Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion

Feces samples from 80 patients with cholesterol GS and 49 gallstone-free controls (GSF) were collected. The fecal microbiota composition of both groups was analyzed by 16s rRNA sequencing. The microbiota composition showed a higher abundance of Desulfovibrionales order in GS patients than in GSF controls (Fig. 1A), which suggested an association between Desulfovibrionales and gallstone susceptibility.

Donor feces enriched in Desulfovibrionales from gallstone patients (GS) or feces from gallstone-free (GSF) controls were transplanted to gallstone-resistant AKR/J mice (GS-R mice). No gallstone formed in mice subjected to transplantation of feces from GSF controls (0/12) after 8-week LD. Desulfovibrionales were not detected in these mice. However, gallstone formed in 73% (8/11) of the mice receiving feces from the GS donors (Fig. 1B). The profiles of gut microbiota suggested the successful introduction of Desulfovibrionales to these mice (Fig. 1C). Biliary composition analysis showed the increased cholesterol molar and cholesterol saturation index (CSI) (Fig. 1D) in the gallbladder bile in mice carrying Desulfovibrionales. Serum high-density lipoprotein, low-density lipoprotein cholesterol (Fig. S1A), and hepatic cholesterol (Fig. S1B) levels were higher as well. The results demonstrated that Desulfovibrionales from gallstone patients were capable to induce gallstone formation.

Comparing the gut microbiota between the gallstone-susceptible mice C57BL/6J (GS-S mice), and the gallstone-resistant mice AKR/J (GS-R mice), we found that Desulfovibrionales order was also markedly abundant in C57BL/6J mice (Fig. 2A) as well as shown by LEfSe algorithm (Fig. 2B and Fig. S2). Desulfovibrio fairfieldensis, piger, desulfuricans, and vulgaris were identified to be the major species (Fig. 2C) in GS-S mice, which were barely detected in GS-R mice.

Co-housing (Fig. 3A) successfully modified the gut microbiota in GS-R mice from the original mice (Fig. 3B) with significant enrichment of Desulfovibrionales order in the co-housing GS-R mice (Fig. 3D). As a result, gallstone formation increased to 70% in co-housing GS-R mice (Fig. 3A). Profiles of gallbladder bile composition from co-housing GS-R mice turned to resemble GS-S mice, presenting as elevated biliary cholesterol molar percentage and CSI (Fig. 3C), a condition favoring gallstone formation.

A major role of Desulfovibrionales is to reduce substrates such as taurine into H₂S, which was reported to be a necessary grow factor for 7α-dehydroxylating bacteria¹⁹. Interestingly, comparing the multi-gene BA-inducible (Bai) operon encoding genes involved in 7-α/β dehydroxylation of BAs (Fig. 4A), we found that GS-S mice had higher expression of BaiB, CD, and F (Fig. 4B), indicating an active bile acid dehydroxylation in these mice. Accordingly, 7α-dehydroxylation activity in the cecum content was significantly higher in GS-S mice as well as GS-R mice after co-housing (Fig. 4C).

Next, we measured the cecal bile acids. As expected, DCA, the product of bacteria 7a-dehydroxylation and the major component of cecal bile acids, was significantly higher in GS-S mice and co-housing GS-R mice (Fig. 4D).

In gallbladder bile, cholic acid (CA) and DCA, comprised >97% of the total bile acids in GS-S mice (Fig. 4E and Fig. S3A–C). However, GS-R mice retained a considerable amount of hydrophilic bile acids: β-muricholic acid (βMCA), αMCA, and ursodeoxycholic acid (UDCA) in their bile (≈25%), but less hydrophobic DCA. Co-housing led to a 50% decrease of βMCA and an 18% increase of CA in GS-R mice (Fig. 4E and Fig. S3C). The hydrophobicity index (HI) of bile acids was significantly higher in GS-S mice and co-housing GS-R mice (Fig. 4F).

