The Current and Future State of Microbiome Therapeutics in Liver Disease

Patricia P. Bloom, Jasmohan S. Bajaj · 2023 · 10 citationsRead the paper

THE GUT-LIVER CONNECTION The gut microbiome has direct and indirect effects on the liver. Blood from the intestines travels through the portal vein to the liver, allowing gut products to reach the liver directly. Gut bacteria play important roles in carbohydrate, protein, lipid, and bile acid metabolism (1,2), and as such, the composition and function of gut bacteria influences metabolites approaching the liver. Bacteria, viruses, and fungi in the intestine engage with the intestinal immune system, influencing the immune cells and molecules that travel through the portal vein to reach the liver (3). The gut-liver connection is bidirectional. The liver produces bile, which travels through the bile ducts to the duodenum. Bile contents influence bacterial composition and function through detergent properties, inducing antimicrobial peptides and regulating host immunity (4). In cross-sectional studies, many liver diseases have been associated with abnormal gut microbiome composition and function—including metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-related liver disease, primary sclerosing cholangitis, viral hepatitis, cirrhosis, and hepatic encephalopathy (HE) (5–10). In animal studies and prospective human studies, the microbiome has been implicated as playing a causative role in disease pathogenesis (11–14). MICROBIOME THERAPEUTIC TARGETS IN LIVER DISEASE Many of the shared metabolic and immune pathways connecting the gut microbiome to the liver are also potential targets of microbiome therapeutics in liver disease (Figure 1). Primary bile acids are generated in the liver and enter the intestines through the biliary tree, where they are deconjugated and further transformed by intestinal bacteria. The composition of the enteric bile acid pool, as largely determined by microbiota, modulates several aspects of intestinal barrier function including the mucus layer, immune regulation, and tight junction protein integrity (15–18). Short-chain fatty acids (SCFAs) are products of carbohydrate fermentation and are an important energy source for colonic enterocytes (19). Both bile acids and SCFAs, products of bacterial metabolism, are important in regulating the intestinal barrier function and therefore have an impact on the substrates reaching the liver through portal circulation.Figure 1.: Microbiome therapeutic targets in liver disease. LPS, lipopolysaccharide; SCFA, short-chain fatty acid.Several molecules, once able to cross the gut barrier, have been implicated in liver disease. Endotoxins such as lipopolysaccharide are one of many pathogen-associated molecular patterns that can reach the liver, elicit macrophage activation, and likely promote hepatic fibrosis (20). There is increasing evidence for endogenous alcohol production in microbiota using human and animal studies (12,21,22). These include microbiota as diverse as Klebsiella pneumoniae and lactate-producing bacteria (23,24). Finally, ammonia is a product of gut bacterial metabolism and can elicit HE when present at high levels in the systemic circulation. MICROBIOME THERAPIES Diet Specific dietary practices can modulate the microbiome, as well as the development and progression of liver disease. In precirrhotic liver disease, especially MASLD, there is evidence of gut microbial modification through dietary modification in translational and human studies (25). Specific diets such as the Mediterranean diet are associated with improvement in insulin resistance and favorable changes in the microbiome (26), while the reverse is seen with a high-fat Western diet (Table 1) (35). Alcohol intake can also unfavorably change the microbiome, and cessation of alcohol intake improves microbial composition and function (6). As mentioned in the above section, steatotic liver disease could have a microbial origin either through endogenous production of alcohol or external intake of alcohol and fat.Table 1.: Key studies of microbiome therapies in liver diseaseIn patients with cirrhosis, there is evidence from several cohorts of compensated and decompensated patients that diet could modulate microbial composition and function, which in turn can influence hospitalizations. In a comparison between US and Turkish cohorts, there was higher alpha diversity in the microbiome likely due to fermented milk products consumed by Turkish patients compared with the American cohort (28). This was replicated in a Brazilian population, where a diet rich in whole grains and yogurt was also associated with higher alpha diversity and lower hospitalizations compared with American patients (29). Mexican patients, who consumed lower protein, higher carbohydrate, and lower milk intake because of lactulose intolerance, had higher Prevotellaceae and more hospitalizations (36). Although none of these reports are randomized controlled trials (RCTs), they provide insight into how dietary changes across the world could affect patients with cirrhosis. Prebiotics Prebiotics are substrates selectively used by host microorganisms that confer a health benefit (37). Lactulose is the principal prebiotic used in liver disease to treat HE, a common complication of decompensated cirrhosis (38). Lactulose is a synthetic disaccharide, fermented by mainly colonic bacteria into SCFAs. Fermentation of lactulose has multiple consequences: (i) SCFAs produced by lactulose provide nutrition to the intestinal epithelial layer and in so doing likely bolster barrier function and reduce gut translocation (39); (ii) SCFAs lead to acidification of colonic contents, which decreases ammonia production (a culprit in the pathogenesis of HE) from certain bacteria (40); (iii) lactulose enhances the growth of bacteria that able to ferment it, which then pushes