The FASEB Journal article fj.14-253831. Published online November 12, 2014.

The FASEB Journal • Research Communication

Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response Caroline M. W. van Stijn,* Jason Kim,* Aldons J. Lusis,† Grant D. Barish,‡ and Rajendra K. Tangirala*,†,1 *Division of Endocrinology, Diabetes & Hypertension and †Division of Cardiology, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, USA; and ‡Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/ macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40–60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 mg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-a, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by upregulating liver X receptor-a (LXRa) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.—van Stijn, C. M. W., Kim, J., Lusis, A. J., Barish, G. D., Tangirala, R. K. Macrophage polarization phenotype regulates adiponectin receptor expression

ABSTRACT

Abbreviations: ACE, angiotensin-converting enzyme; AdipoR, adiponectin receptor; APN, adiponectin; CRP, C-reactive protein; Egr-1, early growth response 1; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GINF, glucose infusion rate; IR, insulin resistance; IS, insulin sensitive; LXR, liver X receptor; M1, classical activation; M2, alternative activation; MR, mannose receptor; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative RT-PCR; RPMI, Roswell Park Memorial Institute

0892-6638/15/0029-0001 © FASEB

and adiponectin anti-inflammatory response. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: adipokines • inflammation • metabolic disorder macrophage activation • nuclear receptors



A LARGE BODY OF EVIDENCE suggests that chronic, low-grade inflammation in adipose tissue contributes to obesityrelated metabolic disorders, including diabetes and cardiovascular disease (1, 2). Infiltration of proinflammatory macrophages into the adipose tissue has been postulated to induce secretion of inflammatory mediators contributing to insulin resistance (IR) and vascular damage (3–5). Bioactive mediators produced by the adipose tissue, known as adipokines, regulate inflammation, metabolism, and tissue remodeling. APN is a pleotropic adipokine, which unlike other adipokines, exerts anti-inflammatory and antiatherogenic effects in vascular cells, especially macrophages, thus linking adipose tissue to vascular functions (4, 6). Circulating APN levels are inversely correlated with systemic inflammatory markers, C-reactive protein (CRP) and IL-6, as well as with obesity, type 2 diabetes, and cardiovascular disease (7, 8). APN exerts beneficial effects by inhibiting NF-kB signaling, production of proinflammatory cytokine TNF-a (9), expression of scavenger receptor A1, and foam cell formation (10). Furthermore, APN promotes the removal of apoptotic debris (11) and secretion of the anti-inflammatory cytokine IL-10 (12, 13). Recent studies reported that APN induces markers of M2 in human and mouse macrophages (14–16). Monocyte/macrophages play a critical role in innate immunity and regulate inflammatory response and host defense (17, 18). They exhibit marked functional heterogeneity by undergoing reprogramming to acquire distinct polarization phenotypes in response to various cytokine signals in tissues. Recent evidence supports the concept that macrophages develop into a spectrum of distinct, functional phenotypes of which proinflammatory (M1) type and anti-inflammatory (M2) type represent the 1 Correspondence: Division of Cardiology, David Geffen School of Medicine at UCLA, 675 Charles E. Young Drive South, MRL-3-740, Los Angeles, CA 90095, USA. E-mail: [email protected] doi: 10.1096/fj.14-253831

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opposite ends (19–21). Studies in humans and mouse models have shown that obesity is strongly correlated with a reduction of M2 and an increase in M1 macrophages in the adipose tissue (22, 23). Proinflammatory cytokines, such as IFN-g, IL-1b, and the inducer of the TNF-a, LPS, all promote M1 polarization of macrophages. M1 macrophages promote inflammation and production of proinflammatory cytokines (IL-6, TNF-a, IL-12) and reactive oxygen species, which enhance further monocyte recruitment and lipid oxidation, leading to tissue damage (24). In contrast, anti-inflammatory cytokines, such as IL-4 and IL-13, induce M2 polarization of macrophages. M2 macrophages suppress inflammation and induce the antiinflammatory cytokine (IL-10 and TGF-b) secretion and promote scavenging of apoptotic cell debris and tissue repair (17, 25). Several lines of evidence link inflammatory and metabolic signals that induce polarization of macrophages into M1 or M2. APN exerts its pleotropic actions through interaction with two major, specific cell-surface receptors, AdipoR1 and AdipoR2, which belong to the progesterone and AdipoR Q family (26, 27). The molecular structure of these receptors exhibits significant homology containing seventransmembrane domains with intracellular N terminus and extracellular C terminus, which is distinct from GPCRs (26). The AdipoRs are widely expressed in a variety of tissues and exhibit differences in their downstream signaling pathways: AdipoR1 activates AMPK, whereas AdipoR2 activates proliferator-activated receptor (PPAR)a–mediated pathways (28). In addition, AdipoR1 and AdipoR2 mediate APN activation of ceramide signaling, which contributes to anti-inflammatory effects (29). Studies in gene-targeted mice have revealed that biologic activity of APN depends on the expression of AdipoR1 and AdipoR2, thus providing evidence for the critical role of these receptors in glucose and lipid metabolism, inflammation, and oxidative stress (30–32). Although AdipoR expression in liver, skeletal muscle, and adipose tissue has been shown to be regulated by metabolic factors in obesity and diabetes (33–35), regulation and function of AdipoR1 and AdipoR2 in macrophages are poorly understood. Studies in human monocyte/macrophages have shown that AdipoR expression in monocyte-macrophages is regulated by differentiation and nuclear hormone receptor (LXR and PPAR) ligands (36). Previous reports indicate that blood monocytes from Type 2 diabetes and coronary artery disease patients, with reduced APN levels, expressed low AdipoR levels, although AdipoR1 and AdipoR2 levels were elevated in monocytes from obese subjects compared with normal subjects (37, 38). Recently, overexpression of AdipoR1 in macrophages has been shown to reduce inflammatory cytokine production and foam cell formation (39). However, it is unknown whether APN levels and macrophage inflammatory status, as a result of a differential polarized activation phenotype, regulate AdipoR expression and functional responses of macrophages to APN. In this study, we tested the hypothesis that macrophage polarization phenotypes regulate AdipoR1 and AdipoR2 expression and modulate macrophage responses to APN in an inflammatory cytokine environment. We demonstrate that polarization of macrophages into a proinflammatory (M1) phenotype reduced their AdipoR1 and AdipoR2 expression and that with alteration of exogenous 2

