Project 1: Mechanisms of innate sensing and pathogenesis of SARS-CoV-2

了解导致严重COVID-19的免疫反应对于识别可能感染的患者至关重要

Understanding immune responses that contribute to severe COVID-19 is essential to identify patients likely to become critically ill and to discern which pathways to target for therapeutic intervention. The inflammasome is an antiviral and proinflammatory pathway activated by many viruses, but its role in COVID-19 has not been defined fully. Inflammasome activation results in an inflammatory type of cell death called pyroptosis as well as the release of proinflammatory cytokines that include interleukin (IL)-18. Inflammasome cytokines are central to viral control, but excessive or prolonged activation enhances pathogenesis of numerous respiratory virus infections, including avian influenza and SARS-CoV-1. Having extensively studied the role of inflammasome cytokines in the pathogenesis of multiple other viral infections, we measured IL-18 and 36 other cytokines and chemokines in plasma from patients with COVID-19. While most were not significantly different in COVID-19, IL-18 and IL-1 receptor antagonist (RA) levels are elevated in intubated patients with COVID-19 versus non- intubated COVID-19 and hospitalized influenza patients. Incubation of human macrophages with SARS-CoV-2 in vitro produced IL-18, IL-1, IL-RA, IL-6, and IL-8. We used a human macrophage cell-line with various inflammasome genes disrupted to show that caspase-1 and NLRP3 are required for SARS-CoV-2 activation of the inflammasome. We will establish the mechanism by which SARS-CoV-2 activates the inflammasome and determine how inhibition of this pathway alters innate immune signaling using a panel of endocytosis inhibitors, macrophage cell lines with specific inflammasome and other innate sensing genes disrupted, and specific inhibitors of innate sensing in primary human macrophages. Early investigations suggest that antibodies (Abs) modulate innate sensing of SARS-CoV-2. To test whether Abs produced during COVID-19 alter innate signaling, we will incubate SARS-CoV-2 with monoclonal Abs or patient sera, inoculate primary or immortalized macrophages, and measure supernatant cytokines. To investigate cellular function, we developed a flow cytometry-based platform that enables single cell analysis of traditional cell surface markers combined with intracellular staining for proteins involved in metabolic programming. Using this platform, we identified myeloid derived suppressor cells (MDSCs) with distinct metabolic profiles that correlated with severe COVID-19. Prolonged inflammation induces MDSCs in cancer, obesity, and chronic infections. We will use single cell RNA sequencing to characterize these novel MDSCs and assess how cytokines produced in COVID-19 regulate MDSC metabolic programming. The overall goal is to define the mechanism by which SARS-CoV-2 activates inflammatory pathways, Ab modulation, the role of MDSCs, and how they intersect to mediate SARS-CoV-2 immune control and pathology. This will identify targets for therapeutic intervention that minimize inflammatory pathology without impairing antiviral immunity as the foundation of novel clinical trials and markers of disease progression that allow targeting resources to patients most likely to experience severe disease.