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

描述（由申请人提供）：细胞死亡是对病原性感染的免疫应答的组成部分，不同形式的细胞死亡可能具有不同的免疫学后果。例如，细胞凋亡被认为是清除病毒感染的细胞并控制失控的免疫反应，而巨噬细胞的细胞凋亡可导致细胞因子的释放。死亡受体的肿瘤坏死因子受体（TNF-R）家族可以触发细胞凋亡，但也可以触发另一种形式的死亡，称为程序性坏死或坏死性凋亡。坏死性凋亡依赖于RIP 1和RIP 3激酶，但对其生理作用知之甚少。为了解决这个问题，我们已经产生了三个系的组织特异性FADD敲除小鼠。FADD是已知的所有TNF-R死亡受体的衔接蛋白，因此FADD缺陷细胞对死亡受体介导的细胞凋亡具有抗性。然而，FADD缺陷细胞的刺激通过坏死性凋亡导致死亡。这发生在来自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、来自DC特异性FADD缺陷（dcFADD-/-）小鼠的脂多糖（LPS）刺激的树突状细胞（DC）中以及来自巨噬细胞特异性FADD缺陷（mFADD-/-）小鼠的LPS刺激的巨噬细胞中。来自tFADD-/-小鼠的T细胞由于过早坏死性凋亡而具有功能缺陷。然而，FADD缺陷型DC在功能上是正常的，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，它们的一些表型与具有不平衡红细胞和骨髓增生性疾病的DC-少小鼠惊人地相似。同时，dcFADD-/-小鼠表现出适度的炎症增加。与其他DC特异性抗肿瘤小鼠相比，老年dcFADD-/-小鼠不患有自身免疫性，但它们似乎具有增强的免疫系统。我们假设坏死性凋亡是先天免疫细胞在受体诱导的凋亡被阻断的情况下刺激免疫系统的策略之一。在dcFADD-/-小鼠中，坏死性DC释放炎性内容物可导致针对病原性感染的免疫力提高。这一假设将在目标1中得到检验。使用DC特异性FADD-/-/MyD 88-/-和dcFADD-/-/RIP 3-/-小鼠，我们将解决通过TLR对DC的组成性刺激是否导致坏死性凋亡和炎性内容物的释放。为了检查dcFADD-/-小鼠的可能增强的免疫力，将用流感和弓形虫攻击它们并进行详细研究。将使用RIP 3-/-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，我们将阐明TLR刺激引起的坏死性凋亡中涉及FADD的生化信号通路。这些目标的成功完成将大大提高我们对宿主-病原体相互作用和坏死性凋亡的生理作用的理解。! ! !

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. ! ! !