Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice.
Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice.
复制标题
白细胞介素 35 在正常条件下诱导小鼠 Th2 和 Tregs 偏向
作者：
Zhang X;Zhang Z;He Z;Ju M;Li J;Yuan J;Jing Y;Li K;Liu Y;Li G
目的IL-35治疗疾病的益处已在多种动物模型中得到验证，但其在正常情况下对T细胞免疫的影响仍有待阐明。本研究旨在探讨体内和体外调节IL-35水平对正常小鼠T细胞反应的影响以及对T细胞亚群的影响。方法构建携带两个亚基连接的小鼠线性IL-35片段的质粒pMSCV-IL-35-GFP，并证实其异源二聚体的表达。正常小鼠随机分为三组，分别静脉注射PBS、pMSCV-GFP和pMSCV-IL-35-GFP。72h后，取脾组织和外周血进行分析。同时，分离脾T细胞，分别用10、30、50 ng/mL重组IL-35因子和抗cd3 /CD28体外孵育24 h。采用荧光活化细胞分选法（FACS）检测t细胞亚群，实时荧光定量PCR检测相关细胞因子及效应分子。结果转染pMSCV-IL-35-GFP质粒的HEK 293T细胞裂解液中出现52 kDa条带，IL-35成功表达。IL-35的两个亚基Ebi3和IL-12A在注射DNA 72 h后被鉴定出来。体内IL-35上调可有效抑制CD4+、CD8+ T细胞增殖及Th1细胞因子分泌。CD8+ T细胞的效应分子也被显著抑制。相反，高水平IL-35显著诱导CD4+ CD25+ Tregs和Th2增强。体外研究也提供了类似的结果。结论正常情况下IL-35对Th1和CD8+ T细胞有抑制作用，对Th2和Tregs有偏倚作用，这是其治疗潜力的部分原因。
Objective The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice. Methods A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR. Results Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results. Conclusion The results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.
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