PROJECT SUMMARY Regulation of Innate Immunity and Inflammation Through Nuclear Reprogramming We propose to advance our mechanistic studies of innate immunity and inflammation in severe infections that evolve into their end stage, septic shock, thereby threatening the lives of wounded soldiers on the battlefield, our Veterans, and the civilian population. Those Veterans (and non-Veteran patients) between the ages of 65 to 85 are particularly prone to septic shock with a 45% mortality rate while many survivors suffer debilitating cognitive decline. We are pursuing our long-term goal to decode innate immunity and inflammation with a new class of cell-penetrating Nuclear Transport Modifiers (NTMs) that reprogram the inflammatory regulome. One of these anti-inflammatory peptides achieved an unprecedented enhancement of innate immunity-mediated bacterial clearance in the lungs (700-fold), spleen (300-fold), and blood (200-fold), all taking place before antimicrobial therapy. Concurrently, it improved the blood markers of microvascular endothelial injury, including thrombocytopenia. When combined with anti-microbial therapy, NTM increased the survival rate to 55%, compared to a 30% survival rate in the antibiotic-only group. This NTM peptide, cSN50.1, simultaneously targets two nuclear transport shuttles, importins α5 and β1. To unravel the mechanism of innate immunity and inflammation that underlie microbial and metabolic disorders, we designed, produced, and tested two novel mono-selective NTMs: (i) Importin α5-selective cSN50.1α, and (ii) Importin β1-selective cSN50.1β. Strikingly, the importin α5-selective peptide produced an 80% survival rate in endotoxin shock induced by lipopolysaccharide (LPS), one of the most potent virulence factors of Gram-negative bacteria responsible for two thirds of septic shock cases. This new NTM also effectively controlled the Western Diet-induced hypersensitivity to lethal endotoxin shock while preventing liver glycogenolysis, which causes hyperglycemia and hypertriglyceridemia linked to the nuclear transport of Carbohydrate Responsive Elements-Binding Proteins (CHREBPs). In turn, the Imp β1-selective peptide suppressed lipogenesis mediated by the nuclear transport of the Sterol Regulatory Element-Binding Proteins (SREBPs) that control over 30 genes involved in the production of triglycerides, cholesterol, and fatty acids, a hallmark of metabolic syndrome. We hypothesize that the proposed use of selective NTMs with antimicrobial agent(s) would untangle multiorgan damage and mortality in severe infections. Furthermore, we posit that the Importin α5/SRTFs/CHREBPs and Imp β1/SREBPs pathways are interdependent, thereby predisposing individuals with metabolic syndrome to hyperacute responses to severe infections. Hence, in Aim 1, we will investigate the mechanism of the NTM’s beneficial action in polymicrobial peritonitis (i.e. the striking enhancement of the innate immunity-mediated clearance of bacteria in the lungs, spl...