香烟烟雾提取物抑制树突状细胞诱导CD4^+T细胞分化为CD4^+CD25^+Foxp3^+调节性T细胞

Depressive effect of cigarette smoke extracts on dendritic cells inducing differentiation of CD4^+T cells into CD4^+CD25^+Foxp3^+Tregs

目的探讨香烟烟雾提取物(CSE)暴露及抑制CD40-CD40L路径对小鼠髓源性树突状细胞(BMDC)诱导CD4^+T细胞分化为CD4^+CD25^+Foxp3^+调节性T细胞(Treg)的影响。方法使用40 ng/m L重组小鼠粒细胞-巨噬细胞集落刺激因子(rm GM-CSF)和10 ng/m L重组小鼠白细胞介素4(rm IL-4)联合诱导无特定病原体级健康雄性BALB/c小鼠骨髓来源的单个核细胞分化为树突状细胞(DC),流式细胞术检测小鼠BMDC表面CD40分子的表达,再采用免疫磁珠分选的方法从BALB/c小鼠脾脏细胞分离出CD4^+T细胞。将所培养出的小鼠BMDC与正常对照组小鼠脾脏细胞分选的CD4^+T细胞共培养,加入CSE及拮抗性CD40抗体,作用24 h;流式细胞术观察各组细胞CD4^+CD25^+Foxp3^+Treg的变化,液相芯片技术检测细胞上清液白细胞介素10(IL-10)、IL-6的水平。结果 BMDC与CD4^+T淋巴细胞共培养可以促进CD4^+CD25^+Foxp3^+Treg分化,经CSE刺激后,流式细胞术检测显示小鼠BMDC与CD4^+T细胞共培养后分化的CD4^+CD25^+Foxp3^+Treg数量降低,细胞上清液IL-10降低、IL-6的水平升高;而加入拮抗性CD40抗体细胞共培养组CD4^+CD25^+Foxp3^+Treg增加,液相芯片检测细胞上清液IL-10上升、IL-6的水平下降。结论 CSE减少CD4^+CD25^+Foxp3^+Treg数量,体外使用拮抗性抗CD40抗体阻断CD40-CD40L通路可以促进CD4^+T细胞分化为CD4^+CD25^+Foxp3^+Treg。

Objective To explore the impact of both cigarette smoke extracts（ CSE） and CD40-CD40 L pathway blocking on mouse myeloid dendritic cells（ DCs） inducing CD4~＋T cells differentiation into CD4~＋CD25~＋Foxp3~＋regulatory T cells（ Tregs）. Methods A combination of recombinant mouse granulocyte-macrophage colony stimulating factor（ rm GM-CSF,40 ng / m L） and recombinant mouse IL-4（ rm IL-4,10 ng / m L） was applied in vitro to induce the differentiation of BALB / c mouse myeloid monocytes into DCs. DCs were detected for the expression of CD40 molecules through flow cytometry. CD4~＋T cells were sorted out from BALB / c mouse spleen cells with magnetic activated cell sorting technique. Myeloid DCs were co-cultivated with CD4~＋T cells separated from the control-group for 24 hours in the presence of CSE or antagonistic CD40 antibody. CD4~＋CD25~＋Foxp3~＋Tregs were quantified with flow cytometry,and concentrations of IL-10 and IL-6 in cell supernatants were detected with liquid phase chip technology. Results The co-cultivation of DCs and CD4~＋T cells promoted CD4~＋CD25~＋Foxp3~＋Tregs differentiation. However,after stimulated by CSE,the number of CD4~＋CD25~＋Foxp3~＋Tregs differentiated by co-cultivation of DCs and CD4~＋T cells was reduced,accompanied by the decrease of IL-10 concentration and the increased of IL-6 concentration. In contrast,with the intervention of antagonistic CD40 antibody,the number of CD4~＋CD25~＋Foxp3~＋Tregs increased,IL-10 concentration rose and IL-6 dropped. Conclusion CSE can substantially reduce CD4~＋CD25~＋Foxp3~＋Tregs,but blocking CD40-CD40 L pathway in vitro by antagonistic CD40 antibody can promote the process of CD4~＋T cells differentiating into CD4~＋CD25~＋Foxp3~＋Tregs.