other bacteria out of the ecological niche, including potentially harmful bacteria with lipopolysaccharide production (40–42); (iv) the growth of probiotic taxa, stimulated by lactulose fermentation, uses ammonia as a substrate (40,43–45); and (v) the acidification of colonic contents may lead to ammonia translocation across the intestinal epithelial layer into the colon lumen, trap ammonia as an ammonium ion, and expel it in stool (46,47). Lactitol has similarly been used as a prebiotic to treat HE and found to have similar efficacy in a systematic review (27). Inulin and fructooligosaccharides have also shown some benefit in animal studies of fatty liver disease, although data in humans are still required (48,49). Antibiotics Patients with cirrhosis and chronic liver disease are often prescribed antibiotics for prevention and treatment of specific complications of cirrhosis such as HE and infections such as spontaneous bacterial peritonitis (SBP). The use of antibiotics can differentially affect the microbiome that is unique to each antibiotic. Rifaximin is a nonabsorbable antibiotic with activity against a broad range of bacteria but a high threshold for transmissible resistance. Treatment with rifaximin is efficacious in prevention of HE recurrence, and improvement in cognitive function and quality of life in minimal HE. However, the impact on microbial composition is relatively subtle with major changes in microbial function, mucus-degrading bacteria, and microbial-viral interactions focused on ammonia-producing Streptococcus. A water-soluble form of rifaximin (rifaximin SSD) showed promise by inhibiting ammonia production even in germ-free conditions, and a larger trial to prevent HE is ongoing. Using appropriate antibiotics for SBP is important to prevent acute kidney injury and acute on chronic liver failure. However, the use of primary and secondary prophylaxis using ciprofloxacin and trimethoprim-sulfamethoxazole may have major implications on the microbiome. In patients using SBP prophylaxis, there is an increased relative abundance of Gram-positive organisms such as Enterococcus and their interactions with bacteriophages (50). The opposite was seen in patients not on SBP prophylaxis, where Escherichia coli and K. pneumoniae were predominant. Antimicrobial resistance (AMR) is epidemic in cirrhosis because of multiple hospitalizations and instrumentation, an impaired mucosal-immune barrier, and use of antibiotic prophylaxis (51,52). This has deadly consequences, with greater organ failure and death in patients with cirrhosis (53–55). Moreover, this burden is not reversed but exacerbated with liver transplant (56). The gut microbiota is one of the biggest reservoirs for AMR, which is greater in cirrhosis than with renal failure and diabetes (57). AMR carriage in the gut microbiota of patients with cirrhosis independently predicts death (57). There is evidence that fecal microbiota transplant (FMT) can reduce this in cirrhosis (58) and specific microbial therapeutics are being developed, but until then it is critical to reduce the spectrum and duration of antibiotic use when possible (52), reconsider primary prophylaxis for SBP in cirrhosis (59), and use nonantibiotic options as much as possible (60). Probiotics Probiotics in cirrhosis and chronic liver disease have a mixed record. There is a relatively poor quality of evidence in precirrhotic conditions, specifically related to MASLD and alcohol-related liver disease. Current evidence does not point toward meaningful changes in clinically relevant outcomes using probiotics in chronic liver disease. In cirrhosis, probiotics have been studied in the greatest detail in HE. Although smaller scale studies have shown improvements in microbial function with probiotics such as Lactobacillus GG in cirrhosis, larger studies have focused more on clinical outcomes without studying changes in microbiota. Open-label studies using bacteria found in the previous formulation of VSL#3 have shown efficacy in outpatients with cirrhosis and minimal HE. One double-blind RCT from India showed that the probiotic above was associated with lower all-cause but not HE-related hospitalizations. Probiotics are also fraught with issues related to batch-to-batch variability and lack of standardization because of their classification as dietary supplements, not drugs, in the United States. There has been some early-stage investigation into genetically engineered probiotics to treat liver conditions. An E. coli Nissle engineered to metabolize ammonia failed to lower blood ammonia levels in patients with cirrhosis (61). A Lactobacillus reuteri, genetically engineered to produce interleukin-22, reduced liver injury in mice fed alcohol (62). Fecal microbiota transplant FMT is an effective method to change microbiota composition and function by introducing new communities of microbes. In liver disease, FMT trials have been performed in cirrhotic and precirrhotic stages with varying levels of success (63). In precirrhotic nonalcoholic steatohepatitis, FMTs have been largely unsuccessful in meaningfully changing clinical outcomes but have been noted as safe (64). As a corollary, smaller studies showed that FMT could improve insulin resistance, which was not sustained in larger experiences (65–68). FMT has also been studied in primary sclerosing cholangitis (PSC) without IBD in an open-label study, where 30% of patients experienced decreases in their alkaline phosphatase, a surrogate marker (69). In cirrhosis and more advanced liver disease, FMT has shown promise in phase 1 and phase 2 trials (61). In patients with HE who are already on medications, enema FMT after antibiotics was safe in a phase 1 RCT with potential improvements in cognition and hospitalizations (31). A similar safety profile with FMT capsules was seen without preprocedure antibiotics in HE (32). FMT has been