低剂量Pam3Csk4预处理诱导鼠腹腔巨噬细胞对肺炎克雷伯菌的低反应性

Pretreatment with low-dose TLR2 agonist attenuate the response of murine peritoneal macrophage to Klebsiella pneumonia

目的:初步探讨TLR2激动剂Pam3Csk4预处理后,小鼠腹腔巨噬细胞对肺炎克雷伯菌(Klebsiella pneumoniae,K.p)的免疫反应性。方法:1μg/ml Pam3CSK4于0小时作用鼠腹腔巨噬细胞,24小时后以不同剂量热灭活肺炎克雷伯菌刺激细胞,ELISA检测细胞培养上清中TNF-α、IL-6含量,比色法检测NO含量,流式检测小鼠腹腔巨噬细胞对热灭活K.p的吞噬能力;RT-PCR和流式检测细胞表面TLR2表达。结果:肺炎克雷伯菌刺激后,Pam3Csk4预处理组TNF-α、IL-6、NO分泌量显著低于未处理组(P<0.05),Pam3Csk4预处理组细胞对K.p吞噬能力显著降低(P<0.05),巨噬细胞表面TLR2及其基因水平表达量均降低。结论:Pam3Csk4预处理小鼠腹腔巨噬细胞能降低其对肺炎克雷伯菌的免疫反应。

Objective：To investigate the immune response of murine peritoneal macrophage pretreated with TLR2 agonist Pam3Csk4 to Klebsiella pneumoniae （K. p）. Methods： Murine peritoneal macrophages were pretreated with Pam3Csk4 （I μg/ml）. Following pretreatment 24 h later, heat-killed K. p （HK-K. p ） was added and incubated for 12 hours. The concentrations of TNF-α, IL-6 and nitric oxide （NO） were determined by ELISA and colorimetry, respectively. Meanwhile, the expression of TLR2 was assessed by RT-PCR and flow cytometry. To estimate phagocytosis of macrophage, HK-K. p labeled with FITC （FITC-HK-K. p） was added into the well and incubated for 30 rain. After washing 5 times with PBS, intracellular FITC-HK-K. p was detected by flow cytometry. Re- sults ： Compared with the control, the secretion of TNF-α, IL-6 and NO released from pretreated cells was diminished respectively, and the phagocytosis was reduced in macrophages pretreated with Pam3Csk4 after stimulated with HK-K. p. The macrophages pretreated with Pam3Csk4 had lower TLR2 expression. Conclusion： Murine peritoneal macrophages were hypo-responsive to Klebsiella pneumo- nia after pretreatment with low-dose TLR2 agonist.