Original article HIV-1 Tat protein impairs adipogenesis and induces the expression and secretion of proinflammatory cytokines in human SGBS adipocytes

Transcription

1 Antivirl Therpy 2012; 17: (oi: /IMP2021) Originl rtile HIV-1 Tt protein impirs ipogenesis n inues the expression n seretion of proinflmmtory ytokines in humn SGBS ipoytes Juliet Díz-Delfín 1 *, Pere Domingo 2, Mrtin Witsh 3, Mrt Girlt 1, Frnes Villrroy 1 1 Deprtment e Bioquími i Biologi Moleulr, Institut e Biomeiin e l Universitt e Brelon (IBUB), Universitt e Brelon n CIBER Fisioptologi e l Oesi y Nutriión (CIBEROBN), Brelon, Spin 2 Infetious Diseses Unit, Hospitl e l Snt Creu i Snt Pu, Autonomous University of Brelon, Brelon, Spin n Re e Investigión en SIDA (RIS), Instituto e Slu Crlos III, Spin 3 Deprtment of Peitris n Aolesent Meiine, University of Ulm, Ulm, Germny *Corresponing uthor e-mil: izelfin_j@yhoo.om Bkgroun: HIV-1 Tt protein hs een shown to ply multiple roles in the pthogenesis of AIDS; however, there is no informtion urrently ville on its effets on ipose tissue ltertions. We hve stuie the effets of Tt on SGBS ipoytes to gin insight on its role on the evelopment of lipoystrophy. Methos: SGBS preipoytes were expose to Tt uring n fter ifferentition. Aquisition of ipoyte morphology, expression of gene mrkers of ipogenesis n inflmmtion, relese of ipokines n ytokines to the meium, n gluose uptke were mesure. The tion of Tt on tumour nerosis ftor (TNF)--regulte messenger RNA expression ws etermine in ifferentite ipoytes. The pity of rosiglitzone, resvertrol n prthenolie to influene the tion of Tt ws lso ssesse. Results: Tt tretment reue the numer of SGBS preipoytes tht quire ipoyte morphology. It lso le to repression of ipogeni gene expression n inue the oorinte expression n relese of proinflmmtory ytokines in humn ipose ells. Moreover, omine tretment with Tt n TNF- proue n itive effet on the repression of ipoyte genes. The oserve effets of Tt on gene trnsription in ipoytes were ue, in prt, to TNF- tht ws serete s onsequene of intrellulr exposure to Tt. lusions: Tt impirs ipogenesis in humn SGBS preipoytes n inreses the expression n relese of proinflmmtory ytokines. Positive rosstlk etween Tt n TNF- ontriutes to the nti-ipogeni n proinflmmtory effets. HIV-1 Tt protein my ply role in the ipose tissue ltertions tht ultimtely le to lipotrophy n systemi metoli isturnes oserve in HIV-1-infete ptients. Introution Lipoystrophy n metoli isturnes reminisent of the metoli synrome (tht is, insulin resistne n yslipiemi) re frequently oserve in HIV-1-infete ptients unergoing ntiretrovirl tretment. The ltertions in ipose tissue in these ptients re thought to ontriute to the systemi metoli isturnes. Thus, it hs een reporte tht HIV-1-infete ptients who present with lipoystrophy hve normlly low levels of iponetin n, in some ses, leptin in the loo. Systemi n lol inreses in the levels of proinflmmtory ytokines in ipose tissue hve lso een reporte [1,2]. These ltertions pper to evelop s onsequene of omplex set of pthogeni insults. Some ntiretrovirl rugs, suh thymiine nlogue inhiitors of reverse trnsriptse, re espeilly prone to eliit peripherl ipose tissue trophy, wheres protese inhiitors pper to e ssoite primrily with systemi metoli isturnes [3,4]. However, severl oservtions point to role for HIV-1 infetion per se in the evelopment of lipoystrophy n ssoite metoli ltertions. Aoringly, mil ltertions in ipose tissue istriution [5] n gene expression [6] re oserve in HIV-1-infete ptients efore ntiretrovirl tretment hs een initite. Some of the ipose tissue ltertions in HIV-1-infete ptients my e ttriutle to proinflmmtory environment in 2012 Interntionl Meil Press (print) (online) 529

