Liver X Receptor-Mediated Induction of Cholesteryl Ester Transfer Protein Expression Is Selectively...

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7272 Greenville Avenue, Dallas, TX 72514Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.

DOI: 10.1161/ATVBAHA.109.193201 published online Aug 13, 2009; Arterioscler Thromb Vasc Biol

Gambert, Jacques Grober, Bernard Bonnotte, Eric Solary and Laurent Lagrost PhilippeAngélique Chevriaux, Magalie Raveneau, Nicolas Ogier, Anh Thoai Nguyen,

Daniela Lakomy, Cédric Rébé, Anne-Laure Sberna, David Masson, Thomas Gautier,Expression Is Selectively Impaired in Inflammatory Macrophages

Liver X Receptor�Mediated Induction of Cholesteryl Ester Transfer Protein

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Liver X Receptor–Mediated Induction of Cholesteryl EsterTransfer Protein Expression Is Selectively Impaired in

Inflammatory MacrophagesDaniela Lakomy, Cedric Rebe, Anne-Laure Sberna, David Masson, Thomas Gautier,

Angelique Chevriaux, Magalie Raveneau, Nicolas Ogier, Anh Thoai Nguyen, Philippe Gambert,Jacques Grober, Bernard Bonnotte, Eric Solary, Laurent Lagrost

Objective—Cholesteryl ester transfer protein (CETP) is a target gene for the liver X receptor (LXR). The aim of this studywas to further explore this regulation in the monocyte-macrophage lineage and its modulation by lipid loading andinflammation, which are key steps in the process of atherogenesis.

Methods and Results—Exposure of bone marrow–derived macrophages from human CETP transgenic mice to theT0901317 LXR agonist increased CETP, PLTP, and ABCA1 mRNA levels. T0901317 also markedly increased CETPmRNA levels and CETP production in human differentiated macrophages, whereas it had no effect on CETP expressionin human peripheral blood monocytes. In inflammatory mouse and human macrophages, LXR-mediated CETP geneupregulation was inhibited, even though ABCA1, ABCG1, and SREBP1c inductions were maintained. The inhibitionof CETP gene response to LXR agonists in inflammatory cells was independent of lipid loading (ie, oxidized LDLincreased CETP production in noninflammatory macrophages with a synergistic effect of synthetic LXR agonists).

Conclusion—LXR-mediated induction of human CETP expression is switched on during monocyte-to-macrophage differen-tiation, is magnified by lipid loading, and is selectively lost in inflammatory macrophages, which suggests that inflammatorycells may not increase the circulating CETP pool on LXR agonist treatment. (Arterioscler Thromb Vasc Biol. 2009;29:00-00.)

Key Words: liver X receptor � cholesteryl ester transfer protein � macrophage � monocyte � inflammation

Macrophages play a central role in the formation of athero-sclerotic lesions. These cells produce the cholesteryl ester

transfer protein (CETP), which accounts for the enrichment ofcirculating apoB-containing lipoproteins with cholesteryl esters,but can also contribute to reverse cholesterol transport.1 Asshown by bone marrow transplantation experiments in mice,macrophages, including liver macrophages (Kuppfer cells), con-tribute significantly to plasma CETP and PLTP pools.2–5 CETPmRNA was identified mainly in nonparenchymal sinusoidalcells of the liver in cynomolgus monkeys.6 In humans, choles-terol transfer activity was observed in the culture supernatant ofhuman macrophages,7,8 and CETP was localized in macrophage-derived foam cells by immunohistochemistry or in situ hybrid-ization analysis of atherosclerotic lesions.9,10 CETP produced inthe vicinity of the vascular bed may prevent cholesterol accu-mulation in lesion macrophages,9,11 an event involved in bothearly and late stages of the disease.12 However, under unfavor-able conditions (eg, when apoB-containing lipoprotein clearanceis defective), the deleterious effect of systemic CETP couldovercome this local beneficial effect. Accordingly, hyperlipid-

emic LDLr-KO mice transplanted with CETP transgenic(CETPTg) bone marrow cells display a proatherogenic plasmalipoprotein profile and accelerated development of atheroscle-rotic lesions.2

