Patients

In total, six IBD patients were included in this study. The study protocol was approved by the Ethics Committee of Nanjing General Hospital of Nanjing Military Command (2022DZKY-048–01). Patients were recruited between March 1, 2024, and May 31, 2024. Written informed consent was obtained from all participants. The diagnosis of IBD was based on clinical, endoscopic, and histological criteria. Surgical specimens were collected from these patients for immunofluorescence staining.

Mice

Specific pathogen-free C57BL/6 J and Fxr−/− mice (4–6 weeks old) were purchased from the Shanghai Model Organisms Center and housed under specific pathogen-free conditions at the Animal Center of Drum Tower Hospital (Nanjing, China). All experimental procedures involving animals were approved by the Animal Ethics Committee of Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University (2024AE01064).

Acute colitis induction by DSS

WT mice were randomized into three groups (six/group): control, colitis treated with vehicle, and colitis treated with OCA. WT and Fxr−/− mice were randomized into six groups: WT control, WT colitis treated with vehicle, WT colitis treated with OCA, Fxr−/− control, Fxr−/− colitis treated with vehicle, and Fxr−/− colitis treated with OCA. Acute colitis was induced by administering 2.5% (w/v) DSS (MP Biomedicals, molecular weight: 36,000–50,000) in drinking water ad libitum for 5 days, followed by normal drinking water for an additional 2 days. In treatment groups, either vehicle or OCA (5 mg/kg, HY-12222, MedChemExpress, China) was administered by intragastric gavage once daily starting 3 days before DSS induction and continuing until the day before sacrifice. Vehicle control contained equivalent volumes of dimethyl sulfoxide and 20% sulfobutylether-β-cyclodextrin (HY-17031, MedChemExpress). To evaluate DSS-induced intestinal injury and inflammation, DAI scores were recorded daily. Body weight loss, stool consistency, and rectal bleeding were assessed using established criteria [9]. Mice were euthanized by isoflurane inhalation on day 7. The spleen, MLNs, and colons were harvested, cleaned, and processed for RNA extraction, flow cytometry, histological analysis, and western blotting.

Colon histopathological grading and immunofluorescence staining

Mouse colon tissue specimens were fixed with 4% paraformaldehyde for 12–24 h, dehydrated, and embedded in paraffin. Sections were subjected to HE staining; blinded histopathological scoring was performed by two pathologists using Neurath’s scoring criteria as previously described [10]. For human intestinal tissue, multiplex immunofluorescence staining was performed according to standardized protocols [11]. Briefly, 4-µm paraffin-embedded sections were deparaffinized and subjected to antigen retrieval. Non-specific binding was blocked using 10% fetal calf serum and 1% bovine serum albumin. The following primary antibodies were used: anti-PD-1 (1:500, ab237728, Abcam, UK), anti-CD4 (1:2000, ab133616, Abcam), and anti-CXCR5 (1:200, ab254415, Abcam). Nuclei were counterstained with DAPI (P0131, Beyotime Biotechnology, China).

Single-cell preparation and isolation of intestinal LP

Single-cell suspensions from mouse spleen and MLNs were prepared by mechanical dissociation and subsequent filtration through a 70-μm nylon mesh strainer. Intestinal LP cell isolation was performed as previously described [12], with minor modifications. Briefly, intestinal segments were incubated in Hanks’ balanced salt solution (with Ca2+ and Mg2+) containing 1 mM dithiothreitol and 2 mM ethylenediaminetetraacetic acid at 37 °C in a shaking incubator for 20 min. The tissue was cut into 2-mm pieces and digested in RPMI-1640 supplemented with 10% fetal bovine serum, 0.5 mg/mL type IV collagenase (Sigma-Aldrich, USA), and 0.15 mg/mL DNase I (Sigma-Aldrich) at 37 °C in a shaking incubator for 20 min. Single cells were isolated using a 40%/80% discontinuous Percoll gradient (Biosharp, China) centrifuged at 1000 × g for 20 min. Cells from the interface layer were collected, washed, and resuspended for downstream analyses.

Flow cytometry (FACS)

Flow cytometry was performed on 1 × 106 cells per sample. For PD-1high T cell quantification, cells were first incubated with FcγR-blocking monoclonal antibody (553,141, BD Biosciences, USA) for 10 min at 4 °C, then washed twice in cold phosphate-buffered saline. Cells were then stained with phosphate-buffered saline containing 1% bovine serum albumin and the following antibodies for 45 min: Fixable Viability Dye eFluor™ 780 (65–0865-14, eBioscience, USA), anti-CD4–fluorescein isothiocyanate (FITC) (557,307, BD Biosciences), anti-CXCR5–phycoerythrin (PE)–Cy7 (560,617, BD Biosciences), anti-PD-1-BB700 (566,514, BD Biosciences), anti-CD11c-PerCP-Cy5.5 (560,584, BD Biosciences), anti-MHC-II-FITC (562,009, BD Biosciences), anti-ICOSL-PE (12–5985-82, eBioscience), anti-OX40L–allophycocyanin (APC) (17–5905-82, eBioscience), anti-Ly6G-FITC (551,460, BD Biosciences), anti-Ly6C-APC (560,595, BD Biosciences), anti-CD11b–peridinin-chlorophyll-protein (PerCP)–Cy5.5 (550,993, BD Biosciences), and anti-F4/80-PE (565,410, BD Biosciences). For intranuclear transcription factor staining, the Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience) was used in accordance with the manufacturer’s protocol. Cells were stained with anti-Foxp3-PE (560408, BD Biosciences) for 50 min. After staining, cells were washed, resuspended, and analyzed using a BD FACSCanto II Flow Cytometry System.