2 J Díz-Delfín et l. ipose tissue use y the infetion of ells within ipose epots, suh s mrophges n, possily, lymphoytes [2]. Inee, inrese levels of tumour nerosis ftor (TNF)- expression hve een oserve in ipose tissue from HIV-1-infete tretment-nive ptients [6]. The possiility tht ipoytes might e infete y HIV-1 hs een mtter of ete [7 9], n reent t inite tht suh infetions my e signifint in the ontext of n environment with high levels of TNF- [10]. HIV-1 genome-enoe proteins might e mong the gents tht potentilly eliit ltertions in ipose tissue. To te, only virl protein R hs een reporte to e ple of influening ipoyte funtion, whih it omplishes vi repression of peroxisome prolifertortivte reeptor (PPAR)-g funtion [11]. In the present stuy, we hve nlyse the role of Tt on ipoyte ifferentition n funtion. Tt is n HIV-1 regultory protein require for effiient virl gene expression [12]. It interts with the HIV-1 genome long-terminl repet to enhne trnsription n RNA proessing [13]. In ition to its effets on virl replition, Tt is serete extrellulrly y infete ells n n enter uninfete ells n lter multiple ellulr funtions [14,15]. Moreover, Tt is present t sustntil levels in the serum of infete ptients [16,17], n my exert prrine effets on ells in the neighourhoo of infete ells s well s on istnt ells n tissues [18]. In ition, Tt moultes host ellulr mehnisms, therey ontriuting to immune system mlfuntion. For exmple, Tt hs een shown to ffet the proution of ytokines, suh s interleukin (IL)-10, TNF- n tumour growth ftor-, in ifferent ell systems [19 21] n to inue poptosis of T-ells [18]. The extrellulr n intrellulr HIV Tt hs een shown to ply multiple roles in the pthogenesis of AIDS [22]; however, there is no informtion urrently ville on the effets of HIV Tt protein on the ipoyte environment, whih is ruil for the evelopment of lipoystrophy. In the present stuy, we nlyse the role of the HIV-1 Tt protein in the ifferentition n funtion of SGBS ipose ells. Our results provie the first iret eviene tht Tt lters ipogenesis n ipokine relese, n promotes ytokine proution in ipose ells. Methos Mterils Duleo s moifie Egle meium (DMEM)/F12 meium n fetl ovine serum were from Gio (Life Tehnologies Corp., Grn Isln, NY, USA). All other regents use for ipoyte ell ultures were from Sigm (St Louis, MO, USA), with the exeption of rosiglitzone, whih ws from Alexis Biohemils (Enzo Life Sienes In, Frmingle, NY, USA). TNF- neutrlizing ntioy ws purhse from R&D Systems (Minnepolis, MN, USA). Cell ulture n ifferentition Humn SGBS preipoytes were ulture n ifferentite into mture ipoytes s previously esrie [23]. In rief, SGBS preipoytes were mintine in DMEM/ F12 ontining 10% fetl ovine serum n ntiiotis t 37 C in humiifie 5% CO 2 environment. After ells h eome onfluent, ifferentition ws initite y first inuting ells for 4 ys in DMEM/F12 serum-free ontining 20 nm insulin, 0.2 nm triioothyronine n 100 nm ortisol, supplemente with 25 nm exmethsone, 500 mm 3-isoutyl-methyl-xnthine n 2 mm rosiglitzone (Quikiff meium). Susequently, the ells were swithe to ipogeni meium (ontining insulin, triioothyronine n ortisol only) n mintine for up to 16 ys. Cell tretment n enovirl trnsution Experiments were performe using two experimentl settings. For stuies on the effets of Tt on ipoyte ifferentition, tretments with reominnt Tt (70 nm; Dithev, Fno, Itly) or TNF- (1 ng/ml) were initite on y 0 n ontinue throughout the entire ifferentition proess. The levels of Tt in HIV- 1-infete ptients were reporte to e in nm rnge [16,17]. However, the reliility of in vivo mesurements of Tt is mtter of isussion, mong other resons euse Tt in vivo might e sequestere y enogenous nti-tt ntioies [15,17,22,24], n it is possile tht Tt onentrtions surrouning HIV-1-infete ells re higher thn those mesure using stnr ville ntioies [25]. Previous stuies on the effets of Tt in vriety of ell types report the inution of proinflmmtory pthwys t 100 nm [26 29]. Therefore, the present stuy use similr onentrtions of Tt, whih re t the lower rnge of the Tt oses use in omprle stuies, n whih hve een foun to e pproprite to moel in vitro the effets of Tt in vivo. Previous pilot experiments using 10 nm Tt i not yiel sustntil effets on quisition of SGBS ipoyte morphology or expression of mrker genes of ipogenesis n inflmmtion (t not shown). For stuies on the effets of Tt on ifferentite ipoytes, ifferentite SGBS ipoytes were trnsue with n enovirl vetor expressing Tt (ACMV- Tt) or ontrol enovirl vetor (ACMV-GFP) t multipliity of infetion of 100 for 4 h in serum-free DMEM/F12, n ells were inute for further 48 h in fresh ipogeni meium. Tt levels were mesure in pellete ells from enovirl trnsution experiments using Tt ELISA ssy (Dithev) n the onentrtion ttine ws 54 ±18 nm. When inite, fter inuting for n itionl 24 h in fresh ipogeni Interntionl Meil Press

