Project description:Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Persistent NAFLD can progress to nonalcoholic steatohepatitis (NASH) with inflammation and fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor that modulates glycolipid homeostasis, cell differentiation and tumor progression. MTL-CEBPA, a first-in-human small activating RNA therapeutic, has been used to overexpress CEBPA and treat hepatocellular carcinoma by targeting myeloid cells in clinical trials. However, whether and how hepatocyte-specific CEBPA modulates NASH are unknown. Here we found that CEBPA expression was inhibited in the livers of high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed mice, NASH patients and palmitic/oleic acid-treated primary hepatocytes. Liver-specific Cebpa knockout mice (Cebpa-dLiv) and liver-specific tamoxifen-inducible ERT2-Cre mice (Cebpa-dLiv-ERT2) were then generated. Both Cebpa-dLiv and Cebpa-dLiv-ERT2 mice (treated with tamoxifen) developed enhanced NASH and fibrosis compared to control mice. Global transcriptome analyses demonstrated hepatic secreted phosphoprotein 1 (Spp1) encoding the fibrosis-promoting factor SPP1, was markedly induced by hepatocyte CEBPA knockout in HFCFD-fed mice. Consistently, hepatic SPP1 was upregulated by NASH and negatively correlated with CEBPA expression in humans. Hepatocyte loss of CEBPA enhanced hepatic SPP1 expression and serum SPP1 levels both at the early and late stage of NASH in mice. CEBPA-knockout primary hepatocytes released more SPP1 to the culture medium than wild-type mouse hepatocytes, resulting in enhanced fibrogenesis of hepatic stellate cells, a phenotype that was rescued by the Spp1 shRNA. Hepatocyte-directed AAV8-deliverey of Spp1 shRNA rescued the fibrosis in HFCFD-fed Cebpa-dLiv mice. Mechanistically, CEBPA overexpression reduced, while CEBPA knockout enhanced, SPP1 expression in primary hepatocytes in vitro. The Spp1 promoter had functional CEBPA response elements, while CEBPA modulated histone acetylation to restrict SPP1 expression in a novel feedback loop. Furthermore, overexpression of CEBPA in hepatocytes with AAV8-TBG-CEBPA, inhibited hepatic SPP1 expression and reduced fibrosis in HFCFD-fed wild-type mice. This study demonstrates a novel hepatocyte CEBPA-SPP1 axis involved in modulating NASH fibrosis that supports hepatocyte CEBPA as an anti-NASH target.

Project description:Background & Aims: Alcohol-associated liver disease (ALD) heavily contributes to global alcohol related mortality. Alcohol can synergize with other causes of liver disease such as metabolic syndrome induced by western diet (high fat, high fructose, high cholesterol). However, the role of individual western diet components in ALD development is not fully understood. We aimed to study the role of cholesterol in alcohol pathogenesis. Previous studies have shown that hepatic cholesterol metabolism is dysregulated with alcohol use resulting in altered lipoprotein levels. We aimed to study the role of cholesterol in ALD development using a mouse model. Approach and results: 8-week-old male mice were fed ad libitum with low fat high cholesterol diet (Research Diets, cat# D24120501, 1% cholesterol) in combination with 20% EtOH in the drinking water for 10 weeks. Compared to high-cholesterol diet alone, alcohol treated mice showed elevated levels of serum ALT and AST, INR/PT, increased liver inflammation, fibrosis, ductular reaction and signs of liver failure such as reduced HNF4α and albumin production. To assess the mechanism of alcohol-induced pathology, we performed spatial transcriptomic analysis using 5k gene Xenium panel (10x Genomics). We found that the liver adapted to high cholesterol diet by increasing cholesterol metabolism in hepatocytes. In contrast, alcohol impaired liver adaptation to high cholesterol diet by reducing cholesterol metabolism, HDL production, and hepatic cholesterol secretion, thus driving inflammation and fibrosis. Hepatocyte specific Kdm5b knockout mice were partially protected from alcohol effects by restoring cholesterol secretion in part through an upregulation of Abcg8. Accordingly, these mice were protected against alcohol-induced fibrosis development and inflammation. Conclusion: In summary, alcohol impairs liver adaptation to high cholesterol diet. Increasing liver cholesterol secretion by KDM5B inhibition protects liver from ALD development.

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Hepatocyte-specific CEBPA-ORM1 axis restricts alcohol-associated liver disease

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