The present study was approved by the Ethics Committees of Union Hospital, Tongji Medical College (Wuhan, China; approval no. 2016-LSZ-S180) and Shanxi Children's Hospital (Taiyuan, China; approval no. 2RB-SB-2021-005), and was registered in the Chinese Clinical Trial Registry (registration ID: ChiCTR2000039619). Written informed consent was obtained from the guardians of all participants. Liver tissues were obtained from 27 patients with BA (BA group) undergoing Kasai portoenterostomy between May 2021 and December 2023, and from 23 age-matched patients with choledochal cyst undergoing surgery during the same period as the patients with BA. The BA group included 10 male patients and 17 female patients, with a median age at surgery of 54 days (range, 28-90 days), whereas the control group consisted of 8 male patients and 15 female patients, with a median age at surgery of 60 days (range, 25-98 days). Fresh liver samples were processed immediately after collection. Liver tissues for flow cytometry and magnetic bead-based cell enrichment were maintained on ice and utilized within 4 h, whereas the remaining samples were either fixed in 10% formalin (24 h at room temperature) for preparation of paraffin-embedded tissue sections or stored at −80°C for mRNA isolation.

Adult wild-type (WT) Balb/c mice (n=48; 16 male mice and 32 female mice; age, 8-10 weeks; weight, 20-25 g) were purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. The Tcrδ^−/−, Tlr2^−/− and Tlr4^−/− Balb/c mice (n=2 male mice and 4 female mice per genotype; age, 8-10 weeks; weight, 20-25 g) were produced by Shanghai Model Organisms Center, Inc., using a proprietary CRISPR-Cas9 gene targeting platform. All mice were maintained in specific pathogen-free laminar-flow cages at a temperature of 23°C and humidity of 50%, under a 12 h light/dark cycle with free access to standard chow and water. All procedures were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; approval no. 4296; 2024). Experimental BA was induced in neonatal Balb/c mice (the WT offspring from the aforementioned adult WT mice; the Tcrδ^−/− offspring from adult Tcrδ^−/− mice; all mated in-house) by intraperitoneal injection of 1.5×10⁶ ffu rhesus rotavirus (RRV; provided by Professor C.L. Mack, University of Colorado, Denver, CO, USA) in 20 μl within 24 h of birth [n=25 for RRV group (11 male, 14 female); n=52 for Tcrδ^−/− + RRV group (24 male, 28 female); n=54 for WT + RRV group (26 male, 28 female); n=48 for Tcrδ^−/− + RRV + RPMI 1640 (cat. no. 11875093; Gibco; Thermo Fisher Scientific, Inc.) group (25 male, 23 female); n=51 for Tcrδ^−/− + RRV + IL-17A⁺ γδT group (24 male, 27 female)] (23), whereas control mice received minimum essential medium (MEM; cat. no. 11095080; Gibco; Thermo Fisher Scientific, Inc.) (n=25 for MEM group). Additionally, an independent cohort of neonatal WT mice (n=96; 45 male and 51 female; the offspring from the aforementioned adult WT mice) was used for γδT cell extraction following RRV injection. Separate cohorts of neonatal WT mice (n=18; 8 male and 10 female; the offspring from the aforementioned adult WT mice), Tlr2^−/− mice (n=8; 4 male and 4 female; the offspring from the aforementioned adult Tlr2^−/− mice), and Tlr4^−/− mice (n=8; 5 male and 3 female; the offspring from the aforementioned adult Tlr4^−/− mice) were used for γδT cell extraction. The mice were sacrificed on days 3, 7 and 14 for serum and tissue examination. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg/kg), followed by cardiac puncture for blood collection (50-150 μl per mouse). Subsequently, the mice were euthanized by cervical dislocation. Death was confirmed by the cessation of heartbeat and respiration, loss of reflexes and dilated pupils.

For inhibitor treatment, mice were intraperitoneally injected with glycyrrhizin (HMGB1 inhibitor; 50 mg/kg; cat. no. NSC 167409; Selleck Chemicals) on days 1, 3, 5 and 7 after RRV injection, respectively (n=20 for RRV + Glycyrrhizin group). The control group received an intraperitoneal injection of an equal volume of DMSO (n=20 for RRV + DMSO group). For macrophage depletion, mice were intraperitoneally administered with GdCl₃ (cat. no. G7532; Sigma-Aldrich; Merck KGaA) 24 h before RRV infection (n=15 for RRV + GdCl₃ group), whereas control mice were administered PBS (n=15 for RRV + PBS group).

