Biochemical and Biophysical Research Communications 446 (2014) 663–668

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Transcriptional regulation and functional characterization of the oxysterol/EBI2 system in primary human macrophages Inga Preuss a, Marie-Gabrielle Ludwig a, Birgit Baumgarten a, Frederic Bassilana a, Francois Gessier b, Klaus Seuwen a, Andreas W. Sailer a,⇑ a b

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland

a r t i c l e

i n f o

Article history: Available online 27 January 2014 Keywords: EBI2 GPR183 GPCR Oxysterols CH25H Macrophages

a b s t r a c t Oxysterols such as 7 alpha, 25-dihydroxycholesterol (7a,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein-coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses. Activation of EBI2 by specific oxysterols leads to chemotaxis of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adaptive immune response is established by a stromal cells subset here we investigate the involvement of the oxysterol/EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes needed for ligand production such as cholesterol 25-hydroxylase (CH25H), sterol 27-hydroxylase (CYP27A1), and oxysterol 7a-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharides (LPS) triggers a strong up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Stimulation of EBI2 expressed on macrophages leads to calcium mobilization and to directed cell migration. Supernatants of LPS-stimulated macrophages are able to stimulate EBI2 signaling indicating that an induction of CH25H, CYP27A1, and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is a study characterizing the oxysterol/EBI2 pathway in primary monocyte-derived macrophages. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Inflammation is the protective reaction of the body to infection, injury, or irritation with the aim to remove harmful stimuli such as pathogens, damaged cells, or allergic irritants and to initiate the healing process. Inflammatory abnormalities play a crucial role in the pathogenesis of many human diseases including autoimmune disorders such as type 1 diabetes, rheumatoid arthritis, or multiple sclerosis as well as cardiovascular diseases such as atherosclerosis or metabolic disease. Monocyte-derived macrophages are key mediators of the innate immune response and are central for the inflammation process (for recent reviews see [1,2]). Based on functional properties macrophage phenotypes fall within a spectrum of two extremes [3]. First, pro-inflammatory macrophages (M1 phenotype or classically-activated macrophages) secrete proinflammatory cytokines and chemokines, thereby initiating and

⇑ Corresponding author. Address: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, WSJ-355.4025.8, CH-4002 Basel, Switzerland. Fax: +41 61 6968714. E-mail address: [email protected] (A.W. Sailer). 0006-291X/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2014.01.069

sustaining inflammation in order to fight microbes and infected cells. Second, the anti-inflammatory, pro-resolution macrophages (M2 phenotype or alternatively-activated macrophages) which are induced by anti-inflammatory factors such as IL-4 or IL-13 and promote resolution events such as clearance of apoptotic cells and tissue repair. For mounting an effective immune response by secretion of pro- or anti-inflammatory mediators and phagocytosis of invading pathogens or cellular debris during an infection, deployment of specific subsets of macrophages to the site of inflammation must be temporally and spatially strictly regulated. In a healthy environment this goal is achieved by a highly concerted production and release of different chemokines and the expression of their corresponding cognate receptors. Imbalances between the pro- and anti-inflammatory processes have been demonstrated to underlie the pathophysiology of autoimmunity and autoinflammation [4,5]. In this study we have explored a functional role of the oxysterol/EBI2 system in this process. In 2011 we and others reported the discovery of oxysterols as ligands for EBI2 [6,7]. Oxysterols are metabolites generated by hydroxylation of cholesterol and have been linked to a variety of fundamental physiological processes

