Chlamydia Pneumonia-induced Foam Cell Formation In THP-1-derived Macrophages Via Interfering With MAPK-PPARα/γ Crosstalk

PartⅠ Chlamydia pneumoniae infection caused THP-1macrophage turn into foam cellObjective Observe Chlamydia pneumoniae (C.pn) caused foam cell formation of THP-1-derived macrophages.Methods We used Hep-2cells to proliferated C.pn, and used PMA to turn THP-1cells into macrophages, next they were randomly divided into3groups:(1) control group;(2) concentrations group:1×105IFU,4×105IFU,5×105IFU, or1×106IFU C.pn;(3) time group:C.pn for0h,24h,48h or72h. Each group was incubated with50μg/ml LDL. C.pn infection on Hep-2cells and THP-1macrophages were observed using the light microscope. We used oil red O staining, to observe lipid droplets, as well as the number of foam cells.Results THP-1differentiating into macrophage and C.pn infecting Hep-2cells and THP-1macrophages were observed under the light microscope. Compared with uninfected macrophages (control group), C.pn infection increased the accumulation of intracellular Oil Red O-stained lipid droplets (>10) especially induced foam cell formation at higher doses of C.pn infection (5×105IFU-1×106IFU)(p<0.05). In addition, Oil Red O-stained lipid droplets were steadily increased by1×106IFU C.pn infection in72h (p<0.05).Conclusion Higher concentrations (5×105and1×106IFU) of C.pn infection induced foam cell formation of THP-1-derived macrophages exposed to LDL in48h and72h. Part Ⅱ Effects of PPAR α/γ pathways in Chlamydia pneumoniae-induced foam cell formation via regulating the cholesterol metabolism genes expressionsObjective To investigate the involvement of PPARα and PPARγ signaling pathways in C.pn infection caused THP-1macrophage turn into foam cell by regulating the expressions of CD36, SR-A1, ACAT1, ABCA1and ABCG1.Methods THP-1macrophages were transiently transfected with50nM siRNA interference vectors of PPARα, PPARγ or negative control by using lipofectamine2000for6h. The RNA interfering mRNA and protein expressions of PPARα/γ were evaluated by by real-time quantitative PCR (RT-PCR) and Western blotting, respectively.THP-1macrophage were randomly divided into9groups:(1) control group;(2) C.pn infection group:1×106IFU C.pn infection for48h;(3) fenofibrate/rosiglitazone (PPAR α and PPAR γ agonists)+C.pn:fenofibrate (50μmol/L) or rosiglitazone (20μmol/L) for2h, then1×106IFU C.pn infection for48h;(4) fenofibrate/rosiglitazone group:pretreatment with fenofibrate (50μmol/L) or rosiglitazone (20μmol/L) for48h;(5) MK886/GW9662(PPAR α and PPAR γ inhibitors)+C.pn:MK886/GW9662(1,10or20μmol/L) for2h, then1×106IFU C.pn infection for48h;(6) MK886/GW9662group:pretreatment with MK886/GW9662(20μmol/L) for48h;(7) negative control siRNA+C.pn:negative control sequence transfected for6h, then1×106IFU C.pn infection for48h;(8) PPARα/γ siRNA1+C.pn:PPARα/γ siRNA1sequence transfected for6h, then1×106IFU C.pn infection for48h;(9) PPARα/γ siRNA2+C.pn:PPARα/γ siRNA2sequence transfected for6h, then1×106IFU C.pn infection for48h; Each group was incubated with50μg/ml LDL. We used oil red O staining, to observe lipid droplets, as well as the number of foam cells. The mRNA and protein expressions of CD36, scavenger receptor A-1(SR-A1), Acyl-CoA cholesterol acyltransferase-1(ACAT1) and ATP-binding cassette transporter A1/G1(ABCA1/G1) were evaluated by RT-PCR and Western-blot, respectively.Results In the first part, The transfection efficiency of PPARα/γ siRNA1,2all greater than80%(p<0.05). PPARα/γ siRNA inhibited PPARα/γ gene expression (p<0.05). C.pn could not only up-regulated the expression of SR-A1and ACAT1but also down-regulated the