Liu Xingxing, Hu Hui, Fan Heng, Zuo Dongmei, Shou Zhexing, Liao Yi, Nan Zhen, Tang Qing
Department of Integrated Chinese and Western Medicine Department of Rheumatology and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
Medicine (Baltimore). 2017 Apr;96(17):e6615. doi: 10.1097/MD.0000000000006615.
This study aimed to investigate the role of aryl hydrocarbon receptor (AhR) and STAT3 gene during the differentiation of cluster of differentiation (CD)4 T cells into T helper (Th)17 and T regulatory (Treg) cells.
First, CD4 T cells were isolated from the spleen of BALB/c mice. Then, stable CD4 T cells expressing STAT3 shRNA were constructed. CD4 T cells were assigned to one of the following treatments: Th17 group: antibodies against CD3 and CD28, 2.5 ng/mL transforming growth factor β (TGF-β)1, 30 ng/mL interleukin (IL)-6, and 30 ng/mL IL-23; 6-formylindolo[3,2-b]carbazole (FICZ) group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 100 nM FICZ; FICZ + STAT3 RNAi group (shSTAT3 group): antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, 100 nM FICZ, and STAT3 RNAi; naphthoflavone group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 3 μM naphthoflavone; 5) no antibodies were added in the control group. Later, the proportions of Th17 and Treg cells in each group were measured by flow cytometry; phospho-STAT3 and -STAT5 levels were measured by western blotting; and AhR, STAT3, STAT5, receptor-related orphan nuclear receptor γt (RORγt), FOXP3, T-cell receptor (TCR), CD25, IL-6R, IL-10, and IL-17 mRNA levels were also measured by real-time PCR.
Th17 cells showed a rise and Treg cells showed a decrease in the FICZ group, but revised in the shSTAT3 group and the naphthoflavone group. Significant differences were observed in CD25, IL-6R, IL-10, and IL-17 mRNA levels among different groups.
STAT3 may cooperate with AhR to regulate the differentiation of both Th17 and Treg cells.
本研究旨在探讨芳烃受体（AhR）和信号转导与转录激活因子3（STAT3）基因在分化簇（CD）4 T细胞分化为辅助性T细胞（Th）17和调节性T细胞（Treg）过程中的作用。
首先，从BALB/c小鼠脾脏中分离出CD4 T细胞。然后，构建稳定表达STAT3短发夹RNA（shRNA）的CD4 T细胞。将CD4 T细胞分为以下处理组之一：Th17组：抗CD3和抗CD28抗体、2.5 ng/mL转化生长因子β（TGF-β）1、30 ng/mL白细胞介素（IL）-6和30 ng/mL IL-23；6-甲酰基吲哚并[3,2-b]咔唑（FICZ）组：抗CD3和抗CD28抗体、2.5 ng/mL TGF-β1、30 ng/mL IL-6、30 ng/mL IL-23和100 nM FICZ；FICZ + STAT3 RNA干扰组（shSTAT3组）：抗CD3和抗CD28抗体、2.5 ng/mL TGF-β1、30 ng/mL IL-6、30 ng/mL IL-23、100 nM FICZ和STAT3 RNA干扰；萘黄酮组：抗CD3和抗CD28抗体、2.5 ng/mL TGF-β1、30 ng/mL IL-6、30 ng/mL IL-23和3 μM萘黄酮；5）对照组不添加抗体。之后，通过流式细胞术检测每组中Th17和Treg细胞的比例；通过蛋白质免疫印迹法检测磷酸化STAT3和STAT5水平；通过实时聚合酶链反应（PCR）检测AhR、STAT3、STAT5、受体相关孤儿核受体γt（RORγt）、叉头框蛋白P3（FOXP3）、T细胞受体（TCR）、CD25、IL-6受体（IL-6R）及IL-10和IL-17的信使核糖核酸（mRNA）水平。
FICZ组中Th17细胞增加而Treg细胞减少，但在shSTAT3组和萘黄酮组中情况有所改变。不同组间CD25、IL-6R、IL-10和IL-17的mRNA水平存在显著差异。
STAT3可能与AhR协同调节Th17和Treg细胞的分化。