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including sterol transportation, bile acid biosynthesis as well as immune cell function [8,9]. For example oxysterols are ligands for several nuclear hormone receptors (e.g. LXR, RORa, RORc) which play a role in the immune process. While LXR and their ligands are negative regulators of macrophage inflammatory gene expression [10] and constitute a metabolic checkpoint for immune cell proliferation [11], RORct is the key transcription factor to orchestrate differentiation of pro-inflammatory Th17 cells [12]. 7a,25-OHC has been identified as the most potent natural ligand for the EBI2 receptor and there are two hydroxylases required for its synthesis. CH25H adds a hydroxyl group to carbon 25 and CYP7B1 catalyzes the hydroxylation at position 7. Alternatively, CYP27A1 hydroxylates carbon 27 before CYP7B1 carries out the second hydroxylation. While less potent 7a,27-OHC is also a very powerful EBI2 ligand able to direct cell migration of immune cells. Degradation of 7a,25-OHC and 7a,27-OHC occurs via enzymatic action of HSD3B7 which moves the double bond from ring B to ring A and oxidizes the hydroxyl group at position 3 to a ketone, rendering the oxysterol unable to interact with the EBI2 receptor. For mouse macrophages it was reported that they up-regulate CH25H expression in a time-dependent manner upon immune challenge with LPS. The increase in mRNA and protein levels leads to a subsequent production of 25-OHC which is released into the cellular environment [13,14]. In contrast, EBI2 mRNA transcripts are down-regulated after LPS treatment [15,16]. While the oxysterol/EBI2 complex in murine B cells is well studied its role in primary human cells of the innate immune system, in particular macrophages, has not yet been investigated. In order to develop an integrated understanding of the transcriptional regulation and function of the oxysterol/EBI2 pathway here we describe the expression of the oxysterol-metabolizing enzymes CH25H, CYP27A1, CYP7B1, HSD3B7, and the receptor itself following inflammatory stimuli and show a functional role of this ligand/ receptor pair in monocyte-derived macrophages. 2. Materials and methods Details on materials and methods can be found in the Supplemental material section. 3. Results 3.1. Primary human monocytes and macrophages express enzymes necessary for oxysterol synthesis and degradation In order to ensure correct migration of EBI2-expressing cells, the oxysterol gradient has to be tightly regulated. A number of oxysterol-metabolizing enzymes are responsible for the maintenance of this gradient by either synthesizing (CH25H, CYP27A1, and CYP7B1) or degrading (HSD3B7) the most potent EBI2 agonists 7a,25-OHC and 7a,27-OHC. In mouse models, it could be shown that stromal cells are the predominant source for generating the

oxysterol gradient which plays a crucial role for EBI2 in controlling the positioning of B cells within secondary lymphoid tissues during adaptive responses [17]. Here, we investigated whether primary human cells of the innate immune system also express enzymes needed for oxysterol formation and degradation. Using Affymetrix gene chips we determined mRNA expression levels of CH25H, CYP27A1, CYP7B1, and HSD3B7 during differentiation of monocytes to distinct macrophage subtypes (Table 1). Exclusive treatment of monocytes with M-CSF yields a macrophage population which we termed M0. In contrast, combination of M-CSF with IFNc or IL-4 leads to M1 and M2 macrophage subtypes, respectively. Unexpectedly, in contrast to the almost completely down-regulated CH25H mRNA levels in M0 and M1 macrophages, the mRNA amount of CH25H is strongly up-regulated in M2 macrophages during the differentiation process from monocytes, starting at day 1 and continuing up to day 5. In comparison to freshly isolated monocytes, CYP27A1 transcripts are elevated up 10-fold in macrophages with hardly any differences between the specific subtypes. Same is true for HSD3B7 expression. There is a strong increase in mRNA levels observable in macrophages with no further regulation during polarization. No present calls were detected for CYP7B1. We next determined a time course of CH25H, CYP27A1, CYP7B1, and HSD3B7 mRNA levels in monocytes and M0 macrophages by qRT-PCR using TaqMan probes (Fig. 1A). Freshly isolated PBMCderived monocytes express all four enzymes. M0 macrophages differentiated with M-CSF for 1, 3, 5, or 7 days have reduced amounts of CH25H mRNA as already shown in the Affymetrix microarray analysis. CYP7B1 transcript levels are decreased during the first 3 days of differentiation but return to levels found in monocytes on day 5. While we observe an increased level of CYP27A1 starting at day 5 in M0 macrophages HSD3B7 mRNA was only transiently elevated. These results indicate that primary human monocytes and macrophages express enzymes necessary for oxysterol production and metabolism and that mRNA levels are differentially regulated during the differentiation process. 3.2. Expression of oxysterol-metabolizing enzymes is highly regulated upon immune challenge In the murine system it has been shown that upon immune challenge bone marrow (BM)-derived macrophages highly up-regulate CH25H via a pathway that includes TLR and interferon receptors and JAK/STAT signaling [13,14]. TLR agonists such as LPS and IFNb lead to a time-dependent induction of CH25H expression. To examine how the expression levels of CH25H, CYP27A1, CYP7B1, and HSD3B7 are regulated after immune challenge in primary human macrophages M0 cells were treated with LPS for different time points (Fig. 1B). Similar to mouse macrophages we found that CH25H mRNA levels of monocyte-derived human macrophages are strongly up-regulated but only in a transient manner. CH25H expression peaks after 2 h and is then rapidly down-regulated. For CYP7B1 the expression profile is comparable to that observed in murine cells. An increase of CYP7B1 mRNA can be