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proinflammatory stimuli, macrophages largely regained their AdipoR1 and AdipoR2 expression. Furthermore, APN induced AdipoR expression in human monocytes and mouse macrophages under proinflammatory (M1) conditions. We also demonstrate that polarization of macrophages into M1 and M2 phenotypes produced contrasting macrophage responses to APN. In M1 macrophages, APN induced a proinflammatory response marked by the induction of proinflammatory cytokines, TNF-a and IL-12, despite enhancing the AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine, IL-10, without affecting the AdipoR levels. Thus, APN actions in macrophages are dependent on their polarized phenotype that ultimately influences their inflammatory and atherogenic properties. MATERIALS AND METHODS Isolation of human blood monocytes Human blood monocytes were isolated from IR and insulinsensitive (IS) male Mexican-American subjects, identified by euglycemic clamp and expressed as the glucose infusion rate (GINF; IR, GINF , 4.0; IS, GINF . 7.5). The study was approved by the Institutional Review Board, and consent was obtained. From 20 ml blood, purified monocytes were by negative immunoselection by use of the RosetteSep human monocyte enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada), according to the manufacturer’s guidelines. In brief, 1 mM EDTA and 50 ml/ml RosetteSep cocktail were added to the whole blood and incubated for 20 min at room temperature. The blood was then diluted with an equal volume of PBS, supplemented with 2% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1 mM EDTA, layered on Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuged for 20 min 1200 3 g. The purified monocytes at the interface were collected, washed 2 times with PBS + 2% FBS, and suspended in RIPA buffer (SigmaAldrich, St. Louis, MO, USA) for protein analysis or TRIzol (Invitrogen, Grand Island, NY, USA) for mRNA analysis. THP-1 cells and mouse macrophages Human THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% FBS, penicillin, streptomycin, and 2 mM L-glutamine. Cells plated into 6-well plates were stimulated with 10 mg/ml recombinant fulllength human APN (EC50 5 mg/ml to induce macrophage secretion of tissue inhibitor of matrix proteinase-1; R&D Systems, Minneapolis, MN, USA) or 10 ng/ml TNF-a (R&D Systems). After incubation, cells were suspended in RIPA buffer for protein analysis or TRIzol (Invitrogen) for mRNA analysis. Bone marrow cells were isolated from femurs of male C57/Bl6 mice (The Jackson Laboratory, Bar Harbor, ME, USA). In brief, hind legs were surgically removed, and clean femurs were prepared. Bone marrow was flushed out, and the cells were collected by centrifugation. Red blood cells were depleted with ammoniumchloride-potassium lysis buffer (Gibco). After adding RPMI-1640 medium, bone marrow cells were spun down after 2 rinses, then layered on a Ficoll-Paque gradient, and after a 30 min centrifugation step (400 3 g), the interface containing the bone marrow cells was removed and suspended in a medium containing 49% DMEM (Gibco), 30% L929 supernatant, 20% FBS, and 1% penicillin and streptomycin. The cells were plated at a density of 4 million/30 ml in 15 cm cell-culture plates, and 30 ml penicillin/ streptomycin/amphotericin was added to each plate. After 3 days, cells were rinsed, and fresh medium containing either M1

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(2 ng/ml IFN-g, R&D Systems; and 10 ng/ml LPS from Escherichia coli, Sigma-Aldrich) or M2 (20 ng/ml IL-4 or 20 ng/ml IL-10; R&D Systems) was added and incubated for an additional 2 days. For the treatment with APN, 10 mg/ml recombinant full-length mouse APN (EC50 5 mg/ml; R&D Systems) was added to the M1or M2-stimulated cells at the start of the incubation. After 48 h, cells were rinsed and harvested in TRIzol for mRNA analysis. Thioglycollate-elicited macrophages were obtained from 10week-old male C57/Bl6 mice. Four days after thioglycollate (1 ml i.p.; Difco; BD Biosciences, San Jose, CA, USA) injection, peritoneal macrophages were isolated by peritoneal lavage with 10 ml PBS, centrifuged at 1400 rpm for 10 min, resuspended in RPMI1640 media supplemented with 10% FBS, and incubated in 6-well plates for 3 h. Nonadherent cells were washed, and adherent cells were stimulated for 24 h to induce M1 (2 ng/ml IFN-g and 10 ng/ml LPS from E. coli) or M2 (20 ng/ml IL-4 or 20 ng/ml IL-10) phenotype in RPMI supplemented with 10% FBS, penicillin, streptomycin, and 2 mM L-glutamine. For the washout experiments, cells were rinsed after the first 24 h, and new medium without the M1 or M2 stimuli was added and incubated further for an additional 24 h, with or without 10 mg/ml mouse APN. Cells were harvested in RIPA buffer for protein or TRIzol for mRNA isolation and gene expression analyses. Animal studies were carried out in accordance with the University of California Los Angeles Animal Research Committee guidelines.

