Inhibition of HMGB1/NF-κB Signaling Restores Th17/Treg Balance via Dendritic Cell Modulation in Liver Transplant Rejection

Linyu Li¹Jie Wang¹Liyun Huang²Yi Chen²Lihong Chen^1,3*

• ¹Department of Pathology and Institute of Oncology, The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
• ²Department of Pathology, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, China
• ³Diagnostic Pathology Center, Fujian Medical University, Fuzhou, China

Background: Acute rejection (AR) remains a major challenge in liver transplantation (LT) despite advances in immunosuppression. High-mobility group box 1 (HMGB1) has emerged as a critical driver of immune activation; however, its role in dendritic cell (DC)-mediated T helper 17 (Th17)/regulatory T cell (Treg) imbalance during AR is unclear.Orthotopic LT was performed in rats assigned to sham, isograft, and allograft groups. Liver injury, HMGB1 expression, and hepatic DC infiltration were assessed by histopathology, immunohistochemistry, and CD11c immunofluorescence staining (IF), respectively, while serum levels of ALT, AST, and total bilirubin (TBIL) were measured to evaluate graft function. Th17/Treg populations were analyzed by flow cytometry to assess immune imbalance. RNA sequencing (RNAseq) was conducted to explore transcriptional changes in bone marrow-derived DCs stimulated with HMGB1 or PBS. DC maturation, cytokine secretion (ELISA), antigen uptake, and metabolic activity (CCK-8 assay) were assessed. A DC-CD4⁺ T cell coculture system was used to evaluate the ability of DCs to drive T cell proliferation and polarization. NF-κB signaling activation was examined by Western blot and immunofluorescence, and the NF-κB inhibitor helenalin was used to assess pathway relevance.Results: Allograft recipients displayed elevated serum ALT/AST/TBIL, accompanied by aggravated liver injury, increased Rejection Activity Index (RAI) scores, and upregulated HMGB1 expression. While CD11c IF demonstrated a pronounced increase in hepatic DC infiltration. Th17 cell frequencies and the Th17/Treg ratio were markedly increased, while Treg proportions were reduced. RNA-seq of DCs revealed HMGB1-induced transcriptional reprogramming, with nominal enrichment of NF-κB signaling. HMGB1 stimulation promoted DC maturation, enhanced proinflammatory cytokine production, and impaired antigen uptake and metabolic function. These activated DCs further facilitated CD4⁺ T cell proliferation and skewed differentiation toward the Th17 lineage while suppressing Treg induction. Notably, helenalin treatment effectively attenuated DC activation, restored their antigen uptake and metabolic activity, and reversed the Th17/Treg imbalance mediated by HMGB1-activated DCs. Conclusion: HMGB1 drives DC-mediated Th17/Treg imbalance during LT rejection through NF-κB activation. Targeting this pathway may offer a novel immunomodulatory strategy for managing AR.