Mononuclear cells were isolated from the liver tissues of patients with BA (n=6) by density gradient centrifugation using Ficoll-Paque PLUS (cat. no. 17-1440-02; Cytiva) according to the manufacturer's instructions. The cells were cultured in RPMI-1640 complete medium and stimulated with 1 μg/ml anti-CD3 monoclonal antibody (mAb; cat. no. 300437; BioLegend, Inc.) and 10 μg/ml anti-CD28 mAb (cat. no. 302933; BioLegend, Inc.) at 37°C for 48 h. Subsequently, digoxin (100 nM; cat. no. S4290; Selleck Chemicals) or AM80 (100 nM; cat. no. 3507; Tocris Bioscience) was added to selectively inhibit T helper 17 (Th17) cells or γδT cells, respectively, followed by incubation at 37°C for 72 h. Cell supernatants were then collected for IL-17A detection by ELISA.

Flow cytometry was performed using an LSRII cytometer (BioLegend, Inc.). Liver tissues from patients [BA group (n=5), control group (n=5)] and mice [MEM group (n=5 per time point), RRV group (n=5 per time point), RRV + DMSO group (n=5 per time point), RRV + Glycyrrhizin group (n=5 per time point)] were homogenized into single-cell suspensions and washed in PBS containing 0.3% fetal calf serum (FCS; cat. no. 10100147; Gibco; Thermo Fisher Scientific, Inc.) and 4 mM EDTA. Dead cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Staining kit (cat. no. L34957; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

For extracellular staining at 4°C for 30 min in the dark, human liver single cells were incubated with PE anti-human CD45 (cat. no. 368509), Alexa Fluor^® 700 anti-human CD3 (cat. no. 300423), Brilliant Violet 421™ anti-human TCR γ/δ (cat. no. 331217) (all from BioLegend, Inc.), BB700 anti-human CD56 (cat. no. 566574; BD Biosciences), FITC anti-human CD4 (cat. no. 344604) and PE/Cyanine7 anti-human CD8 (cat. no. 344711) (both from BioLegend, Inc.). Th17 cells were identified as CD3⁺CD4⁺IL-17A⁺ T cells.

Mouse liver single cells were incubated with APC/Cyanine7 anti-mouse CD45 (cat. no. 103115), Brilliant Violet 421™ anti-mouse CD3 (cat. no. 100227) and PE anti-mouse TCR γ/δ (cat. no. 118107) (all kits from BioLegend, Inc.).

For intracellular cytokine staining, the aforementioned human and mouse liver single cells were incubated for 2 h at 37°C in MEM containing 5% FCS and Golgi Plug/Golgi Stop (cat. no. 554724; BD Biosciences). After fixation and permeabilization using the Cytofix/Cytoperm™ Fixation/Permeabilization kit (cat. no. 554714; BD Biosciences) according to the manufacturer's protocol, intracellular staining at 4°C for 30 min in the dark was performed with APC anti-human IL-17A (cat. no. 512333) and Brilliant Violet 605™ anti-mouse IL-17A (cat. no. 506927) (both from BioLegend, Inc.). Analyses were performed using FlowJo v10.8.1 software (FlowJo; BD Biosciences).

