Neutrophil Heterogeneity and Immunopathogenesis of COVID-19 ARDS

Project Summary Coronavirus disease 2019 (COVID-19) is a potentially life-threatening disease caused by the novel viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of the 10%-20% of COVID-19 patients hospitalized, approximately 1/3 develop acute respiratory distress syndrome (ARDS). COVID-19- induced ARDS results from a combination of virally induced lung injury and the rapid influx of immune cells that release inflammatory mediators leading to a hyper-activated state known as cytokine storm. Understanding the pathophysiology of COVID-19 ARDS is critical to finding effective therapeutic interventions. Accumulating evidence indicates critical roles for neutrophils in both ARDS and immunothrombosis in COVID-19. Multi-omics studies identified dramatic neutrophil heterogeneity in COVID-19, including emergence of a large number of low density neutrophils (LDN). We reported a novel population of LDN present in both the circulation and lungs of severe COVID-19 patients which expresses intermediate levels of CD16 (CD16Int LDN). CD16Int LDN spontaneously form neutrophil extracellular traps (NETs), activate platelets in vivo and in vitro, and have heightened degranulation. Our preliminary data show that LDN subsets from severe COVID-19 patients are functionally distinct from mature, normal density neutrophils (NDN). Based on these findings, we postulate that differential functional responses of heterogeneous neutrophil populations collaborate to induce systemic and pulmonary inflammation leading to ARDS in COVID-19 patients. Three specific Aims are proposed to further dissect the underlying mechanisms. Aim 1 will comprehensively characterize neutrophil subsets from severe/critical COVID-19 patients using proteomics and transcriptomics approaches. The information gained from those studies will be used to refine our CyTOF antibody panel and prospectively measure dynamic changes of neutrophil subsets over the disease course in COVID-19 patients. Aim 2 will determine neutrophil subset functional changes during disease progression and their contributions to dysregulated inflammatory response and coagulopathy in severe/critical COVID-19 patients. Neutrophil degranulation, NET formation, phagocytosis, chemotaxis, T cell suppressive activity, and cytokine/chemokine release will be examined. We will also determine if LDN subsets and NDN or their derived products promote coagulopathy in COVID-19 patients. Aim 3 will determine the relative contributions of neutrophil host defense systems to development of acute lung injury. We will also use a hACE2 Tg mouse model to determine the roles of neutrophil respiratory burst, granule exocytosis, and NET formation to SARS-CoV-2-induced acute lung inflammation. Successful completion of this proposal will provide novel insights into COVID-19-induced ARDS pathophysiology by defining the functional roles of different neutrophil subsets and by establishing which host defense systems of neutrophils as a therapeutic target for inhibiting inflammatory lung injury and immunothrombosis in COVID-19.