Pulmonary Arterial Hypertension Patients Have a Proinflammatory Gut Microbiome and Altered Circulating Microbial Metabolites

Abstract

Rationale: Inflammation drives pulmonary arterial hypertension (PAH). Gut dysbiosis causes immune dysregulation and systemic inflammation by altering circulating microbial metabolites; however, little is known about gut dysbiosis and microbial metabolites in PAH. Objectives: To characterize the gut microbiome and microbial metabolites in patients with PAH. Methods: We performed 16S ribosomal RNA gene and shotgun metagenomics sequencing on stool from patients with PAH, family control subjects, and healthy control subjects. We measured markers of inflammation, gut permeability, and microbial metabolites in plasma from patients with PAH, family control subjects, and healthy control subjects. Measurements and Main Results: The gut microbiome was less diverse in patients with PAH. Shannon diversity index correlated with measures of pulmonary vascular disease but not with right ventricular function. Patients with PAH had a distinct gut microbial signature at the phylogenetic level, with fewer copies of gut microbial genes that produce antiinflammatory short-chain fatty acids (SCFAs) and secondary bile acids and lower relative abundances of species encoding these genes. Consistent with the gut microbial changes, patients with PAH had relatively lower plasma concentrations of SCFAs and secondary bile acids. Patients with PAH also had enrichment of species with the microbial genes that encoded the proinflammatory microbial metabolite trimethylamine. The changes in the gut microbiome and circulating microbial metabolites between patients with PAH and family control subjects were not as substantial as the differences between patients with PAH and healthy control subjects. Conclusions: Patients with PAH have proinflammatory gut dysbiosis, in which lower circulating SCFAs and secondary bile acids may facilitate pulmonary vascular disease. These findings support investigating modulation of the gut microbiome as a potential treatment for PAH.

Keywords: dysbiosis; metabolites; microbiome; pulmonary arterial hypertension.

Figure 1.

Gut microbiome diversity is reduced and distinct in patients with pulmonary arterial hypertension (PAH) compared with control subjects. (A and B) Shannon diversity (A) and Chao1 (B) indices in healthy and family (Fam) control subjects and patients with PAH. (C) Bray-Curtis pairwise dissimilarity indices are shown in a PCoA plot for healthy control subjects, Fam control subjects, and patients with PAH. Analysis was performed of similarity statistics of Bray-Curtis pairwise dissimilarity indices, comparing all groups (table shows R and P values). (D) Bray-Curtis dissimilarity between PAH–Fam pairs that cohoused (true) was significantly less than the difference between randomly generated PAH–Fam pairs (random). Circles denote healthy control subjects, squares denote Fam control subjects, and triangles denote patients with PAH. PCoA = principal coordinate analysis.

Figure 2.

Patients with pulmonary arterial hypertension (PAH) have a distinct gut microbiome signature at the phylogenetic level compared with healthy and family control subjects. (A and B) Linear discriminant analysis of effect size of shotgun metagenomic data between (A) healthy control subjects and patients with PAH and (B) family control subjects and patients with PAH. Gray bars denote healthy control subjects, blue bars denote family control subjects, and red bars denote patients with PAH. CAG = coabundance gene.

Figure 3.

Shannon diversity index correlates with the severity of pulmonary vascular disease but not with right ventricular function in patients with pulmonary arterial hypertension. (A–C) There are significant negative linear correlations between Shannon diversity index and (A) mPAP, (B) PVR, and (C) PAC. (D–F) There are no significant linear correlations between Shannon diversity index and (D) RVFAC, (E) CO, and (F) RA pressure. CO = cardiac output; mPAP = mean pulmonary arterial pressure; PAC = pulmonary arterial compliance; PVR = pulmonary vascular resistance; R² = coefficient of determination; RA = right atrial; RVFAC = right ventricular fractional area change; WU = Wood units.

Figure 4.