3 HIV-1 Tt protein inues proinflmmtory ytokines in humn ipoytes meium, ipoytes expressing Tt or GFP were trete for 24 h with TNF- (10 ng/ml), rosiglitzone (10 mm), resvertrol (50 mm) or prthenolie (5 mm). Quntifition of ipoyte-serete ipokines n ytokines The effets of Tt on the relese of iponetin, IL-8, IL-6 n monoyte hemottrtnt protein (MCP)-1 uring the ipoyte ifferentition proess ws etermine using 25 ml of ulture meium ollete efore hrvesting ells. The levels of these ftors in the meium were quntifie using multiplex nlysis system employing fluoresently lelle mirosphere es linke to speifi ntioies (Lino Reserh/Millipore, Sint Chrles, MO, USA). Fluoresene ws etete using Luminex100IS v2 system (Luminex Corp., Austin, TX, USA). Assessment of ytotoxiity Potentil ytotoxi effets of Tt or TNF- on ifferentiting SGBS preipoytes or on ipoytes when lrey ifferentite were etermine y the Cyto- Tox96 kit (Promeg, Mison, WI, USA) following the mnufturer s instrutions. Quntittive rel-time reverse trnsription PCR RNA ws extrte using n RNesy Mini Kit (Qigen, Hilen, Germny). Reverse trnsription (RT) ws performe in totl volume of 20 ml using rnom hexmer primers (Applie Biosystems, Foster City, CA, USA) n 0.5 mg totl RNA. PCR ontine 1 ml omplementry DNA, 10 ml TqMn Universl PCR Mster Mix (Applie Biosystems), 250 nm proes n 900 nm primers from Assys-on-Demn Gene Expression Assy Mix (TqMn; Applie Biosystems) in totl volume of 20 ml, n were onute using n ABI/Prism 7700 Sequene Detetor System (Applie Biosystems). Assy-on-Demn proes for the following trgets were use: TNF- (Hs ), PPAR-g (Hs ), iponetin (Hs ), MCP-1 (Hs ), IL-6 (Hs ), IL-8 (Hs ), gluose trnsporter type 4 (GLUT4; Hs ), n 18S riosoml RNA (Hs ). trols lking RNA or primers were inlue in eh set of experiments. Eh smple ws run in uplite n the men vlue of the uplite ws use to lulte the reltive mount of iniviul trgets. Eh men vlue of messenger RNA (mrna) ws normlize to tht of 18S riosoml RNA using the omprtive (2 -DCT ) metho, following the mnufturer s instrutions. Prllel lultions using the RPLP0 referene gene (Hs ) were performe n results were essentilly the sme. Determintion of 3 H-lelle 2-eoxygluose uptke Prior to ssying for gluose uptke, ipoytes were trnsue with GFP or Tt enovirl vetors, inute for 24 h, then inute for nother 24 h in the presene or sene of TNF-. Next, ells were inute in Kres-Ringer phosphte uffer (137 mm NCl, 4.7 mm KCl, 2.5 mm CC1 2, mm MgS0 4 n 20 mm HEPES ph 7.4) supplemente with 2 mm soium pyruvte n 0.2% ovine serum lumin. Therefter, 2-eoxy-D-[ 3 H] gluose (Amerin Riolele Chemils, In., St louis, MO, USA) ws e t finl onentrtion of 0.1 mm (1 mci) n ssys were stoppe fter 10 min y pling the ells on ie n ing the stop solution (50 mm gluose in phosphteuffere sline). After terminting the trnsport ssy with three wshes of stop solution, eh monolyer ws soluilize in 1 M NOH-0.1% soium oeyl sulfte. A 0.2 ml liquot ws remove for etermintion of riotivity y liqui sintilltion ounting. Mesurements were me in triplite n orrete for non-speifi iffusion, n normlize to totl protein ontent s etermine y Brfor nlysis. Sttistis The istriution of t ws ontrolle for normlity. Unpire Stuent s t-test ws use to test the level of signifine of the ifferenes etween mens, n to test whether men ws signifintly ifferent from zero (in the se of non-etetle vlues). Results Tt inhiits SGBS ipoyte ifferentition n inues the expression of proinflmmtory genes The influene of Tt on ipogenesis ws exmine uring onversion of SGBS preipoytes into ipoytes. SGBS ells, whih pper funtionlly similr to norml humn ipoytes, re not immortlize ut retin high pity for ifferentition into mture ipoytes over mny genertions [23]. To evlute the effets of Tt on ipoyte ifferentition, we expose ells to 70 nm reominnt Tt or 1 ng/ml TNF- (for omprtive purposes) uring the entire ifferentition proess. These tretments with Tt or TNF- were not ytotoxi to SGBS ells (t not shown). Representtive mirosopy imges of Tt n TNF--trete ells show the morphologil hnges ssoite with the quisition of the ipoyte phenotype uring the in vitro ifferentition proess (Figure 1A). Tt tretment reue the numer of ells tht quire ipoyte morphology. The numer of ells tht umulte lipi vuoles ws even more mrkely reue y TNF- tretment. To etermine whether Tt moulte gene expression in SGBS ells, we quntifie mrna levels of iponetin, PPAR-g, GLUT4, IL-8, IL-6 n MCP-1 using rel-time RT-PCR. sistent with the oserve morphologil effets, Tt erese the expression of gene mrkers of ipogeni ifferentition, Antivirl Therpy

