Atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages through p38 mitogen-activated protein kinase-dependent peroxisome proliferator-activated receptor

International Immunopharmacology 26 (2015) 58–64

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International Immunopharmacology

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Atorvastatin promotes human monocyte differentiation towardalternative M2 macrophages through p38 mitogen-activatedprotein kinase-dependent peroxisome proliferator-activatedreceptor γ activation

Ou Zhang a,b, Jinying Zhang a,⁎a Cardiovascular Department, First Hospital Affiliated to Zhengzhou University, Henan 450000, Chinab Coronary care unit, First Hospital Affiliated to Nanyang Medical College, Henan 473058, China

⁎ Corresponding author. Tel.: +86 371 67967641; fax:E-mail address: [email protected] (J. Zhang).

http://dx.doi.org/10.1016/j.intimp.2015.03.0051567-5769/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 10 January 2015Received in revised form 17 February 2015Accepted 5 March 2015Available online 18 March 2015

Keywords:AtorvastatinMonocyteM2 macrophagesp38 MAPKPPARγAtherosclerosis

M1 and M2 macrophages are detectable in human atherosclerotic lesions, and M2 macrophages are present atlocations distant from the lipid core in more stable zones of the plaque and appear to exert anti-inflammatoryproperties on M1 macrophages. Peroxisome proliferator-activated receptor (PPAR) γ promotes the differentia-tion of monocytes into anti-inflammatory M2 macrophages. Although both statins and PPARγ ligands havebeen reported to protect against the progression of atherosclerosis, no data are currently available regardingthe implication of statins in the alternative differentiation of humanmonocytes. In the present study, we hypoth-esized that atorvastatin may exert novel effects to prime human monocytes toward an anti-inflammatory alter-nativeM2 phenotype. To this aim,wefirst found that abundantM2markerswere expressed inhuman circulatingmonocytes after atorvastatin treatment. Moreover, atorvastatinwas able to induce PPARγ expression and activa-tion in humanmonocytes in vivo and in vitro, resulting in priming primary humanmonocytes differentiation intoM2macrophages with a more pronounced paracrine anti-inflammatory activity in M1 macrophages. Additionaldata with molecular approaches revealed that p38 mitogen-activated protein kinase (MAPK) but not extracellu-lar signal-regulated kinase (ERK) 1/2 activation was involved in atorvastatin-mediated PPARγ activation and en-hanced alternative M2 macrophage phenotype. Collectively, our data demonstrated that atorvastatin promoteshuman monocyte differentiation toward alternative M2 macrophages via p38 MAPK-dependent PPARγactivation.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Atorvastatin, a most widely used member of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, havebeen shown to reduce the incidence of cardiovascular events anddeath in several mega trials [1–3].The reasons why cardiovascularevents were decreased with atorvastatin therapy are reported to bedue to many pleiotropic effects, for instance, inhibition of the prolif-eration and migration of endothelial cells, smooth muscle cells, andmacrophages [4].

Atherosclerosis is well established as a chronic inflammatory disorderinvolving many immune cells [5]. Among those cell types, macrophageswere the first inflammatory cells to be identified within atheroscleroticplaques [6], exerting an important impact on lesion progression in allstages of atherogenesis. A crucial step in this inflammatory process isthe infiltration of monocytes into the subendothelial space of large

+86 371 67967646.

arteries and their differentiation into tissue macrophages [7], which area heterogeneous cell population adapting their activation state and func-tions to the microenvironment [8,9]. Therefore, macrophages exhibit aclassical M1 activation profile in the presence of lipopolysaccharide(LPS) or interferon gamma (IFNγ). M1 macrophages are associated withinflammation and tissue destruction, produce proinflammatory cytokinessuch as 2TNF-α, interleukin (IL)-6, and monocyte chemotactic protein(MCP)-1 [10], and increase the production of reactive oxygen species sus-taining the process of atherogenesis [11]. In contrast, IL-4 or IL-13 inducedan alternative activation, knownasM2.M2macrophages dampen inflam-matory process by producing anti-inflammatory mediators such as IL-10and transforming growth factor-β, scavenging debris, and promotingangiogenesis, tissue remodeling and repair [10].

