Hepatic pIgR-mediated secretion of IgA limits bacterial translocation and prevents ethanol-induced liver disease in mice

Abstract

Objective: Alcohol-associated liver disease is accompanied by microbial dysbiosis, increased intestinal permeability and hepatic exposure to translocated microbial products that contribute to disease progression. A key strategy to generate immune protection against invading pathogens is the secretion of IgA in the gut. Intestinal IgA levels depend on the polymeric immunoglobulin receptor (pIgR), which transports IgA across the epithelial barrier into the intestinal lumen and hepatic canaliculi. Here, we aimed to address the function of pIgR during ethanol-induced liver disease.

Design: pIgR and IgA were assessed in livers from patients with alcohol-associated hepatitis and controls. Wild-type and pIgR-deficient (pIgR^-/- ) littermates were subjected to the chronic-binge (NIAAA model) and Lieber-DeCarli feeding model for 8 weeks. Hepatic pIgR re-expression was established in pIgR^-/- mice using adeno-associated virus serotype 8 (AAV8)-mediated pIgR expression in hepatocytes.

Results: Livers of patients with alcohol-associated hepatitis demonstrated an increased colocalisation of pIgR and IgA within canaliculi and apical poles of hepatocytes. pIgR-deficient mice developed increased liver injury, steatosis and inflammation after ethanol feeding compared with wild-type littermates. Furthermore, mice lacking pIgR demonstrated increased plasma lipopolysaccharide levels and more hepatic bacteria, indicating elevated bacterial translocation. Treatment with non-absorbable antibiotics prevented ethanol-induced liver disease in pIgR^-/- mice. Injection of AAV8 expressing pIgR into pIgR^-/- mice prior to ethanol feeding increased intestinal IgA levels and ameliorated ethanol-induced steatohepatitis compared with pIgR^-/- mice injected with control-AAV8 by reducing bacterial translocation.

Conclusion: Our results highlight that dysfunctional hepatic pIgR enhances alcohol-associated liver disease due to impaired antimicrobial defence by IgA in the gut.

Keywords: alcoholic liver disease; immune-mediated liver damage; liver immunology; mucosal immunology.

Figure 1.. H&E staining and immunofluorescent detection of IgA and pIgR in alcohol-associated liver disease.

A) Representative images of H&E staining and staining for IgA (red) and pIgR (cyan) of non-alcoholic control liver slides. Low amounts of pIgR in canaliculi and apical poles of hepatocytes, and minimal IgA in hepatocytes. Large amounts of pIgR in the the cytoplasm of cholangiocytes. B) Representative images of low magnification view of H&E staining and staining for IgA (red) and pIgR (cyan) in patients with alcohol-associated hepatitis. Increased IgA and pIgR staining localizing to zones of active fibroplasia, and away from hepatocytic nodules and end-stage fibrosis. C) High magnification view of increased IgA and pIgR staining in patients with alcohol-associated hepatitis, with both IgA and pIgR localizing to canaliculi and apical poles of hepatocytes. N, hepatocytic nodule. F, end-stage fibrosis. Green fluorescence, autofluorescence. Blue fluorescence, nuclei. Red fluorescence, IgA. Cyan fluorescence, polymeric immunoglobulin receptor (pIgR).

Figure 2.. pIgR-deficient mice develop more steatohepatitis after chronic-binge ethanol feeding.

A) Schematic of chronic-binge ethanol feeding study in female and male pIgR^−/− mice (red) and wildtype littermates (grey). B) Fecal IgA levels at the end of the study.

C) Plasma IgA levels at the end of the study.

D) Liver to body weight ratio. E) Plasma ALT levels. F) Representative images showing H&E staining of liver sections. G) Hepatic triglyceride content. H) Quantification of immunohistochemical staining for lipids using Oil Red O. I) Representative images showing Oil Red O staining of liver sections. J-M) mRNA levels of indicated genes (Cxcl1, Cxcl2, Ccr2, Cd11b) in livers of ethanol-fed mice as shown in Figure 2A, assessed by qPCR. Data are shown relative to the respective wildtype mice and normalized to 18S. N) Quantification of immunohistochemical staining for infiltrating macrophages and neutrophils in the liver using Mac-1. O) Representative images showing Mac-1 staining of liver sections. Data shown as mean ± SEM of n=12–18/group from 6 independent experiments. * indicates p≤0.05, ** p≤0.01, *** p≤0.001. Exact p-values are provided in Table S2.

