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

项目摘要/摘要 甲型流感病毒(IAV)杀死了它们在细胞培养和感染中复制的大多数细胞类型 活体内的肺。虽然受调控的细胞死亡代表了一种宿主防御机制，它限制了病毒的传播和 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 尽管控制了体内的病毒复制，细支气管肺泡上皮的降解和随后的死亡。 事实上，感染高致病性IAV毒株后的严重疾病与广泛传播密切相关。 人类肺上皮细胞死亡与细支气管肺泡组织损伤。尽管如此，令人惊讶的是， 已知IAV激活相关肺细胞类型的细胞死亡的分子机制。因此(1) 了解IAV引发细胞死亡的机制，(2)确定IAV的身份和重要性 体内IAV感染过程中通过这些机制死亡的肺细胞类型；以及(3)确定是否具有药理学意义 操纵细胞死亡是呼吸系统IAV治疗的新切入点 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IAV- 激活了感染的呼吸道上皮细胞的死亡。当DAI蛋白感觉到IAV时，这一途径就开始了 基因组RNA，并核化RIPK3。然后，RIPK3激活程序性坏死的平行通路 (坏死性下垂)，以及细胞凋亡。RIPK3下游的坏死下垂依赖于MLKL和FADD上的细胞凋亡。 使得DAI、RIPK3或MLKL+FADD缺失使小鼠对呼吸道IAV异常易感 复制和杀伤力。值得注意的是，单独消除MLKL没有明显的效果，表明 FADD凋亡轴可以完全补偿MLKL的丢失和坏死性下垂。据我们所知，这些发现 代表了专门的IAV激活的细胞死亡途径的第一次描述，DAI作为一种 RNA病毒传感器，并首次鉴定出同时引发细胞凋亡和坏死性下垂的病毒 RIPK3下游。坏死下垂、细胞凋亡和IAV清除的冗余也提供了一种 在坏死性死亡与IAV发病机制有关的病例中出现意想不到的治疗机会。基于 这些和其他观察，这项建议的目标是：(1)确定分子机制，通过 DAI-RIPK3轴识别IAV并激活细胞死亡；(2)利用尖端的鼠标报告模型 分离和鉴定通过RIPK3诱导的细胞凋亡和坏死性下垂而屈服于IAV的肺细胞类型，以及 确定在这些细胞类型中，RIPK3信号对病毒控制很重要；以及(3)测试是否有选择性 在感染高致病性IAV毒株后，阻断坏死性下垂将有临床益处。 这些目标的成功完成有可能改变我们对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.