For magnetic bead enrichment, liver mononuclear cells from human [BA group (n=6)] and mouse [RRV-infected WT mice (n=96), WT mice (n=18), Tlr2^−/− mice (n=8), Tlr4^−/− mice (n=8)] liver tissues were used for γδT-cell enrichment. Because the yield of γδT cells from each neonatal mouse liver was limited, a relatively large number of RRV-infected WT mice was required to obtain sufficient viable γδT cells for subsequent in vitro cell experiments and adoptive-transfer experiments in the Tcrδ^−/− + RRV + IL-17A⁺ γδT group (n=51). By contrast, γδT cells isolated from WT mice, Tlr2^−/− mice and Tlr4^−/− mice were used only for in vitro co-culture experiments. Human BA samples were processed individually, whereas mouse liver mononuclear cells were pooled, when necessary, only within the same mouse group in each experimental batch. The cells from different mouse groups were not mixed with each other. The cells were separately incubated with biotinylated antibodies (biotin anti-human TCR γ/δ antibody, 1:100, cat. no. 331206; biotin anti-mouse TCR γ/δ antibody, 1:200, cat. no. 118103; both from BioLegend, Inc.) for 15 min at room temperature, followed by anti-biotin microbeads (20 μl per 10⁷ cells; Miltenyi Biotec, Inc.) for 15 min at 4°C and isolated through two rounds of magnetic separation using MS columns (designed for the positive selection of cells; Miltenyi Biotec, Inc.) to obtain γδT cells (live mononuclear cells, CD3⁺ and TCRγδ⁺). The purity of CD3⁺ TCRγδ⁺ cells isolated from liver tissues was >90% for both human and mouse samples (Fig. S1A and B). For activation of γδT cells from human and mouse liver tissues, subsets were separately stimulated with 10 ng/ml IL-1β (recombinant human IL-1β, cat. no. 579402; recombinant mouse IL-1β, cat. no. 575102; both from BioLegend, Inc.) and 10 ng/ml IL-23 (recombinant human IL-23, cat. no. 574102; recombinant mouse IL-23, cat. no. 589002; both from BioLegend, Inc.) at 37°C for 48 h, and stimulated with 1 μg/ml anti-CD3 mAb (purified anti-human CD3, cat. no. 300437; purified anti-mouse CD3, cat. no. 100339; both from BioLegend, Inc.) and 10 μg/ml anti-CD28 mAb (purified anti-human CD28, cat. no. 302933; purified anti-mouse CD28, cat. no. 102115; both from BioLegend, Inc.) at 37°C for 48 h.

After RRV infection in neonatal Tcrδ^−/− mice, 10⁶ IL-17A⁺ γδT cells isolated from RRV-infected WT mouse liver tissue, suspended in 50 μl RPMI 1640 medium (cat. no. 11875085; Gibco; Thermo Fisher Scientific, Inc.) were injected intraperitoneally within 24 h for 3 consecutive days. Control mice received medium alone and the relevant indexes were assessed: Hepatic IL-17A levels, liver histopathology, extrahepatic bile duct morphology, BA incidence, survival rate and hepatic pro-inflammatory cytokine levels.

The purified human (n=3) γδT cells isolated from liver tissues were stimulated with HMGB1 (20 ng/ml; cat. no. 1690-HMB-050; R&D Systems, Inc.) for 48 h at 37°C, then collected for flow cytometric analysis, whereas the supernatants were collected for IL-17A measurement.

All cells were cultured using RPMI 1640 medium containing 10% FCS, 2 mmol/l L-glutamine and 100 U/ml penicillin-streptomycin. Immortalized BECs were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., whereas THP-1 cells were purchased from Pricella^®; Elabscience Bionovation, and were differentiated into macrophages with phorbol 12-myristate 13-acetate (cat. no. P8139; Sigma-Aldrich; Merck KGaA) at 37°C for 24 h. RRV [multiplicity of infection (MOI)=20] was added to the culture medium of BECs and macrophages for incubation, and the release of HMGB1 was dynamically detected by immunofluorescence confocal microscopy in BECs, and by ELISA in the supernatants of both BECs and macrophages after 0, 12, 24 and 36 h at 37°C.

BECs or macrophages (4×10⁴ cells/well) were cultured in 24-well plates. After placing the Transwell chamber (pore size, 0.4 μm; cat. no. 3470; Corning, Inc.) in a 24-well plate, 200 μl medium containing WT/Tlr2^−/−/Tlr4^−/− γδT cells (2×10⁴ cells/chamber) (isolated from the liver tissues of WT, Tlr2^−/− or Tlr4^−/− mice) was added to the chamber, along with anti-mouse CD3 mAb (1 μg/ml) and anti-mouse CD28 mAb (10 μg/ml), to establish a co-culture system. RRV (MOI=20) was added, whereas the control group received medium alone without RRV. Where indicated, WT γδT cells were pretreated with 10 μM Bay 11-7082 (NF-κB inhibitor; cat. no. S2913; Selleck Chemicals) for 1 h at 37°C before co-culture. After 36 h of co-culture, the cell supernatant was collected for IL-17A detection.

