Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation

Cytokine xxx (2013) xxx–xxx

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Cytokine

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Maraviroc reduces cytokine expression and secretion in human adipose cellswithout altering adipogenic differentiation

Julieta Díaz-Delfín a,⇑, Pere Domingo b,c, Marta Giralt a, Francesc Villarroya a

a Department Biochemistry and Molecular Biology, Institute of Biomedicine, University of Barcelona, CIBER Fisiopatologia de la Obesidad y Nutrición (CIBEROBN),08028 Barcelona, Spainb Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spainc Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 June 2012Received in revised form 17 December 2012Accepted 18 December 2012Available online xxxx

Keywords:MaravirocCytokinesLipodystrophyAdipocytesInflammation

1043-4666/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.cyto.2012.12.013

⇑ Corresponding author. Address: Department ofBiology, Faculty of Biology, University of BarcelonaBarcelona, Spain. Tel.: +34 934021549; fax: +34 9340

E-mail address: [email protected] (J. Díaz-D

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Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patientswith CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequentlyappear in HIV-infected patients under HAART, there is no information available on the effects of MVC onadipose tissue. Here we studied the effects of MVC during and after the differentiation of humanadipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured theacquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity,adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally,we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokineexpression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed noalterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistentwith this, there were no changes in the mRNA levels of PPARc or SREBP-1c, two master regulators ofadipogenesis. In addition, MVC caused a significant decrease in the gene expression and release ofpro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflamma-tion-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes indifferentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rathershows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatorycytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissuedevelopment, metabolism, and inflammation, and thus could be a potentially beneficial component ofantiretroviral therapy.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Highly active antiretroviral therapy (HAART) has improved theprognosis of patients with HIV-1 by dramatically suppressingHIV-1 viral load, increasing CD4+ cell counts, and reducing oppor-tunistic infections associated with AIDS [1,2]. However, a high pro-portion of patients on antiretroviral therapy develop metabolicalterations, lipodystrophy, or both [3–5]. In these patients, the adi-pose tissue alterations appear to be related to the appearance of apro-inflammatory status in fat, arising from a complex combina-tion of HIV-1 infection-related events and HAART [6]. Antiretrovi-ral drugs may induce pro-inflammatory processes in adiposetissue, and EFV, some NRTIs and some PIs have been shown to in-duce the expression and release of pro-inflammatory cytokines,

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Biochemistry and Molecular, Avda Diagonal 643, 0802821559.elfín).

et al. Maraviroc reduces cytokdx.doi.org/10.1016/j.cyto.2012.

such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6),in cultured adipocytes [7,8]. Moreover, inflammatory processeswithin adipose tissue depots involve adipocytes along with othercell types (e.g. vascular endothelial cells and macrophages) thatact as both targets and effectors of pro-inflammatory signaling [9].

The inflammatory process that occurs in adipose tissue hasmultiple origins. A recent study showed that the complicationsassociated with HIV-1 infection include the translocation of bacte-ria from the gastrointestinal tract into the circulation [10]. Consis-tent with this, significant amounts of bacterial lipopolysaccharide(LPS), which exerts powerful pro-inflammatory effects, have beenfound in the blood of HIV-1 patients. The role of the potent pro-inflammatory stimulus of LPS in many clinical and infectious disor-ders has been well described for immune cells, such as monocytes,but it is notable that adipocytes are also immuno-reactive cellsthat respond to both LPS and microorganisms [11,12].

In recent years, a considerable research on novel therapeuticagents has focused on the discovery and development of orallyavailable inhibitors of HIV-1 entry into cells. These include

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antagonists of CCR5 [13,14] and CXCR4 [15], which have antiviralproperties, as well as inhibitors of the binding between gp120and CD4 [16]. Maraviroc (MVC), which is a selective antagonistof the CCR5, receptor that HIV-1 uses to enter the cells, has provento be a potent inhibitor of HIV-1 activity and shows favorable phar-macological properties [17]. MVC binds to CCR5, preventing HIV-1from binding to this receptor; when CCR5 is unavailable, CCR5-tro-pic HIV-1 cannot engage with CD4+ cells, which is a prerequisitefor infection. The CCR5-tropic variant of the virus is common inearly HIV-1 infection, whereas viruses adapted to enter via theCXCR4 receptor gradually become dominant as HIV infection pro-gresses [18]. MVC has been approved for use as a part of HAARTin treatment-experienced HIV-1 patients with CCR5-tropic virus[19].

