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. 2013 Dec;94(6):1309–1315. doi: 10.1189/jlb.0113043

Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages

Balázs Koscsó *, Balázs Csóka *, Endre Kókai *,†, Zoltán H Németh ‡, Pál Pacher §, László Virág †, S Joseph Leibovich ‖, György Haskó *,†,¶,1
PMCID: PMC3828607  PMID: 23922379

Adenosine signaling augments IL-10-induced STAT-3 dependent gene expression in macrophages, suggesting its role in M2c polarization.

Keywords: arginase, TIMP-1, alternative activation

Abstract

The alternatively activated macrophage phenotype induced by IL-10 is called M2c. Adenosine is an endogenous purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances, hypoxia, inflammation, physical damage, or apoptosis. As adenosine is known to regulate classically activated M1 and IL4- and IL-13-activated M2a macrophages, the goal of the present study was to explore its effects on M2c macrophages. We found that adenosine augmented the IL-10-induced expression of TIMP-1 and arginase-1 by the mouse macrophage cell line RAW 264.7 and by mouse BMDMs. The effects of AR stimulation on IL-10-induced TIMP-1 or arginase-1 expression were lacking in A2BAR KO macrophages. The role of A2BAR on TIMP-1 production of RAW 264.7 cells was confirmed with specific agonist BAY606583 and antagonist PSB0788. AR stimulation augmented IL-10-induced STAT3 phosphorylation in macrophages, and pharmacological inhibition or silencing of STAT3 using siRNA reduced the stimulatory effect of AR stimulation on TIMP-1 production. In contrast to its stimulatory effect on IL-10-induced STAT3 activation, adenosine inhibited IL-6-induced STAT3 phosphorylation and SAA3 expression. In conclusion, adenosine enhances IL-10-induced STAT3 signaling and M2c macrophage activation.

Introduction

Macrophages acquire particular phenotypes depending on the activation signals and cytokine environment to which they are exposed. The M2c macrophage phenotype arises in the presence of the anti-inflammatory cytokine IL-10 [1]. The M2c phenotype is characterized by low expression of inflammatory cytokines, such as IL-12 or TNF-α, poor ability for antigen presentation and phagocytosis, and high expression of anti-inflammatory mediators, such as IL-10 and TGF-β [2]. M2c macrophages participate in immunoregulation, matrix deposition, and tissue remodeling [3, 4]. TIMP-1 is a prominent marker that is up-regulated during M2c macrophage activation [5, 6]. TIMP-1 inhibits matrix metalloproteinases and thus, decreases inflammatory tissue destruction [7, 8]. SOCS-3, IL-1 receptor antagonist, BCL-3, and arginase-1 are also up-regulated in M2c macrophages. Increasingly, activation of multiple markers is used to identify M2c macrophages unequivocally in the context of responses to different antigens and environments [2].

IL-10 binds to the IL-10R, which is composed of two chains: IL-10R1 and IL-10R2 [9]. IL-10 binding to the receptor activates the tyrosine kinase JAK1; this process leads to the phosphorylation of transcription factor STAT3. STAT3 then dimerizes, enters the nucleus, and activates the transcription of anti-inflammatory genes [10]. IL-10 also signals through the PI3K pathway [9, 11]. The STAT3 pathway is also activated by IL-6; however, IL-6-induced STAT3 activation leads to the transcription of proinflammatory genes, including that of acute phase proteins [12]. SAA denotes a family of acute-phase proteins that are released in an inflammatory environment and are used as biomarkers of inflammation [13, 14]. SAAs are involved in atherosclerosis by promoting plaque formation and recruiting macrophages [14, 15] and rheumatoid arthritis by stimulating the production of inflammatory cytokines and cartilage-degrading proteinases [16–18]. In contrast to other members of the family, which are produced mostly by hepatocytes, SAA3 is produced by nonhepatic cells, such as macrophages [19] and adipocytes [20]. SAA3 functions as a chemotactic agent for phagocytes, and its level is up-regulated in metastatic lung cancer [21] and in the adipose tissue of obese mice [20].

