Mechanisms of immunopathology of COVID-19/ARDS, and strategies to mitigate detrimental inflammatory responses

SARS-CoV-2的出现迅速成为全球大流行，需要了解疾病的机制，并开发疫苗和治疗方法。这些努力受到以下事实的阻碍，即在这些努力中最有用的实验模型，小鼠，由于用于病毒进入的细胞受体ACE 2的不相容序列而不易于感染。因此，我们已经开始了两项主要的努力来开发小鼠模型，这些模型可以用于理解与严重病毒性肺炎相关的先天性和适应性免疫失调。最终目标是定义这些过程，因为它们与SARS-CoV-2相关，以确定可以治疗的炎症反应的干预点。 第一个主要举措是开发严重SARS-CoV-2感染的小鼠模型。为了实现这一目标，我们与杰克逊实验室合作，对表达人源化ACE 2基因的小鼠进行基因工程改造，使病毒能够在组织中复制。我们目前正在测试4种新的小鼠模型，这些模型反映了在内源基因座或作为转基因使ACE 2人源化的不同策略。我们还测试了小鼠背景的易感性，包括协作交叉的8只创始小鼠，我们的初步数据表明，不同的小鼠品系可以反映人类疾病的某些方面，包括男性偏见和血栓形成的并发症。终极目标将是充分表征宿主反应，因为它们与这些模型中的病理学有关，然后利用这些模型来测试阻断炎症级联中各种事件的生物制剂。 第二项重大举措是采用致命的流感感染作为严重病毒性肺炎的模型，可用作更好地了解其他病毒性肺部感染（如SARS-Cov 2引起的COVID-19）的试验平台。正在进行的研究包括（i）在可能具有临床实用性的致命流感模型中测试干预措施，以及（ii）旨在更好地理解组织损伤的潜在机制以及为什么限制病毒复制或先天免疫的干预措施通常在感染早期但在宿主死亡之前失败的细胞和分子研究。使用绕过早期鼻咽复制并导致快速肺部深部感染的严重流感感染模型，我们发现只有非常早期的抗病毒治疗（磷酸奥司他韦-达菲）才能预防感染动物的死亡。没有其他药物或抗炎治疗明显改变了疾病的进程，认为要么涉及多种破坏活动，仅钝化一种不足以产生临床效果，要么早期发生不可逆的组织损伤，一旦发生，干扰病毒复制或宿主免疫力不会在肺功能丧失中发挥主要作用。目前的工作利用淋巴细胞生物学部分，LISB，NIAID，NIH开发的高度多重成像方法，在干预影响死亡率的关键窗口期间定量探测肺中关键细胞和结构的状态，以确定可能的损伤部位，同时正在测试涉及抗病毒和抗免疫药物配对的治疗策略的协同作用。

The emergence of SARS-CoV-2 rapidly became a global pandemic necessitating the need to understand the mechanisms of disease, and develop vaccines and therapeutics. These efforts are hampered by the fact that the most useful experimental model in these efforts, the mouse, is not susceptible to infection due to an incompatible sequence of the cellular receptor for virus entry, ACE2. Therefore we have initiated two major efforts to develop mouse models that can be utilized in the efforts to understand dysregulation of innate and adaptive immunity associated with severe viral pneumonia. The ultimate goal is to define these processes as they relate to SARS-CoV-2 to identify points of intervention of inflammatory responses that can be targeted therapeutically. The first major initiative is to develop mouse models of severe SARS-CoV-2 infection. To achieve this, we have partnered with Jackson Laboratories to genetically engineer mice that express a humanized ACE2 gene to enable virus replication in tissues. We are currently testing 4 novel mouse models that reflect different strategies to humanize ACE2 at the endogenous locus, or as a transgene. We are also testing mouse backgrounds for susceptibility, including the 8 founder mice of the Collaborative Cross, and our preliminary data suggests that different mouse strains can reflect some aspects of human disease, including a male bias and complications from thrombosis. The ulimate goal will be to fully characterize the host responses as they relate to pathology in these models, and then utilize the models for testing biologics that block various events in inflammatory cascades. The second major initiative is the employment of a lethal influenza infection as a model for severe viral pneumonia that can be used as a test bed for better understanding other viral pulmonary infections such COVID-19 caused by SARS-Cov2. Ongoing studies involve (i) tests of interventions in the lethal influenza model that might have clinical utility and (ii) cell and molecular studies aimed at better understanding the underlying mechanism(s) of tissue damage and why interventions that constrain viral replication or innate immunity often fail after an early point in infection but well before death of the host. Using a severe influenza infection model that bypasses early nasopharyngeal replication and leads to rapid deep lung infection, we found that only very early treatment with the anti-viral (oseltamivir phosphate - Tamiflu) could prevent death of the infected animals. No other drug or anti-inflammatory treatments tested altered the course of disease appreciably, arguing that either multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, or that irreversible tissue damage occurs early and once this occurs, interfering with viral replication or host immunity does not play a major role in loss of pulmonary function. Current work utilizes the highly multiplex imaging methods developed in the Lymphocyte Biology Section, LISB, NIAID, NIH to quantitatively probe the state of key cells and structures in the lung during the critical window in which intervention affects death rates to identify possible sites of damage, while treatment strategies involving pairing of anti-virial and anti-immune drugs are being tested for synergy.