Perturbing gut microbiota early in life can lead to the development of autoimmunity. We are just beginning to unravel how early immune programming by microbiota may have long-term effects on noncommunicable diseases. In this thesis, we lay groundwork for programming of the immune system by commensal bacteria early in life through our studies on the induction of early endogenous neonatal IgA, and we evaluate Limosilactobacillus reuteri's characteristics as an inducer. Garnering attention for use a probiotic, L. reuteri has many proven health promoting benefits, such as IgA induction, but emerging evidence also links specific strains to autoimmune disease. "Super-induction" of neonatal IgA can be achieved through cross-fostering immunocompetent pups on immunocompromised dams. We found that this phenomenon was categorically due to transferal of microbes from dam to offspring. By comparing strain CF48-3A to the non-gastric-related organism L. oris, we discovered that L. reuteri is a microorganism that can enhance early neonatal IgA induction. Further investigations revealed that the ability to induce neonatal IgA is not ubiquitous in all L. reuteri strains, as ATCC PTA 6475 did not significantly elevate IgA. We discovered that 6475 has the antigenic ability to stimulate B cell differentiation and IgA production, but it is suppressed by a mechanism related to differences in surface architecture of this strain. L. reuteri strains also vary in their potency of aryl hydrocarbon receptor (AhR) stimulation. In mice, activation of AhR during gestation by a potent prototypical ligand, TCDD, leads to development of autoimmunity offspring. We found that TCDD exacerbated autoimmunity in adult mice using a strain of mice with similar AhR affinity to humans. Further investigations can clarify whether differential AhR ligand expression between L. reuteri strains contributes to the relationship between L. reuteri and autoimmunity. Overall, we conclude that differences between strains of L. reuteri have profoundly different immunological consequences that contribute to our understanding of the linkage between strains and autoimmunity. / Doctor of Philosophy / Differences in microbes transferred to infants through maternal routes shapes the early development of the immune system. In general, transferred microbes are healthy for the infant, and studies suggest that disruption of healthy microbes in the infant gut is linked to long-term health consequences, like autoimmune diseases. We found that a particular difference in maternally transferred microbes increases the early appearance of immunoglobulin A (IgA, a gut-related antibody) in neonatal mice, which is an antibody important for protecting against gut-related infections. We were able to link this early IgA production to a probiotic species Limosilactobacillus reuteri. Within the species classification as L. reuteri, several genetically different strains are health-promoting and broadly marketed over-the-counter for use in probiotic supplements for infants, children, and pregnant and nursing mothers. Emerging scientific evidence also points to a potential connection between other L. reuteri strains and autoimmune disease. Secreted products of genetically different L. reuteri strains have been discovered to activate aryl hydrocarbon receptor (AhR) with various potency. We used a prototypical AhR ligand and found exacerbation of autoimmune disease in adult mice. Thus, we have concluded that different strains of L. reuteri have broadly different effects on immune system development, and strain variability may explain the different effects on autoimmunity that have been observed.

