C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

Abstract

Macrophage inflammation and foam cell formation are critical events during the initiation and development of atherosclerosis (AS). C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel adipokine with anti-inflammatory and cardioprotection properties; however, little is known regarding the influence of CTRP3 on AS. As macrophages play a key role in AS, this study investigated the effects of CTRP3 on macrophage lipid metabolism, inflammatory reactions, and phenotype transition, as well as underlying mechanisms, to reveal the relationship between CTRP3 and AS. CTRP3 reduced the number of lipid droplets, lowered cholesteryl ester (CE), total cholesterol (TC), and free cholesterol (FC) levels, reduced the CE/TC ratio, and dose-dependently inhibited TNFα, IL-6, MCP-1, MMP-9 and IL-1β release in lipopolysaccharide (LPS)-stimulated THP-1 macrophages and mouse peritoneal macrophages. Pretreatment with CTRP3 effectively increased macrophage transformation to M2 macrophages rather than M1 macrophages. Western blotting showed that the specific NF-κB pathway inhibitor ammonium pyrrolidine dithiocarbamate (PDTC) or siRNA targeting PPARγ/LXRα markedly strengthened or abolished the above-mentioned effects of CTRP3, respectively. These results show that CTRP3 inhibits TLR4-NF-κB pro-inflammatory pathways but activates the PPARγ-LXRα-ABCA1/ABCG1 cholesterol efflux pathway. Taken together, CTRP3 participates in anti-lipid accumulation, anti-inflammation and macrophage phenotype conversion via the TLR4-NF-κB and PPARγ-LXRα-ABCA1/ABCG1 pathways and, thus, may have anti-atherosclerotic properties.

Keywords: C1q/tumor necrosis factor-related protein-3 (CTRP3); atherosclerosis; cholesterol efflux; inflammation; macrophage phenotype.

Figure 1. CTRP3 reduces ox-LDL-induced foam cell formation and lipid deposition in LPS-stimulated macrophages

(A) Impacts of increasing CTRP3 concentration on the viability of THP-1 differentiated macrophages and mouse peritoneal macrophages. Cells were suspended into 1 × 10⁴ cells /100μL/well; THP-1 cells were induced to differentiate into macrophages by culture with 100 ng/mL PMA for 48 h. Mouse peritoneal macrophages were extracted and cultured as mentioned above, after which increasing doses of CTRP3 (0-10 μg/mL) were added; 24 h later, the CCK-8 assay was used to detect viability (mean ± SD). (B-C) Impacts of CTRP3 on LPS-triggered macrophage lipid deposition and foam cell formation in both kinds of macrophages (1 × 10⁶ cells/mL/well). The cholesterol content was detected using a cholesterol quantification kit to evaluate lipid deposition in LPS-triggered macrophages (B, mean ± SD, * : compared to the control group, P < 0.05; # : compared to the ox-LDL and LPS treatment group, P < 0.05). Oil red O staining was performed to analyze the effect of CTRP3 on foam cell formation (C, THP-1 cells: 200×, mouse peritoneal macrophages: 400×). At least three independent experiments were performed.

Figure 2. CTRP3 decreases inflammatory factors produced by LPS-triggered foam cells

THP-1 cells and mouse peritoneal macrophages (1 × 10⁶ cells/mL/well) were preincubated with different concentrations of CTRP3 (0-1.0 μg/ mL) for 30 min. After LPS and ox-LDL stimulation, the supernatant protein levels of TNFα, IL-6, MCP-1, IL-1β and MMP-9 were evaluated by ELISA. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS and ox-LDL treatment group, P < 0.05).

Figure 3. Impacts of CTRP3 on LPS-induced cell phenotype transition in macrophages

Cells (1 × 10⁶ cells /mL/well) were treated, and expression of specific M1 and M2 markers on the cell surface was detected by FCM to observe macrophages polarization. For THP-1 cells, cells expressing CD68 but not CD206/MR (CD68⁺CD206⁻) were considered to be M1 macrophages; cells co-expressing CD68 and CD206 (CD68⁺CD206⁺) were considered to be M2 macrophages. For mouse peritoneal macrophages, cells expressing CCR7 and CD86 were considered to be M1 macrophages; cells expressing CD163 and CD206/MR were considered to be M2 macrophages. The macrophage phenotype ratio was calculated as (CCR7 plus CD86 expression)/(CD163 plus CD206 expression). At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS but without CTRP3 treatment group, P < 0.05).