Table 1 CH25H, CYP27A1, HSD3B7, and EBI2 mRNA expression in PBMC-derived monocytes and macrophages during differentiation. Gene chip analysis of primary human monocytes, M0, INFc-driven M1, or IL-4-driven M2 macrophages were cultured for 1, 3, or 6 days. Expression was normalized to MAS5 and the median values of three different donors are shown.

CH25H CYP27A1 HSD3B7 EBI2

Day 0

Day 1

Mono

M0

M1

M2

Day 3 M0

M1

M2

Day 6 M0

M1

M2

47 373 0 5365

5 2789 1753 841

0 2521 1852 287

211 2555 1449 917

0 3662 1879 1040

0 2180 1165 206

613 2910 1284 884

0 2872 1605 1332

2 3733 1458 1137

685 3490 1130 767

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0.002

0.000

0

1

3

5

7

Mono M0 MΦ

60 40 20 0

0

1

3

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2

4

LPS [h]

0.010

0.005

0.000

0

1

6

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2

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80

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relative expression to HPRT1

relative expression to HPRT1

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0.004

80

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0.006

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relative expression to HPRT1

A

0.4 0.3 0.2 0.1 0.0

0

2

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LPS [h]

Fig. 1. Basal and LPS-regulated mRNA expression of oxysterol-metabolizing enzymes in PBMC-derived monocytes and M0 macrophages analyzed by qRT-PCR. (A) Basal mRNA expression of CH25H, CYP27A1, CYP7B1, and HSD3B7 during differentiation of monocytes to M0 macrophages using TaqMan probes. (B) LPS-stimulated (100 ng/mL) mRNA expression of CH25H, CYP27A1, CYP7B1, and HSD3B7 in M0 macrophages using TaqMan probes. The HPRT1-normalized median of six experimental values from 1 representative donor is shown.

observed over time of LPS treatment. In contrast, CYP27A1 and HSD3B7 transcripts are time-dependently down-regulated within 2 or 6 h, respectively. Our data demonstrate a very dynamic regulation of the enzymes necessary for oxysterol synthesis and degradation under immune challenge. 3.3. EBI2 is expressed in primary human monocytes and macrophages An effective immune response is dependent on a functional migration process and a correct localization of the cells. Not only the production of the chemoattractant has to be strictly controlled, also the expression of the cognate receptor is critical. EBI2 expression and function on murine B cells has been shown to prevent movement to the germinal center of lymphoid follicles in which the antibody maturation takes place [18,19]. Expression of the EBI2 receptor in PBMC-derived macrophages has not yet been described. Affymetrix microarray analysis revealed that primary human monocytes express substantial levels of EBI2 which are rapidly decreased after 1 day of differentiation to macrophages (Table 1). After being down-regulated EBI2 transcripts stay at the same level over time with only minor differences between M0, M1, and M2 macrophages. Taken together, primary human macrophages express EBI2 mRNA but only at approximately 15% of the levels found in their monocytic precursor cells. 3.4. EBI2 expression is quickly up-regulated in a transient manner after LPS treatment Previous work suggested that EBI2 mRNA levels are down-regulated in bone marrow (BM)-derived macrophages and selected mouse macrophage cell lines (e.g. RAW264.7) upon immune challenge [15,16]. Information on the receptor regulation in primary