for 2 h, spun down, and washed 2 times with 75% ethanol. After washing, mRNA pellets were left to dry for 10 min and taken up in RNase-free water. RNA was then reverse transcribed into cDNA by use of TaqMan Reverse Transcription Reagent Kit (Applied Biosystems, Foster City, CA, USA). To determine the effects of APN on inflammatory gene expression induced by TNF-a, RNA samples isolated from human THP-1 cells were converted to cDNA and hybridized to low-density, biased gene arrays (SuperArray, OHS-038; SABiosciences/Qiagen, Valencia, CA, USA) to identify TNF-a–regulated genes that were altered by APN. Chemiluminescent hybridization signals captured by autoradiography (Hyperfilm ECL; Kodak) were analyzed by use of SuperArray’s GEArray Expression Analysis Suite to identify genes whose expression levels were increased ($1.5-fold) or decreased (#0.067-fold) between the 2 samples. qRT-PCR (SYBR Green) was performed with an ABI Prism 7500 system (Applied Biosystems) in a total volume of 20 ml by use of a TaqMan PCR Core Reagent Kit (Applied Biosystems). The reaction profile was 2 min 50°C, 10 min 95°C, and 40 cycles of 15 min 95°C and 1 min 60°C, followed by 15 s 95°C, 1 min 60°C, and 15 s 95°C. Each sample was analyzed in duplicate and normalized to the value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. The primer sequences of genes used for the quantification of mRNA levels are shown in Table 1. Western blotting

Gene expression analyses Macrophage gene expression was measured by quantitative RTPCR (qRT-PCR). For RNA isolation, macrophages were harvested in TRIzol (Invitrogen), and mRNA was isolated, according to the manufacturer’s guidelines. In brief, 200 ml chloroform was added to 1 ml TRIzol (Invitrogen), and samples were shaken and left for 15 min. After centrifugation (15 min 12,000 g), the mRNAcontaining aqueous phase was taken off and transferred to a new tube, and 500 ml isopropanol was added. Samples were incubated

Cell lysates (10 mg protein) were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked (5% fat-free milk in Tris-buffered saline/0.01% Tween 20) and exposed to the primary antibodies against mouse AdipoR1 and AdipoR2, which cross-react with human AdipoRs (Alpha Diagnostics, San Antonio, TX, USA), AMPK, phospho-AMPK, p38, phospho-p38, or b-actin (Abcam, San Francisco, CA, USA) overnight at 4°C. The membrane was washed, and a secondary

TABLE 1. Sequences of real-time PCR primers used for quantification of mRNA levels Gene product

Human AdipoR1 Human AdipoR2 Human GAPDH Mouse AdipoR1 Mouse AdipoR2 Mouse GAPDH Mouse IL-12 Mouse mannose receptor (MR) Mouse IL-10 Mouse IL-6 Mouse LXRa Mouse LXRb Mouse PPARg Mouse p38 MAPK

Forward and reverse primers (59→ 39)

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

MACROPHAGE POLARIZATION PHENOTYPE AND ADIPONECTIN RECEPTORS

TTC TTC CTC ATG GCT GTG AGT GGA CAA AGG CTG CTG ATA GTC TCC CAG TGG GAC AGG ATC CGG GCA GCA TAC ATG GGG AAG GTG AAG GTC GGG GTC ATT GAT GGC AAC Mm01291331_m1 (ABI) Mm01184030_m1 (ABI) AGG TCG GTG TGA ACG GAT TGT AGA CCA TGT AGT TGA TGG TTT GCC ATC GTT TTG ACA GGT GAG GTT CAC TGT CTC TGT TCA GCT ATT GGA CGG AAT TTC TGG GAT TCA GCT CTT ACT GAC TGG CAT CGC AGC TCT AGG AGC ATG GGT GCC CTG CCA GTA TTC GGC TCC CAA CAC AGG ATG AAT GCC AGG AGT GTC GAC CTT GCC GCT TCA GTT TCT CAG GAG ATT GTG GAC TTT TTG TAG CGT CTG GCT GTT TAT GGA GTG ACA TAG AGT CCA CTT CAA TCC ACC CAG CGAAATGACCGGCTACGTGG CACTTCATCGTAGGTCAGGC

ATG T CCA ATG A G AAT A TTG GGT CTG TTC CGC GCT GAG TG TC A TT TC GC TC GTG AAA

CA T TC

CT G

3

antibody conjugated with horseradish peroxidase was added [Anti-Rabbit-HRP (R&D Systems)] for 1.5 h at room temperature. The membrane was developed with Amersham ECL Prime (GE Healthcare, Piscataway, NJ, USA), according to the manufacturer’s guidelines, and the band intensity was measured with ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). Statistical analyses All data are expressed as mean 6 SD. Statistical significance was determined by one-way ANOVA with Newman-Keuls post-test and unpaired Student’s t-test by use of GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

RESULTS AdipoR expression in IR and IS human monocytes To determine whether increased systemic inflammation as a result of IR alters AdipoR expression in human monocytes, blood monocytes were isolated from prediabetic IR and control IS subjects. The IR subjects had significantly higher body mass index (BMI; 32.5 6 1.2 vs. 22.5 6 0.4 kg/m2, P , 0.01), fasting insulin (21.3 6 1.7 vs. 9.0 6 0.4 mU/ml, P , 0.01), and plasma glucose (97.8 6 2.3 vs. 83.8 6 1.4 mg/dl, P , 0.01) levels compared with IS subjects. The IR subjects, although not diabetic, had significantly higher plasma high-sensitivity CRP levels than those in IS subjects (3.0 6 0.4 vs. 0.9 6 0.2 mg/L, P , 0.01), indicating enhanced systemic inflammatory status as a result of IR. Furthermore, plasma APN levels of IR subjects were significantly lower compared with those in the IS subjects (5.8 6 0.3 vs. 7.6 6 0.4 mg/ml, P , 0.01). To determine the impact of systemic effects of insulin resistance on monocyte AdipoR (AdipoR1 and AdipoR2) expression, mRNA levels of these receptors were measured by use of qRT-PCR. Results showed that the mRNA levels of AdipoR1 and AdipoR2 were increased significantly in IR monocytes compared with the levels in IS monocytes (Fig. 1). These results in IR monocytes were in contrast to a previous report that monocytes from coronary artery disease patients with reduced APN levels expressed reduced AdipoR1 and AdipoR2 levels (37).