Despite ongoing concerns over the potential adverse effects ofnovel anti-HIV-1 therapeutic agents on metabolism and adiposetissue, there has been no evidence to date that MVC causes overtlipoatrophy or profound effects on the metabolism of treatedHIV-1 patients. However, lymphocytes, macrophages and adipo-cytes all express CCR5 [20], and the possibility that MVC mightinterfere with the physiological functions of CCR5 as a chemokinereceptor in adipose cells cannot be excluded. Studies on the effectsof antiretroviral drugs on adipose cells have proven useful for ini-tial in vitro assessment of the potential for such drugs to disturbadipose tissue and lipid metabolism. In this study, we investigatedthe effects of MVC on adipocyte differentiation, and the expressionand release of adipokines and cytokines by human adipocytes. Fur-thermore, we investigated whether the anti-inflammatory proper-ties of MVC reduced the expression of LPS-activated genes in thesecells.

Fig. 1. Effects of MVC on human pre-adipocytes differentiating in culture. (A)Representative micrographs of human pre-adipocytes differentiated in culture inthe presence of the indicated concentrations of MVC or 4 lM EFV. (B) Bars representthe means ± SEMs of the extent of morphological adipocyte differentiation of three(0.1 lM and 1 lM MVC) or four (Control, 4 lM EFV, 4 lM MVC) independentcultures treated with the indicated drug concentrations, and are expressed relativeto values from untreated control cells, which were defined as 100% (�P < 0.05 forcomparisons of MVC and EFV at the same concentration).

2. Materials and methods

2.1. Materials

Dulbecco’s modified Eagle medium (DMEM)/F12 medium andfetal bovine serum (FBS) were from Gibco (Life Technologies Corp.,Grand Island, NY, USA). All other reagents used for adipocyte cellcultures were from Sigma (St. Louis, MO, USA), with the exceptionof rosiglitazone, which was from Alexis Biochemicals (Enzo LifeSciences Inc., Farmingdale, NY, USA). MVC was supplied by Pfizerand EFV was from Bristol-Myers-Squibb.

2.2. Cell culture and differentiation

Human adipocyte precursor cells from healthy individuals,were obtained from Advancell (Barcelona, Spain) and cultured aspreviously reported [21]. In brief, human pre-adipocytes weremaintained in DMEM/F12 containing 10% FBS and antibiotics at37 �C in a humidified 5% CO2 environment. After the cells reachedconfluence, differentiation was initiated by incubation in DMEM/F12 serum-free containing 20 nM insulin, 0.2 nM triiodothyronine(T3), 100 nM cortisol, 25 nM dexamethasone, 500 mM 3-isobutyl-methyl-xanthine and 2 lM rosiglitazone. After 4 days, the cellswere switched to adipogenic medium (DMEM/F12 containinginsulin, T3, and cortisol only) and maintained for up to 15 days.The extent of morphological differentiation under the variousexperimental conditions was quantified by measuring the percent-age of cell culture surface occupied by adipocytes compared tocontrols (defined as 100%).

2.3. Cell treatment

Two experimental settings were used. For studies on the effectsof MVC on adipocyte differentiation, treatment with MVC or EFV

Please cite this article in press as: Díaz-Delfín J et al. Maraviroc reduces cytokpogenic differentiation. Cytokine (2013), http://dx.doi.org/10.1016/j.cyto.2012

was initiated on day 0 and was maintained throughout the differ-entiation process. Fresh drugs (dissolved in DMSO) were includedwith each change of medium. For studies on the effects of MVCon differentiated adipocytes, differentiated human adipocyteswere incubated with 1 lM MVC and/or 100 ng/mL LPS for 24 h.

2.4. Assessment of cytotoxicity

The potential cytotoxic effects of MVC and EFV on differentiat-ing human pre-adipocytes or differentiated adipocytes weredetermined using a CytoTox96 kit (Promega, Madison, WI, USA)following the manufacturer’s instructions.

