Low Density Neutrophils Link Inflammation and Coagulopathy in COVID-19

项目摘要 2019年冠状病毒病（COVID-19）是一种由新型病毒引起的可能危及生命的疾病， 病原体，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）。约20%的COVID-19 患者经历严重的疾病，通常表现为双侧肺炎，约5%进展为 急性呼吸窘迫综合征（ARDS）。急性呼吸窘迫综合征是由病毒引起的肺损伤和 释放炎症介质的免疫细胞的快速流入导致称为 细胞因子风暴COVID-19 ARDS因独特的弥漫性凝血病导致血栓而进一步恶化 静脉和动脉循环中的血栓形成以及肺毛细血管中的微血栓。诱发因素 这种凝血病包括纤维蛋白原增加，激活凝血级联反应，血小板活化， 炎症、中性粒细胞胞外陷阱（NET）形成和内皮细胞损伤。了解 COVID-19凝血病和ARDS的病理生理学对于寻找有效的治疗干预至关重要。 越来越多的证据表明，中性粒细胞在COVID-19的ARDS和免疫血栓形成中发挥着关键作用。 我们的初步研究确定了一种新的低密度中性粒细胞（LDN）群体， 在COVID-19患者中，CD 16（CD 16 Int LDN）水平处于中等水平。CD 16 Int LDN的数量与 疾病严重程度、炎性细胞因子IL-6/TNF-α水平、D-二聚体水平和临床结果。此外，本发明还提供了一种方法， CD 16 Int LDN显示自发性NET形成和体内血小板活化和颗粒形成的证据。 胞吐作用基于这些发现，我们推测CD 16 Int LDN在诱导人肝癌细胞凋亡中起关键作用。 严重和危重COVID-19患者的凝血病和肺部炎症。三个具体目标 旨在进一步剖析潜在的机制。目标1将全面表征LDN 使用蛋白质组学和转录组学方法的子集。从这些研究中获得的信息将 用来完善我们的CyTOF抗体组我们将使用这个面板来跟踪不同的中性粒细胞簇， 纵向患者样本。目的2：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-1患者中对炎症反应失调和凝血障碍的作用， 19名患者。中性粒细胞脱粒、NET形成、吞噬作用、趋化性、凋亡和细胞因子 释放将被审查。我们还将确定LDN是否会促进COVID-19患者的凝血功能障碍。目标3将 确定使用我们的新型TAT融合蛋白抑制剂抑制中性粒细胞颗粒胞吐是否 防止活化的嗜酸性粒细胞介导的功能变化和高凝状态。我们还将使用hACE 2 Tg 小鼠模型，以确定TAT融合蛋白对肺部炎症和受损功能的体内功效。 该提案的成功完成将通过定义COVID-19病理生理学提供新的见解， 中性粒细胞的独特亚群的作用，并通过建立中性粒细胞脱粒作为治疗靶点， 抑制COVID-19中的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.