Adenosine is a purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances and other types of insults, which include inflammation, physical damage, and apoptosis [22–24]. The cellular effects of adenosine are mediated by four GPCRs: the A1-, A2A-, A2B-, and A3AR [25]. Adenosine decreases the expression of proinflammatory cytokines, such TNF-α and IL-12; chemokines, such as MIP-1α; and the release of NO by classically activated or M1 macrophages [26–29]. We have shown recently that adenosine enhances IL-4- or IL-13-induced M2a macrophage activation [30]. However, the role of AR signaling in M2c macrophages has not been explored. Thus, the goal of present study was to characterize further the effect of adenosine on M2 macrophage polarization by examining the role of AR stimulation in controlling IL-10-induced M2c macrophages.

MATERIALS AND METHODS

Drugs and reagents

Adenosine was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The selective A2BAR agonist BAY606583 was a kind gift from Dr. Holger Eltzschig (University of Colorado, Denver, CO, USA). The nonselective AR agonist NECA, selective A2AAR agonist CGS21680, selective A2AAR antagonist 4-{2-[7-amino-2-(2-furyl)[1.2.4]triazolo[2.3-a][1.3.5]-triazin-5-ylamino] ethyl} phenol, selective A2BAR antagonist PSB0788, and JAK/STAT inhibitors AG490, ZM39923, and cucurbitacin were purchased from Tocris Cookson (Ellisville, MO, USA). The p38 MAPK pathway inhibitor SB203580, and PI3K inhibitor LY294002 were purchased from Calbiochem (San Diego, CA, USA). IL-6 and IL-10 were purchased from PeproTech (Rocky Hill, NJ, USA).

Experimental animals and cell cultures

A2AAR and A2BAR KO mice on the C57BL/6J genetic background were bred as described previously [31]. All mice were maintained in accordance with the recommendations of the “Guide for the Care and Use of Laboratory Animals”, and the experiments were approved by the Institutional Animal Care and Use Committee of the New Jersey Medical School.

BMDMs were isolated from femurs and tibias of WT, A2AAR, or A2BAR KO mice. BMs were triturated with a 26-gauge needle and passed through a 70-μm nylon mesh cell strainer. Cells were cultured in DMEM, supplemented with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 50 ng/ml M-CSF (PeproTech) for 7 days, during which period culture medium was replaced once on Day 3. BMDMs were scraped in cold 0.1% EDTA-PBS solution, and then, the cells were counted and placed in 96-well cell-culture plates at 2 × 105 cells/well.

Peritoneal macrophages were isolated and cultured as described previously [32]. RAW 264.7 cells (American Type Culture Collection, Manassas, VA, USA) were cultured as described previously [32].

ELISA for determining TIMP-1 production

RAW 264.7 cells or BMDMs placed in the wells of 96-well plates were treated with adenosine or AR agonists, followed immediately by the addition of IL-10 (10 ng/ml) for 6 or 24 h, after which period, the supernatants were frozen and stored. AR antagonists or inhibitors were administered 30 min before NECA and IL-10. TIMP-1, TNF-α, or IL-12 levels in supernatants of RAW 264.7 cells or BMDMs were determined using the ELISA DuoSet kit (R&D Systems, Minneapolis, MN, USA).

RNA extraction, cDNA synthesis, and real-time PCR

Total RNA was prepared from cells using the RNeasy Mini Kit, according to the manufacturer's protocol (Qiagen, Valencia, CA, USA). RT and real-time PCR were performed as described previously [30]. The following primers were used: TIMP-1 5′-TCCTCTTGTTGCTATCACTGATAGCTT-3′ (forward), 5′-CGCTGGTATAAGGTGGTCTCGTT-3′ (reverse); arginase-1 5′-CAGAAGAATGGAAGAGTCAG-3′ (forward), 5′-CAGATATGCAGGGAGTCACC-3′ (reverse); SAA3 5′-TGCCATCATTCTTTGCATCTTGA-3′ (forward), 5′-CCGTGAACTTCTGAACAGCCT-3′ (reverse); SOCS-3 5′-AGCTCCAAAAGCGAGTACCA-3′ (forward), 5′-TGACGCTCAACGTGAAGAAG-3′ (reverse); BCL-3 5′-GACCTGGAGGTTCGCAATTA-3′ (forward), 5′-CACCATGTTCAGGCTGTTGT-3′ (reverse); 18S 5′-GTAACCCGTTGAACCCCATT-3′ (forward), 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse).

