可诱导共刺激分子信号介导的免疫应答对血吸虫性肝纤维化形成的影响

Effect of Immune Response Mediated by ICOS Signaling Pathway on Hepatic Fibrosis in Mice Infected with Schistosoma japonicum

目的探讨可诱导共刺激分子（inducibleCO—stimulator，ICOS）信号介导的Th2极化对日本血吸虫所致肝纤维化的影响。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，分别于感染前（0周）和感染后4、7、12、16和20周采集小鼠血清、脾淋巴细胞和肝脏。脾淋巴细胞用可溶性虫卵抗原（SEA）诱导培养72h后，应用ELISA法分别检测细胞培养上清中细胞因子1干扰素（IFN一1）、白细胞介素12（IL一12）、IL一4和IL．13水平．血清中SEA特异性抗体IgG及其亚类IgGl和IgG2a的水平．以及血清中透明质酸（HA）和羟脯氨酸（HYP）的含量。应用免疫组化法检测各期感染小鼠肝脏中α-平滑肌肌动蛋白（d．SMA）、转移生长因子B1（TGF—B1）和I型胶原蛋白（collagen—I）水平。分别应用苏木素．伊红（HE）染色法和胶原纤维Masson染色法动态观察感染小鼠肝脏虫卵肉芽肿病变及纤维化程度。结果ICOS．Tg小鼠Th2细胞因子IL一4和IL一13水平自感染后7～20周均显著高于野生型小鼠（P〈0．05），Thl细胞因子IFN-γ和IL-12两组之间差异均无统计学意义（P〉0．05）。ICOS—Tg小鼠的1’h2分化指数亦高于野生型小鼠．在感染后7～20周差异均有统计学意义（P〈0．05或P〈0．01）。ICOS—Tg小鼠的SEA特异性抗体IgG、IgGl和IgG2a水平均显著高于野生型小鼠（P〈0．05或P〈0．01，感染后4～7周IgG水平除外），IgGl／IgG2a比值均高于野生型小鼠，其中感染后12和16周（ICOS．Tg小鼠为5．75±0．94和4．96±0．98，野生型小鼠为4．31±0．81和3．41±0．83）差异有统计学意义（P〈0．05）。ICOS—Tg小鼠血清中HA和HYP的水平均高于野生型小鼠，分别在感染后7～20周及12～20周两组差异有统计学意义（P〈0．05或P〈0．01）。免疫组化结果显示，ICOS．Wg小鼠于感染后7～20周肝脏仪一SMA和TGF—B1蛋白表达水平均显著高于野生型小鼠（P〈0．05或P〈0．01）；collagen-I的表达水平亦高于野生型小鼠，但仅在感染后20周两组差异有统计学意义（P〈0．05）。肝组织HE染色结果显示，ICOS-Tg小鼠于感染后7、12和16周的单卵肉芽肿体积『分别为（28．72±6．68）×106、（20．47±5．09）×106和（12．77±4．86）×106μm3]，均显著大于同期野生型小鼠『分别为（18．04±6．21）x106、（15．28±4．87）×106和（11．24±4．38）×10^6μm3]（P〈0．05）。Masson染色结果显示．ICOS—Tg小鼠的肝脏纤维化程度较高．但两组的纤维化程度评分差异无统计学意义（P〉0．05）。结论感染日本血吸虫ICOS．Tg小鼠的Th2免疫应答显著上调．且其肝纤维化程度和相关指标均增强．表明ICOS信号介导的Th2极化与感染日本血吸虫导致的肝纤维化有关。

Objective To investigate the effect of Th2 polarization mediated by ICOS signaling pathway on hepatic fibrosis in mice infected with Schistosoma japonicum. Methods ICOS transgenic(ICOS-Tg) mice and wild-type FVB/NJ mice were used as experimental schistosomiasis model. The sera, livers and spleen lymphocytes of mice were collected, and spleen lymphocytes were stimulated with SEA for 72 h on the day before infection(0 week), and at 4, 7,12, 16 and 20 weeks post-infection. The concentrations of Th1 cytokines(IFN-γ and IL-12) and Th2 cytokines(IL-4and IL-13) in the culture supernatants were measured with sandwich ELISA kit. The levels of SEA-specific antibodies of IgG and its subtypes(IgG1 and IgG2a) in mice sera were measured by ELISA. The concentrations of hyaluronic acid(HA) and hydroxyproline(HYP) in mice sera were measured with sandwich ELISA kit. The expression of α-SMA, TGF-β1 and collagen-Ⅰ in livers from ICOS-Tg/wild-type mice were assessed by immunohistochemical staining. Liver granulomatous pathology and fibrosis level in ICOS-Tg/wild-type mice was dynamically observed with hematoxylin-eosin(HE) staining and Masson trichrome staining, respectively. Results The levels of Th2-type cytokines(IL-4 and IL-13)of ICOS-Tg mice were significantly higher than that of wild-type FVB/NJ mice on 7, 12, 16, and 20 weeks post-infection(P<0.05). However, Th1 cytokines IFN-γ and IL-12 showed no significant difference between the two groups(P>0.05).Th2 differentiation index of ICOS-Tg mice was significantly higher than that of wild-type mice on 7, 12, 16 and 20weeks post-infection(P<0.05 or P<0.01). Compared with wild-type mice, the levels of SEA-specific antibodies of IgG and its subtypes(IgG1 and IgG2a) in ICOS-Tg mice increased significantly(P<0.05 or P<0.01, except IgG on 4 and 7 weeks post-infection). Moreover, the ratio of IgG1/IgG2a in ICOS-Tg mice(5.75±0.94, 4.96±0.98) were significantly higher than that of wild-type mice(4.31±0.81, 3.41±0.83) on 12 and 16 weeks post-infection(P<0.05). The levels of HA on 7, 12,16, and 20 weeks post-infection(P<0.05 or P<0.01) and HYP on 12, 16, and 20 weeks post-infection(P<0.05) in ICOS-Tg mice were significantly higher than that of wild-type mice. Immunohistochemical staining showed, from 7 to 20weeks post-infection, α-SMA and TGF-β1 expression in liver of ICOS-Tg mice was significantly higher than that of wildtype mice(P<0.05 or P<0.01); collagen-Ⅰ level was also higher than wild-type mice. However, there was a significant difference in collagen-Ⅰ level between the two groups on 20 weeks post-infection(P<0.05). Furthermore, HE staining showed, on 7, 12, and 16 weeks post-infection, single-egg granuloma volume of ICOS-Tg mice [(28.72 ±6.68) ×106,(20.47±5.09)×106and(12.77±4.86)×106μm3] was significantly higher than that of wild-type mice [(18.04±6.21)×106,(15.28±4.87)×106and(11.24±4.38)×106μm3]. Masson staining showed that level of hepatic fibrosis in ICOS-Tg mice were higher than that of wild-type mice, but the fibrosis scores showed no statistically significant difference between the two groups(P>0.05). Conclusion Th2 immune response is up-regulated in ICOS-Tg mice infected with S. japonicum,and the degree of hepatic fibrosis and related indicators increase. These findings suggest that Th2 polarization mediated by ICOS signaling plays a role in hepatic fibrosis formation in mice infected with S. japonicum.