IL-10 is a critical cytokine for homeostatic responses in the intestinal mucosa, both against orally ingested antigens and against commensal microflora. IL-10 can exert its effects through the induction of DC that prime tolerogenic responses, referred to as tolerogenic DC. It is proposed that tolerogenic DC prime type-1 regulatory (Tr1) cells, a subset of T cell that produce high amounts of IL-10 and suppress inflammation. However, the molecular mechanism of tolerogenic DC and (Tr1) cell generation in the intestine remain poorly understood. The goal of this proposal is to determine how IL-10-producing cells are generated in the intestinal immune tissue using Y. enterocolitica infection as a model system. What makes Y. enterocolitica an ideal choice for examining induction of IL-10-mediated mucosal responses is because of its tropism for immune sites of the small intestine, specifically the Peyer's patches and the MLN, both sites of immune cell induction and T cell priming. More importantly, Y. enterocolitica harbors the Low Calcium Response V Antigen (LcrV) an essential virulence factor found in all three pathogenic Yersinia species and which has been shown to induce IL-10 from antigen presenting cells (APC). Using systemic Y. pestis infection we found that LcrV induced tolerogenic DC and primed a Tr1 cell response in a TLR6 dependent manner. Furthermore, our initial studies suggest that TLR6 is uniquely involved in the generation of tolerogenic DC and Tr1 through activation of the MAPK JNK. We hypothesize that TLR6 and JNK will play a key role in the generation of mucosal intestinal tolerogenic DC and Tr1 cells. Furthermore, we anticipate that TLR6 and JNK play an important role in Y. enterocolitica pathogenesis.
Our specific aims will investigate [sic], 1) Evaluate the induction of tolerogenic DC and Tr1 cells using mucosal DC from TLR-deficient and JNK-deficient mice stimulated in vitro by LcrV or infected with Y. enterocolitica. 2) Evaluate the role TLR6 and JNK play during pathogenesis of oral Y. enterocolitica infection. In this aim we will utilize knockout mice as well as by using IL-10-T cell reporter mice bred to TLR-deficient and JNK-deficient mice in hopes to identify the mucosal sites of IL-10 induction and evaluate whether mucosal CD4 T cells induced during infection can transfer tolerance and spread tolerance to a second antigen. 3) Use monoclonal antibodies against LcrV and LcrV deletion mutants to identify specific residues within LcrV responsible for inducing IL-10 and activating JNK.
Understanding the molecular mechanism that Y. enterocolitica induces intestinal Tr1 and tolerogenic DC, may give us critical insight into understanding how intestinal tissue generates regulatory responses homeostatically.
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