The human monocytic THP-1 (cat no. TIB-202) and murine macrophage RAW264.7 cell lines (cat no. SC-6003) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and 0.1% nonessential amino acids in a 5% CO₂ chamber at 37°C. Next, the cells were treated with 160 nmol/l phorbol 12-myristate 13-acetate (Sigma-Aldrich; Merck, Darmstadt, Germany) for 12 h. Subsequently, the medium was replaced by a serum-free medium containing 50 µg/ml ox-LDL for 48 h in order to obtain macrophage-derived foam cells prior to the following experiments.

In order to examine the dose-dependent and time-dependent effects of TLR2 on cholesterol efflux, the THP-1 and RAW264.7 macrophage-derived foam cells were incubated with 0, 50, 100 and 200 ng/ml TLR2 for 24 h or with 100 ng/ml TLR2 for 0, 6, 12, 24 and 48 h.

THP-1 and RAW264.7 macrophage-derived foam cells were divided into 3 groups: Control group (cells were incubated in RPMI-1640 medium for 24 h), LPS group [cells were cultured in 10 ng/ml lipopolysaccharides (LPS; Biosea Biotechnology Co., Ltd., Beijing, China) for 24 h] and LPS+PDTC group [cells were incubated with 50 µM pyrrolidine dithiocarbamate (PDTC; Sigma-Aldrich; Merck KGaA), and 10 ng/ml LPS for 24 h]. The cells were then harvested.

The lipid accumulation of macrophages following treatment with ox-LDL was examined by ORO staining as described previously (20). Briefly, cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 10% formalin in PBS for 1 h. Next, cells were washed with water for three times, dried and stained with ORO (Sigma-Aldrich; Merck) for 15 min. Subsequently, 70% ethanol was used to remove excess stain and the stained cells were washed with water. Images of the cells were captured using a light microscope (Olympus CX23; Olympus Corporation, Tokyo, Japan).

THP-1 and RAW264.7 macrophage-derived foam cells (2×10⁶ cells/well) were seeded in 96-well plates. For knockdown of TLR2, the cells were transfected with siRNA-TLR2 (GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and siRNA-scramble (GenePharma Co., Ltd.) was used as the control. After transfection for 48 h, the cells were harvested and used in further experiments.

For analysis of the cholesterol efflux, cells were initially labeled with 0.2 µCi/ml [³H] cholesterol (PerkinElmer, Inc., Waltham, MA, USA). Following cultivation for 72 h, cells were washed with PBS and incubated in RPMI-1640 medium supplemented with 0.1% (w/v) bovine serum albumin in order to allow for equilibration of the [³H] cholesterol in all the cellular pools. Equilibrated [³H] cholesterol-labeled cells were then washed with PBS and incubated in 2 ml serum-free RPMI-1640 containing 0.1% bovine serum albumin (fraction V, fatty acid free; EMD Millipore, Billerica, MA, USA). Next, 150 µl efflux medium was obtained after a 6 h incubation and passed through a 0.45-µm filter to remove any floating cells. The monolayers were subsequently washed twice in PBS, and cellular lipids were extracted with isopropanol. A liquid scintillation counting method was performed to measure the medium and cell-associated [³H] cholesterol (21). The percentage of efflux was calculated as follows: Cellular cholesterol efflux=[total media counts/(total cellular counts + total media counts)] ×100% (22).

The lipid analysis was conducted by HPLC as described previously (23). Briefly, the protein concentrations in the cell solution were measured using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.), and 0.1 ml cell lysate was used to measure the free cholesterol (FC) and total cholesterol (TC) levels. Next, the samples were dissolved in 100 µl isopropanol-acetonitrile (v/v, 20:80; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), followed by an ultrasound water bath for 5 min. Subsequently, the samples were used for HPLC analysis (Agilent 1100; Agilent Technologies, Inc., Santa Clara, CA, USA). The cholesterol was eluted with isopropanol-acetonitrile solution (v/v, 20:80) at a speed of 1 ml/min and then detected in terms of the absorbance at 210 nm. The levels of cholesteryl ester (CE) were calculated according to the following formula: CE = TC – FC.

Total RNA was extracted from the cells by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The quality of total RNA was assessed by spectrophotometry (A260/280 ratio: 1.8–2.0). cDNA was reverse transcribed from 100 ng RNA using a First-Strand RT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was then performed using a SYBR Green qRT-PCR kit (Thermo Fisher Scientific, Inc.) on an Applied Biosystems 7900HT Real-Time PCR system (Thermo Fisher Scientific, Inc.). The primers used for qPCR were as follows: ABCA1 forward, 5′-GATTGGCTTCAGGATGTCCATGTTGGAA-3′ and reverse, 5′-GTATTTTTGCAAGGCTACCAGTTACATTTGACAA-3′; ABCG1 forward, 5′-CAGTGACAGCCATCCCGGTGCT-3′ and reverse, 5′-CGATGAAGTCCAGGTACAGCTTGGC-3′; SR-B1 forward, 5′-GCTGTCTGCTGGGAGAGTC-3′ and reverse, 5′-TTCTGCCCGTGCCTGGAGTC-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The PCR conditions for quantification were as follows: 10 min at 95°C, 40 cycles of 10 sec at 95°C, 20 sec at 58°C, and 30 sec at 72°C. qPCR was performed using 2 µl diluted cDNA products, 12.5 µl SYBR Green (Thermo Fisher Scientific, Inc.), 0.5 µl forward and reverse primers (10 µM) and 9.5 µl nuclease-free water in a final volume of 25 µl. GAPDH was used as an internal control and the relative expression of mRNA was calculated using the 2^−ΔΔCq method (24).