human macrophages remains limited. Therefore, we determined EBI2 transcripts after treatment with LPS for different time points in M0 macrophages differentiated for 3, 5, or 7 days (Fig. 2A). In contrast to the murine system, we observed a dramatic up-regulation at 2 h of LPS stimulation which continued to increase until day 7 (Fig. 2A). Dependent on the donor, the maximal up-regulation extends to Ct values below those of the house keeping gene b-actin which corresponds to a relative expression to our endogenous control HPRT1 of more than 100. Remarkably, EBI2 is like CH25H only transiently up-regulated and comes back to basal levels after 4 h of LPS challenge. In order to have a closer look into the kinetics of LPS-induced EBI2 up-regulation we performed a detailed time course of the receptor expression with 30 min intervals (Fig. 2B). Regulation of EBI2 is unusually fast. The increase in EBI2 mRNA levels begins already after 30 min of LPS treatment with a peak at 1.5 h before it returns to basal levels. As there are many chemokine receptors expressed on macrophages we examined expression of other GPCRs at an early time point after immune challenge. Commercially available and customized micro fluidic cards of a human GPCR panel were used for comparison of EBI2 expression in control and 1.5 h LPS-treated samples (Fig. 2C and D). Surprisingly, out of nearly 380 tested GPCRs EBI2 is the receptor with the highest expression at 1.5 h post LPS treatment (relative expression to HPRT1 = 140) (Fig. 2D). In terms of the log2 fold changes between LPS-stimulated and unstimulated macrophages EBI2 belongs to the top 10 GPCRs with the strongest upregulation (log2 fold: LPS/ctl = 2.5) (Fig. 2C). The chemokine receptor CCR7 is the most vigorously regulated receptor (log2 fold: LPS/ctl = 7) although the expression levels are far below that of EBI2 transcripts (relative expression to HPRT1 = 0.16). Our results show that EBI2 mRNA expression exhibits the most dynamic profile compared to other GPCRs in PBMCderived macrophages upon immune challenge. In addition, it is

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A

C

EBI2

relative expression to HPRT1

EBI2

HCAR3

50

ctl 2h LPS 4h LPS 6h LPS

40 30

CCRL2 ADRB2 HCAR2

20

CXCR4 ctl

PTGER4

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GPR132

0 Day 3

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30

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GPR84

relative expression to HPRT1

EBI2 CCR7

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GPR31

20 GPRC5A

15

SSTR2

10

ADRA2B ctl

CXCR7

5

LPS

HRH1

0 0

1

2 3 LPS [h]

4

5

0.0

0.1

0.2

0.3

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relative expression to HPRT1

Fig. 2. LPS-regulated mRNA expression of EBI2 in M0 macrophages analyzed by qRT-PCR. (A) LPS-stimulated (100 ng/mL) EBI2 mRNA expression during differentiation of M0 macrophages using TaqMan probes. (B) Time course of EBI2 mRNA expression after LPS treatment (100 ng/mL) using TaqMan probes. The HPRT1-normalized median of 6 experimental values from 1 representative donor is shown. (C, D) mRNA expression levels of various GPCRs after LPS treatment (100 ng/mL, 1.5 h) using TaqMan micro fluidic cards. Only genes with a log2 fold ratio > 1 are listed. The HPRT1-normalized average values of 3 replicates from 1 donor are displayed.

unrivaled as the highest expressed GPCR mRNA in human macrophages after a short-term LPS stimulation.

3.5. Functional activation of EBI2 and oxysterol-directed migration of primary human macrophages After having shown that EBI2 is expressed in significant amounts on primary human macrophages we investigated whether the receptor is functional. Recent studies demonstrated an effect of 7a,25-OHC on calcium signaling in CHO cells stably expressing EBI2 [6]. Here, we stimulated PBMC-derived macrophages with the EBI2 agonist 7a,25-OHC and monitored mobilization of intracellular calcium. 7a,25-OHC treatment triggers a concentrationdependent increase in cytosolic calcium (Fig. 3A). This activation can be blocked by NIBR189, a potent and selective EBI2 antagonist (Fig. 3B). Activation of EBI2 on mouse B cells leads not only to stimulation of second messenger cascades but also results in directed cell migration towards the source of oxysterol production [6,7]. This functional property is critical for correct positioning of mouse B cells within lymphoid organs. To investigate whether 7a,25-OHC also induces chemotactical responses in PBMC-derived macrophages we performed xCelligence migration experiments. Treatment with 7a,25-OHC directs movement of M0 macrophages in a bell-shaped concentration-dependent manner with a peak at 40 nM (Fig. 3C). The EBI2 antagonist NIBR189 can inhibit this effect demonstrating that the 7a,25-OHC-induced chemotaxis is due to receptor activation (Fig. 3D). Primary human macrophages are able to migrate towards a 7a,25-OHC gradient in a receptor-dependent

