Mechanisms and pathogenicity of SARS-CoV-2-induced neutrophil extracellular traps

项目概要/摘要 急性呼吸窘迫综合征 (ARDS) 于 1967 年首次被正式描述，但它很可能是 几个世纪以来，病毒感染是导致大流行性病毒感染死亡的主要原因。现在，世界正面临着一个新的挑战 SARS-CoV-2 或 COVID-19 引起的大流行，已感染全球 1 亿多人，导致 > 200 万人死亡。 ARDS 免疫发病机制的核心是中性粒细胞和中性粒细胞的作用 激活，包括中性粒细胞染色质释放到细胞外空间，这一过程称为 中性粒细胞胞外陷阱（NET）。最初被描述为一种灭活病原体的宿主防御形式， NET 已成为对感染的潜在适应不良反应，造成大量旁观者伤害 到组织并作为凝血病灶。我们已经证明 NET 的产生是为了响应这两者 无菌和病原体引起的急性肺损伤（包括甲型流感），当中和后，肺损伤 在不影响微生物遏制的情况下减少。我们还表明，NETs 在 ARDS 患者的血浆与更严重的 ARDS 和 ARDS 死亡率相关。新出现的报告 表明 NET 也存在于 COVID-19 患者的血液和肺部中。我们现在的定位是 在此应用中快速测试 NET 响应 SARS-CoV-2 的作用，并开发新方法 中和 NET 以达到治疗目的。在目标 1 中，我们将用 SARS-CoV-2 病毒或 刺突蛋白以确定 NET 的产生和 NET 诱导分子途径。我们假设 分泌的中性粒细胞蛋白酶和 NET 本身会切割刺突蛋白，以引发增强的病毒感染 发病机制。我们还将利用质谱法对中性粒细胞蛋白进行公正的研究 确定 SARS-CoV-2（包括中性粒细胞）引起的中性粒细胞激活的新途径 与 COVID-19 患者隔离。在目标 2 中，我们将把注意力转向 COVID-19 小鼠模型，其中 表达人类 ACE2 的小鼠品系将面临 SARS-CoV-2 和肺损伤、NET 和 测量的系统效应。我们假设 NET 将被生产并主要本地化 肺泡空间。我们将使用 NET 通路（PAD4-/-、DNase1- /-、DNase1L3-/-）以测试其在此模型中的致病性。在这些研究中，我们还将测试一种新型 DNase 将 NET 中和成无毒的单核小体，可以快速部署到 COVID-19 患者身上。 最后，我们将测试 NET 相关血小板激活、补体激活和凝血的作用 反应和减轻这些系统性影响的治疗策略。总之，这些研究将建立 关于中性粒细胞激活的新知识以及 NET 在 COVID-19 中的明确作用并确定治疗方法 在 SARS-CoV-2 诱导的 ARDS 中靶向 NET 的方法。

Project Summary/Abstract The acute respiratory distress syndrome (ARDS) was first formally described in 1967, but it has likely been the predominant cause of death in pandemic viral infections for centuries. Now, the world is at grips with a new pandemic from SARS-CoV-2, or COVID-19, which has infected over 100 million people worldwide resulting in >2 million deaths. Central to the immunopathogenesis of ARDS is the role of neutrophils and neutrophil activation, including the release of neutrophil chromatin into the extracellular space in a process termed neutrophil extracellular traps, or NETs. Originally described as a form of host defense to inactivate pathogens, NETs have emerged as a potentially maladaptive response to infections, producing substantial bystander injury to tissues and serving as a nidus for coagulation. We have shown that NETs are produced in response to both sterile and pathogen-induced acute lung injury (including Influenza A), and when neutralized, lung injury is reduced without compromising microbial containment. We have also shown that NETs are increased in the plasma of patients with ARDS and associate with more severe ARDS and ARDS mortality. Emerging reports indicate that NETs are also present in the blood and lungs of COVID-19 patients. We are now positioned to rapidly test the role of NETs in response to SARS-CoV-2 in this application, and to develop novel approaches to neutralize NETs for therapeutic purposes. In Aim 1, we will challenge neutrophils with SARS-CoV-2 virus or spike protein to determine the production of NETs and NET-induction molecular pathways. We hypothesize that secreted neutrophil proteases and NETs themselves will cleave spike protein to prime for enhanced viral pathogenesis. We will also embark on unbiased studies of neutrophil proteins using mass spectrometry to determine novel pathways of neutrophil activation resulting from SARS-CoV-2, including from neutrophils isolated from COVID-19 patients. In Aim 2, we will turn our attention to a mouse model of COVID-19 in which mouse strains expressing human ACE2 will be challenged with SARS-CoV-2 and lung injury, NETs, and systemic effects measured. We hypothesize that NETs will be produced and predominantly localize to the alveolar spaces. We will use mice with gain and loss of function mutations in NET pathways (PAD4-/-, DNase1- /-, DNase1L3-/-) to test for their pathogenicity in this model. In these studies, we will also test a novel DNase to neutralize NETs into non-toxic mononucleosomes, which could be rapidly deployed to COVID-19 patients. Finally, we will test for the role of NET-associated platelet activation, complement activation, and coagulation responses, and therapeutic strategies to mitigate these systemic effects. In summary, these studies will establish new knowledge on neutrophil activation and the definitive role of NETs in COVID-19 and identify therapeutic approaches to target NETs in SARS-CoV-2-induced ARDS.