Gene silencing using siRNA

RAW 264.7 cells were transfected with STAT3-specific and NT siRNA using the Amaxa Nucleofector (Lonza, Basel, Switzerland), as described in the manufacturer's protocol, and the cells were incubated for 24 h. ON-TARGETplus SMARTpool STAT3 siRNA and ON-TARGETplus NT pool were purchased from Dharmacon (Lafayette, CO, USA).

Western blot

Protein isolation from RAW 264.7 cells and Western blotting was performed, as described previously [33]. The membranes were probed with rabbit anti-mouse primary mAb raised against STAT3, phospho-STAT3, JAK1, phospho-JAK-1, JAK2, phospho-JAK2, JAK3, or phospho-JAK3 (Cell Signaling Technology, Danvers, MA, USA). Thereafter, the membranes were incubated with a secondary HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated polyclonal goat anti-β-actin antibody, to assess equal loading, was used from Santa Cruz Biotechnology. Bands were detected using chemiluminescent HRP detection reagent (Denville Scientific, South Plainfield, NJ, USA). X-ray films were exposed for 1–15 min.

Statistical analysis

Values in the figures are expressed as mean ± the sem of the indicated number of observations. Statistical analyses of the data were performed using Student's t-test or one-way ANOVA, followed by Dunnett's test, as appropriate.

RESULTS

Adenosine augments TIMP-1 production by M2c macrophages through an A2BAR-mediated process

To assess the effect of adenosine on M2c macrophages, we treated RAW 264.7 macrophages with adenosine and/or IL-10 and determined TIMP-1 concentrations from the supernatants using ELISA. Our results showed that whereas adenosine and IL-10 alone augmented basal TIMP-1 production slightly, the combination of adenosine and IL-10 induced a synergistic and marked augmentation of TIMP-1 production (Fig. 1A). To determine which AR is responsible for this effect, we next treated RAW 264.7 cells with NECA, CGS21680, or BAY606583, together with IL-10. We found that NECA and BAY606583 were more efficacious than CGS21680 in augmenting TIMP-1 production in conjunction with IL-10 (Fig. 1B). To further study the role of A2 receptors, we treated BMDMs isolated from WT, A2AAR, or A2BAR KO mice with NECA or NECA and IL-10. NECA and IL-10 markedly augmented TIMP-1 production by macrophages from WT and A2AAR KO mice but failed to do so by macrophages obtained from A2BAR KO mice (Fig. 1C). In addition, pretreatment of RAW 264.7 cells with PSB0788 inhibited the augmenting effect of NECA and IL-10 on TIMP-1 secretion (Fig. 1D), further implicating the A2B receptor. Next, we tested if IL-10 treatment had any effect on the release of proinflammatory cytokines and markers of classically activated macrophages, TNF-α and IL-12. We found that treatment with IL-10 alone or with IL-10 and adenosine failed to induce the release of TNF-α and IL-12 in macrophages (data not shown).

Figure 1. Effect of AR agonists and IL-10 on TIMP-1 production by macrophages.

Figure 1.

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TIMP-1 production by RAW 264.7 cells treated with 100 μM adenosine (ado; A), 0.1 μM NECA, CGS21680, or BAY606583 (B), and/or 10 ng/ml IL-10 for 24 h. *P < 0.05, **P < 0.01 versus vehicle #P < 0.05; ###P < 0.001 versus IL-10. ve, Vehicle. (C) TIMP-1 production by BMDMs isolated from WT, A2AAR, or A2BAR KO mice and treated with 1 μM NECA and/or 10 ng/ml IL-10 for 6 h. *P < 0.05, ***P < 0.001. (D) TIMP-1 production by RAW 264.7 cells treated with increasing concentrations of PSB0788, 30 min before treatment with 1 μM NECA and 10 ng/ml IL-10 for 24 h. ###P < 0.001 versus vehicle (NECA+IL-10). All results (mean±sem) are representative of at least three independent experiments (n=5 in each experiment).

