肺炎嗜衣原体热休克蛋白10经TLR2及TLR4调控THP-1分泌TNF—α

Secretion of TNF-a induced by heat shock protein 10 of Chlamydophila pneumoniae were mediated by TLR2 and TLR4 in THP-1

目的探讨肺炎嗜衣原体（Cpn）热休克蛋白10（HSP10）诱导人单核细胞分泌炎症因子的作用及Toll样受体（TLR）2、TLR4与此作用的相关性。方法制备CpnHSP10（CHSP10）纯化蛋白，去内毒素活性后用不同浓度（0．5、1、5、10、20、30μg/ml）刺激THP-1细胞0、6、12、24、36、48、60h，并比较蛋白不同处理组别中TNF-α的水平差异；以间接免疫荧光及RT-PCR鉴定THP-1细胞上的TLR4及TLR2；分离C3H系野生型和TLLR4缺陷型小鼠巨噬细胞，以CHSP10刺激后检测TNF-α水平；用抗TLR2／TLR4单克隆抗体预孵育细胞，ELISA检测CHSP10刺激细胞前后TNF-α的变化。结果CHSP10刺激THP-1细胞引起上清液炎症因子TNF—α水平显著增加，经加热等处理后蛋白诱生TNF-α的作用明显降低。THP-1细胞可检测到TLR2及TLR4的mRNA及蛋白表达，CHSP10诱生C3H系野生型小鼠细胞分泌的TNF-α明显高于TLR4缺陷型小鼠细胞，TLR2／4经单克隆抗体作用后均可显著降低CHSP10诱导THP-1分泌TNF-α的水平。结论CHSPl0可能作为炎症相关蛋白参与了Cpn对宿主细胞的致炎作用；并且TLR2及TLR4在该炎症刺激信号的传递过程中发挥一定的作用。

Objective To investigate the effect of heat shock protein 10 （HSP10） of Chlamydophila pneumoniae in inducing TNF-α on THP-1 cells and the roles of TLR4 and TLR2 involved in it. Methods Purified native recombinant HSP10 from Cpn（CHSP10） were produced and inactivated the en- dotoxin contamination, then different concentration （ 0. 5,1,5,10,20,30 μg/ml ） of CHSP10 were used to stimulate THP-1 for different time （0,6,12,24,36,48,60 h）. TNF-α were measured by using human TNF-ot ELISA kit and compared among different groups. THP-1 were collected and analyzed for TLR2 and TLR4 mRNA levels and protein expression by RT-PCR and immunofluorescence. Peritoneal macrophages isolated from wide-type （C3H/HeN） and TLR4-defieient mice （C3H/HeJ） were stimulated with endotoxin-free pro- teins respectively, and the TNF-ct were measured. Furthermore, neutralizing anti-human TLR2/TLR4 McAb as a blocking Ab was preincubated with THP-1, after stimulation with CHSP10, ELISA was used to detect the concentration of TNF-α. Results TNF-ot can be induced with CHSP10 in THP-1, while it significantly de- creased with heated or deproteinized CHSP10. Both TLR2 and TLR4 mRNA and protein were detected in THP-I. Macrophages from C3H/HeN mice displayed higher TNF-α compared with it from C3H/HeJ mice af- ter stimulation with CHSP10. The CHSP10-induced TNF-αwould obviously decline when treated with anti- TLR2/TLR4 McAb. Conclusion As a potential inflammation related protein, CHSP10 are involved in the pathogenesis of Cpn inducing inflammation cytokine TNF-α. TLR2 and TLR4 appear to be involved in CHSP10-mediated expression of TNF-α.