Characterization of exosomes derived from Toxoplasma gondii and their functions in modulating immune responses.
Characterization of exosomes derived from Toxoplasma gondii and their functions in modulating immune responses.
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弓形虫外泌体的表征及其在调节免疫反应中的功能
DOI：
10.2147/ijn.s151110
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发表时间：
2018
影响因子：
8
通讯作者：
Zhou H
中科院分区：
文献类型：
--
作者：
Li Y;Liu Y;Xiu F;Wang J;Cong H;He S;Shi Y;Wang X;Li X;Zhou H
引入外体是大多数细胞类型通过多囊泡体与质膜融合而分泌的纳米颗粒膜结合囊泡。其中一些外切体定义良好，已知具有免疫调节特性，并在细胞间通讯中发挥关键作用。在本研究中，我们对弓形虫来源的外切体及其在免疫应答方面的功能进行了研究。方法采用电子显微镜、纳米粒子追踪分析和Western blotting等方法分离鉴定弓形虫外切体。用细胞计数试剂盒(CCK-8)检测外切体对巨噬细胞RAW264.7细胞活性的影响。用荧光染料PKH67标记RAW264.7细胞，检测RAW264.7细胞摄取弓形虫外切体的情况。体外培养的RAW264.7细胞经Exosome刺激后，用酶联免疫吸附试验检测细胞产生IL-12、肿瘤坏死因子-α、干扰素-γ和IL-10的情况。免疫BALB/c小鼠后，用双抗体夹心法和流式细胞仪检测抗体、细胞因子和CD4+、CD8+T细胞百分率。用弓形虫速殖子攻击小鼠，评价其保护效果。结果成功分离并鉴定了弓形虫外切体。在功能上，高浓度(16 0μg/m L)的外切体对巨噬细胞RAW 2 6 4.7的活性有显著影响。巨噬细胞产生IL-12、肿瘤坏死因子-α和干扰素-γ增加，IL-10水平降低。此外，弓形虫外切体免疫的BALB/c小鼠表现出体液免疫和细胞免疫应答，且存活时间延长。结论弓形虫外切酶体能在体外调节巨噬细胞的活化，并能激发体液免疫和细胞免疫反应，对小鼠的急性寄生虫感染有一定的保护作用，提示外切酶体有可能成为弓形虫病的候选抗体。
Introduction Exosomes are nanograde membrane-bound vesicles secreted from most cell types through the fusion of multivesicular bodies with plasma membranes. Some of these exosomes are well defined, and are known to have immunomodulatory properties as well as play critical roles in intercellular communications. In this study, we characterized the exosomes derived from Toxoplasma gondii and their functions in aspect of immune responses. Methods T. gondii exosomes were isolated and identified using electron microscopy, nanoparticle tracking analysis, and Western blotting. The viability of macrophage RAW264.7 cells affected by exosomes was evaluated using a Cell Counting Kit (CCK-8). Then the uptake of T. gondii exosomes by RAW264.7 cells was detected by labeling with fluorescent dye PKH67. After exosomes stimulation, in vitro the production of interleukin (IL)-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10 in RAW264.7 cells were investigated using enzyme-linked immunosorbent assay (ELISA). In immunized BALB/c mice, the antibodies, cytokines as well as the percentage of CD4+ and CD8+ T cells were determined using ELISA and flow cytometric analysis. Protective efficacy was evaluated by challenging intraperitoneally with tachyzoites of T. gondii. Results We successfully isolated and characterized the exosomes derived from T. gondii. Functionally, the viability of macrophage RAW264.7 cells was significantly affected by exosomes at a high concentration (160 μg/mL). The production of IL-12, TNF-α and IFN-γ in macrophage cells were increased, and the level of IL-10 was decreased. Furthermore, BALB/c mice immunized with T. gondii exosomes showed both humoral and cellular immune responses and also exhibited a prolonged survival time. Conclusion T. gondii exosomes could modulate macrophage activation in vitro and trigger humoral and cellular immune responses and partial protection against acute parasite infection in mice, which suggested that exosomes may serve as a potential candidate against toxoplasmosis.