Similarly, a co-culture system was established using BECs or macrophages with human γδT cells isolated from liver tissues of patients with BA (n=3). Briefly, BECs or macrophages (4×10⁴ cells/well) were cultured in 24-well plates. Human γδT cells (2×10⁴ cells/chamber) were added to the Transwell chamber (pore size, 0.4 μm), and stimulated with anti-human CD3 mAb (1 μg/ml) and anti-human CD28 mAb (10 μg/ml). Where specified, BECs or macrophages were pretreated with 40 μM glycyrrhizin for 2 h at 37°C prior to co-culture, followed by the addition of RRV after co-culture establishment, with controls lacking RRV. After 36 h of co-culture, the IL-17A in the cell supernatant was detected.

BECs were immobilized using 4% paraformaldehyde for 30 min at room temperature, washed with PBS, and blocked with 3% bovine serum albumin (cat. no. BS114; Biosharp Life Sciences) at room temperature for 1 h. The cells were then incubated overnight with rabbit anti-HMGB1 antibody (1:800; cat. no. ab18256; Abcam) at 4°C, followed by incubation with Cy3-conjugated goat anti-rabbit secondary antibody (1:100; cat. no. SA00009-2; Proteintech Group, Inc.) at room temperature for 1 h. After nuclear staining with DAPI at room temperature for 10 min, the cells were observed using a confocal fluorescence microscope (Olympus Corporation).

Cell culture supernatants were collected and then centrifuged at 300 × g for 10 min at 4°C to remove cellular debris, and the clarified supernatants were stored at −80°C until detection. IL-17A and HMGB1 in liver homogenates [Humans: BA group (n=27), control group (n=23); Mice: MEM group (n=5 per time point), RRV group (n=5 per time point), WT + RRV group (n=5 per time point), Tcrδ^−/− + RRV group (n=5 per time point), Tcrδ^−/− + RRV + RPMI 1640 group (n=5 per time point), Tcrδ^−/− + RRV + IL-17A⁺ γδT group (n=5 per time point), RRV + DMSO group (n=5 per time point), RRV + Glycyrrhizin group (n=5 per time point), RRV + PBS group (n=5 per time point), RRV + GdCl₃ group (n=5 per time point)] and cell culture supernatants were detected according to the instructions of the IL-17A (cat. nos. ab216167 and ab199081; Abcam) and HMGB1 (cat. nos. SP14752 and SP11733; Wuhan Saipei Biotechnology Co., Ltd.) ELISA kits. A microplate reader (Spectramax Plus; Molecular Devices, LLC) was employed for plate analysis.

Liver tissue samples from patients [BA group (n=5), control group (n=5)] were fixed in 10% formalin at room temperature for 24 h, embedded in paraffin and cut into 4-μm sections. Slices of paraffin-embedded samples were prepared, followed by dewaxing with xylene, rehydration in graded alcohols and antigen restoration using EDTA at 95-100°C for 20 min. The sections were sequentially stained with six primary antibodies (incubated at room temperature for 1 h) using the Opal seven-color immunohistochemistry detection kit (cat. no. NEL821001KT; PerkinElmer, Inc.) according to the manufacturer's instructions, followed by incubation with HRP-conjugated secondary antibodies (included in the Opal seven-color immunohistochemistry detection kit) at room temperature for 10 min and then DAPI nuclear staining. A tyramine signal amplification system was used after each round of primary antibody and HRP-conjugated secondary antibody staining, and antibody stripping was performed between each staining cycle according to the manufacturer's protocol. The primary antibodies and dyes were cytokeratin (CK)19 (1:400; cat. no. ab52625; Abcam)/green, CD3 (1:200; cat. no. A26443; ABclonal Biotech Co., Ltd.)/red, CD4 (1:600; cat. no. ab133616; Abcam)/magenta, IL-17A (1:200; cat. no. A12454; ABclonal Biotech Co., Ltd.)/cyan, NKG2D (1:1,000; cat. no. MA5-51623; Invitrogen; Thermo Fisher Scientific, Inc.)/grey and TCRγδ (1:500; cat. no. ab313573; Abcam)/yellow. The stained slides were scanned for panoramic imaging using a Vectra Polaris slide digital scanner (PerkinElmer, Inc.), and the results were evaluated by two pathologists.

