Altered Gut Microbial Metabolism of Essential Nutrients in Primary Sclerosing Cholangitis

Abstract

Background & aims: To influence host and disease phenotype, compositional microbiome changes, which have been demonstrated in patients with primary sclerosing cholangitis (PSC), must be accompanied by functional changes. We therefore aimed to characterize the genetic potential of the gut microbiome in patients with PSC compared with healthy controls (HCs) and patients with inflammatory bowel disease (IBD).

Methods: Fecal DNA from 2 cohorts (1 Norwegian and 1 German), in total comprising 136 patients with PSC (58% with IBD), 158 HCs, and 93 patients with IBD without PSC, were subjected to metagenomic shotgun sequencing, generating 17 billion paired-end sequences, which were processed using HUMAnN2 and MetaPhlAn2, and analyzed using generalized linear models and random effects meta-analyses.

Results: Patients with PSC had fewer microbial genes compared with HCs (P < .0001). Compared with HCs, patients with PSC showed enrichment and increased prevalence of Clostridium species and a depletion of, for example, Eubacterium spp and Ruminococcus obeum. Patients with PSC showed marked differences in the abundance of genes related to vitamin B6 synthesis and branched-chain amino acid synthesis (Q_fdr < .05). Targeted metabolomics of plasma from an independent set of patients with PSC and controls found reduced concentrations of vitamin B6 and branched-chain amino acids in PSC (P < .0001), which strongly associated with reduced liver transplantation-free survival (log-rank P < .001). No taxonomic or functional differences were detected between patients with PSC with and without IBD.

Conclusions: The gut microbiome in patients with PSC exhibits large functional differences compared with that in HCs, including microbial metabolism of essential nutrients. Alterations in related circulating metabolites associated with disease course, suggesting that microbial functions may be relevant for the disease process in PSC.

Keywords: Branched Chain Amino Acids; Microbiome; Primary Sclerosing Cholangitis; Vitamin B.

Figure 1. Overall bacterial taxonomic composition and gene richness in PSC patients and controls.

Non-constrained ordination of genus-level Bray-Curtis dissimilarities in the microbiome of (A) patients with PSC, as well as healthy and IBD controls, in the merged cohort, and in the (B) the German and (C) the Norwegian cohorts separately, showing shifts in the global microbiome composition. (D) Number of observed genes in PSC compared to controls. All group centroids in A-C represent the arithmetic mean of the groups' respective points in the ordination, ellipses represent 95% confidence level of the multivariate t-distributions. Red horizontal bar in D represents the median. GER, Germany; HC, healthy control; IBD, inflammatory bowel disease; NOR, Norway; MDS, multiple dimension scale; PSC, primary sclerosing cholangitis.

Figure 2. Differences in species composition in PSC compared to healthy controls.

(A) Species that were differently abundant and (B) species with different prevalence in PSC and healthy controls. Species are colored by phyla affiliation. (C) Bacterial abundance co-correlation network in healthy controls and (D) PSC patients. In A&B the triangle/bar in the forest plot indicate log(OR)/SE. Red and blue lines in C&D indicate positive and negative correlations between species, respectively. Node-size reflects the square-root transformed median relative abundances in each phenotype, and encircled nodes mark articulations points. Only correlations with r>0.4 are shown, and increasing correlation coefficient are reflected by increasing line thickness. PSC, primary sclerosing cholangitis; HC, healthy controls. *Q _fdr<.05, ** Q _fdr<.005, *** Q _fdr<.0005.

Figure 3. Alterations in metabolic pathways in the microbiome of PSC.

Heatmap and forest plot showing significant differences in microbiome metabolic pathways (Metacyc pathways, Q _fdr<.05) between PSC and healthy controls from the individual cohorts (two left columns in the heatmap) and the meta-analysis (right column in the heatmap and the forest plot). Triangle/bar in forest plot indicate log(OR)/SE. HC, healthy controls; PSC, primary sclerosing cholangitis. *Q _fdr<.05, **Q _fdr<.005, ***Q _fdr<.0005.

Figure 4. Genetic potential of the gut microbiome in patients with PSC.

Dot-plots showing relative abundance of enzyme families (level-4 Enzyme Commission (EC) categories) related to microbial metabolism of branched chain amino acids (A) valine, (B) leucine and (C) isoleucine, (D) the active form of vitamin B6 (pyridoxal 5’-phosphate, PLP) and (E) the active forms of vitamin B2, flavin mononucleotide (FMN), and (F) flavin adenine dinucleotide (FAD), and (G) synthesis of riboflavin (vitamin B2) and related metabolites, (H-I) ammonia production and (J) antibiotic resistance. Red bar marks the median. EC, level-4 Enzyme Commission; FAD, flavin adenine dinucleotide; HC, healthy controls; IBD, inflammatory bowel disease; PLP, pyridoxal 5’-phosphate; PSC, primary sclerosing cholangitis. *Q _fdr<.05, **Q _fdr<.01, ***Q _fdr<.001.

Figure 5. Microbiome-related plasma metabolites and liver-transplantation free survival.

(A) The active vitamin B6 metabolite pyridoxal 5’-phosphate (PLP), (B) isoleucine, (C) leucine and (D) valine in plasma of patients with PSC compared to healthy controls. Kaplan-Meier plots showing that low levels (1^st quartile) of (E) PLP, (F) isoleucine, (G) leucine and (H) valine were all associated with reduced liver-transplantation free survival in PSC. HC, healthy controls; PLP, pyridoxal 5’-phosphate; PSC, primary sclerosing cholangitis.

Figure 6. Separation of clinical phenotypes using random forest classification.

(A) PSC vs. healthy controls and (B) PSC vs. IBD. Results using all available species (red line) or only the significant species hits (blue line). AUC, area under the curve; F1, F1 measure; IBD, inflammatory bowel disease; MCC, Matthew’s correlation coefficient (range: -1– +1); PSC, primary sclerosing cholangitis.