PPAR (peroxisome proliferator-activated receptor) γ, a member ofthe nuclear hormone receptor family of ligand-dependent transcriptionfactors, has been well characterized as potent anti-inflammatory prop-erties that modulate the immune inflammatory response [12]. PPARγis abundantly expressed inmacrophages, where its expression is rapidlyinduced upon differentiation of monocytes into macrophages [13]. It is

59O. Zhang, J. Zhang / International Immunopharmacology 26 (2015) 58–64

demonstrated that M2 macrophages are present in human atheroscle-rotic lesions, where expression of M2 markers including CD206 andCCL-18 (chemokine (C–C motif) ligand 18) are positively correlatedwith the expression levels of the nuclear receptor PPARγ [14,15].More importantly, monocytes can be primed by PPARγ activation toan enhanced anti-inflammatory M2 macrophages in atherosclerosis[14,16]. Interestingly, statinswere reported to activate PPARγ inmacro-phages via extracellular signal-regulated kinase (ERK) 1/2 and p38mitogen-activated protein kinase (MAPK) activation and increase theDNA-binding activity of PPARγ to PPAR-response elements in mono-cytes [17]. However, it is not clear whether statins-induced PPARγactivation exerts novel effects to prime human monocytes toward ananti-inflammatory alternative M2 phenotype. If the hypothesis is true,the regulatory mechanisms of statin-induced PPARγ activation inmonocytes are required to be further elucidated.

Therefore, in the present study, we first evaluated M2 markers ex-pression in human circulating monocytes before and after atorvastatintreatment, and then investigated whether atorvastatin has the capacityto activate PPARγ to promote human monocyte differentiation towardalternative M2 macrophages and also to examine their underlyingmechanisms. The data presented here demonstrated that atorvastatinwas able to induce PPARγ expression and activation in human mono-cytes in vivo and in vitro, resulting in priming primary human mono-cytes differentiation into M2 macrophages with a more pronouncedparacrine anti-inflammatory activity in M1 macrophages. Additionaldata with molecular approaches revealed that p38 MAPK but notERK1/2 activationwas involved in atorvastatin-mediated PPARγ activa-tion and enhanced alternative M2 macrophage phenotype.

2. Materials and methods

2.1. Patients

Twenty non-diabetic patients with newly diagnosed coronary arterydisease were enrolled from a single center and were treated with 20 to40 mg atorvastatin p.o. daily. During the time they were receiving theatorvastatin, no other lipid lowering drugs were used. Peripheral venousblood (10 ml) was collected from each patient one day before and twomonths after atorvastatin treatment. This study was approved by theinstitutional ethics committee of First Hospital affiliated to ZhengzhouUniversity (Henan, China), and written informed consent was obtainedfrom every patient. The study was undertaken in full accordancewith the Declaration of Helsinki, and other bioethical principles.

2.2. Cell preparation and culture

Human peripheral blood mononuclear cells (PBMCs) were isolatedfrom patients with newly diagnosed Coronary artery disease and fromhealthy donors by density gradient centrifugation using Ficoll-Hypaqueas an established protocol [18].

Isolated PBMCs were washed twice and suspended in RPMI 1640culture medium (GBICO) supplemented with 10% (v/v) human serum,penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells wereseeded at a density of 5 × 106 cells/well in six-well plates and incu-bated for 3 h in an atmosphere of 5% CO2 and 95% air. Nonadherentcells were discarded, and adherent monocytes were used for indicatedtests or maintained in culture medium for 7 days to differentiate intoresting macrophages (RM). RM were activated in M1 macrophages bythe addition of LPS (100 ng/mL). Alternatively, differentiated macro-phages (M2) were obtained by incubating freshly isolated monocyteswith IL-4 (15 ng/ml) for 7 days. In some experiments, monocyteswere coincubated for 7 dayswith IL-4 (15 ng/ml) and indicated concen-trations of atorvastatin or its vehicle.

For the stimulation or inhibition assay, rosiglitazone (Sigma),T0070907 (Selleck Chemicals), PD98059 (Cell Signaling), and SB203580(Sigma) were used.

