C. pneumoniae fails to induce foam cell formation in peritoneal macrophages from TLR2^−/− mice. Lipid droplet levels in peritoneal macrophages from wild-type (WT) (A) and TLR2^−/− (B) mice were enumerated. Cells were left untreated (open bars), infected with C. pneumoniae (C. pn; MOI = 1) (solid bars), or treated with the TLR4 ligand E. coli LPS (500 ng/ml) (diagonal bars) or the TLR2 ligand Pam (2 μg/ml) (gray bars) in the presence or absence of LDL (100 μg/ml) for 24 h. Lipid droplet levels were normalized to cell numbers. The data are representative of three experiments and are means of triplicates with standard deviations. An asterisk indicates a statistically significant difference compared with uninfected samples cultured with LDL (P < 0.05; t test).

Effects of GW3965 on foam cell formation in RAW 264.7 macrophages. (A) Lipid droplet levels in untreated RAW264.7 macrophages (open bars) or cells treated with the TLR4 ligand E. coli LPS (500 ng/ml) (diagonal bars) or the TLR2 ligand Pam (2 μg/ml) (gray bars) or infected with C. pneumoniae (C. pn; MOI = 1) (solid bars). Cells were incubated with or without LDL (100 μg/ml) in the presence or absence of 2 μM GW3965 (GW) for 24 h. Lipid droplet levels were normalized to cell numbers. Levels of inhibition are shown. The data are representative of three experiments and are reported as means of triplicates with standard deviations. An asterisk indicates a statistically significant difference compared with C. pneumoniae-infected or Pam-treated samples cultured with LDL (P < 0.05; t test). Double asterisks indicate a statistically significant difference compared with E. coli LPS-treated samples cultured with LDL (P < 0.01; t test). (B) CE levels in untreated (open bars), TLR4 ligand E. coli LPS (500 ng/ml)-treated (diagonal bars), C. pneumoniae (MOI = 1)-infected (solid bars), and TLR2 ligand Pam (2 μg/ml)-treated (gray bars) RAW 264.7 macrophages. Cells were incubated with or without LDL (100 μg/ml) in the presence or absence of 1 or 2 μM GW3965 (GW1 or GW2) for 24 h. CE levels were normalized to protein content. The data shown are representative of three experiments and are means of triplicates with standard deviations. An asterisk indicates a statistically significant difference compared with foam cells induced by E. coli LPS, C. pneumoniae, and Pam when cultured with LDL, respectively (P < 0.05; t test). (C to E) Micrographs of Oil Red O-stained macrophages containing cytoplasmic neutral lipid droplets (magnification, ×300; light microscopy). (C) Cells infected with C. pneumoniae plus LDL. (D) Cells treated with E. coli LPS plus LDL in the presence of 2 μM GW3965. (E) Cells treated with C. pneumoniae plus LDL in the presence of 2 μM GW3965. The arrow indicates lipid droplets stained by Oil Red O.

C. pneumoniae induces foam cell formation and CE accumulation in RAW 264.7 macrophages when cocultured with LDL. (A, B) Micrographs of Oil Red O-stained macrophages treated with LDL (100 μg/ml) (A) or infected with C. pneumoniae (C. pn; MOI = 1) in the presence of LDL for 24 h (B) (magnification, ×300; light microscopy). (C) Lipid droplet levels were quantified in uninfected (open bars) and infected (solid bars) cells. Cells were incubated with LDL (100 μg/ml) for 24 h. Lipid droplet levels were normalized to cell numbers. (D) CE levels were determined after the same treatment described for panel C. CE levels were normalized to protein content. The data are representative of three experiments and are means of triplicates with standard deviations. An asterisk indicates a statistically significant difference compared with uninfected samples cultured with LDL (P < 0.05; t test).

C. pneumoniae induces foam cell formation in the TLR4-mutant murine macrophage GG2EE cell line. Lipid droplet levels in untreated GG2EE cells (open bars) or cells treated with the TLR4 ligand E. coli LPS (500 ng/ml) (diagonal bars) or the TLR2 ligand Pam (2 μg/ml) (grey bars) or cells infected with C. pneumoniae (C. pn; MOI = 1) (solid bars) are represented. Cells were incubated with or without LDL (100 μg/ml) for 24 h. Lipid droplet levels were normalized to cell numbers. The data are representative of three experiments and are means of triplicates with standard deviations. An asterisk indicates a statistically significant difference compared with uninfected samples cultured with LDL (P < 0.05; t test).