Role of Dock8 in Mucosal Immunity

项目总结 人类DOCK8缺乏症会导致严重的免疫缺陷。DOCK8的临床表现 免疫缺陷包括反复感染、过敏和恶性肿瘤。DOCK8缺陷患者 反复发生的细菌感染，如金黄色葡萄球菌和口腔或皮肤的真菌感染 假丝酵母菌，提示TH17细胞功能障碍。尽管有人建议DOCK8 可能协调细胞骨架的排列、细胞的脱落和调节细胞的迁移，其确切作用是 细胞中的DOCK蛋白在很大程度上仍然未知。我们最近报道了DOCK8是 对轮状芽胞杆菌的保护性免疫是必不可少的。DOCK8基因缺陷小鼠迅速屈服于C. 轮齿感染。DOCK8基因缺陷小鼠体内产生IL-22的γ细胞数量非常少 与WT小鼠比较。DOCK8缺陷的RoRγ细胞在IL-7介导的信号转导方面存在缺陷，更容易发生 与WT小鼠相比，小鼠体内IL-22的产生明显减少。我们还发现在TH17细胞的产生过程中 轮状念珠菌感染受到选择性损害，而TH1细胞的生成在 DOCK8基因缺陷小鼠与WT小鼠的比较。DOCK8是一种非常大的蛋白质，已被证明 作为鸟嘌呤核苷酸交换因子(GEF)，结合并激活小GTP酶 Rho/RAC/CDC42家族。为了确定DOCK8是否在TH17细胞的产生中起作用 取决于它对Cdc42的全球环境基金活性，或者它与黄蜂的相互作用，黄蜂是一种在 肌动蛋白细胞骨架的组织和功能，我们感染了cdc42或is是 在T细胞中被特异性消除。而DOCK8基因缺陷的小鼠不能建立强大的TH17细胞 感染后，CDC42 T细胞缺陷或T细胞缺陷小鼠产生TH17细胞反应 像WT老鼠一样健壮。从这项研究中我们得出结论，至少在TH17细胞的发育过程中，DOCK8 很可能是作为一种支架蛋白，而不是对Cdc42的环境基金，或者通过它与黄蜂的相互作用。因此，它 DOCK8可能作为一种支架蛋白，对未知因子的激活起重要作用 是TH17细胞分化所必需的。这里我们假设DOCK8调节的功能是 通过与一组在TH17细胞而不是在TH1中选择性表达的特定蛋白质相互作用 细胞。为了确定在体内与DOCK8相互作用的蛋白质，我们产生了一种新的Knockin小鼠 其中内源DOCK8与AVI标签、FLAG和GFP报告基因融合。AVI标签技术将允许 美国将对数量相对较少的初级T细胞进行蛋白质组学和质谱分析， 鉴于GFP将允许我们进行共聚焦显微镜和跟踪DOCK8亚细胞定位 ILCs和TH17细胞。总体而言，我们提出的研究将帮助我们理解为什么DOCK8缺陷会导致 对免疫系统有如此深远的影响。

PROJECT SUMMARY DOCK8 deficiency in humans leads to severe immunodeficiency. The clinical manifestations of DOCK8 immunodeficiency include recurrent infections, allergies, and malignancies. DOCK8 -deficient patients suffer from recurrent bacterial infections such as Staphylococcus aureus and fungal infections of the mouth or skin with Candida, which are suggestive of TH17 cell dysfunction. Although it has been suggested that DOCK8 might coordinate cytoskeletal arrangement, cellular detachment and regulate cell migration, the precise role of DOCK proteins in the cell remains for the most part unknown. We have recently reported that DOCK8 is essential for the protective immunity against C. rodentium. DOCK8-deficient mice succumb rapidly to C. rodentium infection. DOCK8-deficient mice have very low numbers of IL-22-producing RORγt+ ILCs in comparison to WT mice. DOCK8-deficient RORγt+ ILCs are defective in IL-7-mediated signaling, more prone to apoptosis and produce less IL-22 than WT mice. We have also found that the generation of TH17 cells during C. rodentium infection is selectively impaired, whereas the generation of TH1 cells is dramatically increased in DOCK8-deficient mice in comparison to WT mice. DOCK8 is a very large protein that has been shown to function as guanine nucleotide exchange factors (GEFs) that binds and activates small GTPases of the Rho/Rac/Cdc42 family. In order to determine whether DOCK8 function in the generation of TH17 cells is dependent on its GEF activity for CDC42, or its interaction with WASp, a protein that plays an important role in the organization and function of the actin cytoskeleton, we infected mice in which CDC42 or WAS was specifically eliminated in T cells. Whereas DOCK8-deficient mice were unable to mount a robust TH17 cell response upon infection, CDC42 T cell-deficient or WAS T cell-deficient mice developed a TH17 cell response as robust as WT mice. From this study, we concluded that at least for the development of TH17 cells, DOCK8 is likely acting as a scaffolding protein rather than a GEF for CDC42, or via its interaction with WASp. Thus, It is possible that DOCK8 might act as a scaffolding protein that is important for the activation of unknown factors necessary for the differentiation of TH17 cells. Here we hypothesize that DOCK8 regulates the function of TH17 cells by interacting with a specific set of proteins selectively expressed in TH17 cells and not in TH1 cells. In order to identify proteins that interact with DOCK8 in vivo, we have generated a novel Knockin mouse in which endogenous DOCK8 was fused to AVI tag, Flag and GFP reporter. The AVI tag technology will allow us to perform proteomics and Mass spectrometry analysis in a relatively small number of primary T cells, whereas the GFP will allow us to perform confocal microscopy and track DOCK8 subcellular localization in both ILCs and TH17 cells. Overall, our proposed studies will help us understand why DOCK8 deficiency has such a profound effect on the immune system.