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

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.