The CETP gene promoter can be transactivated in vitro and invivo by liver X receptors (LXR).13,14 These oxysterol-activatednuclear receptors interact with a DR4 element in the promoter oftarget genes, such as CETP and ABCA1.15 LXR are abundantlyexpressed in macrophages16 where they are involved in thecontrol of lipid homeostasis and inflammation.17 The atheropro-tective actions of LXR agonists have been clearly demonstratedin LDL receptor– and apolipoprotein E–deficient mice,18–20 andbone marrow transplantation studies demonstrated that thiseffect relies to a large extent on LXR activity in macro-phages.20,21 Lipid entry and proinflammatory changes in macro-phages are consecutive, but distinct processes and scavengerreceptor–mediated lipid loading could be harmful only whenassociated with inflammation.22–24 Interestingly, lipopolysaccha-ride (LPS)-mediated inflammation in CETPTg mice results in aprofound decrease in hepatic CETP mRNA and plasma CETPconcentration.25

Received January 26, 2009; revision accepted July 24, 2009.From INSERM UMR866-Universite de Bourgogne (D.L., C.R., A.L.S., D.M., T.G., N.O., A.T.N., P.G., J.G., B.B., E.S., L.L.), IFR Sante-STIC (D.L.,

C.R., A.L.S., D.M., T.G., A.C., M.R., N.O., A.T.N., P.G., J.G., B.B., E.S., L.L.), and Centre Georges-Francois Leclerc (CR) Dijon Cedex, France.D.L. and C.R. contributed equally to this study.Correspondence to Laurent Lagrost, INSERM UMR866, Faculte de Medecine, BP87900, 21079 Dijon Cedex, France. E-mail laurent.

[email protected]© 2009 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.109.193201

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It remains unknown whether the regulation of CETP expres-sion differs between macrophages and foam cells, and whetherCETP gene is equally responsive to synthetic LXR agonists inearly foam cells and in inflammatory cells that accumulate laterin injured vascular wall. Here, we determined the expressionpattern of CETP during monocyte-to-macrophage differentiationand its modulation by LXR agonists and inflammatory stimuli.

Materials and MethodsA complete Materials and Methods section is available in the supple-mental materials (available online at http://atvb.ahajournals.org). Hu-man peripheral blood monocytes were obtained from healthy donors,and macrophage differentiation was induced by M-CSF. C57Bl6 miceexpressed the human CETP under the control of its natural flankingregions, and peritoneal macrophages were harvested after thioglycollateinjection. Mouse bone marrow–derived macrophages were obtained byincubation of bone marrow cells with M-CSF.

ResultsUpregulation of CETP Expression by an LXRAgonist in CETPTg Mouse Macrophages DerivedFrom Bone Marrow CellsBone marrow cells were recovered from transgenic mice inwhich the human CETP gene has been placed under the control

of its natural flanking regions, including the LXR�-responsiveDR4 element.15 The differentiation of bone-marrow monocytesinto macrophages was triggered by exposure to M-CSF andconfirmed by expected changes in the cell morphology (supple-mental Figure IA), flow cytometry expression of F4/80 (aspecific marker of mouse macrophage lineage; supplementalFigure IB), and mRNA expression of the mouse colony stimu-lating factor 1-receptor (CSF1-R; supplemental Figure IC). Asshown in Figure 1A, treatment of differentiated macrophages for24 hours with 1 �mol/L of the T0901317 LXR agonist produceda significant increase in the mRNA level of LXR target genesincluding CETP, PLTP, and ABCA1.

CETP Gene Upregulation by LXR Agonist IsInhibited in Inflammatory Macrophages FromCETPTg MiceCETPTg mouse macrophages that had been activated in situby the intraperitoneal injection of thioglycollate were treatedwith the T0901317 LXR agonist. The LXR agonist enhancedthe levels of PLTP and ABCA1 mRNA but did not induceany change in CETP mRNA levels (Figure 1B). LXR-induced upregulation of ABCA1 and PLTP was also ob-served in bone marrow–derived macrophages treated with