2.5. Quantitative real-time RT–PCR

RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden,Germany). Reverse transcription (RT) was performed in a total vol-ume of 20 ll using random hexamer primers (Applied Biosystems,Foster City, CA, USA) and 0.5 lg total RNA. For polymerase chainreaction (PCR), each 20-ll reaction contained 1 ll cDNA, 10 llTaqMan Universal PCR Master Mix, 250 nM probes and 900 nMprimers from the Assays-on-Demand Gene Expression Assay Mix(TaqMan, Applied Biosystems, Foster City, CA, USA). PCR wasconducted using an ABI/Prism 7700 Sequence Detector System(Applied Biosystems, Foster City, CA, USA). Assay-on-Demandprobes for the following targets were used: peroxisome prolifera-tor-activated receptor-c (PPARc; Hs00234592), sterol regulatoryelement-binding protein-1 (SREBP-1; Hs00231674), adiponectin(Hs00605917), leptin (Hs00174877), monocyte chemoattractantprotein 1 (MCP-1; Hs00234140), IL-6 (Hs00174131), IL-8

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(Hs00174103), cytochrome c oxidase subunit IV (COXIV;Hs00266371), and ribosomal protein, large, P0 (RPLP0;Hs99999902). The primers and probe used or the detection ofcytochrome c oxidase subunit II (COII) and assessment ofmitochondrial DNA (mtDNA) abundance were designed using theAssay-by-Design system (Custom TaqMan Gene Expression Assays,Applied Biosystems) and were as follows: 50-CAA ACC ACT TTC ACCGCT ACA C-3’ (forward primer), 50-GGA CGA TGG GCA TGA AACTGT-30 (reverse) and 50-AAA TCT GTG GAG CAA ACC-30 (FAM-labeled probe). The levels of mtDNA were quantified using thisprimer/probe set and expressed relative to the abundance of nucle-ar DNA, which was determined by amplification of the intronlessgene C/EBPa. Controls lacking RNA or primers were included ineach set of experiments. Each sample was run in duplicate andthe mean value of the duplicates was normalized with respect tothat of RPLP0 mRNA and used to calculate the relative amount ofthe individual targets. Each mean value was calculated using thecomparative (2�DCT) method. Parallel calculations were performedusing the 18S rRNA (Hs99999901) as a reference gene, and yieldedthe same results.

2.6. Quantification of adipocyte-secreted adipokines and cytokines

The effects of MVC on the release of adiponectin, leptin, MCP-1,IL-6, IL-8, total plasminogen activator inhibitor type-1 (PAI-1),hepatocyte growth factor (HGF), and nerve growth factor (NGF)during the adipocyte differentiation process were determinedusing 25 ll of culture medium collected prior to cell harvesting.The levels of these factors in the medium were quantified using a

Fig. 2. Effects of MVC on the expression of genes related to adipogenic function andadipocytes were differentiated in culture in the presence of the indicated concentrations(B) inflammatory genes; and (C) mitochondrial toxicity markers were assessed. The data4 lM EFV, 4 lM MVC) independent cultures treated with the indicated drug concentratdefined as 1. (�P < 0.05 versus controls; §P < 0.05, for comparisons of MVC and EFV at th

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multiplex analysis system employing fluorescently labeledmicrosphere beads linked to specific antibodies (Linco Research/Millipore, Saint Charles, MO, USA). Fluorescence was detectedusing a Luminex100IS v2 system (Luminex Corp., Austin, TX, USA).

2.7. Statistics

Statistical analyses were performed using the Student’s t-test.Differences with P-values < 0.05 were considered statisticallysignificant.

3. Results

3.1. Effects of MVC on adipocyte differentiation status and expressionof inflammatory cytokines

We first investigated the effects of different doses of MVC (0.1,1, and 4 lM) on adipocyte cell differentiation. For comparativepurposes and as a positive control for inhibition of adipogenic dif-ferentiation, cells were treated with 4 lM EFV [21]. MVC and EFVwere not cytotoxic to human adipose cells at any of the concentra-tions tested (data not shown). As shown in Fig. 1A, which presentsrepresentative microscopic images of drug-treated cells, and aspreviously reported [22,23], 4 lM EFV blocked the conversion ofpre-adipocytes to adipocytes. In contrast, the differentiation ofcells treated with MVC was indistinguishable from that of controlcells at all tested concentrations. A quantitative assessment ofthe extent of adipogenic differentiation in drug-treated humanpre-adipocytes relative to non-treated cells is shown in Fig. 1B.

inflammatory actions in human adipocytes differentiating in culture. Human pre-of MVC (black bars) or 4 lM EFV (gray bars). (A) Adipogenesis and adipokine genes;are presented as means ± SEMs from three (0.1 lM and 1 lM MVC) or four (Control,ions, and are expressed relative to values from untreated control cells, which weree same concentrations.)

