淋球菌经NLRP3途径调节小鼠脾细胞Th17/Treg平衡

Neisseria Gonorrhoeae Regulates Th17/Treg Balance in Murine Splenocytes Via NLRP3 Pathway

目的研究NLRP3对小鼠脾细胞淋球菌感染模型中Th17/Treg平衡的调节作用。方法体外分离、培养小鼠脾细胞后,分为如下4组:空白对照组、NLRP3拮抗剂(MCC950)组、淋球菌感染组、淋球菌+MCC950组。培养48 h后收集细胞,采用细胞内染色流式细胞术检测Th17/Treg细胞比例,利用实时荧光定量PCR检测NLRP3、Th17/Treg特异性转录因子RORγt、Foxp3 mRNA表达水平,Western blot检测NLRP3、RORγt、Foxp3蛋白表达。ELISA法检测脾细胞培养上清中Th17/Treg相关细胞因子IL-17、TGF-β、IL-10水平。结果脾细胞在淋球菌的刺激下,Th17细胞和Treg细胞比例增多,细胞内NLRP3、Th17/Treg特异性转录因子RORγt、Foxp3 mRNA和蛋白表达水平升高,细胞培养上清中IL-17、TGF-β、IL-10含量增高(P<0.01);加入MCC950预处理后,Th17细胞比例、细胞内NLRP3、RORγt mRNA和蛋白表达、细胞培养上清中IL-17含量明显降低,Treg细胞比例、细胞内Foxp3 mRNA和蛋白表达、细胞培养上清中TGF-β、IL-10含量明显增高,与淋球菌感染组比较差异有统计学意义(P<0.01)。结论淋球菌通过NLRP3途径诱导小鼠脾细胞向Th17细胞分化,调节Th17/Treg细胞平衡。

Objective To investigate the regulatory effect of NLRP3 on the balance of Th17/Treg in murine splenocytes infected with Neisseria gonorrhoeae.Methods Murine splenocytes were isolated and cultured in vitro,and divided into four groups:control group,NLRP3 antagonist(MCC950)group,Neisseria gonorrhoeae group,Neisseria gonorrhoeae+MCC950 group.The splenocytes were collected after 48 hours of culture,the proportions of Th17/Treg cells were detected by intracellular staining using flow cytometry.The mRNA expressions of NLRP3,Th17/Treg-specific transcription factors RORγt,Foxp3 were detected by real-time PCR.The protein expression levels of NLRP3,RORγt,Foxp3 were determined by Western blot.The concentrations of Th17/Treg related cytokines IL-17,TGF-β,IL-10 in the supernatant were detected by ELISA.Results Under the stimulation of Neisseria gonorrhoeae,the proportions of Th17 cells and Treg cells,the mRNA and protein levels of NLRP3,Th17/Treg-specific transcription factors RORγt,Foxp3 in the splenocytes and the concentrations of IL-17,TGF-β,IL-10 in the supernatant were increased than those in the controls(all P<0.01).After pretreated with MCC950,the proportion of Th17 cells,the mRNA and protein levels of NLRP3,RORγt in the splenocytes and the concentration of IL-17 in the supernatant were significantly lower than those in Neisseria gonorrhoeae group.The proportion of Treg cells,the mRNA and protein levels of Foxp3 in the splenocytes and the concentrations of TGF-β,IL-10 in the supernatant were significantly higher than those in Neisseria gonorrhoeae group(all P<0.01).Conclusion Neisseria gonorrhoeae induces the differentiation of Th17 cells in murine splenocytes via NLRP3 pathway,and regulates the balance of Th17/Treg cells.