Regulatory cells, by virtue of their capacity to control the vigor of immune responses are essential to the maintenance of host homeostasis. Several types of CD4 regulatory T cells exist some of which are induced in response to infectious challenge and some of which are judged as natural regulators (natural Treg). Inducible Treg cells such as Tr1 or TH3 cells can develop from conventional CD4 T cells that are exposed to specific stimulatory conditions. Natural Treg cells, however, arise during the normal process of maturation in the thymus, obey defined rules and express a specific set of markers. Recently, studies also indicate that a unique transcription factor Foxp3 is required for the generation of natural Treg cells and this represents, to date, their most specific marker. Natural Treg play a central role in the control of autoimmunity, a function that is associated with their capacity to recognize self-antigen. Whether or not they also recognize foreign antigens and the extent of their repertoire for such antigens remain unknown. We and others have shown that natural Treg also play a critical role in the outcome of microbial infections. Natural Treg help limit collateral tissue damage caused by vigorous antimicrobial immune responses. These cells can also limit the magnitude of effector responses which result in failure to adequately control infection. Furthermore, there are clear evidences that the efficiency of vaccines can also be hampered by the presence of natural Treg. Thus, strategies aimed to manipulate natural Treg cells function or number, have clearly high therapeutic potential. To develop rational strategies, there is an urgent need to better characterize the function of natural Treg during infections. Using two models of unicellular gastrointestinal parasitic infection (Cryptosporidium muris and Encephalitozoon cuniculi) and one model of cutaneous infection (Leishmania major) we are exploring the antigen specificity of natural Treg that accumulate at sites of infections as well as the conditions that favor their retention and function.? ? We have analyzed Foxp3 Treg that can be found in the gut associated lymphoid tissue (GALT), and in particular in the lamina propria. It is unclear whether GALT Treg, which are in contact with self-antigen and foreign antigen, are naturally occurring Treg that derive from the thymus or whether they are induced Treg that have been converted into a regulatory phenotype. We observe conversion of CD4 Foxp3- T cells into Foxp3 Treg in GALT only, suggesting that GALT Treg may not be thymically derived. Such conversion is further enhanced when mice are infected with C. muris. Our results also suggest that lamina propria Treg (LP Treg), as opposed to peripheral Treg, are IL-2 independent. Furthermore, LP Treg have better suppressive capacity than peripheral Treg. Additionally, we found that LP Treg are able to reconstitute peripheral lymph nodes. Together, our results suggest that the gut constitutes a unique environment that is able to generate and maintain a reservoir of functional Foxp3 Treg.? ? The constant exposure of intestinal epithelial cells and mucosal immune cells expressing several Toll like receptors (TLRs) to enteric bacteria provides a rationale for considering the immunophysiological impact of bacterial DNA on Treg from the GALT. We examined whether the inability to signal via TLR-9 could modulate Treg homeostasis in the GALT and how such effects could influence the immune response to the gastrointestinal parasite E. cuniculi. Naive TLR-9 deficient (TLR-9 --) mice had significantly higher percentages of Foxp3-positive Treg compared to wild type mice in the GALT but not in the periphery. Such an increase is further enhanced upon oral infection with E. cuniculi. The over-expression of Treg in the GALT of TLR-9 -- mice leads to impaired protective Th1 response against the parasite and increased parasite expansion. We showed that this control was asociated with the interation between baterial DNA from the gut flora and TLR9 on hematopoietic cells in the gut. Our results suggest that TLR-9 signaling provided by the intestinal commensal flora can negatively modulate GALT regulatory T cells and that gut flora is a natural adjuvant of immune responses in the gut. ? ? Several lines of evidence suggest that unique DC subsets or DCs in specific maturation states can prime and induce various population of regulatory T cells. But while previous work has examined the ability of peripheral DC populations to initiate regulatory or effector responses, this question remains to be addressed in DC from the intestinal lamina propria (LP). We have identified a unique population of DCs in the LP compartment, which are CD11bCD8alpha-. When compared to splenic DCs (SPDC), though both secrete IL-6 and TNF in response to LPS, LP DCs also secrete the regulatory cytokine IL-10 rather than IL-12 following stimulation. During oral infection with C. muris and E. cuniculi, substantial IL-10 secreting CD4 T cells and DC can be found in LP compartment. Upon transfer, LPDCs can directly induce IL-10 but not IFN-gamma from T cells. Yet, in vitro assays indicated that LPDCs also have the capacity to prime naove T cells into Th1 cells when exposed to IL-12. However, in contrast to SPDC, LPDCs also induced high amounts of IL-10 secretion during Th1 polarization. In particular LPDCs can induce a population of IFN-gammaIL-10 double producing cells. These results suggest that the same population of DC in the intestinal LP has the flexibility to induce both regulatory and effector responses. ? ? A recently described subset of CD4 T cells (Th17), which secrete the inflammatory cytokine, IL-17, has been implicated in the pathogenesis of colitis and several auto-immune disorders. Several reports suggested a dichotomy between Th17 cells and peripherally generated Foxp3 regulatory T cells. This dichotomy is attributed to the conditions surrounding their priming and activation. While the cytokine, transforming growth factor beta (TGF-beta), appears to be essential for the differentiation of both of these subsets, Th17 differentiation is favored in an inflammatory setting (e.g IL-6). Yet, at steady state conditions and during infection, little is known about the generation and function of these subsets. Moreover, the nature of the antigen presenting cell and sites that favor the initiation of either subset in vivo remains to be addressed. We have shown that in a non-infectious setting, the induction of Treg cells is limited to the gut, which is enriched for TGF-beta and serves as the main port of entry for a large number of pathogens. We found that dendritic cells (DC) from the lamina propria of the gut (LPDCs) are more efficient inducers of Treg differentiation than splenic DCs (SPDCs). Conversely, SPDCs are more efficient inducers of Th17 induction than LPDCs. Our data suggest that the capacity of a cell to differentiate into a Th17 or an Treg is not limited to the presence of inflammatory cytokines but that the site and the nature of the antigen presenting cell involved are also critical to the fate of these cells.? We showed that peripheral conversion of CD4 T cells to Treg occurs primarily in gut associated lymphoid tissue (GALT) following both oral exposure to antigen and in a lymphopenic environment. Dendritic cells (DC) purified from the lamina propria (LpDC) of the small intestine were found to promote a high level of Treg conversion relative to lymphoid organ derived DC. This enhanced conversion by LpDC was dependent on TGF- and retinoic acid, a vitamin A metabolite highly expressed in GALT. Together these data demonstrate that the intestinal immune system has evolved a self-contained strategy to promote Treg neo-conversion and potentially extend the repertoire of natural Treg
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