Os primeiros 30 dias pós-nascimento do bezerro, chamado período neonatal, é caracterizado por grande desenvolvimento imunológico. O sistema imune começa a se desenvolver ainda no início da gestação, porém, após o nascimento, mesmo que morfologicamente desenvolvido, não se apresenta totalmente funcional pela ausência de estímulos antigênicos. Ademais as particularidades anatômicas e fisiológicas da placentação dos bovinos são um impediente à transferência de imunidade durante a gestação, assim sendo o feto se desenvolve sem a influência das imunoglobulinas maternas, ficando altamente dependente ao início da vida extrauterina da transferência de imunidade passiva via colostro. As características do sistema imune do bezerro em seu período neonatal fazem com que este seja muito susceptível a doenças. Conhecer o comportamento imunitário dos bezerros recém-nascidos pode auxiliar a diminuir a incidência de doenças e o custo desse animal, além de aumentar o seu bem-estar. Deste modo, buscou-se avaliar o padrão de resposta imunitária do neonato nos primeiros 30 dias de vida, por meio de avaliações: da atividade fagocítica e do metabolismo oxidativo de neutrófilos circulantes e imunoenotipagem de linfócitos T (CD3+) e suas subpopulações (CD4+) e CD8+)) por citometria de fluxo e a expressão gênica das citocinas IL-4, IL-10, IL-12 e IFN-ϒ por PCR em Tempo Real. O exame físico, o hemograma e avaliação de transferência de imunidade passiva foram considerados como critério de inclusão para garantir a sanidade dos animais durante o período experimental. Com base nos resultados obtidos nesta pesquisa para pode se concluir que: a atividade de fagocitose dos granulócitos foi constante nos 30 dias avaliados; a produção de espécies reativas de oxigênio por granulócitos foi observada nos ensaios basal e estimulado; e com comportamento semelhante, apresentando os dois ensaios, maiores porcentagens nos dias 1, 25 e 30 p.n.; os linfócitos CD3+) e suas subpopulações:CD4+), CD8+), estes apresentaram porcentagens semelhantes àquelas encontradas nos bovinos adultos; e a relação CD4+) /CD8+) aumentou aos 30 dias de vida, pelo aumento de CD4+). Não foi possível mensurar a expressão gênica das citocinas IL-4 e IFN-ϒ em nenhum dos momentos avaliados; no entanto, foi verificada a expressão gênica de citocinas IL-10 e IL-12, com uma inclinação para o perfil Th2 induzido pela expressão mais frequente de IL-10, observando-se influência da expressão gênica das citocinas IL-10 e IL-12 no leucograma, na atividade dos granulócitos, e nas subpopulações de linfócitos T. / The first 30 days post-birth of the calf, called the neonatal period, is characterized by large immunological development. The immune system begins to develop even at the beginning of gestation, but after birth, even if morphologically developed, it is not fully functional due to the absence of antigenic stimuli. In addition, the anatomical and physiological characteristics of bovine placentation are an impediment to the transfer of immunity during gestation, so the fetus develops without the influence of maternal immunoglobulins, being highly dependent at the beginning of the extrauterine life of the transference of passive immunity via colostrum. The characteristics of the immune system of the calf in its neonatal period make it very susceptible to diseases. Knowing the immune behavior of newborn calves can help reduce the incidence of diseases and the cost of this animal, and increase their well-being. The aim of this study was to evaluate the immune response pattern of the neonate in the first 30 days of life through evaluations of phagocytic activity and oxidative metabolism of circulating neutrophils and T lymphocyte (CD3 +) immuno-typing and its subpopulations (CD4 + and CD8 +) by flow cytometry and the gene expression of the IL-4, IL-10, IL-12 and IFN-ϒ cytokines by Real-Time PCR. Physical examination, blood count and passive immunity assessment were considered as inclusion criteria to guarantee the health of the animals during the experimental period. Based on the results obtained in this research it can be concluded that: granulocyte phagocytosis activity was constant in the 30 days evaluated; the production of reactive oxygen species by granulocytes was observed in the basal and stimulated assays; and with similar behavior, presenting the two trials, highest percentages on days 1, 25 and 30 p.n .; the CD3 + lymphocytes and their subpopulations: CD4 +, CD8 +, these presented percentages similar to those found in adult bovines; and the CD4 + / CD8 + ratio increased at 30 days of life, due to the increase in CD4 +. It was not possible to measure the gene expression of IL-4 and IFN-ϒ cytokines in any of the evaluated moments; However, IL-10 and IL-12 cytokine gene expression was observed, with an inclination to the Th2 profile induced by the more frequent expression of IL-10, with the influence of the gene expression of the cytokines IL-10 and IL- 12 on leukogram, granulocyte activity, and T lymphocyte subpopulations.