4 J Díz-Delfín et l. Figure 1. Tt inhiits SGBS ipoyte ifferentition n lters the expression n relese of genes relte to ipogenesis n inflmmtion A Tt TNF-α B Aiponetin mrna PPAR-γ mrna GLUT4 mrna Tt TNF-α Tt TNF-α Tt TNF-α IL-8 mrna IL-6 mrna MCP-1 mrna Tt TNF-α Tt TNF-α Tt TNF-α C Aiponetin, ng/ml IL-8, pg/ml 2,000 1, IL-6, pg/ml 1,600 1, Tt TNF-α Tt TNF-α Tt TNF-α Tt TNF-α MCP-1 ng/ml SGBS preipoytes were ulture n ifferentite into mture ipoytes in the presene of 70 nm exogenous Tt or 1 ng/ml tumour nerosis ftor (TNF)-. (A) Representtive mirogrphs. (B) Effets on iponetin, peroxisome prolifertor-tivte reeptor (PPAR)-g, gluose trnsporter type 4 (GLUT4), interleukin (IL)-8, IL-6 n monoyte hemottrtnt protein (MCP)-1 messenger RNA (mrna) levels. Totl RNA ws isolte, n smples were nlyse y quntittive reverse trnsription PCR. (C) Effets of Tt n TNF- on the relese of iponetin n proinflmmtory ytokines into the ell ulture meium y SGBS ipoytes. Results re expresse s protein onentrtion. Dt re presente s men ±sem from four inepenent experiments, n re expresse reltive to vlues from untrete ontrol () ells: P<0.05, P<0.001 n P<0.01 versus Interntionl Meil Press

5 HIV-1 Tt protein inues proinflmmtory ytokines in humn ipoytes inluing iponetin (2.2-fol), PPAR-g (1.3-fol) n GLUT4 (1.7-fol; Figure 1B). In ition, Tt inue n inrese in the expression of mrnas for genes involve in proinflmmtory tions, inluing IL-8 (4.1-fol), IL-6 (1.6-fol) n MCP-1 (1.8-fol; Figure 1B). Similrly, IL-8, IL-6 n MCP-1 mrna levels were up-regulte y TNF- tretment, n iponetin, PPAR-g n GLUT4 mrna levels were own-regulte (Figure 1B). Thus, our t inite tht Tt oorintely represses the expression of genes enoing ipogeni proteins n uses oorinte inution of proinflmmtory proteins in ifferentiting ipose ells. Tt n TNF- similrly moulte the relese of ipokines from SGBS ipoytes The effets of Tt or TNF- on the relese of regultory proteins y SGBS ipoytes were stuie using multiplex nlysis system (Figure 1C). Tt erese the relese of iponetin (1.4-fol) n use signifint inrese in the levels of IL-8 (1.3-fol), IL-6 (1.9-fol) n MCP-1 (1.4-fol) in the ulture meium ompre with tht in meium onitione y untrete ipoytes. TNF- use similr, leit more mrke, hnges in seretion, inuing 6.7-fol erese in the level of iponetin, n inrese the levels of IL-8, IL-6 n MCP-1 in the ulture meium y 30-fol, 4.2-fol n 2.2-fol, respetively. Tt ifferentilly moultes the expression of genes relte to inflmmtory tions n ipogeni funtion in ifferentite SGBS ipoytes We next unertook prllel stuy of the short-term effets of Tt on the expression of inflmmtory n ipogenesis genes in mture, ifferentite SGBS ipoytes. Pilot stuies inite tht 48-h tretment with reominnt 70 nm Tt proue very mil effets, for exmple ner to twofol inution of MCP-1 expression n no signifint effet on mrker genes of ipogenesis suh s iponetin or PPAR-g (t not shown). To improve effiy, we rete n experimentl setting tht fvoure intrellulr Tt exposure, employing gene trnsfer protool using n enovirl vetor to rive Tt expression. Differentite SGBS ipoytes were trnsue with ontrol (ACMV-GFP) or the Tt (ACMV-Tt) enovirl vetors on y 16 fter inititing the ifferentition proess, t whih time >95% of the ells ontine multiple lipi roplets. More thn 90% of mture ipoytes exhiite GFP fluoresene, initing effiient trnsution. This experimentl setting resulte in Tt onentrtions in the intrellulr frtion in the 50 nm rnge (see Methos setion). The mrna levels of IL-8 n MCP-1 were signifintly inrese in ells enogenously expressing Tt (Figure 2A). Although sl levels of TNF- mrna were very