Atherosclerosis is a chronic inflammatory disease, which is triggered by lipid retention. Toll-like receptor 2 (TLR2) is a novel target for therapeutic intervention in atherosclerosis. In addition, nuclear factor-κB (NF-κB) serves important roles in stress response and inflammation. The present study investigated whether TLR2 is involved in the activation of cholesterol efflux in macrophages by regulating the NF-κB pathway. The human monocytic THP-1 cell line and murine macrophage RAW264.7 cell line were treated with 50 µg/ml oxidized low-density lipoprotein (ox-LDL) for 48 h in order to obtain macrophage foam cells. The cholesterol efflux of the cell lines under exogenous TLR2 treatment was assessed by liquid scintillation counting. Furthermore, the protein and mRNA expression levels of ATP binding cassette transporter A1 (ABCA1), ABCG1 and scavenger receptor B1 (SR-B1) were examined by western blot and quantitative polymerase chain reaction assays, respectively. To detect the effect of NF-κB on cholesterol efflux, the cells were divided into three groups, including the control, 10 ng/ml lipopolysaccharides (LPS; 24 h) and 10 ng/ml LPS + 50 µM pyrrolidinedithiocarbamate (PDTC; 24 h) groups. The results indicated that ox-LDL induced foam cell formation in the THP-1 and RAW264.7 cells, while TLR2 significantly decreased the cholesterol efflux in dose- and time-dependent manners. Accordingly, TLR2 reduced ABCA1, ABCG1 and SR-B1 expression at the transcriptional and translational levels in a dose-dependent manner. In addition, application of PDTC (an NF-κB specific inhibitor) markedly suppressed the LPS-induced downregulation of cholesterol efflux. These data revealed that TLR2 may be involved in the activation of cholesterol efflux in macrophages by regulating the NF-κB signaling pathway.
Atherosclerosis is a chronic inflammatory disease, which is triggered by lipid retention in the arterial wall (
In order to prevent cholesterol accumulation, an efficient cholesterol efflux mechanism exists in macrophages. High-density lipoprotein (HDL) and its apolipoproteins participate in the transfer of cholesterol from the peripheral tissues and cells to the liver, through a process known as reverse cholesterol transport (RCT) (
Toll-like receptors (TLRs) are a family of type I transmembrane glycoproteins that containan extracellular domain with leucine-rich repeat motifs and a Toll/interleukin-1 receptor signaling domain (
Nuclear factor-κB (NF-κB) serves important roles in stress response and inflammation (
In present study, it was hypothesized that TLR2 may be involved in the cholesterol efflux in macrophages. Therefore, the study initially examined the dose-dependent and time-dependent effect of TLR2 on cholesterol efflux in THP-1 and RAW264.7 macrophage-derived foam cells. Subsequently, the dose-dependent effect of TLR2 on the expression levels of genes linked to cholesterol efflux, including ABCA1, ABCG1 and SR-B1, was explored. Finally, the regulatory mechanisms of TLR2 on NF-κB in cholesterol efflux were investigated. The present study provided novel insights for evaluating the potential roles and mechanisms of the TLR2/NF-κB pathway in the development of atherosclerosis.
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 (
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 (
The lipid analysis was conducted by HPLC as described previously (
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 (
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 (
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
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
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
ABCA1, ABCG1 and SR-B1 are critical proteins in the regulation of cellular cholesterol homeostasis (
It has been demonstrated that NF-κB is a downstream molecule of TLR2 (
To detect the effect of NF-κB on cholesterol efflux, liquid scintillation counting was performed. As observed in
Macrophages take up ox-LDL and other lipids to form foam cells, resulting in early atherosclerosis (
TLR2 represents an attractive therapeutic target in atherosclerosis (
Cholesterol efflux transport is mediated by specific proteins, and has been recently demonstrated to be mediated by ABCA1, ABCG1 and SR-B1 (
TLR2 has been demonstrated to use the downstream adaptor MyD88 for signal transmission, and the MyD88-dependent pathway gives rise to activation of the NF-κB transcription factor, which controls proinflammatory gene expression (
In conclusion, the present study provided a novel insight into the role of TLR2 on suppression of cholesterol efflux via downregulation of ABCA1, ABCG1 and SR-B1 expression levels, and indicated that the TLR2 effect is mediated by the NF-κB signaling pathway. Thus, TLR2 may be a potential therapeutic target for the prevention of atherosclerosis.
