Higher levels of BA synthesis in the subgroup of IBS-D patients with excessive BA excretion. A total of 345 IBS-D adults, defined by predominant bowel habits based on the Bristol stool scale and defecation frequency (34, 35), were recruited at 2 Chinese medical clinics affiliated with the School of Chinese Medicine, Hong Kong Baptist University. Of these participants, 290 patients completed all biochemical tests and consented to voluntarily provide biospecimens (blood and stools) for the study. Referring to the approximately 30% of pooled prevalence of excess BA excretion in the IBS-D population estimated from published studies (9–12), 91 healthy controls (HCs) were recruited and 89 provided biospecimens. As shown in Table 1, the IBS-D cohort displayed a significant increase in defecation frequency and total fecal BAs, whereas it exhibited decreased fecal consistency compared with that of HC subjects. The level of total fecal BA excretion showed a skewed distribution (P < 0.05 by the Shapiro-Wilk test) in the IBS-D and HC groups (Figure 1A).

Figure 1 Alteration of fecal BA profiles and serum BA synthetic indicators in IBS-D patients. (A) Histogram of the distribution of total fecal BA levels in healthy controls (n = 89) and IBS-D patients (n = 290). Based on the 90th percentile of healthy total fecal BA level, 25% of IBS-D patients (n = 71) with excessive BA excretion were grouped as BA+IBS-D and the other patients (n = 219) were classified as BA–IBS-D. (B and C) Concentrations of serum 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19). (D–F) The severity of bowel symptoms between IBS-D subgroups assessed by defecation frequency (D), Bristol stool scale (E), and IBS severity scoring system (IBS-SSS) (F). (G and H) Absolute contents of fecal dominant BAs. (I) Proportions of fecal dominant BAs. Only BAs constituting greater than 1% of the total BA pool are shown in the legend. Differences in phenotypic scores between IBS-D subgroups were analyzed by the Mann-Whitney test, and BA-related indices were evaluated among 3 groups by the Kruskal-Wallis test. The box-and-whisker plots show the mean (horizontal lines), 5th–95th percentile values (boxes), and SEM (whiskers). *P < 0.05, ***P < 0.005 compared with the HC group; #P < 0.05, ##P < 0.01, ###P < 0.005 compared with the BA–IBS-D group. TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; GCA, glycocholic acid; GCDCA, glycocheno-deoxycholic acid; GUDCA, glycoursodeoxycholic acid; GHDCA, glycohyodeoxycholic acid; GDCA, glycodeoxycholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; 7-KDCA, 7-ketodeoxycholic acid; UDCA, ursodeoxycholic acid; HDCA, hyodeoxycholic acid; KLCA, ketolithocholic acid; HCA, hyocholic acid; ωMCA, ω-muricholic acid; isoLCA, isolithocholic acid; ACA, allocholic acid.

Table 1 The demographics and clinical characteristics of IBS-D patients based on total fecal BA excretion

Twenty-five percent of IBS-D patients (71 of 290) were found to have an excess of total BA excretion in feces (≥10.61 μmol/g) by the 90th percentile cutoff value as determined from the HC group. These patients were classified as BA+IBS-D. The others with normal fecal BA excretion (<10.61 μmol/g) were grouped as BA–IBS-D. Compared with the HC and BA–IBS-D groups, BA+IBS-D patients also exhibited increased C4 and decreased FGF19 in sera, as well as increased severity of diarrheal symptoms (Table 1 and Figure 1, B–F). Correlation analysis revealed that the total fecal BA levels were positively associated with serum C4 levels and scores of diarrheal symptoms (Bristol stool scale and defecation frequency) but inversely correlated with serum FGF19 levels in the BA+IBS-D group (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI130976DS1). These results demonstrate that enhanced BA synthesis exists in IBS-D patients, accompanied by excessive BA excretion and increased severity of diarrheal symptoms.

