Low Density Neutrophils Link Inflammation and Coagulopathy in COVID-19

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)。新冠肺炎约20%的股份 患者经历严重的疾病，典型的表现为双侧肺炎，大约5%的患者进展到 急性呼吸窘迫综合征(ARDS)。ARDS是由病毒引起的肺损伤和 免疫细胞的快速涌入，释放炎症介质，导致一种被称为 细胞因子风暴。新冠肺炎急性呼吸窘迫综合征因一种导致血栓的独特弥漫性凝血障碍而进一步加重 静脉和动脉循环中的形成和肺毛细血管内的微血栓。易感因素 这种凝血障碍包括纤维蛋白原升高、激活的凝血级联反应、血小板活化、 炎症、中性粒细胞胞外陷阱(Net)的形成和内皮细胞的损伤。了解 新冠肺炎凝血障碍和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。 越来越多的证据表明，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。 我们的初步研究发现了一种新的低密度中性粒细胞(LDN)群体，它表达 新冠肺炎患者CD16(CD16Int LDN)的中等水平CD16Int LDN的数量与 疾病严重程度、炎性细胞因子IL-6/肿瘤坏死因子-α、D-二聚体水平以及临床结果。此外, CD16Int LDN显示自发性网状结构及体内血小板活化和颗粒的证据 胞吐。根据这些发现，我们推测CD16Int LDN在诱导 新冠肺炎危重患者的凝血障碍和肺部炎症。三个具体目标 建议进一步剖析其潜在的机制。目标1将全面描述LDN 使用蛋白质组学和转录组学方法的亚集。从这些研究中获得的信息将是 用来改进我们的细胞周期蛋白抗体面板。我们将使用这个面板来跟踪不同的中性粒细胞群 纵向病人样本。目标2将确定LDN亚群在疾病进展过程中的功能变化 以及它们在严重和危重COVID患者失调的炎症反应和凝血障碍中的作用- 19例患者。中性粒细胞脱颗粒、网状形成、吞噬、趋化、凋亡和细胞因子 将对释放进行检查。我们还将确定低密度脂蛋白是否会促进新冠肺炎患者的凝血障碍。目标3将 确定使用我们的新型TAT融合蛋白抑制剂是否抑制中性粒细胞颗粒胞吐 防止激活的中性粒细胞介导的功能变化和高凝状态。我们还将使用hACE2 TG 以小鼠为模型，测定TAT融合蛋白对肺部炎症和功能损害的体内疗效。 这一提议的成功完成将为新冠肺炎的病理生理提供新的见解，通过定义 一个独特的中性粒细胞亚群的作用以及通过建立中性粒细胞脱颗粒作为治疗靶点 新冠肺炎抑制炎性肺损伤和免疫血栓形成。

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). Approximately 20% of COVID-19 patients experience severe disease, typically presenting with bilateral pneumonia, and about 5% progress to acute respiratory distress syndrome (ARDS). 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. COVID-19 ARDS is further exacerbated by a unique diffuse coagulopathy leading to thrombus formation in the venous and arterial circulations and microthrombi in capillaries of the lungs. Predisposing factors for this coagulopathy include increased fibrinogen, activated coagulation cascade, platelet activation, hyper- inflammation, neutrophil extracellular trap (NET) formation, and endothelial cell damage. Understanding the pathophysiology of COVID-19 coagulopathy and ARDS is critical to finding effective therapeutic interventions. Accumulating evidence indicates critical roles of neutrophils in both ARDS and immunothrombosis in COVID-19. Our preliminary studies identified a novel population of low-density neutrophils (LDN) which expresses intermediate levels of CD16 (CD16Int LDN) in COVID-19 patients. The number of CD16Int LDN correlated with disease severity, levels of inflammatory cytokines IL-6/TNF-a, D-dimer levels, and clinical outcomes. In addition, CD16Int LDN showed spontaneous NET formation and evidence of in vivo platelet activation and granule exocytosis. Based on these findings, we postulate that CD16Int LDN play a critical role in the induction of coagulopathy and pulmonary inflammation in severe and critical COVID-19 patients. Three specific Aims are proposed to further dissect the underlying mechanisms. Aim 1 will comprehensively characterize LDN subsets using proteomics and transcriptomics approaches. The information gained from those studies will be used to refine our CyTOF antibody panel. We will use this panel to track differential neutrophil clusters in longitudinal patient samples. Aim 2 will determine LDN subsets functional changes during disease progression and their contributions to dysregulated inflammatory response and coagulopathy in severe and critical COVID- 19 patients. Neutrophil degranulation, NET formation, phagocytosis, chemotaxis, apoptosis, and cytokine release will be examined. We will also determine if LDN promote coagulopathy in COVID-19 patients. Aim 3 will determine whether inhibition of neutrophil granule exocytosis using our novel TAT-fusion protein inhibitors prevents activated neutrophil-mediated functional changes and hypercoagulation. We will also use a hACE2 Tg mouse model to determine the in vivo efficacy of TAT-fusion proteins on lung inflammation and impaired function. Successful completion of this proposal will provide novel insights into COVID-19 pathophysiology by defining the role of a unique subset of neutrophils and by establishing neutrophil degranulation as a therapeutic target for inhibiting inflammatory lung injury and immunothrombosis in COVID-19.