Signaling mechanisms involved in the priming effects of lipopolysaccharide on Staphylococcus aureus-induced nitric oxide production in macrophages / 中国病理生理杂志

ABSTRACT

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 cells.The RAW264.7 cells 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.