The proteins were isolated from the cells using radioimmunoprecipitation assay lysis and extraction buffer (containing 150 mM NaCl, 25 mM Tris-HCl, pH 7.6, 1% sodium deoxycholate, 1% NP-40, protease inhibitor and 0.1% SDS). The BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) was used to calculate the total protein concentration. The total proteins (50 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked with 5% non-fat dry milk in PBS with 5% Tween-20. Following 3 washes in PBS with 5% Tween-20, the membranes were incubated with primary antibodies against TLR2 (1:1,000; ab108998; Abcam, Cambridge, UK), SR-B1 (1:1,000; MAB8114; R&D Systems, Inc., Minneapolis, MN, USA), ABCG1 (1:500; NB400-132), ABCA1 (1:1,000; NB400-105) (both from Novus Biologicals, LLC, Littleton, CO, USA), p-NF-κB (1:500; 8214; Cell Signaling Technology, Inc., Danvers, MA, USA), NF-κB (1:500; MAB72261; R&D Systems, Inc.) and GAPDH (1:200; 4670; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated IgG secondary antibodies for 1 h at room temperature. The bands were subsequently visualized by enhanced chemiluminescence detection reagents (GE Healthcare Life Sciences, Little Chalfont, UK), and the images were analyzed by the NIH ImageJ software (version 1.47t; National Institutes of Health, Bethesda, MD, USA).

The data are demonstrated as the mean ± standard deviation. All experiments were performed at least three times. Comparisons between two groups were evaluated by Student's t-test. Statistical analysis was performed using the SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered as an indicator of statistically significant differences.

Macrophages are known to transform into foam cells when incubated with ox-LDL (25). To evaluate the formation of foam cells, ORO staining and the intracellular cholesterol contents were measured. THP-1 and RAW264.7 cells were incubated with 50 µg/ml ox-LDL for 48 h prior to staining with ORO. As shown in Fig. 1A, THP-1 and RAW264.7 cells treated with ox-LDL exhibited significant accumulation of lipid droplets.

The contents of TC, FC and CE in normal cells and foam cells were also detected following incubation with ox-LDL for 48 h. As shown in Fig. 1B, the results revealed that the TC, FC and CE contents were significantly increased in THP-1 macrophage-derived foam cells treated with ox-LDL for 48 h when compared with those in untreated cells. In addition, the contents of TC and CE were markedly upregulated in RAW264.7 macrophage-derived foam cells treated with ox-LDL compared with those in untreated cells (Fig. 1C). These results demonstrated that ox-LDL induced foam cell formation in the THP-1 and RAW264.7 cells.

In order to investigate the role of TLR2 in mediating the efflux of cholesterol, the cellular cholesterol efflux was measured by liquid scintillation counting. As shown in Fig. 2, addition of TLR2 significantly decreased the cholesterol efflux of THP-1 and RAW264.7 cells in dose- and time-dependent manners. These data suggested that TLR2 was a negative regulator of cholesterol efflux in THP-1 and RAW264.7 macrophage-derived foam cells.

To further confirm whether TLR2 is a negative regulator of cholesterol efflux, THP-1 and RAW264.7 macrophage-derived foam cells were transfected with TLR2 siRNA. As demonstrated in Fig. 3A and B (left panels), the cells transfected with TLR2 siRNA presented inhibited TLR2 protein expression in comparison with those transfected with scramble siRNA, which confirmed the knockdown of TLR2. Furthermore, knockdown of TLR2 by siRNA significantly increased the cholesterol efflux (Fig. 3; right panels). Thus, these results supported the involvement of TLR2 in the downregulation of cholesterol efflux.

ABCA1, ABCG1 and SR-B1 are critical proteins in the regulation of cellular cholesterol homeostasis (9). In the present study, the effect of TLR2 on the mRNA and protein expression levels of ABCA1, ABCG1 and SR-B1 in THP-1 and RAW264.7 macrophage-derived foam cells was examined by RT-qPCR and western blot analysis, respectively. As shown in Fig. 4, TLR2 reduced ABCA1, ABCG1 and SR-B1 expression at the transcriptional and translational levels in a dose-dependent manner.

It has been demonstrated that NF-κB is a downstream molecule of TLR2 (26). Thus, in the present study, it was hypothesized that NF-κB may be involved in the role of TLR2 in the downregulation of cholesterol efflux. As shown in Fig. 5A and B, western blot analysis revealed that THP-1 and RAW264.7 macrophage-derived foam cells transfected with TLR2 siRNA exhibited reduced phosphorylation levels of NF-κB (p65). By contrast, overexpression of TLR2 increased the phosphorylation levels of NF-κB (p65). Moreover, upregulation or downregulation of TLR2 had no effect on the protein expression levels of NF-κB.

To detect the effect of NF-κB on cholesterol efflux, liquid scintillation counting was performed. As observed in Fig. 5C and D, the cholesterol efflux was markedly reduced following treatment with lipopolysaccharides (LPS) in THP-1 and RAW264.7 macrophage-derived foam cells. By contrast, the application of PDTC, an NF-κB specific inhibitor, significantly suppressed the LPS-induced downregulation of cholesterol efflux.