CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

Background: Liver fibrosis caused by chronic inflammation remains the major driver of various liver diseases. However, limited effective therapies have been identified for liver fibrosis. Herein, we elucidated the complicated molecular mechanisms underlying liver fibrosis. Methods: Primary hepatocytes were co-cultured with JS-1 cells. Inflammatory cytokine levels were assessed by ELISA. Liver fibrosis markers and target molecular levels were detected by western blotting and immunohistochemical staining. Molecular mechanisms were analyzed by Acyl-biotin exchange (ABE) assay, Co-IP, proximity ligation assay, biotin pull-down, and GST pull-down assays. Co-localization and subcellular localization of molecules were observed by immunofluorescent staining. Liver fibrosis was induced by CCl 4 in mice and determined by Masson Trichrome and Sirius Red staining. Liver injury was evaluated by HE staining, serum ALT, and AST levels. Results: High mobility group box 1 (HMGB1) bound to toll-like receptor 4 (TLR4) to facilitate palmitoylation of stimulator of interferon genes (STING), which led to hepatocyte inflammation and JS-1 cell activation in vitro. Furthermore, cysteine-rich secreted protein LCCL domain protein 2 (CRISPLD2) blocked HMGB1/TLR4 axis–mediated palmitoylation of STING, and subsequent liver fibrosis. Mechanistically, CRISPLD2 recruited 78 kDa glucose-regulated protein (GRP78) to trigger TLR4 degradation via an autophagic–lysosomal pathway. CRISPLD2 treatment alleviated CCl 4 -induced inflammation and liver fibrosis in mice via inactivation of the HMGB1/TLR4/STING pathway. Conclusions: CRISPLD2 mitigated hepatocyte inflammatory response and fibrosis via interaction with GRP78 to inactivate the HMGB1/TLR4 pathway and consequently restrain STING palmitoylation.