CRAMP deletion exacerbates chronic‐binge alcohol‐induced liver steatosis and injury via regulation of gut microbiota and hepatic inflammation
Authors: Li Fengyuan , Shao Tuo , McClain Craig , Feng Wenke 
Published: November 2023

Background

Antimicrobial peptides (AMPs) expressed in epithelial and immune cells are largely designated for defense against invading microorganisms. LL‐37 is the only known member of human cathelicidin antimicrobial peptide family, known as CRAMP in mouse. With growing understanding of its properties, we know that LL‐37/CRAMP functions also as an immune‐regulator in various diseases, including metabolic diseases. Alcoholic liver disease (ALD) is characterized by gut dysbiosis and intestinal and hepatic inflammation. Previous work in our lab has shown a decreased expression of CRAMP mRNA level in the intestine in a mouse model of ALD, indicating that CRAMP may play an important role in ALD. However, the mechanism underlying the effects of CRAMP in ALD is unclear.

Methods

CRAMP KO mice and their control wild type (WT) mice were subjected to Lieber DeCarli liquid diet containing 5% alcohol for 24 days, a bolus of alcohol (5g/kg) was gavaged to the mice 6 hours before harvesting on the last day. A group of alcohol‐exposed mice were treated with synthetic CRAMP peptide at a dose of 100 μg via IP injection for 3 times in the last week. Mice in pair‐fed groups were fed an isocaloric maltose dextrin solution. Serum samples were collected for ALT, AST and LPS measurement using commercial kits. Liver and intestine tissue samples were collected and processed for histological analysis. In vitro study used the mouse macrophage cell line, Raw 264.7, to determine the effect of CRAMP on LPS‐induced inflammatory activation.

Results

Alcohol feeding significantly increased serum levels of ALT and AST in both WT and CRAMP KO mice, but these elevations were more pronounced in the KO mice. Analysis of gut microbiota by pyrosequencing of fecal and cecal samples revealed a more severe dysbiosis in the KO mice. Endotoxin levels in the serum of the KO mice were higher than in the WT mice. Histological study by HE staining on liver tissues showed hepatic lipid accumulation increased after alcohol exposure in WT mice and was further elevated in CRAMP KO mice. Liver triglyceride measurement further confirmed the histological results for hepatic steatosis. Furthermore, hepatic pro‐inflammatory cytokine expression was increased in the KO mice compared to the WT mice. CAE and F4/80 staining of liver tissues showed a significantly elevated neutrophils/macrophage infiltration in CRAMP KO mice. In vitro study using RAW264.7 cell line demonstrated that treatment with synthetic CRAMP peptide significantly blocked the LPS‐induced mRNA expression of pro‐inflammatory cytokines IL‐6, IL‐1β and TNFα in a dose‐dependent manner.

Conclusions

CRAMP deficiency exacerbates ALD through multiple mechanisms, including dysbiosis and endotoxemia and increases hepatic inflammation. Synthetic CRAMP peptide treatment reduces alcohol exposure‐induced liver steatosis and injury. CRAMP pathway may represent a therapeutic approach for ALD.

Support or Funding Information

Supported by NIH and Veterans Administration