Exploring the Functional Roles of Resident Bacteria in Primary Sclerosis Cholangitis

Primary sclerosis cholangitis (PSC) is a rare, progressive disorder that damages the bile ducts and liver. The chronic inflammation in the bile ducts causes scarring that eventually narrows them and reduces the flow of bile. This blockage causes progressive damage to the liver. The disease is rare, unpredictable and unpreventable, and there are not any current therapies that can halt the disease progression. In addition, patients with PSC often have chronic biliary inflammation, cirrhosis and variable progression to liver failure.

Several studies have indicated that microbiotal alterations are associated with PSC, but there is little information about the functions and mechanisms of resident bacteria and their metabolites. A recent study appearing in Gut provides insight into the roles of these complex resident microbes and their association relevant to PSC patients by examining the microbial profiles of PSC patients in murine models.

“In our study, we used gnotobiotic and antibiotic approaches in specific pathogen-free (SPF) mdr2−/− mice to study the functional roles of resident bacteria in cholestatic liver disease,” explains Muyiwa Awoniyi, MD, a hepatologist at Cleveland Clinic and lead author of the study. “We identified functionally protective — Lachnospiraceae — and pathogenic resident bacteria — E. faecalis and E. coli, and we also used complementation studies to define in vivo mechanisms. We believe the insights we gained from this study could help target donor selection in future PSC-FMT trials, and hopefully guide selective fecal enrichment/depletion approaches so that we can improve the efficacy of future personalized microbial therapies.”

Study design

The study included germ-free mdr2−/− mice as a PSC murine model. The mice received either combined or single antibiotics (0.5 mg/mL vancomycin (Hospira), 1 mg/mL neomycin (Medisca) and 50 mg/kg metronidazole (G.D. Searle) in their drinking water. The mice were able to consume the water ad libitum, and they consumed on average 6–7 mL/mouse/day. The antibiotic mixture was diluted in deionized H₂O, sterilized through a 0.2 μm filter and replaced twice weekly for seven to 14 days.

The authors used the murine model to measure several markers of PSC including weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, fecal 16S rRNA gene profiling and metabolomic endpoints.

Findings

The authors found that relative to their control counterparts (wild-type [WT] specific pathogen-free [SPF] control C57BL/6 mice), the 6–7-week-old germ-free (GF) mdr2−/− mice had decreased weight gain, twice the amount of alkaline phosphatase and 15 times the amount of total bilirubin. The GF mdr2−/− mice had rapid onset of cholestasis beyond six weeks (which was not observed in the control mice). By eight weeks, the GF mdr2−/− mice had 100% mortality with increasing hepatic bile acid (BA) accumulation and cholestasis. However, early fecal microbial transfer (FMT) by four weeks reduced the severity of periportal inflammation and fibrosis in the GF mdr2−/− mice.

“We found that early life early life exposure to resident microbiota in the mice promoted survival and decreased hepatic inflammation, ductular reaction and fibrosis,” says Dr. Awoniyi. “The 100% mortality was due to lack of microbial modulation of toxic progressive hepatic and plasma bile accumulation. However, the fact that the mice could be rescued with fecal microbiota transfer from syngeneic specific confirmed the importance of resident microbiota and its protective role in this model.”

Exploring further

To validate the potential protective role of the host microbiota, beneficial bacterial groups and metabolites, the authors developed an antibiotic depletion model in the SPF mdr2−/− mice. The antibiotic was administered using a broad-spectrum antibiotic cocktail that targeted resident Gram-positive, Gram-negative and anaerobic bacteria for 14 days. The antibiotic similarly depleted microbial abundance in mdr2−/− and WT control mice by α-diversity and fecal qPCR.

Using liquid chromatography-mass spectrometry, the authors analyzed the metabolome of serum and cecal contents from antibiotic-treated versus untreated mdr2−/− mice to identify any potential metabolic profiles that would have protective microbial effects. They used those metabolic profiles to identify some of the functional properties of the bacteria, Clostridiaceae and Lachnospiraceae, that could rescue inflammation/fibrosis driven by antibiotic depletion of protective microbial subsets.

“Antibiotic-induced dysbiosis of SPF mdr2−/− worsened hepatobiliary disease by non-bacteremic hepatic translocation of Enterococcus faecalis and Escherichia coli,” explains Dr. Awoniyi. “This exacerbation resulted in a faster accumulation of detrimental hepatic bile acid, but the FXR signaling did not change. Inhibition of ileal bile salt transporter, Asbt, helped reduce hepatic inflammation and fibrosis in antibiotic-treated SPF mdr2−/− mice, which validated our microbial modulation of bile acid homeostasis. Based on these findings, we wanted to determine if certain antibiotics have different effects on mdr2−/− hepatobiliary disease.”