low in SGBS ipoytes in the ontrol onition (pproximtely yle threshol [CT] in the quntittive RT-PCR ssy), Tt inrese TNF- mrna expression signifintly (Figure 2A). To stuy the tion of Tt on the expression of ipogeni mrkers, we nlyse the levels of iponetin, PPAR-g n GLUT4 mrna. In ontrst to its vrile effets on proinflmmtory ytokine genes, Tt use signifint own-regultion in the mrna levels of ll three ipogeni mrker genes (Figure 2A). sistent with the gene expression results, overexpression of Tt signifintly inrese seretion of IL-8 n MCP-1 into the meium (Figure 2B) n iponetin levels in the meium were not signifintly ltere y Tt. Colletively, these t inite tht Tt represses the overll expression of ipogeni genes n inues oorinte hnge in the expression n relese of proinflmmtory ytokines in humn ipose ells. Comine tretment with Tt n TNF- hs generlly itive effets on ytokine n ipokine expression in ifferentite SGBS ipoytes In ition to stuying the effets of Tt lone on mture ipoytes, we lso nlyse the tion of Tt on TNF--regulte mrna expression. Tretment of SGBS ipoytes with TNF- lone use oorinte up-regultion of proinflmmtory ytokines, inresing the mrna levels of IL-8 n MCP-1 y 1,400-fol n 50-fol, respetively, n it lso inue rmtilly the expression of TNF- mrna itself (Figure 3). This is in greement with previous reports on SGBS ipoytes [30]. The ition of Tt use further signifint inrese in IL-8 n TNF- mrna levels eyon tht inue y TNF- lone (Figure 3A). Like Tt, TNF- lone own-regulte ipogeni genes, reuing iponetin, PPAR-g n GLUT4 mrna to levels tht were 0.37, 0.39 n 0.19, respetively, of those in untrete ontrols. Tt over-expression further reue the expression of ipoyte genes, eresing their mrna levels to one-hlf the lrey low vlues proue y TNF- tretment (Figure 3A). Tt ereses gluose uptke in SGBS ipoytes To etermine whether Tt ffete gluose trnsport, we trnsue mture SGBS ipoytes with ACMV-Tt or ACMV-GFP, inute ells in the sene or presene of TNF-, n then etermine [ 3 H]-eoxygluose uptke. As shown in Figure 3B, Tt over-expression lone (ACMV-Tt inute without TNF-) use signifint reution in gluose uptke y ipoytes; similr results were oserve in ACMV-GFP-trnsue ells inute with TNF- lone. Comine exposure to Tt n TNF- further erese gluose uptke (Figure 3B). These t suggest tht the effets of Tt in ipoytes impire ipogeni gene expression, n tht inution of proinflmmtory genes re Antivirl Therpy

6 J Díz-Delfín et l. ssoite with ltere metoli funtions, suh s impire gluose uptke. The effets of Tt on ipoytes re prtilly meite y TNF- As shown ove, Tt signifintly inue TNF- gene expression n proue effets on mture SGBS ipoytes tht were similr to those oserve for TNF-. We hypothesize tht the effets of Tt might e use, in whole or in prt, y the utorine tion of TNF- relese in response to Tt. To test whether the TNF- serete y Tt-trnsue ipoytes is involve in Tt-inue hnges in gene expression, we trete ells with TNF- neutrlizing ntioy. Figure 2. Effet of Tt on the expression of genes relte to inflmmtory tions n ipogeni funtion in ifferentite SGBS ipoytes A 21 IL-8 mrna 3.0 MCP-1 mrna 4.5 TNF-α mrna Tt Tt Tt Aiponetin mrna PPAR-γ mrna GLUT4 mrna Tt Tt Tt Aiponetin, ng/ml B IL-8, pg/ml MCP-1, ng/ml Tt Tt Tt Differentite SGBS ipoytes (y 16 post-inution of ifferentition) were trnsue with enovirl vetors ACMV-GFP (ontrol) or ACMV-Tt. (A) Effet of Tt on iponetin n proinflmmtory ytokine messenger RNA (mrna) levels. Totl RNA ws isolte, n smples were nlyse y quntittive reverse trnsription PCR. (B) Effet of Tt on the relese of iponetin n proinflmmtory ytokines into the ell ulture meium y SGBS ipoytes. Results re expresse s protein onentrtion. Dt re presente s men ±sem from six inepenent experiments, n re expresse reltive to vlues from untrete ontrol ells: P<0.01 n P<0.05 versus ontrols. GLUT4, gluose trnsporter type 4; IL, interleukin; MCP, monoyte hemottrtnt protein; PPAR-g, peroxisome prolifertor-tivte reeptor-g Interntionl Meil Press

