IL‑17A⁺ γδT cell activation via the HMGB1‑TLR2/4‑NF‑κB signaling pathways in biliary atresia

IL‑17A⁺ γδT cells are involved in biliary atresia (BA) inflammatory injury; however, the mechanism underlying their activation remains unclear. The present study aimed to elucidate the mechanism by which γδT cells are activated to secrete IL‑17A in BA. Clinical samples from 27 patients with BA and 23 control individuals were collected and analyzed using reverse transcription‑quantitative PCR (RT‑qPCR), western blotting, ELISA, flow cytometry, Opal multiplex immunohistochemistry and immunohistochemistry. In addition, wild‑type (WT) and Tcrδ^‑/‑ BA murine models were developed through infection with rhesus rotavirus (RRV) and assessed by RT‑qPCR, western blotting, ELISA, flow cytometry, immunohistochemistry and hematoxylin and eosin staining. The release of high‑mobility group box‑1 (HMGB1) from RRV‑infected biliary epithelial cells (BECs) and macrophages was investigated by ELISA and immunofluorescence confocal microscopy. In addition, co‑culture systems of γδT cells with BECs or macrophages were established. The results revealed that the levels of hepatic IL‑17A were higher in patients with BA, with γδT cells serving an important role in producing IL‑17A. In the BA murine model, the IL‑17A levels increased from day 3 to 14, and IL‑17A⁺ γδT cells peaked on day 7. The Tcrδ^‑/‑ BA murine model exhibited lower IL‑17A levels, improved liver and bile duct morphology, and lower BA incidence compared with in the WT BA murine model, which was reversed through adoptive transfer of murine IL‑17A⁺ γδT cells. HMGB1, and Toll‑like receptor (TLR)2 and 4 were upregulated in liver tissues from both patients with BA and the BA murine model. By contrast, hepatic IL‑17A levels were decreased in the BA murine model treated with the HMGB1 inhibitor. In addition, HMGB1 was released from RRV‑infected BECs or macrophages, and exogenous HMGB1 stimulation enhanced IL‑17A production in γδT cells. The Tlr2^‑/‑ and Tlr4^‑/‑ γδT cell co‑culture system inhibited IL‑17A production and NF‑κB activation, whereas NF‑κB inhibition abolished HMGB1‑induced IL‑17A production in WT γδT cells. In conclusion, HMGB1 released from injured BECs or macrophages may activate IL‑17A⁺ γδT cells to mediate BA inflammatory injury via the HMGB1‑TLR2/4‑NF‑κB pathways. The present study was registered with the Chinese Clinical Trial Registry (registration ID: ChiCTR2000039619; registered November 3, 2020).