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

Originalmente, a teoria de nicho ecológico fundamenta-se em indivíduos de uma espécie sendo ecologicamente equivalentes por utilizarem os recursos de forma similar. Portanto, o nicho de uma espécie é definido em termos do uso médio de recursos. Porém, a qualidade e abundância de recursos consumidos, sexo, idade ou morfotipo do consumidor influenciam o comportamento alimentar individual. Estudos recentes sobre forrageamento ótimo tem foco de interesse na variação interindividual no uso de recursos. Ao longo da sua área de distribuição são reconhecidas populações da toninha Pontoporia blainvillei (Cetacea) e no litoral paulista, possivelmente hajam três subpopulações dessa espécie: norte, centro e sul. Meu primeiro objetivo foi investigar se há variação na dieta entre essas três subpopulações de toninha. Adicionalmente, investiguei quais fatores entre sazonalidade, sexo e idade dos indivíduos poderiam estruturar a dieta em uma dessas subpopulações (central). Utilizei 58 indivíduos provenientes de captura acidental no litoral paulista, com representantes das regiões norte, centro e sul. A amostra incluiu juvenis e adultos de ambos os sexos, capturados em todas as estações do ano. Identifiquei as espécies nos conteúdos estomacais usando os otólitos de peixes e os bicos de lulas. Estimei o tamanho das presas utilizando regressões com as medidas dessas estruturas e investiguei a estruturação da dieta usando um índice de variação da dieta. Houve uma clara variação na dieta ente as subpopulações paulistas, possivelmente devido à diferença espacial na disponibilidade de presas. Para a subpopulação central não houve variação na dieta decorrente da sazonalidade ou do sexo. A ausência de variação sazonal pode ser devido à pouca alteração na abundância da principal presa, P. harroweri, no ambiente ao longo do ano. A ausência de variação decorrente do sexo pode ser devido a seleção de presa estar mais relacionada a características de corpo mole e fácil digestão e não ao tamanho da presa, sendo essa seleção comum para ambos os sexos. Indivíduos de idades diferentes possuem dietas distintas, com juvenis consumindo mais espécies que os adultos. Essa mudança de nicho alimentar pode ser devida ao período de aprendizado. Finalmente, mesmo descontando os efeitos da disponibilidade temporal e espacial de presas, do sexo e da idade, houve variação interindividual da dieta na subpopulação central. Somente estudos com marcadores isotópicos poderão investigar se essa variação é devida à especialização individual em toninhas / Ecological niche theory is originally based on the assumption that individuals of a species use similar resources and therefore are ecologically equivalents. Under this framework, the niche of the species can be defined in terms of average resource use. However, factors such as quality and quantity of prey resources in the environment, consumer gender, age or morphotype may influence the individual feeding behavior. Considering that possible interindividual variation, recently studies under optimal foraging theory have variation among individuals as a focus of interest. The species P. blainvillei is partionated in populations along its range and probably, subpopulations can be found in the Sao Paulo state coast: northern, central and southern. Hence, my first goal was to investigate if there is diet variation among the three franciscana subpopulations from Sao Paulo coast. Additionally, I searched for which factors would be the diet structure defined within one of the subpopulations (the central one). I have considered the seasonality, individual gender and sex as possible factors influencing the diet structure within the central subpopulation. I had 58 franciscana specimens obtained from bycatch in the Sao Paulo coast, being them from northern, central and southern regions. They were juveniles and adults of both genders and bycaught in all the seasons. I identified the prey species from the gut contents with fish otholits and squid beaks. I estimated prey length and weight using regressions with that structures measures and I investigated the diet structure using a diet variation index. Within the central subpopulation I found no diet variation based on seasonality or individual gender, instead I found diet variation for individuals from different ages. Probably, most of the diet variation