Glycogen Synthase Kinase 3 Activity Enhances Liver Inflammation in MASH

Background Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSEC) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a pro-inflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear. Methods: Primary mouse LSEC isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multiomics profiling. Mice with established MASH due to a choline-deficient high-fat diet (CDHFD) or FFC diet were treated with two structurally distinct GSK3 inhibitors [LY2090314, elraglusib (9-ING-41)]. Results: Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phospho-proteome identified GSK3β as the major kinase hub in MASH. GSK3β activating phosphorylation was increased in primary human LSEC treated with the toxic lipid palmitate and human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC and CDHFD-fed mice, respectively. Immunophenotyping of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib using cytometry by time-of-flight showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells. Conclusion: GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and may serve as a potential therapeutic strategy for treating human MASH. Impact and implications Liver sinusoidal endothelial cells under lipotoxic stress in MASH develop a pro-inflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identifies Glycogen Synthase Kinase (GSK)3 as the major driver of LSEC endotheliopathy, examines its pathogenic role in myeloid cell-associated liver inflammation, and defines the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver disease and MASH, as well as those interested in drug development.