约氏疟原虫感染小鼠树突状细胞对Th17分化和功能的调控作用

摘要/Abstract

摘要：

目的探讨约氏疟原虫（Plasmodium yoelii）17XL株（Py17XL）感染小鼠过程中树突状细胞（DCs）对辅助性T细胞17型（Thl7）细胞分化和功能的调控作用及机制。方法将12只雌性BALB/c小鼠随机分为感染组（Py17XL）、Toll样受体4（TLR4）阻断组（Py17XL + TLR4）、Toll样受体9（TLR9）阻断组（Py17XL + TLR9）和TLR4联合TLR9阻断组（Py17XL + TLR4 + TLR9）,每组3只。感染前1 d,腹腔注射10 μg 抗-TLR4单抗（TLR4阻断组）或50 μg 抗-TLR9单抗（TLR9阻断组）阻断DCs的功能,注射体积为0.4 ml,对照组注射等量的PBS。4组小鼠均腹腔注射1 × 10⁶ Py17XL感染的红细胞。感染后第0、3和5天计数红细胞感染率,同时制备脾细胞悬液,流式细胞仪检测脾脏细胞中CD11c⁺ TLR9⁺ 和Th17细胞数量变化,ELISA检测脾细胞培养上清中干扰素-γ（IFN-γ）和白细胞介素-10（IL-10）水平的变化。结果流式细胞仪检测表明,DCs阻断模型构建成功。感染后第5天,感染组、TLR4阻断组、TLR9阻断组和TLR4联合TLR9阻断组小鼠原虫血症分别为28.0%、29.0%、31.0%和16.3%;感染后第7天,原虫血症分别为43.3%、47.5%、32.5%和8.0%,感染组和TLR4阻断组小鼠全部死亡,其余阻断组小鼠于感染后第6天开始出现死亡。与感染组比较,TLR9阻断组和TLR4联合TLR9阻断组小鼠原虫血症上升缓慢,生存期明显延长至11 d和15 d。流式细胞仪检测结果显示：感染后第5天,TLR9阻断组和TLR4联合TLR9阻断组Th17细胞数量分别为1.2%和1.4%,与感染组（1.9%）相比,数量明显减少（P < 0.05,P < 0.01）。ELISA检测结果显示：感染后第5天,TLR4联合TLR9阻断组小鼠IFN-γ和IL-10水平分别为（232.4 ± 15.5）pg/ml和（1 791.2 ± 58.2）pg/ml,与感染组[（90.7 ± 50.1）pg/ml和（962.6 ± 409.0）pg/ml]相比,差异有统计学意义（P < 0.05,P < 0.01）。结论 Py17XL感染小鼠过程中DCs调控了Thl7细胞的分化和功能。DCs阻断（Py17XL + TLR4 + TLR9）后,感染小鼠原虫血症降低,生存期延长。

关键词: 约氏疟原虫, 树突状细胞, 辅助性T细胞17型, 分化

Abstract:

Objective To investigate the regulatory effect of dendritic cells (DCs) on Th17 cell differentiation and function during mouse infection with Plasmodium yoelii 17XL strain (Py17XL) and the underlying mechanisms. Methods Twelve female BALB/c mice were randomly assigned into the infection group (Py17XL), the TLR4 blocking group (Py17XL + TLR4), TLR9 blocking group (Py17XL + TLR9), and TLR4 and TLR9 combined blocking group (Py17XL + TLR4+TLR9)(n=3 in each group). Mice in the Py17XL + TLR4 or the Py17XL + TLR9 group received intraperitoneal injection of 10 μg anti-TLR4 or 50 μg anti-TLR9 antibody (both 0.4 ml) to block DCs function at one day before infection. The Py17XL group received same volume of PBS. All groups were then given intraperitoneal injection of 1×10⁶ red blood cells (RBCs) infected with Py17XL. The RBC infection rate was calculated on days 0, 3 and 5 after infection, and spleen cell suspension was prepared, in which the CD11c⁺TLR9⁺ and Th17 cells were counted by flow cytometry. The levels of IFN-γ and IL-10 in supernatant of spleen cell culture were determined by ELISA. Results Flow cytometry showed that DCs were successfully blocked. On day 5 after infection, 28%, 29%, 31% and 16.3% mice showed parasitemia in the Py17XL group, the Py17XL + TLR4 group, the Py17XL + TLR9 group, and the Py17XL + TLR4 + TLR9 group, respectively, and on day 7, the proportions were 43.3%, 47.5%, 32.5% and 8%. Mice in the Py17XL group and the Py17XL + TLR4 group all died, while those in other groups began to die from day 6. There was a slow rise of parasitemia rate in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group compared with the Py17XL group, with a significant extension of survival to days 11 and 15. Results of flow cytometry showed that the proportions of Th17 cells were 1.2% and 1.44% in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group on day 5, both sighificantly decreased compared with the Py17XL group (1.9%)(P < 0.05, P < 0.01). ELISA revealed that the levels of IFN-γ and IL-10 on day 5 in the Py17XL + TLR4 + TLR9 group [(232.4 ± 15.5） pg/ml and（1 791.2 ± 58.2） pg/ml, respectively] were significantly higher than those in the Py17XL group[(90.7 ± 50.1) pg/ml and (962.6 ± 409.0) pg/ml](P < 0.05, P < 0.01). Conclusions The differentiation and function of Th17 cells are regulated by DCs during Py17XL infection. Blockade of DCs decreases parasitemia and extends lifetime of mice. Further studies are needed to clarify the exact mechanisms.

Key words: Plasmodium yoelii, Dendritic cell, T helper 17, Differentiation

中图分类号:

R382.319

陈光, 刘蕾, 王芳芳, 毕胜, 罗兰, 苏菊香, 张慧明, 蔡连顺, 宫梓琳. 约氏疟原虫感染小鼠树突状细胞对Th17分化和功能的调控作用[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 8-12.

Guang CHEN, Lei LIU, Fang-fang WANG, Sheng BI, Lan LUO, Ju-xiang SU, Hui-ming ZHANG, Lian-shun CAI, Zi-lin GONG. The regulatory effect of dendritic cells on Th17 cell differentiation and function in mouse infected with Plasmodium yoelii[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 8-12.

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