Summary/Abstract Pancreatitis is a potentially fatal disease of exocrine pancreas, with obscure pathogenesis and no specific or efficient treatment available. Excessive/unresolving inflammation is a major determinant of pancreatitis severity but its mechanisms are poorly understood. Recent findings in experimental and genetic models have uncovered key pathogenic roles of impaired autophagy and mitochondrial dysfunction in acinar cell damage driving pancreatitis; disordering of these pathways is also prominent in human disease. Blockade/impairment of pancreatic autophagy initiates inflammation in genetic models, such as pancreas-specific knockout of the key autophagy mediator ATG5. Conversely, enhancing autophagic efficiency (or normalizing mitochondrial function) ameliorates the inflammatory response in experimental pancreatitis. These findings strongly indicate that normal autophagy restricts inflammation in pancreatitis; however, the underlying mechanisms are unknown. We hypothesize that a key mechanism linking impaired autophagy to inflammation in pancreatitis is acinar cell mitochondrial dysfunction; and further, that this mechanism is mediated by the increase in acinar cell mitochondrial (mt)ROS and mtDNA release. We posit that these effectors activate major proinflammatory pathways: NF-κB, triggering the expression of cytokines/chemokines in acinar cells and thus initiating the inflammatory response; the DNA-sensing cGAS-STING-TBK1 pathway in macrophages, resulting in production of type I IFNs; and inflammasome activation in macrophages, resulting in massive production of IL-1β and IL- 18. The proposed studies will use genetic and pharmacologic approaches to elucidate the roles of these pathways. The hypothesis will be tested in 3 Specific Aims: 1). Determine the effects of impaired autophagy on mitochondrial dysfunction and pancreatic inflammation in genetic and experimental models of pancreatitis. 2). Investigate macrophage cGAS-STING-TBK1 pathway activation caused by impaired autophagy in genetic and experimental models of pancreatitis. 3). Investigate the role of (canonical) inflammasome pathway in macrophage activation caused by impaired autophagy and mitochondrial dysfunction. Our proposed studies will determine the mechanisms linking impaired autophagy and mitochondrial dysfunction to macrophage-driven inflammatory response of pancreatitis. The studies will identify molecular targets in these pathways that could be amenable for pharmacologic intervention to alleviate the inflammatory response of pancreatitis.