The Effect Of NF-κB Singnal Pathways And Inflammatory Cytokines Expression In THP-1Macrophages Induced By Staphylococcus Aureus Panton-Valentine Leukocidin (PVL)

Objective: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted byStaphylococcus aureus that has been the main pathogenic factor. Polymorphonuclearneutrophils (PMNs) and macrophages of human and rabbit were sensitive to PVL.The aims of this project（:1）Study whether rPVL does damage to THP-1macrophagesor not;(2) To investigate the effect of rPVL inducing inflammatory cytokines throughNF-κB singnal pathway on THP-1macrophages.Methods:(1) we chose optimal concentration by using different concentrations (1nM,5nM,10nM,20nM,40nM and80nM) of rPVL on THP-1macrophages. After1,3,6h,the cell vitality was detected by CCK-8.(2) The THP-1macrophages were incubatedwith different concentrations (5nM,20nM and40nM) of rPVL. After1,3,6h, therates of cells apoptosis/necrosis were detected by flow cytometry.(3) ELISA wasperformed to test the expression of IL-8, IL-6and TNF-α from THP-1macrophagesafter1,3and6h hours treated with5nM and20nM rPVL.(4) The expression of IL-6,TNF-α, IL-8, TLR2, TLR4and CD14mRNA in THP-1macrophages was quantifiedby semi-quantitative RT-PCR analysis after1,3and6h treated with20nM rPVL.Total cellular RNA was isolated from cells by using reagent.(5)Western blotting andimmunocytochemistry methods were used to test the expression of NF-κB protein inTHP-1macrophages induced by rPVL.(6) In addition, THP-1macrophages culturedtogether with NF-κB inhibitor (PDTC) before rPVL treated, then the expression ofIL-8and IL-6were assayed by RT-PCR and ELISA. Results:(1) After treatment with rPVL, the cell vitality decreased in time-andconcentration-dependent manners.(2)5nM rPVL treatment group had higherapoptosis rate than that of control and40nM rPVL treatment groups had highernecrosis rate than that of control. And both of then were in the time-dependentmanner.(3) rPVL could induce the expression of IL-6and IL-8on THP-1macrophages in time-and concentration-dependent manners. The peak time of theexpression of TNF-α was after1h treated with rPVL.(4) The results ofimmunocytochemistry method showed that NF-κB exists in the cytoplasm stainedwith well-distributed light dye in all inactive form in normal condition. IncreasedNF-κB activity with nuclear localization was observed after stimulated by rPVL.Western blotting method showed that PVL was able to induce expression of NF-κB inTHP-1macrophages in time-and concentration-dependent manners.(4) PDTC couldsignificantly diminish the rPVL-induced IL-8and IL-6production in THP-1macrophages (P≦0.05).Conclusion:(1) Low dose rPVL induces apoptosis of human alveolar macrophages,and high dose rPVL directly induces necrosis.(2) PVL can activate the expression ofTLR4/NF-κB signals, and increased the high expression of inflammatory cytokines.(3) The inhibitor of NF-κB, PDTC, can decrease the secretion of pro-inflammatory inTHP-1macrophages by rPVL.