among subpopulations is due to prey availability spatial difference. The lack of diet variation based on seasonality may be due to little seasonal variation in the abundance of the main prey, P. harroweri. The lack of variation due to gender is probably related to the prey selection on soft body and easy digestion instead of prey size, being this type of selection common for both genders. Juveniles franciscana preyed on more species than the adults and that niche shift as an age effect can be consequence of forraging skills development. Enclosing, even discarding the effects of the spacial and temporal prey availability, individual gender and age, I found interindividual-level diet variation within the central subpopulation, which can points out to franciscana individual specialization, but isotopic studies are required to infer that specialization

A maioria dos estudos da interação parasito-hospedeiro tem focado na interação de um único patógeno. Porém, o hospedeiro em um ambiente natural é comumente exposto a múltiplos patógenos sequencialmente ou mesmo simultaneamente. Diversos estudos têm utilizado o modelo de Ligadura e perfuração do Ceco (CLP) para estudar a sepse, mas nenhum deles apresentou modelo de coinfecção ou estudo avaliando o papel de infecções prévias no desfecho da sepse polimicrobiana experimental. Neste contexto, nossa hipótese é de que a infecção crônica por parasitos poderia alterar o curso da resposta durante a sepse polimicrobiana. Para testar essa hipótese, animais C57BL/6 ou BALB/c foram infectados com 5 ou 20 cistos da cepa ME 49 de Toxoplasma gondii e 40 dias após a infecção os animais foram induzidos à sepse polimicrobiana. Em nosso estudo, 100% dos animais cronicamente infectados por T. gondii morreram num período de 24 horas após CLP. O mesmo não foi observado quando animais foram infectados cronicamente com os parasitos Leishmania major e Trypanosoma cruzi ou com o fungo Paracoccidioides brasiliensis. Um dado interessante em nosso estudo foi que, nos animais previamente infectados com T. gondii, constatamos melhora na eliminação de bactérias liberadas pela CLP e aumento do recrutamento celular para o sítio da infecção. Apesar de esses animais apresentarem melhora na resposta contra as bactérias, verificamos a presença de lesão intestinal e maior infiltrado inflamatório neste órgão, associado a um aumento da produção de citocinas pró-inflamatórias (IFN-, TNF-, IL-6 e IL-1) e consequente aumento de óxido nítrico (NO), num período de 24 horas depois da CLP. Verificamos que as células TCD4+ e TCD8+ são responsáveis pela produção de IFN- e TNF- nesse modelo de coinfecção, e em modelo in vitro, que macrófagos podem ser responsáveis pela produção de IL-1 dependente de ativação do inflamassoma NLRP3/ASC/Caspase 1. Neste estudo, observamos que a rápida resposta contra a CLP acontece em função da presença de células de memória de padrão Th1, induzidas na infecção por T. gondii. Dessa forma, esse trabalho mostra que a infecção crônica por T. gondii agrava a sepse polimicrobiana subletal, por aumentar a produção de citocinas pró-inflamatórias IL-6, TNF- e IL-1, com a indução de hipotensão, predispondo ao choque séptico. / Most studies of parasite-host interaction have focused on the interaction of a single pathogen with cells or organism of the host. However, in a natural enviroment, the host is commonly exposed to multiple pathogens sequentially or even simultaneously. Several studies have used the model of cecal ligation and puncture (CLP) to study sepsis, but none of them evaluated the effect of the presence of previous infections to the outcome of polymicrobial sepsis. In this context, we hypothesized that chronic infection with Toxoplasma gondii could alter the course of host response against polymicrobial sepsis. To test this hypothesis, C57BL/6 or BALB/c mice were orally infected with 5 or 20 cysts of ME-49 strain of T. gondii and 40 days post infection, they were subjected to CLP. When mice were chronically infected with T. gondii, 100% of the animals died within 24 hours after CLP. The same phenomenons were not observed in animals previously infected with other parasites, such as Leishmania major and Trypanosoma cruzi or the fungus Paracoccidioides brasiliensis. Interestingly, when we evaluated the response against the CLP in animals that were infected with T. gondii, we found an improvement in the killing