Role of JNK2 and TLR6 during Y. enterocolitica induced mucosal immune responses

描述(由申请人提供)： IL-10是肠粘膜内平衡反应的关键细胞因子，既能对抗口服抗原，又能对抗共生菌群。IL-10可通过诱导DC启动耐受性反应发挥其作用，称为耐受性DC。有研究认为，耐受性DC启动1型调节细胞(TR1)，这是一种T细胞亚群，能产生高水平的IL-10并抑制炎症。然而，在肠道中产生耐受性DC和(TR1)细胞的分子机制仍然知之甚少。这项建议的目标是以小肠结肠炎耶尔森菌感染为模型系统，确定肠道免疫组织中如何产生产生IL-10的细胞。小肠结肠炎耶尔氏菌是检测IL-10介导的粘膜反应诱导的理想选择，这是因为它对小肠的免疫部位，特别是Peyer氏结和MLN，免疫细胞诱导和T细胞启动的部位有趋向性。更重要的是，小肠结肠炎耶尔森氏菌含有低钙反应V抗原(LcrV)，这是在所有三种致病耶尔森菌中发现的一种基本毒力因子，已被证明能诱导抗原提呈细胞(APC)产生IL-10。利用系统性鼠疫杆菌感染，我们发现LcrV诱导耐受树突状细胞，并以TLR6依赖的方式启动TR1细胞反应。此外，我们的初步研究表明，TLR6通过激活MAPK JNK独特地参与了耐受性DC和TR1的产生。我们推测TLR6和JNK在肠粘膜耐受来源的DC和TR1细胞的产生中起关键作用。此外，我们预计TLR6和JNK在小肠结肠炎耶尔森菌的致病过程中起重要作用。我们的具体目标将研究[SIC]，1)评估TLR缺陷和JNK缺陷小鼠粘膜DC在体外经LcrV刺激或感染小肠结肠炎耶尔森菌后诱导产生耐受性DC和TR1细胞的情况。2)探讨TLR6和JNK在小肠结肠炎耶尔森菌口腔感染中的作用。在这一目标中，我们将利用基因敲除小鼠以及培育成TLR和JNK缺陷小鼠的IL-10-T细胞报告小鼠，希望确定IL-10诱导的粘膜部位，并评估感染过程中诱导的粘膜CD4T细胞是否可以将耐受性转移和传播到第二个抗原。3)用抗LcrV和LcrV缺失突变体的单抗鉴定LcrV内负责诱导IL-10和激活JNK的特异性残基。 公共卫生相关性：了解小肠结肠炎耶尔森菌诱导肠道TR1和耐受性DC的分子机制，可能会给我们提供关键的洞察力，了解肠道组织如何在体内稳态地产生调节反应。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.