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: 8997965
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8997965
• 关键词: Address; Affinity Chromatography; Antibiotics; Antigens; Apoptosis; Apoptotic; Autoimmunity; Bacteria; Binding Proteins; Biochemical; CASP8 gene; 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; RIPK1 gene; 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; TNF gene; Testing; Time; Tissues; Toll-like receptors; Toxoplasma gondii; Toxoplasmosis; Transfection; Transgenic Mice; Tumor Necrosis Factor Receptor; Vaccines; Viral; Western Blotting; adapter protein; aged; 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.

描述（由申请人提供）：细胞死亡是针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以产生不同的免疫学后果。例如，细胞凋亡被认为可以清除病毒感染的细胞并控制失控的免疫反应，而巨噬细胞的焦亡可以导致细胞因子的释放。死亡受体的肿瘤坏死因子受体 (TNF-R) 家族可以引发细胞凋亡，但也可以引发另一种死亡形式，称为程序性坏死或坏死性凋亡。坏死性凋亡依赖于 RIP1 和 RIP3 激酶，但对其生理作用知之甚少。为了解决这个问题，我们培育了三系组织特异性 FADD 敲除小鼠。 FADD 是所有 TNF-R 死亡受体的已知衔接蛋白，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，刺激 FADD 缺陷细胞会导致坏死性凋亡。这种情况发生在来自 T 细胞特异性 FADD 敲除 (tFADD-/-) 小鼠的 T 细胞受体刺激的 T 细胞中，以及来自 DC 特异性 FADD 缺陷 (dcFADD-/-) 的脂多糖 (LPS) 刺激的树突细胞 (DC) 中。小鼠和 LPS 刺激的巨噬细胞特异性 FADD 缺陷 (mFADD-/-) 小鼠的巨噬细胞。 tFADD-/- 小鼠的 T 细胞由于过早坏死性凋亡而存在功能缺陷。然而，FADD 缺陷的 DC 功能正常，并且在受到刺激时可以分泌细胞因子。对 dcFADD-/- 小鼠的分析表明，它们的一些表型与患有红细胞不平衡和骨髓增殖性疾病的无 DC 小鼠惊人地相似。同时，dcFADD-/- 小鼠表现出适度的炎症增加。与其他 DC 特异性抗凋亡小鼠相比，老年 dcFADD-/- 小鼠不会遭受自身免疫，但它们似乎具有增强的免疫系统。我们假设，当受体诱导的细胞凋亡被阻断时，坏死性凋亡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 释放的炎症内容物可以提高对病原体感染的免疫力。这一假设将在目标 1 中进行检验。使用 DC 特异性 FADD-/-/MyD88-/- 和 dcFADD-/-/RIP3-/- 小鼠，我们将解决通过 TLR 对 DC 的组成性刺激是否会导致坏死性凋亡和释放炎症内容物。为了检查 dcFADD-/- 小鼠可能增强的免疫力，他们将受到流感和弓形虫的攻击并进行详细研究。将使用 RIP3-/- 小鼠评估针对这两种病原体的免疫是否需要坏死性凋亡。在目标 2 中，我们将揭示涉及 FADD 的生化信号通路在响应 TLR 刺激的坏死性凋亡中的作用。成功完成这些目标将极大地增强我们对宿主-病原体相互作用和坏死性凋亡的生理作用的理解。