7 HIV-1 Tt protein inues proinflmmtory ytokines in humn ipoytes As expete, inution of TNF--trete (0.25 ng/ml) ontrol ells with the TNF- neutrlizing ntioy for 24 h rmtilly impire TNF--inue expression of IL-8, MCP-1 n TNF- mrna (Figure 4), initing tht the onentrtion of ntioy use ws suffiient to ompletely inhiit the iologil tivity of exogenously e TNF- (ompre TNF- n TNF- plus nti- TNF- in Figure 4). When pplie to ells over-expressing Tt, the TNF- neutrlizing ntioy inhiite the Tt-inue inrese in IL-8 n MCP-1 mrna expression y pproximtely 50%. The TNF- neutrlizing ntioy lso ompletely rogte the Tt-inue inrese in TNF- mrna (Figure 4). Interestingly, the TNF- neutrlizing ntioy i not signifintly ffet Tt-inue own-regultion of the ipogeni genes, iponetin or GLUT4, lthough it i effetively lok the reution in iponetin n GLUT4 mrna levels proue y tretment with TNF- (Figure 4). Thus, our t inite tht TNF- serete s onsequene of intrellulr exposure to Tt prtilly ontriutes to the effets of Tt on overll gene trnsription in ipoytes. Rosiglitzone, resvertrol n prthenolie moulte Tt-epenent inution of proinflmmtory genes We nlyse the pity of rugs with known ntiinflmmtory tion to prevent the Tt-epenent Figure 3. Effets of Tt on TNF--regulte gene expression in ifferentite SGBS ipoytes A 8,000 IL-8 mrna 80 MCP-1 mrna 20 TNF-α mrna 6,000 4,000 2, TNF-α TNF-α/Tt TNF-α TNF-α/Tt TNF-α TNF-α/Tt Aiponetin mrna PPAR-γ mrna GLUT4 mrna e TNF-α TNF-α/Tt TNF-α TNF-α/Tt TNF-α TNF-α/Tt B Deoxygluose uptke, fol inrese 1.6 TNF-α Tt TNF-α/Tt SGBS ells were ulture s esrie in Figure 2 n trete with 10 ng/ml tumour nerosis ftor (TNF)- for 24 h. (A) Effet of Tt on iponetin n proinflmmtory ytokine messenger RNA (mrna) levels. Dt re presente s men ±sem from six inepenent experiments, n re expresse reltive to vlues from ontrol () ells. (B) Effet of Tt on 2-eoxygluose uptke in SGBS ipoytes fter trnsution n tretment with TNF- for 24 h. Dt re presente s men ±sem of triplite eterminnts, n re expresse reltive to vlues from ells: P<0.01 versus, P<0.05 for omprisons etween TNF-- n Tt-trete ells; P<0.05 versus ; P<0.001 versus. e P<0.001 for omprisons etween TNF-α n Tt-trete ells. GLUT4, gluose trnsporter type 4; IL, interleukin; MCP, monoyte hemottrtnt protein; PPAR-g, peroxisome prolifertor-tivte reeptor-g. Antivirl Therpy

8 J Díz-Delfín et l. Figure 4. Effets of TNF- loke on the tion of Tt in ifferentite SGBS ipoytes 100 IL-8 mrna 24 MCP-1 mrna e e Tt Tt+ nti-tnf-α TNF-α TNF-α+ nti-tnf-α Tt Tt+ TNF-α TNF-α+ nti-tnf-α nti-tnf-α 8 TNF-α mrna 1.5 Aiponetin mrna f e Tt Tt+ TNF-α nti-tnf-α TNF-α+ nti-tnf-α Tt Tt+ TNF-α TNF-α+ nti-tnf-α nti-tnf-α 1.5 GLUT4 mrna Tt Tt+ TNF-α nti-tnf-α TNF-α+ nti-tnf-α SGBS ells were ulture n trnsue with ACMV-Tt or ACMV-GFP (ontrol) s in Figure 2. Where inite, ells were trete with 0.25 ng/ml tumour nerosis ftor (TNF)- for 24 h. A TNF- neutrlizing ntioy ws e t 1 mg/ml. Dt re presente s men ±sem from three inepenent experiments, n re expresse reltive to vlues from ontrol () ells: P<0.01 versus ; P<0.05, for omprisons etween TNF-- n Tt-trete ells; P<0.001 versus ; P<0.05 versus. e P<0.001 for omprisons etween TNF-α n TNF-α plus nti-tnf-α. f P<0.01 for omprisons etween Tt n Tt plus nti-tnf-α. GLUT4, gluose trnsporter type 4; IL, interleukin; MCP, monoyte hemottrtnt protein; mrna, messenger RNA. inution of proinflmmtory genes in SGBS ipoytes. First, we teste rosiglitzone, n nti-ieti rug with nti-inflmmtory properties n known tivtor of PPAR-g [31]. Tretment of ells with rosiglitzone for 24 h signifintly inhiite Tt-inue expression of IL-8, MCP-1 n TNF-, lthough it i not lter the sl levels of these mrnas (Figure 5). Seon we teste resvertrol, polyphenol with nti-inflmmtory properties [32] known to tivte the SIRT1 eetylse [33]. Resvertrol tretment signifintly inhiite Interntionl Meil Press

