Alzheimer’s disease (AD) is the most common form of dementia and a growing disease burden that has seen pharmacological interventions primarily fail. Instead, it has been suggested that preventative measures such as a healthy diet may be the best way in preventing AD. Prebiotics are one such potential measure and are fermented into metabolites by the gut microbiota and acting as gut-brain axis components, beneficially impact the brain. However, the impact of prebiotics in AD prevention is unknown. Here we show that the prebiotic inulin increased multiple gut-brain axis components such as scyllo-inositol and short chain fatty acids in the gut, periphery, and in the case of scyllo-inositol, the brain. We found in E3FAD and E4FAD mice fed either a prebiotic or control diet for 4-months, that the consumption of the prebiotic inulin can beneficially alter the gut microbiota, modulate metabolic function, and dramatically increase scyllo-inositol in the brain. This suggests that the consumption of prebiotics can beneficially impact the brain by enhancing metabolism, helping to decrease AD risk factors.

This thesis presents the first randomised controlled trial (RCT) to investigate whether supplemented probiotic bacteria-"live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Sanders, 2008)-affect mood and other psychological outcomes in people presenting with low mood. Seventy-nine participants with at least moderate symptoms of depression were randomised in a double-blind manner to receive either a probiotic preparation containing Lactobacillus helveticus and Bifidobacterium longum or a matched placebo for eight weeks. The RCT phase was followed by an open label extension in which all participants were offered the active study product for a further 8 weeks. Participants were followed up at 6 months post-study. Based on the existing evidence from gut-brain axis research, and on models linking depression with inflammation, immune activation, low vitamin D levels, and the gut microbiota (outlined in Chapters 1 and 2), it was hypothesised that: the overall sample would have elevated levels of inflammatory biomarkers and low levels of vitamin D at baseline, and that this would be associated with scores on psychological and irritable bowel syndrome (IBS) outcome measures; that group differences (active treatment versus placebo) would be observed in scores on psychological outcome measures after eight weeks of probiotic intervention; that group differences would also be observed in blood levels of proinflammatory cytokines, hsCRP, vitamin D and BDNF, and scores on a measure of gut function/IBS, and that levels of these variables may predict or impact on treatment response; and that group differences would be observed on outcome measures at the point of the 6-month follow-up between those who continued to take the probiotic and those who discontinued probiotic use. In total, 58 of the 77 participants who provided baseline blood samples (75%) had at least one marker of inflammation elevated outside the normal reference range at baseline. Baseline vitamin D was approaching the deficient level, displayed a seasonal pattern, and was associated with severity on one measure of cognition. No significant differences were found between the active treatment and placebo groups on any psychological outcome measure, the measure of gut function or in the level of any blood-based biomarker in the randomised phase. Baseline vitamin D level was found to moderate treatment effect on several outcome measures. The results of the open label extension supported the lack of efficacy observed in the randomised phase, and also allowed for the comparison of efficacy over intervention periods of varying durations. The results of the follow-up at 6 months post-trial indicated that, while mean scores on psychological outcome measures remained lower than baseline, there was regression on some outcome measures after the study. When the participants who replied to the 6 month follow-up questionnaire were divided into groups based on their self-reported dominant treatment since the trial (probiotics/nutrition, standard treatment or no treatment) there was no difference in mood or other psychological outcomes among the groups at 6 months. The current trial found no evidence that this probiotic formulation is effective in treating the symptoms of depression or IBS, or in moderating the levels of inflammatory and other biomarkers in a sample recruited with moderate depression. This finding does not support the theory proposed in several narrative reviews which suggests probiotics as a possible intervention for depression and other mental health outcomes, but is supported by the systematic review of human probiotics studies presented in Chapter 3 which found overall limited evidence of probiotic efficacy for psychological outcomes. Future studies in the area should attempt to further broaden this field, in particular by recruiting samples with mild and/or non-chronic depression for interventional studies, or by approaching probiotics as a preventative or adjuvant treatment strategy for depression.