of bacteria released by CLP and an increase in recruitment of inflammatory cells to the site of infection. However, despite the fact that these animals have improved response against the bacterial infection, they presented intestinal damage and increased inflammatory infiltrate in this organ. The animals also had increased pro-inflammatory cytokines (IFN-, TNF-, IL-6 and IL-1), and nitric oxide (NO) detected within 24 hours after CLP. We also found that the TCD4+ and TCD8+ cells were responsible to produce IFN- and TNF-, and, using an in vitro model, we verified that macrophages are primarily responsible for the production of IL-1 in a pathway dependent on the activation of NLRP3/ASC/Caspase 1 inflamassoma. In this study, we found that early response against CLP happens due to the presence of mainly Th1 memory cells, induced by T. gondii infection. Finally, we found that chronic infection with T. gondii aggravates sublethal polymicrobial sepsis by increasing the cytokines IL-6, TNF- and IL-1, with induction of hypotension that predispose to septic shock.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-12-13T14:00:15Z No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-14T10:15:36Z (GMT) No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-14T10:15:36Z (GMT). No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Gut microbiota is changed by various factors, being the diet which has the highest influence, as well as the modulating additives of the microbiome. The purpose of this work was to evaluate the effects of growth promoting additives in the diet on the percentage of goblet cells with acid and neutral mucin in small intestine of pigs, the percent of gut cells in proliferation by the technique of immunohistochemistry by PCNA, to certify and quantify the presence of Paneth’s cells by specific staining and by immunohistochemistry, since that cell has a great importance in the immunity and in the gut microbiota. The experiment was conducted in a certified swine breeding farm (CSBF), applying the CRD (completely randomized design). Four hundred and eighty castrated male piglets were used, in five treatments, with ten piglets by cage, eight repetitions, and in the basal treatment 16 repetitions were used, being T1=basal diet, denominated negative control, T2=T1+antimicrobial agent, T3=T1+probiotic, Saccharomyces cerevisiae, T4=T1+prebiotic MOS, T5=T1+probiotic Saccharomyces cerevisiae + the prebiotic MOS. The diet was based on corn and soybean meal plus a supplement of minerals and vitamins. There were changes with higher increase in quantity of goblet cells, calculated by millimeter of gut mucosa with neutral mucus compared to those with acid mucus and to the negative control. There were no expressive changes for cells with acid mucus. The percentage of proliferating cells in small intestine by the immunehistochemistry technique for PCNA did not show significant changes and the presence of Paneth’s cells was confirmed by special staining and by immunehistochemistry. The quantity of Paneth’s cells of duodenum changed according to additives used, in comparison with the negative control which had a greater quantity. With the addition of antimicrobial agent there was a lesser quantity. With the prebiotics and synbiotics occurred milder reduction and this suggests that immunomodulation occurred. Besides promoting a better gut health due to a healthier and more appropriate microbiota to the rearing of piglets in the nursery phase, the addition of growth promoters in feeds favors significant gains and can be recommended, since the results are compensatory in productivity. / A microbiota intestinal é alterada por diversos fatores, sendo a dieta o que mais influência, assim como os aditivos moduladores do microbioma. Objetivou-se, neste estudo, avaliar os efeitos dos aditivos promotores de crescimento na dieta, sobre a porcentagem de células caliciformes com mucina ácida e neutra no intestino delgado de suínos, por modularem a microbiota e atuar na imunidade intestinal, avaliou a porcentagem de células intestinais em proliferação pela técnica de imunoistoquímica por PCNA, certificar e quantificar a presença de células de Paneth por coloração específica e por imunoistoquímica, relatos da literatura cita a ligação de probióticos com o epitélio intestinal via células de Paneth e caliciformes. O experimento foi conduzido em uma GRSC, aplicando