2.3. Real-time PCR

Total RNA was extracted using Trizol reagent according to themanufacturer's instructions, and 1 μg of total RNA was converted tocDNA by SuperScript™ III First-Strand Synthesis System for RT-PCR(Invitrogen, Life Technologies). PCR was performed on ABI Prism7000 using corresponding primers and SYBR Green PCR Master Mix(Invitrogen). The primer sequences were provided in SupplementaryTable 1. Template cDNA was denatured at 95 °C for 10 min followedby 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The quantificationdata were analyzed with ABI Prism 7000 SDS software. The cycle timevalues were normalized to GAPDH of the same sample. The expressionlevels of themRNAswere then reported as fold changes versus the indi-cated control.

2.4. Flow cytometry

Monocytes were collected by gentle scrapping andwashedwith ice-cold phosphate-buffered saline containing 1% BSA. The cells were thenFc-blocked by treatment with 1 μg of human IgG/105 cells for 30 minat 4 °C. Next, the cells were incubated at 4 °C for 30 min with monoclo-nal antibodies against CD206 (Allophycocyanin (APC), clone 685641,R&D systems) and CD163 (Phycoerythrin (PE), clone 215927, R&Dsystems) following the manufacturers' instructions. Finally, thecells were washed and analyzed on a FACSCaliburi flow cytometer(Becton Dickinson). CD206 and CD163 expression levels are referredto as mean fluorescence intensity.

2.5. Enzyme-linked immunosorbent assay (ELISA)

2.5.1. ELISA for TNF-α and MCP-1TNF-α and MCP-1 were measured in the supernatants using ELISA

according to the manufacturer's instruction (eBioscience).

2.5.2. ELISA for active PPARγThe amount of activated PPARγ in nuclear extract (5 μg) was

assessed by a sensitive ELISA assay for active PPARγ (TRANS-AM,Active Motif). The PPARγ ELISA kits contain a 96-well plate, whichhas been immobilized oligonucleotide containing the PPARγ consen-sus site (5′-AACTAGGTCAAAGGTCA-3′). The active form of PPARγcontained in nuclear-cell extract specifically binds to this oligonucle-otide. The primary antibodies used to detect PPARγ recognize an epi-tope on PPARγ that is accessible only when PPARγ is activated andbound to its target DNA. A horseradish peroxidase (HRP)-conjugatedsecondary antibody provides a sensitive colorimetric readout that isquantified by spectrophotometry by reading within 5 min the absor-bance at 450 nm.

2.5.3. ELISA for total and phosphorylated ERK and p38 MAPKTotal and phosphorylated ERK and p38MAPK were measured using

ERK1/2 (Total/Phospho (Thr202/Tyr204, Thr185/Tyr187)) InstantOne™

ELISA and p38 MAPK (Total/Phospho (Thr180/Tyr182)) InstantOne™

ELISA (eBioscience) separately following themanufacturers' instruc-tions. Cells were lysed in 100 μl per well of 1× Cell Lysis Mix (ELISAkit component) by incubation for 10 min at room temperature withshaking (300 rpm). Total soluble protein was quantified by BCA Pro-tein Assay Kit and the same amount of protein was loaded per well(about 1 μg/well). Fifty microliters of antibody cocktail (captureantibody + detection antibody reagents from the ELISA kit) wasadded per well and incubated for 1 h at room temperature. Afterthree washes with 200 μL 1× wash buffer per well, 100 μL of DetectionReagent (kit component) was added per well and incubated for 30 minat room temperature. Finally, the reaction was terminated by adding100 μl of Stop Solution (kit component) and the plate was read bymea-suring absorbance at 450 nm.

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2.6. Western blot

Whole-cell lysates were prepared and subjected to western blotanalysis. Equal amounts of the cell lysates were resuspended in 5×Tris-glycine SDS sample buffer, electrophoresed on 12% SDS–PAGE,and transferred to nitrocellulose membranes (Amersham Pharmacia).The detection of proteinswas performedwith anti-p38MAPK antibody,anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody, anti-Phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187) antibody, anti-Erk1/2 anti-body, and anti-β-actin antibody, followed by corresponding IDRysecond antibody. All antibodies were from Cell Signaling Technology.The blots were scanned using an Odyssey Imaging System (LI-CORBioscience, USA).

