Mechanisms Of THP-1-derived Foam Cell Formation Induced By Chlamydia Pneumoniae

PartⅠEffect of Chlamydia pneumoniae infection on the formation ofTHP-1-derived foam CellsBackground A typical pathological hallmark of atherosclerosis is the excessiveaccumulation of cholesteryl esters in macrophages,leading to their conversion to foam cells.Objective To observe the effect of Chlamydia pneumoniae (C.pn) infection on theformation of THP-1-derived foam cells in vitro.Methods C.pn was propagated in Hep-2 cells.Human monocytic cell line (THP-1)were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h,and then were randomly allocated into four groups:①negative control group,RPMI 1640medium containing 50μg / ml low density lipoprotein (LDL) for 48 h;②positive controlgroup,RPMI 1640 medium containing 50μg / ml oxidized low density lipoprotein (ox-LDL)for 48 h;③different concentrations of C.pn infection group,50μg / ml LDL plus 1×10~5,4×10~5,5×10~5 and 1×10~6 IFU C.pn infection for 48 h;④different time of C.pn infectiongroup,50μg / ml LDL plus 1×10~6 IFU C.pn infection for 0,24,48 and 72 h.The infection ofC.pn on Hep-2 and THP-1 macrophages were assessed by transmission electron microscopeand polymerase chain reaction (PCR).Lipid droplets in cytoplasm were observed by oil red Ostaining.The contents of intracellular total cholesterol (TC) and cholesteryl esters (CE) weredetected by enzymic chromatometry. Results Compared with negative control,there were significantly increases in theaccumulation of lipid droplets and the ratio of CE to TC (more than 50 percent) in positivecontrol (p＜0.05).C.pn infection increased the contents of intracellular lipid droplets,TC andCE in concentration-dependent and time-dependent manner.Higher concentrations of C.pninfection (5×10~5 and 1×10~6 IFU) group also obviously increased the accumulation of lipiddroplets and the ratio of CE to TC (more than 50 percent) compared with negative control (p＜0.05).However,the ratio of CE to TC in C.pn infection 72 h group (68.6 %) and 48 h group(66.4 % ) had no difference (p＞0.05).Compared with positive control,there was nodifferences in the number of lipid droplets and the ratio of CE to TC in C.pn infection (1×10~6IFU) group (p＞0.05).Conclusion In the presence of LDL,higher concentrations of C.pn infection (5×10~5 and1×10~6 IFU) induce the formation of THP-1-derived foam cells.PartⅡEffect of Chlamydia pneumoniae infection on the expressionsof key genes involved in macrophage cholesterol metabolismBackground The imbalance of cholesterol metabolism homeostasis in macrophagecontributes to foam cell formation.Objective To investigate the roles of scavenger receptor A1 (SR-A1),B (CD36),lectin-like oxidized low density lipoprotein receptor-1(LOX-1),acyl-coenzyme A:cholesterolacyltransferase 1 (ACAT1),ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1)in Chlamydia pneumoniae (C.pn)-induced foam cell formation.Methods THP-1 monocytes were induced into macrophages by 160 nmol/L PMA for48 h,and then were randomly allocated into four groups:①control group,RPMI 1640medium containing 50μg / ml low density lipoprotein (LDL) for 48 h;②differentconcentrations of C.pn infection group,50μg / ml LDL plus 1×10~5,4×10~5,5×10~5 and 1×10~6IFU C.pn infection for 48 h;③different time of C.pn infection group,50μg / ml LDL plus 1×10~6 IFU C.pn infection for 0,24,48 and 72 h;④mouse anti-human CD36 monoclonalantibody plus C.pn infection group,pre-treatment with different dilutions of mouseanti-human CD36 monoclonal antibody (1:100,1:200,1:400) for 1 h,50μg / ml LDL plus1×10~6 IFU C.pn infection for 48 h.The mRNA and protein expressions of SR-A1,CD36,LOX-1,ACAT1,ABCA1 and ABCG1 from group①to group③were determined byRT-PCR and Western-blot,respectively.Intracellular lipid droplets were observed by oil red Ostaining.The contents of intracellular total cholesterol (TC) and cholesteryl esters (CE) weredetected by enzymic chromatometry.Results C.pn