OM85-BV Induced The Productions Of Cytokines Via TLR4and TLR2Mediated ERK1/2/NF-κB Pathway In RAW264.7Cells

Objective Broncho-Vaxom (OM85-BV) is an extract mixture derived from eight of the most common bacteria strains in upper respiratory tract infection (URI), including Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae, Klebsiella ozeanae, Staphylococcus aureus, Streptococcus viridans, Streptococcus pyogenes and Neisseria catarrhails. It plays an important role in anti-infection immune response by regulating macrophages activity and cytokines productions. In this study, we evaluated the effects of OM85-BV on the productions of cytokines in RAW264.7cells and explored the mechanism.Methods RAW264.7murine macrophage cells were cultured in vitro,(1) RAW264.7cells were stimulated with OM85-BV for different time and concentrations respectively. The expressions of IL-1β、IL-6and TNF-a mRNA were analyzed by RT-PCR and the levels of cytokines in supernatants were detected by ELISA. Immunocytochemistry staining was used to test the expressions of TLR4and TLR2in RAW264.7cells. After knockdowning TLR4, TLR2, MyD88and TRAM gene with siRNA individually, the expressions of IL-1β, IL-6and TNF-a mRNA and protein were detected with RT-PCR and ELISA.(2) The activations of ERK1/2, P38and AKT in response to OM85-BV were examined by Western Blot. RAW264.7cells were pretreated with U0126, SB203580and LY294002, the specific inhibitor of ERK1/2, P38and AKT, and then the expressions of IL-1β, IL-6and TNF-a mRNA and protein were detected with RT-PCR and ELISA. After knockdowning TLR4, TLR2, MyD88and TRAM gene with siRNA individually, the level of ERK1/2, P38and AKT phosphorylation was detected by Western Blot.(3) RAW264.7cells were co-cultured with OM85-BV for different time, the level of P65and IκB phosphorylation and cytoplasmic and nucleoprotein P65were detected by Western Blot. Cells were pretreated with Bayl1-7082, the specific inhibitor of NF-κB, and then the expressions of IL-1β, IL-6and TNF-a mRNA and protein were detected with RT-PCR and ELISA. After knockdowning TLR4, TLR2, MyD88and TRAM gene with siRNA individually, the level of ERK1/2, P38and AKT phosphorylation was detected by Western Blot.Results (1) Exposure of RAW264.7cells to OM85-BV up-regulated the expressions of IL-1β, IL-6and TNF-a at the mRNA and protein levels in a time-and does-dependent manner. Application of TLR4, TLR2, MyD88or TRAM siRNA into RAW264.7cells could decrease the productions of cytokines at the mRNA and protein levels.(2) OM85-BV induced ERK1/2phosphorylation, but no effect on AKT, P38phosphorylation. Pretreatment with U0126could decrease IL-1β, IL-6and TNF-a production at the mRNA and protein levels induced by OM85-BV. Application of TLR4, TLR2, MyD88or TRAM siRNA into RAW264.7cells could down-regulate ERK1/2phosphorylation.(3) OM85-BV induced P65and IκB phosphorylation as well as the translocation of P65. Pretreatment with Bayl1-7082could decrease IL-1β, IL-6and TNF-a production at the mRNA and protein levels induced by OM85-BV. Application of TLR4, TLR2, MyD88or TRAM siRNA into RAW264.7cells could down-regulate P65and IκB phosphorylation.Conclusions Our study demonstrated that the productions of IL-1β, IL-6and TNF-a induced by OM85-BV in RAW264.7cells were through TLR4and TLR2signaling pathway mediated activation of ERK1/2and NF-κB.