Figure 1. Expression of monocyte AdipoRs, AdipoR1 and AdipoR2, is altered in IR (GINF , 4.0) subjects compared with IS (GINF . 7.5) subjects. IS is determined by euglycemic clamp and expressed as the GINF. Data are mean 6 SD, n = 5/group; *P , 0.01 vs. IS.

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TABLE 2. APN regulates inflammatory and atherogenic genes in TNF-a–treated human THP-1 monocytes

Gene name

ACE Adipocyte differentiation-related protein (ADRP) Chemokine ligand 2 CD36 antigen [CD36/fatty acid translocase (FAT)] CD44 antigen (CD44) Caspase 8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator Connective tissue growth factor (CTGF) Heparin-binding epidermal growth factor (EGF)–like growth factor EGR-1 IL-1a IL-1b IL-8 Integrin a2 Integrin a5 Oxidized low-density lipoprotein receptor-1 (LOX-1) Platelet-derived growth factor-b (PDGF-b) Prostaglandin endoperoxide synthase-1

Fold reduction vs. TNF-a

3.6 1.5 3.8 2.0 1.5 1.9 3.7 4.2 1.5 14.1 2.4 1.8 2.0 1.6 1.9 1.7 1.9

RNA isolated from human THP-1 cells treated with TNF-a (20 ng/ml) or TNF-a (20 ng/ml) + APN (10 mg/ml) was analyzed by use of gene arrays (SuperArray, OHS-038) to identify APN-repressed genes in TNF-a–treated monocytes.

These results suggest that altered systemic inflammatory status and/or APN levels in insulin-resistant subjects can potentially regulate monocyte/macrophage AdipoR expression and thus, modulate their response to APN.

APN up-regulates AdipoR1 and AdipoR2 expression in human and mouse macrophages To determine the effect of APN on AdipoR (AdipoR1 and AdipoR2) and inflammatory gene expression, human THP-1 monocytes were incubated for 24 h with APN or TNF-a, which opposes APN effects and is a critical modulator of IR in the metabolic syndrome. To identify TNFa–regulated genes altered by APN, we performed gene expression analysis by use of biased gene arrays (SuperArray, OHS-038). The data revealed that APN repressed angiotensin-converting enzyme (ACE), monocyte chemotactic protein 1, early growth response 1 (Egr-1), multiple integrins, and several proinflammatory cytokines, including IL-1b and IL-8 (Table 2). Thus, APN exerts anti-inflammatory and antiatherogenic effects in TNF-atreated THP-1 monocytes. Determination of AdipoR mRNA levels in untreated control THP-1 cells showed the expression of AdipoR1 and AdipoR2, with AdipoR1 levels relatively higher than those of AdipoR2, consistent with previous reports on human monocytes (37). Notably, we found that APN treatment significantly increased the mRNA and protein levels of AdipoR1 and AdipoR2 compared with untreated controls (Fig. 2B, C). The induction

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Figure 2. APN up-regulates AdipoR1 and AdipoR2 expression in human monocytes. A) APN-induced phosphorylation of AMPK and p38 MAPK. Cells were incubated with 5 mg/ml APN for 30 min, and cell lysates were separated by SDS-PAGE and immunoblotted with antibodies to phospho-AMPK and phospho-p38 MAPK. B) AdipoR1 and AdipoR2 mRNA levels in human THP-1 monocytes treated with 10 mg/ml APN or 10 ng/ml TNF-a. Data are mean 6 SD, n = 5/group; *P , 0.01 vs. control; **P , 0.01 vs. TNF-a. C) Western blot analysis of AdipoR1 and AdipoR2 protein expression in human THP-1 monocytes treated with APN or TNF-a. APN signaling in human THP-1 monocytes.

of AdipoR2 (80% increase) expression was more prominent than that of AdipoR1 (50% increase; Fig. 2B). In contrast to APN, TNF-a suppressed the mRNA levels of AdipoR1 and AdipoR2 compared with the levels in untreated cells (Fig. 2B). Although TNF-a significantly suppressed the AdipoR1 protein levels, this effect was not seen in the case of AdipoR2 (Fig. 2C). To determine cellular signaling mediated by APN in human THP-1 monocytes, AMPK and p38 phosphorylation were analyzed by Western blot analysis. As shown in Fig. 2A, APN treatment increased the levels of phospho-AMPK and phospho-p38 in THP-1 monocytes. These data demonstrate that APN enhances, whereas TNF-a suppresses, AdipoR1 and AdipoR2 mRNA expression in human monocytes. To confirm further that the APN-mediated upregulation of AdipoR1 and AdipoR2 expression is not restricted to human monocytes but also occurs in mouse macrophages, we performed additional studies by use of thioglycollate-elicited mouse peritoneal macrophages stimulated for 24 h with APN. As in human THP-1 cells, APN treatment significantly increased AdipoR1 and AdipoR2 mRNA and protein levels in mouse peritoneal macrophages (Fig. 3A, B). As with human monocytes, TNF-a treatment significantly suppressed AdipoR1 mRNA and protein levels (Fig. 3A, B) in mouse macrophages. In the case of AdipoR2, TNF-a significantly inhibited AdipoR2 mRNA levels but not the protein levels (Fig. 3A, B). These results demonstrate that APN induces the expression of its own receptors, AdipoR1 and AdipoR2, whereas

