沙眼衣原体pORF5质粒蛋白激活NALP3炎性复合体诱导THP-细胞产生IL-1β和IL-18

pORF5 plasmid protein of Chlamydia trachomatis induces IL-1β and IL-18 production through NALP3 inflammasome activation

目的:探讨沙眼衣原体(Chlamydia trachomatis,Ct)pORF5质粒蛋白对IL-1β和IL-18等促炎性细胞因子的诱生作用,并初步分析其分子机制。方法:用0、3、6、12、24、36μg/ml不同浓度的pORF5蛋白刺激THP-1细胞,并于0、8、16、24、36 h收集上清及细胞,ELISA检测IL-1β和IL-18的含量;Realtime-PCR检测NALP3炎性复合体mRNA表达水平,Western blot鉴定Caspase-1活化状态;分别用NALP3 siRNA、ASC siRNA转染THP-1细胞24 h或用Caspase-1特异性抑制剂(Z-YVADFMK)预处理THP-1细胞30 min后,再用24μg/ml的pORF5质粒蛋白刺激THP-1细胞24 h,ELISA分析各处理因素对IL-1β和IL-18产生的影响。结果:pORF5质粒蛋白以浓度依赖的方式刺激THP-1细胞产生IL-1β和IL-18。当pORF5质粒蛋白浓度为24μg/ml时,IL-1β和IL-18的表达水平最高,分别为491 pg/ml、186 pg/ml;同时pORF5质粒蛋白以时间依赖的方式刺激THP-1细胞产生IL-1β和IL-18,两者分别在24 h和16 h达到峰值。pORF5质粒蛋白可增强THP-1细胞NALP3、ASC和Caspase-1 mRNA的表达,促进Caspase-1的活化;NALP3-siRNA、ASC-siRNA及Caspase-1抑制剂处理组IL-1β和IL-18的产生水平均显著低于对照组(P<0.05)。结论:pORF5质粒蛋白通过激活NALP3炎性复合体诱导THP-1细胞分泌IL-1β和IL-18。

Objective：To investigate whether pORF5 plasmid protein of Chlamydia trachomatis （Ct） induces 1 L-1β and 1L-18 production in THP-I cells, and its potential molecular mechanism. Methods： pORF5 plasmid protein was used to stimulate THP-1 cells at different concentrations（0,3,6,12,24,36 μg/ml） ,then the inflammatory cytokines IL-18 and IL-1β were detected by ELISA at the time of 0,8,16,24,36 h;The mRNA expression of NALP3 inflammasome were detected by Realtime-PCR, and Caspase-1 activity was determined by Western blot analysis. THP-1 ceils were transfected with siRNA targeting NALP3 and ASC gene for 24 h or pretreated with Caspase-I inhibitor（Z-YVAD-FMK） for 30 rain, and subsequently stimulated with pORF5 （24 I^g/ml） for 24 h, then secretion of IL-115 and IL-18 were analyzed by ELISA. Results： The pORF5 plasmid protein induced THP-1 cells to secrete IL-115 and IL-18 by dose-and time-dependent manners, production of IL-115 and IL-18 reached their peaks （491 pg/ml and 186 pg/ml） at concentration of 24 μg/ml,and the peak amount of IL-1β and IL-18 occurred at 24 h and 16 h post-stimulation respectively, pORF5 plasmid protein in- creased mRNA expression of NALP3 inflammasome and activated Caspase-1 in THP-1 cells. NALP＇3 siRNA, ASC siRNA and Z-YVAD- FMK reduced pORF5-induced IL-1β and IL-18 production when compared with control groups （P〈0.05）. Conclusion： pORF5 plasmid protein could induce THP-1 cells to produce IL-1β and IL-18 through NALP3 inflammasome activation, which may play an important role in the pathogenesis in Ct infection.