日本血吸虫感染可诱导共刺激分子配体基因敲除小鼠CD154和CD40动态变化及对Th1/Th2偏移的影响

Dynamic alteration of CD154 / CD40 and its effects on Th1 / Th2 polarization in inducible co-stimulator ligand knockout mice infected with Schistosoma japonicum

目的:探讨可诱导共刺激分子(inducible costimulator,ICOS)-ICOS配体(ICOS ligand,ICOSL)信号通路对感染日本血吸虫小鼠的CD154、CD40表达及Th1/Th2偏移的影响。方法:建立ICOSL基因敲除(ICOSL knockout,ICOSL-KO)小鼠及野生型C57BL/6J小鼠感染日本血吸虫疾病模型,于感染前和感染后4、7、12、16、20周,应用流式细胞术、免疫组织化学法分别检测小鼠脾细胞与肝虫卵肉芽肿周围炎性浸润细胞CD154、CD40的水平;应用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测小鼠脾细胞经可溶性虫卵抗原(soluble egg antigen,SEA)诱导72 h后培养的上清液中γ-干扰素(interferon gamma,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)水平及小鼠血清中SEA特异性抗体Ig G、Ig G1、Ig G2a的水平;应用HE染色观察小鼠肝虫卵肉芽肿病变动态变化。结果:感染后4、7、12、16、20周,ICOSL-KO小鼠脾细胞CD154、CD40水平与野生型小鼠相比,显著降低[CD154为(18.62±4.76)%vs.(27.91±3.94)%、(22.44±4.67)%vs.(40.86±5.21)%、(25.50±6.81)%vs.(43.81±8.41)%、(20.22±5.28)%vs.(40.95±7.34)%、(17.87±4.59)%vs.(33.16±6.31)%,皆P<0.01;CD40为(19.43±3.26)%vs.(24.37±3.59)%、(23.00±4.47)%vs.(31.80±5.86)%、(24.46±5.01)%vs.(35.85±5.32)%、(23.42±4.69)%vs.(33.30±6.14)%、(22.85±3.78)%vs.(30.88±5.94)%,皆P<0.05]。感染后7、12、16、20周,ICOSL-KO小鼠肝虫卵肉芽肿炎性浸润细胞CD154、CD40水平与野生型小鼠相比,亦显著降低[CD154为(0.319±0.066)vs.(0.488±0.086)、(0.389±0.067)vs.(0.596±0.082)、(0.378±0.064)vs.(0.543±0.072)、(0.348±0.069)vs.(0.523±0.076),皆P<0.01;CD40为(0.398±0.066)vs.(0.546±0.079)、(0.461±0.085)vs.(0.618±0.076)、(0.453±0.087)vs.(0.587±0.074)、(0.449±0.065)vs.(0.565±0.082),皆P<0.05]。感染后,ICOSL-KO小鼠IFN-γ表达显著高于野生型小鼠(P<0.05),而IL-4表达水平则显著低于野生型小鼠(P<0.05),其Th2分化指数亦显著低于野生型小鼠(P<0.01)。ICOSL-KO小鼠血清SEA特异性抗体Ig G、Ig G1、Ig G2a的水平与Ig G1/Ig G2a的比值亦显著低于野生型小鼠(P<0.01),且ICOSL-KO小鼠的肝虫卵肉芽肿体积显著小于野生型小鼠(P<0.01)。结论:ICOS-ICOSL信号通路在血吸虫病免疫病理中起重要调控作用,其对Th1/Th2偏移的影响可能是通过调控CD154-CD40信号通路来实现的。

