Li Linyu, Wang Jie, Huang Liyun, Chen Yi, Chen Lihong
Department of Pathology and Institute of Oncology, The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
Department of Pathology, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian, China.
Front Immunol. 2025 Sep 4;16:1649366. doi: 10.3389/fimmu.2025.1649366. eCollection 2025.
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 alanine aminotransferase (ALT), aspartate aminotransferase (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 (RNA-seq) 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 (WB) and IF, and the NF-κB inhibitor helenalin was used to assess pathway relevance.
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, which was further confirmed by WB and IF. HMGB1 stimulation promoted DC maturation, enhanced pro-inflammatory 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.
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.
尽管免疫抑制取得了进展，但急性排斥反应（AR）仍是肝移植（LT）中的一项重大挑战。高迁移率族蛋白B1（HMGB1）已成为免疫激活的关键驱动因素；然而，其在AR期间树突状细胞（DC）介导的辅助性T细胞17（Th17）/调节性T细胞（Treg）失衡中的作用尚不清楚。
对分配至假手术、同基因移植和同种异体移植组的大鼠进行原位肝移植。分别通过组织病理学、免疫组织化学和CD11c免疫荧光染色（IF）评估肝损伤、HMGB1表达和肝DC浸润，同时测量血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和总胆红素（TBIL）水平以评估移植物功能。通过流式细胞术分析Th17/Treg细胞群以评估免疫失衡。进行RNA测序（RNA-seq）以探索用HMGB1或PBS刺激的骨髓来源DC中的转录变化。评估DC成熟、细胞因子分泌（ELISA）、抗原摄取和代谢活性（CCK-8测定）。使用DC-CD4 T细胞共培养系统评估DC驱动T细胞增殖和极化的能力。通过蛋白质印迹（WB）和IF检测NF-κB信号激活，并使用NF-κB抑制剂海伦alin评估通路相关性。
同种异体移植受体的血清ALT/AST/TBIL升高，伴有肝损伤加重、排斥活动指数（RAI）评分增加和HMGB1表达上调。而CD11c IF显示肝DC浸润明显增加。Th17细胞频率和Th17/Treg比率显著增加，而Treg比例降低。DC的RNA-seq显示HMGB1诱导的转录重编程，NF-κB信号有明显富集，WB和IF进一步证实了这一点。HMGB1刺激促进DC成熟，增强促炎细胞因子产生，并损害抗原摄取和代谢功能。这些活化的DC进一步促进CD4 T细胞增殖，并使分化偏向Th17谱系，同时抑制Treg诱导。值得注意的是，海伦alin治疗有效减弱了DC激活，恢复了它们的抗原摄取和代谢活性，并逆转了由HMGB1活化DC介导的Th17/Treg失衡。
HMGB1通过激活NF-κB在肝移植排斥反应期间驱动DC介导的Th17/Treg失衡。靶向该通路可能为管理AR提供一种新的免疫调节策略。
