Effect of cholesterol  overload on gene expression of hepatic macrophages isolated from normal and NASH livers.
Hepatocyte death plays a critical role in the disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH). We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated free cholesterol was accumulated in macrophages around dead hepatocytes containing cholesterol crystals. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH. Overall design: Hepatic macrophages were isolated from normal livers of wild-type mice fed a standard diet or steatotic livers of MC4R-KO mice fed a Western diet for 6 to 10 weeks using anti-F4/80 antibodies and magnetic separation columns. Macrophages were treated with cholesterol crystals at a dose of 500 µg/ml for 24 hours. RNA sequencing analysis was performed to compare the response to lysosomal stress induced by cholesterol crystals.