Mechanism, Function, and Exploitation of Influenza A Virus-Activated Cell Death

项目摘要/摘要 无论是在细胞培养物中还是在感染的细胞中，甲型流感病毒（IAV）都会杀死其复制的大多数细胞类型 肺在体内虽然受调节的细胞死亡代表了限制病毒传播和感染的宿主防御机制， 宿主免疫病理学在感染早期，无节制的细胞死亡，特别是坏死，可导致严重的 尽管在体内控制了病毒复制，细支气管肺泡上皮细胞的降解和随后的死亡率。 事实上，感染高致病性IAV株后的严重疾病与广泛的 肺上皮细胞死亡和细支气管肺泡组织损伤。尽管如此， 已知IAV激活相关肺细胞类型中细胞死亡的分子机制。因此（1） 了解IAV触发细胞死亡的机制，（2）确定IAV的身份和重要性， 在体内IAV感染期间通过这些机制死亡的肺细胞类型;和（3）确定是否药理学上 操纵细胞死亡代表了呼吸道IAV的新治疗切入点， 目标.我们最近发现了一种细胞死亡的机制，似乎可以解释几乎所有的IAV- 感染的气道上皮细胞的活化死亡。当蛋白质DAI感知IAV时，该途径被启动 基因组RNA和成核激酶RIPK 3。然后RIPK 3激活程序性坏死的平行途径 （坏死性凋亡）以及凋亡。RIPK 3下游的坏死性凋亡依赖于MLKL，而凋亡依赖于FADD， 使得DAI、RIPK 3或MLKL+FADD缺失使得小鼠对呼吸道IAV格外敏感 复制和致命性。值得注意的是，单独消除MLKL没有明显的效果，这表明， FADD凋亡轴可以完全补偿MLKL的丢失和坏死性凋亡。据我们所知，这些发现 代表了一个专门的IAV激活细胞死亡途径的第一个描述，DAI作为一种免疫抑制剂的第一个含义， RNA病毒的传感器，并首次鉴定出一种引发细胞凋亡和坏死性凋亡的病毒 RIPK 3的下游。坏死性凋亡与细胞凋亡对IAV清除的冗余也提供了一个新的途径。 在坏死性死亡与IAV发病机制有关的情况下，这是意想不到的治疗机会。基于 这些和其他观察，本建议的目标是：（1）确定分子机制， DAI-RIPK 3轴识别IAV并激活细胞死亡;（2）采用尖端小鼠报告模型， 通过RIPK 3驱动凋亡与坏死性凋亡分离和鉴定死于IAV的肺细胞类型，和 确定这些细胞类型中的哪一种是对病毒控制重要的RIPK 3信号传导;以及（3）测试是否具有选择性 在感染高致病性IAV株后，阻断坏死性凋亡将具有临床益处。 这些目标的成功实现有可能改变我们对IAV发病机制的理解， 直接的临床后果。

PROJECT SUMMARY/ABSTRACT Influenza A viruses (IAV) kill most of the cell types in which they replicate, both in cell culture and in infected lungs in vivo. While regulated cell death represents a host defense mechanism that limits both virus spread and host immunopathology early in an infection, unbridled cell death, particularly necrosis, can lead to severe degradation of bronchioalveolar epithelia and consequent mortality despite control of virus replication in vivo. Indeed, severe illness following infection with highly pathogenic strains of IAV is well-correlated with widespread pulmonary epithelial cell death and bronchioalveolar tissue damage in humans. Despite this, remarkably little is known of the molecular mechanisms by which IAV activates cell death in relevant lung cell types. Thus (1) understanding the mechanisms by which IAV triggers cell death, (2) determining the identity and importance of lung cell types that die by these mechanisms during IAV infection in vivo; and (3) determining if pharmacological manipulation of cell death represents a new therapeutic entry-point for respiratory IAV are each important unmet objectives. We have recently discovered a mechanism of cell death that appears to account for almost all IAV- activated death in infected airway epithelial cells. This pathway is initiated when the protein DAI senses IAV genomic RNA and nucleates the kinase RIPK3. RIPK3 then activates parallel pathways of programmed necrosis (necroptosis), as well as apoptosis. Necroptosis downstream of RIPK3 relies on MLKL and apoptosis on FADD, such that deletion of DAI, RIPK3, or MLKL+FADD renders mice extraordinarily susceptible to respiratory IAV replication and lethality. Remarkably, eliminating MLKL alone has no discernible effect, demonstrating that the FADD apoptosis axis can fully compensate for loss of MLKL and necroptosis. To our knowledge, these findings represent the first description of a dedicated IAV activated cell death pathway, the first implication of DAI as a sensor of RNA viruses, and the first identification of a virus that triggers both apoptosis and necroptosis downstream of RIPK3. The redundancy of necroptosis with apoptosis to IAV clearance also provides an unexpected therapeutic opportunity in cases where necrotic death is implicated in IAV pathogenesis. Based on these and other observations, the goals of this proposal are to: (1) identify the molecular mechanisms by which the DAI-RIPK3 axis recognizes IAV and activates cell death; (2) employ cutting-edge mouse reporter models to isolate and identify lung cell types that succumb to IAV by RIPK3-driven apoptosis versus necroptosis, and determine in which of these cell types is RIPK3 signaling important for virus control; and (3) test if selective blockade of necroptosis will have clinical benefit following infection with highly-pathogenic strains of IAV. Successful completion of these Aims has the potential to transform our understanding of IAV pathogenesis, with immediate clinical ramifications.