9 HIV-1 Tt protein inues proinflmmtory ytokines in humn ipoytes oth sl n Tt-inue MCP-1 mrna expression ut, in ontrst to rosiglitzone, signifintly enhne the effets of Tt on IL-8 n TNF- mrna expression (Figure 5). Thir, ells were expose to prthenolie, n nti-inflmmtory gent tht inhiits nuler ftor (NF)-kB tivity [34]. Prthenolie tretment signifintly inhiite Tt-inue expression of IL-8, MCP-1 n TNF-, n it lso lowere the sl levels of IL-8 mrna expression. Disussion In the urrent stuy, we provie the first esription of the effets of Tt in SGBS ipoytes. Tt use mrke inution in the expression n seretion of proinflmmtory ytokines, inluing IL-8, TNF- n MCP-1. Moreover Tt impire humn ipogenesis n reue iponetin relese. Hypoiponetinemi is ommonly foun in HIV-1-infete ptients with lipoystrophy, n proinflmmtory lol environment hs lso often een oserve in the ipose tissue of HIV-1-infete ptients [6]. In relte oservtions, Tt hs previously een shown to up-regulte numer of ytokines, the HIV-1 oreeptor CCR5, n the IL-2 reeptor (CD25) in HIV-1-infete non-ipose ells [35]. Moreover, severl reports hve inite enhne expression of proinflmmtory ytokine genes in ipose tissue from ptients with lipoystrophy n even in ft from HIV-infete, tretment-nive ptients [6,36]. Thus, the oserve in vitro effets of Tt on severl spets of ipoyte funtion re onsistent with the ltertions tht pper in HIV-1-infete ptients who evelop lipoystrophy, even mong those with mil ltertions prior to ntiretrovirl tretment. By ontrst, Tt reue the expression of PPAR-g. PPAR-g plys n essentil role in the evelopment n norml funtion of white ipoytes n ontrols the proution n seretion of iponetin [37]. In HIV- 1-infete ptients with lipoystrophy, there is erese in PPAR-g expression in ipose tissue, s well s inrese expression of inflmmtory ytokines, suh s IL-6 n TNF-, n erese expression n irulting levels of iponetin, whih is involve in regulting insulin sensitivity n lso plys n nti-inflmmtory role [38]. From our results, it is tempting to speulte tht impire PPAR-g expression my ontriute to the nti-ipogeni n proinflmmtory effets of Tt, n further nlysis woul e require to estlish it. In ny se, even if PPAR-g levels were reue y Tt, they re enough to llow for the tion of rosiglitzone reuing Tt-epenent tivtion of proinflmmtory genes. We lso explore the potentil of the ntioxint resvertrol, nturl polyphenol tht hs een reporte to meliorte insulin resistne in niml moels [39,40], n to protet ginst protese-inhiitor-inue Figure 5. Effets of rosiglitzone, resvertrol n prthenolie on Tt-inue expression of proinflmmtory ytokine genes IL-8 mrna Rosi Resv Prth Tt Tt+ Rosi MCP-1 mrna Rosi Resv Prth Tt TNF-α mrna Rosi Resv Prth Tt Tt+ Rosi Tt+ Rosi Tt+ Resv Tt+ Resv Tt+ Resv Tt+ Prth Tt+ Prth Tt+ Prth SGBS ipoytes were ulture n trnsue s in Figure 2, n were further trete with rosiglitzone (Rosi; 10 mm), resvertrol (Resv; 50 mm) or prthenolie (Prth; 5 mm) for 24 h. Dt re presente s men ±sem from three inepenent experiments, n re expresse reltive to vlues from ontrol () ells: P<0.01 n P<0.05 versus ; P<0.05 versus Tt-trnsue ells. P<0.001 versus ontrols. e P<0.01 versus Tt-trnsue ells. IL, interleukin; MCP, monoyte hemottrtnt protein; mrna, messenger RNA; TNF, tumour nerosis ftor. oxitive stress in ells [41]. Our t inites tht resvertrol enhnes the effets of Tt on the expression of proinflmmtory ytokines suh s IL-8 n TNF. This my e ue to the tion of eetylse SIRT1, whih is tivte y resvertrol [33] n hs een reporte to enhne Tt trnsriptionl tivity vi eetyltion [42]. These finings, in ition to reports of the inhiition of PPAR-g tivity y SIRT1 [43,44] my le to the onsiertion of resvertrol s n unlikely phrmologil tool for plliting the effets of Tt, in ontrst with rosiglitzone. Aitionlly, the inhiition of Tt inue MCP-1 expression y resvertrol is in e e Antivirl Therpy

10 J Díz-Delfín et l. goo greement with reent stuy tht emonstrte n nti- inflmmtory effets of resvertrol on HIV-1 Tt inue MCP-1 proution in the hippompus [45]. However, the tion of resvertrol lowering Tt-inue MCP-1 shoul eserve further reserh to explore potentil enefiil effets on speifi inflmmtory pthwys. We lso exmine effets of prthenolie in the HIV-1 Tt-meite up-regultion of ytokines in SGBS ipoytes. It is well known tht NF-kB plys ritil role in the trnsriptionl regultory mehnisms of vrious inflmmtory genes [46]. In ition, HIV-1 Tt protein hs een reporte to tivte NF-kB in vriety of ells types, suh s enothelil ells, lymphoytes, stroytes n humn rest ner ells [26,47 49]. In the present stuy, we oserve for the first time tht exposure to n NF-kB inhiitor n signifintly ttenute Tt-inue up-regultion of proinflmmtory ytokines in SGBS ipoytes. A remrkle fining of the urrent