PROJECT SUMMARY/ABSTRACT Controlling inflammation and cellular damage is key to preventing and treating multiple organ failure (MOF) following trauma. However, many of the mechanisms that regulate inflammation and cell death in specific organs remain unknown. So despite advances in supportive care for MOF patients, there have been few advances in MOF treatments, or in our ability to adequately prevent MOF onset. Our overarching goal is to ultimately develop new therapeutics for trauma patients based on regulating inflammatory responses and effects on cell death following trauma and hemorrhagic shock with resuscitation (HS/R). In this proposal we will continue to investigate downstream cell and tissue-specific effects of caspase-4 (human)/11 (mouse) activation after HS/R. We will also assess the ability of a novel caspase-4/11 inhibitor to reduce HS/R- mediated effects on systemic inflammation, cell death and end-organ injury. Understanding the multiple effects of caspase-11 on inflammation and organ damage will enable us to assess the translational potential of targeted caspase-11-inhibition for improved patient outcomes after trauma and hemorrhage. The most recent work on this grant defined a novel mechanism of caspase-4/11 activation (non- canonical inflammasome) in trauma/HS/R. Caspase-4/11 is the intracellular receptor for lipopolysaccharide (LPS) and is an important mediator of inflammation during infection and sepsis. Our work showed activation of caspase-4/11 in non-LPS-driven models of inflammation, and we defined a novel mechanism of activation of caspase-11 by endogenous oxidized cardiolipin (CLox) on the outside of stressed mitochondria. CLox is regulated by cytochrome c, and specific inhibition of cytochrome c inhibits caspase-11 activation and is highly organ protective in a mouse model of HS/R. However, the mechanistic basis of detrimental effects of caspase- 11 activation in HS/R are not clear, and may be multifactorial given its multiple cell-specific inflammatory effects, including induction of pyroptosis (inflammatory cell death), active release of inflammatory HMGB1 in extracellular vesicles (EV) from hepatocytes after HS/R, and more recently-described effects on coagulation through enhanced binding of tissue factor (TF) on endothelial cells (EC) in sepsis. Therefore, our main hypothesis is that caspase-4/11 activation is detrimental during HS/R through multiple cell-specific effects. In this proposal we will: 1: determine the role of caspase-4/11-mediated inflammation on organ injury during HS/R; 2: determine the effects of caspase-4/11-activation on coagulation and organ injury during HS/R; 3: determine safety and effectiveness of caspase-11-specific inhibition in mouse models of HS/R. We expect to show specific inhibition of caspase-4/11 in HS/R is organ protective and the mechanisms of protection are multi-factorial. We also expect to show inhibition of caspase-4/11 is an attractive therapeutic target to reduce trauma-ind...