Chlamydia Pneumoniae Invades Into Vascular Smooth Muscle Cells Through The CXCR4-β-arrestin 2 Pathway Via TLR2

Objective: Using the model of vascular smooth muscle cell(VSMC)infected with Chlamydia pneumoniae(C.pneumoniae)in vitro,to observe the roles of TLR2/CXCR4 interaction in C.pneumoniae invasion into VSMCs,and to demonstrate that C.pneumoniae invades into VSMCs through the CXCR4-β-arrestin 2 pathway via TLR2.Methods: 1.VSMCs were infected with C.pneumoniae in vitro after the culture and propagation of C.pneumoniae.2.Western blot was used to determine the expression levels of TLR2,CXCR4 and CXCR7 in VSMCs after C.pneumoniae infection.3.Western blot were used to detect the protein levels of TLR2,CXCR4 and CXCR7 in plasma membrane and cytoplasm in VSMCs at different time potins after C.pneumoniae infection.4.The effects of C.pneumoniae infection on the distribution of TLR2,CXCR4 and CXCR7 in VSMCs were observed by using a confocal laser scanning microscopy(CLSM).5.The expression of TLR2,CXCR4,CXCR7 and β-arrestin 2 in VSMCs were down regulated by means of RNA interference,Western blot was then used to detect the expressions of these proteins to evaluate the effects of siRNA-mediated gene silencing of TLR2,CXCR4,CXCR7 and β-arrestin 2.6.The expressions of TLR2,CXCR4,CXCR7 and β-arrestin 2 in VSMC were down regulated by means of RNA interference.And immunofluorescence assay was then used to dertermine C.pneumoniae infection rate.7.The effects of C.pneumoniae infection on the co-localization of TLR2 and CXCR4,TLR2 and CXCR7 in VSMCs were observed by using CLSM.8.Co-immunoprcipation was performed to determine the interactions of TLR2 and CXCR4,TLR2 and CXCR7 in VSMCs infected with C.pneumoniae.9.The effects of C.pneumoniae infection on the interactions of TLR2 and CXCR4,TLR2 and CXCR7 in VSMCs were observed by using the proximity ligation assay(PLA).10.The effect of CXCR4 on recruietment of β-arrestin 2 in VSMC was observed by using PLA.11.The expression of TLR2 in VSMCs was downregulated by means of RNA interference,and Western blot were used to detect the distribution of CXCR4 in plasma membrane and cytoplasm.12.After the expression of TLR2 was downregulated by means of RNA interference,changes in roles of TLR2 in the recruietment of β-arrestin 2 to CXCR4 induced by C.pneumoniae infection was detected by using PLA.Results: 1.The expression level of TLR2 in VSMCs began to increase 15 min after C.pneumoniae infection,and sustained to 30 min,60 min,120 min and 180 min after infection compared with the control group(P < 0.05).2.The expression of TLR2 on plasma membrane was upregulated at 60 min postinfection compared with the control group(P < 0.05),and returned to normal level at 180 min postinfection.In the cytoplasm,TLR2 expression was downregulated at 60 min postinfection compared with the control group(P < 0.05)and returned to normal level at 180 min postinfection.The distribution of TLR2 in VSMCs by using CLSM was consistent with the above results.3.C.pneumoniae infection rate was significantly reduced in the group of C.pneumoniae + TLR2 siRNA compared with the group of C.pneumoniae + scr RNA(P< 0.05).4.There were no statistical differences in the expression level of CXCR4 after C.pneumoniae infection compared with the control group.The expression of CXCR4 on plasma membrane began to increase at 15 min postinfection,and reached to a peak at 60 min postinfection,then downregulated at 180 min postinfection compared with the control group(P < 0.05).In the cytoplasm,CXCR4 expression was downregulated at 15 min postinfection,and sustained to 180 min postinfection compared with the control group(P < 0.05).The distribution of CXCR4 in VSMCs by using CLSM was consistent with the above results.5.C.pneumoniae infection rate was significantly reduced in the group of C.pneumoniae + CXCR4 siRNA compared with the group of C.pneumoniae + scr RNA(P< 0.05).6.The expression level of CXCR7 was significantly increased at 15 min postinfection,and sustained to 30 min,60 min,120 min and 180 min after C.pneumoniae infection compared with the control group(P < 0.05).The expression of CXCR7 on plasma membrane began to increase at 15 min postinfection,but decreased below the normal level at 60 min postinfection,and then upregulated again at 180 min postinfection(P < 0.05).In the cytoplasm,CXCR7 expression began to increase at 15 min postinfection,and sustained to 180 min postinfection compared with the control group(P< 0.05).The distribution of CXCR7 in VSMCs by using CLSM was consistent with the above results.7.C.pneumoniae infection rate was significantly reduced in the group of C.pneumoniae + CXCR7 siRNA compared with the group of C.pneumoniae + scr RNA(P< 0.05).8.The results from CLSM,co-immunoprecipitation assay and PLA all showed that the increased interaction of TLR2 with CXCR4,but not CXCR7 was observed in VSMCs infected with C.pneumoniae.9.The effect of CXCR4 on recruietment of β-arrestin 2 in VSMCs was enhanced by C.pneumoniae infection by using PLA.10.C.pneumoniae infection rate was significantly reduced in the group of C.pneumoniae + β-arrestin 2 siRNA compared with the group of C.pneumoniae + scr RNA(P< 0.05).11.No significant changes in the protein expression of CXCR4 in the plasma membrane and cytoplasm were detected in the group of C.pneumoniae + TLR2 siRNA.The effect of CXCR4 on recruietment of β-arrestin 2 induced by C.pneumoniae infection was decresed in the group of C.pneumoniae + TLR2 siRNA.Conclsion: C.pneumoniae invades into VSMCs possibly by enhancing the interaction of TLR2 and CXCR4.Morever,C.pneumoniae invades into VSMCs through the CXCR4-β-arrestin 2 pathway via TLR2.