Anti-IL-12/IL-23 p40抗体对实验性自身免疫性葡萄膜炎的抑制作用及其机制

Therapeutic effect of anti-IL-12/IL-23 p40 on experimental autoimmune uveitis and its mechanism

目的探讨Anti-白细胞介素(IL)-12/IL-23 p40抗体对实验性自身免疫性葡萄膜炎(EAU)的抑制作用及其机制。方法选取SPF级健康无眼疾6~8周龄雌性C57BL/6N小鼠66只,其中24只采用光感受器维生素A类结合蛋白(IRBP)651-670诱导小鼠EAU模型,分别在免疫前及免疫后第3、12、18天各取6只小鼠,流式细胞术检测各时间点小鼠脾脏、淋巴结和眼球中IL-17A^(+)γ干扰素(IFN-γ)^(+)CD4^(+)T细胞比例。取6只小鼠制作EAU模型,免疫后18 d采用小动物成像仪进行眼底拍照并行光相干断层扫描(OCT)检查。检查完成后处死小鼠,摘取眼球,采用苏木精-伊红染色法检测小鼠视网膜炎症反应和组织结构形态学改变;取淋巴结行流式细胞术检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例,按照表达数量不同分为IL-17A^(+)IFN-γ^(+)细胞高表达组和IL-17A^(+)IFN-γ^(+)细胞低表达组,比较2个组小鼠视网膜损伤情况。取36只小鼠制作EAU模型,采用随机数字表法分成Anti-IL-12/IL-23 p40组和IgG组,每组18只,分别尾静脉注射Anti-IL-12/IL-23 p40和IgG,每3天1次。免疫后第12天和第18天每组各取6只小鼠,分别取淋巴结和眼球组织,采用流式细胞仪检测T细胞亚群比例。免疫后第24天,每组各取6只小鼠,摘取眼球,采用苏木精-伊红染色法观察视网膜损害情况;采用流式细胞仪检测CD4^(+)T细胞体外诱导分化情况;采用酶联免疫吸附测定(ELISA)法检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞诱导分化后IL-17和IFN-γ表达情况;采用实时荧光定量PCR法检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞诱导分化后Th1细胞转录因子T-bet和Th17细胞转录因子维甲酸相关孤核受体γt(ROR-γt)相对表达量。结果免疫前和免疫后第3、12、18天,淋巴结、脾脏、眼球中IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例总体比较差异均有统计学意义(H=9.642、16.531、10.385,均P<0.05),其中与免疫前相比,EAU小鼠免疫后第12天淋巴结IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例明显升高;免疫后第18天脾脏、眼球中IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例明显升高,差异均有统计学意义(均P<0.05)。IL-17A^(+)IFN-γ^(+)细胞高表达组小鼠视网膜严重水肿、视网膜脱离、重度炎性细胞浸润和广泛视网膜褶皱;IL-17A^(+)IFN-γ^(+)细胞低表达组小鼠视网膜轻度水肿、局灶性炎性细胞浸润、轻度视网膜褶皱。Anti-IL-12/IL-23 p40组免疫后18 d,眼球中CD3和IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例低于IgG组,差异均有统计学意义(t=15.304、8.080,均P<0.05);免疫后12 d,Anti-IL-12/IL-23 p40组淋巴结中IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例为(0.33±0.18)%,明显低于IgG组的(4.83±0.45)%,差异有统计学意义(t=15.974,P<0.001)。与IgG组相比,Anti-IL-12/IL-23 p40组Th1、Th17、IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞百分比及IL-17、IFN-γ、T-bet、ROR-γt表达量明显降低,差异均有统计学意义(均P<0.05)。结论Anti-IL-12/IL-23 p40可通过抑制IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞发挥对EAU的治疗作用。

