Adiponectin promotes macrophage polarization toward an anti-inflammatory phenotype

Abstract

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.

FIGURE 1.

Adiponectin deficiency promotes peritoneal macrophage activation. A, quantitative Western blot of arginase-1 and α-tubulin (Tubulin) in peritoneal macrophages from WT and APN-KO mice. B, the mRNA levels of anti-inflammatory M2 markers arginase-1, Mgl-1, and IL-10 and inflammatory M1 markers TNF-α, MCP-1, and IL-6 in quantitative RT-PCR methods. All results were normalized to 36B4. *, p < 0.05 versus WT (n = 6 in each group). C, Ad-β-galactosidase (Ad-β-gal) or Ad-APN (4 × 10⁸ plaque-forming units each) was intravenously injected into WT and APN-KO mice 7 days prior to collection of peritoneal macrophages. The left panel shows Western blot of arginase-1 from WT and APN-KO (KO) mice after the injection of Ad-β-galactosidase or Ad-APN. The right panel shows the mRNA levels of arginase-1 from WT and KO mice after the injection of Ad-β-galactosidase or Ad-APN. *, p < 0.05 versus Ad-β-galactosidase-treated WT mice (n = 4 in each group). Data are presented as mean ± S.E.

FIGURE 2.

Adiponectin deficiency promotes SVF cell activation. A, quantitative Western blot of arginase-1/α-tubulin (Tubulin) and the mRNA levels of anti-inflammatory M2 markers arginase-1, Mgl-1, and Chi3l3 and inflammatory M1 markers, TNF-α, MCP-1, IL-6, and iNOS in SVF cells from WT and APN-KO mice. All mRNA data were normalized to 36B4. *, p < 0.05 versus WT (n = 6 in each group). B, Ad-β-galactosidase (Ad-β-gal) or Ad-APN (4 × 10⁸ plaque-forming units each) was intravenously injected into WT and APN-KO mice 7 days prior to collection of peritoneal macrophages. The left panel shows Western blot of arginase-1 from WT and APN-KO (KO) mice after the injection of Ad-β-galactosidase or Ad-APN. The right panel shows the mRNA levels of arginase-1 from WT and KO mice after the injection of Ad-β-galactosidase or Ad-APN. *, p < 0.05 versus Ad-β-galactosidase-treated WT mice (n = 4 in each group). Data are presented as mean ± S.E.

FIGURE 3.

Adiponectin promotes an anti-inflammatory phenotype in freshly isolated murine peritoneal macrophages. A, left panel, shows Western blot of arginase-1 and α-tubulin (Tubulin) under a 48-h treatment of recombinant APN (30 μg/ml) and IL-4 (10 ng/ml). The right panel shows mRNA levels of arginase-1. *, p < 0.05 versus vehicle (n = 4 in each group). The mRNA levels of arginase-1 were normalized to 36B4. B, left panel, shows Western blot of arginase-1 and tubulin under the treatment of APN, IL-4, or both. The right panel shows mRNA levels of arginase-1 under the treatment of APN, IL-4, or both. *, p < 0.05 and **, p < 0.01 (n = 4 in each group). The mRNA levels of arginase-1 were normalized to 36B4. C, the mRNA levels of anti-inflammatory M2 markers, Mgl-1 and IL-10 and inflammatory markers, TNF-α and MCP-1 in quantitative RT-PCR methods. *, p < 0.05 and **, p < 0.01 versus vehicle (n = 4 in each group). All results were normalized to 36B4. D, macrophage response to different levels of adiponectin in the culture media. *, p < 0.05 for APN 0 μg/ml treatment (n = 3 in each group). All results were normalized to 36B4. E, the mRNA levels of ROS-related genes under the treatment of APN or IL-4. *, p < 0.05 and **, p < 0.01 (n = 4 in each group). F, anti-inflammatory effects of APN and IL-4 against LPS-induced M1 phenotypic change in peritoneal macrophages. The mRNA levels of TNF-α, MCP-1, and iNOS in quantitative RT-PCR methods. **, p < 0.01 versus vehicle; #, p < 0.05 and ##, p < 0.01 versus LPS treatment (n = 4 in each group). All results were normalized to 36B4. Data are presented as mean ± S.E.

FIGURE 3.

Adiponectin promotes an anti-inflammatory phenotype in freshly isolated murine peritoneal macrophages. A, left panel, shows Western blot of arginase-1 and α-tubulin (Tubulin) under a 48-h treatment of recombinant APN (30 μg/ml) and IL-4 (10 ng/ml). The right panel shows mRNA levels of arginase-1. *, p < 0.05 versus vehicle (n = 4 in each group). The mRNA levels of arginase-1 were normalized to 36B4. B, left panel, shows Western blot of arginase-1 and tubulin under the treatment of APN, IL-4, or both. The right panel shows mRNA levels of arginase-1 under the treatment of APN, IL-4, or both. *, p < 0.05 and **, p < 0.01 (n = 4 in each group). The mRNA levels of arginase-1 were normalized to 36B4. C, the mRNA levels of anti-inflammatory M2 markers, Mgl-1 and IL-10 and inflammatory markers, TNF-α and MCP-1 in quantitative RT-PCR methods. *, p < 0.05 and **, p < 0.01 versus vehicle (n = 4 in each group). All results were normalized to 36B4. D, macrophage response to different levels of adiponectin in the culture media. *, p < 0.05 for APN 0 μg/ml treatment (n = 3 in each group). All results were normalized to 36B4. E, the mRNA levels of ROS-related genes under the treatment of APN or IL-4. *, p < 0.05 and **, p < 0.01 (n = 4 in each group). F, anti-inflammatory effects of APN and IL-4 against LPS-induced M1 phenotypic change in peritoneal macrophages. The mRNA levels of TNF-α, MCP-1, and iNOS in quantitative RT-PCR methods. **, p < 0.01 versus vehicle; #, p < 0.05 and ##, p < 0.01 versus LPS treatment (n = 4 in each group). All results were normalized to 36B4. Data are presented as mean ± S.E.

FIGURE 4.

Adiponectin promotes an anti-inflammatory phenotype in human circulating monocyte-derived macrophages, SVF cells from human subcutaneous fat pads, and a murine alveolar macrophage cell line. A, anti-inflammatory effects of APN against LPS-induced M1 phenotypic change in MH-S cells, a murine alveolar macrophage cell line. TNF-α concentration in cell culture media as measured by enzyme-linked immunosorbent assay (R&D Systems). mRNA levels of MCP-1 by quantitative RT-PCR methods normalized to β-actin. **, p < 0.01 versus vehicle; #, p < 0.05 and ##, p < 0.01 versus LPS treatment (n = 3 in each group). B, the mRNA levels of anti-inflammatory M2 markers, mannose receptor (Mannose R), IL-10, and CD163 and inflammatory M1 markers, TNF-α and MCP-1 in quantitative RT-PCR methods. C, the mRNA levels of M2 marker, mannose receptor (mannose R), and M1 markers, TNF-α and MCP-1 in quantitative RT-PCR methods. All results were normalized to β-actin. *, p < 0.05; **, p < 0.01 versus vehicle (n = 4 in each group). Data are presented as mean ± S.E.