Alteration of individual BA levels was also observed in the sera and feces of BA+IBS-D patients. Serum BA profiles revealed that glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GUDCA), and chenodeoxycholic acid (CDCA) were significantly elevated in both absolute amounts and relative proportions in BA+IBS-D patients compared with those of the HC group (Supplemental Figure 1). In addition, BA+IBS-D patients had an increased absolute level of ursodeoxycholic acid (UDCA) and a reduced relative proportion of glycohyodeoxycholic acid (GHDCA) in sera. The fecal BA pool of all recruits was largely composed of free BAs, as previously described (36), of which cholic acid (CA), CDCA, deoxycholic acid (DCA), lithocholic acid (LCA), 7-ketodeoxycholic acid (7-KDCA), UDCA, and ω-muricholic acid (ωMCA) showed significant increases in their absolute amounts in the BA+IBS-D group compared with the HC group (Figure 1, G and H). Meanwhile, the proportions of CA, CDCA, UDCA, and 7-KDCA increased in total fecal BAs, whereas the proportions of LCA and 12-KLCA decreased in the BA+IBS-D group (Figure 1I). In addition, among the abundant conjugated BAs, GCDCA was slightly increased in feces of BA+IBS-D patients, while GUDCA showed no difference. However, BA–IBS-D patients showed similar serum and fecal BA compositions as those of the HC subjects. These metabolic results reveal an altered composition of microbiota-derived BAs (e.g., GUDCA, UDCA, 7-KDCA, etc.) in sera and feces of BA+IBS-D patients, indicating that an abnormality in BA-transforming gut microbiota might contribute to the cause of this condition.

Association of a Clostridia-rich microbiota with increased BA synthesis and excretion in BA+IBS-D patients. Fecal metagenomic data were successfully obtained from 84 HC subjects, 70 BA+IBS-D patients, and 207 BA–IBS-D patients. An average of 6.04 gigabases of high-quality sequencing reads was obtained from each sample, and an average of 63.7% reads per sample were successfully mapped (Supplemental Table 2). In comparison with the HC and BA–IBS-D groups, fecal microbial communities exhibited a higher Bray-Curtis dissimilarity in the BA+IBS-D group, but without differences in total gene count and Shannon index (Figure 2A and Supplemental Figure 2, A and B). These results indicate the unchanged microbial richness within all included subjects, but a larger instability of the enteric ecosystem in the BA+IBS-D subgroup.

Figure 2 The association between Clostridia-rich microbiota and the levels of BA synthesis and excretion in the IBS-D cohort. (A) Microbial β-diversities measured by Bray-Curtis dissimilarity. (B) The ratio of Firmicutes to Bacteroidetes (F/B). (C–E) The relative abundances of BA-transforming genomes and bacteria. (F and G) Spearman’s correlation between bacterial abundances and biochemical indices in the IBS-D cohort. The metagenomic data set was obtained from 277 IBS-D and 84 HC fecal samples. Differential taxa and genes among 3 groups were analyzed with the Benjamin-Hochberg method. The box-and-whisker plots show the mean (horizontal lines), 5th–95th percentile values (boxes), and SEM (whiskers). *P < 0.05, ***P < 0.005 compared with the HC group; #P < 0.05; ##P < 0.01 compared with the BA–IBS-D group. Statistical significance for Spearman’s correlation was defined as P < 0.05. ALT, alanine aminotransferase; TG, triglycerides; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TC, total cholesterol; cgh, gene coding choloylglycine hydrolase; hdhA, gene encoding 7α-hydroxysteroid dehydrogenase; bai A/B/CD/E/F/H/I, BA-inducible genes A/B/CD/E/F/H/I; C. scindens, Clostridium scindens; C. leptum, Clostridium leptum; R. torques, Ruminococcus torques; R. obeum, Ruminococcus obeum; R. gnavus, Ruminococcus gnavus; B. hansenii, Blutia hansenii; F. mortiferum; Fusobacterium mortiferum; F. varium, Fusobacterium varium; E.coli, Escherichia coli; B. xylanisolvens, Bacteroides xylanisolvens; B. intestinalis, Bacteroides intestinalis.

Principal coordinate analysis revealed an overlap of fecal microbial communities among IBS-D and HC subjects (Supplemental Figure 2C); however, a different microbial profile was found in BA+IBS-D patients in comparison with either HC or BA–IBS-D subjects at different taxonomic levels (Supplemental Tables 3–5). Specifically, the relative abundances of the phyla Firmicutes, Actinobacteria, Fusobacteria, and Proteobacteria were increased, while that of Bacteroidetes was decreased in BA+IBS-D patients (Supplemental Figure 2D). Accordingly, the ratio of Firmicutes to Bacteroidetes was significantly elevated (Figure 2B). At the genus level, the abundances of Clostridia bacteria, including Ruminococcus, Clostridium, Eubacterium, and Dorea were significantly increased in BA+IBS-D fecal microbiota (Supplemental Figure 2E). The abundances of Bifidobacterium, Escherichia, and Bilophila were also increased. However, the abundances of Alistipes and Bacteroides were reduced significantly compared with those of HC or BA–IBS-D fecal microbial communities.