Effect of AR stimulation on gene expression in IL-10-treated macrophages

We next assessed the effect of AR stimulation on gene expression of several M2c markers. Similar to its effect of TIMP-1 protein, adenosine augmented TIMP-1 mRNA expression in IL-10-treated macrophages (Fig. 2A). AR stimulation also enhanced arginase-1 mRNA expression; the effect was enhanced markedly in IL-10-treated cells (Fig. 2B). In addition, AR stimulation augmented arginase-1 mRNA levels in WT and A2AAR KO macrophages but failed to do so in A2BAR KO macrophages (Fig. 2C), again implicating the A2BAR. NECA failed to augment significantly the expression of IL-10-induced SOCS3 in WT, A2AAR KO, or A2BAR KO macrophages (Fig. 2D) or the expression of IL-10-induced BCL-3 (Fig. 2E), indicating that AR stimulation does not up-regulate gene expression globally in M2c macrophages.

Figure 2. AR stimulation augments the IL-10-induced expression of TIMP-1 and arginase-1 mRNA.

Figure 2.

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(A) TIMP-1 mRNA expression in RAW 264.7 cells treated with 10 ng/ml IL-10 or 100 μM adenosine and 10 ng/ml IL-10 for 6 h. ###P < 0.001 versus IL-10 (n=6). (B) Arginase-1 (Arg-1) mRNA expression of RAW 264.7 cells treated with 1 μM NECA or 10 ng/ml IL-10 or 1 μM NECA and 10 ng/ml IL-10 for 6 h. *P < 0.05, versus vehicle; ##p < 0.01 versus IL-10 (n=6). (C) Arginase-1 mRNA expression of BMDMs, isolated from WT, A2AAR, or A2BAR mice, which were treated with 1 μM NECA and/or 10 ng/ml IL-10 for 6 h. *P < 0.05, **P < 0.01, ***P < 0.001 (n=3). (D) SOCS-3 mRNA expression of WT, A2AAR, or A2BAR KO BMDMs treated with 10 ng/ml IL-10 or 1 μM NECA and 10 ng/ml IL-10 for 6 h. **P < 0.01, ***P < 0.001 versus vehicle (n=6). Results are the summary of two independent experiments. (E) BCL-3 mRNA expression of RAW 264.7 cells treated with IL-10 or 1 μM NECA and 10 ng/ml IL-10 for 6 h. **P < 0.01 versus vehicle (n=6). All results (mean±sem) are representative of at least three independent experiments.

STAT3 activation participates in the effect of AR stimulation on M2c polarization

We next studied the intracellular signaling pathways mediating the up-regulation of TIMP-1 production by NECA in M2c macrophages. As IL-10 is a well-known activator of STAT3 [10], we first determined the effect of NECA on STAT3 activation, as assessed by evaluating STAT3 phosphorylation. Treatment with IL-10 augmented STAT3 phosphorylation in RAW 264.7 cells, and AR stimulation with NECA increased this STAT3 phosphorylation further (Fig. 3A). NECA alone failed to stimulate STAT3 phosphorylation (data not shown). We then silenced STAT3 using transfection with specific siRNAs to study its contribution to the augmenting effect of AR stimulation on TIMP-1 expression in IL-10-treated RAW 264.7 cells (Fig. 3B). We treated STAT3-silenced or control macrophages with NECA or NECA and IL-10, and found that cells transfected with a NT siRNA responded to NECA and IL-10 treatment with significantly higher TIMP-1 release than macrophages transfected with STAT3-specific siRNA (Fig. 3C). A pharmacological approach confirmed the role of STAT3, as the STAT3 pathway inhibitors AG490, ZM39923, or cucurbitacin all inhibited the effect of NECA (Fig. 3D). To explore the role of further signaling pathways, previously linked to IL-10 or adenosine signaling, we treated RAW 264.7 cells with SB203580 or LY294002 before treatment with NECA. Our results showed that both inhibitors reduced the TIMP-1-inducing effect of NECA/IL-10 (Fig. 3E), suggesting a role of p38 and PI3K/Akt pathways, respectively, in the process.

Figure 3. AR stimulation augments IL-10-induced STAT3 phosphorylation.

Figure 3.