Effect of ox-LDL on foam cell formation in THP-1 and RAW264.7 cells. (A) Lipid accumulation by THP-1 and RAW264.7 macrophages following treatment with 50 µg/ml ox-LDL for 48 h was detected by oil red O staining (magnification, ×200). Effect of ox-LDL on the cholesterol content in (B) THP-1 and (C) RAW264.7 macrophage-derived foam cells. High-performance liquid chromatography was performed to determine the content of cellular TC, FC and CE. *P<0.05 vs. control group. ox-LDL, oxidized low-density lipoprotein; TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester.
Dose-dependent and time-dependent effects of TLR2 on the cholesterol efflux in THP-1 and RAW264.7 macrophage-derived foam cells. (A) THP-1 and RAW264.7 macrophage-derived foam cells were exposed to various concentrations of TLR2 (50, 100 and 200 ng/ml) for 24 h. (B) THP-1 and RAW264.7 macrophage-derived foam cells were treated with 100 ng/ml TLR2 for 0, 6, 12, 24 and 48 h. Subsequently, cholesterol efflux was analyzed using the liquid scintillation counting method. *P<0.05 vs. control group (0 ng/ml or 0 h). TLR2, Toll-like receptor 2.
Effect of knockdown of TLR2 on the cholesterol efflux in THP-1 and RAW264.7 macrophage-derived foam cells. The TLR2 protein expression levels and cholesterol efflux were detected by western blot analysis and the liquid scintillation counting method, respectively, in (A) THP-1 and (B) RAW264.7 macrophage-derived foam cells transfected with siRNA-TLR2 and siRNA-scramble. *P<0.05 vs. scramble group. TLR2, Toll-like receptor 2; siRNA, small interfering RNA.
Dose-dependent effects of TLR2 on ABCA1, ABCG1 and SR-B1 expression in THP-1 and RAW264.7 macrophage-derived foam cells. (A) mRNA and (B) protein expression levels of ABCA1, ABCG1 and SR-B1 are shown in THP-1-derived foam cells treated with 0, 50, 100 and 200 ng/ml TLR2. (C) mRNA and (D) protein expression levels are demonstrated in RAW264.7-derived foam cells. mRNA expression was measured by reverse transcription-quantitative polymerase chain reaction, while protein expression was measured by western blot assays. *P<0.05 vs. 0 ng/ml group. TLR2, Toll-like receptor 2; ABC, ATP binding cassette transporter; SR-B1, scavenger receptor B1.
Effect of TLR2 treatment on NF-κB activation. The activation of NF-κB inhibited cholesterol efflux in THP-1 and RAW264.7 macrophage-derived foam cells. Western blot analysis reveals the total NF-κB protein expression and the phosphorylation of NF-κB in (A) THP-1 and (B) RAW264.7 macrophage-derived foam cells transfected with siRNA-TLR2, TLR2 overexpression vector and control vector. The graph bars represent the densitometry results of p-NF-κB (p65) protein expression. Cholesterol efflux was detected by liquid scintillation counting in (C) THP-1 and (D) RAW264.7 macrophage-derived foam cells treated with 10 ng/ml LPS or with 10 ng/ml LPS + 50 µM PDTC for 24 h. *P<0.05 vs. control group; #P<0.05 vs. LPS group. TLR2, Toll-like receptor 2; siRNA, small interfering RNA; NF-κB, nuclear factor-κB; p-, phosphorylated; LPS, lipopolysaccharides; PDTC, pyrrolidine dithiocarbamate.