The alteration in the bacterial composition of the BA+IBS-D group was also associated with variation in BA-transforming genomes (Figure 2C and Supplemental Table 6). Reduced abundances of Alistipes and Bacteroides were mainly associated with a decreased abundance of the cgh gene (Figure 2D), which encodes the BA-deconjugating enzyme choloylglycine hydrolase (36). However, an elevated abundance of the hdhA gene, encoding 7α-hydroxysteroid dehydrogenase (7α-HSDH) (36), was attributed to increases in Escherichia, Fusobacterium, Blautia, Ruminococcus, and Clostridium species (Figure 2E). Moreover, Clostridium scindens and an unclassified Lachnospiraceae species largely contributed to higher abundances of the genes baiCD and baiH (Figure 2E), which are known to encode 7-dehydroxylases (36). Correlation analysis revealed that the abundances of Clostridia genera and C. scindens species were positively correlated with the concentrations of total fecal BAs and serum C4, but negatively correlated with the serum FGF19 level (Figure 2, F and G). These results suggest a specific Clostridia-rich microbiota in the BA+IBS-D group, with different genomes for BA deconjugation, C7 isomerism, and dehydroxylation. Given the importance of gut microbiota in maintenance of host BA metabolism (22), the significant associations between gut bacteria and BA indices in the IBS-D cohort suggest the possibility that the Clostridia-rich microbiota may influence patients’ BA synthesis and excretion.

Enhancement of BA synthesis and excretion in pseudo-germ-free mice transplanted with Clostridia-rich fecal microbiota. To investigate the effects of the Clostridia-rich microbiota on BA synthesis and excretion, transplantation of BA+IBS-D fecal microbiota to pseudo-germ-free mice was conducted (Figure 3A and Supplemental Figure 3A). One week after microbial transplantation, BA+IBS-D microbiota mouse recipients displayed shortened GI transit time and increased fecal water content (Figure 3B) that were similar to the diarrheal symptoms of their donors. Fecal profiles of BA-transforming bacteria in recipients were similar to those of BA+IBS-D donors, with a reduced abundance of Bacteroidetes and elevated abundances of Firmicutes, Clostridium cluster XIVa, and C. scindens (Figure 3C). An in vitro transforming-activity assay with detection of the ratio of BA products to substrates found that the cecal microbiota isolated from mouse recipients exhibited a decreased deconjugating capability but a slight increase in 7-HSDH and 7α-dehydroxylation (Supplemental Figure 3B). These results are consistent with the BA-transforming activities in the fecal microbiota of the human BA+IBS-D donors (Supplemental Figure 3C).

Figure 3 Excessive BA synthesis and excretion in mouse recipients receiving BA+IBS-D fecal microbiota. (A) Experimental procedure for fecal microbiota transplantation (FMT) in antibiotic cocktail–induced (ABX-induced) pseudo-germ-free mice (n = 6/group). Mice that received fecal microbiota of HC donors were grouped as ABX+HC, and mice treated with fecal microbiota from BA+IBS-D and BA–IBS-D donors were classified as ABX+BA+ and ABX+BA–, respectively. (B) The GI transit time and fecal water contents of mouse recipients. (C) Relative levels of BA-related bacteria in feces of donors and mouse recipients based on qPCR analysis. (D and E) The levels of total fecal BAs and serum C4 in mouse recipients. (F) Hepatic BA profiles of mouse recipients. (G) Relative gene expression of BA synthetic regulators in the hepatic tissues of mouse recipients. Differential BA-related phenotypes, bacteria, and genes are shown as mean ± SEM. BA metabolites are expressed with 5th–95th percentile values. Differences were assessed with the Kruskal-Wallis test. *P < 0.05, **P < 0.01 compared with the ABX+HC group. Cyp7a1, Cyp8b1, Cyp7b1, Cyp27a1, Fxr, Shp, Fgfr4, and Klb represent the mRNAs for the proteins cholesterol 7α-hydroxylase, sterol 12α-hydroxylase, steroid 7α-hydroxylase, sterol 27-hydroxylase, farnesoid X receptor, small heterodimer partner, fibroblast growth factor receptor 4, and Klothoβ, respectively.