Une consommation élevée de sel a été associée à beaucoup de maladies et à un risque accru de décès. Plusieurs mécanismes sous-jacents, y compris le stress oxydatif, ont été étudiés. Mais la salinité dans l'intestin et l'altération possiblement associée de son microbiote, récemment identifiées comme un symbiote critique de la santé et de la maladie, n'ont pas encore été explorées chez l'homme. En testant 1334 prélèvements de selles, nous avons montré qu'une salinité élevée était associée à une diminution de la diversité globale et à l'émergence de populations microbiennes halophiles dans l'intestin. La salinité fécale était associée au régime alimentaire salé et à l'obésité, conformément aux données épidémiologiques. Aucun procaryote halophile n’a été cultivé en dessous d'un seuil de salinité fécale de 1,5 %. Au-delà de ce seuil, nous avons découvert une diversité inattendue de microbiote halophile humain dont la richesse était corrélée avec les concentrations de sel; 64 espèces différentes ont été isolées, dont 21 nouvelles espèces et 43 espèces connues dans l'environnement mais non chez les humains. Trois procaryotes extrêmement halophiles ont été isolés, dont deux Archaea appartenant au genre Haloferax, avec une nouvelle espèce Haloferax massiliensis, et un nouveau genre bactérien, Halophilibacterium massiliense. D'autres études devraient spécifier les facteurs qui conduisent à la salinité intestinale et préciser si les altérations de microbiota intestinal associées à des niveaux élevés de sel peuvent être liées à des causes humaines / High salt intake has been linked with many diseases and an increased risk of death. Several underlying mechanisms, including oxidative stress, have been investigated, but salinity in human gut and the possible associated alteration of its microbiota recently identified as a critical symbiote of health and disease, have not yet been investigated in humans. Here, by testing 1,334 stools, we have shown that high salinity is associated with a decrease in overall diversity but the emergence of halophilic microbial populations in the intestine. Fecal salinity was associated with saline diet and obesity, according to epidemiological data. No halophilic prokaryote can be grown below a fecal salinity threshold of 1.5%. Beyond this threshold, we discovered an unexpected diversity of human cultured halophilic microbiota whose richness was correlated with salt concentrations; 64 different species were isolated, including 21 new unknown species and 43 known species in the environment but not in humans. Three extremely halophilic prokaryotes were isolated, including two Archaea belonging to the genus Haloferax, with a new species Haloferax massiliensis, and a new bacterial genus, Halophilibacteriums massiliense. Further studies should specify the factors driving gut salinity, and clarify if the gut microbiota alterations associated with high salt levels could be causally related to human diseas

Introdução: O trato gastrointestinal humano é composto por uma comunidade microbiana diversificada que atua no controle da saúde. Estudos recentes demonstraram que o equilíbrio da microbiota intestinal é afetado na doença renal crônica (DRC), ocasionando o quadro de disbiose intestinal. Estes estudos sugeriram uma associação da disbiose intestinal com complicações metabólicas como acúmulo de toxinas urêmicas, progressão da DRC, inflamação e risco cardiovascular. Diante disso, medidas com o objetivo de restaurar o equilíbrio da microbiota intestinal são sugeridas, tais como a ingestão oral de probióticos, mas poucos estudos têm abordado o efeito destes suplementos na progressão da DRC e no risco cardiovascular destes pacientes. Objetivo: Avaliar o efeito do consumo de probióticos em fatores associados com progressão da DRC e risco cardiovascular de pacientes com DRC. Material e métodos: Trata-se de um estudo clínico controlado por placebo registrado no Clinical Trials NCT03400228. O estudo incluiu 30 pacientes adultos com DRC nos estágios 3 a 5 não em diálise, com função renal estável e proteinúria igual ou