Role of " Apoptotic proteins" Regulation Innate Immunity

“凋亡蛋白”调节先天免疫的作用

• 批准号: 7081706
• 负责人: ASTAR WINOTO
• 金额: $ 31.15万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081706
• 关键词: Orthomyxoviridae; RNA interference; Toxoplasma gondii; apoptosis; bioterrorism /chemical warfare; cellular immunity; disease /disorder model; fibroblasts; fluorescence microscopy; genetically modified animals; host organism interaction; immunogenetics; immunoregulation; interferons; laboratory mouse; lymphocytic choriomeningitis virus; macrophage; mass spectrometry; microarray technology; microorganism immunology; nuclear factor kappa beta; parasite infection mechanism; protein structure function; toll like receptor; tumor necrosis factor alpha; vaccinia virus; virus infection mechanism

Mammalian cells have evolved multiple sensing pathways to detect foreign invasion. Recent evidence indicated that several proteins previously implicated in apoptosis also participate in innate immunity through Toll-Like receptors (TLR)-dependent and independent pathways. These include TRAIL-R, an apoptosis-inducing member of the tumor necrosis factor receptor family and FADD, a death-domain containing adapter protein. TRAIL can be induced through TLR-> interferon pathway. Subsequent activation of TRAIL-R by TRAIL results in negative feedback loop of NF-kappaB transcription factor. TRAIL-R-/- dendritic cells/macrophages stimulated with TLR-3/4 ligands display enhanced cytokine levels and loss of NF-kappaB homeostatic regulation. FADD, reminiscent of the Drosophila Imd-FADD innate immune response system, was found to be crucial for intra-cellular dsRNA-activated gene expression in human/mouse. In the presence of interferon, FADD-/- fibroblasts were not able to clear RNA viruses that include Influenza. Thus, FADD is part of an alternative TLR-independent mammalian pathogen-sensing pathway. In this application, we hypothesize that TRAIL-R and FADD play significant but distinct roles in the innate immune responses against a variety of viruses and selected parasite. In Aim 1, viruses from various families, including several in the bio-defense category like Influenza, Vaccinia and LCMV (with Project 3) and the intracellular parasite Toxoplasma gondii (with Project 1), will be used to determine the role of FADD and TRAIL-R in regulating host responses. We will first examine FADD-/- and TRAIL-R-/- fibroblasts for their ability to support viral replication. Microarray analysis will then be done to assess altered global gene expression, if any, in these cells. Dendritic cells/macrophage-specific FADD-/- mice will be generated. The host responses of these and TRAIL-R-/- mice against selected pathogens (Influenza, Toxoplasma, Cytomegalovirus) will be examined. Two-photon imaging studies in fluorescent transgenic mice in either TRAIL-R-/- or FADD tissue-specific deficient alleles will be used to assess host-pathogen interaction. In Aim 2, the signal transduction pathway leading to negative regulation of NF-KB by TRAIL will be examined. Signaling proteins involved in FADD-mediated innate immunity will also be identified. Microarray analysis will be performed to examine gene expression profile of TRAIL-R-/- macrophages. Mass spectrometry will then be used to identify TRAIL-R- or FADD-associated proteins in experiments involving Fas/TRAIL-R chimeric protein, tandem-affinity-protein technology and co-immunoprecipitation. Finally, RNAi knockdown approach will be used to assess the functional significance of any newly identified TRAIL-R or FADD associated proteins in innate immunity against selected bio-defense organisms.

哺乳动物细胞已经进化出多种传感途径来检测外来入侵。最近的证据 表明先前与细胞凋亡有关的几种蛋白质也参与先天免疫 通过 Toll 样受体 (TLR) 依赖性和独立途径。其中包括 TRAIL-R， 肿瘤坏死因子受体家族的凋亡诱导成员和 FADD（死亡结构域） 含有接头蛋白。 TRAIL可以通过TLR->干扰素途径诱导。随后的 TRAIL 激活 TRAIL-R 会导致 NF-kappaB 转录因子的负反馈环路。踪迹-R-/- 用 TLR-3/4 配体刺激的树突状细胞/巨噬细胞表现出细胞因子水平增强和 NF-kappaB 稳态调节。 FADD，让人想起果蝇 Imd-FADD 先天免疫反应 系统，被发现对于人类/小鼠的细胞内 dsRNA 激活基因表达至关重要。在 由于存在干扰素，FADD-/- 成纤维细胞无法清除包括流感在内的 RNA 病毒。 因此，FADD 是另一种不依赖于 TLR 的哺乳动物病原体感应途径的一部分。在这个 应用中，我们假设 TRAIL-R 和 FADD 在 针对多种病毒和选定寄生虫的先天免疫反应。在目标 1 中，病毒来自 不同的家族，包括生物防御类别中的几个家族，如流感、牛痘和 LCMV（ 项目 3) 和细胞内寄生虫弓形虫 (Toxoplasma gondii)（与项目 1）将用于确定 FADD 和 TRAIL-R 在调节宿主反应中的作用。我们首先检查 FADD-/- 和 TRAIL-R-/- 成纤维细胞具有支持病毒复制的能力。然后将进行微阵列分析来评估 这些细胞中的全局基因表达（如果有的话）发生了改变。树突状细胞/巨噬细胞特异性 FADD-/- 小鼠将 被生成。这些小鼠和 TRAIL-R-/- 小鼠对选定病原体（流感、 弓形虫、巨细胞病毒）将被检查。荧光转基因中的双光子成像研究 TRAIL-R-/- 或 FADD 组织特异性缺陷等位基因的小鼠将用于评估宿主病原体 相互作用。目标2，TRAIL对NF-KB负调控的信号转导通路 将接受检查。参与 FADD 介导的先天免疫的信号蛋白也将被鉴定。 将进行微阵列分析以检查 TRAIL-R-/- 巨噬细胞的基因表达谱。 然后，质谱法将用于在实验中鉴定 TRAIL-R 或 FADD 相关蛋白 涉及Fas/TRAIL-R嵌合蛋白、串联亲和蛋白技术和免疫共沉淀。 最后，RNAi 敲低方法将用于评估任何新的功能意义 鉴定出 TRAIL-R 或 FADD 相关蛋白在针对选定生物防御的先天免疫中 有机体。