The Study On TLRs Involved In The RBmpA Initiated Storm Of Pro-inflammatory Chemokines Produced By Immune Cells

Objective:To explore the Toll-like receptors that involved in rBmpA initiated storm of pro-inflammatory chemokines of immune cells.Human monocytic leukemia cell line THP-1 and recombinant Borrelia burgdorferi membrane protein A(rBmpA)were used.Methods:1.The rBmpA was produced in E.coli using the bacterial expression vector pGEX-6P-1.The recombinant E.coli expression system conceived in our laboratory was cultured in LB medium.Purification and enzymatic cleavage of glutathione transferase fusion protein were carried out using Glutathione Sepharose 4B column affinity chromatography.The expressed proteins were analyzed by SDS-PAGE and the concentration was detected.2.Human monocytic leukemia cell line THP-1 were cultured in 96-well microplates and differentiated into macrophages with the pretreatment of PMA.After respectively incubated with TLR1 antibody,TLR2 antibody,TLR5 antibody and TLR6 antibody,THP-1 cells were stimulated with rBmpA for 24 hours.Cellular supernatants were collected after 24 hours stimulation and were detected by Human Chemokine Antibody Array.3.Human monocytic leukemia cell line THP-1 were cultured in 6-well microplates and differentiated into macrophages with the pretreatment of PMA.After stimulated with rBmpA for 0.5 hours,1 hours,5 hours,10 hours and 20 hours,cell lysates were prepared using Trizol.The mRNA expressions of TLR1,TLR2,TLR5 and TLR6 were assessed by QRT-PCR.4.Human monocytic leukemia cell line THP-1 were cultured in 6-well microplates and differentiated into macrophages with the pretreatment of PMA.After stimulated with rBmpA for 5 hours,10 hours and 20 hours,cell lysates were prepared using RIPA lysis Buffer.The level of protein expression of TLR1,TLR2,TLR5 and TLR6 were analyzed by Western blotting.Results:1.The results of the Human Chemokine Antibody Array showed that,compared with control group,8 kinds of cytokines increased in rBmpA group.Compared with rBmpA group,5 kinds of cytokines decreased in TLR1 antibody treated group,7 kinds of cytokines decreased in TLR2 antibody treated group,5 kinds of cytokines increased in TLR5 antibody treated group,and 6 kinds of cytokines increased in TLR6 antibody treated group.2.After 1 hours,5 hours,and 10 hours of culture,the relative mRNA expression of TLR1 and TLR2 of THP-1 cells increased while the relative mRNA expression of TLR5 and TLR6 had no change.3.After 5 hours,10 hours,and 20 hours of culture,the protein expression of TLR1 and TLR2 of THP-1 cells increased while the protein expression of TLR5 and TLR6 had no change.Conclusion:1.After the stimulation of rBmpA,8 kinds of cytokines in the culture supernatants increased.2.TLR1 and TLR2 neutralizing antibody treated THP-1 cells showed a decreased trend in chemokines while the chemokines showed an increased trend in TLR5 and TLR6 antibody treated group.3.After the stimulation of rBmpA,the expression of TLR1,TLR2 of THP-1 cells increased while TLR5 and TLR6 expression showed no change.4.TLR1 and TLR2 were closely with the storm of pro-inflammatory chemokines stimulated by rBmpA.