Reducing Intestinal Inflammation With Secondary Bile Acids

The relationship between dysbiosis, inflammation and disease is a burgeoning area of research ^{[reviewed in 1-4]}. A recently published paper by Sinha and colleagues examines the role of dysbiosis in Ulcerative Colitis^[5]. Their work highlights how a change in the relative abundance of a particular family of bacteria can influence intestinal inflammation, through perturbation of primary bile acid (PBA) metabolism by the gut microbiota. Furthermore, the insight provided opens up the potential for new treatment options for intestinal inflammation.

Primary bile acids (chendeoxycholic acid and cholic acid in humans; muricholic acid and cholic acid in mice) are synthesised in the liver from cholesterol, by a number of different cytochromes P450, with the last step in the synthetic pathway being conjugation with the amino acids glycine (most commonly in humans) or taurine (predominant in mice)^[6]. The conjugated PBAs are secreted from hepatocytes into the bile duct, via the bile salt export protein and from here may be transported into the duodenum or stored in the gall bladder. When PBAs reach the terminal ileum, they are reabsorbed by the apical sodium-dependent bile acid transporter (ASBT) expressed in the epithelium and recycled back to the liver.

In addition to their role as emulsifiers that facilitate the uptake of fats and fat-soluble dietary components, PBAs also have an anti-bacterial role, either through the direct disruption of bacterial cell walls^[7], or though transcriptional modulation of genes coding for anti-microbial proteins by intestinal epithelial cells, via the farsenoid X receptor^[8]. As a consequence of this, bacteria in the intestine need to be adapted to colonise and establish stable populations in the presence of high concentrations of PBAs; therefore, bacterial species that are found in the intestinal microbiome (predominantly Firmicutes and Bacteroidites phyla) express bile salt hydrolase (which deconjugates the PBA) in order to mitigate PBA toxicity.

Deconjugated bile acids are not recirculated by the ASBT, but instead progress into the large bowel. It is here that they are further metabolised by the microbiota, resulting in production of secondary bile acids (SBAs), including deoxycholic acid (DCA) and lithocholic acid (LCA) in humans and, murideoxycholic acid (MDCA) in mice. Clostridium and Eubacterium (both Firmicutes) possess the ability to produce SBAs. High colonic concentrations of SBAs have been proposed to promote inflammation and carcinogenesis^[9]. In contrast, perhaps, a reduction in Firmicutes (and an increase in Proteobacteria) has been associated with dysbiosis in IBD patients^[10], conditions which could reduce PBA metabolism and levels of SBAs. Like PBAs, SBAs can also modulate host cell function by interaction via receptor-mediated mechanisms. DCA and LCA are both potent ligands for the transmembrane G‑protein-coupled receptor 5 protein (TGR5)^[11] that is expressed by a range of cell types including colonic epithelial cells, brown adipose tissue cells, haematopoetic cells and skeletal muscle cells [6]. One specific function of SBA signalling through the TGR5 receptor is the secretion of glucagon-like peptide 1 (GLP-1) by L-cells in the colonic epithelium; it is thought that colonic L‑cells are “energy sensors”, modulating intestinal transit time via GLP‑1 secretion, in order to optimise nutrient absorption in the intestine^[12].

Sinha and colleagues’ initial work was to determine bile acid profiles (using high-performance liquid chromatography – mass spectrometry) in stool from patients with Ulcerative Colitis, and compare them with those from a control cohort; in this case, patients with Familial Adenomatous Polyposis Coli (FAP). Specifically, they studied patients (for both disease cohorts) that had undergone total colectomy and had surgery to form an ileo-anal pouch. Unfortunately, for many UC patients that have undergone this procedure, they continue to suffer with active inflammatory disease in the pouch mucosa (pouchitis); in contrast, FAP patients hardly ever demonstrate pouchitis. What the researchers found was that levels of the secondary bile acids, DCA and LCA, were significantly lower (almost absent) in UC patients compared to FAP patients. In contrast to this, the UC patients demonstrated significant elevation of the primary bile acid, CDCA. This bile acid profile was found to be consistent between UC patients that had pouchitis and those that did not. Meaningful differences in the profiles of other bile acids and metabolically-important molecules such as short-chain fatty acids were not identified.

It was concluded that the reduction in the two SBAs and increase in the PBA were the result of reduced metabolism of PBA, but that this was not based on overall bacterial abundance, which was similar between both groups of patients. UC patients, however, did show a reduced diversity of the microbiome in pouch stool samples (as determined by 16S rRNA marker gene sequencing), which was associated with a specific reduction in the Ruminococcaceae family, a member of the Firmicutes phylum. As mentioned previously, Firmicutes are responsible for SBA synthesis (from deconjugated PBAs) in the large bowel. The genes coding for the necessary enzymes for metabolism of PBAs to SBAs are grouped together in the bai (bile acid-inducible) operon. Metagenomic and metatranscriptomic analysis on the pouch samples was able to demonstrate that a reduction in bai operon gene expression was associated with UC (relative to FAP).