Objective： To analyze effect on the CD154-CD40 signaling pathway and Th1 / Th2 polarization by deficient inducible co-stimulator（ ICOS）-ICOS ligand（ ICOSL） signaling in mice infected with Schistosoma japonicum. Methods： ICOSL knockout（ ICOSL-KO） mice and wild-type C57 BL /6J mice were used as experimental Schistosomiasis model infected with Schistosoma japonicum. The expressions of CD154 and CD40 on splenocytes and on inflammatory cells around granulomatous infiltration of liver in mice infected with Schistosoma japonicum were analyzed by flow cytometry,immunohistochemical staining,respectively,on the day before infection（ 0 week） and at the end of 4,7,12,16 and 20 weeks postinfection. The splenocytes of the mice were stimulated with soluble egg antigen（ SEA） for 72 hours,then the concentrations of interferon gamma（ IFN-γ） and interleukin-4（ IL-4） in the culture supernatants were measured by sandwich enzyme-linked immunosorbent assay（ ELISA） kits. The levels of SEA-specific antibodies of Ig G and Ig G1 and Ig G2 a were measured in the mice sera by ELISA. The granulomatous pathology in the mice liver was dynamically observed by hematoxylin and eosin（ HE） staining. Results：Compared with the wild-type C57 BL /6J mice, the expressions of CD154 on CD4＋T splenocytes[（ 18. 62 ± 4. 76） % vs.（ 27. 91 ± 3. 94） %,（ 22. 44 ± 4. 67） % vs.（ 40. 86 ± 5. 21） %,（ 25. 50 ±6. 81） % vs.（ 43. 81 ± 8. 41） %,（ 20. 22 ± 5. 28） % vs.（ 40. 95 ± 7. 34） %,（ 17. 87 ± 4. 59） % vs.（ 33. 16 ± 6. 31） %,all P〈0. 01] and of CD40 on CD19＋B splenocytes [（ 19. 43 ± 3. 26） % vs.（ 24. 37 ± 3. 59） %,（ 23. 00 ± 4. 47） % vs.（ 31. 80 ± 5. 86） %,（ 24. 46 ± 5. 01） % vs.（ 35. 85 ±5. 32） %,（ 23. 42 ± 4. 69） % vs.（ 33. 30 ± 6. 14） %,（ 22. 85 ± 3. 78） % vs.（ 30. 88 ± 5. 94） %,all P〈0. 05] in the ICOSL-KO mice significantly decreased at the end of 4,7,12,16 and 20 weeks postinfection. Moreover,the expressions of CD154[（ 0. 319 ± 0. 066） vs.（ 0. 488 ± 0. 086）,（ 0. 389 ± 0. 067）vs.（ 0. 596 ± 0. 082）,（ 0. 378 ± 0. 064） vs.（ 0. 543 ± 0. 072）,（ 0. 348 ± 0. 069） vs.（ 0. 523 ± 0. 076）,all P〈0. 01] and CD40 [（ 0. 398 ± 0. 066） vs.（ 0. 546 ± 0. 079）,（ 0. 461 ± 0. 085） vs.（ 0. 618 ±0. 076）,（ 0. 453 ± 0. 087） vs.（ 0. 587 ± 0. 074）,（ 0. 449 ± 0. 065） vs.（ 0. 565 ± 0. 082）,all P〈0. 05] on inflammatory cells around granulomatous infiltration in liver from the ICOSL-KO mice were significantly lower than those of the wild-type C57 BL /6J mice at the end of 7,12,16 and 20 weeks post-infection. The levels of IFN-γ of the ICOSL-KO mice were significantly higher than those of the wild-type C57 BL /6J mice at the end of 4,7,12,16 and 20 weeks post-infection（ P〈0. 05）. However,the levels of IL-4 of the ICOSL-KO mice were significantly lower than those of the wild-type mice（ P〈0. 05）. Compared with the wild-type C57 BL /6J mice,the levels of SEA-specific antibodies of Ig G and Ig G1 and Ig G2 a in the sera of the ICOSL-KO mice significantly decreased（ P〈0. 01）. Moreover,The Th2 differentiation index of the ICOSL-KO mice was significantly lower than that of the wild-type mice in post-infection（ P〈0. 01）. Also,the ratio of Ig G1 / Ig G2 a of the ICOSL-KO mice were significantly lower than that of the wild-type mice at the end of 7,12 and 16 weeks post-infection（ P〈0. 05）. And the volume of liver egg granulomas of the ICOSL-KO mice was significantly smaller than that of the wild-type mice（ P〈0. 01）. Conclusion： These findings suggest that there is obvious down-regulation in the expressions of CD154 and CD40 and impairment of Th2 immune response in the ICOSL-KO mice infected with Schistosoma japonicum,accompanying with notedly reduced hepatic granulomatous pathology. The ICOS-ICOSL signaling has a regulatory effect on CD154-CD40 signaling pathway,and may play an important role in the hepatic egg granuloma formation of Schistosomiasis.