The role of CD4+CD25+ regulatory T cells in macrophage-derived foam-cell formation

Abstract

Cluster of differentiation (CD)4+CD25+ regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis, but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect macrophages foam-cell formation. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxidized LDL (oxLDL) for 48 h to transform foam cells. After co-culture with Tregs, macrophages showed a decrease in lipid accumulation, which was accompanied by a significantly downregulated expression of CD36 and SRA but no obvious difference in ABCA1 expression. Tregs can inhibit the proinflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that both cell-to-cell contact and soluble factors are required for Treg-mediated suppression on macrophage foam-cell formation. Cytokines, interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta) are the key factors for these suppressive functions.

Fig. 1.

Effects of Tregs on oxLDL uptake in peritoneal macrophages. Macrophages(3–4 × 10⁶/well) were cultured alone (no T), with Tregs (CD25+, 5 × 10⁵/well), or CD4+CD25− T cells (CD25−, 5 × 10⁵/well) for 40 h in the presence of anti-CD3 antibody (50 ng/ml), after which the different cultures were stimulated for 48 h with oxLDL (50 μg/ml). (A) Staining with oil red O allowed visualization of cytoplasmic lipid accumulation. (200× magnification). (B) Photometric measurement of lipid stained with oil red O from differentiated macrophage foam cells. (# is indicated for CD25+ versus no T; * is indicated for CD25+ versus CD25− [## = P < 0.01; ** = P < 0.01]). (C) Foam-cell formation was quantified by the total cellular cholesterol and cholesterol ester measurement. Data are presented as mean ± SEM of at least three separate experiments. (# is indicated for CD25+ versus no T; * is indicated for CD25+ versus CD25− [## = P < 0.01; ### = P < 0.001; ** = P < 0.01; *** = P < 0.001]). (D) Murine peritoneal macrophages were treated with Tregs at indicated concentrations (1.25 × 10⁵ cells; 2.5 × 10⁵ cells; 5 × 10⁵ cells) in the presence of oxLDL (50 μg/ml) for 48 h. Foam-cell formation was quantified by cholesterol ester measurement. Data are presented as mean ± SEM of at least three separate experiments (* is indicated for versus no T [* = P < 0.05; ** = P < 0.01]).

Fig. 2.

Effect of Tregs on rate of cholesterol efflux in macrophage foam cells. Macrophages (3–4 × 10⁶/well) were incubated with oxLDL and [3H] cholesterol, then co-cultured with or without Tregs (5 × 10⁵/well). The efflux of cholesterol was initiated by addition of ApoA-I. The percentage of efflux was expressed as the percentage of the radioactivity released from the cells in the medium relative to the total radioactivity in cells plus medium. Data are presented as mean ± SEM of at least three separate experiments.

Fig. 3.

Effects of Tregs on CD36, SRA, and ABCA1 mRNA expression in macrophage foam cells. (A) Murine peritoneal macrophages were treated with CD4+CD25+ Tregs at indicated concentrations (1.25 × 10⁵ cells; 2.5 × 10⁵ cells; 5 × 10⁵ cells) in the presence of oxLDL (50 μg/ml) for 48 h. Total RNA was collected and CD36 and SRA mRNA were analyzed by semi-quantitative PCR. GADPH served as endogenous control. (B) Cells were cultured as described as above. Quantitative realtime PCR analysis of CD36 and SRA mRNA relative to GAPDH was performed with equal loading of samples from indicated cultures. Data are presented as mean ± SEM of triplicate wells and are representative of at least three independent experiments. (* is indicated for versus no T [* = P < 0.05; *** = P < 0.001]). Co-cultures were set up as described in Fig. 1. Total RNA was collected. CD36, SRA, and ABCA1 mRNA were analyzed by semi-quantitative PCR (C) and realtime PCR (D). Data are presented as mean ± SEM of triplicate wells and are representative of at least three independent experiments. (# is indicated for CD25+ versus no T; * is indicated for CD25+ versus CD25− [## = P < 0.01; ### = P < 0.001; ** = P < 0.01; *** = P < 0.001]).

Fig. 4.

Effects of Tregs on CD36, SRA, and ABCA1 protein expression. Co-culture was set up as described in Fig. 1. (A) CD36, SRA, and ABCA1 protein was analyzed by Western blotting. (B)The intensities of CD36, SRA, and ABCA1 protein level were normalized with β-actin. Data are presented as mean ± SEM of at least three independent experiments. (# is indicated for CD25+ versus no T; * is indicated for CD25+ versus CD25− [# = P < 0.05; ## = P < 0.01; * = P < 0.05; *** = P < 0.001]).

Fig. 5.

Tregs inhibit the proinflammatory response of macrophages to oxLDL. Macrophages were cultured as described in the Fig. 1. After 48 h of incubation with oxLDL, cytokine/chemokine production was measured by ELISA (TNF-α, MCP-1, MMP-9, IL-10, and TGF-β). The average production of six independent experiments ± SEM of proinflammatory (A) and anti-inflammatory (B) cytokines/chemokines are shown for the three cultures. (# is indicated for CD25+ versus no T; * is indicated for CD25+ versus CD25−; + is indicated for CD25− versus no T [# = P < 0.05; ## = P < 0.01; ### = P < 0.001; * = P < 0.05; ** = P < 0.01; *** = P < 0.001; + = P < 0.05]).

Fig. 6.

Inhibitory mechanisms of Tregs on macrophage foam-cell formation. Macrophages (3–4 × 10⁶/well) were cultured with or without Tregs (CD25+, 5 × 10⁵/well) as described in Fig. 1. In some experiments, Transwell inserts were used to separate macrophages from Tregs. Neutralizing IL-10 antibody and/or TGF-β antibody or nonblocking IgG anti-mouse control antibody were added at the start of co-culture system. (A) Cellular cholesterol ester measured from macrophages cultured alone (no T), in Transwell insert (TW) or in the co-culture system (CC). Data are presented as mean ± SEM of at least three independent experiments. (# is indicated for TW versus no T; * is indicated for TW versus CC; + is indicated for CC versus no T [* = P < 0.05; ### = P < 0.001; *** = P < 0.001]). (B) Cellular cholesterol ester was measured in the presence of nonblocking IgG anti-mouse antibody, neutralizing anti-IL-10, and/or anti-TGF-β antibody in the co-culture. Data are presented as mean ± SEM of at least three independent experiments. (* is indicated for versus control antibody [* = P < 0.05, ** = P < 0.01, *** = P < 0.001]). (C) Quantitative real-time PCR was performed to measure the expression of CD36 and SRA mRNA in neutralizing experiment. GADPH served as endogenous control. Data are presented as mean ± SEM of at least three independent experiments. (* is indicated for versus control antibody [* = P < 0.05, ** = P < 0.01, *** = P < 0.001]).