高迁移率族蛋白B1对小鼠调节性T细胞Foxp3表达的影响

Effect of high mobility group box-1 protein on Foxp3 expression in spleen regulatory T cells in mice

目的观察高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)对调节性T细胞(regulatory Tcell,Treg)叉头翼状螺旋转录因子(forkhead/winged helix transcription factorp3,Foxp3)基因及蛋白表达的影响,并对其机制进行初步探讨。方法免疫磁珠法分离正常BALB/c小鼠脾脏Treg。采用固相包被抗-CD3及可溶性CD28辅助活化,给予HMGB1刺激,观察HMGB1刺激与Foxp3基因及蛋白表达的时间-效应关系及剂量-效应关系。结果经HMGB1刺激后的TregFoxp3蛋白表达于24～72h明显下调(P<0.05,P<0.01),其中以作用48、72h后表达下调尤为显著(P<0.01);给予不同剂量HMGB1刺激72h后,10、100、1000ng/ml的HMGB1均可诱导Foxp3表达减弱(P<0.05,P<0.01),其中HMGB1的浓度在1000ng/ml时Foxp3表达下调最明显。Foxp3mRNA表达呈现出与蛋白表达相同的时间、剂量依赖关系。结论HMGB1通过诱导TregFoxp3mRNA表达下调,进一步影响其蛋白产物合成,从而影响Treg免疫调节活性。

Objective Intranuclear forkheacL/winged helix transcription factor p3 （ Foxp3 ） plays a key role in T cell-mediated immunosuppression. The present study was performed to investigate the effects of high mobility group box-1 protein （HMGB1） on Foxp3 gene as well as protein expressions in splenic regulatory T cells （Tregs） and their potential regulating mechanisms in mice. Methods CD4^＋ CD25^＋ Tregs isolated from the spleens of male BABL/c mice by magnetic beads were seeded on 96-well （ 1 × 105 cells/well ） cell culture plates coated with anti-CD3 （1 ug/ml） and soluble anti-CD28 （1 ug/ml）, and the cells were stimulated with HMGB1 at various intervals or at different concentrations. After being stimulated, the Foxp3 mRNA/protein expressions in the Tregs were determined. The time-dependent and dose-dependent responses between HMGB1 and intranuclear Foxp3 expression were analyzed by flow cytometry, and the expressions of Foxp3 mRNA of Tregs were analyzed by quantitative PCR of SYBR GREEN. Results After stimulation with HMGB1, the intranuclear Foxp3 protein and mRNA expressions of splenic Tregs in mice were markedly down-regulated in 24 h to 72 h （P 〈0.05 or 0. 01 ）, and the expression levels of Foxp3 protein and Foxp3 mRNA were lowest at 72 h （P 〈0.01）. When Tregs were cultured in the presence of 10, 100, and 1 000 ng/ml HMGB1 for 72 h, the expressions of Foxp3 protein and mRNA were significantly down-regulated （ P 〈 0. 05 or O. 01 ） , and values of this molecules were lowest in 1 000 ng/ml HMGBl-treated group （P 〈0.01 ）. Conclusion These data indicate that HMGB1 stimulation can result in significant down-regulation of the Foxp3 mRNA and protein expressions in mouse splenic Tregs. HMGB1 appears to be a potential immunoregulatory signal that influences the function of Tregs by inhibiting CD4^＋ CD25^＋ Treg activity.