Paneth Cell Dysfunction Mediates Alcohol-related Steatohepatitis Through Promoting Bacterial Translocation in Mice: Role of Zinc Deficiency

Abstract

Background and aims: Microbial dysbiosis is associated with alcohol-related hepatitis (AH), with the mechanisms yet to be elucidated. The present study aimed to determine the effects of alcohol and zinc deficiency on Paneth cell (PC) antimicrobial peptides, α-defensins, and to define the link between PC dysfunction and AH.

Approach and results: Translocation of pathogen-associated molecular patterns (PAMPs) was determined in patients with severe AH and in a mouse model of alcoholic steatohepatitis. Microbial composition and PC function were examined in mice. The link between α-defensin dysfunction and AH was investigated in α-defensin-deficient mice. Synthetic human α-defensin 5 (HD5) was orally given to alcohol-fed mice to test the therapeutic potential. The role of zinc deficiency in α-defensin was evaluated in acute and chronic mouse models of zinc deprivation. Hepatic inflammation was associated with PAMP translocation and lipocalin-2 (LCN2) and chemokine (C-X-C motif) ligand 1 (CXCL1) elevation in patients with AH. Antibiotic treatment, lipopolysaccharide injection to mice, and in vitro experiments showed that PAMPs, but not alcohol, directly induced LCN2 and CXCL1. Chronic alcohol feeding caused systemic dysbiosis and PC α-defensin reduction in mice. Knockout of functional α-defensins synergistically affected alcohol-perturbed bacterial composition and the gut barrier and exaggerated PAMP translocation and liver damage. Administration of HD5 effectively altered cecal microbial composition, especially increased Akkermansia muciniphila, and reversed the alcohol-induced deleterious effects. Zinc-regulated PC homeostasis and α-defensins function at multiple levels, and dietary zinc deficiency exaggerated the deleterious effect of alcohol on PC bactericidal activity.

Conclusions: Taken together, the study suggests that alcohol-induced PC α-defensin dysfunction is mediated by zinc deficiency and involved in the pathogenesis of AH. HD5 administration may represent a promising therapeutic approach for treating AH.

Figure 1.

AH is associated with PAMP translocation. (A) Immunofluorescence (IF) staining of CD11b⁺ and (B) CD68⁺ cells, (C) quantification and immunohistochemistry (IHC) staining of LCN2, (D) quantification and IHC staining of IL-8, (E) endotoxin levels, (F) total 16S bacterial rRNAs, E. coli, and H. hepaticus DNAs relative to β-globin in the livers of patients with SAH (n=5) and healthy controls (n=5). Scale bar, 20 μm (A,B) and 50 μm (C,D). *P<0.05, ^**P<0.01, ^***P<0.001. SAH, severe alcoholic hepatitis.

Figure 2.

PAMPs are responsible for hepatic LCN2 and CXCL1 induction. (A) Experimental design. Black arrows represent antibiotic (Abx) treatments. (B) Relative hepatic Lcn2 and Cxcl1 levels in mice of the Abx experiment (n=6). (C) Experimental design. Black arrow indicates LPS injection that was given 4 hours before sample collection. (D) Relative hepatic Lcn2 and Cxcl1 levels in mice of the LPS experiment (n= 6). (E) Expressions of Lcn2 and Cxcl1 in Hepa1c1c7 cells treated with LPS for 6 h (n=3). (F) Expressions of Lcn2 and Cxcl1 in Hepa1c1c7 cells pre-treated with alcohol toxicants for 3 d followed by LPS for 6 h (n=3). *P<0.05, ^**P<0.01, ^***P<0.001 except for (M) and (N), where * represents the difference between control and LPS treatments (white bar vs. gray bar), and # represents difference between AcH and other treatments (EtOH, H₂O₂, and 4-HNE; gray bars). PF, pair-fed; AF, alcohol-fed; Ctrl, control; EtOH, ethanol; AcH, acetaldehyde.

Figure 3.

Chronic alcohol feeding causes systemic bacterial dysbiosis and Paneth cell dysfunction in mice. (A) Cladogram and (B) family-classified taxonomic abundances of bacteria in the livers and cecal contents of mice (n=4). P, phylum. (C) Transmission electron microscopy (TEM) of the ileal crypts of mice (n=3). Arrowheads indicate PCs, and stars indicate secretary granules. Scale bar, 10 μm (upper), 2 μm (lower). (D) Relative ileal mRNA levels of α-defensins in mice (n=6). *P<0.05, ^**P<0.01. (E) IF staining of DEFA5 (arrowheads). Scale bar, 20 μm. PF, pair-fed; AF, alcohol-fed.

Figure 4.