superior a 500 mg. A coleta de dados ocorreu entre novembro de 2015 até dezembro de 2017. O protocolo do estudo constou de período de washout de 4 semanas e randomização dos pacientes para o grupo de intervenção (GI, suplemento com probiótico) ou para o grupo controle (GC, maltodextrina). Foi realizado avaliação basal e após 24 semanas de consumo de probiótico ou placebo. Todos os pacientes receberam a orientação de consumir 2 sachês por dia do probiótico ou do placebo (maltodextrina). Foram avaliadas variáveis demográficas, clínicas, nutricionais, hábito intestinal e exames laboratoriais com amostras sanguíneas e urinárias. Resultados: Dos 30 pacientes incluídos, 20 completaram as 24 semanas do estudo, sendo 10 no grupo intervenção e 10 no grupo placebo. Após o uso de probiótico houve aumento na taxa de filtração glomerular estimada (p<0,001) e diminuição nos níveis séricos de creatinina (p<0,001), ureia (p=0,015), proteína C reativa (p=0,03), hormônio da paratireóide (p=0,03) e potássio (p=0,012), em comparação ao grupo placebo. Os efeitos positivos do probiótico na taxa de filtração glomerular estimada e na diminuição dos níveis séricos de creatinina e ureia permaneceram após análise de regressão multivariada. Não houveram diferenças significativas nos parâmetros urinários entre os grupos. Sintomas de constipação (p<0,001) e consistência fecal (p=0,016) apresentaram melhora no grupo intervenção versus placebo. Conclusão: A suplementação de probióticos melhorou os marcadores de função renal e reduziu inflamação, além de auxiliar na melhora dos sintomas de constipação intestinal em pacientes com DRC. / Introduction: The human gastrointestinal tract is colonized by a diversified microbial community that acts in control of health. Recent studies have shown that intestinal microbiota balance is affected in chronic kidney disease (CKD) leading to intestinal dysbiosis. These studies have suggested association of intestinal dysbiosis with several metabolic disorders such as accumulation of uremic toxins, progression of CKD, inflammation and cardiovascular risk. Therefore, interventional measurement that improve intestinal microbiota balance are suggested such as supplementation of probiotics, however few studies evaluated the effect of these supplements on the progression of CKD and cardiovascular risk in CKD patients. Aim: The purpose of the study was to evaluate the effects of probiotic supplementation on the factors associated with progression of CKD and cardiovascular risk in patients with CKD. Desing and Methods: This was a randomized, double-blind, placebo-controlled study. Thirty patients with CKD stages 3 to 5 not on dialysis, with stable renal function and protein-creatinine ratio > 0.50 were included. Data collection was between November 2015 and December 2017. Study protocol was 4-week washout period, patients randomized to intervention group (IG, probiotic supplement) or control group (CG, maltodextrin), and follow for 24 weeks. Renal function, C-reactive protein (CRP), bone and mineral metabolism, nutritional, and lipid profile markers and intestinal habit were measured at baseline and 24 weeks of study. Results: From 30 patients included in this study, 20 completed the 24 study weeks, 10 in the TG and 10 in PG. After probiotic supplementation, there was increase in estimated glomerular filtration rate (p<0.001) and decrease in serum creatinine 8 (p<0.001), urea (p=0.015), C-reactive protein (p=0.030), parathyroid hormone (p=0.03), and potassium (p=0.012) levels compared to CG. The beneficials effects of probiotics on estimated glomerular filtration rate and serum creatinine, urea, and Creactive protein remained after multivariate linear regression. There were no significant differences in the urinary parameters between the two groups. Symptoms of constipation (p<0.001) and stool consistency (p=0.016) improved in IG compared to CG. Conclusion: Probiotic supplementation improved markers of renal function and reduced inflammation. In addition, it improved the symptoms of intestinal constipation in patients with CKD.

