The role of Fas-associated death domain in necroptosis in vivo

Fas相关死亡结构域在体内坏死性凋亡中的作用

• 批准号: 8295838
• 负责人: ASTAR WINOTO
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-02 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8295838
• 关键词: Address; Affinity Chromatography; Antibiotics; Antigens; Apoptosis; Apoptotic; Autoimmunity; Bacteria; Binding Proteins; Biochemical; Caspase; Cell Aging; Cell Death; Cell Death Signaling Process; Cells; Cessation of life; Co-Immunoprecipitations; Complex; Data; Death Domain; Dendritic Cells; Erythrocytes; Exhibits; Fas Signaling Pathway; Human; Immune; Immune response; Immune system; Immunity; Infection; Inflammation; Inflammatory; Influenza; Injection of therapeutic agent; Interphase Cell; Knock-out; Knockout Mice; Lead; Ligands; Lipopolysaccharides; Mass Spectrum Analysis; Mediating; Modeling; Mus; Mutant Strains Mice; Myeloproliferative disease; Necrosis; Parasites; Pathway interactions; Phenotype; Phosphotransferases; Physiological; Play; Predisposition; Proliferating; Proteins; RIPK3 gene; Resistance; Role; Running; Salmonella infections; Serum; Shapes; Signal Pathway; Signal Transduction; Stimulus; Syndrome; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; TLR4 gene; Testing; Time; Tissues; Toll-like receptors; Toxoplasma gondii; Toxoplasmosis; Transfection; Transgenic Mice; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-alpha; Vaccines; Viral; Western Blotting; adapter protein; aged; caspase-8; cell age; cell type; cytokine; improved; in vivo; macrophage; member; mouse development; mouse model; neutrophil; pathogen; premature; programs; receptor; response; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Cell death is an integral part of the immune responses to pathogenic infection, and different forms of cell death can have different immunological consequences. For example, apoptosis is thought to clear viral infected cells and to control run-away immune responses whereas pyroptosis of macrophages can result in the release of cytokines. The Tumor Necrosis Factor Receptor (TNF-R) family of death receptors can trigger apoptosis but also an alternative form of death called programmed necrosis or necroptosis. Necroptosis is dependent on the RIP1 and RIP3 kinases but little is known on its physiological role. To address this, we have generated three lines of tissue- specific FADD knockout mice. FADD is a known adapter protein for all the TNF-R death receptors and thus FADD-deficient cells are resistant to death receptor-mediated apoptosis. However, stimulation of FADD-deficient cells leads to death through necroptosis. This occurs in T-cell receptor stimulated T cells from T-cell specific-FADD knockout (tFADD-/-) mice, in lipopolysaccharide (LPS) stimulated dendritic cells (DCs) from DC-specific-FADD deficient (dcFADD-/-) mice and in LPS stimulated macrophages from macrophage-specific FADD deficient (mFADD-/-) mice. T cells from tFADD-/- mice are functionally defective due to premature necroptosis. However, FADD-deficient DCs are functionally normal and can secrete cytokines when stimulated. Analysis of dcFADD-/- mice showed that some of their phenotypes are surprisingly similar to DC-less mice with imbalance erythrocytes and myeloproliferative disease. At the same time, dcFADD-/- mice exhibit a modest increase of inflammation. In contrast to other DC-specific apoptosis-resistant mice, aged dcFADD-/- mice don't suffer from autoimmunity but they appear to have an enhanced immune system. We hypothesize that necroptosis is one of the strategies for innate immune cells to stimulate the immune system in cases when receptor-induced apoptosis is blocked. In dcFADD-/- mice, necroptotic DCs releasing inflammatory contents can lead to improved immunity against pathogenic infection. This hypothesis will be tested in aim 1. Using DC-specific FADD-/-/MyD88-/- and dcFADD-/-/RIP3-/- mice, we will address whether constitutive stimulation of DCs through TLRs leads to necroptosis and release of inflammatory contents. To examine the possible enhanced immunity of dcFADD-/- mice, they will be challenged with influenza and Toxoplasma gondii and studied in details. Whether necroptosis is required for immunity against these two pathogens will be assessed using RIP3-/- mice. In Aim 2, we will unravel the biochemical signaling pathways involving FADD in necroptosis in response to TLR stimulation. Successful completion of these aims will greatly enhance our understanding of the host- pathogen interaction and the physiological role of necroptosis. ! ! ! PUBLIC HEALTH RELEVANCE: Cell death, including apoptosis, pyroptosis, autophagic cell death and necrosis, is an integral part of development of the mouse/human immune system and/or responses to pathogenic infection. Members of the tumor necrosis factor (TNF) death receptors can initiate apoptosis and a new form of necrosis called necroptosis but the physiological role of necroptosis is poorly understood; we propose in this application to understand the role of necroptosis in infection by studying mouse models deficient in an adapter protein required for the TNF cell death signals.