found to be largely safe in cirrhosis in other experiences from the United States and India using different modes of administration and multiple donors (33,70), although there has been 1 reported case of FMT-transmitting drug-resistant E. coli bacteremia to a patient with cirrhosis (71). Currently, FMT is being studied extensively for improving outcomes in larger studies across the world. In alcohol-related cirrhosis, FMT was found to be safe and demonstrated a reduction in short-term and long-term consequences of alcohol use (72). This was transferable to germ-free mice with similar changes in behavior focused on SCFA-producing microbiota but not with supernatants (73). In alcohol-associated hepatitis, open-label phase 1 and 2 trials have shown that nasojejunal tube FMT administration improved outcomes compared with standard-of-care groups and historical controls (74,75). FMT has some challenges, namely inconsistent efficacy in most conditions, small risk of infection or other “off target” effects, and challenges with regulation, production, and distribution (76). Defined bacterial consortium products Given the limitations of FMT described above, there has been productive investigation in the last decade of narrow spectrum microbiome therapeutics to treat Clostridioides difficile infection (CDI) (76). Narrowed compositions have been similarly effective to treat recurrent and refractory CDI, and some are clonally produced or not reliant on a human donor—advantages over FMT. Several of these products increase secondary bile acid and SCFA production (76), biological pathways implicated in liver disease pathogenesis. There is 1 ongoing trial of VE303, a defined consortium of 8 clostridial strains, to treat HE (77). Postbiotics Postbiotics are the bioactive products of bacteria, such as SCFAs and secondary bile acids. Although postbiotics have been studied in other gastrointestinal conditions (78), they do not always have consistent or uniformly positive effects (79–81). There are mixed data on the benefits of SCFA supplementation in animal models of liver disease, and no clinical trials to date in humans (82). Bacteriophages Bacteriophages are viruses that infect and replicate within bacteria. Lytic bacteriophages lyse, and therefore kill, the bacterial host after viral replication is complete. Many bacteriophages have a tail that recognizes specific bacterial hosts and therefore can be targeted in their infecting and lysing (83). The potential therapeutic benefit of bacteriophages has recently been highlighted in 2 liver diseases: alcohol-associated liver disease and PSC. Cytolysin is an exotoxin, produced by Enterococcus faecalis, and causes hepatocyte death and liver injury when it reaches the liver (14). Patients with alcohol hepatitis have higher abundance of cytolysin-producing E. faecalis in their stool. In the work by Duan and colleagues, a bacteriophage that targeted cytolysin-producing E. faecalis was able to selectively eliminate this bacteria, reduce cytolysin levels, and substantially reduce alcohol-induced liver injury in mice (14). In PSC, there is a high abundance of K. pneumoniae in the stool, and in a mouse model, this bacteria translocates to mesenteric lymph nodes and contributes to hepatic inflammatory changes (84). Ichikawa et al have developed a lytic phage cocktail that targets this K. pneumoniae, eliminates the bacteria, and decreases subsequent liver inflammation in a mouse model (34). A similar effect was found in MASLD in a mouse study with phages against K. pneumoniae (85). Finally, bacteriophage abundance associates with the presence of HE in patients with cirrhosis, although bacterial composition appears more relevant to clinical outcomes (86). THE FUTURE The microbiome plays a significant role in human nutrient metabolism and immune regulation and is able to directly affect the liver through the portal vein. Some microbiome-targeted therapeutics are already used in liver disease, namely lactulose, rifaximin, and certain antibiotics. There is promise for microbiome-targeted therapeutics in treating or preventing liver disease, but future research is required in many areas before they are ready to use in patients (Table 2). The most promising microbiome therapeutics, such as FMT, defined consortium products, bacteriophages, and genetically engineered probiotics, should move forward to rigorous randomized clinical trials. Future research is required to better understand the efficacy, mechanism of action, and optimal delivery method of these microbiome-targeted therapeutics in liver disease. One trial studying the mode of delivery and dose in patients with HE (ClinicalTrials.gov NCT03796598) has completed enrollment, but results are not yet available. Even within 1 liver disease, patients have a diversity of microbiome compositions. Trial design and therapy selection will need to take this heterogeneity into account, either with prespecified microbiome analysis and stratification or post hoc analysis of the interaction between therapy and baseline microbiome.Table 2.: Considerations for future investigation of microbiome therapies in liver diseaseCONFLICTS OF INTEREST Guarantor of the article: Jasmohan S. Bajaj, MD, MS, FACG. Specific author contributions: P.P.B. and J.S.B.: planning, writing, editing. Financial support: This article appeared as part of the ACG Monograph on Microbiome Therapeutics. Unrestricted educational grants to support the monograph have been provided to the ACG Institute for Clinical Research and Education from Nestlé Health Science and Seres Therapeutics and Ferring Pharmaceuticals Inc. Potential competing interests: P.P.B.: research grant from Vedanta Biosciences, consulting for Nexilico. J.S.B.: research grants from Bausch, Cosmo, Grifols, and Mallinckrodt, consulting for Seres and Merz.

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