Deficiency of active α-defensins exaggerates alcohol-induced intestinal antibacterial dysfunction, dysbiosis, and barrier disruption. (A) Experimental design. (B) TEM of ileal crypts of mice. Scale bar, 10 μm. (C) Relative ileal mRNA levels of α-defensins in mice (n=6). (D) Bactericidal activity of isolated small intestinal crypts against E. coli ML35. Data are presented as percentage of remaining live bacteria (n=6). (E) Alpha-diversity measurement of observed number of OTUs, PCoA plot showing dissimilarity in bacterial community structures based on Bray-Curtis distances, and barplot showing the bacterial composition at family level of the intestinal microbiome from mice cecal contents (n=8). Legend of prominent families is shown at right. (F) IF staining of ileal ZO-1 and Western blot of ileal ZO-1 and claudin-1. White arrowheads indicate disarranged ZO-1. Scale bar, 20 μm. *P<0.05, ^**P<0.01, ^***P<0.001 except 4F where letters indicate difference at P<0.05. PF, pair-fed; AF, alcohol-fed.

Figure 5.

Deficiency of active α-defensins promotes alcohol-induced PAMP translocation and hepatic inflammation. (A) Plasma and hepatic endotoxin levels, and relative hepatic 16S bacterial rRNAs in WT and Mmp7^−/− mice fed control or alcohol diet for 8 wk (n=8). (B) mRNA and protein levels of LCN2 and CXCL1 (n=6). (C) Plasma ALT levels (n=8). (D) H&E staining of the liver sections (n=3). Arrowheads indicate lipid accumulation, and arrows indicate inflammatory cells. Scale bar, 50 μm. (E) IF staining of hepatic F4/80⁺ cells (n=3). Scale bar, 20 μm. (F) Flow cytometry analysis of activated macrophages and neutrophils (n=3). *P<0.05, ^**P<0.01, ^***P<0.001. PF, pair-fed; AF, alcohol-fed.

Figure 6.

HD5 treatment alters intestinal microbiome, and reverses alcohol-impaired intestinal bactericidal function and barrier disruption. (A) Experimental design. Green arrows indicate HD5 treatment. (B) Alpha-diversity measurement of observed number of OTUs, PCoA plot showing dissimilarities in bacterial community structures based on Bray-Curtis distances, and barplot showing the bacterial composition at family level of the intestinal microbiome from cecal contents of the AF mice with or without HD5 treatment (n=8). Legend of prominent families is shown at right. (C) Relative abundance of A. muciniphila determined by qPCR (n=4). (D) Relative mRNA levels of ileal α-defensins (n=6). (E) IF staining of ileal ZO-1 and Western blot of ZO-1 and claudin-1 (n=3). Arrowheads indicate disarranged ZO-1. Scale bar, 20 μm. Letters indicate difference at P<0.05. (F) Relative mRNA levels of Il22 and inflammatory cytokines/chemokines in mice ileum. *P<0.05, ^**P<0.01, ^***P<0.001. PF, pair-fed; AF, alcohol-fed.

Figure 7.

HD5 treatment alleviates alcohol-induced liver damage in mice. (A) Plasma endotoxin levels and hepatic 16S bacterial rRNAs relative to β-actin (n=8). (B) mRNA levels of Lcn2 and Cxcl1 (n=6). (C) IHC staining of LCN2 and CXCL1 (n=3). (D) IF staining of hepatic F4/80⁺ and CD11b⁺ cells (n=3). (E) Plasma ALT levels (n=8). (F) H&E staining of liver sections. Scale bar, 50 μm (C,F), 20 μm (D). *P<0.05, ^**P<0.01. PF, pair-fed; AF, alcohol-fed.

Figure 8.

Zinc regulates α-defensin bactericidal activity and stability, and dietary zinc deficiency exaggerates alcohol-induced PC α-defensin dysfunction. (A) Representative image of isolated crypts, bactericidal activity, relative ileal mRNA levels of Defa5 and Lyz1 of mice treated with or without dithizone (n=6); and bactericidal activity of released AMPs from isolated small intestinal crypts after direct zinc chelation by TPEN (n=6). (B) E. coli ML35 killing of HD5 with or without zinc determined by flow cytometry (n=6). (C) Analytical nUHPLC-MS traces of HD5 with or without zinc. (D) Experimental design of chronic alcohol feeding with zinc adequate (ZnA) or zinc deficient (ZnD) diets. (E) TEM of ileal crypts of mice (n=3). Scale bar, 10 μm. (F) Relative ileal mRNA levels of Defa5 and Mmp7, and bactericidal activity of isolated small intestinal crypts of mice (n=6). *P<0.05, ^**P<0.01. PF, pair-fed; AF, alcohol-fed.