o DIC. Utilizaram-se 480 leitões machos castrados, em cinco tratamentos, com dez leitões por gaiola, com oito repetições e no tratamento basal realizou-se 16 repetições. Sendo, T1=dieta basal, denominado de controle negativo, T2=T1+ antimicrobiano Avilamicina, T3=T1+probiótico Scchacaromyces cerevisae T4=T1+prebiótico MOS, T5=T1+probiótico Scchacaromyces cerevisae + o prebiótico MOS, foram abatidos com 65 dias de idade. A dieta foi à base de milho e farelo de soja mais um suplemento mineral e vitamínico. Ocorreram alterações com maior aumento na quantidade de células caliciformes com muco neutro comparativamente com aquelas de muco ácido e com o controle negativo. Não houve alterações expressivas para as células com muco ácido. A porcentagem de células no intestino delgado em proliferação pela técnica de imunoistoquímica para PCNA não apresentou alterações significativas, confirmou-se a presença de células de Paneth por colorações especiais e por imunoistoquímica. A quantidade de células de Paneth do duodeno alteraram-se em função dos aditivos utilizados, em comparação ao controle negativo que teve a maior quantidade, com a adição de antimicrobiano houve a menor quantidade, com prebiótico e simbiótico ocorreu redução mais branda, o que sugere ter ocorrido uma imunomodulação Os aditivos promotores de crescimento nas rações são tecnologias que favorecem ganhos significativos e podem ser recomendados, pois os resultados são compensatórios em produtividade. Além de promover uma melhor saúde intestinal em função de uma microbiota mais saudável e mais apta à criação de leitões na fase de creche

Le bisphénol A (BPA) est un perturbateur endocrinien couramment employé dans l’industrie agroalimentaire, en particulier pour les matériaux en contact des denrées alimentaires. Son utilisation dans la fabrication de polycarbonates et de résines époxy, recouvrant la face interne des boîtes de conserve et des canettes, en fait un contaminant ubiquitaire de l’alimentation humaine. L’augmentation de l’exposition humaine à ce contaminant a été corrélée avec la récurrence de certains troubles comme l’intolérance alimentaire, l’obésité ou le diabète de type 2. Ces dernières années, une attention particulière a été portée sur sa capacité à perturber différentes fonctions physiologiques, dont celle du système immunitaire, après exposition périnatale à des niveaux environnementaux pertinents pour l’Homme. Dans une première étude, nous avons montré qu’une exposition périnatale au BPA à 50 µg/kg de poids corporel/jour induit une diminution de l’activité anti-microbienne corrélée à une chute de l’expression du lysozyme dans l’iléon de la descendance femelle. La perméabilité intestinale de ces individus augmente en association avec le niveau d’IFN- dans les muqueuses du côlon. De plus, nous observons une diminution des plasmocytes à IgA associée à une perte de sécrétion d’IgA dans les fèces, démontrant un défaut de la fonction barrière et des défenses de l’intestin chez la descendance BPA. De manière intéressante, une diminution de la fréquence des ILC3 intestinaux est observée chez ces individus, avec une augmentation du niveau d’IgG sanguin dirigé contre une E.coli commensale. Ces effets sont associés à un défaut de maturation et de capacité migratoire des cellules de la lamina propria (LP) et de la rate. L’exposition périnatale au BPA provoque une augmentation de la sécrétion d’IFN- et d’IL-17 après re-stimulation in vitro CD3/CD28 des cellules de la LP, et une réponse de type Th17 dans la rate. Réunis, ces effets confortent la capacité du BPA, lors d’une exposition périnatale, à induire une intolérance alimentaire chez la descendance femelle. Dans une seconde étude, nous avons mis en évidence que l’exposition périnatale au BPA, à la même dose, induit des perturbations de l’homéostasie du système immunitaire intestinal et systémique chez la descendance mâle au jour 45, via une diminution de la fréquence des Th1 et Th17 dans la LP et une augmentation de la réponse Th1 et Th17 dans la rate. Ces impacts apparaissent en parallèle avec une altération de la sensibilité au glucose, une diminution de la sécrétion d’IgA dans les fèces et un appauvrissement des bifodobacteria dans le microbiote intestinal de ces individus. L’ensemble de ces évènements précède l’infiltration de macrophages M1 pro-inflammatoires dans le tissu adipeux péri-gonadique, en association avec