Les nouveau-nés (enfants, ruminants) sont particulièrement sensibles à l’infection intestinale par le parasite Cryptosporidium parvum car leur système immunitaire est encore en cours de développement. Peu de solutions de contrôle existent à ce jour. Il n’existe pas de vaccin et seule une molécule l’Halocur™ possède une AMM pour les veaux mais l’utilisation du traitement est contraignante et il peut présenter une toxicité pour l’animal. Le développement de nouvelles alternatives immunoprophylactiques requiert de mieux comprendre les mécanismes immunitaires mis en jeux lors de l’infection. L’immunité innée joue un rôle prépondérant pour le contrôle de la phase aigüe de l’infection et nous avions montré au laboratoire que les phagocytes mononucléés CD11c+ sont des acteurs déterminant dans le processus protection. Lors de cette thèse nous avons confirmé le rôle des cellules dendritiques (DC) CD103+ en utilisant des souriceaux BatF3-/- chez qui le développement des deux sous-populations CD103+CD11b+ et CD103+CD11b- est altéré au niveau intestinal ce qui rend les animaux beaucoup plus sensibles à l’infection. / Newborns (children, ruminants) are particularly susceptible to intestinal infection by the parasite Cryptosporidium parvum because their immune system is still developing. To date, parasite control methods are limited. There is no vaccine and the only molecule which possess a marketing authorization for calves, Halocur ™, presents toxicity at 2 times the therapeutic dose. The development of new immunoprophylactic methods requires better understanding of the immune mechanisms occurring during infection. Innate immunity plays a major role in controlling the acute phase of infection and we previously demonstrated in the laboratory that intestinal mononuclear phagocytes CD11c+ are key players in the protection process. In this thesis, we confirmed the role of dendritic cells (DC) CD103+ using mice BatF3-/- in which the development of the two DC subsets CD103+CD11b+ and CD103+CD11b- is altered in the intestine making these animals more susceptible to infection. This high susceptibility can be partially mitigated by preventive administration of IL-12 to Batf3-/- neonatal mice. Batf3-/- adult mice which are only deficient for the CD103+CD11b- DC subset were transiently susceptible to infection in contrast to conventional mice that are highly resistant.

A la naissance, le système immunitaire des nouveau-nés est encore en plein développement. La première partie du travail de thèse a consisté à étudier les spécificités du système immunitaire intestinal des nouveau-nés qui conduisent à leur plus grande susceptibilité à l’infection par Cryptosporidium parvum. Ce protozoaire constitue un excellent modèle pour étudier les réponses immunitaires mucosales. En effet, son développement est restreint à l’épithélium intestinal et est strictement relié au statut immunitaire de son hôte ce qui explique que cet agent zoonotique affecte tout particulièrement les nouveau-nés et les immunodéficients. Nous avons démontré que les cellules dendritiques (DC) CD103+ étaient indispensables au contrôle de la phase aigüe de l’infection et que leur faible représentation dans la lamina propria de l’iléon chez les nouveau-nés était responsable de leur susceptibilité à l’infection. Nous avons identifié avec précision le mécanisme CXCR3 dépendant permettant le recrutement des DC CD1O3+ dans la muqueuse infecté et leur capacité à produire de l’IL-12 et de l’IFNdz, deux cytokines majeures impliquées dans le mécanisme de protection. La deuxième partie de ce travail a consisté à utiliser une stratégie d’immunostimulation basée sur l’utilisation de ligands de TLR capables d’activer fortement les cellules dendritiques du nouveau-né. Cette approche permet un contrôle rapide et très efficace d’une infection par C. parvum. / At birth, the neonatal immune system is still developing. In the first part of the thesis we investigated the characteristics of the intestinal immune system of neonates that lead to their greater susceptibility to infection by Cryptosporidium parvum. This protozoan is an excellent model for studying mucosal immune responses. Indeed, its development is restricted to the intestinal epithelium and is strictly related to the immune status of its host which explains the particular susceptibility of neonates and immunocompromised to this zoonotic agent. We have demonstrated that CD103+ dendritic cells (DC) are essential for the control of the acute phase of infection and their low representation in the ileal lamina propria of neonates was responsible for their higher susceptibility to infection. We have accurately identified the CXCR3-dependent mechanism for the recruitment of DC CD1O3+ in the infected mucosa and their ability to produce IL -12 and IFNdz, two major cytokines involved in the mechanism of protection. The second part of this work was to use an immunostimulatory strategy based on administration of TLR ligands that can strongly activate neonatal DC of the intestine. This approach allows a fast and highly effective control of an ongoing C. parvum infection.

