TLR2和TLR4在小鼠生殖道沙眼衣原体感染免疫应答中的应用研究

Study of TLR2 and TLR4 in the immune response to a genital tract infection with Chlamydia trachomatis in mice

目的探讨TLR2和TLR4在小鼠沙眼衣原体生殖道感染免疫应答中的作用。方法用沙眼衣原体小鼠肺炎株(MoPn)1×104 IFUs经生殖道感染野生型小鼠(WT,11只)、TLR2基因缺陷小鼠(TLR2KO,14只)和TLR4基因缺陷小鼠(TLR4KO,11只),复制生殖道沙眼衣原体感染模型。于感染后不同时间点取生殖道分泌物,采用免疫荧光法检测衣原体包涵体数量。在感染后第70d处死小鼠,留取血清,采用免疫荧光法检测抗体类型和效价;无菌分离的脾细胞,用经紫外线灭活的衣原体EB刺激培养72h,采用ELISA法测定上清液中细胞因子IFN-γ、IL-17、IL-4和IL-5水平。结果 TLR2KO或TLR4KO小鼠与WT小鼠在每一个检测时间点生殖道带菌量差异无统计学意义(P>0.05),3组小鼠带菌持续时间相同,截止到感染后第38d,所有小鼠下生殖道感染的衣原体均被清除.。3种基因型小鼠脾细胞均产生高水平的IFN-γ和IL-17,且3组间差异无统计学意义(P>0.05);但IL-4及IL-5水平均降低,且3组间差异无统计学意义(P>0.05)。3种基因型小鼠血清IgG2a/IgG1比值均>1,且组间差异无统计学意义(P>0.05)。3种基因型小鼠均发展为Th1偏倚的免疫应答。结论在沙眼衣原体生殖道感染中,TLR2或TLR4介导的信号通路对控制小鼠下生殖道衣原体感染不是必需的,沙眼衣原体诱导的适应性免疫应答既不依赖于TLR2,也不依赖于TLR4。

Objective To evaluate the roles of TLR2 and TLR4in the immune response to a genital tract infection with Chlamydia trachomatis in mice. Methods Wild-type mice（WT,n=11）and mice deficient in TLR2（TLR2KO,n=14）or TLR4（TLR4 KO,n=11）were infected intravaginally with 1×10^4 inclusion forming units（IFUs）of live Chlamydia muridarum（MoPn）to establish a mouse model of genital tract infection with C.trachomatis.Vaginal swabs were collected on different days after intravaginal infection.An immunofluorescence assay was used to monitor live organism shedding in each vaginal swab.The mice were sacrificed on day 70post-infection,and mouse sera were collected to determine the titers of Chlamydia-specific antibody isotypes using an immunofluorescence assay.The harvested splenocytes were re-stimulated with UV-inactivated MoPn EB for 72 h,and the culture supernatants were measured for the cytokines IFN-γ,IL-17,IL-4,and IL-5.All cytokines were measured using ELISA. Results There was no significant difference in the number of IFUs recovered from the vaginal swabs（at any time point post-infection）among WT,TLR2 KO,and TLR4 KO.All mice displayed a similar time course of live organism shedding.By day 38,all mice had cleared the infection.Splenocytes from three groups of mice produced high levels of IFN-γand IL-17 and low levels of IL-4and IL-5,but there was no significant difference in these levels among the three groups of mice（P〉0.05）.The ratio of MoPn-specific serum IgG2a/IgG1 in all three groups of mice was greater than 1,and there was no significant difference inthis ratio among the three groups（P〉0.05）.All mice（regardless of the genotype）developed a Th1-based immune response. Conclusion Neither TLR4 nor TLR2 is required for an adaptive immune response to a genital tract infection with C.trachomatis.