Th17细胞/调节性T细胞(Treg)失衡促进小鼠日本血吸虫病肝虫卵肉芽肿形成

Imbalance of Th17/Tregs promotes egg granuloma formation of liver with Schistosomiasis japonicum in mice

目的探究Th17细胞/调节性T细胞(Treg)失衡在日本血吸虫病肝虫卵肉芽肿形成中的作用。方法采用6~8周龄的BALB/c小鼠建立日本血吸虫感染模型,在感染后第2、4、6、8周处死小鼠,收集肝脾组织和血液。采用HE染色和Masson染色检测小鼠肝组织病变情况;流式细胞术检测肝、脾组织中Th17细胞、Treg百分比变化;实时定量PCR检测肝组织中白细胞介素6(IL-6)、IL-10、IL-17、IL-23和维甲酸相关孤核受体γt(RORγt)、叉头盒转录因子P3(FOXP3)的mRNA水平;ELISA检测血清中IL-6、IL-17、IL-23和转化生长因子β(TGF-β)表达水平。制备日本血吸虫可溶性虫卵抗原(SjSEA),体外刺激小鼠脾细胞,实时定量PCR检测不同时间点RORγt、FOXP3的mRNA水平,ELISA检测培养上清液中IL-6、IL-17、IL-23、TGF-β的水平。结果血吸虫感染第4周开始,肝组织中出现炎性细胞浸润、虫卵肉芽肿形成和胶原蛋白沉积增加,感染时间越长,病变程度越严重。在肝、脾组织中,与正常对照组比较,Th17细胞百分数在感染后第2、4、6周显著性升高;Treg百分数在感染后第4、6、8周的小鼠肝组织中显著性升高,而在小鼠脾脏中则是在感染第2、4周显著性升高。与正常对照组相比,在肝组织中,Th17细胞/Treg比值在感染组均明显降低;在脾组织中,Th17细胞/Treg比值在感染后第2、4周明显降低,第6周出现升高。Th17细胞/Treg比例失衡在肝脏更为明显。感染小鼠肝组织和血清中相关的转录因子和细胞因子表达水平与Th17细胞和Treg的动态变化相吻合。SjSEA刺激小鼠脾细胞能够诱导Th17细胞的分化以及相关细胞因子和转录因子的表达。结论Th17细胞在血吸虫病的肝组织病理损伤中可能起主要作用,Th17细胞/Treg比例失衡与日本血吸虫肝虫卵肉芽肿反应和纤维化的形成密切相关。

Objectives To investigate the effect of the imbalance of Th17/Treg on egg granuloma formation of liver with Schistosomiasis japonicum.Methods The BALB/c mice were infected with Schistosoma japonicum cercariae to establish a model of Schistosomiasis japonica.The blood samples,liver tissues and spleen tissue were harvested at the 2nd,4th,6th,8th week,respectively.HE staining and Masson staining were performed to assess the pathological characteristics of the liver.Flow cytometry(FCM)was conducted to evaluate the proportion of CD4^(+)T cell subsets including Th17 cells and Tregs in liver and spleen tissue.The quantitative real-time PCR(qRT-PCR)was carried out to investigate the mRNA level of cytokines including RORγt,FOXP3,IL-6,IL-17,IL-23 and IL-10 in liver tissues.Finally,ELISA was performed to assess the serum level of cytokines including IL-6,IL-17,IL-23 and TGF-β.Schistosoma japonicium soluble egg antigen(SjSEA)were prepared to stimulate mouse spleen cells in vitro.qRT-PCR was carried out to investigate the mRNA level of cytokine including RORγt and FOXP3 and ELISA was performed to assess the expression level of cytokines including IL-6,IL-17,IL-23 and TGF-β at different time points.Results HE and Masson staining demonstrated that inflammatory cell infiltration,schistosome egggranuloma formation and the collagen deposition increased in the liver tissue after the 4th week.The longer the infection,the more severe the liver pathology.In the liver and spleen tissues,the percentage of Th17 cells of infection group(2nd,4th and 6th weeks)were significantly higher than the healthy group.The percentage of Tregs in the liver tissues of infection group(4th,6th and 8th weeks)were significantly higher than the healthy group,and the percentage of Tregs in the spleen of infection group(2nd and 4th weeks)were significantly higher than the healthy group.Th17/Treg ratios in the liver of infection group were lower than the healthy group.Th17/Treg ratios in the spleen of infection group(2nd and 4th weeks)were lower than the healthy group,while it increased in the 6th week.At the same time,the levels of Th17 cells and Tregs related nuclear transcription factors and cytokines showed similar dynamic changes as the percentages of T cell subsets.SjSEA can induce the differentiation of Th17 and Tregs and the expression of related cytokines and transcription factors.Conclusion Th17 cells may play a major role in liver pathology,and the imbalance of Th17 cells/Tregs was closely related to the schistosome egg granuloma formation.