infection not only up-regulated the mRNA and protein expressions ofSR-A1 and ACAT1,but also down-regulated the mRNA and protein expressions of ABCA1and ABCG1 in concentration-dependent and time-dependent manner in LDL-treated THP-1macrophages (all p＜0.05).However,C.pn infection had no effect on the expression ofLOX-1 at mRNA and protein levels compared with control group (p＞0.05).Compared withcontrol group,the down-regulation of CD36 at mRNA and protein expressions was observedin high concentration of C.pn infection (1×10~6 IFU) group.In addition,from morphologicaland biochemical criteria,C.pn-induced foam cell formation derived from THP-1 had not beeninfluenced after pre-treatment with different dilutions of mouse anti-human CD36 monoclonalantibody.Conclusion C.pn infection may destroy the homeostasis of intracellular cholesterolmetabolism by increasing SR-A1 and ACAT1 expressions,and inhibiting CD36,ABCA1 andABCG1 expressions in LDL-treated THP-1-derived macrophages,which leads to foam cellformation. PartⅢThe roles of peroxisome proliferator-activated receptorspathways in Chlamydia pneumoniae-induced foam cell formationBackground peroxisome proliferator-activated receptors (PPARs) are key nuclearreceptors that regulate macrophage cholesterol metabolism.Objective To investigate the effects of peroxisome proliferator-activated receptorα(PPARα) andγ(PPARγ) on the expressions of SR-A1,CD36,ACAT1,ABCA1 andABCG1 regulated by Chlamydia pneumoniae (C.pn) infection,and to discuss the pathways ofC.pn-induced foam cell formation.Methods THP-1 monocytes were induced into macrophages by 160 nmol/L PMA for48 h.and then were randomly allocated into seven groups:①control group,RPMI 1640medium containing 50μg / ml low density lipoprotein (LDL) for 48 h;②differentconcentrations of C.pn infection group,50μg / ml LDL plus 1×10~5,4×10~5,5×10~5 and 1×10~6IFU C.pn infection for 48 h;③different time of C.pn infection group,50μg / ml LDL plus1×10~6 IFU C.pn infection for 0,24,48 and 72 h;④Fenofibrate (a specific PPARαagonist)plus C.pn infection group,pretreatment with different concentrations of fenofibrate (10,20,50μmol/L) for 2 h,50μg / ml LDL plus 1×10~6 IFU C.pn infection for 48 h;⑤Fenofibrategroup,RPMI 1640 medium containing 50μmol/L fenofibrate for 48 h;⑥Rosiglitazone (aspecific PPARγligand) plus C.pn infection group,pretreatment with different concentrationsof rosiglitazone (1,10,20μmol/L) for 2 h,50μg / ml LDL plus 1×10~6 IFU C.pn infection for48 h;⑦Rosiglitazone group,RPMI 1640 medium containing 20μmol/L rosiglitazone for48 h.The mRNA and protein expressions of PPARα,PPARγ,SR-A1,ACAT1,ABCA1 andABCG1 were determined by RT-PCR and Western-blot,respectively.Lipid droplets incytoplasm were observed by oil red O staining.The contents of intracellular cholesteryl esterswere detected by enzymic chromatometry.Results C.pn infection suppressed the expressions of PPARαand PPARγat mRNAand protein levels in concentration-dependent and time-dependent manner in LDL-treatedTHP-1 macrophages (all p＜0.05).Compared with C.pn infection group,not only the up-regulation of SR-A1 and ACAT1 but also the down-regulation of ABCA1 and ABCG1 atmRNA and protein levels by C.pn infection were concentration-dependently inhibited byfenofibrate and rosiglitazone (all p＜0.05).Compared with C.pn infection group,20μmol/Lrosiglitazone reversed the down-regulation of CD36 by C.pn infection (p＜0.05).In addition,from morphological and biochemical criteria,higher concentrations of fenofibrate (20 and 50μmol/L) and rosiglitazone (10 and 20μmol/L) markedly inhibited THP-1-derived foam cellformation induced by C.pn infection.Conclusion C.pn infection induces THP-1-derived foam cell formation byup-regulating SR-A1 and ACAT1 expressions and down-regulating CD36,ABCA1 andABCG1 expressions partly via PPARαand PPARγ-dependent pathways,which mayprovide new insights for the development and progression of atherosclerosis initiated by C.pninfection.