少根根霉肿胀孢子诱导小鼠树突状细胞产生Th1及Th17适应性免疫应答  

Rhizopus arrihizus swollen spores induce mice dendritic cells to Thl and Th17 differentiation

作　　者：姚丽敏[1] 张淑贞[2] 万喆[1] 李若瑜[1] 余进[1] 

机构地区：[1]北京大学第一医院皮肤性病科、北京大学真菌和真菌病研究中心、北京市皮肤病分子诊断重点实验室,100034 [2]河南省人民医院皮肤性病科,郑州450003

出　　处：《中华微生物学和免疫学杂志》2017年第1期14-21,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81371783）

摘　　要：目的初步探究宿主对少根根霉的免疫识别模式及适应性免疫机制。方法提取野生型及Card9缺陷小鼠骨髓来源细胞（bone marrow-derived cells, BMDCs），将使用和未使用Syk抑制剂的BMDCs与肿胀期及静止期少根根霉孢子孵育，24 h后ELISA法检测细胞上清液IL-23、IL-1β和IL-12分泌情况。提取并分离小鼠na？ve T细胞，并与已被不同时期孢子刺激的BMDCs共孵育，4 d后ELISA法检测细胞上清液中IL-17A和IFN-γ分泌情况，收集细胞使用流式细胞术检测T细胞分化情况。将静止期与肿胀期孢子进行免疫荧光染色，使用Confocal显微镜观察孢子表面β-葡聚糖暴露情况。将肿胀期孢子与可溶性Dectin-1、Dectin-2、TLR2和MMR共孵育，检测结合情况。结果肿胀期而非静止期少根根霉孢子可以刺激野生型小鼠的BMDCs产生细胞因子IL-23、IL-1β和IL-12，并诱导Th17和Th1细胞分化，肿胀期少根根霉孢子刺激T细胞分泌的IL-17A和IFN-γ明显高于静止期孢子。使用Syk抑制剂抑制后，肿胀期孢子刺激野生型小鼠BMDCs产生细胞因子与T细胞分化的能力明显降低。Card9缺陷小鼠的BMDCs经肿胀期孢子刺激后产生细胞因子和T细胞分化的能力低于野生型小鼠。Confocal显微镜显示，在肿胀期而非静止期少根根霉表面出现了β-葡聚糖的暴露。可溶性蛋白结合实验显示肿胀期孢子表面有可以与Dectin-1和TLR2结合的病原相关分子模式（PAMPs）。结论肿胀期少根根霉孢子可能因为表面暴露了能与Dectin-1结合的β-葡聚糖，从而刺激BMDCs分泌细胞因子IL-23、IL-1β和IL-12，诱发T细胞分化并分泌细胞因子IL-17A和IFN-γ，这一反应是Syk-Card9通路依赖的。ObjectiveTo study the mechanism of adaptive immunity against Rhizopus arrihizus （R.arrihizus） infections.MethodsBone marrow derived dendritic cells （BMDCs） were separated from C57BL/6 mice and Card9-/- mice and then were cultured in vitro. Resting spores and swollen spores of R. arrihizus were in vitro co-cultured with BMDCs with or without Syk inhibition. Secretion of cytokines （IL-23, IL-1β and IL-12） was analyzed by ELISA after 24 hours of culture. Na？ve T cells derived from C57BL/6 mice were in vitro co-cultured with spore-stimulated BMDCs for four days. Levels of IL-17A and IFN-γ in supernatants of cell culture were analyzed by ELISA. Flow cytometry was performed to analyze T cell differentiation. Confocal microscopy was used to observe the images of stained β-glucan on the surface of resting and swollen spores. Swollen spores were co-cultured with Dectin-1, Dectin-2, TLR2 and mannose receptor （MMR）, and the binding results were analyzed by flow cytometry.ResultsSwollen spores but resting spores could induce the maturation of BMDCs and promote the secretion of cytokines （IL-23, IL-1β and IL-12）. Co-culturing T cells with swollen spore-stimulated BMDCs enhanced their differentiation to Th17 and Th1. In addition, swollen spores promoted the secretion of Th1-related cytokine （IFN-γ） and Th17-related cytokine （IL-17A）. Adding Syk inhibitor to Card9-/- BMDCs or wild type BMDCs significantly inhibited the secretion of cytokines and T cell differentiation, especially in the Card9-/- group. β-glucan was overserved on the surface of swollen spores, but not on resting spores. On the surface of swollen spores existed pathogen associated molecular patterns （PAMPs） that could bind with Dectin-1 and TLR2.ConclusionSwollen spores of R. arrihizus could active BMDCs to secrete cytokines of IL-23, IL-1β and IL-12 and trigger T cell responses in vitro. The possible mechanism might be associated with β-glucan exposed on the surface of swollen spores that binds with Dectin-1. The respons

关 键 词：骨髓来源细胞 少根根霉 Syk—Card9 

分 类 号：R392[医药卫生—免疫学]