Le microbiote digestif, composé de 1012 à 1014 bactéries par gramme de selle, est dominé par les bactéries anaérobies. Ces dernières, qui dépassent largement les aérobies, ont été découvertes en 1865 par Louis Pasteur dans ses travaux sur la fermentation. Les bactéries anaérobies représentent un intérêt médical particulier par leur implication dans les maladies infectieuses et métaboliques. Les anaérobies sont caractérisés par leur difficulté à être cultivés, car nécessitant une absence ou des concentrations faibles d’oxygène. Ainsi les connaissances sur ces microorganismes étaient limitées. Cependant, avec l’avènement des outils de biologie moléculaire notamment le séquençage à haut débit et le concept culturomics associé à l’identification par spectrométrie de mass MALDI-TOF, la connaissance des microorganismes anaérobies a été accentuée. Dans ce travail, nous nous sommes attelés dans un premier temps à la mise au point d’un milieu de culture efficace pour la culture des bactéries anaérobies et les tests d’activités antimicrobiennes. Nous avons montré dans un deuxième temps que culturomics, associé au MALDI-TOF, était un outil puissant dans l’identification des bactéries anaérobies en microbiologie clinique, mais également dans l’exploration de la diversité microbienne du tube digestif. Cette technique nous a permis d’isoler 19 nouvelles espèces de bactéries anaérobies dont 9 ont été décrites dans la troisième partie de ce travail. / The human gut microbiota is known to contain around 1012 to 1014 bacteria per gram of feces, with the majority being anaerobic. The latters were first discovered in 1865 by Louis Pasteur while working on fermentation. Anaerobic bacteria are known to play an important role in health and diseases and thus have taken a lot of attention in the medical field, especially in infectiousdiseases and metabolism. These bacteria are known for its sophisticated culture system because its growth requires little to no oxygen. Nevertheless, few is known about these type of microorganisms but with the advancement of molecular biology and sequencing techniques, its study became wider. Culturomics, is a recently developed culture-based approach that relies on optimizing culture conditions for bacterial growth and its identification by MALDI-TOF MS and 16S rRNA sequencing. The present work aims to create and optimized culture condition for anaerobic bacteria along with testing its anaerobic activities. Also, we aim to demonstrate the efficiency of Culturomics and MALDI-TOF in culturing, identifying and describing anaerobic bacteria in clinical microbiology and in the human gut. This approach allowed us to isolate 19 new anaerobic species out of which 9 has been described in this work.Keywords: Human gut microbiota, culture of anaerobic bacteria, culturomics, MALDI-TOF.

Introdução: O trato gastrointestinal humano é composto por uma comunidade microbiana diversificada que atua no controle da saúde. Estudos recentes demonstraram que o equilíbrio da microbiota intestinal é afetado na doença renal crônica (DRC), ocasionando o quadro de disbiose intestinal. Estes estudos sugeriram uma associação da disbiose intestinal com complicações metabólicas como acúmulo de toxinas urêmicas, progressão da DRC, inflamação e risco cardiovascular. Diante disso, medidas com o objetivo de restaurar o equilíbrio da microbiota intestinal são sugeridas, tais como a ingestão oral de probióticos, mas poucos estudos têm abordado o efeito destes suplementos na progressão da DRC e no risco cardiovascular destes pacientes. Objetivo: Avaliar o efeito do consumo de probióticos em fatores associados com progressão da DRC e risco cardiovascular de pacientes com DRC. Material e métodos: Trata-se de um estudo clínico controlado por placebo registrado no Clinical Trials NCT03400228. O estudo incluiu 30 pacientes adultos com DRC nos estágios 3 a 5 não em diálise, com função renal estável e proteinúria igual ou superior a 500 mg. A coleta de dados ocorreu entre novembro de 2015 