une diminution de la sensibilité à l’insuline et une augmentation du poids corporel apparaissant avec le vieillissement (jour 170) chez la descendance BPA. Cette étude longitudinale a permis de proposer une séquence d’évènements aboutissant à un phénotype obèse et au T2D lors d’une exposition périnatale au BPA, et ainsi de comprendre le rôle du système immunitaire en lien avec le microbiote intestinal dans le développement de ces désordres métaboliques. Enfin, nous émettons l’hypothèse que les bisphénols S et F, deux analogues structuraux du BPA, peuvent disposer de capacités immunomodulatrices équivalentes à celles du BPA, susceptibles de provoquer les mêmes troubles chez la descendance. Nous avons testé cette hypothèse de manière préliminaire chez la descendance femelle après exposition périnatale au BPS et au BPF. Des résultats préliminaires d’études in vitro sur ces deux composés en comparaison au BPA sont également apportés. Ce travail de thèse contribue à accroître les connaissances relatives aux effets immunotoxiques des bisphénols dans le contexte de l’origine développementale des pathologies chroniques de l’adulte (DOHaD). / The endocrine disruptor bisphenol A (BPA) is commonly found in food industry, more precisely in food contact packaging. BPA is used to manufacture polycarbonate plastics and epoxy resins lining food and beverage cans, becoming an ubiquitous contaminant in human food. The extent of human exposure to this chemical is thought to be correlated with the occurrence of disorders like food intolerance, obesity and type-2 diabetes (T2D). During last years, a particular interest have been raised about its ability to disrupt various physiological functions, including immune system, after perinatal exposure at relevant environmental doses for Human. In the first study, we showed that perinatal exposure to BPA (50 µg/kg body weight/day) decreased anti-microbial activity and ileal lysozyme expression in the female mouse offspring. In those mice, we observed an increased gut permeability, in association with an increase of colonic IFN- level. Moreover, we observed a decrease of IgA+ cells with a loss of IgA secretion into faeces, depicting intestinal barrier and defense function defects in BPA female offspring. Interestingly, a decrease of the intestinal ILC3 frequency associated with an increase of IgG against commensal E.coli in sera have been observed in these individuals. These effects were linked to a defect of maturation and migratory ability of dendritic cells from lamina propria (LP) and spleen. Perinatal exposure to BPA also increased IFN- and IL-17 secretions after in vitro stimulation in the gut and elicited Th17 response in the spleen. Altogether, these effects support the ability of a perinatal exposure to BPA to induce oral intolerance with ageing in female offspring. Secondly, we showed that perinatal exposure to BPA at the same dose led to intestinal and systemic immune system homeostasis disturbances in male mouse offspring at day 45, through a decrease of Th1 and Th17 frequencies in the LP and an increase of Th1 and Th17 response in spleen. These effects were associated with an altered glucose sensibility, a decrease of faecal IgA secretion and a fall of bifidobacteria in the microbiota of these individuals. These BPA-mediated events precede infiltration of pro-inflammatory M1 macrophages in gonadal white adipose tissue, together with a decreased insulin sensitivity and an increased weight gain. This longitudinal study allowed us to better understand the sequential events linked to perinatal exposure to BPA that lead to obesity and T2D, and highlighted the role of immune system linked to gut microbiota in the development of these metabolic disorders. Finally, we hypothesized that two structural analogs of BPA –i.e., Bisphenol S and F- can display similar immune-modulatory effects that could lead to similar developmental disturbances than BPA in exposed-offspring. This hypothesis was tested in a preliminary experiment in female mouse offspring perinatally exposed to BPS and BPF. We also provided preliminary results of these two compounds, compared to BPA, from an in vitro study. This thesis contributes to the increase knowledge about the immunotoxic effects of bisphenol compounds in the context of the Developmental Origins of Health and Disease (DOHaD).

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.

