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感知途径来检测外来入侵。最近的证据 表明一些先前与细胞凋亡有关的蛋白质也参与了先天免疫。 通过Toll样受体(TLR)依赖和独立的途径。其中包括TRAIL-R、 肿瘤坏死因子受体家族中的凋亡诱导成员和死亡结构域FADD 含有适配蛋白。TRAIL可通过TLR-GT；干扰素途径诱导。后续 TRAIL激活TRAIL-R导致转录因子NF-kappaB的负反馈循环。TRAIL-R-/- TLR-3/4配体刺激的树突状细胞/巨噬细胞显示细胞因子水平升高和 核因子-kappaB的动态平衡调节。FADD，让人想起果蝇的IMD-FADD先天免疫反应 系统，被发现是细胞内dsRNA激活的基因在人/鼠中表达的关键。在……里面 在干扰素存在的情况下，FADD-/-成纤维细胞无法清除包括流感在内的RNA病毒。 因此，FADD是TLR非依赖的哺乳动物病原体感知通路的一部分。在这 应用，我们假设TRAIL-R和FADD在 针对各种病毒和选定的寄生虫的先天免疫反应。在目标1中，来自 各种家庭，包括几个生物防御类别，如流感，牛痘和LCMV(与 项目3)和细胞内寄生虫弓形虫(与项目1一起)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 在这些细胞中改变了全球基因表达，如果有的话。树突状细胞/巨噬细胞特异性FADD-/-小鼠 将被生成。这些小鼠和TRAIL-R-/-小鼠对选定病原体(流感、 将对弓形虫、巨细胞病毒)进行检查。荧光转基因技术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被用来评估宿主病原体 互动。在目标2中，导致TRAIL负调控核因子-kB的信号转导途径 将会被检查。参与FADD介导的先天免疫的信号蛋白也将被识别。 基因芯片分析将用于检测TRAIL-R-/-巨噬细胞的基因表达谱。 质谱学将在实验中用于鉴定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，将使用RNAi敲除方法来评估任何新的 特定生物防御天然免疫中TRAIL-R或FADD相关蛋白的鉴定 有机体。