Targeted metabolomic analysis of patients with pulmonary arterial hypertension (PAH) and family (Fam) and healthy control subjects. (A) Hierarchical cluster analysis of targeted metabolomics of circulating concentrations of short-chain fatty acids and bile acids in healthy control subjects, Fam control subjects, and patients with PAH shows a clear separation of patients with PAH from healthy and Fam control subjects. The heatmap displays the group averages of the microbial metabolites for each group. The P values were generated by comparing the individual microbial metabolite plasma concentrations among the study groups using one-way ANOVA with a Tukey post hoc analysis to correct for multiple comparisons or a Kruskal-Wallis test with Dunn’s multiple-comparisons test. (B) There was no significant difference in plasma trimethylamine N-oxide (TMAO) concentrations between healthy control subjects and patients with PAH. Data are not available for TMAO concentrations in Fam controls. *P < 0.05, **P ⩽ 0.01, and ****P ⩽ 0.0001. ns = not significant (P ≥ 0.05).

Figure 5.

Hierarchical cluster analysis of encoding genes for enzymes that produce short-chain fatty acids, bile acid deconjugation, and trimethylamine from shotgun metagenomic data of the gut microbiomes showed clear separation of patients with PAH from healthy control subjects, with family control subjects in the middle. The heatmap displays the group averages of relative gene copies per million for each group. The P values were generated by comparing the relative number of gene copies per million among the study groups using one-way ANOVA with a Tukey post hoc analysis to correct for multiple comparisons or a Kruskal-Wallis test with Dunn’s multiple-comparisons test. *P < 0.05, **P ⩽ 0.01, ***P ⩽ 0.001, and ****P ⩽ 0.0001. CoA = coenzyme A; ns = not significant (P ≥ 0.05); PAH = pulmonary arterial hypertension.

Figure 6.

(A–C) Hierarchical cluster analysis of the relative abundance of species with the encoding genes for the enzyme involved in the synthesis of butyrate, including (A) carboxylesterase, (B) butyrate kinase, and (C) phosphate butyryl transferase. The heatmap displays the group averages of relative abundances of species for each group. The P values were generated by comparing the relative abundances of the species among the groups using one-way ANOVA with a Tukey post hoc analysis to correct for multiple comparisons or a Kruskal-Wallis test with Dunn’s multiple comparisons test. *P < 0.05, **P ⩽ 0.01, ***P ⩽ 0.001, and ****P ⩽ 0.0001. CAG = coabundance gene; Fam = family; ns = not significant (P ≥ 0.05); PAH = pulmonary arterial hypertension.

Figure 7.

(A and B) Hierarchical cluster analysis of the relative abundance of species with the encoding genes for (A) amidase (an enzyme involved in the production of valerate and propionic acid) and (B) propionate CoA transferase (an enzyme involved in acetate production). The heatmap displays the group averages of relative abundances of species for each group. The P values were generated by comparing the relative abundances of the species among the groups using one-way ANOVA with a Tukey post hoc analysis to correct for multiple comparisons or a Kruskal-Wallis test with Dunn’s multiple-comparisons test. *P < 0.05, **P ⩽ 0.01, ***P ⩽ 0.001, and ****P ⩽ 0.0001. CoA = coenzyme A; Fam = family; ns = not significant (P ≥ 0.05); PAH = pulmonary arterial hypertension.

Figure 8.

(A–C) Hierarchical cluster analysis of the relative abundance of species with the encoding genes for (A) betaine reductase and (B) choline trimethylamine-lyase (both enzymes involved in the synthesis of trimethylamine) and (C) choloylglycine hydrolase (an enzyme involved in bile acid conjugation). The heatmap provides the group averages of relative abundances of species for each group. The P values were generated by comparing the relative abundances of the species between the groups using one-way ANOVA with a Tukey post hoc analysis to correct for multiple comparisons or a Kruskal-Wallis test with Dunn’s multiple-comparisons test. *P < 0.05, **P ⩽ 0.01, ***P ⩽ 0.001, and ****P ⩽ 0.0001. Fam = family; ns = not significant (P ≥ 0.05); PAH = pulmonary arterial hypertension.