Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

Abstract

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.

FIGURE 1.

Anti-inflammatory effects of gAcrp and flAcrp are differentially dependent on IL-10 in Kupffer cells from ethanol- and pair-fed rats. Kupffer cells isolated from ethanol- and pair-fed rats were transfected or not with 100 nm IL-10 siRNA or scrambled siRNA and then cultured with or without 45 nm gAcrp or flAcrp for 18 h. Kupffer cells were then stimulated with 100 ng/ml LPS for 60 min, and TNF-α and 18 S mRNA were measured by qRT-PCR. n = 4. Values with different superscripts (a–d) within each treatment group are significantly different from each other (p < 0.05).

FIGURE 2.

HO-1 partially mediates the inhibitory effects of flAcrp on MyD88-dependent and -independent cytokine signatures in RAW264.7 macrophages. A and B, RAW264.7 cells were cultured for 18 h in the absence or presence of 0.5 μm zinc protoporphyrin (ZnPP) in the presence or absence of flAcrp (45 nm). Cells were then stimulated with 100 ng/ml LPS for 4 h and expression of TNFα and IL-6 mRNA (A) and IFN-β and CXCL10 mRNA (B) were normalized to 18 S mRNA. C and D, RAW264.7 cells were transfected or not with 100 nm of HO-1 siRNA or scrambled siRNA and then cultured with or without 45 nm flAcrp for 18 h. Cells were then stimulated with LPS for 4 h. TNFα and IL-6 mRNA (C) and IFN-β and CXCL10 mRNA (D) were normalized to 18 S mRNA. n = 4. LPS-stimulated cells with different superscripts (a–c) are significantly different from each other (p < 0.05).

FIGURE 3.

Chronic ethanol feeding shifts Kupffer cells to an M1 phenotype, and treatment of Kupffer cells with flAcrp promotes M2 macrophage polarization. Primary cultures of Kupffer cells isolated from pair-fed and EtOH-fed rats were cultured overnight in the absence or presence of 45 nm gAcrp or flAcrp for 18 h. Expression of Arg-1, MMR, IL-4Rα, and inducible NOS mRNA relative to 18 S mRNA was measured in Kupffer cells not treated with adiponectin (A) or after 18 h of culture with adiponectin (B). A, values are expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4. + indicates EtOH-fed compared with pair-fed (p < 0.05). B, values are expressed relative to Kupffer cells not treated with adiponectin. Values with different superscripts (a–d) are significantly different from each other (p < 0.05).

FIGURE 4.

Differential effects of gAcrp and flAcrp on expression of M2 macrophage markers in RAW264.7 macrophages. A–I, RAW264.7 macrophages were treated either without or with 4.5–45 nm gAcrp or 4.5–45 nm flAcrp for 18 h. A–H, expression of markers of M2 polarization measured by qRT-PCR. mRNA of interest was normalized to 18 S mRNA. n = 4. + indicates p < 0.05 compared with cells not treated with adiponectin; * indicates p < 0.05 flAcrp-treated cells compared with gAcrp-treated cells. I, surface expression of CD206 was measured by flow cytometry. Histogram is representative of four independent experiments.

FIGURE 5.

Differential contribution of AdipoR1 and AdipoR2 in mediating the effects of gAcrp and flAcrp on expression of M2 macrophage markers. RAW264.7 cells were transfected or not with either 100 nm AdipoR1, AdipoR2 siRNA, or scrambled siRNA and then cultured with or without 45 nm gAcrp or flAcrp for 18 h. Arg-1, Mgl2, and IL-4Rα mRNA were measured by qRT-PCR and normalized to 18 S mRNA. n = 4. + indicates p < 0.05 compared with nontransfected cells treated with gAcrp or flAcrp.

FIGURE 6.

Differential contribution of IL-10, HO-1, and IL-4 to flAcrp-mediated M2 polarization in RAW 264.7 macrophages. A, RAW264.7 macrophages were treated with gAcrp or flAcrp for 18 h, and IL-4 mRNA accumulation was measured by qRT-PCR, and IL-4 protein expression was assessed by Western blot (inset). Kupffer cells isolated from chow-fed control rats were cultured with flAcrp for 18 h, and accumulation of IL-4 protein in the media was assessed by Western blot (inset). flAcrp increased IL-4 protein by 2.0 ± 0.4-fold (n = 5) in Kupffer cells and 1.6 ± 0.2-fold (n = 7) in RAW264.7 macrophages when compared with untreated cells. B–D, RAW264.7 cells were transfected or not with either 100 nm of IL-10, IL-4, HO-1. or scrambled siRNA and then cultured with or without 45 nm flAcrp for 18 h. Arg-1, Mgl2, and IL-4Rα mRNA were measured by qRT-PCR and normalized to 18 S mRNA. n = 4. Values from flAcrp-treated cells with different superscripts (a–c) are significantly different from each other, p < 0.05. E, Kupffer cells were treated with gAcrp or flAcrp for 60 min, and phosphorylation of STAT6 and STAT3 was assessed by Western blot. Expression of STAT6, STAT3, and Hsc70 was measured as loading controls. F, RAW264.7 macrophages were transfected with siRNA against IL-10 or IL-4 and then stimulated with gAcrp or flAcrp for 60 min. Phosphorylation of STAT6 and STAT3 was assessed by Western blot. Expression of Hsc70 was measured as a loading control. E and F, images are representative of at least four independent experiments.

FIGURE 7.

gAcrp and flAcrp differentially modulate M2 polarization of RAW264.7 macrophages. RAW264.7 cells were transfected or not with 100 nm of STAT3, STAT6, or scrambled siRNA and then cultured with or without 45 nm gAcrp or flAcrp for 18 h. Arg-1, Mgl2, and IL-4Rα mRNA were measured by qRT-PCR and normalized to 18 S mRNA. n = 4. Values from adiponectin-treated cells with different superscripts (a–d) are significantly different from each other, p < 0.05.

FIGURE 8.

M2 macrophage polarization by flAcrp is dependent on AMPK in Kupffer cells from ethanol- and pair-fed rats. Kupffer cells isolated from ethanol- and pair-fed rats were transfected or not with 100 nm of AMPK siRNA or scrambled siRNA and then cultured with or without 45 nm flAcrp for 18 h. Arg-1 and 18 S mRNA measured by qRT-PCR. n = 4, values with different superscripts (a–d) within each treatment group are significantly different from each other (p < 0.05).

FIGURE 9.

Differential effects of gAcrp and flAcrp on expression of PPARs and oxidative phosphorylation genes in RAW264.7 macrophages. RAW264.7 macrophages were treated either without or with 45 nm gAcrp or flAcrp for 18 h, and expression of PPARδ, PPARγ, medium chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA oxidase (AOX) was measured by qRT-PCR. mRNA of interest was normalized to 18 S mRNA. n = 4. + indicates p < 0.05 compared with cells not treated with adiponectin.