2.7. Statistical analysis

Data analyses were performed using GraphPad Prism 5.0 software.Results are presented as mean ± standard deviation (SD). Differencesbetween two groups were compared with the Student's t-test. Inter-group comparison of means was performed by ANOVA followed bythe Student's t-test. P values less than 0.05 were considered to besignificant.

3. Results

3.1. Abundant M2 markers are expressed in human peripheral bloodmonocytes after atorvastatin treatment

Previous studies have demonstrated that monocytes can be primedby PPARγ activation to an enhanced anti-inflammatory M2 macro-phages in atherosclerosis [14]. To clarifywhether atorvastatin treatmentaffects M2 markers expression in human peripheral blood monocytes,we performedM2marker RNA analysis of monocytes isolated from pa-tients before and after atorvastatin administration. Interestingly, atorva-statin treatment significantly increased the expression of the M2markers such as CD206, IL-10 and CCL18 (Fig. 1A, C and D). However,atorvastatin therapy only caused a slight but not significant incrementin the expression of the M2 marker CD163 (Fig. 1B), which was in line

Fig. 1. AbundantM2markers expression and PPARγ activation are detected in human periphermonocytes isolated from 20 patients before and after 2 months of atorvastatin treatment. Realevels. (F) Activated PPARγ in nuclear extraction in isolated monocytes were measured usi(*P b 0.05); NS, not significant.

with previous reports of PPARγ agonists treatment [14]. Furthermore,the expression of PPARγ in circulating monocytes was also increased byatorvastatin treatment (Fig. 1E). This observation was concordant withthe strikingly high amount of activated PPARγ protein detected inmono-cytes after atorvastatin therapy for twomonths (Fig. 1F). Taken together,these results suggest that atorvastatin treatment might be capable ofpriming monocytes cells toward an anti-inflammatory M2 phenotypethrough PPARγ activation in vivo.

3.2. Atorvastatin induces PPARγ expression and activation in humanmono-cytes in vitro

We next examined the effect of atorvastatin on the PPARγ mRNAexpression in isolated monocytes. Time course experiments for thePPARγ mRNA expression in monocytes revealed that atorvastatin(10 μM) significantly increased PPARγ mRNA expression levels by ap-proximately 3-fold (versus 0 hour) at 12 and 24 h (Fig. 2A). As shownin Fig. 2B, the PPARγmRNA expression levels were remarkably inducedfollowing atorvastatin treatment in a dose-dependentway. On theotherhand, the nuclear expression of the activated PPARγ of monocytes doseescalation with atorvastatin treatment was also examined and found tobe correspondent to the PPARγ mRNA expression (Fig. 2C). To furtherexplore atorvastatin-mediated PPARγ activation, the mRNA expressionof adipocyte fatty acid binding protein (aP2), a known PPARγ targetgene in monocytes [19], was investigated. As shown in Fig. 2D, aP2mRNA expression was also markedly induced in a dose-dependentmanner by atorvastatin treatment. Furthermore, atorvastatin-inducedPPARγ expression and activation were augmented by PPARγ agonistrosiglitazone and were reversed by PPARγ antagonist T0070907(Fig. 2E-G).

3.3. Atorvastatin promotes alternative differentiation of human monocytesinto macrophages via PPARγ activation and enhances anti-inflammatoryproperties

To determine whether atorvastatin influence M2 differentiation ofhuman monocytes into macrophages, primary human monocyteswere differentiated in vitro into alternative macrophages with IL-4 in

al bloodmonocytes after atorvastatin treatment. RNAwas extracted from peripheral bloodl-time PCR analysis of (A) CD206, (B) CD163, (C) IL-10, (D) CCL18, and (E) PPARγ mRNAng ELISA method. Statistically significant differences between treatments are indicated