Furthermore, GS-R mice receiving FMT from the gallstone patients also had higher cecal 7α-dehydroxylation activity (Fig.), indicating a similar enhancement of secondary bile acids conversion. In line with this, DCA and ωMCA were higher, but βMCA, αMCA, and UDCA were lower in gallbladder bile (Fig.), leading to higher HI in these mice (Fig.). S4A S4B, C S4D

These data collectively indicated that introducing Desulfovibrionales to GS-R mice led the bile acid profile to be more hydrophobic.

To further unravel the impacts of Desulfovibrionale on the hepatic metabolic disorders in promoting gallstone formation, we performed RNA-seq in liver samples from mice in the co-housing experiment (Fig. 5A). Co-housing could further repress the expression of genes related to bile acid synthesis in GS-R mice, especially the expression of the rate-limiting enzymes of bile acid synthesis—cholesterol 7α-hydroxylase (Cyp7a1), the enzyme responsible for cholic acid synthesis—Cyp8b1. qRT-PCR further validated the inhibition of their expression in AKR/J mice after co-housing (Fig. 5B).

The gene expression profile in hepatic cholesterol metabolism was also changed (Fig. 5A). 3α-hydroxy-3-methyl-glutarylcoenzeme A (Hmgcr), the rate-limiting enzyme for cholesterol synthesis, was higher in GS-S mice even under conditions when cholesterol was overloading (Fig. 5A, B). The expression of Hmgcr elevated in GS-R mice after co-housing. The expressions of hepatic cholesterol transporters, ATP binding cassette (Abc) g5 and Abcg8, were elevated GS-R mice after co-housing (Fig. 5A, B).

In the GS-R mice receiving FMT from gallstone patients, similar changes of genes in bile acid and cholesterol metabolism could be observed after carrying Desulfovibrionales (Fig. 5C).

Desulfovibrionales contains genes for sulfate reduction to reduce sulfate to H₂S. In GS-S mice (C57BL/6J) had significantly higher serum H₂S than GS-R mice (AKR/J) and co-housing led to an increase of serum H₂S in GS-R mice (Fig. 6A). H₂S was reported to enhance FXR expression in HepG2 cells²⁰, which in turn inhibited CYP7A1 expression²¹. To collaborate this, we treated mice with GYY4137, a novel H₂S donor releasing H₂S slowly and mimicking the time course of H₂S release in vivo²². We found GYY4137 increased FXR protein and decreased CYP7A1 protein in liver tissues (Fig. 6B). Furthermore, both in the murine hepatocyte, Hepa1-6 (Fig. 6C), and human hepatoma cells, HuH7 (Fig. 6D), a dose-dependent induction of FXR and inhibition of CYP7A1 expression was found. These data suggested the existence of a regulatory pathway of hepatic bile acid metabolism by Desulfobibronale through its product H₂S.

To further validate the role of Desulfovibrio species on gallstone formation, we transplanted three commercially available species of Desulfovibrio piger, desulfuricans, and vulgaris to antibiotics-pre-treated GS-S mice. Depletion of gut microbiota decreased the gallstone incidence to 70% (7/10). In contrast, Desulfovibrio species transplantation restored gallstone incidence to 100% (10/10, Fig. 7A). Similarly, Desulfovibrio species transplantation could induce gallstone formation in 42% (5/12) of the GS-R mice (Fig. S5A), whereas none of the control GS-R mice formed gallstone (0%).

In the Desulfovibrio species transplantation group of mice, serum and hepatic cholesterol levels were significantly elevated in the transplanted mice (Fig. 7B, C). Moreover, expressions of genes in hepatic bile acid synthesis, Cyp7a1, Cyp8b1, Cyp27, and Cyp2c70 were inhibited, and the expressions of Abcg5, Abcg8, Srb1, and Hmgcr were elevated (Fig. 7D and Fig. S5B).

To monitor the influence of Desulfovibrio species on the hepatic secretion of biliary lipids, hepatic bile was collected by common bile duct cannulation. In mice with Desulfovibrio species transplantation, a significant increase of cholesterol and phospholipids but less bile acid output in their hepatic bile was observed (Fig. 7E). This result suggested that carrying Desulfovibrio species promoted the hepatic secretion of cholesterol into bile.