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Fig. 3. Effects of MVC on the release of selected adipokines and cytokines by humanadipocytes differentiating in culture. Human pre-adipocytes were differentiated inculture in the presence of the indicated concentrations of MVC (black bars) or 4 lMEFV (gray bars). Values represent concentrations in the cell culture medium,presented as means ± SEMs from three (0.1 lM and 1 lM MVC) or four (Control,4 lM EFV, 4 lM MVC) independent experiments. (�P < 0.05 versus controls;§P < 0.05 for comparisons of MVC and EFV at the same concentrations.)

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3.2. Effects of MVC on mitochondrial DNA levels and mRNA expressionlevels for marker genes of adipogenesis, inflammation andmitochondrial toxicity in differentiating human adipocytes

The extent of lipid accumulation in MVC-treated cells was con-sistent with the absence of an effect of MVC on mRNA expressionof PPARc and SREBP-1, two master regulators of adipogenesisand lipid accumulation (Fig. 2A). Treatment with MVC at concen-trations up to 4 lM also had no effect on the mRNA levels of adipo-nectin or leptin. In contrast, 4 lM EFV reduced significantly themRNA expression of adipogenesis markers (PPARc, SREBP-1,adiponectin, and leptin). 4 lM MVC decreased significantly themRNA expression levels of MCP-1, IL-6 and IL-8 mRNA, whereasEFV caused an increase in the gene expression levels of these cyto-kine-encoding genes (Fig. 2B). In addition, MVC did not cause mito-chondrial toxicity at any of the concentrations tested, as evidencedby the absence of significant changes in COIV (nuclear DNA-en-coded) and COII (mtDNA-encoded) mRNA levels, and the relativelevels of mitochondrial DNA (Fig. 2C), in contrast with the markeddecrease caused by EFV, a sign of mitochondrial toxicity.

3.3. Effects of MVC on the release of adipokines and inflammatorycytokines by differentiating human adipocytes

We next studied the effects of MVC on the secretion of regula-tory proteins by differentiating human adipocytes. While 4 lM EFVdecreased the release of adiponectin into the medium, 4 lM MVCfailed to alter adiponectin release and decreased that of leptin toa lesser extent than EFV (Fig. 3). MVC (4 lM) decreased signifi-cantly the release of the pro-inflammatory cytokines MCP-1, IL-6and IL-8 into the cell culture medium (Fig. 3) whereas EFV in-creased significantly the secretion of these cytokines. With respectto other regulatory proteins secreted by adipocytes, MVC had noeffect on the release of total PAI-1 or NGF and reduced significantlythe secretion of HGF, whereas EFV increased the secretion of thesefactors (Fig. 3). Thus, unlike EFV, MVC did not alter adipocyte dif-ferentiation and inhibited pro-inflammatory cytokine expressionand secretion, suggesting that MVC exerts anti-inflammatory ef-fects on adipocytes.

3.4. Effects of MVC on the expression and release of cytokines inpreviously differentiated human adipocytes

We next examined the effects of MVC on mature (previouslydifferentiated), human adipocytes. Treatment of differentiated hu-man adipocytes with 1 lM MVC did not alter the mRNA levels ofadiponectin or leptin but decreased significantly the expressionlevels of MCP-1, IL-6, and IL-8 (Fig. 4A). Although MVC did not alterthe secretion of adiponectin by adipocytes, it reduced significantlythe secretion of leptin (Fig. 4B). Consistent with the gene expres-sion results, MVC decreased significantly the release of MCP-1,IL-6, IL-8, and NGF proteins into the adipocyte culture medium(Fig. 4B). Collectively, these data demonstrate that MVC reducesthe pro-inflammatory basal state in human differentiated adipo-cytes, as reflected by its effects on the expression and release ofpro-inflammatory cytokines.