Fig. 2. Atorvastatin induces PPARγ expression and activation in human monocytes in vitro. (A) Time course of PPARγ mRNA expression in monocytes after treatment with 10 μM ator-vastatin. (B) PPARγmRNAexpression, (C) activated PPARγ in nuclear extraction, and (D) aP2mRNA expression inmonocytes after treatmentwith indicated concentrations of atorvastatinfor 24 h. (E) PPARγ mRNA expression, (F) activated PPARγ in nuclear extraction, and (G) aP2 mRNA expression in monocytes after treatment with 10 μM atorvastatin for 24 h in thepresence or absence of T0070907 (10 nM) or rosiglitazone (100 nM). Columns,mean (n=6); bars, SD. §P b 0.05 for atorvastatin versus vehicle. *P b 0.05 for atorvastatin plus the indicatedchemicals versus atorvastatin.

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the presence of different concentrations of atorvastatin added only atthe beginning of the differentiation process. As shown in Fig. 3A, theM2 marker CD206 was strongly induced after IL-4 stimulation and thiseffect was amplified by atorvastatin in a dose-dependent manner. Bycontrast, the expression of CD163 was significantly reduced by IL-4,which was consistent with previous studies [14,20,21]. Atorvastatintreatment enhanced the IL-4-induced decrease of CD163 expression inM2 macrophages. In addition, flow cytometry analysis demonstratedthat the amount of both CD206 and CD163 on the surface of M2macro-phages was correspondent to their mRNA expression (Fig. 3B and C).Finally, coincubation of monocytes undergoing M2 differentiationwith the PPARγ antagonist T0070907 completely abolished the regula-tion of CD206 and CD163 expression by atorvastatin, and an additive ef-fect was observed with PPARγ agonist rosiglitazone treatment (Fig. 3D).

To determinewhether atorvastatin-primedM2macrophages can in-fluence the inflammatory M1 macrophages, indirect coculture experi-ments were performed, and proinflammatory cytokines such as TNF-αand MCP-1 release by M1 macrophages were subsequently quantified.Incubation of M1 macrophages with medium from M2 macrophages-derived culture supernatant resulted in a pronounced inhibition ofTNF-α and MCP-1 (Fig. 3E). Furthermore, this inhibitory effect wasstrongly augmented when atorvastatin were added at the beginningof the alternative differentiation of human monocytes into macro-phages. Moreover, this inhibitory effect by atorvastatin was augmentedby the PPARγ agonist rosiglitazone and completely reversed by thePPARγ antagonist T0070907 (Fig. 3E).

Collectively, these results suggested that atorvastatin-mediatedpromotion of human monocytes differentiation into M2 macrophageseffects is PPARγ dependent.

3.4. p38 MAPK but not ERK1/2 activation is involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2 macrophagephenotype

To further decipher the mechanism involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2 macrophagephenotype, p38 MAPK and ERK1/2 phosphorylations by atorvastatintreatment were assessed. As shown in Fig. 4A and B, atorvastatincould markedly induced phosphorylation of p38 MAPK, however, onlycaused a slight but not significant ERK1/2 activation. Western blot

analysis also confirmed the results of ELISA tests (Fig. 4C and D). Fur-thermore, both atorvastatin-induced PPARγ expression and activationwere abolished by p38 MAPK-specific inhibitor SB203580 in adose-dependent way, whereas no obvious changes were found afterMAPK/ERK kinase-specific inhibitor PD98059 treatment (Fig. 4E andF). Most importantly, SB203580 but not PD98059 treatment blockedPPARγ dependent M2 macrophage differentiation by altering theexpressions of CD206 and CD163 in both mRNA and protein levels(Fig. 4G and H).

