SIRT1/Adenosine Monophosphate-Activated Protein Kinase α Signaling Enhances Macrophage Polarization to an Anti-inflammatory Phenotype in Rheumatoid Arthritis

Abstract

Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) α/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, FcεRII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon γ-mediated NF-κB activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNFα and IL-1β expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPKα signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA.

Keywords: M1/M2 macrophages; SIRT1; adenosine monophosphate-activated protein kinase α; inflammation; macrophage polarization; rheumatoid arthritis.

Figure 1

Effects of SIRT1 activation by resveratrol (RSV) on M1 and M2 markers in rheumatoid arthritis macrophages. (A–D) After pretreatment with RSV (50 µM) for 24 h, cells were incubated with interleukin (IL)-4 (20 ng/ml) for 12 h. The mRNA expression levels of MDC, FcεRII, MrC1, and IL-10 were quantified by qPCR and normalized versus 18S ribosomal RNA. (E–H) After pretreatment with RSV (50 µM) for 24 h, cells were incubated with LPS (1 µg/ml) and/or interferon (IFN)γ (20 ng/ml) for 12 h. The mRNA expression levels of MCP-1, iNOS, IL-12 p35, and IL-12 p40 were quantified by qPCR and normalized versus 18S ribosomal RNA. Values are means ± SEMs. Results are representative of four to six independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated.

Figure 2

Expression of M1/M2 polarization markers in SIRT1 siRNA-transfected rheumatoid arthritis macrophages. (A) Cells were either transfected with the siRNA targeting SIRT1 (200 nM) or with scrambled siRNA duplex (200 nM) for 24 h and then were treated with or without resveratrol (RSV, 50 µM) for 24 h. (B,C) SIRT1 gene knockdown cells were pretreated with RSV (50 µM) for 24 h and were incubated with interleukin (IL)-4 (20 ng/ml) for 12 h, and the mRNA expression levels of MDC and MrC1 were quantified by qPCR. (D,E) These cells were incubated with LPS (1 µg/ml) and/or interferon (IFN)γ (20 ng/ml) for 12 h, and then the mRNA expression levels of MCP-1 and iNOS were quantified by qPCR. These levels were normalized versus 18S ribosomal RNA. Values are means ± SEMs. Results are representative of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated.

Figure 3

Enhanced macrophage M2 polarization by resveratrol (RSV) by SIRT1-mediated p-adenosine monophosphate-activated protein kinase α (AMPKα) upregulation. (A) Effects of SIRT1 on the phosphorylations of AMPKα and acetyl-CoA carboxylase (ACC). Cells were treated with RSV (50 µM) for 0–48 h. (B) The antagonizing effect of sirtinol (a SIRT1 inhibitor): cells were pretreated with sirtinol (20 µM) for 30 min and then with RSV (50 µM) for 24 h. (C) Effect of SIRT1 gene knockdown on the phosphorylations of AMPKα and ACC. SIRT1-gene knockdown cells were treated with RSV (50 µM) for 24 h. Values are means ± SEMs. Results are representative of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus zero time or none; ^#P < 0.05, ^##P < 0.01 versus RSV alone. (D–G) Inhibitions of resveratrol (RSV)-induced M2 macrophages markers by sirtinol and compound C (CC). After pretreatment with RSV (50 µM) for 24 h, cells were stimulated with interleukin (IL)-4 (20 ng/ml) for 12 h with or without sirtinol (20 µM) or CC (1 µM, an AMPK inhibitor) for 30 min. The mRNA expression levels of MDC, FcεRII, MrC1, and IL-10 were quantified by qPCR and normalized versus 18S ribosomal RNA. Values are means ± SEMs. Results are representative of four to five independent experiments. *P < 0.05, **P < 0.01 as indicated.

Figure 4

Suppression of NF-κB signaling by resveratrol (RSV) in the LPS/interferon (IFN)γ-induced M1 macrophage polarization. (A) Effects of RSV on IκBα degradation and nuclear NF-κB expression. After pretreatment with RSV (50 µM) for 24 h, cells were stimulated with LPS (1 µg/ml) plus IFNγ (20 ng/ml) for 1 h. (B) Effects of RSV on the acetylation of p65-K310 and the phosphorylation of p65-serine 536. Cells were pretreated with RSV (50 µM) for 24 h and then with LPS (1 µg/ml) plus IFNγ (20 ng/ml) for 1 h. (C) Effect of SIRT1 gene knockdown on NF-κB signaling. Cells were transfected with SIRT1 siRNA or negative control siRNA at a final concentration of 200 nM. Macrophages were incubated with RSV (50 µM) for 24 h, and then exposed to LPS (1 µg/ml) plus IFNγ (20 ng/ml) for 1 h. Values are means ± SEMs. Results are representative of four to five independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 as indicated. (D) Cells were transfected with basic-pGL3-luc or NF-κB-pGL3-luc using Lipofectamine 2000. After 24 h of transfection, cells were treated with or without sirtinol (20 µM) for 30 min, and then treated with RSV (50 µM) for 24 h followed by LPS (1 µg/ml) + IFNγ (20 ng/ml) for 3 h. The firefly luciferase enzyme activity were detected in cell extracts and normalized to the Renilla luciferase activity. Values are means ± SEMs. Results are representative of five independent experiments. *P < 0.05, **P < 0.01 as indicated.

Figure 5

Upregulation of the M2 phenotype and downregulation of the M1 phenotype in bone marrow-derived macrophage (BMDMs) obtained from SIRT1 transgenic (Tg)-mice. (A–D) Cells were incubated with interleukin (IL)-4 (20 ng/ml) for 12 h and ariginase-1, Fizz, Yim-1, and FcεRII mRNA levels were then quantified with qPCR and normalization versus 18S ribosomal RNA. (E–I) Cells were incubated with LPS (1 µg/ml) or interferon (IFN)γ (20 ng/ml) for 12 h and TNFα, IL-1β, MCP-1, IL-12 p35, and IL-12 p40 mRNA levels were quantified by qPCR and normalization versus 18S ribosomal RNA. Values are means ± SEMs. Results are representative of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated.

Figure 6

Analysis of the pathological severity of the joints of C57BL/6N and SIRT1 transgenic (Tg) mice (n = 5) subjected to collagen-induced arthritis (CIA) (refer to Section “Materials and Methods” for details). (A) Arthritis severity scores of CIA mice were recorded after second immunization with type II collagen. (B) Histological scores of synovitis, pannus formation, and erosion in knee joints. Values are means ± SEMs. Results are representative of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus SIRT1 Tg-CIA mice. (C) Representative H&E staining of joints from CIA mice. Scale bar = 200 µm. (D) Representative sections showing staining for arginase-1, TNF-α, interleukin (IL)-1β, and RANKL, which were markedly reduced in the knee joint tissues of SIRT1 Tg-CIA mice. Tissue sections from knee joints were stained with the indicated antibodies. Staining for each antibody on each cell are shown in dark brown. Scale bars; 25 µm.