stuy is the positive rosstlk etween Tt n TNF- in ipoytes. Tt inue TNF- gene expression in SGBS ipoytes, similr to wht hs een reporte in other ell types [20,50]. This is onsistent with the enhne levels of TNF- expression foun in ipose tissue from HIV-1-infete ptients. Our present t inite tht TNF- inution y Tt use prrine effets in ipoytes tht ontriute to the nti-ipogeni n proinflmmtory effets of Tt. It hs een reporte tht TNF- promotes suseptiility to HIV infetion in ipoytes [10]. Thus, it is likely tht ipoytes in ptients my experiene oth intrellulr exposure to Tt ue to the promotion of HIV-1 infetion y TNF- s well s exposure to Tt relese y other ell types within ipose epots (for exmple, mrophges) tht re highly suseptile to HIV-1 infetion. Our urrent results lso inite tht the effets of Tt on SGBS ipoytes re meite, in prt, through the inution of TNF-. These finings inite viious yle of proinflmmtory n nti-ipogeni effets rising from Tt TNF- rosstlk. erning the ourrene of some TNF--inepenent effets of Tt, it my e speulte tht known effets of Tt on stress-ssoite protein kinses or on trnsription ftors suh s CREB or Sp1 [22] my e involve in suh effets, mtter tht shoul eserve further reserh in the ontext of the ipose ell. In summry, our results inite tht exposure of SGBS pre-ipoytes to Tt n over-expression of Tt in SGBS ipoytes reues ipogenesis n promotes the expression n relese of proinflmmtory ytokines. These sme ltertions re foun in ipose tissue from HIV-1-infete ptients to istint egrees in reltion to the extent of lipoystrophy. Tt my e novel meitor of ipose tissue ltertions, either s onsequene of iret infetion of ipoytes or ue to the exposure of ipoytes to Tt serete y surrouning non-ipose ells. However, the present in vitro stuy hs ovious limittions in reltion to its iret trnsltion to the nlysis of ltertions in HIV-1- infete ptients. In ition to the intrinsi limittions of the in vitro pprohes, it is iffiult to iretly ompre the levels of exposure of ells to Tt in our experimentl settings with the tul exposure of ptient ipoytes to Tt. Further reserh will e neessry to improve the tehnologil omplexities of Tt mesurements in humn smples n to estlish the tul lol onentrtion of Tt in the ipose tissue miroenvironment, in orer to fully estlish the role of Tt in the ipose tissue erngements ourring in HIV-1-infete ptients. If onfirme, ttempts to prevent n/or meliorte metoli ltertions in HIV-1 ptients, inluing lipoystrophy, shoul tke into onsiertion trgeting the eleterious tion of Tt on the humn ipoyte funtion. Aknowlegements The present stuy ws supporte y grnts from Ministerio e Cieni e Innovión (grnt numer SAF ), Instituto e Slu Crlos III (grnt numer PI n Re e Investigión en SIDA) n FIPSE (grnt numer 36610/06), Spin. Dislosure sttement The uthors elre no ompeting interests. Referenes 1. Cron-Derle M, Lgthu C, Bor F, Vigouroux C, Cpeu J. HIV-ssoite lipoystrophy: from ft injury to premture ging. Trens Mol Me 2010; 16: Villrroy F, Domingo P, Girlt M. Lipoystrophy in HIV 1-infete ptients: lessons for oesity reserh. Int J Oes (Lon) 2007; 31: Boy MA, Dixit NM, Singphoe U, et l. Virl ey ynmis in HIV-infete ptients reeiving ritonvir-ooste squinvir n efvirenz with or without enfuvirtie: rnomize, ontrolle tril (HIV-NAT 012). J Infet Dis 2006; 194: Flint OP, Noor MA, Hruz PW, et l. The role of protese inhiitors in the pthogenesis of HIV-ssoite lipoystrophy: ellulr mehnisms n linil implitions. Toxiol Pthol 2009; 37: Visnegrwl F, Rghvn SS, Mullin CM, et l. Sex ifferenes in the ssoitions of HIV isese hrteristis n oy omposition in ntiretrovirl-nive persons. Am J Clin Nutr 2005; 82: Girlt M, Domingo P, Gullr JP, et l. HIV-1 infetion lters gene expression in ipose tissue, whih ontriutes to HIV- 1/HAART-ssoite lipoystrophy. Antivir Ther 2006; 11: Hzn U, Romero IA, Cnello R, et l. Humn ipose ells express CD4, CXCR4, n CCR5 reeptors: new trget ell type for the immunoefiieny virus-1? FASEB J 2002; 16: Dupin N, Buffet M, Mrelin AG, et l. HIV n ntiretrovirl rug istriution in plsm n ft tissue of HIV-infete ptients with lipoystrophy. AIDS 2002; 16: Interntionl Meil Press

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