proinflammatory cytokine, TNF-a, attenuates its mRNA expression in monocyte/macrophages. Next, we investigated mechanisms of APN-induced AdipoR expression in macrophages by determining APN modulation of PPARs and LXRs, the key transcriptional factors that integrate multiple aspects of inflammation and metabolism (40, 41). As LXR activation regulates AdipoRs in monocyte/macrophages (36), we examined whether APN regulated LXRs in bone marrow macrophages. Our studies revealed that APN significantly induced LXRa and LXRb levels in macrophages, which is consistent with the upregulation of AdipoR expression (Fig. 3C). Thus, APN induction of LXRs is at least one mechanism by which APN up-regulates AdipoR levels in macrophages. Macrophage polarization differentially regulates the expression of AdipoR1 and AdipoR2 Both inflammatory status and low APN levels, associated with the metabolic syndrome, diabetes, and atherosclerosis, can profoundly influence macrophage inflammatory phenotype and potentially alter the status of AdipoR expression. Thus, we investigated whether the expression of AdipoRs is differentially regulated by macrophagepolarized phenotypes. First, bone marrow–derived macrophages, isolated from C57/Bl6 mice and cultured for 7 days, were differentiated into M1 macrophages with IFN-g and LPS or M2 macrophages with IL-4 or IL-10. The

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Figure 3. Effect of APN on AdipoR1 and AdipoR2 expression in mouse peritoneal macrophages. A) mRNA levels of AdipoR1 and AdipoR2 in mouse peritoneal macrophages treated with 10 mg/ml APN or 10 ng/ml TNF-a for 24 h. Data are mean 6 SD, n = 5/group; *P , 0.01 vs. control; **P , 0.01 vs. TNF-a. B) Western blot analysis of AdipoR1 and AdipoR2 protein levels in mouse peritoneal macrophages treated 10 mg/ml APN or 10 ng/ml TNF-a for 24 h. C) mRNA levels of LXRa and LXRb in mouse macrophages treated with 10 mg/ml APN or 10 ng/ml TNF-a for 24 h. Data are mean 6 SD, n = 5/group; *P , 0.01 vs. control and TNF-a.

M1-polarized macrophages expressed increased levels of the M1 marker, IL-12p40 mRNA, whereas the mRNA levels of the M2 marker, MR (42), were largely suppressed compared with those in control macrophages. In the IL-4– and IL-10–derived M2 macrophages, there was a significant increase in MR mRNA levels and a substantial suppression of IL-12 mRNA compared with the levels in control macrophages (Fig. 4). Interestingly, AdipoR1 and AdipoR2 mRNA levels were reduced significantly in M1 macrophages compared with those in control macrophages (Fig. 4). However, in the two M2 macrophage subsets, the levels of AdipoR1 and AdipoR2 were significantly higher than in M1 macrophages, although the levels in M2 macrophages were significantly lower than in control macrophages (Fig. 4). These results demonstrate that M1 macrophage activation results in the suppression of AdipoR expression, whereas M2 macrophage activation results in the maintenance of AdipoR levels, although the levels are somewhat lower than in control macrophages. Thus, our studies identify that macrophage polarization is an important regulator of AdipoR expression, which can potentially influence macrophage functional responses to APN. Because bone marrow–derived macrophages are considered relatively immature compared with other tissue macrophages, we also investigated whether similar macrophage activation phenotypes can be induced in mature macrophages. Thus, thioglycollate-elicited peritoneal macrophages, isolated from C57/Bl6 mice, were induced into M1 with IFN-g and LPS or M2 with IL-10 for 24 h. Remarkably, the M1-activated peritoneal macrophages showed substantial up-regulation of IL-12 mRNA levels with concomitant suppression of MR mRNA levels 6

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compared with control macrophages, as shown in M1 bone marrow-derived macrophages (Fig. 5A). In contrast, the M2-activated peritoneal macrophages showed almost complete suppression of IL-12 and significant upregulation of MR levels. As in bone marrow–derived macrophages (Fig. 4), AdipoR1 and AdipoR2 mRNA was down-regulated in M1-activated peritoneal macrophages compared with the levels in control macrophages (Fig. 5A). In M2-activated peritoneal macrophages, AdipoR1 and AdipoR2 levels were significantly higher than those in M2. Remarkably, AdipoR2 levels were significantly upregulated in M2-activated peritoneal macrophages beyond the level in control macrophages (Fig. 5A). Determination of AdipoR1 and AdipoR2 protein levels revealed similar pattern consistent with mRNA levels (Fig. 5B). Notably, in our studies with human and mouse macrophages, we found a tight correlation between mRNA and protein levels of AdipoR1 and AdipoR2. This consistent correlation between mRNA and protein levels suggests a wellcoordinated mRNA transcription and protein translation of AdipoRs in macrophages. Overall, our results from bone marrow macrophages and activated peritoneal macrophages demonstrated that polarized activation of macrophages alters their functional phenotype, differentially regulating AdipoR1 and AdipoR2 levels. Effect of exogenous cytokine environment on AdipoR expression and macrophage polarization status Macrophages shift their functional activation patterns in response to different cytokine environments (18, 43, 44).

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Figure 4. Macrophage-polarized activation regulates the expression of AdipoR1 and AdipoR2. Bone marrow-derived macrophages were differentially activated into M1 (IFN-g/LPS) or M2 (IL-4 or IL-10) for 24 h, and mRNA levels of macrophage polarization markers, IL-12 and MR, as well as AdipoR1 and AdipoR2, were measured by qRT-PCR. Data are mean 6 SD, n = 5; *P , 0.05 vs. control; **P , 0.05 vs. M1. Representative of 3 experiments. ND, Not detected.