The lack of DCA and LCA in UC patient samples prompted the authors to investigate whether supplementation with exogenous SBAs could ameliorate colonic inflammation in three different mouse models: DSS-induced acute colitis; TNBS-induced acute-colitis and naïve T cell transfer-mediated colitis in immunodeficient mice. In the DSS model, 8-week old female C57BL/6 mice (substrain not identified) were treated with 2.5% DSS for 10 days, with rectal instillation of DCA, LCA or CDCA on days 4, 6 and 8. The TNBS colitis model was run using 8 week old female BALB/c mice, with the mice pre-sensitised by dermal application of 1% TNBS (volume not stated) in acetone, followed eight days later by instillation of 2.5mg of TNBS in 50% ethanol (in a 100μl volume). LCA (1mg in 150μl volume) was instilled into the colon on days 1, 3 and 5 post‑TNBS instillation, with a scheduled study endpoint on day 7. In the T cell transfer model, an 8-week old female, Rag2^-/- mice (background not specified) received 500,000 CD4⁺CD45RB^hiCD25^lo T cells (isolated from spleens of wild-type donors; strain not specified) by intravenous injection on study day 0; one group of mice received an additional 100,000 CD4⁺CD45RB^loCD25^hi T cells (regulatory T cells) as a treatment control. LCA (1mg in 150μl volume) was instilled into the colon twice a week from seven days post‑TNBS instillation, with a scheduled study endpoint on day 49.

Both SBAs were able to reduce DSS‑induced pathological changes including amelioration of body weight loss, large bowel shortening and histopathology score; the primary bile acid, CDCA, was not associated with any improvement in large bowel shortening or histopathology score. Levels of pro-inflammatory chemokines/cytokines (CCL5, CXCL10, IL‑17A and TNF‑α) were also reduced in DCA- and LCA-treated mice. Treatment with LCA was associated with similar improvements in disease, for both the TNBS and T cell transfer models of colitis.

In order to address the question of how the anti-inflammatory effect of LCA was mediated, the authors employed TGR5^-/- mice and repeated their studies using the DSS-induced acute colitis model. It was found that the ability of LCA to ameliorate colitis in this model was abolished in the absence of TGR5. Subsequently, a chimeric mouse was generated by irradiating (two doses of 9.5Gy, 4 hours apart) 8-week old C57BL/6 mice (substrain not identified) and subsequently reconstituting them with 2 million bone marrow-derived cells (via retro-orbital injection) isolated from TGR5^-/- donors (age- and sex-matched); a control group of mice that were reconstituted with cells derived from wild-type donors was also included. After allowing eight weeks for engraftment, mice were treated with DSS with or without LCA treatment, as previously. The chimeric mice were still resistant to the anticoagic effect of LCA, demonstrating that TGR5-expressing leukocytes were a key factor mediating LCA efficacy in this model.

In summary, pouchitis in UC patients was found to be associated with greatly reduced levels of secondary bile acids (in pouch stool samples) that are derived from the metabolism of primary bile acids by the intestinal microbiome. A reduction in bacteria of the Firmicutes phylum, and particularly the Ruminococcaceae family, was shown to be associated with this absence of secondary bile acids. As a result of these findings, it was hypothesised that secondary bile acids may have an anti-inflammatory role in the large bowel. This hypothesis was tested in mouse models of colitis, which demonstrated that exogenous lithocholic acid, administered as an enema was able to ameliorate disease, based on improved body weight, gross and microscopic pathological changes in the large bowel and changes in inflammatory chemokines/cytokines. The efficacy of the lithocholic acid was demonstrated to be dependent on the expression of the TGR5 receptor by immune cells.

This study highlights a new approach for treatment of Ulcerative Colitis in the clinic and in this regard, the authors have already initiated a clinical trial to study secondary bile acid function in patients with antibiotic-refractory pouchitis.

Services

Epistem offers preclinical models that replicate aspects of IBD in humans and can be used to generate target validation and mechanism of action data to confirm the efficacy of novel therapeutic agents. We offer acute models of IBD using DSS and TNBS as well as an adoptive T-cell transfer model for chronic IBD.

Epistem has a long history of evaluating a broad range of potential therapeutics with differing mechanisms of action and routes of delivery. All studies can be conducted in comparison to standard of care agents.

We evaluate disease severity and mechanisms of action in our IBD models by:

• Objective assessment of large bowel histopathology
• Measurement of large bowel length:weight ratio
• In-life assessment of body weight and diarrhoea
• Gene expression (RNA-Seq, microarray, qPCR) (including full data analysis package) and RNAscope analysis
• Immunohistochemistry analysis
• Flow Cytometry Analysis

Epistem also offers genomic services to provide a comprehensive assessment of the microbiome present at various sites in the body including the gut, to provide an understanding of the role of microbes in health and disease. Profiling the resident bacterial flora can reveal correlations with treatment response (and hence assist patient selection) and characterise the changes as a result of treatment. We deploy the following techniques to gain a comprehensive assessment of microbial communities:

• Shotgun metagenomic sequencing: Comprehensive DNA sequencing of all genes in all organisms present in each complex microbial sample, enabling both taxonomic and functional analysis.
• 16S rRNA sequencing: Sequencing of the 16S ribosomal RNA gene generating a taxonomic profile of species in a microbial sample.
• Microbial metatranscriptomics: RNA-sequencing of genes expressed within a microbial sample generating functional information such as immune interactions, resistance to therapeutic compounds and disease progression.