PROJECT SUMMARY Chronic inflammation is a major cause of cancer, diabetes, and neurodegeneration. Inflammation is a complex topic, with both positive and negative effects on disease and therapy, and if we are to understand the pathological results and therapeutic benefits we need to better understand the mechanisms of where inflammation originates. The big picture in which this proposal is set is to understand the mechanisms of inflammation as they relate to regulated cell death. This application seeks to substantially advance current approaches, concepts, and technology to delve deeply into the inflammatory consequences of cell death and the connections between the cell death mechanisms. We combine biochemistry, cell biology, genome editing, non-animal models of inflammation, protein engineering, and chemical biology to explore linked pro- and anti- inflammatory cell death mechanisms. We propose that a robust and quantitative approach to defining inflammation is an important innovative quality of this proposal. The main goals of this proposal are to 1) compute the difference in inflammatory mediators released as a result of apoptosis, pyroptosis and necroptosis in macrophages, 2) define the mechanism of cytokine release from cells undergoing inflammatory cell death, and 3) understand the activation mechanisms of caspases in pyroptosis, and synthesize highly selective fluorescent probes to allow for direct visualization of specific caspase activation.