内毒素预处理促进金黄色葡萄球菌刺激的巨噬细胞生成一氧化氮

Signaling mechanisms involved in the priming effects of lipopolysaccharide on Staphylococcus aureus-induced nitric oxide production in macrophages

目的:明确脂多糖(LPS)预处理后巨噬细胞对后续热灭活革兰氏阳性金黄色葡萄球菌(HKSa)刺激的反应性变化,并探讨其机制。方法:Griess法检测细胞培养上清一氧化氮(NO)含量;实时荧光定量PCR和Western blotting技术检测Toll样受体2(TLR2)mRNA和蛋白表达;双荧光报告基因检测法观察细胞内活化T细胞核因子(NF-AT)转录活性。结果:LPS预处理小鼠巨噬细胞系RAW264.7细胞24 h后,该细胞在后续HKSa刺激下NO生成量显著增加,提示LPS可以致敏巨噬细胞、增强其对HKSa的反应性。LPS可以剂量依赖性地增加细胞内TLR2 mRNA和蛋白表达;LPS预处理也可增强TLR2特异性配体肽聚糖刺激巨噬细胞NO生成的效应;TLR2特异性中和抗体可部分阻断LPS的致敏效应。LPS可促进NF-AT转录激活,细胞内钙离子(Ca2+)螯合剂BAPTA/AM和calcineurin抑制剂cyclosporin A(CsA)均可阻断LPS的作用;BAPTA/AM和CsA均能显著抑制LPS的致敏效应。结论:LPS可以致敏巨噬细胞,从而使其在后续HKSa作用下NO生成量增加;模式识别受体TLR2和Ca2+/cal-cineurin/NF-AT信号转导途径可能参与了LPS的这一致敏效应。

AIM： To investigate the signaling mechanisms involved in the priming effects of lipopolysaccharide （LPS） on heat killed Staphylococcus aureus （HKSa） -induced nitric oxide （NO） production in macrophages. METHODS ： Murine macrophage RAW264.7 was used in the experiment. Griess reagent was used to measure the content of nitrite in culture medium. Real - time PCR and Western blot was utilized to examine the mRNA and protein levels of toll - like receptor 2 （ TLR2）, respectively. Dual luciferase reporter assay was used to assess the transcriptional activity of nuclear factor of activated T cells （ NF - AT）. RESULTS： The RAW264.7 cells pretreated with LPS for 24 h significantly enhanced NO production induced by HKSa, suggesting that LPS primed the macrophages and increased the reactivity of the cells to HKSa. LPS increased the mRNA and protein levels of TLR2 in a dose - dependent manner in RAW264.7 ceils. The RAW264.7 ceils pretreated with LPS enhanced NO production induced by peptidoglycan, one of the specific ligand of TLR2. The priming effect of LPS on HKSa - induced NO production was partly blocked by TLR2 neutralizing antibody. LPS significantly enhanced the transcriptional activity of NF - AT, which was inhibited by BAPTA/AM （ a cell - permeable cytosolic calcium chelator） and cyclosporine A （CsA, an inhibitor for calcineurin）. Both BAPTA/AM and CsA inhibited the priming effect of LPS on HKSa - induced NO production in RAW264.7 cells. CONCLUSION ： The present study confirms the priming effect of LPS on the reactivity of RAW264.7 cells to HKSa. Pattern recognition receptor TLR2 and calcium/calcineurin/NF - AT signaling pathway may be involved in the priming process initiated by LPS.