expression of CD36, ABCA1/G1and PPARα/γ, compared with control group. Up-regulation of SR-A1and ACAT1and down-regulation of ABCA1and ABCG1were substantially reversed after rosiglitazone or fenofibrate treatment while MK886, PPARα siRNA, GW9662and PPARγ siRNA showed a similar effect of C.pn. However, rosiglitazone completely abolished the down-regulating effect of C.pn on CD36, PPARγ siRNA and GW9662enhanced it, while fenofibrate, PPARα siRNA and MK886show minor influence on C.pn-induced gene expression changing of CD36. Additionly, from morphological criteria, C.pn infection induced foam cell formation, and this effect was significantly inhibited by fenofibrate and rosiglitazone, whereas strongly enhanced by MK886, PPARα siRNA, GW9662and PPARγ siRNA.Conclusion PPARα/γ siRNA can significantly inhibit PPARα/γ expression in macrophage. PPARα/γ regulated ACAT1, ABCA1, SR-A1, ABCG1and CD36exoression, thus pay a role in C.pn infection caused THP-1macrophage turn into foam cell, which might propose new insights into the mechanism for the occurrence and development of atherosclerosis initiated by C.pn infection. Part ⅢEffects of MAPK and PPAR a/y signaling pathways in C.pn infection caused THP-1macrophage turn into foam cellObjective To investigate whether the interplay between Mitogen-activated protein kinase (MAPK) and PPAR a/y signaling pathways play a role in Chlamydia pneumoniae (C.pn) induced THP-1macrophage-derived foam cell formation.Methods THP-1macrophage were randomly divided into8groups:(1) control group;(2) C.pn infection group:1×106IFU C.pn infection for48h;(3) fenofibrate/rosiglitazone (PPAR a and PPAR y agonists) plus C.pn infection group:pretreatment with50μmol/L fenofibrate or20μmol/L rosiglitazone for2h, then1×106IFU C.pn infection for48h;(4) fenofibrate/rosiglitazone group:pretreatment with50μmol/L fenofibrate or20μmol/L rosiglitazone for48h;(5) MK886/GW9662(PPAR a and PPAR y inhibitors) plus C.pn infection group:pretreatment with20μmol/L MK886/GW9662for2h, then1×106IFU C.pn infection;(6) MK886/GW9662group:pretreatment with20μmol/L MK886/GW9662for2h;(7) MAPK inhibitors plus C.pn infection group:pretreatment with20μmol/L SP600125,50μmol/L PD98059or10μmol/L SB203580for2h, then1×106IFU C.pn infection;(8) MAPK inhibitors group:pretreatment with20μmol/L JNK1/2inhibitor SP600125,50μmol/L ERK1/2inhibitor PD98059or10μmol/L p38inhibitor SB203580for2h; Each group was incubated with50μg/ml LDL. We used oil red O staining, to observe lipid droplets, as well as the number of foam cells. The phosphorylation level of MAPK was assessed by Western-blot. The mRNA and protein expressions of PPAR α and PPAR γ were evaluated by RT-PCR and Western-blot, respectively.Results Compared with control group, C.pn stimulated the phosphorylation of MAPK including c-Jun NH2terminal kinase (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38. However, the phosphorylation of JNK1/2, ERK1/2and p38MAPK by C.pn were substantially reversed after rosiglitazone or fenofibrate treatment while GW9662and MK886enhanced C.pn-induced phosphorylation of JNK1/2, ERK1/2and p38MAPK. In addition, C.pn-induced PPARγ and PPARα down-regulation were significantly suppressed by SP600125and PD98059, but not SB203580. More notably, from morphological criteria, SP600125and PD98059strongly inhibited C.pn-induced accumulation of lipid droplet, whereas SB203580had no obvious change on this phenomenon.Conclusion C.pn induces LDL-treated THP-1macrophage-derived foam cell formation via MAPK-PPAR α/γ crosstalk signal pathways, which may provide a new insight for the mechanism of atherosclerosis initiated by C.pn infection.