até dezembro de 2017. O protocolo do estudo constou de período de washout de 4 semanas e randomização dos pacientes para o grupo de intervenção (GI, suplemento com probiótico) ou para o grupo controle (GC, maltodextrina). Foi realizado avaliação basal e após 24 semanas de consumo de probiótico ou placebo. Todos os pacientes receberam a orientação de consumir 2 sachês por dia do probiótico ou do placebo (maltodextrina). Foram avaliadas variáveis demográficas, clínicas, nutricionais, hábito intestinal e exames laboratoriais com amostras sanguíneas e urinárias. Resultados: Dos 30 pacientes incluídos, 20 completaram as 24 semanas do estudo, sendo 10 no grupo intervenção e 10 no grupo placebo. Após o uso de probiótico houve aumento na taxa de filtração glomerular estimada (p<0,001) e diminuição nos níveis séricos de creatinina (p<0,001), ureia (p=0,015), proteína C reativa (p=0,03), hormônio da paratireóide (p=0,03) e potássio (p=0,012), em comparação ao grupo placebo. Os efeitos positivos do probiótico na taxa de filtração glomerular estimada e na diminuição dos níveis séricos de creatinina e ureia permaneceram após análise de regressão multivariada. Não houveram diferenças significativas nos parâmetros urinários entre os grupos. Sintomas de constipação (p<0,001) e consistência fecal (p=0,016) apresentaram melhora no grupo intervenção versus placebo. Conclusão: A suplementação de probióticos melhorou os marcadores de função renal e reduziu inflamação, além de auxiliar na melhora dos sintomas de constipação intestinal em pacientes com DRC. / Introduction: The human gastrointestinal tract is colonized by a diversified microbial community that acts in control of health. Recent studies have shown that intestinal microbiota balance is affected in chronic kidney disease (CKD) leading to intestinal dysbiosis. These studies have suggested association of intestinal dysbiosis with several metabolic disorders such as accumulation of uremic toxins, progression of CKD, inflammation and cardiovascular risk. Therefore, interventional measurement that improve intestinal microbiota balance are suggested such as supplementation of probiotics, however few studies evaluated the effect of these supplements on the progression of CKD and cardiovascular risk in CKD patients. Aim: The purpose of the study was to evaluate the effects of probiotic supplementation on the factors associated with progression of CKD and cardiovascular risk in patients with CKD. Desing and Methods: This was a randomized, double-blind, placebo-controlled study. Thirty patients with CKD stages 3 to 5 not on dialysis, with stable renal function and protein-creatinine ratio > 0.50 were included. Data collection was between November 2015 and December 2017. Study protocol was 4-week washout period, patients randomized to intervention group (IG, probiotic supplement) or control group (CG, maltodextrin), and follow for 24 weeks. Renal function, C-reactive protein (CRP), bone and mineral metabolism, nutritional, and lipid profile markers and intestinal habit were measured at baseline and 24 weeks of study. Results: From 30 patients included in this study, 20 completed the 24 study weeks, 10 in the TG and 10 in PG. After probiotic supplementation, there was increase in estimated glomerular filtration rate (p<0.001) and decrease in serum creatinine 8 (p<0.001), urea (p=0.015), C-reactive protein (p=0.030), parathyroid hormone (p=0.03), and potassium (p=0.012) levels compared to CG. The beneficials effects of probiotics on estimated glomerular filtration rate and serum creatinine, urea, and Creactive protein remained after multivariate linear regression. There were no significant differences in the urinary parameters between the two groups. Symptoms of constipation (p<0.001) and stool consistency (p=0.016) improved in IG compared to CG. Conclusion: Probiotic supplementation improved markers of renal function and reduced inflammation. In addition, it improved the symptoms of intestinal constipation in patients with CKD.

