanols in plasma, urine, and fecal samples of supplemented animals. In this study, we investigated the pharmacokinetics including excretion of vitamin E forms and the formation of their metabolites after a single dose intragastric administration of tocopherols and tocotrienols in rats. We also measured vitamin E metabolites in the serum obtained from healthy humans after gT supplementation. In the plasma of rat, the pharmacokinetic profiles of γT and δTE are described as the following: γT, Cmax = 25.6 ± 9.1 μM, Tmax = 4 h; δTE, Cmax = 16.0 ± 2.3 μM, Tmax = 2 h. Sulfated CEHCs and sulfated 11’-COOHs were the predominant metabolites in the plasma of rat with Cmax of 0.4-0.5 μM (Tmax ~ 5-7 h) or ~0.3 μM (Tmax at 4.7 h), respectively. In 24-h urine, 2.7% of γT and 0.7% of dTE were excreted as conjugated CEHCs, the major identified urinary metabolites. In the feces, 17-45% of supplemented vitamers were excreted as un-metabolized forms and 4.9-9.2% as metabolites. The majority of metabolites excreted in feces were unconjugated carboxychromanols, among which 13’-COOHs constituted ~50% of total metabolites. Interestingly, 13’-COOHs derived from δTE were 2-fold higher than 13’-COOH from γT. Unlike rats, γ-CEHC is the predominant metabolites found in human plasma, although 11’-COOHs and 13’-COOHs (sulfated and unconjugated) were elevated by >20 folds responding to γT supplement. In this study, we found that tocopherols and tocotrienols, when taken as supplements, are mainly excreted as un-metabolized forms and long-chain carboxychromanols in feces. High fecal availability of 13’-COOHs may contribute to modulating effects on gut health.</p><p><br></p><p>The second project of my dissertation investigated the effect of vitamin E forms, αT and γT, on intestinal barrier function in a cellular model and a mouse colitis model. Inflammatory bowel diseases (IBD) are chronic idiopathic inflammatory conditions characterized by disruption of intestinal barrier integrity. Previous studies by others and us had demonstrated that vitamin E forms, αT and γT, can protect against chemical-induced colitis in animal models. However, the role of these vitamin E forms on intestinal barrier function has not been studied. Herein, we investigated the potential protective effects of vitamin E forms, αT and γT, on intestinal barrier function in a Caco-2 colon epithelial cell model and a dextran sodium sulfate (DSS)-induced colitis mouse model. In Caco-2 cells, pretreatment with 25mM αT and γT attenuated Caco-2 monolayer barrier dysfunction induced by 10 ng/mL TNF-α/IFN-γ, suggesting that these vitamin E forms protect intestinal barrier integrity in this cellular model. In male BALB/c mice, the supplementation of αT (0.05%) or γTmT (0.05%) when given 3 weeks before DSS treatment or at the same time as DSS treatment alleviated DSS-induced fecal bleeding and diarrhea symptoms in mice, and attenuated colon inflammation and colitis-associated damages. Additionally, αT and γTmT supplementation attenuated DSS-induced intestinal barrier dysfunction, as indicated by improving the level of occludin, a tight junction protein, in the colon and reducing lipopolysaccharide-binding protein (LBP) in the plasma. Furthermore, gut microbiota analysis demonstrated that αT and γTmT supplementation could modulate intestinal microbiome composition in mice with DSS treatment. DSS treatment reduced the relative abundance of Lachnospiraceae compared to healthy mice, and supplementation of αT and γT partially reversed this effect. Interestingly, the family Lachnospiraceae has been reported to decrease in IBD patients. Our study demonstrated the protective effects of vitamin E forms on intestinal barrier integrity in a cell-based model and a colitis model in mice. Furthermore, we demonstrated that these vitamin E forms caused favorable changes in the intestinal microbial population under colitis condition.</p><p><br></p><p>The third project of my dissertation evaluated the anticancer efficacy of the combination of aspirin and γT using an azoxymethane (AOM)-induced and colitis-promoted colon tumorigenesis mouse model. Extensive inflammation in the colon promotes the development of colorectal cancer (CRC). Eicosanoid production by pro-inflammatory enzymes, cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) play a critical role in the initiation, progression, and invasion of CRC. Thus, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been recommended for chemoprevention of CRC. However, long-term use of aspirin can cause many side effects, and the anticancer activity of aspirin is very modest. Previously, we have demonstrated that the combination of γT with aspirin prolonged the anti-inflammatory activity of aspirin and alleviated aspirin-associated adverse effects in a carrageenan-induced inflammation model in rats. Additionally, we found that the combination of γT and aspirin has stronger anticancer activity than aspirin or γT alone against HCT-116 human colorectal carcinoma cells. Therefore, we examined the anticancer effect of the combination of 0.025% aspirin and 0.05% γT against AOM-induced and DSS-promoted tumorigenesis in mice. In this study, we have found that the combination of aspirin and γT, but not aspirin or γT alone, suppressed colon tumorigenesis in mice, as indicated by 40% and 50% reduction in the multiplicity of total polyps (P < 0.05) and large adenomatous polyps (>2mm2, P < 0.05), respectively. More strikingly, the combination of aspirin and γT reduced the overall tumor area by 60% (P < 0.05). Noteworthy, the supplementation of γT also alleviated aspirin-induced stomach lesion and appeared to modulate intestinal microbial composition. Our study demonstrated that the combination of aspirin and γT has stronger anticancer activity than aspirin or γT alone while alleviates aspirin-associated adverse effect, suggesting that the combination of γT and aspirin is a more effective and safer chemopreventive agent for CRC than aspirin alone.</p>