CRAMP Deletion Exacerbates Alcohol‐induced Organ Damage via Regulation of Gut Microbiota and Inflammasome Activation
Authors: Li Fengyuan , McClain Craig , Feng Wenke 
Published: June 2021

Background

Recent studies identify that antimicrobial peptides (AMPs) function as immune‐regulators in addition to their antimicrobial property.as CRAMP is the murine ortholog of human LL‐37, the only known member of cathelicidin AMP family. Alcoholic liver disease (ALD) is characterized by gut dysbiosis and intestinal and hepatic inflammation. We previously showed a decreased expression of intestinal CRAMP in a mouse model of ALD, suggesting a potential role of CRAMP in ALD. Gut microbiota‐derived butyrate increases pancreatic CRAMP, which is important in regulating autoimmune diabetes. Here we aimed to test the hypothesis that CRAMP protects the liver and pancreas from alcohol‐induced organ damage by positively modifying the gut microbiota and inhibiting the pro‐inflammatory response.

Methods

CRAMP KO and wild type (WT) mice were subjected to 5% alcohol liquid diet for 24 days and were gavaged with a bolus of alcohol (5g/kg) on the last day. Mice in control groups were fed an isocaloric maltose dextrin solution. Hepatic and pancreatic injury were examined by analyzing serum samples and histological analysis. Mouse fecal samples were collected for metagenomic and metabolomics study. Mouse macrophage Raw 264.7 cells were used to determine the effect of synthetic CRAMP on LPS‐induced inflammatory activation. Results were further examined in CRAMP deficient mouse peritoneal macrophages and bone marrow derived macrophages.

Results

Serum levels of ALT and AST significantly increased by alcohol in both groups but more pronounced in CRAMP KO mice. Alcohol induced an increased and distinct zonal distributed hepatic fat accumulation in CRAMP KO mice compared to WT mice. Furthermore, CRAMP KO mice had increased pro‐inflammatory and decreased anti‐inflammatory cytokines compared to WT mice. In addition, the marker of pancreatitis, serum amylase activity was elevated in CRAMP KO mice, but not in WT mice. Microbiota analysis in mouse fecal samples showed a difference in the abundance of Firmicutes and Bacteroidetes phyla after alcohol exposure between two groups. Metabolomics study showed that major fecal concentrations of short chain fatty acids (SCFAs), in particular, butyric acid, were significantly lower in the CRAMP KO mice than in the WT mice. In vitro study using RAW 264.7 cells demonstrated that sodium salt of butyric acid (NaB) and synthetic CRAMP peptide significantly inhibited LPS‐induced pro‐inflammatory cytokines production in a dose dependent manner, partially through the inhibition of NLRP3 inflammasome activation. Interestingly, ex vivo study demonstrated a reduction of LPS induced IL‐1b expression in CRAMP deficient peritoneal macrophages compared to those from WT mice, suggesting that other compensatory pathways may exist to offset the deleterious effects of CRAMP deficiency.

Conclusions

CRAMP deficiency exacerbates ALD and alcoholic pancreatitis through gut dysbiosis associated decrease in SCFAs and increased hepatic and systemic inflammation. Synthetic CRAMP peptide inhibits endotoxin‐associated inflammasome activation. CRAMP signaling may represent a therapeutic approach for ALD and alcoholic pancreatitis.

Support or Funding Information

Supported by grants from NIH and Veterans Administration

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.