Total RNA was isolated from human liver tissues [BA group (n=5-8), control group (n=5-8)], mouse liver tissues [MEM group (n=5), RRV group (n=5), WT + RRV group (n=5), Tcrδ^−/− + RRV group (n=5), RRV + DMSO group (n=5), RRV + Glycyrrhizin group (n=5)] and γδT cells isolated from mouse liver using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using a PrimeScript™ RT Reagent Kit (Perfect Real Time; TaKaRa Bio, Inc.). The RT reaction was performed at 37°C for 15 min, followed by inactivation at 85°C for 5 sec. qPCR was performed with SYBR Green PCR Master Mix (SYBR Premix Ex Taq™ II; TaKaRa Bio, Inc.). The thermocycling program consisted of initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. The primer sequences are provided in Table SI. Transcript levels were normalized to GAPDH and were calculated via the 2^−ΔΔCq method (24).

Total proteins were extracted from human liver tissues [BA group (n=5-8), control group (n=5-8)], mouse liver tissues [MEM group (n=5), RRV group (n=5)] and γδT cells (isolated from mouse liver) using radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Biotechnology) following the provided protocol. Protein concentrations were determined using a bicinchoninic acid protein assay kit (cat. no. P0010; Beyotime Biotechnology) according to the manufacturer's instructions. Equal amounts of total protein (20-30 μg/lane) were separated by SDS-PAGE on 10 or 12% gels, transferred onto polyvinylidene difluoride membranes, blocked with 5% fat-free milk at room temperature for 45 min and incubated with the following primary antibodies overnight at 4°C according to the recommended dilution method: Anti-IL17A rabbit polyclonal antibody (pAb) (1:2,000; cat. no. A12454; ABclonal Biotech Co., Ltd.), anti-HMGB1 rabbit pAb (1:5,000; cat. no. A2553; ABclonal Biotech Co., Ltd.), anti-TLR2 antibody (1:400; cat. no. ab213676; Abcam), anti-TLR4 antibody (1:10,000; cat. no. ab218987; Abcam), anti-NF-κB (1:10,000; cat. no. A19653; ABclonal Biotech Co., Ltd.), phosphorylated (p)-NF-κB p65/RelA-S536 rabbit mAb (1:10,000; cat. no. AP1294; ABclonal Biotech Co., Ltd.) and anti-GAPDH mAb (1:50,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature (anti-rabbit IgG: 1:5,000; cat. no. RGAR001; anti-mouse IgG: 1:5,000; cat. no. RGAM001; both from Proteintech Group, Inc.). Proteins were visualized with enhanced chemiluminescence detection reagent (cat. no. RM00020P; ABclonal Biotech Co., Ltd.).

Briefly, human [BA group (n=8), control group (n=8)] and mouse liver tissues [MEM group (n=5), RRV group (n=5), WT + RRV group (n=5), Tcrδ^−/− + RRV group (n=5), Tcrδ^−/− + RRV + RPMI 1640 group (n=5), Tcrδ^−/− + RRV + IL-17A⁺ γδT group (n=5), RRV + DMSO group (n=5), RRV + Glycyrrhizin group (n=5)] were fixed in 10% formalin at room temperature for 24 h, followed by paraffin embedding and sectioning. The paraffin-embedded sections (4 μm) were dewaxed using xylene and rehydrated in graded alcohols. After antigen retrieval using either sodium citrate buffer or EDTA at a temperature of 95-100°C for 20 min continuously, endogenous enzyme blocking was performed with 3% H₂O₂ for 10 min at room temperature and then 5% goat serum (cat. no. C0265; Beyotime Biotechnology) was used for blocking (at room temperature for 30 min). According to the manufacturer's instructions, the sections were then incubated with the following primary antibodies overnight at 4°C: Anti-CK19 antibody (1:400; cat. no. ab52625; Abcam), anti-HMGB1 rabbit pAb (1:100; cat. no. A2553; ABclonal Biotech Co., Ltd.), anti-TLR2 antibody (1:200; cat. no. ab213676; Abcam) and anti-TLR4 antibody (1:200; cat. no. ab218987; Abcam), followed by incubation with secondary antibody (HRP-conjugated goat anti-rabbit IgG; 1:50; cat. no. A0208; Beyotime Biotechnology) for 1 h at room temperature. After DAB solution was added for color development, the sections were counterstained with hematoxylin for 1 min at room temperature. In addition, some paraffin-embedded sections underwent H&E staining, using 0.5% hematoxylin for 5 min at room temperature and 0.05% eosin for 1 min at room temperature. All stained sections were observed using a light microscope (Nikon Corporation). The results were assessed by two pathologists.

