Targeting abnormal alveolar immune activation and failed epithelial repair in COVID-19

项目摘要 这一应用直接源于我们最近在《自然》杂志上发表的文章， 采用流式细胞术， 流式细胞分选肺泡巨噬细胞的大量转录组学分析，在一些患者中，单细胞RNA- 测序我们将这些数据与从211名肺炎患者中收集的类似样本进行了比较 在大流行之前和期间继发于其他病原体，目的是确定独特的病理生物学 SARS-CoV-2肺炎的特征利用这些数据，我们提出了SARS-CoV-2导致 一种缓慢展开的、空间受限的肺泡炎，其中肺泡巨噬细胞携带SARS-CoV-2和 交叉反应性T细胞形成正反馈回路，其驱动进行性肺泡炎症。我们 在三个相互关联的具体目标中解决这一假设的关键问题。 目标1.为了确定肺泡巨噬细胞感染是否激活了交叉反应记忆T细胞， 细胞驱动SARS-CoV-2肺炎患者的肺泡巨噬细胞/T细胞回路。我们将研究 交叉反应抗体在介导肺泡巨噬细胞感染SARS-CoV-2中的作用我们还将 研究识别SARS-CoV-2和其他冠状病毒的交叉反应记忆T细胞在 在T细胞和受感染的肺泡巨噬细胞之间建立和维持正反馈回路， 这些信号环在引发持续性肺部和全身炎症中的作用。 目标2.为了确定SARS-CoV-2的包膜蛋白激活的钙通道是否是 人肺泡巨噬细胞中IL-1 β的活化所必需的。我们会感染人类的肺泡 巨噬细胞与突变的SARS-CoV-2病毒缺乏钙通道活性的包膜蛋白， 体外检测其对人肺泡巨噬细胞产生IL-1 β的影响。 目标3.确定CRAC通道激活的药理学抑制剂是否可以减弱 严重SARS-CoV-2肺炎患者的肺泡炎通过破坏感染者之间的回路 肺泡巨噬细胞和交叉反应性T细胞。我们正在积极参与一项临床试验，以确定 使用BAL液的连续分析的CRAC通道激活的小分子抑制剂的生物学效应 收集自需要机械通气的SARS-CoV-2肺炎患者。我们将使用新的数据 科学方法来整合本研究产生的临床和基因组数据。 我们组建了一个独特的研究小组，他们在肺免疫学、临床试验、钙离子代谢、 渠道和病毒学。我们的研究将探讨观察到的疾病严重程度变异的可能原因 在SARS-CoV-2感染后，提供机制证明CRAC通道抑制剂既具有抗炎作用， 和可能的抗病毒治疗严重SARS-CoV-2肺炎患者，并提供了一个框架， 用于未来的临床试验，设计使用来自支气管肺泡灌洗液的生物标志物。

PROJECT SUMMARY This application directly stems from our recent publication in Nature in which we analyzed bronchoalveolar lavage (BAL) fluid from 88 patients with critical SARS-CoV-2 pneumonia using a combination of flow cytometry, bulk transcriptomic profiling of flow sorted alveolar macrophages and, in some patients, single cell RNA- sequencing. We compared these data with analogous samples collected from 211 patients with pneumonia secondary to other pathogens before and during the pandemic with a goal of identifying unique pathobiologic features of SARS-CoV-2 pneumonia. Using these data, we generated the hypothesis that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 and cross-reactive T cells form a positive feedback loop that drives progressive alveolar inflammation. We address key questions from this hypothesis in three interrelated Specific Aims. Aim 1. To determine whether alveolar macrophage infection and activation of cross-reactive memory T cells drive alveolar macrophage/T cells circuits in patients with SARS-CoV-2 pneumonia. We will examine the role of cross-reactive antibodies in mediating alveolar macrophage infection with SARS-CoV-2. We will also examine the role of cross-reactive memory T cells recognizing SARS-CoV-2 and other coronaviruses in establishing and maintaining positive feedback loops between T cells and infected alveolar macrophages and the role of these signaling loops in initiating persistent lung and systemic inflammation. Aim 2. To determine whether calcium channel activation by the envelope protein of SARS-CoV-2 is necessary for activation of IL-1β in human alveolar macrophages. We will infect human alveolar macrophages with mutant SARS-CoV-2 viruses lacking calcium channel activity in the envelope protein and measure their generation of IL-1β in human alveolar macrophage in vitro. Aim 3. To determine whether a pharmacologic inhibitor of CRAC channel activation can attenuate alveolitis in patients with severe SARS-CoV-2 pneumonia by disrupting circuits between infected alveolar macrophages and cross-reactive T cells. We are actively enrolling in a clinical trial to determine the biologic effects of a small molecule inhibitor of CRAC channel activation using sequential analysis of BAL fluid collected from patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. We will use novel data science approaches to integrate the clinical and genomic data generated from this study. We have assembled a unique group of investigators with expertise in lung immunology, clinical trials, calcium channels and virology. Our studies will explore possible reasons for the observed variability in disease severity after SARS-CoV-2 infection, offer mechanisms to credential CRAC channel inhibitors as both anti-inflammatory and possible antiviral therapeutics in patients with severe SARS-CoV-2 pneumonia, and provide a framework for future clinical trials designed using biomarkers derived from bronchoalveolar lavage fluid.