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

项目摘要/摘要 急性呼吸窘迫综合征(ARDS)于1967年首次正式描述，但它很可能是 几个世纪以来大流行性病毒感染的主要死亡原因。现在，世界正面临着一种新的 来自新冠肺炎的大流行，它已经感染了全球超过1亿人，导致 &gt；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，包括将中性粒细胞染色质释放到细胞外间隙的过程 中性粒细胞胞外陷阱，或称Net。最初被描述为一种灭活病原体的宿主防御形式， Net已经成为对感染的潜在不适应反应，产生了严重的旁观者伤害 凝结到组织中，并作为凝结的病灶。我们已经证明，Net是为了应对这两种情况而产生的 无菌和病原体引起的急性肺损伤(包括甲型流感)，当中和时，肺损伤 在不影响微生物遏制的情况下减少。我们还表明，Net在 ARDS患者的血浆水平，并与更严重的ARDS和ARDS死亡率相关。新兴报告 表明新冠肺炎患者的血液和肺中也存在Net。我们现在的定位是 快速测试网络在应对SARS-CoV-2方面的作用，并开发新的方法来 中和Net用于治疗目的。在目标1中，我们将用SARS-CoV-2病毒或 Spike蛋白决定了Net的产生和Net诱导的分子途径。我们假设 分泌的中性粒细胞蛋白酶和Nets本身将裂解Spike蛋白以增强病毒 发病机制。我们还将利用质谱学对中性粒细胞蛋白质进行无偏见的研究，以 确定SARS-CoV-2引起的中性粒细胞激活的新途径，包括中性粒细胞 分离自新冠肺炎患者。在目标2中，我们将把注意力转移到新冠肺炎的一个小鼠模型上 表达人ACE2的小鼠菌株将受到SARS-CoV-2和肺损伤、Net和 测量的系统性影响。我们假设Net将被产生并主要本地化到 肺泡腔。我们将在网络通路(PAD4-/-，DNase1-)中使用功能突变获得和丢失的小鼠 /-，DNase1L3-/-)，以检测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酶来 将Net中和成无毒的单核小体，可迅速部署到新冠肺炎患者身上。 最后，我们将测试网络相关的血小板激活、补体激活和凝血的作用。 反应，以及减轻这些全身影响的治疗策略。总而言之，这些研究将确立 中性粒细胞激活和Net在新冠肺炎中的决定性作用及识别治疗的新认识 SARS-CoV-2诱导的急性呼吸窘迫综合征的靶向网络探讨

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.