Thus, we determined whether alteration of the cytokine environment, inducing M1 and M2 macrophage polarization, displays stability or changes the functional phenotype and AdipoR expression. Peritoneal macrophages treated for 24 h with M1 (IFN-g and LPS) or M2 cytokines (IL-10) were washed free of the cytokines and incubated further for 24 h in cytokine-free medium in the presence or absence of APN. Upon cytokine removal, M1-pretreated macrophages expressed significantly reduced M1 marker, IL-12, mRNA levels than in control macrophages but relatively elevated levels compared with M2-pretreated macrophages (Fig. 6). Notably, the M2 marker, MR expression in the M1-pretreated macrophages, increased compared with control macrophages, indicating a reversal of the M1 to M2 phenotype. In contrast, the M2 phenotype in M2pretreated macrophages was maintained with low IL-12 and high MR expression. These data demonstrate that M1 macrophages are more responsive to external triggers than M2 macrophages as primary responders to acute inflammation. Interestingly, AdipoR1 and AdipoR2 mRNA levels in M1-pretreated macrophages were increased significantly compared with control macrophages, indicating restoration of AdipoR levels comparable with those in M2-pretreated macrophages (Fig. 6). However, in M2pretreated macrophages, the AdipoR expression remained essentially similar to that in the presence of cytokines. Surprisingly, APN had no significant effect on the reversal of the M1 to M2 phenotype or changes in AdipoR expression induced by cytokine removal (Fig. 6).

These results reveal that depletion of the M1 stimulus exerts a significant effect on AdipoR expression, whereas depletion of the M2 stimulus has only a minimal impact on AdipoRs in macrophages. APN induces proinflammatory response and AdipoR expression in M1-polarized macrophages APN exerts anti-inflammatory effects in TNF-a–treated monocytes, as we have shown in this study (Table 2) and up-regulated AdipoR1 and AdipoR2 expression in macrophages (Figs. 2, 3, and 6). Thus, we addressed the important question of whether a differential macrophage polarization phenotype alters the nature of APN actions. Bone marrow–derived macrophages polarized to induce the M1 or M2 phenotype were incubated with or without APN. Surprisingly, APN induced a strong proinflammatory effect in M1-polarized macrophages by substantially elevating the expression of the inflammatory cytokine TNF-a, IL-6, and IL-12 levels compared with control macrophages (Fig. 7A). In contrast, APN induced an anti-inflammatory effect in M2 macrophages, inducing IL-10 but not proinflammatory cytokines (Fig. 7A). These data demonstrate that differential macrophage polarization phenotypes can produce divergent APN responses. These results provide the first evidence that APN produces a strong proinflammatory response in the context of M1 macrophages by marked induction of TNF-a, IL-6, and IL-12 levels

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Figure 5. Regulation of AdipoR expression in differentially polarized mouse peritoneal macrophages, which were activated into M1 (IFN-g/LPS) or M2 (IL-4 or IL-10) for 24 h, and mRNA levels of IL-12, MR, AdipoR1, and AdipoR2 were measured by qRTPCR. Data are mean 6 SD, n = 5; *P , 0.001 vs. Control; **P , 0.001 vs. M1. B) Western blot analysis of AdipoR1 and AdipoR2 protein levels in mouse peritoneal macrophages treated with 10 mg/ml APN or 10 ng/ml TNF-a for 24 h. Data are representative of 3 experiments.

(Fig. 7A). Furthermore, APN induced AdipoR1 and AdipoR2 expression in M1 macrophages, and by comparison, the induction of AdipoR1 was greater than AdipoR2 (Fig. 7A). In the presence of APN, AdipoR1 levels in M1 macrophages were restored almost to the levels of control macrophages. However, APN did not mitigate IL-12 levels in M1 macrophages. In contrast, APN treatment had no effect on AdipoR expression in M2 macrophages (Fig. 7A). Taken together, these results provide evidence that although APN restores AdipoR1 and AdipoR2 levels suppressed by M1 polarization of macrophages, it paradoxically produces a potent proinflammatory cytokine response. In contrast, in M2 macrophages, APN had no major effect on AdipoR1 and AdipoR2 expression but promoted an anti-inflammatory phenotype. Thus, a polarized activation status of macrophages is an important determinant controlling their inflammatory functions in response to APN. Next, to investigate mechanisms responsible for AdipoR repression in M1 and maintenance in M2 macrophages, we determined whether macrophage polarization differentially controls the transcriptional regulators LXRs and PPARg, which regulate AdipoRs. Our studies revealed that M1 macrophages display substantial (.70%) suppression of LXRa and LXRb compared with M2 and control macrophages (Fig. 7B). Likewise, PPARg levels in M1 macrophages were also largely suppressed (.80%) compared with those in M2 or control macrophages (Fig. 7B). As 8

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p38 MAPK activation regulates an APN-mediated inflammatory cytokine response, we determined the effect of macrophage-polarized activation on p38 MAPK expression. In M2 macrophages, p38 MAPK mRNA levels were suppressed significantly compared with M1 or control macrophages (Fig. 7C). These results suggest that suppression of LXRs and PPARg and relatively high levels of p38 MAPK in M1 macrophages, which enhance the macrophage inflammatory status, is at least one mechanism by which AdipoRs are suppressed in M1 macrophages. In contrast, M2 macrophages maintained higher LXR and PPARg and reduced p38 MAPK expression, contributing to prevalence of relatively higher AdipoR levels compared with those in M1 macrophages (Fig. 7A). DISCUSSION Macrophages are an essential component of innate immunity and exhibit functional heterogeneity in response to diverse tissue signals by expressing a spectrum of activation states of which M1 and M2 represent the opposite and distinct polarization phenotypes (19–21). The reprogramming involving their transition from an inflammatoryactivation state to a suppressive-tolerant state is regulated by transcription factors and epigenetic modifications (43, 45). Although APN activates distinct signaling pathways downstream of AdipoR1 and AdipoR2 to modulate

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Figure 6. Exogenous M1/M2 cytokine environment alters macrophage inflammatory status and AdipoR expression. Mouse peritoneal macrophages were activated into M1 (IFN-g/LPS) and M2 (IL-10) for 24 h, after which, the inducer media were removed, and the cells were incubated further for an additional 24 h in the presence or absence of 10 mg/ml APN without the M1/M2 inducer cytokines. IL-12, MR, AdipoR1, and AdipoR2 mRNA levels were measured by qRT-PCR. Data are mean 6 SD, n = 6/group; *P , 0.01 vs. control.

