Dectin-2受体介导宿主抵抗烟曲霉菌感染的研究

Dectin-2 mediates host defense against Aspergillus fumigatus infection

关键词:  Dectin-2  C型凝集素受体  小鼠  肺曲霉菌病  烟曲霉菌

Key words:  Dectin-2  C-type lectin receptors  mice  pulmonary aspergillosis  Aspergillus fumigatus

摘要:

目的探讨C型凝集素受体Dectin-2在烟曲霉菌感染中的作用及机制。方法用紫外线灭活的烟曲霉菌膨胀态分生孢子刺激野生型(wild type, WT)和Dectin-2缺失(Clec4n-/-)小鼠骨髓来源的巨噬细胞(bone marrow derived macrophages, BMDMs)。刺激20min及40min后，利用Western印迹法检测BMDMs中脾酪氨酸激酶(spleen tyrosine kinase, Syk)和核转录因子κB抑制因子(inhibitor-κB, IκBα)的磷酸化水平。刺激16h后，利用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测BMDMs细胞上清液中IL-6、TNF-α和IL-12p40的水平。另一方面，利用压舌感染的方法在WT和Clec4n-/-小鼠中构建烟曲霉菌肺部感染模型。感染2d后，统计小鼠整个肺脏荷菌量，并检测肺脏匀浆液中IL-6和IL-12p40水平。结果体外试验提示，烟曲霉菌刺激后，Dectin-2缺失的BMDMs中Syk和IκBα的磷酸化水平及IL-6、TNF-α和IL-12p40水平显著下降(P＜0.05)。体内试验发现，烟曲霉菌感染后，Dectin-2缺失小鼠中肺脏荷菌量显著升高(P＜0.05)，肺脏匀浆液内IL-6、IL-12p40水平显著降低(P＜0.05)。结论C型凝集素受体Dectin-2激活烟曲霉菌诱导的NF-κB信号通路并介导促炎细胞因子的产生，可在小鼠肺烟曲霉菌病动物模型中发挥保护性作用。

Abstract:

ObjectiveTo investigate the role and mechanism of C-type lectin receptor Dectin-2 in Aspergillus fumigatus infection. MethodsBone marrow-derived macrophages(BMDMs)from wild type(WT) and Dectin-2-deficient(Clec4n-/-) mice were stimulated with UV-inactivated Aspergillus fumigatus swollen conidia. The phosphorylation levels of spleen tyrosine kinase(Syk) and inhibitor-κB(IκBα) in BMDMs of WT and Clec4n-/- mice were detected by Western blot after stimulation for 20 and 40 min. The levels of IL-6, TNF-α and IL-12p40 in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA) after stimulation for 16 h. In addition, WT and Clec4n-/-mice were challenged intratracheally with Aspergillus fumigatus conidia for 2d. The fungal burden of the whole lung was checked, and the levels of IL-6 and IL-12p40 in the lung homogenate were examined in the infected mice. ResultsIn vitro, after stimulation by Aspergillus fumigatus, BMDMs with Dectin-2 deficiency significantly reduced the production of IL-6, TNF-α and IL-12p40 as well as the phosphorylation levels of Syk and IκBα in BMDMs(P＜0.05). In vivo, Clec4n-/- mice exhibited significantly heavier fungal burden, while decreased pro-inflammatory cytokine production in lung homogenate compared with WT mice(P＜0.05). ConclusionC-type lectin receptor Dectin-2 activates Aspergillus fumigatus induced NF-κB signaling pathway and mediates pro-inflammatory cytokine production, which plays a protective role in defense against pulmonary Aspergillus fumigatus infection.