In order to identify the various microbial populations that are responsible for protecting mdr2−/− mice, the authors tested several antibiotic components. Among these were vancomycin, neomycin and metronidazole that target Gram-positive, Gram-negative and anaerobic bacteria, respectively.

“Of the antibiotic components that we tested, vancomycin decreased fecal bacterial α-diversity most significantly,” says Dr. Awoniyi. “It also maintained the most divergent bacterial populations and had the maximum β-diversity separation compared with the untreated controls. Vancomycin and metronidazole both decreased putative protective Clostridiaceae, but only vancomycin weakened Lachnospiraceae. Neomycin did not significantly affect either family. Furthermore, compared to the control mice, we found that vancomycin was the only one that significantly increased histological liver fibrosis scores, hepatic profibrotic co1α1 and timp-1 expression, although it did not increase hepatic hydroxyproline.”

Dr. Awoniyi notes that one of the other big findings from the study was that Clostridium strains did not significantly alter the body weight or liver enzymes in antibiotic-pretreatedmdr2−/− mice. This is despite efficacy of these strains in multiple colitis models. After that finding, the group pivoted to assessing the biological activity of potentially protective Lachnospiraceae, which was also depleted by antibiotics and vancomyocin. They found that serial treatment with a protective colonic 23 strain Lachnospiraceae consortium (Lachno) restored weight gain and reduced histological liver inflammation, ductular reaction and fibrosis but did not decrease serum biochemistries. Furthermore, reconstituting Lachno in GF mdr2−/− mice resulted in reduced liver fibrosis and did not affect mortality or liver inflammation when compared with non-colonized GF mdr2−/− mice.

“We wanted to learn more about the hepatic changes caused by E. faecalis and E. coli, so we isolated hepatic translocated E. faecalis and E. coli in antibiotic-pretreated and gnotobiotic conditions and compared them against controls,” says Dr. Awoniyi. “We found that Lachno treatment prevented E. faecalis and E. coli liver translocation and E. faecalis fecal concentrations. We also assessed the lethality of these bacteria gnotobiotic mdr2−/− mice by orally inoculating E. faecalis and E. coli isolates with and without Lachno. The E. faecalis and E. coli dual-associated mice had earlier mortality relative to the control mice, but survival improved with cocolonization with Lachno relative to control GF mdr2−/− mice. We were able to determine that the short-chain fatty acids produced by Lachno have hepatic antifibrogenic effects.”

In human cohorts, the authors found that fecal E. faecalis and Enterobacteriaceae were positively associated and Lachnospiraceae was negatively associated with PSC clinical severity measured by Mayo risk score. Trends existed towards increased Enterobacteriaceae and reduced Lachnospiraceae in patients with antibiotics compared with those without. “These clinical results are similar to what we observed in our mdr2−/− mice, which helps reinforce the clinical relevance of our murine models,” says Dr. Awoniyi.

Takeaways

Since there is so much conflicting data circulating around PSC, the authors hope the findings from their study will help clinicians better understand the function of complex resident microbiomes and how they relate to tailoring care for patients.

“We know that antibiotics can have both beneficial and detrimental outcomes in clinical and experimental settings, but we didn’t really understand the mechanisms at play there,” says Dr. Awoniyi. “We hope the findings from this study show how antibiotics interact in these microbiomes. Our mdr2−/− results suggest that broad-spectrum antibiotics should be administered with caution in PSC patients due to their potential for exacerbating the disease and contributing to recurrent cholangitis by promoting enterohepatic bacterial translocation.”

The authors believe the findings from their models have direct translational implications for how to approach treatment for patients with PSC. Better patient selection through screening may improve responses to selective depletion of disease-inducing subsets and/or augmentation of protective bacteria or secreted metabolites. They believe their gnotobiotic model can be used to further understand hepatobiliary, microbial impacts of humanized fecal transfer. The study also shed light on the potential in vivo roles of bacteria, such as Lachnospiraceae, which require additional protective resident bacteria to boost various protective effects transferred by fecal microbiota transplantation. Looking ahead, the group hopes to explore the impact of macrophage phenotypes, downstream SCFA targets and other potentially protective bacteria on experimental hepatobiliary inflammation and fibrosis.