Project Summary/Abstract: The pathologic hallmarks of systemic lupus erythematosus (SLE or lupus) are altered immune responses to nuclear autoantigens with autoantibody production and subsequent tissue injury. Studies have suggested a pathogenic role for innate immunity in lupus. Plasmacytoid dendritic cells and monocytes (MO) of innate immunity recognize ssRNA of the self antigen U1-small nuclear ribonucleoprotein (U1-snRNP) and dsDNA via TLRs 7/8 and 9, respectively, leading to the production of IFN-α, IL-1β and IL-18 which are linked to lupus pathogenesis and clinical manifestations. Regulated cell death can occur in distinct forms including pyroptosis and necroptosis. Pyroptosis and necroptosis cause inflammation by releasing inflammatory molecules such as cytokines, chemokines, and damage-associated molecular patterns (DAMPs). Pyroptosis is induced by the activation of the caspase-1-containing inflammasomes that cleave pro-IL-1β and - IL18 into IL-1β and IL-18, as well as gasdermin D (GASDMD) into the active form gasdermin D amino-terminal fragment (GASDMDN), the essential molecule for membrane pore formation in pyroptosis. Necroptosis that occurs independently of caspases is regulated by TNF-α, receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), and downstream substrate pseudokinase mixed-lineage kinase domain-like (MLKL), the critical molecule for membrane disruption with the release of inflammatory molecules in necroptosis. RIPK3 and MLKL can also activate the NLRP3 inflammasome with IL-1β release, raising the possible interface of pyroptosis and necroptosis. Studies, including our own, support the implication of the inflammasome activation, pyroptosis, and necroptosis in lupus. We showed IL-1β and IL-18 production from human MO exposed to lupus U1-snRNP/anti- U1-snRNP antibody (Ab) or dsDNA/anti-dsDNA Ab immune complex (snRNP IC or dsDNA IC, also lupus IC refers to both) through the NLRP3 inflammasome activation which is enhanced by the cytokine macrophage migration inhibitory factor (MIF). However, there is a substantial scientific gap in our understanding of how pyroptic and necroptic pathways become activated, interface, and synergize to promote inflammation in lupus at molecular levels with contributing to unique clinical manifestations. Thus, we will test the hypotheses that lupus IC activate pyroptic and necroptic pathways in MO with synergistic interface in promoting inflammation and tissue injury and that the activation of such pathways alters in lupus contributing to clinicopathologic manifestations. The goal of the proposal is to address this hypothesis with: 1) Aim 1. Elucidate the mechanism and significance of the activation and interface of pyroptic and necroptic molecules in lupus; 2) Aim 2. Elucidate the “functional” alteration in monocytes that enhances pyroptosis- and necroptosis-mediated inflammation in lupus patients; and 3) Aim 3. Elucidate the role of macrophage migration inhibitory factor in promoting in...