Role of Dock8 in Mucosal Immunity

项目概要 人类 DOCK8 缺陷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 缺陷患者遭受痛苦 反复出现的细菌感染，例如金黄色葡萄球菌以及口腔或皮肤的真菌感染 念珠菌，提示 TH17 细胞功能障碍。虽然有人建议DOCK8 可能协调细胞骨架排列、细胞分离和调节细胞迁移，其精确作用 细胞中的 DOCK 蛋白大部分仍然未知。我们最近报道说 DOCK8 是 对于针对啮齿类柠檬酸杆菌的保护性免疫力至关重要。 DOCK8 缺陷小鼠会迅速死于艰难梭菌感染。 啮齿类动物感染。 DOCK8 缺陷型小鼠中产生 IL-22 的 RORγt ILC 数量非常低 与WT小鼠的比较。 DOCK8 缺陷的 RORγt ILC 在 IL-7 介导的信号传导中存在缺陷，更容易发生 细胞凋亡并产生比 WT 小鼠更少的 IL-22。我们还发现 TH17 细胞的产生 C. 啮齿类动物感染选择性受损，而 TH1 细胞的产生显着增加 DOCK8 缺陷小鼠与 WT 小鼠相比。 DOCK8 是一种非常大的蛋白质，已被证明可以 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在TH17细胞生成中的功能是否有效 依赖于其 GEF 对 CDC42 的活性，或其与 WASp 的相互作用，WASp 是一种在 为了研究肌动蛋白细胞骨架的组织和功能，我们感染了 CDC42 或 WAS 的小鼠 在T细胞中被特异性消除。而 DOCK8 缺陷小鼠无法安装强大的 TH17 细胞 感染后的反应，CDC42 T 细胞缺陷或 WAS T 细胞缺陷小鼠产生了 TH17 细胞反应 与 WT 小鼠一样强壮。从这项研究中，我们得出的结论是，至少对于 TH17 细胞的发育来说，DOCK8 可能充当 CDC42 的支架蛋白而不是 GEF，或者通过其与 WASp 相互作用。因此，它 DOCK8 可能充当支架蛋白，对于未知因子的激活很重要 TH17细胞分化所必需的。这里我们假设 DOCK8 调节以下功能： TH17 细胞通过与在 TH17 细胞而非 TH1 中选择性表达的一组特定蛋白质相互作用 细胞。为了鉴定体内与 DOCK8 相互作用的蛋白质，我们生成了一种新型 Knockin 小鼠 其中内源性 DOCK8 与 AVI 标签、Flag 和 GFP 报告基因融合。 AVI 标签技术将允许 我们在相对少量的原代 T 细胞中进行蛋白质组学和质谱分析， 而 GFP 将允许我们进行共聚焦显微镜并跟踪 DOCK8 亚细胞定位 ILC 和 TH17 细胞。总的来说，我们提出的研究将帮助我们理解为什么 DOCK8 缺陷会导致 对免疫系统产生如此深远的影响。

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.