The Roles Of TLR4 In Anti-β2GPI/β2GPI-Induced Tissue Factor Expression On THP-1 Cells

Objective:Toll-like receptors(TLRs) are not only a kind of important receptors which take part in innate immune response,but also the bridges that connect innate immunity and the specificity immunity. Recently,it was found that TLRs and its related signaling molecules were associated with the pathological mechanisms of APS.In this study,the roles of TLR4 in antiphospholipid antibody/antigen complex (anti-β2GPI/β2GPI) -induced tissue factor expression on THP-1 cells was investigated.And whether the effects of TLR4 were relied on another molecule-annexin A2(ANX2) on THP-1 cells was also evaluated.Methods:①The TF expression on THP-1 cells was detected by Real-time PCR or TF activity kit after stimulating with anti-β2GPI/β2GPI complex.②The binding of receptors on THP-1 cells toβ2GPI was analyzed withβ2GPI-Affi-Gel columns.③The TLR4,MyD88(myeloid differentiation factor 88),MD-2(myeloid differentiation protein 2) expression on THP-1 cells induced by anti-β2GPI/β2GPI complex was investigated by Real-time PCR and western blotting.④Whether TLR4 pathway inhibitor(paclitaxel,1μmol/L) could inhibit the effects of anti-β2GPI/β2GPI on THP-1 cells was also evaluated.⑤The ANX2 siRNA lentivirus(LV-RNAi-ANX2) was infected to the THP-1 cells, then the interference effect was detected to construct the RNAi model.⑥The expression of TLR4,MD-2,MyD88 and TF on the ANX2 RNAi-cells was then estimated with anti-β2GPI/β2GPI stimulation.Results:①The TF mRNA expression and TF activity on THP-1 cells could be significantly increased with treatment of anti-β2GPI/β2GPI complex(100μg/ml)(p＜0.05 vs control).②The TLR4 and ANX2 on THP-1 cell membrane could bind toβ2GPI-Affi-Gel columns.③After THP-1 cells was stimulated with anti-β2GPI/β2GPI(100μg/ml) complex,the TLR4,MD-2 and MyD88 expression(both mRNA and protein) on cells was significantly increased(p＜0.05 vs control).④All the stimulating effects of anti-β2GPI/β2GPI on THP-1 cells could be inhibited by paclitaxel(1μmol/L).⑤THP-1 cells transfected with LV-RNAi-ANX2 showed almost knockout of ANX2 both at mRNA and protein levels.The best MOI for THP-1 cells was 100.⑥When the ANX2 RNAi-THP-1 cells were stimulated with the same anti-β2GPI/β2GPI complex,the TLR4,MyD88,MD-2 expression were obviously reduced,and the TF expression was also significantly decreased(p＜0.05 vs uninfected cells).Conclusions:The TLR4,partly dependent on ANX2,plays an important role in anti-β2GPI/β2GPI complex-induced TF expression on THP-1 cells.And the paclitaxel may interfere in this process,indicating the new way for therapy as well as prevention of thrombotic events in APS.