Data were analyzed using FlowJo software (v10.8.1) and GraphPad Prism 8.0 software (Dotmatics). The χ² test was used to compare the BA incidence between two groups. Continuous data between two groups were compared using the unpaired Student's t-test, whereas comparisons across three or more groups were performed using one-way ANOVA, followed by Bonferroni's post hoc test for pairwise comparisons. All experiments were performed in triplicate. Following intraperitoneal injections of RRV or saline within 24 h after birth, the survival and disease status of neonatal mice [n=28 for Tcrδ^−/− + RRV group; n=34 for WT + RRV group; n=32 for Tcrδ^−/− + RRV + RPMI 1640 group; n=34 for Tcrδ^−/− + RRV + IL-17A⁺ γδT group] were assessed and recorded for 20 consecutive days. Day 1 was regarded as the starting point, and the event of death as the endpoint. Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test. The hazard ratio (HR) with a 95% confidence interval (CI) was calculated as part of the log-rank analysis. P<0.05 was considered to indicate a statistically significant difference.

IL-17A is a key pathogenic cytokine in various inflammatory and autoimmune processes (10,22). In the present study, hepatic IL-17A levels were measured using RT-qPCR (Fig. 1A), western blotting (Fig. 1B) and ELISA (Fig. 1C) to investigate its role in BA; the results revealed that IL-17A levels were higher in the BA group compared with those in the control group. Flow cytometric analysis of human liver tissues identified TCRγδ⁺ T cells (γδT cells), CD56⁺ T cells (natural killer T cells), CD4⁺ T cells and CD8⁺ T cells as potential IL-17A sources (Fig. 1D), with CD4⁺IL-17A⁺ T cells (Th17 cells) and IL-17A⁺ γδT cells showing the highest increases in percentages within their respective subsets (Fig. 1D and E). In Fig. 1E, quantitative analysis of the percentages represent the proportion of IL-17A⁺ cells within their respective T cell subsets (for example, CD56⁺ IL-17A⁺ T cells/CD56⁺ T cells), calculated as [(upper-right quadrant)/(upper-right + lower-right quadrants) ×100%] based on the flow cytometry dot plots in Fig. 1D. Opal multiplex immunohistochemistry results revealed that the BA group had higher levels of CK19 markers in the liver, indicating abnormal proliferation of intrahepatic bile ducts, and IL-17A, NKG2D immune activation index, CD3, CD4 and TCRγδ were markedly increased around the bile ducts (Fig. 1F). Quantitative analysis validated higher percentages of IL-17A⁺ γδT cells in γδT cells and Th17 cells in CD4⁺ T cells in BA (Fig. 1G), which was consistent with the flow cytometry results. Functional blockade experiments using digoxin (Th17 cell inhibitor) and AM80 (γδT cell inhibitor) on mononuclear cells isolated from the liver tissues of patients with BA demonstrated γδT cells as an important source of IL-17A, with AM80 treatment causing a more marked reduction in IL-17A levels (Fig. 1H). These results indicated that γδT cells are the major producers of IL-17A driving inflammatory injury in BA, consistent with previous study results (9,10).