L’exposition aux polluants environnementaux a été associée à de nombreux désordres métaboliques, immunitaires et reproductifs ainsi qu’à divers cancers. De plus en plus de travaux, indiquent que le microbiote intestinal, qui joue un rôle majeur dans l’immunité et le métabolisme de l’hôte, interagit avec les xénobiotiques dont les polluants organiques persistants (POPs) et les contaminants néoformés dans les aliments. Cette interaction peut avoir des conséquences toxicologiques importantes via la modification des fonctions du microbiote intestinal mais également via la métabolisation des xénobiotiques, entraînant une potentielle altération de l'homéostasie de l'hôte. Dans le cadre de cette thèse, nous avons démontré, en modèle in vitro, qu’une exposition aigüe du microbiote intestinal humain au benzo[a]pyrène (hydrocarbure aromatique polycyclique) a entraîné une altération des fonctions du microbiote intestinal au niveau du volatolome et du métatranscriptome microbien. Cependant, dans nos conditions expérimentales, aucun impact sur la structure microbienne n'a été observé. L’Homme étant continuellement exposé à un panel de composés chimiques environnementaux, nous avons par la suite étudié l'impact de divers POPs et produits néoformés dans les aliments sur le microbiote intestinal humain. Des familles de gènes ainsi que des composés volatiles microbiens ont été identifiés comme altérés après l’exposition, conduisant à une perturbation de l'activité microbienne. Nous avons finalement démontré que l'interaction microbiote-polluant pourrait conduire à l'établissement d'un état pro-inflammatoire modéré dans l'intestin avec une libération de cytokine IL-8 par les cellules épithéliales intestinales. Ces résultats appuient le concept émergent selon lequel les contaminants alimentaires pourraient altérer les activités du microbiote intestinal. / Exposure to environmental pollutants has been associated with various life-threatening disorders, including dysregulation of the immune and reproductive systems, metabolic diseases and various cancers. Growing evidences indicate that the gut microbiota, which plays major roles in host metabolic and immune functions, interacts with xenobiotics including persistent organic pollutants (POPs) and foodborne chemicals. The toxicological relevance of the gut microbiota-pollutant interplay is of great concern for the host since the chemicals may disrupt the gut microbiota functions leading to a potential impairment of the host homeostasis. During this PhD thesis, we demonstrated that in vitro acute exposure of the human gut microbiota with benzo[a]pyrene (polycyclic aromatic hydrocarbon) led to an impairment of the gut microbiota functions with a specific shift of the microbial volatolome and metatranscriptome. However, in our experimental conditions, no impact on the microbial structure was observed. Since humans are exposed to a wide range of environmental chemicals we investigated the impact of various POPs and foodborne chemicals on the human gut microbiota. We identified microbial volatiles and gene families that shifted after this exposure leading to an imbalance of the microbial activity. Furthermore, we demonstrated that the interaction between the pollutants and the gut microbiota lead to a significant release of pro-inflammatory IL-8 cytokine by the intestinal epithelial cells which may contribute to the establishment of a low-grade inflammatory state in the gut. All together, these data support the emerging concept that food pollutants could alter the gut microbiota activities.

Le système immunitaire muqueux, et plus particulièrement les réponses intestinales IgA sont essentielles non seulement à la défense contre les agents pathogènes, mais aussi au façonnement de la flore intestinale commensale. Dans les modèles murins de déficit en IgA, on observe une dysbiose intestinale majeure associée à une inflammation muqueuse, réversibles après restauration des IgA. Le but de ce travail est de décrire l'impact de l'absence d'IgA chez l'homme sur la composition du microbiote intestinal ainsi que ses conséquences locales et systémiques. L'étude comparative par analyse métagénomique des selles de 17 sujets déficitaires en IgA et de 34 donneurs sains retrouve l'absence de différence majeure en termes de répartition des phyla dominants, de diversité et de richesse génique bactériennes entre les deux groupes. En revanche, en analysant à l'échelon des espèces, on observe dans le déficit en IgA une surreprésentation d'espèces pro-inflammatoires et une sous-représentation d'espèces anti-inflammatoires. En outre, en l'absence d'IgA, nous observons la présence de réponses IgM qui opsonisent partiellement les genres ciblés par l'IgA, mais semblent maintenir la diversité au sein des Actinobactéries. Les patients présentent un biais phénotypique lymphocytaire T circulant (TH17) associé à des stigmates de translocation bactérienne. Enfin, l'absence d'IgA s'associe à une perturbation du réseau bactérien minimal "obligatoire". Ces résultats suggèrent que le déficit en IgA humain s'accompagne d'une dysbiose modérée associée à une altération de l'architecture du réseau bactérien induisant une hyperactivation du système immunitaire, malgré la présence de réponses IgM. / IgA responses play a key role in gut mucosa, defending host against pathogens but also shaping the commensal flora. In order to get insights into the specific contributions of IgA to host/microbial symbiosis in humans, we explored patients that lack only IgA, using gut microbial metagenomics and systems immunology. Microbiota composition was compared between 34 healthy controls and 17 selective