Cathelicidin-related antimicrobial peptide alleviates alcoholic liver disease through inhibiting inflammasome activation

Abstract

Alcoholic liver disease (ALD) is associated with gut dysbiosis and hepatic inflammasome activation. While it is known that antimicrobial peptides (AMPs) play a critical role in the regulation of bacterial homeostasis in ALD, the functional role of AMPs in the alcohol-induced inflammasome activation is unclear. The aim of this study was to determine the effects of cathelicidin-related antimicrobial peptide (CRAMP) on inflammasome activation in ALD. CRAMP knockout (Camp^-/-) and wild-type (WT) mice were subjected to binge-on-chronic alcohol feeding and synthetic CRAMP peptide was administered. Serum/plasma and hepatic tissue samples from human subjects with alcohol use disorder and/or alcoholic hepatitis were analyzed. CRAMP deficiency exacerbated ALD with enhanced inflammasome activation as shown by elevated serum interleukin (IL)-1β levels. Although Camp^-/- mice had comparable serum endotoxin levels compared to WT mice after alcohol feeding, hepatic lipopolysaccharide (LPS) binding protein (LBP) and cluster of differentiation (CD) 14 were increased. Serum levels of uric acid (UA), a Signal 2 molecule in inflammasome activation, were positively correlated with serum levels of IL-1β in alcohol use disorder patients with ALD and were increased in Camp^-/- mice fed alcohol. In vitro studies showed that CRAMP peptide inhibited LPS binding to macrophages and inflammasome activation stimulated by a combination of LPS and UA. Synthetic CRAMP peptide administration decreased serum UA and IL-1β concentrations and rescued the liver from alcohol-induced damage in both WT and Camp^-/- mice. In summary, CRAMP exhibited a protective role against binge-on-chronic alcohol-induced liver damage via regulation of inflammasome activation by decreasing LPS binding and UA production. CRAMP administration may represent a novel strategy for treating ALD. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: ALD; CRAMP; IL‐1β; LPS; uric acid.

Figure 1.

Cathelicidin dysregulation in alcohol-fed animals and ALD patients. (A) Alcohol-induced changes in Camp expression in different organs. PF, pair-fed; AF, alcohol-fed. (B) CRAMP protein immunoblotting of liver and spleen tissues. The bands are composite images of selected bands, and the corresponding GAPDH controls are shown. (C) Serum CRAMP protein levels. Data are expressed as mean SEM (n = 5–9). (D) Hepatic CAMP mRNA expression and (E) plasma LL-37 levels in ALD patients and healthy controls (HC). Data are expressed as mean SEM (n = 5–16). (F) Hepatic CAMP expression correlates with HIF1A and VDR expression in ALD patients.

Figure 2.

Effects of CRAMP deficiency on ethanol-induced liver injury, steatosis, and pro-inflammatory response. (A) Serum levels of AST and ALT. (B) Representative photomicrographs of liver sections with hematoxylin and eosin (H&E) staining (original magnification 200×) (upper panel), Oil Red O staining (original magnification 200×) (middle panel), and TUNEL-positive cell staining (original magnification 100×) (lower panel). Black arrows: TUNEL-positive cells. (C) Hepatic levels of triglycerides and free fatty acids. (D) Hepatic cytokine and chemokine mRNA expression. (E) Representative photomicrographs of paraffin-embedded liver section stained for chloroacetate esterase (CAE) (original magnification 200×) (upper panel) and frozen liver tissue stained using immunofluorescence (IF) for F4/80 (original magnification 100×) (lower panel). Black arrows: infiltrated neutrophils; white arrows: F4/80 positive stained macrophages. Blue: DAPI counterstained nuclei; red: F4/80. Data are expressed as mean SEM (n = 8–10).

Figure 3.

CRAMP is involved in alcohol-induced IL-1β production and inflammasome activation. (A) Serum concentrations of IL-1β. (B) Serum endotoxin levels (left panel) and hepatic mRNA levels of Lbp, Cd14, and Tlr4 (right panel). (C) LPS binding activity shown as FITC intensity of FITC-conjugated LPS-treated RAW264.7 cells. LPS-FITC: 1 μg/ml. Asterisks indicate the significant difference comparing LPS alone and treated groups. (D) Serum uric acid (UA) and ATP levels. Data are expressed as mean SEM (n = 8–10). (E) Correlation of serum levels of UA and IL-1β in AUD patients. ^#Covaried with LBP. Univariate and multivariate regression model was used (n = 14–26). (F) Hepatic mRNA levels of Casp1 and Nlrp3. (G) Representative bands of immunoblotting quantification of molecules in inflammasome activation in liver lysates. Data are expressed as mean SEM (n = 8–10). (H) Left panel: Representative bands of immunoblotting quantification of pro-IL-1β and cleaved IL-1β protein in BMDM lysates. The bands are composite images of selected bands, and the corresponding β-actin controls are shown. Right panel: IL-1β protein levels in culture media of BMDMs. Data are expressed as mean SEM (n = 4–6).

Figure 4.

CRAMP KO mice had increased hepatic oxidative stress by alcohol. (A) Xanthine oxidase (XO) activity in the serum and liver lysates of alcohol-fed mice. (B) Immunoblotting and quantification of CYP2E1 protein. The bands are composite images of selected bands, and the corresponding β-actin controls are shown. (C) Liver ROS production and quantification by DHE staining. Original magnification: 100×. Data are expressed as mean SEM (n = 8–10). (D) Validation of knockdown efficiency by siRNA-Camp transfection in desferrioxamine (DFO)-treated mouse hepatocyte Hepa1–6 cells. (E) Ethanol-induced UA production in Hepa1–6 culture supernatant. siRNA-NT: non-targeting siRNA control; siRNA-Camp: Camp-targeting siRNA; EtOH: ethanol (400 mM for 4 h). Data are expressed as mean SEM (n = 4–6).

Figure 5.

CRAMP peptide administration rescues alcohol-induced liver steatosis and injury. Mice were treated as described in the Materials and methods section. (A) Illustration of feeding and treatment timeline. (B) Serum levels of AST and ALT. (C) Representative photomicrographs of liver sections of H&E (original magnification 100×) and ORO staining (original magnification 200×). (D) Hepatic levels of triglycerides and free fatty acids. (E) Hepatic mRNA expression of Il1b and Mcp1. (F) Hepatic mRNA expression of Il10. Data are expressed as mean SEM (n = 4–8).

Figure 6.

CRAMP peptide administration attenuates alcohol-induced IL-1β production via inhibiting inflammasome activation signaling. (A) Serum IL-1β levels. (B) Serum LPS levels. (C) Hepatic mRNA expression of Lbp and Cd14. (D) Serum UA levels. (E) Serum XO activity. (F) Proposed scheme for the effects of CRAMP on ALD. Data are expressed as mean SEM (n = 4–8).