inflammation in monocyte/macrophages, it does not sufficiently explain various pleiotropic actions of APN (46). Beyond APN levels, control of AdipoR expression provides yet another level regulating the biologic actions of APN (47, 48). In this study, we demonstrated that macrophage polarization is an important regulator of AdipoR expression leading to suppression of AdipoR1 and AdipoR2 levels in M1 macrophages (Fig. 8). Furthermore, we have shown that low AdipoR expression displayed in M1 macrophages is reversible by changing the cytokine environment. Importantly, results from this study demonstrated that APN restores AdipoR expression in M1 macrophages even under proinflammatory (M1) conditions. Our studies with human monocytes from IR subjects revealed increased AdipoR1 and AdipoR2 expression in the presence of elevated systemic inflammation, as indicated by elevated CRP levels and relatively low APN levels. This suggests that factors, including APN and inflammatory mediators, can potentially modulate AdipoR expression in monocytes. Previous studies with human monocytes found increased AdipoR expression in obese subjects compared with normal subjects and found no correlation between plasma APN and monocyte AdipoR levels (38). In this study, we compared monocyte AdipoR expression between IR subjects with elevated CRP levels and IS control subjects. High CRP levels are inversely correlated with APN levels (8). Although CRP levels were higher in our IR subjects, their APN levels were reduced only modestly compared with IS subjects. The paradoxical

increase in monocyte AdipoR expression in IR suggests a more complex pattern of AdipoR regulation under prediabetic conditions. Nevertheless, in the context of AdipoR regulation by APN and proinflammatory conditions, we showed that in human THP-1 monocytes, APN upregulates, whereas TNF-a, a key proinflammatory cytokine, down-regulates mRNA expression of AdipoR1 and AdipoR2. We extended these studies further to a mouse macrophage system to expand the physiologic relevance of these effects in macrophages. Our results confirmed that as in human THP-1 monocytes, APN increases AdipoR1 and AdipoR2 expression in mouse macrophages. Thus, this evidence supports the role of APN in regulating its own receptor levels in human and mouse monocyte/ macrophages. In all of these cell types, we found a consistent correlation between AdipoR1 and AdipoR2 mRNA and protein levels, suggesting that their expression is wellcoordinated at both transcriptional and translation levels. Furthermore, our microarray studies by use of human THP-1 monocytes under a proinflammatory condition revealed that APN exerts an anti-inflammatory effect to inhibit several TNF-a–induced inflammatory and atherogenic genes (Table 2). This is consistent with previous in vitro (9, 49–51) and in vivo (10, 52) anti-inflammatory roles of APN. Because macrophages acquire functionally distinct proor anti-inflammatory-polarized phenotypes (18, 19), we posited that this intrinsic property of macrophages can potentially control their AdipoR expression, modulating

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Figure 7. Macrophage polarization differentially regulates AdipoR1 and AdipoR2 expression and inflammatory cytokine expression in response to APN. Bone marrow macrophages differentially activated into the M1 (IFN-g/LPS)- or M2 (IL-4 or IL10)-polarized phenotype were incubated in the presence or absence of 10 mg/ml APN for 48 h. A) IL-12, MR, TNF-a, IL-6, IL-10, AdipoR1, and AdipoR2. B) LXRb, LXRa, and PPARg. C) p38 MAPK mRNA levels were measured by qRT-PCR. Data are mean 6 SD, n = 6/group; *P , 0.05 control vs. APN-treated macrophages.

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Figure 8. Macrophage polarization regulates AdipoR expression and an APN-mediated inflammatory response. This study provides important evidence that differential polarization of macrophages into M1 or M2 phenotype profoundly alters their AdipoR (AdipoR1 and AdipoR2) expression, leading to divergent inflammatory responses to APN. Profound suppression of LXRs and PPARg, accompanied by activation of p38 MAPK in M1 macrophages, contributes to low AdipoR levels. Relatively high and sustained LXRs and PPARg levels and IL-10 accompanied by reduced p38 MAPK levels contribute to preservation of AdipoR expression in M2 macrophages. APN-mediated up-regulation of AdipoR expression in M1 macrophages is accompanied by marked induction of proinflammatory cytokines, whereas in M2 macrophages, APN increased the anti-inflammatory response without affecting AdipoR expression.

functional responses to APN. Previous studies with human monocyte-derived macrophages have shown that the expression of AdipoRs is reduced during differentiation, and the expression is induced by nuclear hormone receptor (LXR and PPAR) ligands (36). However, regulation of AdipoRs in macrophages under pro- and antiinflammatory activation has not been investigated. In this study, with the use of two distinct mouse macrophage systems—bone marrow-derived macrophages and relatively more activated mouse peritoneal macrophages—we have provided evidence supporting our hypothesis that macrophage polarization controls AdipoR2 and AdipoR2 expression. Importantly, we demonstrated that AdipoR1 and AdipoR2 levels in M1 and M2 macrophages are polarization phenotype dependent, with M1 polarization suppressing AdipoR1 and AdipoR2 levels. Interestingly, we also found that APN treatment largely restored AdipoR levels of M1-polarized macrophages compared with those in nonpolarized macrophages, even in the presence of M1 cytokines. We also found that a potential mechanism by which APN restored AdipoR expression in macrophages is through up-regulation of LXRa, which is a key transcriptional regulator of inflammation and metabolism (53). Further studies to determine cellular pathways linking APN-LXRa-AdipoR axis are required and would provide molecular insight into AdipoR regulation. In contrast to M1 macrophages, M2 macrophages maintained higher AdipoR levels compared with nonpolarized macrophages and exhibited no AdipoR up-regulation in response to APN. Although mechanisms responsible for this effect remain to be fully investigated, a potential mechanism may involve inhibition of p38 MAPK by IL-10 induced by APN. Nevertheless, we found that APN did not induce LXRa and LXRb in M2 macrophages and that their expression