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(A) Phosphorylated STAT3 (pSTAT3) and actin protein expression in RAW 264.7 cells treated with 10 ng/ml IL-10 or 1 μM NECA and 10 ng/ml IL-10 for 60 min. (B) STAT3 and actin protein expression in RAW 264.7 cells transfected with NT or STAT3-specific siRNA. (C) TIMP-1 production by RAW 264.7 cells transfected with NT or STAT3-specific siRNA and treated with 10 ng/ml IL-10 or 1 μM NECA and 10 ng/ml IL-10 for 6 h. *P < 0.05, ***P < 0.001 (mean±sem; n=4). (D) TIMP-1 production by RAW 264.7 cells treated with 10 μM AG490, 10 μM ZM39923, or 1 μM cucurbitacin, 30 min before treatment with 1 μM NECA and 10 ng/ml IL-10 for 24 h. ***P < 0.001 versus vehicle (mean±sem; n=5). (E) TIMP-1 production by RAW 264.7 cells treated with 100 nM SB203580 or LY294002, 30 min before treatment with 1 μM NECA and 10 ng/ml IL-10 for 24 h. Data are shown as percent of control (NECA+IL-10). ***P < 0.001 versus vehicle (mean±sem; n=5). Results are representative of at least three independent experiments.

AR stimulation inhibits IL-6-induced STAT3 signaling

In contrast to the augmenting effect found in IL-10-activated cells, AR stimulation inhibited STAT3 phosphorylation induced by IL-6 (Fig. 4A and B). In addition, NECA inhibited the IL-6-induced expression of SAA3 mRNA in RAW 264.7 cells (Fig. 4C). Interestingly, NECA augmented IL-6-induced TIMP-1 (Fig. 4D) and arginase-1 expression (Fig. 4E). In addition, NECA augmented arginase-1 mRNA only in WT and A2AAR KO and not in A2BAR KO macrophages (Fig. 4F). Finally, we found that similar to IL-10, treatment of macrophages with IL-6 did not have any effect on TNF-α and IL-12 (data not shown).

Figure 4. AR stimulation inhibits IL-6-induced STAT3 phosphorylation.

Figure 4.

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(A) pSTAT3 and actin protein expression in RAW 264.7 cells treated with 10 ng/ml IL-6 or 1 μM NECA and 10 ng/ml IL-6 for 1 h. (B) Densitometric analysis of Western blot results. ***P < 0.001 versus vehicle; ###P < 0.001 versus IL-6. SAA3 (C), TIMP-1 (D), or arginase-1 (E) mRNA expression in RAW 264.7 cells treated with 10 ng/ml IL-6 or 1 μM NECA and 10 ng/ml IL-6 for 6 h. **P < 0.01, ***P < 0.001 versus vehicle; ##P < 0.01, ###P < 0.001 versus IL-6 (mean±sem; n=6). Results are representative of at least three independent experiments. (F) Arginase-1 mRNA expression of BMDMs, isolated from WT, A2AAR, or A2BAR mice, which were treated with 1 μM NECA and/or 10 ng/ml IL-6 for 6 h. *P < 0.05, ***P < 0.001 (n=6). Results are a summary of two independent experiments.

DISCUSSION

Most of the effects of adenosine on M1 macrophages, including inhibition of TNF-α [34–36] or IL-12 production [28] and enhancement of IL-10 production [32], have been found to be mediated by the A2AAR. However, the A2BAR can also affect M1 macrophages; for example, it augments IL-10 production by LPS-treated RAW 264.7 macrophages or microglia [33, 37]. We have shown recently that the A2BAR, with the A2AAR playing a lesser role, augments IL-4- or IL-13-induced M2a macrophage activation [30]. Here, we provide evidence that A2BAR signaling and IL-10 synergistically increase gene expression in M2c macrophages.

STAT3 is widely appreciated as the primary transcription factor mediating IL-10 signaling [6, 10, 38], and the role of STAT3 in TIMP-1 induction has been explored by a number of studies [6, 39]. Our results suggest that adenosine regulates M2c macrophage function, at least in part, by augmenting STAT3 activation, as AR stimulation augmented the IL-10-induced phosphorylation of STAT3, and silencing STAT3 inhibited the effect of AR stimulation on TIMP-1 production. In addition, pharmacological inhibitors of the STAT3 pathway prevented the effect of AR stimulation. As AR stimulation did not influence the IL-10-induced phosphorylation of JAK1, JAK2, or JAK3 (data not shown), further studies will be required to delineate how AR signaling up-regulates STAT3 activation.