Three investigations were conducted in order to investigate the effect of dietary probiotics on tilapia (Oreochromis niloticus) growth performance, intestinal morphology, intestinal microbiology and immunity. The first experiment demonstrated that Bacillus subtilis, Lactobacillus reuteri and Pediococcus acidilactici supplemented individually and as a mixed probiotic (in addition to Enterococcus faecium; AquaStar® Growout) were capable of modulating intestinal microbial populations as determined by culture dependent methods and DGGE. Furthermore, high-throughput sequencing reported that >99% of 16S rRNA reads in the mixed probiotic group belonged to the probiotic genera, predominantly assigned to Enterococcus (52.50%) and Bacillus (45.94%). Tilapia in the mixed probiotic group displayed significantly higher intraepithelial leucocyte (IEL) populations in the mid intestine when compared to the control and L. reuteri treatment. The mixed probiotic also improved microvilli density and had a higher absorptive surface area when compared to the control. In the second trial, after six weeks of supplementing tilapia diets with AquaStar® Growout at 3g kg-1, fish demonstrated significantly higher final weight, weight gain and SGR when compared to that of the control (void of probiotic) treatment or an initial probiotic feed (lasting two weeks) followed by control feeding. Probiotic supplementation at 3g kg-1 also caused an increase in the abundance of intestinal IELs and goblet cells and an up-regulation in the gene expression of intestinal caspase-3, PCNA and HSP70 and immunity genes TLR2, TNFα, IL-1β, TGFβ and IL-10 when compared with the expression of control replicates. These changes were not observed when supplementing tilapia diets with a lower dose (1.5g kg-1), nor when supplementing the probiotic in either a pulsed manner or as an initial feed (two weeks) followed by control feeding. Trial three revealed that the probiotic had a more discrete effect on the intestinal allochthonous microbiota as 16S rRNA reads assigned to probiotic genera only accounted for 5-10% of total reads. Nevertheless, the supplementation of dietary AquaStar® Growout at 3g kg-1 improved the localised immune response in tilapia, through the regulation of immunity genes TLR2, MYD88, NFκB, TNFα, IL-1β, TGFβ and IL-10, larger populations of goblet cells and a higher recruitment of IELs. Furthermore, the probiotic also improved the systemic immune response through the regulation of immunity genes (mentioned above) in the head kidney and significantly higher circulating leucocyte levels in whole blood. The extent of these changes were dependent on the probiotic treatment (i.e. continuously supplemented in feed or alternating weekly between probiotic at 3 g kg-1 and control feeding), the duration of feeding and the parameter investigated. This research demonstrates that B. subtilis, L. reuteri, P. acidilactici and AquaStar® Growout can modulate the intestinal microbiota. In addition, AquaStar® Growout can improve intestinal morphology, growth performance and modulate both the localised and systemic immune responses of tilapia when supplemented through the feed at the appropriate dosage and feeding regime.

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.

