PROJECT SUMMARY During severe COVID-19, immune hyperactivation contributes to tissue injury and worsened patient outcomes. Complement (C’) proteins are believed to centrally mediate COVID-19-related immune hyperactivation. C’ are a conserved set of immune proteins involved in host defense that become activated on various cell types in the lung during severe COVID-19. Upon terminal activation, C’ proteins assemble to form pore-like membrane attack complexes (MACs) that insert into target cell surfaces as transmembrane structures. The presence of MACs in pulmonary tissues strongly correlates with immune hyperactivation, but underlying mechanisms are unknown. The immune effects of MACs have been widely attributed to their cytolytic properties. However, widespread MAC deposition occurs on alveolar endothelial cells (ECs) during severe COVID-19 in the absence of significant EC death or vessel rarefaction. Instead, MAC-bound ECs show signs of EC dysfunction with dysregulated NF-B activation and elaboration of pro-inflammatory cytokines. These patient-level observations suggest immune effects of MACs that are separable from their cytolytic properties. In this application we examine a novel role for intracellular MAC proteins as alarmins. We used a SARS-CoV-2-derived antigen, S protein, to induce non-cytolytic MAC assembly on human ECs. Following assembly on ECs, surface-bound MACs became rapidly internalized and transferred to Rab5+ endosomes. The pool of intracellular, but not extracellular or surface-bound MACs, activated NF-B to induce elaboration of inflammatory cytokines. The intraluminal milieu of Rab5+ endosomes caused a MAC protein, C9, to form insoluble aggregates that stimulated aggrephagy, a specialized form of selective macroautophagy, to activate NF-B. Via proteomic profiling of solubilized MAC complexes (Sc5b-9) in COVID-19 sera, we identified guanylate binding protein 4 (GBP4) as a C9-binding protein regulating aggrephagy and NF-B activity. Based on these exciting preliminary data we propose 2 Specific Aims to explore the hypothesis that intracellular MAC proteins act as alarmins to initiate inflammatory signaling. We will consolidate role(s) for GBP4 as an immune sensor by examining binding interactions with C9 using COVID-19 patient sera. Secondly, we will identify mouse orthologs for human GBP4 that mediate C9 sensing and MAC-induced immune hyperactivation in vivo in response to S protein-induced MAC and following infection by replication competent SARS-CoV-2. Our studies introduce a new paradigm for understanding MAC-related immunity and contribute to our long-term aim of understanding C’-induced inflammation. By doing this, druggable targets ameliorating MAC-induced immune complications of severe COVID-19 may emerge.