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，但它可能是一种常见的呼吸道疾病。 几个世纪以来大流行性病毒感染的主要死亡原因。现在，世界正面临着一个新的 SARS-CoV-2或COVID-19的大流行，已感染全球1亿多人， 超过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 激活，包括中性粒细胞染色质释放到细胞外空间的过程称为 中性粒细胞胞外陷阱或NET。最初被描述为宿主防御病原体的一种形式， NET已经成为一种潜在的对感染的适应不良反应，产生大量的旁观者损伤 并作为凝固的病灶。我们已经表明，NET是响应于 无菌和病原体诱导的急性肺损伤（包括甲型流感），中和后， 在不影响微生物控制的情况下减少。我们还表明，NET在 与ARDS严重程度及病死率相关。新报告 表明NET也存在于COVID-19患者的血液和肺部。我们现在可以 快速测试NET在响应SARS-CoV-2中的作用，并开发新的方法， 中和NET用于治疗目的。在目标1中，我们将用SARS-CoV-2病毒攻击中性粒细胞， 刺突蛋白来确定NET的产生和NET诱导的分子途径。我们假设 分泌的嗜中性粒细胞蛋白酶和NET本身将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无偏研究， 确定由SARS-CoV-2引起的中性粒细胞激活的新途径，包括中性粒细胞 分离自COVID-19患者。在目标2中，我们将把注意力转向COVID-19的小鼠模型， 表达人ACE 2的小鼠品系将用SARS-CoV-2和肺损伤、NET进行攻击， 测量系统效应。我们假设，NET将产生并主要定位于 肺泡腔我们将使用在NET途径中具有获得和丧失功能突变的小鼠（PAD 4-/-，DNase 1-/-， /-，DNase 1 L3-/-）来测试它们在该模型中的致病性。在这些研究中，我们还将测试一种新的DNA酶， 将NET中和成无毒的单核细胞体，可以迅速部署到COVID-19患者身上。 最后，我们将测试NET相关的血小板活化、补体活化和凝血的作用。 反应和治疗策略，以减轻这些全身性影响。总之，这些研究将建立 关于中性粒细胞活化的新知识以及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.