Role of Dock8 in Mucosal Immunity

Dock8 在粘膜免疫中的作用

基本信息

批准号：
10017649
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
项目类别：
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/10017649
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 Immune Immune response Immune system Immunity Immunologic Deficiency Syndromes Impairment Individual Infection Interleukin-7 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

项目摘要

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。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与念珠菌，这表明 TH17 细胞功能障碍，尽管有人认为 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 蛋白大部分仍未知。我们最近报道 DOCK8 是未知的。 DOCK8 缺陷小鼠对 C. rodentium 的保护性免疫至关重要。 DOCK8 缺陷小鼠中产生 IL-22 的 RORγt+ ILC 数量非常低。与 WT 小鼠相比，DOCK8 缺陷的 RORγt+ ILC 在 IL-7 介导的信号传导中存在缺陷，更容易发生我们还发现，TH17 细胞的产生过程中，细胞凋亡并比 WT 小鼠产生更少的 IL-22。 C. 啮齿类动物感染选择性受损，而 TH1 细胞的产生显着增加与 WT 小鼠相比，DOCK8 缺陷小鼠是一种非常大的蛋白质，已被证明可以发挥作用。作为鸟嘌呤核苷酸交换因子（GEF），结合并激活小 GTP 酶为了确定DOCK8在TH17细胞的产生中是否发挥作用。取决于其 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 DOCK8 缺陷小鼠中的 T 细胞被特异性消除，无法建立强大的 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 细胞反应从这项研究中我们得出结论，至少对于 TH17 细胞的发育来说，DOCK8 与 WT 小鼠一样强大。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。 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，在此我们竞争 DOCK8 调节的功能。 TH17 细胞通过与 TH17 细胞（而非 TH1）中选择性表达的一组特定蛋白质相互作用为了识别体内与 DOCK8 相互作用的蛋白质，我们培育了一种新型 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 将使我们能够进行共聚焦显微镜并跟踪 DOCK8 亚细胞定位总体而言，我们提出的研究将帮助我们理解 DOCK8 缺陷的原因。对免疫系统产生如此深远的影响。