fashion. In line with previous findings, these data confirm that the oxysterol/EBI2 complex triggers cell migration. 3.6. Supernatants from LPS-induced macrophages can regulate calcium signaling High expression of the enzymes necessary for oxysterol synthesis raises the question whether the oxysterol are generated in quantities sufficient to attract immune cells. To test this hypothesis we stimulated monocyte-derived macrophages with LPS for 4, 8, or 22 h and subsequently used the supernatants on other macrophages (Fig. 4A and B). Supernatants do indeed trigger the release of intracellular calcium (Fig. 4B). Stimulation with LPS for 22 h leads to the highest increase in cytosolic calcium. Calcium signaling is partially due to EBI2 activation as pre-treatment with the antagonist can block the response by up to 30%. A complete blockade was not expected since LPS induces the production and release of several factors which can result in calcium mobilization and the lipid-containing supernatants were not fractionated into different components. Our results show that oxysterols produced in cell culture can stimulate signal transduction pathways by activating EBI2. 4. Discussion The EBI2 receptor is widely expressed in the immune system. While its role in the control of B cell maturation is beginning to be understood, little is known about the relevance of the receptor in innate immunity. In the current study, we show that PBMC-derived human monocytes and macrophages express EBI2 as well as

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A

C

1.2

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slope (1/h)

dF/F

1.0 0.8 0.6

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0.4 0

-10

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-9 -8 -7 -6 7α,25-OHC [log(M)]

0

-5

40

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7α,25-OHC [nM]

D

1.2

5 4

slope (1/h)

1.0

dF/F

8

0.8 0.6

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****

2 1

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0

-9

-8 -7 -6 -5 NIBR189 [log(M)]

ctl

-4

40 nM 7α,25-OHC 40 nM 7α,25-OHC + 10 μM NIBR189

Fig. 3. 7a,25-OHC induces Ca2+ mobilization and directed cell migration in M0 macrophages. (A) 7a,25-OHC dose titration in M0 macrophages measured by a Ca2+ mobilization assay. (B) Inhibition of 7a,25-OHC-induced (100 nM) Ca2+ mobilization in M0 macrophages by the EBI2 antagonist NIBR189. (C) 7a,25-OHC-directed cell migration of M0 macrophages measuring electrical impedance (xCelligence-type). (D) Inhibition of 7a,25-OHC-directed (40 nM) cell migration of M0 macrophages by the EBI2 antagonist NIBR189 (10 lM). The average values of 4 replicates from 1 representative donor are shown (⁄⁄⁄⁄p < 0.0001).

A

PBMC

B

2.0

hMΦ SN hMΦ SN + 10μM NIBR189

CD14+ / CD16+ negative selection

1.5

dF/F

monocytes 6d M-CSF

MΦ

*** 1.0

*

0.5

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0h, 4h, 8h and 22h LPS

*

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4h LPS

8h LPS

22h LPS

Fig. 4. Supernatants of LPS-stimulated macrophages induce Ca2+ mobilization in M0 cells in an autocrine manner. (A) Experimental set-up. (B) FLIPR-type Ca2+ mobilization assay in M0 macrophages. Cells were stimulated with undiluted supernatants of macrophages treated for 4, 8, and 22 h with LPS (100 ng/mL). Increase in cytosolic Ca2+ was measured with or without EBI2 antagonist pre-treatment (NIBR189, 10 lM). The average values of 6 replicates from 1 representative donor are shown (⁄p < 0.05, ⁄⁄⁄p < 0.001).