4. Discussion

Monocytes are precursors of macrophages, which are prominentcells in the response to lipid accumulation in large arteries that contrib-ute to the development of atherosclerosis and its complications. It ischaracterized that monocytes preferentially migrate to lesions withhigh inflammatory activity and to give rise to the macrophages inatherosclerotic lesions inmice [22,23], indicating that circulatingmono-cytes have the capacity to influence plaque formation. It has been alsoreported that the expression of PPARγ positively correlateswith expres-sion levels of M2 markers, such as CD206, CCL18, and IL-10 in humanatherosclerotic plaques [14,16]. Besides, Pucci et al. demonstrated thatPPARγ expression was higher in coronary plaques and peripheralblood monocytes of statin-treated patients [15]. In the current study,abundant M2markers such as CD206, IL-10, and CCL18 were expressedin human circulatingmonocytes after atorvastatin treatment. In fact, theexpression of PPARγ gene positively correlates with mRNA levels ofM2 markers, such as CD206, CCL18, and IL-10 in peripheral bloodmonocytes of patients in the current study (data not shown). Nota-bly, we also found that increasing expression of PPARγ in mRNAlevel and amount of activated PPARγ protein were detected inmonocytes after atorvastatin administration for 2 months. Previousreports have shown that statins activate PPARγ in monocytes aswell as macrophages [17,24]. Consistent with the previous results,our results confirmed that atorvastatin dose dependently inducedPPARγ expression and activation in human monocytes in vitro. Takentogether, these results demonstrated that atorvastatin-mediatedPPARγ activation in vivo and in vitro, implicating that atorvastatinmight be able to prime monocytes cells toward an anti-inflammatoryM2 phenotype via PPARγ activation.

Fig. 3. Atorvastatin promotes alternative differentiation of humanmonocytes into macrophages via PPARγ activation and enhances anti-inflammatory properties. Primary humanmono-cyteswere differentiated toM2 in the presence or absence of 10 μMatorvastatin added only at the beginning of the differentiation process. (A) Real-time PCR analysis of CD206 and CD163mRNA in RM andM2macrophages. (B) Flow cytometry analysis of CD206 and CD163 on the surface of RM andM2 cells. Data are referred to as mean fluorescence intensity (§P b 0.05 forM2versus RM. *P b 0.05 for indicated concentrations of atorvastatin versus vehicle inM2). (C) Representative figures offlow cytometry. (D) Primary humanmonocyteswere differentiatedto M2 in the presence or absence of T0070907 (10 nM) or rosiglitazone (100 nM) added only at the beginning of the differentiation process. Real-time PCR analysis of CD206 and CD163mRNA in RM and M2 macrophages treated with 10 μM atorvastatin or vehicle. (§P b 0.05 for atorvastatin versus vehicle. *P b 0.05 for atorvastatin plus the indicated inhibitor versusatorvastatin.) (E) TNF-α and MCP-1 were quantified in macrophage supernatants from M1 macrophages, which had been previously exposed to medium from atorvastatin primed-M2macrophage cultureswith orwithout T0070907 (10 nM) or rosiglitazone (100 nM) added only at the beginning of the differentiation process. (§P b 0.05 for atorvastatin versus vehicle.*P b 0.05 for atorvastatin plus the indicated chemicals versus atorvastatin.) Columns, mean (n = 6); bars, SD.

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Fig. 4. p38 MAPK but not ERK1/2 activation is involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2macrophage phenotype. Isolated monocytes were incu-bated with the indicated periods of time with 10 μM atorvastatin. After incubation, cells were then lysed, and (A) p38 MAPK (Total/Phospho (Thr180/Tyr182)) and (B) ERK1/2 (Total/Phospho (Thr202/Tyr204, Thr185/Tyr187)) were quantified by ELISA. Data are referred to as phosphorylated/total protein. Phosphorylation and total levels of (C) p38 MAPK and(D) ERK1/2 were examined. The levels of β-actin served as the loading control. (E) PPARγmRNA expression and (F) activated PPARγ in nuclear extraction in monocytes after treatmentwith 10 μM atorvastatin for 24 h in the presence or absence of various concentrations of SB203580 or PD98059. Primary humanmonocytes were differentiated to M2 in the presence orabsence of SB203580 (10 μM) or PD98059 (10 μM) added only at the beginning of the differentiation process. (G) Real-time PCR analysis and (H) flow cytometry analysis of CD206 andCD163 mRNA in RM andM2macrophages treated with 10 μM atorvastatin or vehicle. Columns, mean (n=6); bars, SD. §P b 0.05 for atorvastatin versus vehicle. *P b 0.05 for atorvastatinplus the indicated inhibitors versus atorvastatin.