Metabolic analysis showed increases in total fecal BAs and serum C4 in the BA+IBS-D microbiota recipients, along with elevated taurine-conjugated BAs (TβMCA, TCA, TCDCA, and TUDCA) in the liver and ileal lumen (Figure 3, D–F and Supplemental Figure 3D). Furthermore, tissue mRNA analysis showed increased expression of hepatic Cyp7a1 and Cyp8b1 but reduced expression of hepatic Shp and ileal Fgf15 in BA+IBS-D microbiota recipients (Figure 3G and Supplemental Figure 3E). Increased expression of hepatic CYP7A1 and decreased expression of ileal FGF15 were also confirmed at the protein level (Supplemental Figure 3F). Additionally, the mRNA analysis of hepatic FGF19/15 receptor complexes (FGFR4 and Klothoβ [KLB]) and ileal BA active transporters (apical sodium bile acid transporter, ASBT; multidrug resistance–associated protein 2 and 3 (MRP2 and -3); and organic solute transporter α/β (OSTA and -B) showed no changes among recipient groups when compared with those of HC microbiota recipients (Figure 3G and Supplemental Figure 3E). These results demonstrate that the Clostridia-rich microbiota of BA+IBS-D donors could induce diarrhea-like phenotypes and enhance BA synthesis and excretion in mouse recipients, but these effects are probably independent of ileal BA absorption and hepatic feedback inhibition.

Enhancement of hepatic BA synthesis and excretion in mice with colonization of Clostridium species. To further clarify the effects of Clostridium species on BA synthesis and excretion, we introduced the C. scindens strain (1 × 108 CFU/mL) and specifically inhibited Clostridium species using vancomycin (0.1 mg/mL) in 2 separate groups of conventional mice (Figure 4A). Colonization of C. scindens significantly decreased the fecal consistency in mice when compared with mice treated with vehicle. Vancomycin treatment significantly attenuated the GI transit (Figure 4B). Moreover, qPCR analysis showed a significant increase in C. scindens abundance in the cecum of C. scindens–colonized mice compared with that of controls (Figure 4C). The transforming-activity assay showed a slightly reduced deconjugating activity and a significantly increased activity of 7-HSDH in the cecal microbiota of C. scindens recipients (Supplemental Figure 4A). In contrast, the abundances of Clostridium species in the cecum of vancomycin-treated mice significantly decreased, along with the reduction of in vitro activities of 7-HSDH and 7-dehydroxylases, and an increase in BA deconjugating capability (Figure 4C and Supplemental Figure 4A).

Figure 4 Dysregulation of BA synthesis and excretion in mice with manipulation of Clostridium species. (A) Manipulation of Clostridium species in conventional mice (n = 5–6/group) through introduction of C. scindens strain (C. s) or administration of vancomycin (0.1 mg/mL). (B) The GI transit time and fecal water contents of Clostridium-treated mice. (C) Relative levels of the fecal Clostridial bacteria (C. XIVa) and C. scindens measured by qPCR analysis. (D and E) The levels of total fecal BAs and serum C4. (F) The BA profile in the mouse liver. (G) Relative gene expression of BA synthetic regulators in the hepatic and ileal tissues. Differential BA-related phenotypes, bacteria, and genes are shown as mean ± SEM. BA metabolites are expressed with 5th–95th percentile values. Statistical significance was determined with the Kruskal-Wallis test. *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the C. scindens group. CTR, control.

Metabolic analysis found that the total fecal BA and serum C4 concentration significantly increased in C. scindens–colonized mice, while these were reduced in vancomycin-treated mice (Figure 4, D and E). Consistently, the concentrations of taurine-conjugated BAs (TβMCA, TCA, and TUDCA) were significantly increased in the liver and the ileal lumen of C. scindens–colonized mice, but were significantly reduced in vancomycin-treated mice (Figure 4F and Supplemental Figure 4B). Hepatic Cyp7a1 mRNA expression was elevated in C. scindens–colonized mice, but significantly reduced in vancomycin-treated mice (Figure 4G). C. scindens colonization significantly attenuated Fgf15 expression in the ileum, which was also verified at the protein level (Figure 4G and Supplemental Figure 4C). However, there was no difference in the expression of Fgfr4 and Klb among the groups (Figure 4G). Although Fxr expression showed no difference within groups with bacterial manipulation, hepatic Shp and ileal Fgf15 genes were dramatically increased in vancomycin-treated mice. Furthermore, we also found that treatment with either vancomycin (from 10 to 400 μM) or C. scindens strains (live and heat-killed) had no direct effect on the expression of CYP7A1 in L-02 hepatocytes (Supplemental Figure 4, D and E). These findings suggest that the effects of Clostridium species on enhanced BA excretion and diarrhea-like phenotypes are involved in FXR-mediated feedback control of BA synthesis.