enzymes necessary for oxysterol metabolism. We further demonstrate that these cells produce significant amounts of EBI2 agonists and are able to respond to oxysterol gradients by chemotaxis. Macrophages are derived from circulating monocytes that enter the tissue on demand. Dependent on their differentiation state they can either act as pro-inflammatory M1 macrophages or as anti-inflammatory, pro-resolution M2 macrophages. Our gene expression profiling demonstrates that the differentiation process of monocytes to macrophage subtypes greatly influences the basal expression levels of CH25H, CYP27A1, and EBI2. In comparison to freshly isolated monocytes CH25H is almost completely down-regulated in M0 and IFNc-driven M1 macrophages whereas stimulating M2 polarization by IL-4 strongly induces CH25H mRNA expression. In contrast, CYP27A1 is well expressed in monocytes and expression is even increased in different macrophage subtypes. CYP7B1, the enzyme that converts 25-OHC and 27-OHC into the di-hydroxylated oxysterols, and HSD3B7, responsible for the

degradation of oxysterols, are also expressed in PBMC-derived monocytes and M0 macrophages, suggesting that both cell types are able to produce potent EBI2 agonists and to control their breakdown. The relative abundance of EBI2 RNA transcripts in primary monocytes is up to 10-fold higher compared to the various macrophage subtypes. This fact likely reflects the need of the monocyte, as a prime migratory cell type in the circulation, to utilize the sensitive oxysterol/EBI2 pathway for its deployment to sites of inflammation at any time. In our experiments we mimicked inflammation by LPS challenge of M0 macrophages. We discovered a strong increase in expression of CH25H and CYP7B1 while CYP27A1 and HSD3B7 are down-regulated over time. CH25H up-regulation is transient with a peak around 2 h. This is similar to the dramatic raise in expression of EBI2, even beyond expression levels of b-actin, which occurs after LPS treatment. Both findings suggest a feedback mechanism by which increasing amounts of oxysterols diminish further

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expression of enzyme and receptor mRNA. Interestingly, this result is in contrast to the murine system where EBI2 transcript levels are strongly down-regulated upon LPS challenge. PBMC-derived macrophages not only express EBI2 but also have the ability to respond to their natural agonist 7a,25-OHC by activating downstream signal transduction pathways. In line with previous findings, our data confirm that receptor stimulation induces calcium mobilization which can be blocked by the EBI2 antagonist NIBR189. As described in several studies for EBI2-expressing immune cells, the main function of the ligand/receptor complex is the promotion of chemotaxis [6,17]. Here, we demonstrate EBI2-dependent movement of macrophages. The observed bellshaped curve, a classical hall mark of directed cell migration in transwell assays, suggests receptor desensitization. Increased expression of oxysterol-producing enzymes lead to an elevation of oxysterol levels. While detection of oxysterols in cell culture systems has been successful [20–22] it remains challenging. Thus, we decided to use a bioassay in which supernatants from LPS-stimulated macrophages were transferred on other cells and the release of intracellular calcium was monitored. Supernatants induced calcium mobilization which is in part mediated by EBI2 as demonstrated through blockade by the receptor antagonist. These results confirm our hypothesis that an inflammatory challenge leads to enhanced generation of oxysterols which act as EBI2 agonists. To determine which specific oxysterol species are produced in this setting analysis by mass spectrometry will be needed. It is interesting to note that Eibinger and colleagues [23] recently reported chemotactic movement to 25-OHC in THP1 cells as well as in primary human monocytes. RNA interference suggested that in part this migration was mediated by EBI2. In summary, our results demonstrate a functional role of the oxysterol/EBI2 system in monocytes and macrophages and encourage the further exploration of pathologies correlated with alterations of the innate immune system. Competing financial interest statement All authors are current or previous employees of Novartis and some of them do hold stock or stock options in their company. Acknowledgments We would like to thank Stéphane Laurent, Marie-Christine Lasbennnes, and Caterina Safina for their excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbrc.2014.01.069. Reference [1] C. Nathan, A. Ding, Nonresolving inflammation, Cell 140 (2010) 871–882. [2] A. Sica, A. Mantovani, Macrophage plasticity and polarization: in vivo veritas, J. Clin. Invest 122 (2012) 787–795.

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