3.5. Effects of MVC on LPS-induced inflammatory gene expression inhuman adipocytes

The anti-inflammatory properties of MVC suggest that this drugmay have a palliative effect on inflammatory processes. It has alsobeen proposed that adipose tissue is a target of the pro-inflamma-tory effects elicited by LPS and studies have shown that pre-adipo-cyte-derived cell lines are sensitive to LPS [24]. Here, we found thattreatment of human adipocytes with LPS increased significantly

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the mRNA expression levels of MCP-1, IL-6, and IL-8 genes(Fig. 5). Treatment with 1 lM MVC reduced significantly theLPS-induced mRNA expression of inflammatory genes in humanadipocytes, abrogating LPS-induced MCP-1 gene expression andreducing the LPS-induced increases in the mRNA expression levelsof IL-6 and IL-8 (Fig. 5). Taken together, our results indicate thatMVC has an anti-inflammatory activity, inhibiting both basal andLPS-stimulated pro-inflammatory chemokine and cytokine expres-sion in adipocytes.

4. Discussion

In the current study, we provide the first description of the ef-fects of MVC on human adipocytes in vitro. Several antiretroviraldrugs have been shown to have direct adverse effects on culturedadipocytes, impairing differentiation, enhancing the production ofpro-inflammatory cytokines, and repressing the release of anti-inflammatory adipokines such as adiponectin [25,26]. It is thoughtthat both HAART and HIV-1 infection-related events, such as theextracellular appearance of the HIV-1-encoded proteins Tat and/

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Fig. 4. MVC inhibits the mRNA expression and release of inflammation-related cytokines in previously differentiated human adipocytes. Differentiated human adipocyteswere exposed to 1 lM of MVC. (A) Effects of MVC on the expression of inflammation-related genes. Data are expressed relative to values from untreated control cells (definedas 1). (B) Effects of MVC on the release of selected cytokines from differentiated human adipocytes. Values are means ± SEMs of the relative mRNA levels (A) and adipokine/cytokine concentrations (B) from four independent experiments (�P < 0.05, versus controls).

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Please cite this article in press as: Díaz-Delfín J et al. Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adi-pogenic differentiation. Cytokine (2013), http://dx.doi.org/10.1016/j.cyto.2012.12.013

Fig. 5. Effects of MVC on LPS-regulated gene expression in human adipocytes.Human adipocytes were treated with 100 ng/mL LPS with or without 1 lM MVC.Data are means ± SEMs from four independent experiments and expressed relativeto values from untreated control cells (�P < 0.05 versus controls; §P < 0.05 for LPS/MVC versus LPS-treated cells).

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or Vpr, act on adipocytes [27,28] to contribute to the release ofinflammatory cytokines and impair the release of adiponectin byadipocytes. Here, we show that MVC treatment reduced theexpression and secretion of pro-inflammatory cytokines, includingMCP-1, IL-8, and IL-6, without affecting the adipocyte differentia-tion status. In addition, MVC decreased the secretion of other adi-pocyte-secreted regulatory proteins known to be associated withthe pro-inflammatory status of adipose tissue, such as NGF andHGF [29,30]. Thus, it appears that MVC exerts anti-inflammatoryeffects that are opposite to the pro-inflammatory action of otherantiretroviral drugs such as EFV. Perhaps consistent with this no-tion, a recent clinical study showed that patients on MVC regimenstend to have earlier, more modest decreases in certain markers ofimmune activation and inflammation (CD38 expression in CD4+ T

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cells, plasma levels of D-dimers and C reactive protein) comparedto those on EFV regimens [31].

Also consistent with its observed effects on adipocytes, MVCtreatment has been shown to play a role in decreasing HIV-associ-ated chronic inflammation by blocking the recirculation and traf-ficking of macrophages and monocyte-derived dendritic cells[32]. In clinical studies, MVC treatment was also shown to triggerlarger increases in CD4+ T-cell counts than could be accountedfor based on its antiviral effects alone [33,34]. Furthermore, a studyof MVC-treated patients showed that this increase in CD4+ T-cellnumbers involved the modulation of T cell activation andinflammation [31]. Collectively, these reports from clinical studiesindicate that MVC possesses anti-inflammatory properties. Theanti-inflammatory actions of MVC on adipocytes observed in thecurrent in vitro study suggest that the beneficial effects of MVCon the inflammatory process in HIV-1-infected patients may alsotake place in the adipocyte cell population. This is especiallyrelevant given the importance of adipose tissue inflammation asa pivotal event in the metabolic alterations ranging from insulinresistance to lipodystrophy [9].

