MAC formation and Cell-Specific Inflammasome Activation in COVID-19 - Program summary: The objective of this training proposal is to determine how enhanced complement activation and increased membrane attack complex (MAC) formation exacerbate severe COVID-19. The complement system, a key component of the innate immunity, can be initiated by three pathways (lectin, alternative, and classical), potentially leading to MAC formation. MAC's primary function is to lyse pathogens via pore formation. However, clinical evidence indicates that complement activation and MAC formation, while normally protective, can be detrimental in SARS-CoV-2 infection and contribute to COVID-19 pathogenesis. Currently, the cellular and molecular mechanisms underlying MAC-accelerated severe COVID-19 remain unexplored. To address this question, we have characterized a murine model of severe COVID-19 using a mouse-adapted strain (MA30) that recapitulates key aspects of SARS-CoV2 infection, including lung edema and epithelial cell tropism. Using this MA30 strain to infect an array of complement deficient mice (C3-/- and C7-/-) and mice deficient in the critical MAC regulator CD59 (mCd59-/-), I demonstrated a causative role of MAC on severe COVID-19. However, the cell targets and molecular mechanism of MAC-mediated severe COVID-19 remain unknown. Our preliminary data show increased activity of Caspase-1, a downstream event of NLRP3 inflammasome formation, in LPS- challenged mCD59-/- mice. Additionally, NLRP3 inflammasome formation and Caspase-1 activation, leading to the release of IL-1β and IL-18, contributes to the cytokine storm and severe immunopathology in COVID-19. Our single-cell and bulk RNA sequencing data show increased transcription of the casp-1, IL-18 and Gsdmd in endothelial cells and macrophages of MA30 infected mice at 2 days-post infection. Therefore, my overarching hypothesis is that MAC targets endothelial cells and macrophages, accelerating severe COVID-19 through the activation of Caspase-1 pathway. To test this central hypothesis, I propose the following aims. Aim 1 is to determine the pathogenic role of MAC-mediated Casp1 activation in severe COVID-19. Aim 2 is to determine the MAC-mediated cellular effects on COVID-19 and associate the disease severity with cell specific casp1 activation. With our prior work we established a causative role for MAC in severe COVID-19 pathogenesis. This proposal advances that work by determining the cellular and molecular mechanisms by which MAC causes severe COVID-19. By elucidating the role of the MAC/Casp1 axis in specific cell types during severe COVID-19, this research will further our understanding of the disease's immunopathology, and better prepare us for new SARS variants and emerging diseases. A better understanding is crucially needed to identify novel therapeutic targets for mitigating cytokine storm and improving outcomes for critically ill patients.