Figure 3.. Increased ethanol-induced liver disease in mice lacking pIgR after chronic ethanol feeding with single gavage of ethanol is associated with elevated bacterial translocation.

A) Schematic of 8 weeks Lieber-DeCarli ethanol diet or isocaloric control diet feeding study with a single gavage of ethanol in female pIgR^−/− mice (red) and wildtype littermates (grey). B) Fecal IgA levels after dietary intervention. C) Plasma IgA levels after dietary intervention. D) Liver to body weight ratio. E) Plasma ALT levels. F) Representative images showing H&E staining of liver sections. G) Hepatic triglyceride content. H) Quantification of immunohistochemical staining for lipids using Oil Red O. I) Representative images showing Oil Red O staining of liver sections. J-O) mRNA levels of indicated genes (Cxcl1, Cxcl2, Ccr2, Cd11b, Col1a1, Timp1) in livers of ethanol-fed mice as shown in Figure 3A, assessed by qPCR. Data are shown relative to the isocaloric control-fed mice and normalized to 18S. P) Quantification of sirius red staining of liver sections. Q) Representative images showing sirius red staining of liver sections. R) Plasma LPS levels after dietary intervention. S) Total hepatic bacteria assessed by 16S levels in ethanol-fed mice as shown in Figure 3A. Data are shown relative to wildtype mice after normalization to 18S. T) Colony forming units of viable E. coli in feces of ethanol-fed mice as shown in Figure 3A (n=6–7 mice/group). U) Representative images showing fecal E. coli cultures in ethanol-fed mice. V) Hepatic E. coli in ethanol-fed mice as shown in Figure 3A. Data are shown relative to wildtype mice after normalization to 16S. Data shown as mean ± SEM of n=4 mice/isocaloric group and n=17–16/group for ethanol-fed mice from 4 independent experiments. * indicates p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001. Exact p-values are provided in Table S3.

Figure 4.. Treatment with non-absorbable antibiotics reduces ethanol-induced liver disease in pIgR^−/− mice.

A) Schematic of intervention with antibiotics (white) or vehicle control (red) during the last two weeks of 8 weeks Lieber-DeCarli ethanol diet plus single gavage of ethanol in female pIgR^−/− mice. B) Fecal IgA levels at the end of the study. C) Plasma IgA levels at the end of the study. D) Liver to body weight ratio. E) Plasma ALT levels. F) Representative images showing H&E staining of liver sections. G) Hepatic triglyceride content. H) Quantification of immunohistochemical staining for lipids using Oil Red O. I) Representative images showing Oil Red O staining of liver sections. J-M) mRNA levels of indicated genes (Cxcl1, Ccr2, Cd11b, Ccl2) in livers of ethanol-fed mice as shown in Figure 4A, assessed by qPCR. Data are shown relative to the control-treated pIgR^−/− mice and normalized to 18S. N) Quantification and representative images of immunohistochemical staining for infiltrating macrophages and neutrophils in the liver using Mac-1. Data shown as mean ± SEM of n=11–13 mice/group from 3 independent experiments. * indicates p≤0.05, ** p≤0.01. Exact p-values are provided in Table S4.

Figure 5.. Hepatic pIgR overexpression restores gut IgA levels and ameliorates steatohepatitis in pIgR^−/− mice.

A) Schematic of study layout. Female pIgR^−/− mice received a single intravenous injection of GFP-expressing AAV8 control vector (red) or pIgR-expressing AAV8 (blue). Female wildtype littermates received GFP-expressing AAV8 control vector (grey). After 2 weeks, mice were placed on Lieber-DeCarli diet for 8 weeks followed by single gavage of ethanol. B) Fecal IgA levels at the end of the study. C) Plasma IgA levels at the end of the study. D) Liver to body weight ratio. E) Plasma ALT levels. F) Representative pictures of H&E staining of liver sections (20x magnification). G) Hepatic triglyceride levels, normalized to liver protein content. H) Quantification of immunohistochemical staining for lipids using Oil Red O. I) Representative images showing Oil Red O staining of liver sections. J-L) mRNA levels of indicated genes (Cxcl1, Cxcl2, Ccr2) in whole liver tissue of mice as shown in Figure 5A, assessed by qPCR. Data are shown relative to the control-treated wildtype mice after normalization to 18S. M) Plasma LPS levels at the study endpoint. Data shown as mean ± SEM of n=13–15 mice per group from 5 independent experiments. * indicates corrected p≤0.05, ** corrected p≤0.01, *** corrected p≤0.001. Exact p-values are provided in Table S5.