Neutrophil Heterogeneity and Immunopathogenesis of COVID-19 ARDS

项目摘要 冠状病毒病2019(新冠肺炎)是一种由新型病毒引起的潜在威胁生命的疾病 病原体，SARS冠状病毒2(SARS-CoV-2)。在新冠肺炎的10%-20%中 住院患者中，约有三分之一发展为急性呼吸窘迫综合征(ARDS)。新冠肺炎-- 诱发性ARDS是由病毒引起的肺损伤和免疫细胞的快速涌入共同作用的结果 释放炎症介质，导致被称为细胞因子风暴的过度激活状态。了解 新冠肺炎急性呼吸窘迫综合征的病理生理学对于找到有效的治疗干预措施至关重要。积累 有证据表明，中性粒细胞在急性呼吸窘迫综合征和新冠肺炎免疫血栓形成中都发挥了关键作用。多元组学 研究发现新冠肺炎中中性粒细胞的戏剧性异质性，包括出现大量 低密度中性粒细胞(LDN)。我们报告了一种存在于循环和肺中的新的LDN种群。 表达中等水平CD16Int LDN的重症新冠肺炎患者。CD16Int LDN 在体内和体外自发形成中性粒细胞外陷阱(Net)，激活血小板，并具有 脱颗粒加重。我们的初步数据显示，重症新冠肺炎患者的低密度脂蛋白亚群 在功能上有别于成熟的、正常密度的中性粒细胞(NDN)。基于这些发现，我们假设 异质性中性粒细胞群体的不同功能反应协同诱导 新冠肺炎患者全身和肺部炎症导致急性呼吸窘迫综合征。三个具体目标是 建议进一步剖析其内在机制。目标1将全面表征中性粒细胞 使用蛋白质组学和转录组学方法从重症/危重新冠肺炎患者中分离出亚群。这个 从这些研究中获得的信息将被用来改进我们的细胞周期蛋白抗体组合，并有望 检测新冠肺炎患者中性粒细胞亚群在病程中的动态变化。目标2将 确定中性粒细胞亚群在疾病进展过程中的功能变化及其对 重症/危重新冠肺炎患者的炎症反应和凝血功能紊乱。中性粒细胞 脱颗粒、网状形成、吞噬、趋化、T细胞抑制活性和细胞因子/趋化因子 将对释放进行检查。我们还将确定LDN子集和NDN或其派生产品是否促进 新冠肺炎患者的凝血障碍。目标3将确定中性粒细胞宿主防御的相对贡献 系统与急性肺损伤的发生有关。我们还将使用hACE2 TG小鼠模型来确定 中性粒细胞呼吸爆发、颗粒胞吐和网络形成在SARS-CoV-2诱导的急性发作中的作用 肺部发炎。这项提案的成功完成将为研究新冠肺炎引发的 通过定义不同中性粒细胞亚群的功能作用和通过建立 哪些中性粒细胞的防御系统是抑制炎性肺损伤的治疗靶点 新冠肺炎中的免疫血栓形成。

Project Summary Coronavirus disease 2019 (COVID-19) is a potentially life-threatening disease caused by the novel viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of the 10%-20% of COVID-19 patients hospitalized, approximately 1/3 develop acute respiratory distress syndrome (ARDS). COVID-19- induced ARDS results from a combination of virally induced lung injury and the rapid influx of immune cells that release inflammatory mediators leading to a hyper-activated state known as cytokine storm. Understanding the pathophysiology of COVID-19 ARDS is critical to finding effective therapeutic interventions. Accumulating evidence indicates critical roles for neutrophils in both ARDS and immunothrombosis in COVID-19. Multi-omics studies identified dramatic neutrophil heterogeneity in COVID-19, including emergence of a large number of low density neutrophils (LDN). We reported a novel population of LDN present in both the circulation and lungs of severe COVID-19 patients which expresses intermediate levels of CD16 (CD16Int LDN). CD16Int LDN spontaneously form neutrophil extracellular traps (NETs), activate platelets in vivo and in vitro, and have heightened degranulation. Our preliminary data show that LDN subsets from severe COVID-19 patients are functionally distinct from mature, normal density neutrophils (NDN). Based on these findings, we postulate that differential functional responses of heterogeneous neutrophil populations collaborate to induce systemic and pulmonary inflammation leading to ARDS in COVID-19 patients. Three specific Aims are proposed to further dissect the underlying mechanisms. Aim 1 will comprehensively characterize neutrophil subsets from severe/critical COVID-19 patients using proteomics and transcriptomics approaches. The information gained from those studies will be used to refine our CyTOF antibody panel and prospectively measure dynamic changes of neutrophil subsets over the disease course in COVID-19 patients. Aim 2 will determine neutrophil subset functional changes during disease progression and their contributions to dysregulated inflammatory response and coagulopathy in severe/critical COVID-19 patients. Neutrophil degranulation, NET formation, phagocytosis, chemotaxis, T cell suppressive activity, and cytokine/chemokine release will be examined. We will also determine if LDN subsets and NDN or their derived products promote coagulopathy in COVID-19 patients. Aim 3 will determine the relative contributions of neutrophil host defense systems to development of acute lung injury. We will also use a hACE2 Tg mouse model to determine the roles of neutrophil respiratory burst, granule exocytosis, and NET formation to SARS-CoV-2-induced acute lung inflammation. Successful completion of this proposal will provide novel insights into COVID-19-induced ARDS pathophysiology by defining the functional roles of different neutrophil subsets and by establishing which host defense systems of neutrophils as a therapeutic target for inhibiting inflammatory lung injury and immunothrombosis in COVID-19.