Role of STING in Cholestatic Liver Injury

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Biliary liver diseases, such as primary sclerosing cholangitis (PSC), are characterized by the damage and proliferation of cholangiocytes that are a key link between biliary injury and the subepithelial fibrosis, manifesting chronic hepatobiliary injury that represents a major clinical challenge. Whereas biliary senescence and inflammatory damage are crucial to the pathogenesis of biliary liver injury, there are significant gaps in understanding the precise mechanisms of ductular reaction and liver fibrosis. Recent evidence demonstrates stimulator of interferon genes (STING) as a mediator that promotes macrophage-stimulated liver inflammation and fibrosis. In various cell types, STING is activated by cyclic GMP-AMP (cGAMP), whose synthesis is catalyzed by cGAMP synthase (cGAS) in response to the aberrant cytosolic presence of double-stranded DNA (dsDNA), including mitochondrial DNA (mtDNA). During the preliminary studies, we obtained exciting data suggesting the following novel findings: 1) STING expression is increased in cholangiocytes, liver macrophages, and hepatic stellate cells (HSCs) in livers of PSC patients; 2) cholangiocyte- and/or myeloid cell-specific STING disruption alleviates ductular reaction and liver fibrosis during cholestasis; 3) while mtDNA is increased in cholangiocytes from cholestatic mice, extracellular vesicles (EVs) isolated from human PSC cholangiocytes promote macrophage activation in a manner involving STING; and 4) STING-driven macrophage factors increase cholangiocyte expression of genes related to senescence-associated secretory phonotype (SASP) and fibrosis. These findings point to a critical role for STING in regulating cholangiocyte damage and dysfunctional cell-cell crosstalk during biliary liver injury. Based on these findings, we hypothesize that during cholestatic liver injury, STING expression/activation is stimulated in cholangiocytes, liver macrophages, and HSCs through autocrine and paracrine manners via EVs containing mtDNA cargo, which in turn results in dysfunctional cell- cell crosstalk to enhance cholangiocyte SASP, macrophage/HSC activation, and consequent ductular reaction and liver fibrosis. To test the central hypothesis, two Specific Aims will be persued. For Aim 1, experiments involving novel mouse models have been proposed to determine the extent to which cholangiocyte-specific STING disruption alleviates cholestatic-induced ductular reaction and liver fibrosis. Also, cell experiments will be performed to determine the extent to which STING-driven cholangiocyte factors promote macrophage proinflammatory activation and HSC fibrogenic activation. For Aim 2, experiments have been designed to determine the extent to which myeloid cell-specific STING disruption alleviates cholestasis-induced ductular reaction, cholangiocyte SASP, and liver fibrosis in mice. Also, cell experiments will be performed to evaluate the extent to which STING-driven macrophage factors promote cholangiocyte SASP and HSC fibrogenic activation. Successful completion of this project will fill knowledge gaps in biliary liver injury and provide the experimental basis for innovative therapeutic strategies based on STING inhibition.