白细胞介素-23受体过表达对实验性自身免疫性葡萄膜炎小鼠辅助性T细胞17/调节性T细胞平衡的影响

Effect of interleukin-23R overexpression on Th17/Treg balance in experimental autoimmune uveitis mice

目的初步探讨白细胞介素(IL)-23受体(IL-23R)过表达对实验性自身免疫性葡萄膜炎(EAU)模型小鼠辅助性T细胞17(Th17细胞)/调节性T细胞(Treg细胞)平衡的影响。方法8周龄雌性C57BL/6J小鼠12只,随机分为LV-Ctrl组、LV-IL-23R组,每组各6只。两组小鼠经尾静脉分别注射LV-Ctrl、LV-IL-23R慢病毒;注射后7 d采用光感受器间维生素A类结合蛋白1-20主动免疫构建EAU小鼠模型。免疫后13 d开始,每2天使用间接检眼镜观察小鼠眼底并进行临床评分。免疫后30 d,苏木精-伊红染色观察小鼠视网膜组织病理学改变。酶联免疫吸附测定检测两组小鼠血清中IL-17水平;流式细胞仪检测Th17细胞和Treg细胞比例;实时荧光定量聚合酶链反应检测IL-23R、IL-17、维甲酸相关孤儿受体γt(RORγt)、IL-10和叉头状转录因子p3(Foxp3)mRNA相对表达量。组间比较采用重复测量方差分析、独立样本Mann-Whitney U检验和独立样本t检验。结果与LV-Ctrl组比较,LV-IL-23R组小鼠视网膜炎症反应更重。免疫后13 d,LV-IL-23R组、LV-Ctrl组小鼠眼底炎症评分差异无统计学意义(t=-2.001,P=0.058);免疫后15~29 d,LV-IL-23R组小鼠眼底炎症评分均高于LV-Ctrl组,差异有统计学意义(t=-4.429、-6.578、-7.768、-10.183、-6.325、-7.304、-4.841、-6.872,P<0.001)。组织病理学检查显示,与LV-Ctrl组比较,LV-IL-23R组眼底炎性细胞浸润增多,视网膜结构破坏更为严重,组织病理学评分显著升高,差异有统计学意义(t=-4.339,P=0.001)。与LV-Ctrl组比较、LV-IL-23R组小鼠脾脏中IL-23R mRNA相对表达量显著升高,差异有统计学意义(Z=2.087,P=0.037);T细胞中IL-17、RORγt mRNA相对表达量增加,IL-10、Foxp3 mRNA相对表达量降低,差异均有统计学意义(t=-6.313、-5.922、4.844、7.572,P=0.003、0.004、0.008、0.002)。与LV-Ctrl组比较、LV-IL-23R组小鼠血清中IL-17水平明显升高,差异有统计学意义(t=-5.423,P=0.002);脾脏和淋巴结中Th17细胞比例明显升高,Treg细胞比例显著降低,差异有统计学意义(t=-4.290、3.700,P=0.002、0.006)。结论IL-23R过表达可促使EAU小鼠的Th17/Treg失衡,加重EAU临床及病理表现。

Objective To investigate the effect of interleukin(IL)-23 receptor(IL-23R)overexpression on the balance of T helper 17(Th17 cells)/regulatory T cells(Treg cells)in experimental autoimmune uveitis(EAU)mice.Methods Twelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group,with 6 mice in each group.Two groups of mice were injected with LV-Ctrl and LV-IL-23R lentiviruses through the tail vein,respectively;7 days after injection,the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors.Starting from 13 days after immunization,the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed;30 days after immunization,hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina.The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay;the proportion of Th17 cells and Treg cells was detected by flow cytometry.The relative mRNA expression of IL-23R,IL-17,retinoic acid-related orphan receptorγt(RORγt),IL-10 and forkhead transcripyion factor p3(Foxp3)were detected by real-time quantitative polymerase chain reaction.Comparisons between groups were performed using repeated measures analysis of variance,independent samples Mann-Whitney U test,and independent samples t test.Results Compared with the LV-Ctrl group,the retinal inflammatory reaction of the LV-IL-23R group was more severe.At 13 days after immunization,there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group(t=-2.001,P=0.058);15-29 days after immunization.The fundus inflammation scores of LV-IL-23R group were higher than those of LV-Ctrl group,and the difference was statistically significant(t=-4.429,-6.578,-7.768,-10.183,-6.325,-7.304,-4.841,-6.872;P<0.001).Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased,the retinal structure was damaged more seriously,and the histopathological score was significantly increased,and the difference was statistically significant(t=-4.339,P=0.001).Compared with the LV-Ctrl group,the relative expression of IL-23R mRNA in the spleen of the LV-IL-23R group was significantly increased,and the difference was statistically significant(Z=2.087,P=0.037).The relative expression of IL-17 and RORγt mRNA increased,while the relative expression of IL-10 and Foxp3 mRNA decreased,and the differences were statistically significant(t=-6.313,-5.922,4.844,7.572;P=0.003,0.004,0.008,0.002).Compared with the LV-Ctrl group,the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased,and the difference was statistically significant(t=-5.423,P=0.002);the proportion of Th17 cells in the spleen and lymph nodes was significantly increased,whereas,the proportion of Treg cells was significantly reduced,and the difference was statistically significant(t=-4.290,3.700;P=0.002,0.006).Conclusion IL-23R overexpression can promote Th17/Treg imbalance in EAU mice,and aggravate the clinical and pathological manifestations of EAU.