Os primeiros 30 dias pós-nascimento do bezerro, chamado período neonatal, é caracterizado por grande desenvolvimento imunológico. O sistema imune começa a se desenvolver ainda no início da gestação, porém, após o nascimento, mesmo que morfologicamente desenvolvido, não se apresenta totalmente funcional pela ausência de estímulos antigênicos. Ademais as particularidades anatômicas e fisiológicas da placentação dos bovinos são um impediente à transferência de imunidade durante a gestação, assim sendo o feto se desenvolve sem a influência das imunoglobulinas maternas, ficando altamente dependente ao início da vida extrauterina da transferência de imunidade passiva via colostro. As características do sistema imune do bezerro em seu período neonatal fazem com que este seja muito susceptível a doenças. Conhecer o comportamento imunitário dos bezerros recém-nascidos pode auxiliar a diminuir a incidência de doenças e o custo desse animal, além de aumentar o seu bem-estar. Deste modo, buscou-se avaliar o padrão de resposta imunitária do neonato nos primeiros 30 dias de vida, por meio de avaliações: da atividade fagocítica e do metabolismo oxidativo de neutrófilos circulantes e imunoenotipagem de linfócitos T (CD3+) e suas subpopulações (CD4+) e CD8+)) por citometria de fluxo e a expressão gênica das citocinas IL-4, IL-10, IL-12 e IFN-ϒ por PCR em Tempo Real. O exame físico, o hemograma e avaliação de transferência de imunidade passiva foram considerados como critério de inclusão para garantir a sanidade dos animais durante o período experimental. Com base nos resultados obtidos nesta pesquisa para pode se concluir que: a atividade de fagocitose dos granulócitos foi constante nos 30 dias avaliados; a produção de espécies reativas de oxigênio por granulócitos foi observada nos ensaios basal e estimulado; e com comportamento semelhante, apresentando os dois ensaios, maiores porcentagens nos dias 1, 25 e 30 p.n.; os linfócitos CD3+) e suas subpopulações:CD4+), CD8+), estes apresentaram porcentagens semelhantes àquelas encontradas nos bovinos adultos; e a relação CD4+) /CD8+) aumentou aos 30 dias de vida, pelo aumento de CD4+). Não foi possível mensurar a expressão gênica das citocinas IL-4 e IFN-ϒ em nenhum dos momentos avaliados; no entanto, foi verificada a expressão gênica de citocinas IL-10 e IL-12, com uma inclinação para o perfil Th2 induzido pela expressão mais frequente de IL-10, observando-se influência da expressão gênica das citocinas IL-10 e IL-12 no leucograma, na atividade dos granulócitos, e nas subpopulações de linfócitos T. / The first 30 days post-birth of the calf, called the neonatal period, is characterized by large immunological development. The immune system begins to develop even at the beginning of gestation, but after birth, even if morphologically developed, it is not fully functional due to the absence of antigenic stimuli. In addition, the anatomical and physiological characteristics of bovine placentation are an impediment to the transfer of immunity during gestation, so the fetus develops without the influence of maternal immunoglobulins, being highly dependent at the beginning of the extrauterine life of the transference of passive immunity via colostrum. The characteristics of the immune system of the calf in its neonatal period make it very susceptible to diseases. Knowing the immune behavior of newborn calves can help reduce the incidence of diseases and the cost of this animal, and increase their well-being. The aim of this study was to evaluate the immune response pattern of the neonate in the first 30 days of life through evaluations of phagocytic activity and oxidative metabolism of circulating neutrophils and T lymphocyte (CD3 +) immuno-typing and its subpopulations (CD4 + and CD8 +) by flow cytometry and the gene expression of the IL-4, IL-10, IL-12 and IFN-ϒ cytokines