The gastrointestinal tract is in constant contact with food proteins, commensals and potentially pathogenic microorganisms. In order to maintain immune homeostasis in this environment, the intestinal immune system has evolved redundant regulatory strategies. In this regard, the gut is home to a large number of regulatory T cells, including Foxp3+ Treg. These cells play a central role in the control of intestinal homeostasis. Additionally, several subsets of DCs with regulatory properties have been described with the capacity to induce IL-10 secretion from T cells or induce oral tolerance at steady state conditions. Previous work also demonstrated that a subset of DC expressing CD103 were necessary for the control of gut homeostasis. We hypothesized that the gut environment and in particular small intestine lamina propria dendritic cells (LpDCs), could potentially mediate extrathymic Treg development. We first observed Treg conversion in a lymphopenic transfer. This system allowed us to isolate and transfer pure T cell populations devoid of Foxp3. We confirmed these findings in complete mice that received Foxp3- OT-II T cells and were placed on an OVA feeding regimen. In this model, converted cells were rapidly found in the Mesenteric LN, Payers Patches and particularly in the small intestine Lamina propria. Remarkably, the intestinal immune system tolerates repeated exposure to resident microflora and food antigens, while maintaining the capacity to mount powerful immune responses against pathogens. Considering the amount of antigens persistently present in the lumen of the intestine, our data suggest that peripheral conversion may represent a significant pathway for the generation of Foxp3+ T cells.? It is becoming clear that nutrient status can impact an individuals susceptibility to intestinal pathologies. In the case of vitamin A, and in particular, its active metabolite, RA, prolonged insufficiency not only disrupts the integrity of the intestinal epithelial barrier but also prevents the proper deployment of effector lymphocytes into the GALT following priming. Indeed, the GALT is a significant producer of RA in the body and some effects of GALT DCs on mucosal immunity are associated with their capacity to synthesize this metabolite. For instance, RA production by GALT DCs can selectively induce molecules, such as CCR9 and a4b7, on conventional T cells and Treg involved in directing gut tropism. The observation that Treg converted in the presence of LpDC expressed high levels of a4b7 suggested that RA might be involved in the conversion process. Consistent with this observation, we found that LpDCs induce Foxp3 de novo on T cells based on their capacity to release RA. We showed that RA acts to enhance signals delivered by TGF-beta and that TGF-beta, but not RA, is the limiting factor in LpDC induced conversion. These results newly identify a food metabolite, retinoic acid as a co-factor in Treg cell generation, providing a mechanism via which functionally specialized GALT APCs can extend the repertoire of Treg cells focused on the intestine. ? Another critical component of the GI tract is its constant exposure to microbial stimuli. The adult human intestine contains up to 10x14 microorganisms. These microflora play a central role in the maintenance and control of host homeostasis. However, in genetically susceptible individuals, some components of the flora can contribute to the pathogenesis of various intestinal disorders including inflammatory bowel disease (IBD). In murine models of IBD, the presence of gut flora leads to the development of pathogenic immune responses that are held in check by Treg. In some cases, the role played by Treg cells is beneficial in limiting pathogenic effector responses, while in others, Treg control is excessive and leads to impaired development of protective responses. To maintain tonic immune responses, the GI tract needs to receive both regulatory and counterregulatory signals which implies that Treg cells have themselves to be controlled. Previous work showed that natural Treg can be controlled by Toll like receptor (TLR) signaling. Some interactions with TLR have been proposed to increase Treg suppressive capacity, while others have been shown to limit Treg function. The constant exposure of the gut immune system to a large number of microbial products in the gut provides a rational to consider the physiologic impact of TLR signaling on Treg homeostasis and function. TLR9 recognizes unmethylated cytosine phosphate guanosine (CpG) dinucleotides, which are abundant in prokaryotic DNA found in gut flora. Previous work demonstrated an association of Crohns disease with a promotor polymorphism in the TLR9 gene in humans. Such association supports the idea of a role for bacterial DNA sensing in the pathophysiology of IBD. To determine if bacterial DNA could modulate Treg homeostasis in vivo, we evaluated the percentage of CD4+Foxp3+ T cells in various compartments of TLR9-/-and WT mice. Nave TLR9-/- mice displayed a significantly higher percentage and absolute number of Foxp3+ Treg compared with nave WT mice in all GALT compartments compared to WT mice but not in other tissues. In addition, the basal levels of IFN-gamma and IL-17 produced by both CD4 and CD8+ T cells was reduced in the GALT of TLR9-/- mice. Such modification of the GALT environment in TLR9-/- mice is associated with a poor response to oral infection with E.cuniculi and enhanced susceptibility to the parasite and impairment of the mice to develop proper immune responses following oral vaccination. ? We next assessed whether bacterial DNA from conventional gut flora has a direct adjuvant effect on the induction of immune responses in the gut. To reduce the impact of other innate signaling induced by gut flora, we treated mice with a cocktail of antibiotics in their drinking water according to a protocol previously shown to remove most of the gut flora. Mice were subsequently treated or not by gavage with bacterial DNA from mouse gut flora. Antibiotic treatment alone increased the percentage of Foxp3+Treg in the GALT but not in other peripheral tissues such as the spleen. Following antibiotic treatment, mice were vaccinated orally with OVA in combination with E. coli LT(R129G) or infected with microsporidia and Treg percentages and immune responses evaluated. Consistent with the increased percentage of Treg in the gut, antibiotic treatment alone reduced the capacity of mice to produce IFN-gamma and IL-17 following oral infection. Treatment of mice with bacterial DNA from conventional gut flora restored the response to a level comparable to control mice in all compartments. Similar data were obtained with CpG but not with LPS or other TLR ligands. To assess the mechanism underlying the increase of Treg and limitation of immune responses in the gut, we tested if bacterial DNA could limit the neo-generation of Treg. Our in vitro data clearly demonstrate that the addition of CpG or bacterial DNA prepared from gut flora but not other TLR ligands dramatically decreases the capacity of T cells to acquire Foxp3 when stimulated with LpDCs in vitro while enhancing their capacity to release IL-17. Thus, our data support the hypothesis that the adjuvant role of bacterial DNA is associated with its capacity to inhibit peripheral conversion while favoring the emergence of effector T cells. Together, our data demonstrate that in the gut commensal bacterial DNA acts as a natural immunological adjuvant and critically controls the balance between Treg and effector cell populations.
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