IgA deficiency (sIgAd) patients. Contrary to what was observed in murine models of IgA deficiency, we show that human sIgAd is not associated with massive perturbations of gut microbial ecology, regarding phyla distribution, bacterial diversity and gene richness. A clear gut microbial signature is however associated to sIgAd: we found 19 over-represented MGS mainly described to be pro-inflammatory, but also 14 under-represented MGS, mainly known to be beneficial. We also explored local consequences of IgA deficiency, particularly whether IgM could replace IgA at host/bacterial interface. Using a combination of bacterial flow sorting and DNA sequencing, we therefore analysed the composition of IgM-coated microbiomes observed in sIgAd. We show that IgM only partially supply IgA deficiency, as not all typical IgA targets can also be opsonized by IgM, but nevertheless contribute to maintain Actinobacteria diversity. IgA deficiency is associated with a skewed circulating CD4+ T cell profile towards TH17, as well as markers of bacterial translocation. Finally, sIgAd is associated with a perturbation of the minimal bacterial network. Altogether our results suggest that human IgA deficiency is associated with a mild dysbiosis associated to systemic inflammation despite the presence of IgM

Le développement de l’allergie peut être influencé par le microbiote intestinal qui est impliqué dans la maturation du système immunitaire de l’hôte lors de la colonisation du tractus digestif dès la naissance. L’objectif de mon travail a été d’étudier l’impact du microbiote intestinal sur le développement d’une sensibilisation allergique à des protéines de lait de vache à l’aide d’un modèle de souris BALB/c gnotoxéniques. Dans une première étude, nous avons montré que les souris axéniques (Ax, sans germe) sont plus réactives que les souris conventionnelles (CV) au potentiel immunogénique et allergénique de la β-lactoglobuline (BLG) et de la caséine (CAS), lorsque ces deux protéines sont injectées intrapéritonéalement sans adjuvant. A l’aide d’un autre modèle de sensibilisation par voie orale au lait, nous avons confirmé que les souris Ax développent des réponses IgE contre la BLG plus fortes que celles des souris CV. Les mécanismes de sensibilisation contre la BLG et la CAS sont alors différemment affectés par la présence ou non d’un microbiote intestinal. Par ailleurs, une colonisation tardive du tractus digestif de souris Ax à l’âge de 6 semaines par le microbiote de souris CV induit chez les souris conventionnalisées (CVd) le développement, après sensibilisation, de réponses humorales toujours plus fortes que celles observées chez les souris CV. A l’inverse, une conventionnalisation des souris Ax au moment du sevrage à l’âge de 3 semaines, induit un niveau de sensibilisation plus faible que celui des souris CV. Dans ce cas, des différences de composition du microbiote intestinal entre souris CV et CVd pourraient jouer un rôle dans le faible niveau de sensibilisation des souris CVd. Nous avons enfin évalué l’impact de l’implantation dès la naissance d’une souche de Lactobacillus casei en monoxénie (souris Mx). La réponse humorale contre la CAS, mais pas contre la BLG, est alors significativement plus élevée chez les souris Mx que chez les souris Ax. Ces différentes études suggèrent que l’influence du microbiote sur le développement d’une sensibilisation aux protéines du lait de vache diffère selon les allergènes et selon le mode d’exposition aux allergènes. Ces résultats soulignent également qu’un retard de colonisation du tractus digestif peut perturber durablement la réactivité du système immunitaire à une sensibilisation contre des antigènes alimentaires. / The development of allergic responses can be influenced by the gut microbiota, which critically stimulates the maturation of the host immune system during colonization of the digestive tract at birth. We thus aimed to study the impact of the gut microbiota on the development of an allergic sensitization to cow's milk proteins by using a gnotobiotic BALB/c mouse model. First, we showed that germ-free (GF) mice are more responsive than conventional mice (CV) to the immunogenic and allergenic potential of β-lactoglobulin (BLG) and casein (CAS) when these proteins are injected intraperitoneally without adjuvant. With another model of oral sensitization to cow’s milk, the development of higher BLG-specific IgE responses in GF mice compared to CV mice was confirmed. We also observed that the mechanisms leading to oral sensitization to BLG and CAS are differentially affected by the absence of gut microbiota. Furthermore, a delayed colonization of the digestive tract of 6-week-old GF mice by a conventional microbiota was studied. The conventionalized mice (CVd) still developed, after sensitization, higher antibody responses than those measured in CV mice. In contrast, GF mice conventionnalized just after weaning, at 3 week of age, displayed a level of sensitization lower than that of CV mice. Differences in the gut microbiota composition evidenced between CVd and CV mice could also play a role in the lower level of sensitization of CVd mice. Finally, we evaluated the impact of the neonatal mono-colonization of mice by a strain of Lactobacillus casei. The antibody responses against CAS, but not against BLG, were then significantly higher in mono-associated mice than in GF mice. These studies suggest that the influence of microbiota on the development of sensitization to cow's milk proteins depends on the nature of the allergens and the mode of exposure. These results also underline that delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.

