ICOS转基因小鼠感染日本血吸虫对CD28/CD86表达及Th1/Th2极化的影响

Effects of Schistosoma japonicum Infection on the CD28/CD86 Signaling Pathway and Th1/Th2 Polarization in ICOS Transgenic Mice

目的探讨上调可诱导共刺激分子(ICOS)信号对感染日本血吸虫小鼠CD28/CD86的表达及Th1/Th2极化的影响。方法建立ICOS转基因(ICOS-Tg)小鼠及野生型FVB/NJ小鼠日本血吸虫病模型,应用流式细胞术分析感染前(0周)和感染后(4～20周)小鼠的脾CD4+T淋巴细胞上CD28与脾CD19+B淋巴细胞上CD86的表达水平。应用免疫组化法检测同期小鼠肝脏虫卵肉芽肿周围炎性浸润细胞上CD28、CD86表达水平变化。取同期小鼠脾淋巴细胞用SEA进行诱导培养72 h后,采用ELISA法检测培养上清中Th1(IFN-γ、IL-12)及Th2(IL-4、IL-13)细胞因子表达水平。采集同期小鼠的血清,应用ELISA法检测血清中SEA特异性抗体IgG及其亚类IgG1、IgG2a的表达水平。应用HE染色法观察小鼠肝脏虫卵肉芽肿病变动态变化。结果ICOS-Tg小鼠脾CD4+T细胞上CD28和脾CD19+B细胞上CD86的水平与同期野生型FVB/NJ小鼠相比明显升高,且分别在感染4周后(50.57±7.54 vs 40.38±5.43,P<0.05)及在感染12周后(76.46±10.55 vs 65.10±10.17,P<0.05)差异显著。ICOS-Tg小鼠肝脏虫卵肉芽肿的CD28、CD86表达亦高于野生型FVB/NJ小鼠的水平,且分别在感染7周后(0.485±0.094 vs 0.357±0.078,P<0.05)及感染12周后(0.649±0.072 vs 0.537±0.064,P<0.05)差异显著。ICOS-Tg小鼠Th2细胞因子(IL-4、IL-13)水平从感染7周后比野生型FVB/NJ小鼠显著升高(皆P<0.05);ICOS-Tg小鼠血清SEA特异性抗体IgG及其亚类IgG1、IgG2a的水平显著高于野生型FVB/NJ小鼠的水平(P<0.05、0.01、0.001不等);ICOS-Tg小鼠Th2分化指数与IgG1/IgG2a的比值亦高于野生型小鼠,分别在感染7、12、16、20周后(P<0.05、0.001)及12、16周后(皆P<0.05)具有显著性差异。在整个病程中,ICOS-Tg小鼠肝脏虫卵肉芽肿体积显著大于野生型FVB/NJ小鼠。结论感染日本血吸虫的ICOS-Tg小鼠CD28、CD86表达及其Th2免疫应答显著上调,并伴随肝虫卵肉芽肿病变增强,表明ICOS信号对CD28-CD86信号通路具有一定的调控作用,且在介导Th2极化及肝虫卵肉芽肿的发生、发展中发挥重要作用。

Objective To analyze effects of ICOS expression on the CD28/CD86 signaling pathway and Th1/Th2 polarization in mice in- fected with Schistosoma japonicum. Methods In this study, ICOS transgenic （ICOS-Tg） mice and wildtype FVB/NJ miee were used as ex- perimental schistosomiasis model. The expression of CD28 on CD4＋T splenocytes and of CD86 on CD19＋B splenocytes from mice infected with Scbistosoma japonicum were analyzed by flow cytometry before infection （0 week ） ,and at 4,7,12,16 and 20 weeks postinfection. The expression of CD28 and CD86 on inflammatory cells around granulomatous infiltration in mice liver were assessed by immunohistochemical staining. The spleen lymphocytes of mice were stimulated with SEA for 72 horns,and then,the concentrations of Thl cytokines （IFN-γ,IL- 12） and Th2 cytokines （IL-4,IL-13） in the culture supernatants were measured by sandwich ELISA kits according to the manufacturer＇s guideline. The levels of SEA-specific antibodies of IgG and its subtypes （IgG1 and IgG2a） were measured in ＇all mice sera by ELISA. The granulomatous pathology in mice liver was dynamically observed by hematoxylin and eosin （HE） staining. Results Compared with wildtype FVB/NJ mice, the expression of CD28 on CD4＋ T splenocytes and CD86 on CD19＋ B splenocytes in ICOS-Tg mice was significantly increased. Inaddition ,the expression of CD28 and CD86 was significantly higher than that of wildtype mice at 4 weeks （50.57±7.54 vs 40.38±5.43, P 〈 0.05 ）, 12 weeks （ 76.46±10.55 vs 65.10±10.17, P 〈 0.05 ） postinfection, respectively. Moreover, the expression of CD28, CD86 on inflammatory cells around granulomatous infdtration in liver from ICOS-Tg mice were significantly higher than that of wildtype FVB/NJ mice at 7 weeks （0.485±0.094 vs 0.357±0.078, P 〈 0.05 ）, 12 weeks （0.649±0.072 vs 0.537±0.064, P 〈 0.05 ） postinfeetion, respectively. The levels of Th2- type cytokines （ IL-4, IL-13 ） of ICOS-Tg mice were significantly higher than that of wildtype FVB/NJ mice in 7,12,16 and 20 weeks posfinfee- tion （all P 〈 0.05 ）. Compared with wildtype FVB/NJ mice, the levels of SEA-specific antibodies of IgG and its subtypes （IgG1 and IgG2a） in sera of ICOS-Tg mice was significantly increased （ P 〈 0.05 or P 〈 0.01 or P 〈 0.001 ）. Moreover, Th2 differentiation index of ICOS-Tg mice was significantly higher than that of wildtype FVB/NJ mice at 7,12, 16 and 20 weeks postinfeetion （ P 〈 0.05 or P 〈 0.001 ）. The ratio of IgG1/IgG2a of ICOS-Tg mice was sigfificanfly higher than that of wildtype FVB/NJ mice at 12 and 16 weeks postinfection （all P 〈 0.05 ）. Throughout the course, the volume of liver egg granulomas of ICOS-Tg mice was significantly higher than that of wildtype FVB/NJ mice. Conelusion There is obviously up-regulated the expression of CD28 and CD86 mad Th2 immune response in ICOS-Tg mice with Schistosoma japonicum infection ac- companying with notably increased hepatic granulomatous pathology. The ICOS signaling has a regulatory effect on CD28/CD86 signaling path- way, and may play an important role in the hepatic egg granuloma formation of schistosomiasis.