by Real-Time PCR. Physical examination, blood count and passive immunity assessment were considered as inclusion criteria to guarantee the health of the animals during the experimental period. Based on the results obtained in this research it can be concluded that: granulocyte phagocytosis activity was constant in the 30 days evaluated; the production of reactive oxygen species by granulocytes was observed in the basal and stimulated assays; and with similar behavior, presenting the two trials, highest percentages on days 1, 25 and 30 p.n .; the CD3 + lymphocytes and their subpopulations: CD4 +, CD8 +, these presented percentages similar to those found in adult bovines; and the CD4 + / CD8 + ratio increased at 30 days of life, due to the increase in CD4 +. It was not possible to measure the gene expression of IL-4 and IFN-ϒ cytokines in any of the evaluated moments; However, IL-10 and IL-12 cytokine gene expression was observed, with an inclination to the Th2 profile induced by the more frequent expression of IL-10, with the influence of the gene expression of the cytokines IL-10 and IL- 12 on leukogram, granulocyte activity, and T lymphocyte subpopulations.

O ácido retinoico (RA), metabolito ativo da vitamina A, exerce ampla atividade biológica sobretudo na modulação da resposta imunológica. A interação do RA com os seus receptores nucleares induz a transcrição de genes que atuam na homeostase de sítios imunológicos, principalmente no tecido linfóide associado à mucosa intestinal (GALT). O RA promove a diferenciação de células T reguladoras CD4&#43CD25&#43Foxp3&#43, a migração de células efetoras para a mucosa intestinal induzindo a expressão de CCR9 e 4&#9467, além de inibir a diferenciação de linfócitos T helper (Th) 17 no intestino, garantindo a homeostase intestinal. Entretanto, eventos mediados pelo RA durante o desenvolvimento do sistema imunológico neonatal ainda não são totalmente conhecidos, principalmente no contexto materno-fetal. Desta forma, o objetivo do trabalho foi avaliar o efeito da suplementação materna com RA, durante a amamentação, no sitema imunológico da prole. Para tanto, camundongos fêmeas C57BL6 Foxp3-GFP receberam 6 doses de RA (1mg/gavagem), durante o período de amamentação, e o grupo controle recebeu apenas óleo vegetal. Os resultados mostram que a suplementação materna com RA foi capaz modular o sistema imunológico da prole aumentando o percentual de linfócitos T reguladores (Treg) esplênicos nas proles com 6 semanas de idade. Além disso, houve aumento percentual de linfócitos Treg, TCD4&#43 e TCD8&#43 que expressam CCR9, tanto no baço quanto nos linfonodos mesentéricos da mães e de suas prole, o que pode proporcionar a migração de células para o intestino. Este efeito foi duradouro nas proles até 6 semanas de idade. A suplementação materna com RA elevou o percentual de linfócitos Treg e linfócitos B IgA&#43 no intestino das proles, e a concentração de imunoglobulina (Ig) A fecal, mas não alterou a composição da microbiota intestinal. Nas mães suplementadas houve redução das concentrações séricas de IgA e IgG. Em contraste com o efeito tolerogênico do RA na lâmina própria do intestino, observamos o aumento sérico de interferon (IFN)- nas proles de mães suplementadas e aumento na secreção de IFN- por esplenócitos induzida por CL097 (agonista de Toll-like receptor 7/8), sugerindo que o RA pode ter um impacto importante na deficiente resposta de perfil Th1 nos neonatos. Para averiguar o efeito modulatório in vivo da suplementação materna de RA, foi avaliada a indução de colite por sulfato de sódio dextrano (DSS) nas proles. Não houve perda de peso acentuado nas proles de mães suplementadas com RA quando comprado às proles de mães controles, além de apresentarem a permeabilidade intestinal conservada e aumento do fator de transformação do crescimento (TGF)- &#946 no homogenato