Depuis plusieurs années, il s’avère de plus en plus clair que le microbiote digestif a un impact remarquable sur la santé humaine. Il est affecté par de nombreux facteurs dont l’alimentation. En effet, en fonction du macronutriment majoritaire d’un régime alimentaire, certaines populations et fonctions bactériennes sont stimulées ou inhibées. Plusieurs pathologies de l’intestin ou liées à des troubles nutritionnels ou métaboliques ont un lien causal avec une altération du microbiote digestif parmi lesquelles la malnutrition sévère aigue. En effet, il a été récemment montré que le microbiote digestif des enfants malnutris était différent et colonisé par des Proteobacteria, des Enterococci, des bacilles Gram-négatifs et des espèces pathogènes. Au cours de nos travaux, une dysbiose est également observée chez nos patients malnutris par métagénomique et par culturomics avec un enrichissement en bactéries aérobies, en Proteobacteria et en espèces potentiellement pathogènes telles que Streptococcus gallolyticus et une perte notable en bactéries anaérobies associée à une perte de la capacité antioxydante du tractus gastro-intestinal révélée par une absence totale de Methanobrevibacter smitii, archeae méthanogène et un des procaryotes les plus sensibles à l’oxygène du tractus gastro-intestinal ainsi que un potentiel redox fécal accru. De plus, une perte de la diversité globale, connue et inconnue, est observée. Enfin, par culturomics et métagénomique, nous avons établi un répertoire des bactéries manquantes chez les malnutris dont treize présentent un potentiel probiotique et pourront être testées comme probiotiques dans un modèle expérimental dans un futur proche. / For the last decade, it has become increasingly clear that the gut microbiota has a tremendous impact on human health. It is affected by several factors among which diet that has a big impact. In fact, according to the major macronutrient in a diet type, specific bacterial populations and functions are stimulated or inhibited. Several pathologies of the gut or linked to nutritional or metabolic disorders among which severe acute malnutrition are causally linked to an alteration of the diversity of the human gut microbiota. In fact, it has recently been shown by several studies that the gut microbiota of malnourished patients was different and colonized by Proteobacteria, Enterococci, Gram-negative bacilli and pathogenic species. The analysis of our data regarding the fecal microbiota of children afflicted with severe acute malnutrition from Niger and Senegal showed a dysbiosis observed through metagenomics and culturomics with an increase of aerobic bacteria, Proteobacteria and pathogenic species such as Streptococcus gallolyticus, and a depletion of anaerobic species associated with a loss of the antioxidant capacity of the gastro-intestinal tract exhibited by a total absence of Methanobrevibacter smithii, a methanogenic archaeon and one the most oxygen sensitive prokaryote of the gut microbiota alongside an increased fecal redox potential. Moreover, a loss of the overall diversity, known and unknown, was observed. Finally, through culturomics and metagenomics, we were able to identify a repertoire of missing microbes in malnourished children among which thirteen presented a probiotic potential and will be tested as such in an experimental model in the near future.