Our study uses an intragastric model of continuous alcohol or isocaloric diet feeding to describe the dynamics of liver disease, bacterial translocation, and intestinal dysbiosis. Steatosis and steatohepatitis occur one week following alcohol administration at a similar time when translocation of live bacteria to the systemic circulation is observed. Intestinal bacterial overgrowth of both aerobic and anaerobic bacteria is evident after 3 weeks of alcohol feeding, which is pronounced in the small intestine. As only 20% of all bacteria can be cultured by conventional techniques (43
), we used 454 pyrosequencing to demonstrate qualitative changes of the microbiome following alcohol administration, characterized by a decrease in Firmicutes and an increase in Bacteroidetes. Alcohol induces a downregulation of the host antimicrobial proteins Reg3b and Reg3g. Finally, we demonstrate that partial restoration of Reg3g protein levels with prebiotics reduces bacterial overgrowth and ameliorates alcoholic steatohepatitis.
There is strong evidence in support of the concept that gut-derived endotoxin as a marker for bacterial translocation plays a central role in the initiation and progression of alcohol induced liver injury. First, plasma endotoxin levels are increased in patients with alcoholic liver disease (1
), and a correlation between alcohol ingestion and increased systemic levels of endotoxin has also been demonstrated in animal models of alcohol-induced liver injury (14
). Second, selective intestinal decontamination with antibiotics prevents experimental alcoholic liver injury (12
). Third, mice with genetic deletions in the LPS signaling pathway are resistant to alcohol-induced liver damage (15
The question arises whether bacterial translocation is dependent on qualitative and/or quantitative changes in the intestinal microbiome. It was noted that increased intestinal permeability with subsequent endotoxemia occurs 4 weeks following daily alcohol administration and prior to the development of alcoholic steatohepatitis in rats (46
). We have shown that translocation of bacteria to the systemic circulation occurs prior to the onset of intestinal bacterial overgrowth. Endotoxin plasma levels and the number of translocated viable bacteria to mesenteric lymph node showed a higher trend in alcohol fed animals after 1 week and 3 weeks. One possibility why we did not observe a statistically significant difference is related to the nature of the mouse model of intragastric tube feeding. The catheter extends through a skin wound into the stomach that makes the animals prone to have transloaction at baseline. However, when bacterial overgrowth in the small intestine is induced experimentally, it results in hepatic injury mediated by translocated bacterial products (47
). Thus, based on these studies, increased intestinal permeability with subsequent translocation of bacterial products or bacteria likely occurs very early in alcohol-induced liver disease. One possible mediator to increase intestinal permeability is ethanol itself, as acute ingestion of alcohol alters the epithelial barrier in the colon through ethanol oxidation into acetaldehyde by the colonic microflora and results in activation of mast cells (48
). As described, bacterial overgrowth occurs three weeks following alcohol administration. As the overall amount of enteric endotoxin load increases, one could speculate that plasma endotoxin levels subsequently increase given a preexisting disrupted mucosal barrier. This might contribute to liver disease progression. Thus, alcohol-induced dysbiosis does not cause intestinal permeability with resulting endotoxemia, but it might increase systemic levels of endotoxin to perpetuate later disease stages.
Recently, gut bacterial microbiota fingerprinting using length heterogeneity PCR was applied to an animal model of alcoholic steatohepatitis. Consistent with our results, there is evidence for qualitative changes in the composition of the intestinal microflora in the colon following daily alcohol consumption in rats (41
). However, the identity of bacteria has not been addressed in this study. We have advanced these findings by performing 454 massively parallel pyrosequencing of the intestinal contents of mice following an isocaloric diet or alcohol feeding for 3 weeks. Technical advances have helped to characterize the gastrointestinal microflora in its deep biodiversity and its functional contribution to the host biology by examination of the 16S ribosomal RNA genes used in taxonomical classification of bacteria. We found striking qualitative changes in the overall composition of the enteric microbiome associated with alcohol consumption. Following alcohol feeding there was an overall decrease in Firmicutes, while the relative abundance of Bacteroidetes and Verrucomibrobia increased in mice fed alcohol. Interestingly, Lactobacillus
was strongly suppressed and almost absent in mice fed alcohol for 3 weeks as compared to control fed animals. This now provides a rationale for the beneficial effect of various probiotic Lactobacillus
strains in experimental models of alcoholic liver disease. Feeding a Gram-positive probiotic Lactobacillus
strain with subsequent displacement of Gram-negative bacteria protected mice from ethanol-induced liver injury with a concurrent decrease in systemic endotoxin levels (40
). This is a very promising example of how directed manipulation of the microbiome, and specifically of one microorganism within the gastrointestinal tract, may yield health benefits. This also demonstrates how remodeling of microbial communities associated with disease can be used for prevention or therapy.
The exact reason for intestinal bacterial overgrowth and enteric dysbiosis is unknown. Several factors contribute to the homeostasis of the intestinal microbial community, such as gastric acid secretion and small and large bowel motility. Intestinal epithelial cells are the main interface between the host and the intestinal microflora. Epithelial cells have many important functions in maintaining a symbiotic relationship between the host and the microflora. One of these functions is the secretion of antimicrobial effector molecules as part of the mucosal innate immune system (49
). As we have reported, Reg3g is a secreted c-type lectin with potent bactericidal activity that is expressed in intestinal epithelial and Paneth cells, with highest levels found in the ileum (22
). Similarly, Reg3b has antimicrobial activity and has been implicated in intestinal homeostasis (50
). Here we demonstrate that chronic alcohol exposure suppresses the gene and protein expression of Reg3b and Reg3g, which might contribute to quantitative and qualitative changes in the enteric flora following chronic alcohol feeding. Interestingly, the lowest levels of Reg3b and Reg3g were observed in the proximal small intestine, where the bacterial overgrowth was most pronounced and luminal alcohol concentrations are highest. Restoration of Reg3g levels using prebiotics decreases intestinal bacterial overgrowth and ameliorates alcoholic steatohepatitis. This dysregulation of the mucosal innate immune system demonstrates a novel link between alcohol and enteric dysbiosis.
Ongoing research initiatives including those by our group are expected to provide new insights into the metagenomics, transcriptomics, and metabolomics in alcoholic liver disease. The gut transcriptome and metabolome will help identify key substrates and signaling mediators to explain microbe-microbe and microbe-host interactions. This knowledge will facilitate rationale attempts to manipulate commensal microflora and to target specific microorganisms and metabolites by dietary supplements such as probiotics, antibiotics, prebiotics, and synbiotics.