T cell-derived iNOS switches off TH17 cell differentiation in inflammation

描述(申请人提供)：虽然已经证明iNOS在宿主抵抗微生物病原体方面发挥着重要作用，但iNOS在T细胞和慢性炎症性疾病中的确切功能尚未确定。Th17细胞分泌IL-17和IL-22，是最近发现的不同于TH1和TH2亚群的CD4+T细胞亚群，RORgt已被确定为TH17细胞分化的关键转录因子。越来越多的证据表明，TH17免疫反应参与了多种自身免疫性/炎症性疾病的发病机制。因此，阻断TH17细胞的激活可能导致开发治疗慢性炎症性疾病的新策略。我们发现，iNOS基因敲除小鼠表现出更强劲的TH17细胞分化，而对TH1或TH2细胞谱系没有重大影响。我们发现诱导型一氧化氮合酶在活化的CD_4~+T细胞中被诱导，并且使用诱导型一氧化氮合酶选择性抑制剂L-NIL显著增加WT细胞培养中产生IL-17的CD_4~+T细胞的百分率，而NO供体SNAP则剂量依赖地抑制WT和iNOS-/-T细胞中IL-17的产生。此外，RORgt蛋白的酪氨酸残基被硝化，导致 抑制RORgt介导的IL-17启动子激活。最后，将iNOS-/-CD4+CD45Rbhi细胞转移到RAG-/-小鼠体内，与对照CD4+CD45Rbhi细胞相比，诱导了更严重的结肠炎，并且用iNOS-/-细胞重组的小鼠产生IL-17的细胞比例显著高于对照小鼠。结果表明，T细胞来源的iNOS负性调节TH17免疫反应的发展，从而控制炎症。这项建议围绕三个目标构建：1)我们将表征T细胞来源的iNOS调节TH17细胞分化的分子机制。2)我们将研究T细胞来源的iNOS在人TH17、TH1、TH2和Treg细胞发育中的作用。3)分析巨噬细胞、树突状细胞、T细胞等不同细胞亚群中iNOS在结肠炎发生发展中的作用。这些拟议的研究将定义一种新的TH17细胞分化转录抑制因子，并强调T细胞来源的iNOS作为治疗慢性炎症性疾病的新靶点的重要性。

DESCRIPTION (provided by applicant): Although it has been demonstrated that iNOS plays an important role in host defense against microbial pathogens, the exact function of iNOS in T cells and chronic inflammatory diseases has not been defined. TH17 cells, which secrete interleukin 17 (IL-17) and IL-22 comprise a recently identified subset of CD4+ T cells distinct from the TH1 and TH2 subsets and RORgt has been identified as a key transcription factor for TH17 cell differentiation. Increasing evidence indicates that TH17 immune responses are involved in the pathogenesis of various autoimmune/inflammatory diseases. Thus, blocking TH17 cell activation could lead to the development of novel strategies for the treatment of chronic inflammatory diseases. We show that iNOS knock out mice display more robust TH17 cell differentiation without major effects on either TH1 or TH2 cell lineages. We demonstrated that iNOS protein was induced in activated CD4+ T cells and the use of an iNOS selective inhibitor L-NIL significantly increased the percentage of IL-17-producing CD4+ T cells in WT cell cultures, while, an NO donor, SNAP, dose-dependently suppressed IL-17 production in WT and iNOS-/- T cell cultures. In addition, tyrosine residues of RORgt protein were nitrated resulting in the inhibition of RORgt-mediated IL-17 promoter activation. Finally, transfer of iNOS-/- CD4+CD45Rbhi cells into RAG-/- mice induces more severe colitis compared to control CD4+CD45Rbhi cells and mice reconstituted with iNOS-/- cells had a significantly higher percentage of IL-17-producing cells than control mice. The results suggest that T cell-derived iNOS negatively regulates the development of TH17 immune responses resulting in the control of inflammation. This proposal is structured around three aims: 1) We will characterize the molecular mechanisms involved in the regulation of TH17 cell differentiation by T cell-derived iNOS. 2) We will characterize the function of T cell- derived-iNOS on human TH17, TH1, TH2, and Treg cell development. 3) We will analyze the roles of iNOS in different cell compartments including macrophages, dendritic cells, and T cells in the development of colitis. The proposed studies will define a novel transcriptional inhibitor of TH17 cell differentiation and highlight th importance of T cell-derived iNOS as a novel therapeutic target for the treatment of chronic inflammatory diseases.