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

项目总结 这一应用直接源于我们最近在《自然》杂志上发表的文章，在该文章中，我们分析了支气管肺泡 流式细胞术联合检测88例重症SARS-CoV-2肺炎患者的BAL液 流式分选的肺泡巨噬细胞的批量转录图谱，在一些患者中，单细胞RNA- 测序。我们将这些数据与从211名肺炎患者收集的类似样本进行了比较。 在大流行前和大流行期间次于其他病原体，目的是确定独特的病理生物学 SARS-CoV-2肺炎的特点。使用这些数据，我们产生了SARS-CoV-2引起的假设 一种缓慢展开的空间受限的肺泡炎，其中携带SARS-CoV-2的肺泡巨噬细胞和 交叉反应的T细胞形成一个正反馈循环，推动进行性肺泡炎。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。 目的1.探讨肺泡巨噬细胞感染与交叉反应性记忆T细胞激活之间的关系 SARS-CoV-2肺炎患者的细胞驱动肺泡巨噬细胞/T细胞回路。我们将研究 交叉反应抗体在介导SARS-CoV-2感染肺泡巨噬细胞中的作用我们还将 检测识别SARS-CoV-2和其他冠状病毒的交叉反应记忆T细胞在 在T细胞和感染的肺泡巨噬细胞之间建立和维持正反馈环 这些信号环在启动持续性肺和全身炎症中的作用。 目的2.确定SARS-CoV-2囊膜蛋白激活钙通道是否 是激活人肺泡巨噬细胞IL-1β所必需的。我们会感染人的肺泡 SARS-CoV-2变异病毒感染的巨噬细胞膜蛋白和钙通道活性缺失 测定其体外培养人肺泡巨噬细胞产生IL-1β的能力。 目的3.确定CRAC通道激活的药物抑制剂是否可以减弱 肺泡炎患者重症SARS-CoV-2肺炎感染间电路中断 肺泡巨噬细胞和交叉反应T细胞。我们正在积极地进行临床试验，以确定 应用BAL液序贯分析研究CRAC通道激活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。我们将使用新颖的数据 整合这项研究产生的临床和基因组数据的科学方法。 我们已经组建了一支独特的研究团队，他们在肺部免疫学、临床试验、钙离子 频道和病毒学。我们的研究将探索观察到的疾病严重程度变化的可能原因 SARS-CoV-2感染后，提供机制证明CRAC通道抑制剂既抗炎 和可能的抗病毒治疗药物治疗严重的SARS-CoV-2肺炎，并提供了一个框架 用于使用从支气管肺泡灌洗液中提取的生物标志物设计的未来临床试验。

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.