Feces samples were provided by patients who underwent laparoscopic cholecystectomy due to GS (GS, n = 80) or gallstone-free volunteers who underwent health examination (GSF, n = 49). Only the patients with cholesterol type of gallstone were included by classification as typical cholesterol by visual inspection of cut-surface of gallstones and when necessary, by chemical analysis in the laboratory^37,38. All the controls were confirmed to be gallstone-free by B-type ultrasonography. Subjects who had been taken antibiotics during the past 3 months prior to sampling were excluded. Subjects with metabolic disorders, such as obesity, diabetes mellitus, hyperlipidemia, or chronic bowel disease, or chronic diarrhea, or constipation, were also excluded.

Male C57BL/6J mice were bought from Shanghai SLAC Laboratory Animal Company Ltd. (Shanghai, China) and AKR/J mice from Jackson Laboratory (Bar Harbor, ME, USA). Male mice (at age of 7–8 weeks) were fed with LD (containing 1.25% cholesterol and 0.5% cholic acid, synthesized by Trophic Animal Feed High-Tech Co. Ltd, Nantong, China) for 8 weeks before sacrifice. The mice were bred at the Animal Care Facility on a 12-h light/12-h dark cycle in a controlled temperature (22.5 ± 2.5 °C) and humidity (50 ± 5%) environment.

In human fecal microbiota transplantation (HuFMT) study, AKR/J mice were orally gavaged with donor human feces (dissolved in phosphate-buffered saline (PBS) 1.5 ml/200 mg of feces weight) once after depletion of gut microbes by antibiotics cocktails (containing 0.5 g/l vancomycin, 1 g/l neomycin sulfate, 1 g/l metronidazole, 1 g/l ampicillin³⁹) in drinking water ad libitum for 3 weeks after weaning (at the age of 4 weeks). Donor feces were selected from the patient cohort in result 1 (Fig. 1A). The gallstone-patient donors were the 3 patients whose feces were the top 3 with enrichment of Desulfovibrionales as determined by 16s rRNA sequencing and GSF donors were 3 gallstone-free subjects whose feces had no detection of Desulfovibrionales. Feces sample from each donor was gavaged to 4 recipient mice (1 donor: 4 recipient mice). All mice were then fed with LD for 8 weeks (one mouse in the FMT from the gallstone patient donor group died after gavage).

In the co-housing study, AKR/J mice were given a cocktail of antibiotics for 3 weeks after weaning. Afterward, the AKR/J mice were co-housing with conventional C57BL/6 mice at a ratio of 1:1 for 1-week and then fed with LD for 8 weeks.

In bacteria inoculation study, Desulfovibrio vulgaris (ATCC-29579), Desulfovibrio desulfuricans (ATCC-29577), and Desulfomonas pigra (ATCC-29098) were purchased from ATCC, and cultured in anaerobically sterilized ATCC medium 1249 under strict anaerobic conditions overnight. C57BL/6J or AKR/J mice were depleted of gut microbes by antibiotics cocktails for 3 weeks after weaning and then oral gavaged with sterile PBS or live mixed Desulfovibrio (1:1:1) at a dose of 5 × 10⁸ colony forming units/100 μl in sterile PBS, respectively, three times a week for 4 weeks. Upon starting of gavage, mice were fed with LD for 8 weeks. After 4-week feeding, bile duct cannulation was performed in a subset of mice according to the procedure as described⁴⁰. In brief, the common bile duct of mice fasted overnight was ligated and the common bile duct was cannulated with a PE-10 polyethylene catheter below the entrance of the cystic duct. The cystic duct was doubly ligated and a cholecystectomy was performed and the first-hour hepatic bile was collected.