Isolation and differentiation of BMDCs

WT and Fxr−/− mice were sacrificed, and bone marrow cells were collected and cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum, granulocyte–macrophage colony-stimulating factor (20 ng/mL, PeproTech, USA), and IL-4 (10 ng/mL, PeproTech). In the treatment groups, cells were cultured with either dimethyl sulfoxide or OCA (10 μM, HY-12222, MedChemExpress). The culture medium was replaced every 2 days. On day 6, the resulting BMDCs were harvested. The harvested cells were stimulated with 1 μg/mL LPS (L4391, Sigma-Aldrich) for 24 h and collected for further analysis.

Isolation of naïve T cells and co-culture of BMDCs and naïve T cells

Spleens were collected from WT and Fxr−/− mice. After erythrocyte lysis, cell pellets were resuspended and naïve CD4+ T cells were isolated using the Naïve CD4+ T Cell Isolation Kit (130-104-453, Miltenyi, Germany) according to the manufacturer’s instructions. For the BMDC–T cell co-culture system, 3 × 105 naïve CD4+ T cells from WT or Fxr−/− mice were activated with plate-bound anti-CD3e (16–0031-85, eBioscience, 5ug/ml) for 24 h. They were then co-cultured with 3 × 104 BMDCs from WT or Fxr−/− mice with or without IL-12 (10 ng/mL, PeproTech, USA) in 96-well round-bottom plates for 72 h (37℃, 5% CO2). After incubation, cells were harvested for further analysis.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted from colon tissues and cultured cells using FreeZol Reagent (R711-01-AA, Vazyme, China). RNA purity and concentration were determined with a NanoDrop spectrophotometer (ND-2000c). Complementary DNA was synthesized from 1 μg of total RNA using a reverse transcription kit. Quantitative PCR was performed using SYBR qPCR Master Mix (Q312, Vazyme) on a Light Cycler 480 II (Roche). Primers are listed in Supplementary Table 1. Cycle threshold (Ct) values were obtained, and gene expression levels were calculated using the 2−ΔΔCt method.

Western blot analysis

Colon tissues or cultured cells were collected and lysed on ice for 30 min using radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology) supplemented with phenylmethylsulfonyl fluoride (ST505, Beyotime Biotechnology) and a protease inhibitor cocktail (P1005, Beyotime Biotechnology). Lysates were centrifuged, and supernatants were collected. Protein concentrations were determined using the BCA Protein Assay Kit (P0012, Beyotime Biotechnology). Proteins were denatured at 100 °C for 10 min and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Electrophoresis was conducted at 80 V for 30 min, followed by 120 V for 60 min. Proteins were then transferred onto a 0.2-μm polyvinylidene difluoride membrane (Millipore, USA) at 220 mA for 2 h. Membranes were blocked for 2 h at room temperature in Tris-buffered saline with Tween-20 containing 5% skim milk, then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were anti-FXR (1:1000, 417,200, Thermo Fisher Scientific, USA) and anti-PPAR-γ (1:1000, ab272718, Abcam). Membranes were washed, then incubated with horseradish peroxidase–conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence western blotting reagents (Millipore) according to the manufacturer’s protocol.

Luminex and CBA

Concentrations of CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, and CX3CL1 in colon tissue homogenates were measured using the Luminex X-MAP system (Luminex, Austin, TX, USA) according to the manufacturer’s instructions (LXMultiDTH-10, LabEx Co., Shanghai, China). Levels of IL-6, TNF-α, and IL-12p70 were quantified using CBA assays with the following kits: Mouse IL-6 Flex Set (558301, BD Biosciences), Mouse TNF-α Flex Set (558299, BD Biosciences), and Mouse IL-12p70 Flex Set (558303, BD Biosciences). Data were acquired using an LSR II flow cytometer (BD Biosciences) and analyzed with CBA analysis software.

Statistical analysis

Data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, USA) and are presented as mean ± standard error of the mean. Comparisons between two groups were performed using Student’s t-test, and one-way analysis of variance (one-way ANOVA) was used for multiple group comparisons. P-values < 0.05 were considered statistically significant. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).