Oral tolerance mechanisms and the role of the microbiota

Abstract: The hygiene hypothesis state that microbial stimulation is necessary to fully activate and mature the immune system and to sustain tolerance mechanisms. Oral tolerance maintains immune homeostasis in the gut and prevents adverse reactions towards the comensal flora and Ags from the food and thus protects from development of allergies and inflammatory bowel diseases (IBD). The mechanisms underlying oral tolerance remain elusive but it has become clear that regulatory T cells are important mediators of oral tolerance although, little is known on how and where these regulatory T cells are induced. In order to induce oral tolerance the Ag (Ag) must be processed in the intestine. Ags present in the lumen are sampled by intestinal epithelial cells (IECs) and enter an endosomal/ lysosomal pathway that leads to loading of Ag peptides on major histocompability class class II molecules (MHCII), present on vesicular structures that are released from the IECs. Our research group was the first to identify that these vesicular structures named tolerosomes are present in serum after oral Ag administration. Tolerosomes isolated from serum shortly after a feed of Ag induce Ag specific tolerance when transferred into naive recipients. Mature intestines with MHCII expression in the IECs are necessary for development of oral tolerance and this maturation requires the presence of a comensal flora. The aim of this thesis was to study mechanisms of oral tolerance with focus on the generation of tolerosomes and what role the microbiota play in this process, and to investigate the influence of a comensal flora on the maturation of the immune system including generation of regulatory T cells. We found that oral administration of ovalbumin (OVA) result in activation of Ag specific T cells in the gut associated lymphoid tissue (GALT) as well as at peripheral lymphoid sites, including the lymph nodes draining the liver i.e. celiac lymph nodes (CLN). The proportion of Ag-specific cells with regulatory T cells were highest in CLN and that suggests that CLN could be an important site during development of oral tolerance. In addition, co-administration of Cholera toxin and OVA, preserved oral tolerance in the fed mice and lead to an increase of T cells with regulatory T cell phenotype, especially in the CLN. We confirmed, that tolerosomes can be generated in a mouse system and show that the ability to generate tolerosomes is dependent on expression of MHCII in the IECs and furthermore that the function of tolerosomes is restricted by their MHC haplotype. Tolerosomes induce Ag specific T cells activation, both in vivo and in vitro and show phenotypical similarities with in vitro generated exosomes, derived from cultured IECs. Germ free (GF) mice are unable to generate tolerosomes and neither colonization with Escherichia coli or Lactobacilli, could restore this dysfunction or induce expression of MHCII in the IECs of the former GF mice. We also demonstrate that GF mice in general have a less developed immune system with malfunctioning regulatory T cells and reduced levels of the suppressive cytokine IL-10. In conclusion, these results agree with the hygiene hypothesis that suggests that the microbiota play a crucial role for maturation of the immune system and that microbes are essential for the proper function of the tolerance mechanisms that protects us from adverse reactions to harmless environmental Ags.

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