In exogenous H₂S study, 8-week-old C57BL/6J male mice were administered with either vehicle alone (normal saline) or vehicle containing GYY4137 (133 μmol/kg/day) (Cat No.B7458, APExBIO Technology, Houston, USA) for 7 days by intraperitoneal injection. GYY4137 is a novel H₂S donor that could release H₂S slowly and mimic the time course of H₂S release in vivo²².

All mice were fasted 8 h before sacrifice. The blood sample was obtained by heart puncture. Liver, gallbladder, intestine, and cecal contents were collected and stored at −80 °C under analysis.

Mouse hepatocytes, Hepa1-6 cells, were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-high glucose (Cat No. 12430054, Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate (Cat No. 11360070, Gibco, New York, USA). Human hepatoma cells, HuH-7 cells, were cultured in DMEM-high glucose (Cat No. 11960044, Gibco, New York, USA) supplemented with 10% FBS, 1 mM sodium pyruvate (Cat No. 11360070, Gibco, New York, USA), and GlutaMAX™ (Cat No. 35050061, Gibco, New York, USA). Cells with 80–85% confluence were incubated with or without GYY4137 (300 or 600 μM) and GW4064 (5 μM) (Cat No. B1527, APExBIO Technology, Houston, USA).

Total bile acids, phospholipids, and cholesterol concentration in the gallbladder or hepatic bile were measured by enzymatic methods as previously described^38,41 using commercial purchased kits (cholesterol: Cat No. 11491458216, from Roche Diagnostics GmbH, Germany; total bile acids: Cat No. BI2672 from RANDOX, UK; and phospholipids: phospholipid LabAssay kit, Cat No. WAKO 296-63801, from Fuji Film WAKO Pure Chemical Co, Japan). The relative concentrations of biliary lipids were expressed as molar percentages of the total biliary lipids. The CSI was calculated according to Carey’s critical table⁴².

Twenty-five microliters of serum were diluted and analyzed for cholesterol level on HITACHI automated biochemical analyzer. Lipids in liver tissue (50 mg) were extracted by chloroform:methanol solution (2/1, v/v), and total cholesterol levels were determined by enzymatic kit as previously described⁴³. Serum campesterol and β-sitosterol levels were measured by gas-liquid chromatography-mass spectrometry as described⁴⁴.

Serum H₂S was assayed by spectrophotometry as described⁴⁵. Briefly, 75 μl serum was reacted with 250 μl zinc acetate (1%), 425 μl distilled water, N,N-dimethyl-p-phenylenediamine sulfate in 7.2 M HCl (133 μl, 20 mM) and FeCl₃ in 1.2 mM HCl (133 μl, 30 mM). The sample was incubated at room temperature for 10 min and then the reaction was stopped by adding 10% v/v trichloroacetic acid (250 μl). After centrifugation, the absorption of the supernatant was measured at 670 nm by spectrophotometry. NaHS (2.5–200 μM) was used as a standard.

Gallbladder bile, liver tissue homogenates, or cecal content were diluted with internal standard, vortexed, and purified by the sedimentation plate. Samples were then lyophilized and dissolved with 25% acetonitrile. After centrifugation, the supernatants were collected for measuring the levels of various bile acids. Bile acids profiles were analyzed on an Acquity UPLC system coupled to a Waters Xevo TQ-S MS (Waters, Manchester, UK), equipped with a C18 reverse-phase column with 1.7 mm particle size (Waters Corp., Milford, MA, USA). Analytes were detected by electrospray ionization and quantified by internal standard methods as described⁴⁶. The HI of bile acids in gallbladder bile was calculated as described⁴⁷.

Activities of the gut flora were measured according to the procedure as described^48,49. One gram of cecal contents of each mouse was dissolved in 3 ml sterilized solution A (containing 20 % glycerol and 1.8 % sodium chloride) which was diluted in polyPeptone Yeast Extract Medium (PY culture medium) and then cultured at 37 °C and 200 rpm for 12 h to allow anaerobic bacteria to proliferate. Afterward, 200 μl tauro-cholic acid (200 μg/ml) was added in 1 ml the mixture and incubated for another 1 h at 37 °C in the anaerobic chambers and the reaction was stopped by 100 μl of 1 M HCl. The generation of cholic acid and deoxycholic acid were measured by Acquity UPLC system coupled to a Waters Xevo TQ-S MS for the calculation of gut flora activities.

