Treponema Pallidum Enhances Human Monocyte Migration And Invasion By Dysregulating The MMP/TIMP Balance

Objectives:Syphilis is a long-term,multivisceral disease caused by the spirochete Treponema pallidum(T.pallidum),the pathogenic mechanism is still unclear.Recent studies have found that the infiltration of monocytes into local lesions of syphilis was facilitate the clearance of T.pallidum.Extracellular Matrix is the scaffold of cells,and the chemotaxis of immune cells(especially monocytes)to inflammatory sites is influenced by many factors including matrix metalloproteinases(MMPs),and then tissue inhibitors of metalloproteinases(TIMPs)are also involved.This research examines the molecular mechanism of MMP/TIMP imbalance,which regulated by T.pallidum induced the secretion of MMPs an TIMPs in THP-1 cells,and suggesting that the MMP/TIMP imbalance induced by T.pallidum is an important factor which could facilitate monocyte migration and invasion.Methods:Firstly,to analyze the effects of T.pallidum on the expression of MMPs and TIMPs in THP-1 cells,multiplicities of infection(MOI)represents the amount of T.pallidum.THP-1 cells were incubated with T.pallidum(MOI 100)for 24 h,and PBS-treated samples was control group.Secondly,THP-1 cells were stimulated by T.pallidum at MOI(25,50,75 and 100)for 24 h or stimulated by T.pallidum(MOI 100)for multiple time periods(0,6,12,24,36 and 48 h),RT-PCR detected the expressions of MMP-1,MMP-9,TIMP-1,TIMP-2 mRNA,and ELISA was used to inspect protein content.Whereafter,the MMP-1/TIMP-1,MMP-1/TIMP-2,MMP-9/TIMP-1 or MMP-9/TIMP-2 ratios were calculated.Third,Western blot and immunofluorescence staining were used to assess the signaling pathway activation condition,including MAPK and NF-κB pathway.To further confirm that T.pallidum induced the MMP/TIMP imbalance,THP-1 cells were preprocessed with 10 μmol/L U0126(a MEK inhibitor),10 μmol/L PD98059(an ERK inhibitor),20 μmol/L SP600125(a JNK inhibitor),10 μmol/L SB203580(a p38 inhibitor),10 μmol/L BAY 11-7082(an NF-κB inhibitor)or 100 μmol/L PDTC(an NF-κB inhibitor)for 1h and then incubated with T.pallidum(MOI 100)for 24 h.Finally,when MMP/TIMP imbalance induced by T.pallidum,the changes of motility in THP-1 cells analysed by cell migration and invasion assays in vitro.Results:T.pallidum(MOI 100)stimulated the secretions of MMP-1,MMP-9,TIMP-1 and TIMP-2 in THP-1 cells supernatants.THP-1 cells induced by different concentrations of T.pallidum increased the expressions of MMP-1,MMP-9 mRNA and protein in a concentration dependent manner.After T.pallidum(MOI 100)stimulated THP-1 cells in different time periods,the protein secretion levels of MMP-1,MMP-9 and TIMP-1 increased continuously,reaching the peak in 48 hours,while the expression of TIMP-2 began decrease at 36h and attained minimun in 48h.The ratios of MMP-1/TIMP-1,MMP-1/TIMP-2,MMP-9/TIMP-1 and MMP-9/TIMP-2 in cell supernatants were increased by T.pallidum.Western blot and immunofluorescence staining showed that T.pallidum induced the expression of MMP-1,MMP-9 and TIMP-1 by activating MAPK and NF-κB signaling pathway,and then influenced the ratio of MMP/TIMP in THP-1 cells supernatants.THP-1 cells supernatants which stimulated by different concentrations of T.pallidum could significantly increase the number of migrated and invaded cells,MAPK and NF-κB signaling pathway inhibitors significantly inhibited the number of THP-1 cells migrating and invading.Conclusions:T.pallidum affected the expression of MMP-1,MMP-9,TIMP-1 and TIMP-2 by activating MAPK and NF-κB signaling pathway of THP-1 cells,which induced the imbalance of MMP/TIMP by the increase the ratios of MMP-1/TIMP-1,MMP-1/TIMP-2,MMP-9/TIMP-1 and MMP-9/TIMP-2 in THP-1 cells supernatants.MMP/TIMP imbalance induced by T.pallidum changed the capacities of migration and invasion on THP-1 cells,indicating that T.pallidum enhance human monocytic cells migration and invasion through dysregulating MMP/TIMP balance.