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）依赖性和非依赖性途径。其中包括TRAIL-R， 肿瘤坏死因子受体家族的凋亡诱导成员和死亡结构域FADD 含有衔接蛋白。TRAIL可以通过TLR->干扰素途径诱导。后续 TRAIL对TRAIL-R的激活导致NF-κ B转录因子的负反馈环。TRAIL-R-/- 用TLR-3/4配体刺激的树突状细胞/巨噬细胞显示出增强的细胞因子水平和细胞因子的丧失。 NF-κ B稳态调节。FADD，让人想起果蝇Imd-FADD先天免疫反应 系统，被发现是至关重要的细胞内dsRNA激活的基因表达在人/小鼠。在 干扰素的存在下，FADD-/-成纤维细胞不能清除包括流感病毒在内的RNA病毒。 因此，FADD是另一种TLR非依赖性哺乳动物病原体感应途径的一部分。在这 应用中，我们假设TRAIL-R和FADD发挥重要但不同的作用， 针对多种病毒和选定寄生虫的先天免疫应答。在目标1中，来自 各种家族，包括生物防御类别中的几种，如流感，牛痘和LCMV（ 项目3）和细胞内寄生虫弓形虫（项目1），将用于确定 FADD和TRAIL-R在调节宿主反应中的作用。我们将首先检查FADD-/-和TRAIL-R-/- 成纤维细胞支持病毒复制的能力然后进行微阵列分析以评估 改变了这些细胞中的整体基因表达，如果有的话。树突状细胞/巨噬细胞特异性FADD-/-小鼠将 生成。这些小鼠和TRAIL-R-/-小鼠对所选病原体（流感， 将检查弓形虫、巨细胞病毒）。荧光转基因的双光子成像研究 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 互动在目的2中，研究了导致TRAIL对NF-κ B负调节的信号转导途径。 将被审查。还将鉴定参与FADD介导的先天免疫的信号蛋白。 将进行微阵列分析以检查TRAIL-R-/-巨噬细胞的基因表达谱。 然后将在实验中使用质谱法来鉴定TRAIL-R或FADD相关蛋白质 包括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。 在针对所选生物防御的先天免疫中鉴定的TRAIL-R或FADD相关蛋白 有机体