Role of " Apoptotic proteins" Regulation Innate Immunity

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

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.