intestinal, indicando menor dano no tecido epitelial do intestino. Apesar disto, o RA não foi capaz de inibir totalmente o processo inflamatório na colite. No conjunto, os achados evidenciam que a suplementação materna com RA foi importante no desenvolvimento da imunidade de mucosa e na manutenção da homeostase intestinal, sendo um importante metabólito para atenuar respostas inflamatórias. A indução sérica de IFN- e após estímulo com CL097 pode indicar o uso de RA como estratégia para potencializar respostas Th1, crucial contra infecções virais e bacterianas no período neonatal. / Retinoic acid (RA), the active metabolite of vitamin A, exerts extensive biological activity mainly in the modulation of the immune response. The interaction of RA with its nuclear receptors induces the transcription of genes that acts on the homeostasis of immunological sites, especially in gut associated lymphoid tissue (GALT). RA promotes the differentiation of CD4&#43CD25&#43Foxp3&#43 regulatory T cells, migration of effector cells to the intestinal mucosa through gut-homing receptors CCR9 and 4&#9467, besides inhibiting the differentiation of T helper (Th) 17 cells in the gut, guaranteeing intestinal homeostasis. However, events mediated by RA during the development of the neonatal immune system are not totally known, especially in the maternal-fetal context. Thus, the aim of the study was to evaluate the effect of maternal RA supplementation during breastfeeding on the immune system of offspring. For this, C57BL/6 Foxp3-GFP female mice with 8-10 weeks-old received 6 doses of RA (1mg / gavage) during the breastfeeding period, and the control group received only vegetable oil. The results show that maternal RA supplementation was able to modulate the offspring immune system by increasing the percentage of splenic regulatory T (Treg) cells in offspring at 6 weeks of age. In addition, there was an enhancement in the CCR9 expression on regulatory T cells and CD4&#43 and CD8&#43 T cells, in the spleen and in the mesenteric lymph nodes of mothers and their offspring, which can provide migration of cells into the gut. This effect was long-term in offspring up to 6 weeks of age. Maternal RA supplementation also increasing the percentage of regulatory T cells and B IgA&#43 cells in the offspring´s gut, beside increasing the fecal immunoglobulin (Ig) A concentration, but did not alter the composition of the intestinal microbiota. In the supplemented mothers, serum concentrations of IgA and IgG were reduced. In contrast to the tolerogenic effect of RA on intestinal lamina propria, we observed a serum increase of interferon (IFN)- in offspring and increasing in CL097(Toll-like receptor 7/8 agonist)-induced IFN- secretion by splenocytes, suggesting that RA may have a significant impact on the deficient Th1 profile response in neonates. To investigate the modulatory effect in of RA maternal supplementation, was evaluated the induction of colitis by dextran sodium sulfate (DSS) in the offspring. There was no significant weight loss in the offspring from mothers supplemented with RA in comparison with the offspring from control mothers, as well as having preserved intestinal permeability and increased transforming growth factor (TGF)- &#946 in the intestinal homogenate, indicating less damage to intestinal epithelial tissue. Despite this, RA was not able to totally inhibit the inflammatory process in colitis. Taken together, the findings show that maternal RA supplementation was important in the development of mucosal immunity and maintenance of intestinal homeostasis, being an important metabolite to attenuate inflammatory responses. Induction of serum IFN- after TLR7/8 (CL097) stimulation may indicate the use of RA as a strategy to potentiate Th1 responses, crucial against viral and bacterial infections in the neonatal period.