A mouse model of BA was established to elucidate the role of IL-17A⁺ γδT cells in BA progression. Dynamic analysis revealed a time-dependent increase in IL-17A levels in liver homogenate supernatants from day 3 to 14 (Fig. 2A), whereas the proportion of IL-17A⁺ γδT cells in the liver tissue increased from day 3 and peaked at day 7 (Figs. 2B and S1C), suggesting their pathogenic role in initiating an early inflammatory response in BA. The murine BA model on day 7 was used for further analysis. Mice with BA exhibited elevated hepatic mRNA levels of IL-1β, IL-6 and TNF-α compared with those in the control group (Fig. S2A), reflecting a pro-inflammatory state. Moreover, compared with in the WT murine model of BA, the Tcrδ^−/− murine model of BA exhibited reduced hepatic IL-17A levels (a 61.89% reduction at day 7; Fig. 2C), improved liver inflammation, reduced intrahepatic bile duct proliferation and improved extrahepatic bile ducts (Fig. 2D). The incidence of BA was markedly decreased from 82.4% (28/34) in WT mice to 35.7% (10/28) in Tcrδ^−/− mice (Fig. 2E), with a concomitant increase in 20-day survival rate from 29.4% (10/34) to 71.4% (20/28) [HR for WT vs. Tcrδ^−/−=4.147; 95% CI, 2.055-8.369; Fig. 2E]. Concomitant reductions in IL-1β, IL-6 and TNF-α in Tcrδ^−/− mice compared with in WT mice further validated the role of IL-17A⁺ γδT in orchestrating inflammatory responses (Fig. S2B). By contrast, adoptive transfusion of IL-17A⁺ γδT cells into the Tcrδ^−/− murine BA model (n=34) notably exacerbated these parameters compared with in the Tcrδ^−/− murine BA model receiving RPMI-1640 medium (n=32) (Fig. 2F-H). These data indicated that γδT cell-derived IL-17A may be critical for BA progression.

HMGB1, as a DAMP, triggers early inflammatory responses in various pathologies and is important in the pathogenesis of BA (12,13). Therefore, the expression of HMGB1 in BA and its role in IL-17A⁺ γδT cell activation were explored in the present study. Immunohistochemical staining results showed elevated HMGB1 expression in the hepatic portal area of patients with BA and in the BA mouse model (Fig. 3A and B). Similarly, the mRNA and protein expression levels of HMGB1 were markedly higher in BA (Fig. 3C-F). After inhibiting HMGB1 with glycyrrhizin, the BA mouse model showed a significant decrease in the proportion of hepatic IL-17A⁺ γδT cells and IL-17A levels in the liver homogenate supernatant on days 3 and 7 (Figs. 3G, H and S1D). Meanwhile, compared with that in the untreated BA mice, HMGB1 inhibition reduced pro-inflammatory cytokine levels (IL-1β, IL-6 and TNF-α; Fig. S2C), which is consistent with the role of HMGB1 in BA hepatic inflammation. These changes were accompanied by improvements in liver inflammation, intrahepatic bile duct proliferation and extrahepatic bile duct obstruction (Fig. 3I). These results suggested that HMGB1 may be critical in IL-17A production by γδT cells in BA. Subsequently, 48-h exogenous HMGB1 in vitro stimulation of hepatic γδT cells from patients with BA increased the percentage of IL-17A⁺ γδT cells (Figs. 3J and S1E) and IL-17A levels (Fig. 3K), further demonstrating the importance of HMGB1 in IL-17A⁺ γδT cell activation.

To determine the cellular sources of HMGB1 in BA, the present study continued to focus on BECs and macrophages, which are known targets of RRV infection that have a key role in hepatic inflammation (13,20). The capacity of BECs or macrophages to release HMGB1 after RRV infection was explored in vitro to validate their role in HMGB1 production. The immunofluorescence confocal assay (Fig. 4A) showed that HMGB1 protein was located in the nucleus of BECs at 0 h of RRV infection and began to be released from the nucleus at 12 h. A large amount of nuclear HMGB1 was accumulated in the cytoplasm and extracellular areas of BECs at 24 and 36 h. Meanwhile, the observed low nuclear signals of HMGB1 at 24 and 36 h likely reflect that the rate of HMGB1 release exceeds that of its synthesis, resulting in a reduction in nuclear HMGB1 staining. Correspondingly, BEC supernatants had higher HMGB1 levels at 24 and 36 h after RRV infection (Fig. 4B). Similar findings were observed in macrophages (Fig. 4C), suggesting both BECs and macrophages contribute to HMGB1 release in BA. Consistent with previous studies, HMGB1 was actively released from BECs after RRV infection in the experimental BA model (13,25). In vivo, macrophage depletion via GdCl₃ reduced serum HMGB1 at days 7 and 14 (Fig. 4D), and hepatic IL-17A concentrations at days 3 and 7 (Fig. 4E) in the BA murine model, confirming macrophages as important sources of HMGB1.

