Role of Dock8 in Mucosal Immunity

Dock8 在粘膜免疫中的作用

• 批准号: 10440281
• 负责人: Richard Goff James
• 金额: $ 28.83万
• 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/10440281
• 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 DOCK 8缺陷会导致严重的免疫缺陷。DOCK 8的临床表现 免疫缺陷包括复发性感染、过敏和恶性肿瘤。DOCK 8缺陷患者 复发性细菌感染，如金黄色葡萄球菌和口腔或皮肤真菌感染 与念珠菌，这是提示TH 17细胞功能障碍。尽管有人认为DOCK 8 可能协调细胞骨架排列，细胞分离和调节细胞迁移， 细胞中的DOCK蛋白大部分仍是未知的。我们最近报道说，DOCK 8是 对C.啮齿动物。DOCK 8缺陷小鼠迅速死于C. 啮齿类感染DOCK 8缺陷小鼠在正常小鼠中具有非常低数量的产生IL-22的RORγt+ ILC。 与WT小鼠比较。DOCK 8缺陷型RORγt+ ILC在IL-7介导的信号传导中有缺陷，更倾向于 细胞凋亡和产生比WT小鼠更少的IL-22。我们还发现，TH 17细胞的产生在 C.啮齿类感染是选择性受损，而TH 1细胞的产生显着增加， 与WT小鼠相比，DOCK 8缺陷型小鼠。DOCK 8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）起作用，其结合并激活细胞的小GTP酶。 Rho/Rac/Cdc 42家族。为了确定DOCK 8在TH 17细胞产生中的功能是否被破坏， 依赖于其对CDC 42的GEF活性，或其与WASp的相互作用，WASp是一种在 肌动蛋白细胞骨架的组织和功能，我们感染了小鼠，其中CDC 42或WAS是 在T细胞中特异性消除。而DOCK 8缺陷小鼠不能安装强大的TH 17细胞， 在感染后的应答中，CDC 42 T细胞缺陷型或WAS T细胞缺陷型小鼠产生了TH 17细胞应答 与WT小鼠一样强壮。从这项研究中，我们得出结论，至少对于TH 17细胞的发育，DOCK 8 可能作为支架蛋白而不是CDC 42的GEF，或通过其与WASp的相互作用。因此 DOCK 8可能充当支架蛋白，对于未知因子的激活很重要 这是TH 17细胞分化所必需的。在这里，我们假设DOCK 8调节了 TH 17细胞通过与选择性表达于TH 17细胞而非TH 1中的一组特定蛋白质相互作用 细胞为了鉴定与DOCK 8在体内相互作用的蛋白质，我们已经产生了一种新的敲入小鼠 其中内源性DOCK 8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和跟踪DOCK 8亚细胞定位， ILC和TH 17细胞。总的来说，我们提出的研究将帮助我们理解为什么DOCK 8缺乏症 对免疫系统产生如此深远的影响。

项目成果

Regulation of lymphocyte trafficking in central nervous system autoimmunity.

中枢神经系统自身免疫中淋巴细胞运输的调节。

• DOI: 10.1016/j.coi.2018.09.008
• 发表时间: 2018
• 期刊: Current opinion in immunology
• 影响因子: 7
• 作者: Oukka,Mohamed;Bettelli,Estelle
• 通讯作者: Bettelli,Estelle

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

• DOI: 10.1016/j.jaci.2021.07.014
• 发表时间: 2022-03
• 期刊: The Journal of allergy and clinical immunology
• 作者: Saha SS;Samanas NB;Miralda I;Shubin NJ;Niino K;Bhise G;Acharya M;Seo AJ;Camp N;Deutsch GH;James RG;Piliponsky AM
• 通讯作者: Piliponsky AM