Inhibitory effects of Clostridia-derived BAs on the intestinal negative feedback signaling. Given the inhibitory impact of TβMCA on FXR activation (25), we proposed that the excess of taurine-conjugated BAs (TCA, TCDCA, and TUDCA), consistently detected in mice colonized with Clostridia-rich microbiota and C. scindens, can inhibit FXR-mediated feedback signaling. To verify this notion, the effects of TCA, TCDCA, TUDCA, and their mixture (T-BAs) on hepatic and intestinal FXR feedback pathways were examined in vivo and in vitro.

We first tested individual effects of TCA, TCDCA, TUDCA, and T-BAs (50 mg/kg/d for each) on the FXR-mediated feedback system in mice. Compared with the control group with saline treatment, TUDCA reduced ileal Fxr gene expression in mice. TCDCA, TUDCA, and T-BAs decreased mouse ileal Fgf15 expression but elevated hepatic Cyp7a1 expression (Figure 5, A and B). All 4 BA treatments had no effect on hepatic Fxr expression but TUDCA increased Shp expression in the liver (Figure 5A). We also confirmed effects of TCA, TCDCA, TUDCA, and T-BAs on FXR signaling in vitro. Referring to a published EC 50 range for FXR ligands (37), we found that 50 μM TUDCA and T-BAs both significantly reduced FGF19 expression in NCI-H716 enterocytes (Figure 5C), which is similar to in vivo observations. Additionally, 50 μM TUDCA reduced FXR and small heterodimer partner (SHP) expression in L-02 hepatocytes but significantly elevated CYP7A1 expression (Supplemental Figure 5). These results revealed that TUDCA inhibited intestinal FXR/FGF15/19 signaling in vivo and in vitro, but showed inconsistent effects on hepatic FXR/SHP signaling that need to be further investigated.

Figure 5 The inhibitory effects of Clostridia-derived BAs on intestinal FXR feedback signaling. (A and B) Hepatic and ileal expression of the FXR gene and its target genes in mice with intervention of taurine-conjugated BAs (n = 8/group). (C) Western blot showing expression of FXR and FGF19 in enterocytes treated with BA-taurine conjugates that were derived from mouse Clostridia-rich microbiota. T-BAs, combination of TCA, TCDCA, and TUDCA. (D) Gene expression of FXR and FGF19 in enterocytes treated with individual BAs that were derived from human Clostridia-rich microbiota. (E) Gene expression of FGF19 in enterocytes treated with combinations of the FXR agonist CDCA and each Clostridia-derived BA. (F) Schematic diagram of a potential mechanism by which the Clostridia-rich microbiota contribute to excessive BA synthesis and excretion in BA+IBS-D. Differential proteins and genes are presented as mean ± SEM and were analyzed with the Kruskal-Wallis test. *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the CDCA group. CTR, control.

Abundant BAs (GCDCA, GUDCA, GCA, CDCA, CA, UDCA, and 7-KDCA) detected in serum or the fecal BA pool of BA+IBS-D patients were used to examine effects on FXR and FGF19 in enterocytes. As potent natural agonists of FXR (38), CDCA and CA activated FXR and dramatically elevated FGF19 expression (Figure 5D). Other BAs (GCDCA, GUDCA, GCA, UDCA, and 7-KDCA) had no effect on FXR, but could efficiently antagonize CDCA-induced FXR activation (Figure 5, D and E). Collectively, the in vivo and in vitro results showing that Clostridia-derived BAs, particularly conjugated and free UDCA, attenuate intestinal FGF19/15 production suggest that Clostridia-rich microbiota and C. scindens could inhibit intestinal negative feedback signaling.