描述(由申请人提供)：细胞死亡是对病原体感染的免疫反应的一个组成部分，不同形式的细胞死亡可能会有不同的免疫学后果。例如，细胞凋亡被认为可以清除受病毒感染的细胞，控制失控的免疫反应，而巨噬细胞的松弛可以导致细胞因子的释放。肿瘤坏死因子受体(TNF-R)家族的死亡受体可以触发细胞凋亡，但也可以引发另一种形式的死亡，称为程序性坏死或坏死性下垂。坏死性上睑下垂依赖于RIP1和RIP3激酶，但对其生理作用知之甚少。为了解决这个问题，我们培育了三种组织特异性FADD基因敲除小鼠。FADD是已知的所有肿瘤坏死因子受体死亡受体的适配蛋白，因此FADD缺陷细胞对死亡受体介导的细胞凋亡具有抵抗力。然而，对FADD缺陷细胞的刺激会导致坏死性下垂死亡。这发生在T细胞特异性FADD基因敲除(tFADD-/-)小鼠的T细胞受体刺激的T细胞、DC特异性FADD缺陷(dcFADD-/-)小鼠的内毒素刺激的树突状细胞(DC)和巨噬细胞特异性FADD缺陷小鼠(mFADD-/-)的内毒素刺激的巨噬细胞中。来自tFADD-/-小鼠的T细胞由于过早的坏死性下垂而出现功能缺陷。然而，FADD缺陷的DC在功能上是正常的，在受到刺激时可以分泌细胞因子。对dcFADD-/-小鼠的分析表明，他们的一些表型与缺乏DC的红细胞失衡和骨髓增生性疾病的小鼠惊人地相似。与此同时，dcFADD-/-小鼠的炎症反应略有增加。与其他DC特异性抗凋亡小鼠不同，老年dcFADD-/-小鼠不会受到自身免疫的影响，但它们的免疫系统似乎得到了增强。我们假设，当受体诱导的细胞凋亡被阻断时，坏死性下垂是先天免疫细胞刺激免疫系统的策略之一。在dcFADD-/-小鼠中，坏死性DC释放炎症内容物可提高对病原性感染的免疫力。这一假设将在目标1中得到验证。使用DC特异性的FADD-/-/MyD88-/-和dcFADD-/-/RIP3-/-小鼠，我们将研究通过TLRs对DC的结构性刺激是否会导致坏死性下垂和炎性物质的释放。为了检测dcFADD-/-小鼠可能的增强免疫力，将用流感和弓形虫攻击它们，并进行详细的研究。对这两种病原体的免疫是否需要坏死性下垂将使用RIP3-/-小鼠进行评估。在目标2中，我们将揭开FADD参与坏死性下垂对TLR刺激的反应的生化信号通路。这些目标的成功完成将极大地提高我们对宿主-病原体相互作用和坏死性下垂的生理作用的了解。！！！ 公共卫生相关性：细胞死亡，包括细胞凋亡、下垂、自噬细胞死亡和坏死，是鼠/人免疫系统发育和/或对病原体感染反应的一个组成部分。肿瘤坏死因子死亡受体的成员可以启动细胞凋亡和一种称为坏死性下垂的新形式的坏死，但对坏死性下垂的生理作用知之甚少；在这一应用中，我们建议通过研究缺乏肿瘤坏死因子细胞死亡信号所需的适配蛋白的小鼠模型来了解坏死性下垂在感染中的作用。