Subsequently, the present study investigated whether HMGB1 directly participated in γδT cell activation. BECs and macrophages were co-cultured with γδT cells derived from patients with BA, and IL-17A levels in the cell supernatant were measured under the intervention of RRV and glycyrrhizin (HMGB1 inhibitor) separately or in combination (Fig. 4F). Co-culture of RRV-infected BECs or macrophages with γδT cells derived from patients with BA significantly increased IL-17A secretion compared with co-culture with BECs or macrophages uninfected with RRV, which was suppressed by pre-incubating BECs or macrophages with glycyrrhizin (HMGB1 inhibitor) before co-culture (Fig. 4F).

Due to the interaction of HMGB1 with TLRs on various cell surfaces, including TLR2 and TLR4 (20,26), and the importance of TLR2 and TLR4 in γδT cell activation (27), the present study aimed to verify whether HMGB1 activates γδT cells through the TLR2/4-NF-κB pathways. The expression levels of TLR2 and TLR4 were notably upregulated in patients with BA (Fig. 5A-C) and localized near the hepatic portal area (Fig. 5C), indicating activation of the TLR2 and TLR4 pathways. In addition, the BA murine model showed similar findings, with higher expression of TLR2 and TLR4 compared with in control mice receiving MEM (Fig. 5D-F). The extraction of γδT cells from human liver tissues in the control group was insufficient, making it difficult to detect TLR2 and TLR4 on these cells. Therefore, the present study investigated the TLR pathway in γδT cells in the BA murine model, and the results showed higher mRNA and protein levels of TLR2 and TLR4 in IL-17A⁺ γδT cells compared with in γδT cells (Fig. 5G and H).

To further demonstrate the effect of TLR2 and TLR4 pathways on γδT cell activation, γδT cells were extracted from WT, Tlr2^−/− and Tlr4^−/− mice, and were co-cultured with RRV-infected BECs or macrophages in vitro. After stimulation with RRV for 36 h, the IL-17A secretion levels of the Tlr2^−/− and Tlr4^−/− γδT cells were significantly lower than those of WT γδT cells (Fig. 6A and B). These findings indicated the importance of TLR2 and TLR4 on γδT cells in IL-17A secretion. The reduced IL-17A production by Tlr2^−/− and Tlr4^−/− γδT cells in co-culture with RRV-infected BECs or macrophages (releasing HMGB1) confirms that the HMGB1-TLR2/4 pathways mediates IL-17A⁺ γδT cell activation.

To investigate the downstream mechanism, the present study focused on the NF-κB pathway, a canonical downstream effector of TLR signaling (21,22). The IL-17A⁺ γδT cells from the BA murine model exhibited elevated NF-κB p65 and p-NF-κB p65 levels compared with in γδT cells (Fig. 5G and H), confirming the activation of NF-κB signaling. NF-κB p65 upregulation may ensure sufficient substrate availability for phosphorylation, whereas increased p-NF-κB p65 indicates functional activation, consistent with previous reports on NF-κB signaling regulation (28,29). The co-culture system of Tlr2^−/− or Tlr4^−/− γδT cells with RRV-stimulated BECs or macrophages exhibited attenuated NF-κB activation, as evidenced by reduced levels of NF-κB p65 and p-NF-κB p65 compared with those in WT γδT cells (Fig. 6C-E). After stimulation of γδT cells with HMGB1 released by RRV-infected BECs or macrophages, the IL-17A level was significantly increased compared with that from BECs or macrophages uninfected with RRV (Fig. 4F). However, these effects were abolished by pretreatment with the NF-κB inhibitor Bay 11-7082 (Fig. 6F and G), demonstrating that HMGB1-TLR2/4 signaling depends on NF-κB to activate IL-17A⁺ γδT cells.