An interesting finding in the present study is MVC-inducedreduction in leptin secretion by human adipocytes. The fact thatit was associated with mild, not statistically significant reductionsin leptin mRNA expression, may be related to cumulative nature ofthe leptin levels determined in the culture medium after exposureto drugs. In HIV-1-infected patients undergoing HAART, leptin lev-els are mostly dependent on the amount of body fat, and lipoatro-phy and lipohypertrophy appear to be associated with reduced andelevated leptin levels, respectively [35,36]. When not accompaniedby fat redistribution, the current HAART protocols do not seem toaffect serum leptin, despite earlier observations suggesting a tran-sient increase in leptin levels during the early phase of HAART [36].Although the synthesis of leptin is up-regulated by inflammatorymediators [37,38], the mechanism by which leptin secretion is in-duced in acute inflammation remains unclear. Several authors havecorrelated leptin increases during inflammation with the direct ac-tions of different pro-inflammatory cytokines on fat cells [39].Thus, it is possible that the down-regulation of leptin observed inthe present study was secondary to the MVC-induced reductionof pro-inflammatory cytokines.

Notably, our data indicate that MVC moderates the LPS-inducedenhancement in pro-inflammatory gene expression and release byadipose cells. Furthermore, it is known that LPS induces IL-6, TNF-a, and resistin in human adipose tissue in vivo [40]. Examining ofinnate immunity on adipokines is particularly interesting in lightof evidence suggesting that adipokine signaling modulates themetabolic complications associated with HIV-1 infection. TheMVC-induced reduction in LPS-dependent activation of MCP-1,IL-6, and IL-8 expression in adipocytes is consistent with MVChaving a potential modulatory effect on the relationship betweeninnate immunity and adipose tissue metabolism.

There are obvious limits on the translation of our adipocyte cellculture results to the clinic. First, the present study examined theaction of MVC only in adipocytes, and while this is the predomi-nant cell type in adipose tissue depots, other target cells of MVC(e.g. infiltrating macrophages and lymphocytes) are present in adi-pose tissue and may complicate the response of the tissue to MVC.Another potential limitation concerns the concentration of MVCused to treat adipocytes in vitro versus MVC concentrations thathave been achieved clinically in vivo. Reports on MVC concentra-tions in plasma from HIV-1-infected patients receiving MVC as partof HAART have indicated that plasma MVC levels can range from0.01–0.05 lM to 1 lM [41–43], although there are reports ofpatients with plasma MVC concentrations as high as 5 lM [44].Here, we found that MVC did not exert cytotoxic nor anti-adipo-genic effects even at concentrations exceeding the usual plasma

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levels of MVC; the concentration range of MVC found to reducepro-inflammatory gene expression and release was in the lowmicromolar range. Although this is at the upper range of the typicalplasma MVC levels reported to data, it cannot be excluded thatadipocytes in vivo are exposed to high local concentrations ofMVC (i.e. approaching the low micromolar range). In fact, severalantiretroviral drugs from different drug families are reported tobe present at a higher local concentration in adipose tissue thanin systemic blood [45]. Unfortunately, no such on MVC concentra-tions in adipose tissue from treated patients are available. Thus,while the beneficial effects of MVC on inflammatory signalingobserved herein may be relevant in vivo, further studies will beneeded to confirm this possibility.

In summary, the present results demonstrate that MVC did notalter adipocyte differentiation, but rather inhibited the expressionand secretion of pro-inflammatory cytokines, and thus showedanti-inflammatory properties. These findings suggest that MVCtreatment could be envisaged as a potentially beneficial compo-nent of HAART with the potential to minimize adverse effects onadipose tissue development, metabolism and inflammation.

Acknowledgements

The study was supported by grants from Ministerio de Ciencia eInnovación (SAF2008-01896), Fondo de Investigaciones Sanitarias(PI11-00376) and Red de Investigación en SIDA (RD06/006/0022),Instituto de Salud Carlos III, Spain, and an independent grant fromPfizer. This company had no role in the study design, data collec-tion, and interpretation of data or manuscript preparation.

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