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Since the presence of both M1 and M2 macrophage markers hasbeen demonstrated in human atherosclerotic lesions, the concept ofmacrophage heterogeneity has entered the field of atherosclerosisresearch [25,26]. M2 macrophages are present at locations distantfrom the lipid core in more stable zones of the plaque and appear toexert anti-inflammatory properties on M1 macrophages. Intriguingly,PPARγ expression in anti-inflammatory macrophages positively corre-lates with the expression of M2markers [14]. In our hands, atorvastatinwas also shown to induce PPARγ expression and activation in humanmonocytes in vivo and in vitro, thus prompted us to examine whetheratorvastatin plays a role in the differentiation program of monocytesinto the M2 phenotype. As hypothesized, monocytes could beprimed by atorvastatin toward an enhanced M2 phenotype in thepresence of IL-4 stimuli. Furthermore, atorvastatin-activated M2macrophages dampened the inflammatory status of surroundingM1 macrophages and thus exerted a paracrine anti-inflammatory

effect on M1 macrophages. In addition, these phenomena were aug-mented by PPARγ agonist rosiglitazone and completely reversed byPPARγ antagonist T0070907, supporting the notion that an additionalmechanism by which atorvastatin can exert anti-inflammatory activi-ties in macrophages mainly via PPARγ activation. Most importantly,this presents yet another molecular pathway through which statins ex-hibit its anti-inflammatory effects upon the vasculature, inhibitingplaque formation and stabilizing established atherosclerotic plaques[27,28].

As discussed above, the mechanism via which atorvastatin promotedmonocytes toward the M2 phenotype involves PPARγ activation. In fact,PPARγ is negatively regulated by mitogen-activated protein kinase(MAPK) via its phosphorylation [29]. However, atorvastatin does not in-duce serine phosphorylation of PPARγ in humanmonocyte/macrophage(THP-1) cells [30]. Thus, atorvastatin-induced PPARγ activation cannotbe mediated by inhibition of MAPK-dependent serine phosphorylation.

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Previous reports also note that statins activate ERK1/2 and p38 MAPK toinduce PPARγ activation in murine macrophages [17]. Nevertheless, ourresults proved that atorvastatin significantly induced phosphorylationof p38 MAPK while sparing ERK1/2 phosphorylation in human mono-cytes.Moreover, both atorvastatin-induced PPARγ expression and activa-tion were abolished by p38 MAPK-specific inhibitor SB203580, whereasno significant changes were found inMAPK/ERK kinase-specific inhibitorPD98059 treatment. Most importantly, SB203580 but not PD98059treatment blocked atorvastatin priming monocytes cells toward ananti-inflammatory M2 phenotype. The underlying mechanisms behindwhy atorvastatin only activated p38 MAPK but not propelled ERK1/2phosphorylation to cause PPARγ activation and thus enhance alternativeM2 macrophage phenotype remains unknown.

In conclusion, we first found that abundant M2 markers wereexpressed in human circulatingmonocytes after atorvastatin treatment.Moreover, atorvastatinwas able to induce PPARγ expression and activa-tion in human monocytes in vivo and in vitro, resulting in primingprimary human monocytes differentiation into M2 macrophages witha more pronounced paracrine anti-inflammatory activity in M1 macro-phages. Additional molecular approaches revealed that p38 MAPK butnot ERK1/2 activation was involved in atorvastatin-mediated PPARγactivation and enhanced alternative M2 macrophage phenotype. Ourdata provide additional insight into how atorvastatinmight be involvedin attenuating vascular inflammation and atherosclerosis, at least inpart, lead to generation of a macrophage population with enhancedanti-inflammatory properties and highlight an entirely novel biologicalbasis for the atheroprotective effects in atorvastatin treatment.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.intimp.2015.03.005.

Conflict of interest

The authors have no conflict of interest.

Acknowledgements

We thank Dr. Ye Nan for helpful suggestions in discussions andDr. Bing Li for excellent technical assistance.

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