Figure 4. CTRP3 alters mRNA expression of M1 and M2 markers in macrophages

PMA-induced THP-1 macrophages (1 × 10⁶ cells/mL/well) were divided into three groups: the control group was treated with PBS for 24 h; the LPS group was treated with 100 ng/mL LPS for 24 h; the LPS+CTRP3 group was treated with 1 μg/mL CTRP3 for 30 min before LPS stimulation for 24 h. mRNA expression of specific M1 marker and M2 markers was assessed by qRT-PCR. Relative mRNA concentrations were calculated using the ΔΔCT method, and the expression level was normalized to that of endogenous β-actin. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05).

Figure 5. Impacts of CTRP3 on levels of TLR4-NF-κB and PPARγ-LXRα-ABCA1/ABCG1 pathway proteins in THP-1 macrophages

Different concentrations of CTRP3 were added before cells (1 × 10⁶ cells/mL/well) were exposed to LPS and ox-LDL, and protein expression of TLR4, MyD88, p-NF-κB p65 and NF-κB p65 was assessed by WB (A-D). Protein expression of PPARγ, LXRα, ABCA1 and ABCG1 was assessed by WB (E-I) (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS and ox-LDL treatment group, P < 0.05).

Figure 6. Effectiveness of si-ctrl, si-PPARγ, si-LXRα and PDTC, as verified by WB

THP-1 macrophages (1 × 10⁶ cells/mL/well) were treated as mentioned above, and the protein levels of PPARγ, LXRα, p-p65 and t-p65 were detected by WB (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the si-ctrl or LPS treatment group, P < 0.05).

Figure 7. CTRP3 has an inhibitory effect on the TLR4-NF-κB pathway similar to that of the pathway inhibitor PDTC in macrophages

PMA-induced THP-1 macrophages (1 × 10⁶ cells/mL/well) were divided into five groups:the control group was treated with PBS; the LPS group was treated with 100 ng/mL LPS for 24 h prior to 100 μg/mL ox-LDL for 24 h; the LPS+CTRP3 (CT3) group was pretreated with 1 μg/mL CTRP3 for 30 min before LPS and ox-LDL stimulation; the LPS+PDTC group was pretreated with 100 μM PDTC for 1 h and then stimulated with LPS and ox-LDL; the LPS+PDTC+CT3 group was treated with 100 μM PDTC for 1 h and then 1 μg/mL CTRP3 for 30 min before LPS and ox-LDL stimulation. Expression of TLR4, MyD88, NF-κB p-p65, t-p65 and β-actin was detected by WB (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Figure 8. Transfection of si-PPARγ and si-LXRα reduces the function of the LXRα-ABCA1/ABCG1 cholesterol efflux pathway in macrophages

PMA-induced THP-1 macrophages (1 × 10⁶ cells/mL/well) were divided into six groups: the first three group were treated as mentioned above, and the other three groups were transfected with si-ctrl, si-PPARγ or si-LXRα for 48 h, and then treated with 1 μg/mL CTRP3 for 30 min, and finally stimulated with LPS and ox-LDL. Expression of PPARγ, LXRα, ABCA1, ABCG1 and β-actin was detected by WB (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Figure 9. Suppression of the TLR4-NF-κB pathway and activation of the PPARγ-LXRα-ABCA1/ABCG1 pathway are responsible for the inhibitory effect of CTRP3 on the inflammatory response

Cells (1 × 10⁶ cells/mL/well) were treated as mentioned above, and the supernatant protein levels of TNFα, IL-6, MCP-1, IL-1β and MMP-9 were assessed by ELISA. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Figure 10. Suppression of the TLR4-NF-κB pathway and activation of the PPARγ-LXRα-ABCA1/ABCG1 pathway are responsible for the inhibitory effect of CTRP3 on lipid accumulation in macrophages

Cells (1 × 10⁶ cells/mL/well) were treated as mentioned above, and the levels of CE, TC, and FC, and the CE/TC ratio were evaluated using a cholesterol quantification kit. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Figure 11. Schematic summary of the proposed mechanism by which CTRP3 modulate the function of macrophages

Arrows drawn with a full line show the proposed mechanism verified in our research, and arrows drawn with a dotted line show the proposed mechanism summarized by previous research.