Total genomic bacterial DNA was extracted from cecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. PCR amplification of bacteria DNA was performed as previously described⁴⁶. Amplicons were extracted from 2% agarose gel, purified by the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, USA), and quantified by QuantiFluorTM-ST (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Then purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq platform following standard instructions by the commercial service of Genergy Biotechnology Co. Ltd. (Shanghai, China).

Raw Illumina fasta files were de-multiplexed, quality filtered, and analyzed using the QIIME software according to standardized criteria as described⁵⁰. Operational taxonomic units (OTUs; 97% sequence similarity) were clustered using the UPARSE software (version 7.1, http://drive5.com/uparse/↗), and chimeric sequences were identified and removed using the UCHIME program. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed using the Ribosomal Database Project Classifier tool (version 11.1, http://rdp.cme.msu.edu/↗) against the SILVA (SSU115) 16S rRNA database (http://www.arb-silva.de↗) using a confidence threshold of 70%. Alpha diversity was used to describe the microbial richness, diversity, and evenness within samples with four parameters: two richness estimators (Chao1 and the abundance-based coverage estimator) and two diversity indices (Shannon and Simpson indices). Jackknifed beta diversity analysis (between-sample diversity comparisons) was calculated using weighted and unweighted unifrac distances between samples, and principal coordinates were also computed to compress dimensionality into two-dimensional principal coordinate analysis plots. Observed species alpha rarefaction of filtered OTU tables was also performed to confirm that the sequence coverage was adequate to capture the species diversity observed in all samples. Identification of microbial genes involved in BA biotransformation was performed using methods as reported⁵¹.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was examined by gel electrophoresis and with a Nanodrop spectrophotometer (Thermo, Waltham, MA, USA). cDNA library generation with the TruSeqRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Clusters were generated with the TruSeq SR Cluster Kit v2 according to the reagent preparation guide.

The RNA sequencing was performed using the HiSeq X Ten platform by the commercial service of Genergy Biotechnology Co. Ltd. (Shanghai, China). The raw data was handled by Perl and data quality was checked by FastQC 0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/↗). RNA-Seq reads were aligned to the reference data downloaded from UCSC (version hg19), and the RPKM method was utilized to normalize the reads that exclusively mapped to a gene, to quantify the transcript levels⁵².

Total protein from frozen livers or cultured cells were extracted and used with western immunoblotting as described previously⁵³. The following antibodies were used for western blot: anti-FXR (1:1000, Abcam, ab235094), anti-CYP7A1 (1:1000, Abcam, ab65596), anti-CYP8B1 (1:1000, Abcam, ab191910), anti-GAPDH (1:1000, Proteintech, 60004-1-Ig). Images were quantified using the Image Pro Plus 6.0 software.

Power Mix Sybr Green Master Mix (Applied Biosystems) was used for quantitative real-time PCR to determine the target gene expressions. The relative mRNA expression was calculated with the ΔΔCt method using GAPDH as the internal control as described⁴³. Primer sequences are listed in Supplementary Table 1.

Quantitative data are expressed as mean ± SEM and qualitative data as a ratio. The difference between the two groups was compared by t test and among multiple groups by analysis of variance followed by least significant difference test for post hoc analysis between the groups. Two-sided P < 0.05 was regarded as statistical significance. All the statistics were performed using the SPSS 12.0 software.

Further information on research design is available in thelinked to this article. Nature Research Reporting Summary

All the data are available upon request. The raw data of 16s rRNA sequencing generated in this study has been deposited at NCBI SRA BioProject (accession no: PRJNA773136↗) and that of RNA-seq at EMBL-EBI (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8550↗). Source data are provided with this paper.

Scripts for plots under python are available at the website https://github.com/zhajia2019/Python_Scripts↗.