L’étude de la composition de la flore digestive ainsi que son implication avec la santé et les maladies est devenue un enjeu majeur. Nous avons étudié la flore de malades traités par de très nombreux antibiotiques, à la fois par culturomics et par pyroséquençage. Ce travail a montré une diminution importante du nombre de bactéries différentes colonisant le tube digestif après traitement. Cette diminution était d’autant plus importante que le traitement était prolongé. Les bactéries offrant une protection immunitaire contre certains pathogènes, il est à présent proposé des vaccinations contres des microorganismes invasifs après traitement antibiotique au long cours. Par ailleurs, les techniques de séquençage ont montré pour deux des échantillons un haut taux de colonisation par Akkermansia muciniphila, un microorganisme appartenant au phylum des Verrucomicrobia. Ce résultat a par ailleurs été confirmé en utilisant des techniques d’hybridation de fluorescence in situ (FISH). Les tentatives de culture ce microorganisme se sont révélées infructueuses. Au final ce travail nous aura permis de cultiver 7 nouvelles espèces que nous avons décrites en utilisant la taxonogénomique, une approche incluant des données phénotypiques et le séquençage du génome.Nous avons également étudié la flore de patients infectés par le virus du VIH par métagénomique et avons observé une augmentation considérable des bactéries qui supportent l’oxygène, alors que les bactéries intolérantes étaient très diminuées. Ces changements étaient associés aux marqueurs de progression de la maladie, ouvrant la voie à une supplémentation en antioxydants. / The study of the composition of the intestinal flora as well as its involvement with health and disease has become a major issue.We studied the flora of patients treated by broad-spectrum antibiotics by both culture and pyrosequencing. This work showed a significant decrease in the number of different bacteria colonizing the digestive tract after treatment. This decrease was even more important that treatment was extended. Bacteria interacting with immune protection against some pathogens, it is now proposed vaccinations against invasive microorganisms after prolonged antibiotic treatment. Furthermore, the sequencing techniques have shown for two samples a high-level colonization by Akkermansia muciniphila a microorganism belonging to the phylum Verrucomicrobia. Despite the successive failures of culture attempt of this organism, this finding was also confirmed using fluorescence in situ hybridization technique (FISH). Ultimately this work has enabled us to discover 7 new species that have been described using the taxonomogenomics approach which includes phenotypic data and genome sequencing.We also studied the flora of HIV-infected patients by metagenomics, and observed a significant increase in bacteria that withstand oxygen, while intolerant bacteria were deceased. These changes were associated with markers of disease progression, opening the way for antioxidant supplementation.

Les relations entre bactéries intestinales, aliments et hôte jouent un rôle crucial dans lemaintien de la santé humaine. La caractérisation fonctionnelle d’Uhgb_MP, une enzyme dela famille 130 des glycoside hydrolases découverte par métagénomique fonctionnelle, arévélé une nouvelle fonction de dégradation par phosphorolyse des polysaccharides de laparoi végétale et des glycanes de l'hôte tapissant l'épithélium intestinal. Les déterminantsmoléculaires de la spécificité d’Uhgb_MP vis-à-vis des mannosides ont été identifiés grâce àla résolution de sa structure cristallographique, sous forme apo et en complexe avec sesligands. Un nouveau procédé de synthèse par phosphorolyse inverse d'oligosaccharidesmannosylés à haute valeur ajoutée, a aussi été développé. Enfin, la caractérisationfonctionnelle de la protéine BACOVA_03624 issue de Bacteroides ovatus ATCC 8483, unebactérie intestinale hautement prévalente, a révélé que la famille GH130 comprend à la foisdes glycoside-hydrolases et des glycoside-phosphorylases capables de dégrader lesmannosides et les galactosides, et de les synthétiser par phosphorolyse inverse et/outransglycosylation. L’ensemble de ces résultats, ainsi que l’identification d’inhibiteurs desenzymes de la famille GH130, ouvrent de nouvelles perspectives pour l'étude et le contrôledes interactions microbiote-hôte / The interplay between gut bacteria, food and host play a key role in human health. Thefunctional characterization of Uhgb_MP, an enzyme belonging to the family 130 of glycosidehydrolases, discovered by functional metagenomics, revealed novel functions of plant cellwall polysaccharide and host glycan degradation by phosphorolysis. The moleculardeterminants of Uhgb_MP specificity towards mannosides were identified by solving itscrystal structure, in apo form and in complex with its ligands. A new process of high addedvalue mannosylated oligosaccharide synthesis by reverse-phosphorolysis was alsodeveloped. Finally, the functional characterization of the BACOVA_03624 protein fromBacteroides ovatus ATCC 8483, a highly prevalent gut bacterium, revealed that GH130 familyboth contains glycoside phosphorylases and glycoside hydrolases, which are able to degrademannosides and galactosides, and to synthesize them by reverse-phosphorolysis and/ortransglycosylation. All these results, together with the identification of GH130 enzymeinhibitors, open new perspectives for studying, and potentially also for controlling,interactions between host and gut microbes