Objective To explore the therapeutic effect of anti-interleukin(IL)-12/IL-23 p40 antibody on experimental autoimmune uveitis(EAU)and its mechanism.Methods Sixty-six SPF female C57BL/6N mice aged 6-8 weeks were selected.EAU model was established in 24 mice through immunization with the interphotoreceptor retinoid-binding protein(IRBP)651-670.The 24 mice were sacrificed before immunization,and on the 3rd,12th,and 18th day after immunization,with 6 at each time point.Flow cytometry was used to detect the proportion of IL-17A^(+)interferon-γ(IFN-γ)+CD4^(+)T cells in the spleen,lymph nodes and eyeballs.Another 6 mice were selected to establish EAU model,and fundus images of the mice were taken with a small animal imaging instrument and optical coherence tomography(OCT)18 days after immunization.The 6 mice were sacrificed after OCT examination and the eyeballs were collected.Hematoxylin-eosin staining was used to observe the retinal inflammation and morphological changes in tissue structure.Flow cytometry was employed to detect the proportion of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells in lymph nodes.The 6 mice were divided into IL-17A^(+)IFN-γ^(+)highly expressed group and IL-17A^(+)IFN-γ^(+)lowly expressed group according to flow cytometry results,and the retinal injury was compared between the two groups.EAU model was established in another 36 mice,which were divided into anti-IL-12/IL-23 p40 group and IgG group by random number table method,with 18 mice in each group.Anti-IL-12/IL-23 p40 or IgG was injected by tail vein at a 3-day inteval according to grouping.On the 12th and 18th day after immunization,6 mice were selected from each group to collect lymph nodes and eyeballs,and the proportion of T cell subsets was detected by flow cytometry.Eyeballs of 6 mice in each group were extracted on the 24th day after immunization and retinal damage was observed by hematoxylin-eosin staining.The induced differentiation of CD4^(+)T cells in vitro was assayed by flow cytometry.The expressions of IL-17 and IFN-γwere detected by enzyme-linked immunosorbent assay(ELISA)after induced differentiation of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells.The relative expression levels of Th1 transcription factor T-bet and Th17 transcription factor retinoid acid-related orphan nuclear receptorγt(ROR-γt)after induced differentiation of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells were detected by real-time quantitative PCR.The use and care of animals followed the ARVO statement and this study protocol was approved by an Ethics Committee of Experimental Animals of Tianjin Medical University Eye Hospital(No.TJYY2019111019).Results There were significant differences in the proportion of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells in lymph nodes,spleen and eyeballs between wild-type mice and EAU mice at the 3rd,12th and 18th day after immunization(H=9.642,16.531,10.385;all at P<0.05).Compared with before immunization,the proportion of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells was significantly increased in lymph nodes of EAU mice on the 12th day following immunization and was significantly increased in spleen and lymph nodes on day 18 after immunization(all at P<0.05).Severe retinal exudation,retinal detachment,severe inflammatory cell infiltration and extensive retinal folds were detected in IL-17A^(+)IFN-γ^(+)highly expressed mice.Mild retinal edema,focal inflammatory cell infiltration and mild retinal folds were found in IL-17A^(+)IFN-γ^(+)lowly expressed mice.The proportion of CD3 and IL-17A^(+)IFN-γ^(+)CD4^(+)T cells in the eyeballs of anti-IL-12/IL-23 p40 group was lower than that in IgG group at the 18th day after immunization,and the differences were statistically significant(t=15.304,8.080;both at P<0.05).On day 12 after immunization,the percentage of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells in anti-IL-12/IL-23 p40 group was(0.33±0.18)%,which was significantly lower than(4.83±0.45)%in IgG group(t=15.974,P<0.001).Compared with IgG group,the percentage of Th1,Th17,IL-17A^(+)IFN-γ^(+)CD4^(+)T cells and the expression levels of IL-17,IFN-γ,T-bet,ROR-γt in anti-IL-12/IL-23 p40 group were significantly decreased,with statistical significances(all at P<0.05).Conclusions Anti-IL-12/IL-23 p40 has a therapeutic effect on EAU by inhibiting IL-17A^(+)IFN-γ^(+)CD4^(+)T cells.