COVID-19: Understanding The Role of Corona Virus InducedDisruption Of Alveolar Type 2 Cell Function And SurfactantHomeostasis In The Pathogenesis Of COVID-19 AcuteRespiratory Distress Syndrome

摘要 严重急性呼吸系统综合症相关冠状病毒2型是新冠肺炎的病原体 以难治性急性肺损伤为特征的综合征，导致严重的缺氧性呼吸衰竭 和高死亡率。尽管新型冠状病毒造成了反复的健康和经济破坏 (NCoV)过去20年，包括SCoV2及其前身SCoV1和MERS-CoV， 仍然是一个重要的未得到满足的需求，无论是更清楚地了解病毒与宿主细胞的相互作用，还是 确定新的治疗靶点。以改善健康为最终目标来解决这些问题 和新冠肺炎老兵的成果，我们汇聚了一支国际公认的科学家团队 在冠状病毒病毒学和肺部生物学方面的专业知识，再加上我们之前工作的坚实基础 在表面活性物质生物学、肺损伤和纤维化修复方面，部分由退伍军人事务部功绩审查计划资助。利用 这项建议旨在填补nCoV发病机制中存在的巨大知识空白 导致呼吸衰竭。这项调查的动机是最近认识到 肺远端肺泡2型(AT2)上皮细胞已成为SCOV的重要入口。 2.这一应用的中心假设是感染nCoV的AT2细胞获得表面活性物质缺陷 生物合成/代谢，激活细胞应激途径，并导致祖细胞功能的改变 其中促进缺氧性呼吸衰竭、持续性肺部炎症和损伤，并影响康复 通过对上皮细胞修复能力的影响。为了测试这一点，我们将利用已建立的小鼠冠状病毒模型 具有肺部表型的感染(MHV-1)与体外感染支持的还原研究相结合 从已携带SARS-CoV-2的人肺管道中获得的原代人类AT2细胞 隔离。我们的实验方法将使用可用的工具和试剂来询问这些临床前模型 在我们的计划中，绘制冠状病毒感染对远端肺细胞群的影响图，重点是识别和 翻译AT2功能被破坏的分子机制与改变的表面活性物质生物学和 牙槽窝内促炎症/促纤维细胞的相互作用。在具体目标1中，我们将首先定义时间 AT2动态平衡紊乱的个体发生过程中远端肺上皮内表型的变化 通过病毒感染使用具有良好特征的MHV-1肺部感染的小鼠模型。使用两者不偏不倚 诸如转录图谱以及经典细胞生物学和生物化学等方法将达到这一目标 研究冠状病毒诱导的表面活性物质代谢/生物物理活性、AT2细胞应激(即内质网应激、 蛋白酶体功能障碍、自噬功能障碍、线粒体动力学改变//生物能量学)和AT2 祖细胞功能。在特定的目标2中，配备了这种“功能图”的小鼠冠状病毒-肺和异常 在2行为，我们将翻译识别的铅目标和机制到人类AT2细胞利用这两个 原代AT2培养以及感染SARS-CoV-2的精密切割肺切片(PCL)。研究设计 涉及病毒依赖的AT2表面活性物质蛋白和脂类代谢的分析，AT2细胞的询问 SCOV-2感染株AT2祖细胞功能表型的质控途径和功能评价 人类上皮细胞。通过了解nCoV导致上皮损伤/功能障碍的途径，其机制 使用这些模型识别受影响的肺细胞群将提高对sCOV2/COVID- 19证候，促进识别新的靶点通路，并可以交叉用途来测试新出现的 针对当前和未来新冠状病毒大流行的治疗方法。

ABSTRACT The Severe Acute Respiratory Syndrome (SARS)-associated coronavirus 2 (SCoV2) is the cause of COVID-19 syndrome which is marked by a refractory acute lung injury that results in dramatic hypoxic respiratory failure and high mortality. Despite the recurrent health and economic devastation produced by novel coronaviruses (nCoV) over the past 20 years including SCoV2 as well as its predecessors SCoV1, and MERS-CoV, there remains a significant unmet need both for a clearer understanding of virus-host cell interactions as well as identification of new therapeutic targets. To address these issues with the ultimate goal of improving the health and outcomes of veterans with COVID-19, we have assembled a team of internationally recognized scientists with expertise in coronavirus virology and in lung biology coupled with a strong foundation of our own prior work on surfactant biology, lung injury, and fibrotic repair funded, in part, by the VA Merit Review program. Utilizing this expertise, this proposal is directed at filling in large knowledge gaps that exist in the pathogenesis of nCoV induced respiratory failure. The motivation for this investigation has been fueled by the recent recognition that the alveolar type 2 (AT2) epithelial cell of the distal lung has emerged as an important portal of entry for SCoV- 2. The central hypothesis of this application is that AT2 cells infected with nCoV acquire defects in surfactant biosynthesis/metabolism, activate cellular stress pathways, and develop alterations in progenitor cell function all of which promote hypoxic respiratory failure, persistent lung inflammation and injury, and impact recovery through effects on epithelial repair capacity. To test this, we will leverage an established murine model of CoV infection (MHV-1) with a pulmonary phenotype combined with reductionist studies supported by ex vivo infection of primary human AT2 cells obtained from a robust human lung pipeline with an already in hand SARS-CoV-2 isolate. Our experimental approach will interrogate these preclinical models using tools and reagents available in our program, to map the effect of CoV infection on distal lung cell populations with a focus on identifying and translating molecular mechanisms linking the disrupted AT2 function with altered surfactant biology and proinflammatory/profibrotic cell cross talk in the alveoilar niche. In Specific Aim 1, we will first define temporal changes in distal lung epithelial endophenotypes focusing on the ontogeny of the disruption of AT2 homeostasis by viral infection using a well characterized mouse model of MHV-1 lung infection. Using both unbiased approaches such as transcriptomic profiling as well as classical cell biology and biochemistry this aim will investigate CoV induced changes in surfactant metabolism/biophysical activity, AT2 cell stress (i.e. ER stress, proteasome dysfunction, autophagy malfunction, changes in mitochondrial dynamics/ / bioenergetics) and AT2 progenitor cell function. In Specific Aim 2, armed with this “functional map” of the mouse CoV-lung and aberrant AT2 behaviors, we will translate the identified lead targets and mechanisms to human AT2 cells utilizing both primary AT2 cultures as well as precision cut lung slices (PCLS) infected with SARS-CoV-2. The research design involves the analysis of virus-dependent AT2 surfactant protein and lipid metabolism, interrogation of AT2 cell quality control pathways, and functional evaluation of AT2 progenitor function phenotypes in the SCoV-2 infected human epithelial cells. By understanding the path to epithelial injury / dysfunction from nCoV, the mechanisms and affected lung cell populations identified using these models will improve the understanding of sCOV2/COVID- 19 syndrome, promote identification of new target pathways, and can be cross-purposed to test emerging therapies for both the current and future nCoV pandemics.