PROJECT DESCRIPTION: Hemorrhagic shock (HS) is a significant cause of death in the US and is frequently followed by infection and sepsis. As a result, many patients suffering from HS or HS+sepsis develop acute lung injury (ALI), a life-threatening complication. We have previously discovered that eCIRP, a new DAMP, released after HS or sepsis worsens disease severity and lethality. Our preliminary studies have confirmed elevated levels of eCIRP in the circulation of patients and animals after HS, as well as in the supernatant of macrophages exposed to hypoxia/reoxygenation (H/R). We have also shown that H/R or endotoxin promotes gasdermin D (GSDMD) cleavage into pore-forming subunits. The release of eCIRP after endotoxin was decreased by a GSDMD pore formation inhibitor and in cells from GSDMD knockout mice, implicating an important role of GSDMD in eCIRP’s active release. We have demonstrated that eCIRP activates stimulator of interferon genes (STING) in vitro and in vivo, leading to the upregulation and release of type I interferons (IFNs). Furthermore, we have shown that STING activation is critical to the development of systemic inflammation, tissue injury, ALI, and mortality after HS. We have also demonstrated that eCIRP promotes mitochondrial (mt) DNA fragmentation, thus providing a plausible mechanism for how eCIRP activates STING. In addition, we have shown for the first time that type I IFNs play a pivotal role in the development of ALI after HS. Based on these novel findings, we hypothesize that eCIRP, released through GSDMD pores, activates STING via fragmented mtDNA and increases the release of type I IFNs, ultimately leading to ALI and death after HS or HS+sepsis. Moreover, we have predicted in silico and confirmed in vitro that our newly discovered RNA mimic A12 binds to eCIRP with high affinity, in- hibiting eCIRP’s ability to induce TNFα release from macrophages. A12 had a long half-life in the circulation and reduced inflammation and tissue injury after HS. Therefore, we further hypothesize that eCIRP inhibition with A12 attenuates ALI and mortality in mice subjected to HS or HS+sepsis. In this project, we plan to establish the role of GSDMD pore formation on the active release of eCIRP, determine the detailed mechanism by which eCIRP activates STING, and examine the beneficial effects of the novel eCIRP inhibitor A12 on ALI and survival after HS or HS+sepsis. These studies shall provide novel mechanistic insights into the pathogenesis of ALI and organ injury after HS or HS complicated by sepsis, as well as a new potential therapeutic strategy to treat patients under those conditions.