Our findings, that the PI3K inhibitor LY294002 blocked the effect of NECA and IL-10 on TIMP-1 production, are in agreement with data published previously, showing a role of the PI3K-Akt pathway in IL-10-induced gene expression [11, 38, 40] and specifically, in TIMP-1 expression [41]. The p38 MAPK has also been described to participate in TIMP-1 induction [42]. Our data support this concept, as p38 inhibition reversed the NECA- and IL-10-induced up-regulation of TIMP-1 release.

In addition to IL-10, IL-6 uses the STAT3 pathway for intracellular signaling [43]. In contrast to IL-10-treated cells, AR stimulation inhibited STAT3 activation in macrophages activated with IL-6. In agreement with the role of STAT3 signaling in mediating IL-6-induced SAA3 gene expression [44], our results showed that IL-6-induced SAA3 gene expression was blocked by AR stimulation. Thus, AR stimulation differentially regulates IL-10- and IL-6-induced STAT3 activation. In addition, we found that AR stimulation augments TIMP-1 and arginase-1 expression in IL-6-treated macrophages. Activation of A2BAR was responsible for the stimulatory effects of NECA on IL-6-induced arginase-1, which is in agreement with our data regarding the role of A2BAR in macrophages stimulated with IL-10. However, the fact that AR stimulation inhibited STAT3 phosphorylation in macrophages treated with IL-6 makes the role of STAT3 in this process unlikely and suggests that A2BAR augments IL-10- and IL-6-induced arginase-1 expression through different pathways. As ERK1/2, MAPK, and PI3K have been linked to IL-6 signaling previously [45], these pathways may mediate the effect of AR stimulation on IL-6-induced arginase-1 expression. Thus, our results suggest, that adenosine has STAT3-dependent, as well as independent, regulatory functions on macrophages, depending on the cytokine environment during activation.

It is well-appreciated that the concentration of IL-10 is elevated in tumor surroundings and that IL-10 contributes to the polarization of tumor-associated macrophages. By acquiring an M2-like phenotype, these tumor-associated macrophages promote tumor progression by maintaining immune tolerance and driving metastases [2, 46–48]. As A2BAR activation has been shown to promote tumor progression [49, 50], and STAT3 activation and increased arginase-1 expression have been linked to tumor progression and angiogenesis [51, 52], our observations that AR activation increases STAT3 activation and arginase gene expression in M2c macrophages indicate that the stimulatory effect of AR stimulation on IL-10 induced-STAT3 signaling in macrophages may contribute to tumor progression.

In conclusion, we have shown that adenosine differentially regulates IL-10- and IL-6-induced STAT3 phosphorylation and the expression of STAT3-driven genes. As STAT3 activation is widely implicated in tumor formation and progression, our results further underscore the therapeutic potential of targeting A2BARs in cancer treatment [50, 53].

ACKNOWLEDGMENTS

This work was supported by the U.S. National Institutes of Health grants R01GM66189 and R01GM068636, U.S. Army Medical Research and Materiel Command Grant 09065004, and Hungarian Scientific Research Fund (OTKA) grant K 109178 to G.H. and by a Hungarian Scientific Research Fund grant (Human MB08-1-2011-0015; OTKA 84685) to E.K.

Footnotes

AG490
(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide
AR
adenosine receptor
BAY606583
2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide
BCL-3
B cell lymphoma 3-encoded protein
BMDM
bone marrow-derived macrophage
CGS21680
4-[2-{[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino}ethyl]benzenepropanoic acid hydrochloride
KO
knockout
LY294002
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride
NECA
5′-(N-ethylcarboxamido)adenosine
NT
nontargeting
PSB0788
8-{4-[4-(4-chlorobenzyl)piperazide-1-sulfonyl phenyl]}-1-propylxanthine
SAA3
serum amyloid A3
SB203580
4-{5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl}pyridine
siRNA
small interfering RNA
SOCS3
suppressor of cytokine signaling 3
ZM399223
3-benzylisopropylamino-1-naphthalen-2-yl-propan-1-one hydrochloride

AUTHORSHIP

B.K. designed and performed experiments, analyzed data, and wrote the manuscript. B.C., E.K., and Z.H.N. performed experiments and analyzed data. P.P., L.V., and S.J.L. contributed to analysis and interpretation of data. G.H. conceived of and supervised the project, analyzed data, and wrote the manuscript.

DISCLOSURES

The authors disclose no conflict of interest.

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