Role of Dock8 in Mucosal Immunity

Dock8 在粘膜免疫中的作用

• 批准号: 10198713
• 负责人: Richard Goff James
• 金额: $ 28.86万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198713
• 关键词: Actins; Affect; Apoptosis; Bacterial Infections; Binding; Biology; CDC42 gene; Candida; Cells; Citrobacter rodentium; Clinical; Collaborations; Confocal Microscopy; Cytoskeleton; Data; Development; Family; Functional disorder; Gastrointestinal tract structure; Generations; Guanine Nucleotide Exchange Factors; Helper-Inducer T-Lymphocyte; Human; Hypersensitivity; IL7 gene; Immune; Immune response; Immune system; Immunity; Immunologic Deficiency Syndromes; Impairment; Individual; Infection; Knock-in Mouse; Knockout Mice; Lymphoid Cell; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Molecular; Monomeric GTP-Binding Proteins; Mucosal Immunity; Mus; Mycoses; Patients; Play; Proteins; Proteomics; Recurrence; Reporter; Reporting; Role; Scaffolding Protein; Set protein; Signal Transduction; Skin; Staphylococcus aureus; Suggestion; T-Lymphocyte; Technology; Testing; Th1 Cells; Time; Wasps; base; cell motility; conditional knockout; cytokine; in vivo; interleukin-22; novel; oral infection; pathogen; recurrent infection; response; rho GTP-Binding Proteins; selective expression

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.

项目摘要 人类的Dock8缺乏会导致严重的免疫缺陷。 DOCK8的临床表现 免疫缺陷包括复发性感染，过敏和恶性肿瘤。 Dock8-缺陷患者患者 从复发性细菌感染（例如金黄色葡萄球菌）和口腔或皮肤的真菌感染 与Th17细胞功能障碍有关的念珠菌。尽管有人建议Dock8 可能会协调细胞骨架布置，细胞脱离和调节细胞迁移，这是 细胞中的码头蛋白在大多数情况下仍然是未知的。我们最近报告说Dock8是 防御性免疫对啮齿动物的免疫力至关重要。 Dock8缺陷小鼠迅速屈服于C。 啮齿动物感染。 Dock8缺陷小鼠的IL-22产生RORγT+ ILC数量很少 与WT小鼠进行比较。 DOCK8缺陷RORγT+ ILC在IL-7介导的信号中有缺陷，更容易容易 凋亡比WT小鼠产生的IL-22少。我们还发现，在 C.啮齿动物感染有选择性地受损，而Th1细胞的产生显着增加 与WT小鼠相比，DOCK8缺陷小鼠。 Dock8是一种非常大的蛋白质，已显示为 充当鸟嘌呤核丁基交换因子（GEFS），该因子结合并激活了小的GTPases RHO/RAC/CDC42家庭。为了确定dock8在产生Th17细胞中是否功能是 取决于其对CDC42的GEF活性，或与WASP相互作用的蛋白质，该蛋白在 肌动蛋白细胞骨架的组织和功能，我们感染了Cdc42或所在的小鼠 在T细胞中特别消除。而Dock8缺陷小鼠无法安装强大的Th17单元 感染后的反应，CDC42 T细胞缺陷型或T细胞缺陷小鼠会形成Th17细胞反应 像WT小鼠一样坚固。从这项研究中，我们得出结论，至少对于Th17细胞的发展，Dock8 可能充当脚手架蛋白，而不是用于CDC42的GEF，或者通过其与黄蜂的相互作用。那，它 Dock8可能充当脚手架蛋白，这对于激活未知因素很重要 分化Th17细胞所必需的。在这里，我们假设Dock8调节了 Th17细胞与在Th17细胞中选择的一组特定蛋白质相互作用，而不是在Th1中 细胞。为了鉴定与体内与Dock8相互作用的蛋白质，我们生成了一种新型的敲蛋白小鼠 其中内源性DOCK8与AVI标签，FLAG和GFP记者融合在一起。 AVI标签技术将允许 美国在相对较少的原代T细胞中执行蛋白质组学和质谱分析， 而GFP将使我们能够执行共聚焦显微镜并跟踪Dock8亚细胞定位 ILC和Th17细胞均被ILC。总体而言，我们拟议的研究将帮助我们了解为什么Dock8缺乏症 对免疫系统的深刻影响。