Intestinal REG3 Lectins Protect against Alcoholic Steatohepatitis by Reducing Mucosa-Associated Microbiota and Preventing Bacterial Translocation

Abstract

Approximately half of all deaths from liver cirrhosis, the tenth leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease.

Figure 1. Exacerbated alcohol-induced liver disease in Reg3b^−/− mice

WT mice and their Reg3b^−/− littermates were fed an oral control diet (n=4–7) or ethanol diet (n=9–19). (A, B) Immunoblot and immunohistochemical analysis of REG3B in the small intestine. (C) Plasma levels of ALT. (D) Representative liver sections after hematoxylin and eosin staining. (E) Hepatic triglyceride content. (F) Representative Oil Red O-stained liver sections. (G) Immunofluorescent analysis of F4/80 in the liver; positively stained area was quantitated by image analysis software. (H) Hepatic expression of mRNAs encoding chemokines. (I) Hepatic expression of TNFα protein. Scale bars, 50 μm. *P<.05. See also Figure S1.

Figure 2. REG3B controls bacterial colonization of intestinal epithelial cells and reduces bacterial translocation after chronic alcohol feeding

WT and Reg3b^−/− mice were fed an oral control diet (n=4–7) or ethanol diet (n=7–17). (A) Fecal albumin content. (B) Plasma levels of LPS. (C) Principal component analysis (PCA) of microbiomes was performed. (D) Total bacteria in the lumen and mucus layer, and bacteria associated with epithelial cells, were assessed by qPCR. (E) FISH analysis using a universal probe for eubacteria. Arrowheads indicate bacteria present on epithelial surfaces and within the mucosa. (F) Colony-forming units (CFUs) were counted on anaerobic culture plates of mesenteric lymph nodes (MLN). (G) Total bacteria in the liver as assessed by qPCR. Scale bars, 50μm. *P<.05. See also Figure S1 and S2.

Figure 3. Chronic administration of ethanol exacerbates liver disease in Reg3g^−/− mice

WT and their Reg3g^−/− littermates were fed an oral control diet (n=4–6) or ethanol diet (n=8–13). (A) Plasma level of ALT. (B) Representative liver sections after hematoxylin and eosin staining. (C) Hepatic triglyceride content. (D) Representative Oil Red O-stained liver sections. (E) Immunofluorescent analysis of F4/80 in the liver; positively stained area was quantitated by image analysis software. (F) Hepatic expression of mRNAs encoding chemokines. (G) Hepatic expression of TNFα protein. Scale bars, 50 μm. *P<.05. See also Figure S3 and S4.

Figure 4. Mucosa-associated bacterial colonization and bacterial translocation is increased in Reg3g^−/− mice following chronic alcohol feeding

WT mice and their Reg3g^−/− littermates were fed an oral control diet (n=4–6) or ethanol diet (n=11–18). (A) Fecal albumin content. (B) Plasma level of LPS. (C) Principal component analysis (PCA) of microbiomes was performed. (D) Total bacteria in the lumen and mucus layer, and associated with epithelial cells, as assessed by qPCR. (E) FISH analysis using a universal probe for eubacteria. Arrowheads indicate bacteria present on epithelial surfaces and within the mucosa. (F) CFUs were counted on anaerobic culture plates of mesenteric lymph nodes (MLN). (G) Total bacteria in the liver, assessed by qPCR. Scale bars, 50μm. *P<.05. See also Figure S3 and S4.

Figure 5. Intestine-specific overexpression of REG3G protects mice from ethanol-induced liver disease

WT mice and their Reg3g-Tg littermates were fed an oral control diet (n=5–9) or ethanol diet (n=10–18). (A, B) Immunoblot and immunohistochemical analyses of REG3G in the small intestine. (C) Plasma level of ALT. (D) Representative liver sections after hematoxylin and eosin staining. (E) Hepatic triglyceride content. (F) Representative Oil Red O-stained liver sections. (G) Immunofluorescent analysis of F4/80 in the liver; positively stained area was quantitated by image analysis software. (H) Hepatic expression of mRNAs encoding chemokines. (I) Hepatic expression of TNFα protein. Scale bars, 50μm. *P<.05. See also Figure S5 and S6.

Figure 6. Reg3g-Tg mice have lower intestinal bacterial loads and bacterial translocation after ethanol feeding

WT mice and their Reg3g-Tg littermates were fed an oral control diet (n=5–9) or ethanol diet (n=10–18). (A) Fecal albumin content. (B) Plasma levels of LPS. (C) Principal component analysis (PCA) of microbiomes was performed. (D) Total bacteria in the lumen and mucus layer, and associated with epithelial cells, as assessed by qPCR. (E) FISH analysis using a universal probe for eubacteria. Arrowheads indicate bacteria present on epithelial surfaces and within the mucosa. (F) CFUs were counted on anaerobic culture plates of mesenteric lymph nodes (MLN). (G) Total bacteria in the liver as assessed by qPCR. Scale bars, 50μm. *P<.05. See also Figure S5 and S6.

Figure 7. Alcohol abuse increases the number of mucosa-associated bacteria in humans

Total bacteria in duodenal biopsies from controls without alcohol dependency (n=5) and alcohol dependent patients (n=8). *P<0.05. See also Table S1.