Transplanting a fibrogenic microbiota
Authors: Bernd Schnabl
Journal: Hepatology (2014)

In this edition, De Minicis et al.'s article1 provides exciting evidence for a transmissible microbiota contributing to cholestatic liver fibrosis. Liver disease is associated with quantitative (intestinal bacterial overgrowth) and qualitative (dysbiosis) changes in the intestinal microflora. Dysbiosis implies not only alterations in the microbial taxonomy, but it can also be viewed as a loss of the symbiotic relationship between the microflora and the host, which results in disease.2 High-fat diet (HFD) exacerbates liver damage and fibrosis in bile-duct–ligated rats.3, 4 A synergistic effect of cholestatic liver damage with HFD was suggested. In the current study, De Minicis et al. confirmed that HFD increases cholestatic liver fibrosis in mice.1 Surprisingly, toxin-induced liver fibrosis was not affected by feeding an HFD. Using deep pyrosequencing, bile-duct–ligated mice fed HFD showed intestinal dysbiosis with a relative increase in Gram-negative microbes and, in particular, in the phylum Proteobacteria, and the family Enterobacteriaceae, as compared with bile-duct–ligated mice on a regular chow diet. Cholestatic mice fed HFD demonstrated a leakier gut with increased bacterial translocation, as compared to cholestatic mice fed a regular chow diet. The investigators then performed fecal transplantation experiments: Mice were fed a regular chow diet or HFD for 1 month, the gastrointestinal (GI) tract was then sterilized with antibiotics for another month, followed by bile duct ligation (BDL) and maintaining mice on a regular chow diet. Most interestingly, the investigators demonstrated that transplantation of fecal HFD flora to mice exacerbates BDL-induced liver fibrosis independently of the diet that had been given before the intestinal decontamination. Thus, even mice fed a regular chow diet, but transplanted with HFD flora, had exacerbated liver disease. Bile-duct–ligated recipients of HFD flora showed a similar microbiome composition, as compared with nontransplanted bile-duct–ligated mice on HFD. Transplantation of Gram-negative isolates from HFD microflora, mainly Proteobacteria, resulted in a significant increase in BDL-induced liver fibrosis, when compared with mice transplanted with Gram-positive bacteria, or feces from mice fed a regular chow diet. Taken together, worsening of cholestatic liver fibrosis under an HFD cannot be simply explained by a synergistic effect of two liver-damaging insults. This study elegantly demonstrates that HFD-induced dysbiosis with "fibrogenic" bacteria contributes to this exacerbation. The question arises of how HFD-induced dysbiosis mediates its fibrogenic effect. The importance of bacterial translocation from the intestinal lumen to the liver has been appreciated for quite some time. Liver fibrosis is dependent on microbial products, in particular, lipopolysaccharide (LPS), as part of the cell wall from Gram-negative bacteria.2 As demonstrated by the investigators of the current study, a shift toward a more Gram-negative microbiome results in more translocation of bacterial LPS and, in endotoxemia, causing more liver damage. However, one would expect that toxic liver fibrosis would be similarly affected, which is not the case, as shown by the investigators. Factors other than simple HFD-induced dysbiosis must be involved that are transmissible and mediate fibrogenic effects. One important difference between microbiome changes during different types of experimental liver disease is intestinal bacterial overgrowth. Whereas BDL results in an increase in the bacterial burden along the entire GI tract within 24 hours, up to 12 injections of CCl4 (or 5-6 weeks of treatment) will not result in intestinal bacterial overgrowth.5 Therefore, it is conceivable that the absence of bile acids triggers amplification of HFD-induced "fibrogenic" bacteria, which is not the case in the toxic liver fibrosis model. Changes in the transcriptome and metabolome of intestinal bacteria might be more important than changes in the bacterial taxonomy. A transmissible microbial metabolite might mediate the fibrogenic aspects observed in this study. Alternatively, the absence of a protective metabolite might confer the liver more susceptible to damage. One could speculate on HFD-induced alteration of bile acids as being one of these metabolites. Beyond their bacteriostatic role, bile acids are known to be ligands for farnesoid X receptor (FXR) and the G-protein-coupled receptor, TGR5. Interestingly, cholestatic liver fibrosis induced by BDL is lower in FXR-deficient mice, but the absence of FXR had no effect on toxin-induced liver fibrosis in mice.6 A dual FXR/TGR5 agonist reduced liver injury in a mouse model of cholangiopathy, further highlighting the importance of these two bile acid receptors in experimental cholestatic liver disease.7 In addition, the FXR agonist, obeticholic acid, reduced markers of liver inflammation and fibrosis in patients with type 2 diabetes mellitus and non-alcoholic fatty live disease (NAFLD) in a phase II clinical trial.8 Thus, HFD-induced changes in microbial transformation of bile acids with altered FXR/TGR5 signaling might contribute to the transmissible host phenotype described in the current article. The investigators further demonstrate that expression of inflammasome components, such as caspase-1 and interleukin (IL)-18, were decreased in intestinal epithelial cells in bile-duct–ligated mice fed an HFD, as compared to bile-duct–ligated mice on a regular chow diet. This was confirmed in their transplantation experiments. Bile-duct–ligated recipients of HFD flora showed decreased intestinal inflammasome components, as compared with nontransplanted bile-duct–ligated mice on an HFD. Inflammasomes are cytoplasmic multiprotein complexes composed of nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRPs). Inflammasomes govern cleavage of effector proinflammatory cytokines, such as pro-IL-1β and pro-IL-18.9 Deletion of NLRP3 and NLRP6 inflammasomes results in lower intestinal IL-18 levels and is associated with intestinal dysbiosis in mice. Inflammasome-induced dysbiosis results in subclinical inflammation of the colon and enhanced bacterial translocation driving progression from NAFLD to nonalcoholic steatohepatitis (NASH) in experimental mouse models. Importantly, this dysbiosis-mediated NASH phenotype can be transferred by cohousing of mice.9 In the current study, bile-duct–ligated mice on an HFD showed the strongest suppression of intestinal inflammasome components among all groups. One could speculate that lower intestinal inflammasome activity might contribute to dysbiosis through decreased levels of IL-18. Alternatively, inflammasome inhibition could potentially be a counter-regulation of the body to stabilize a leaky gut barrier and maintain intestinal homeostasis by decreasing IL-1β, which is involved in tight junction disruption. It will be important to test whether the intestinal inflammasome is causatively related to the induction of dysbiosis and cholestatic liver disease using tissue-specific knockout mice. Taken together, this is an important study demonstrating the existence of a transmissible intestinal microbiome with fibrogenic features. Understanding the exact mechanisms by which HFD-induced dysbiosis results in exacerbation of cholestatic liver fibrosis will greatly enhance our ability to design preventive and therapeutic interventions in humans. Bernd Schnabl, M.D. Department of Medicine, University of California San Diego, La Jolla, CA