The Role And Underlying Mechanism Of Macrophage CGAS In Non-alcoholic Steatohepatitis

Objective: Non-alcoholic steatohepatitis(NASH)is an advanced form of fatty liver disease that is a worldwide epidemic and brings a heavy burden to health.However,no effective drugs are currently approved for NASH treatment.Macrophages,which not only secrete inflammatory factors to promote inflammation and fibrosis,but can also timely and effectively remove apoptotic cells through efferocytosis,play a complicated and crucial role in NASH.The underlying mechanisms by which the pro-and anti-inflammation functions of macrophages in NASH development and pathogenesis remain elusive.Cyclic GMP-AMP synthase(c GAS)is a double-stranded DNA sensor of the innate immune system.Previous studies have shown that the activation of the c GASSTING signaling pathway promotes inflammation and contributes to several metabolic diseases.However,the roles of c GAS in macrophages and the pathogenesis of NASH remain to be determined.Methods: We generated systemic and macrophage-specific c GAS knockout mice,and induced NASH or fibrosis in these mice models by feeding the mice with the NASH diet or treating the mice with CCl4.The liver inflammation,lipid deposition,and collagen accumulation were detected by western blot,quantitative PCR,and histopathological staining.Next,resident Kupffer cells and recruited monocytes derived macrophages from the liver of control and macrophage-specific c GAS knockout mice were isolated by fluorescence-activated cell sorting,and RNA sequencing was performed to explore the differentially expressed genes.Next,phagocytosis function in macrophages was determined by in vitro and in vivo phagocytosis assay.Live cell imaging was used to observe the dynamic process of phagocytosis in WT,c GAS-and STING-knockout macrophages.By using yeast two-hybridization and Co-IP experiments,we explored the proteins that regulate phagocytosis and interact with c GAS.Results: In this study,we found both systemic and macrophagespecific c GAS knockout promoted the development of NASH or fibrosis.The RNA-seq results showed that phagocytosis-related genes were significantly down-regulated in macrophages purified from the c GASknockout mice(p < 0.05).Phagocytosis assay verified that knockout of c GAS or STING impaired the phagocytosis function of macrophages.Of note,c GAS knockout delayed the formation of the phagocytic cup and phagosome,while STING knockout had no effect on the early processes but decreased the phagocytotic rate at the later stage.We found c GAS dynamically interacted with phosphatidylinositol 4,5-diphosphate(PIP2)and splenic tyrosine kinase(SYK)on the cell membranes during phagocytosis.Yeast two-hybridization and Co-IP showed the N-terminal of c GAS interacted with SYK.Blocking the interaction between c GAS and PIP2/SYK or diminishing the c GAS membrane location inhibits the SYK activation and impairs the early-stage phagocytotic function of macrophages.Conclusion: Deleting macrophage c GAS aggravates hepatic inflammation,liver injury,and fibrosis.c GAS regulates efferocytosis in STING-dependent and independent manners,which guarantees the timely removal of dead cells and prevents liver fibrosis.This study provides a novel therapeutic potential for targeting macrophage c GAS to treat NASH.