levels were significantly higher than in M1 macrophages. Previous studies have reported that APN induces M2 polarization-inducing expression of M2 markers in human and mouse macrophages (14, 16). In contrast, a recent study using human macrophages found that APN does not promote M2 macrophage polarization but instead, induces a proinflammatory response (54). In this study, APN treatment of bone marrow and peritoneal macrophages produced no significant change in the expression of the M1 (IL-12) or M2 (MR and IL-10) marker. Nevertheless, in both types of macrophages, APN consistently induced the expression of AdipoR1 and AdipoR2. Thus, despite no significant changes in macrophage polarization markers in our study, APN significantly increased AdipoR expression in bone marrow–derived and peritoneal macrophages. The ability of macrophages to maintain pro- and antiinflammatory homeostasis is of critical importance to regulate inflammatory response. The clearance/suppression of proinflammatory cytokines in the system thus plays a critical role to maintain this balance. In this study, we demonstrated that an altered cytokine microenvironment allows macrophages to display a distinct, functional M1 and M2 pattern. Our results showed that cytokine depletion from the environment markedly affected M1 macrophages by suppressing IL-12 production and up-regulating MR expression, whereas this had no effect on M2 macrophages. Clearly, M1 macrophages respond to their environmental signals to reverse their phenotype more readily than M2 macrophages. This rapid response of proinflammatory (M1) macrophages to their microenvironment is critical for the homeostatic macrophage function controlling the inflammatory response preventing tissue damage. Interestingly, after cytokine depletion, AdipoR1

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and AdipoR2 levels in M1 macrophages were increased to levels comparable with M2 macrophages. Although APN consistently induced AdipoR1 and AdipoR2 expression in nonpolarized macrophages, it had no impact on the receptor expression in the M1 or M2 macrophage, beyond the levels restored as a result of M1/M2 cytokine depletion. Thus, in M1-polarized macrophages, either inhibiting proinflammatory stimuli or increasing APN levels can compensate for reduced AdipoR expression under proinflammatory activation. Overall, these data support the notion that proinflammatory suppression of AdipoR levels in macrophages is reversible and that interventions to inhibit or neutralize inflammatory cytokines in tissues can restore and even up-regulate the AdipoR expression. Based on studies that showed APN inhibition of proinflammatory cytokines release in activated monocyte/ macrophages, APN was considered an anti-inflammatory molecule (9, 49, 51, 55). The anti-inflammatory effects of APN in human monocytes were shown to be independent of the anti-inflammatory cytokine, IL-10 induction (49). Nevertheless, APN is known to induce inflammatory cytokines in monocyte/macrophages and thus, considered to be a modulator of macrophage immune function (54–57). Paradoxically, increased APN levels have been reported in chronic inflammatory and autoimmune disorders, such as rheumatoid arthritis and systemic lupus erythematosus, and it is unclear whether APN exerts differential effects depending on the inflammatory context (58–60). In this study, we showed that APN exerts a strong proinflammatory response by inducing IL-12, IL-6, and TNF-a, preferentially in M1 macrophages. Notably, this proinflammatory APN response was absent in nonpolarized and M2 macrophages. Thus, these results provide novel evidence that APN preferentially exerts robust proinflammatory effects in M1 macrophages, whereas it induces anti-inflammatory IL-10 in M2 macrophages. One potential mechanism responsible for the strong APN inflammatory response in M1 macrophages appears to be the suppression of anti-inflammatory factors, such IL-10, PPARg, and LXRs, as we have shown, which inhibit p38 MAPK. The absence of a proinflammatory response in M2 macrophages appears to be linked to IL-10 induction, which suppresses MAPK signaling pathways. Recently, APN has been shown to promote inflammatory activation of human macrophages by induction of mostly proinflammatory rather than anti-inflammatory genes and macrophage tolerance to limit inflammation. This notion is supported by the ability of APN to induce antiinflammatory factors, including A20, suppressor of cytokine signaling 3, and TNF receptor-associated factor 1 (49). In the present study, we provide the first evidence that the macrophage polarization status is an important determinant controlling the proinflammatory response of APN. The differential inflammatory responses of macrophages to APN are determined by their functional phenotype, as well as the context of their inflammatory status (Fig. 8). As resolution of inflammation requires the induction anti-inflammatory factors to promote macrophage tolerance, robust but acute induction of TNF-a, as we have shown in this study, appears to be one potential mechanism to promote tolerance in M1 macrophages. Recently, TNF-a has been shown to play a regulatory function in macrophage tolerance (61). 12

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Our studies provide important evidence that macrophage polarization contributes to differential AdipoR expression and contrasting inflammatory responses to APN. Remarkably, in M1-polarized macrophages, APN restores AdipoR expression but induces proinflammatory responses marked by the induction of proinflammatory cytokines, TNF-a, IL-6, and IL-12 (Fig. 8). Overall, these results provide the first evidence that macrophage polarization is a key determinant regulating the AdipoR expression and differential APN-mediated inflammatory responses that can profoundly influence their pathogenic role in inflammatory and cardiometabolic disorders. The authors thank Dr. Peter Tontonoz for helpful discussion and Dr. Joey Liu, Diana Becerra, and Rima Boyajdian, for technical assistance. This work was supported by U.S. National Institutes of Health Grant R01-HL086566 (to R.K.T.); and from Dr. Alan Fogelman, Department of Medicine, David Geffen School of Medicine at University of California Los Angeles. The authors also acknowledge support from the Carl and Roberta Deutsch Family Foundation.

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Received for publication March 20, 2014. Accepted for publication October 1, 2014.

VAN STIJN ET AL.

Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response.

Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), A...
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