Figure 1. Effect of inflammation on LXR targetgene expression in stimulated CETPTg mousemacrophages. A, CETPTg mouse bone marrow–derived macrophages (BMC-Mac). B, CETPTgmouse intraperitoneal macrophages were treatedin vitro for 24 hours with 1 �mol/L of theT0901317 LXR agonist or not. Mean�SD of tripli-cate determinations. *P�0.05 vs control. C, BMC-Mac from CETPTg mice were left untreated (Co) orincubated for 6 hours with 20 ng/mL of TNF� or 1�g/mL of LPS or the vehicle (�), then exposed for24 hours to 1 �mol/L T0901317. Mean�SD of trip-licate determinations. *P�0.05 LXR alone vs con-trol, #P�0.05 vs LXR agonist (�). D and E,CETPTg mice were intraperitoneally injected withLPS (LPS - 5 mg/kg body weight) or saline (Co)and then treated by gavage with T0901317 (10mg/kg body weight) or vehicle (�) for 24 hours.Mean�SD of 5 distinct animals. *P�0.05 LXRalone vs control without LXR agonist treatment;#P�0.05 vs LXR agonist without LPS treatment.

2 Arterioscler Thromb Vasc Biol November 2009



TNF� or LPS (20 ng/mL and 1 �g/mL, respectively; Figure1C). Under proinflammatory conditions, the expression levelof MCP1 was magnified whereas the LXR-mediated induc-tion of the CETP gene was abolished (Figure 1C). Treatmentof CETPTg mice with one single dose of the T0901317 LXRagonist produced significant increases in both CETP mRNAlevels in liver and cholesteryl ester transfer activity in plasma(Figure 1D and 1E). Most importantly, intraperitoneal injec-tion of LPS in CETPTg mice produced no change in basallevels of liver CETP mRNA and plasma CETP activity butcompletely abolished the LXR-mediated upregulation ofCETP (Figure 1D and 1E).

LXR Agonist Differentially Affects CETP GeneExpression in Human Monocytesand MacrophagesHuman peripheral blood monocytes (Mo) were obtained fromhealthy donors, and their differentiation into macrophages(Mac) was triggered by exposure to M-CSF. Sphericalnonadherent monocytes acquired a typical fibroblast-likestructure after 6 days of culture (Figure 2A). Their differen-tiation into macrophages was confirmed by the increased

expression of CD71, the specific activation of caspase-8 asmeasured by using FAM-LETD.fmk as a substrate (Figure2B) and the induction of the scavenger receptor A-I (SRA-I)gene (Figure 2C). As previously described,26 the differentia-tion of monocytes into macrophages induced an increase inthe expression of LXR� (Figure 2D). The expression of LXRtarget genes such as CETP and PLTP was also increasedduring the differentiation process. Exposure of peripheralblood monocytes to the LXR agonist T0901317 for 24 hoursincreased the expression of ABCA1 and LXR� mRNAwithout significantly affecting the level of CETP and PLTPmRNAs. In contrast, treatment of macrophages strikinglyincreased the mRNA levels of the four studied LXR targetgenes (Figure 2D).

LXR Agonist Induced CETP Protein Synthesisand Secretion in Human MacrophagesWestern blot and lipid transfer activity analyses coincidentlyrevealed that neither human CETP nor human PLTP could bedetected in the supernatant of peripheral blood monocytes,whether the cells were treated or not with the LXR agonist(Figure 3). In contrast, both PLTP and CETP were identifiedin the supernatant of differentiated human macrophages andtheir protein mass and activity levels markedly increased onexposure to the LXR agonist (Figure 3).

Lipid Loading Upregulates CETP GeneExpression in Human MacrophagesOxidized LDL induced the accumulation of cholesterol intodifferentiated human macrophages as judged by lipid engulf-

Figure 2. Expression of LXR target genes in human peripheralblood monocytes and differentiated macrophages. Human mono-cytes (Mo) were treated for 6 days with 100 ng/mL of M-CSF, anddifferentiation of macrophages (Mac) was assessed by morphologi-cal criteria (A), CD71, and FAM-LETD-fmk flow cytometry detection(white histograms, unlabeled; gray histrograms, labeled; B), andexpression of SRA-I (mean�SD of triplicate determinations;*P�0.05 vs Mo; C). D, Cells were treated for 24 hours with1 �mol/L T0901317 or not. Mean�SD of triplicate determinations.*P�0.05 vs Mo; #P�0.05 vs corresponding control.

Figure 3. Effect of the LXR agonist on secretion of CETP andPLTP by human monocytes/macrophages. Human monocytesand macrophages were treated for 24 hours with indicated con-centrations of T0901317. Means�SD of triplicate determina-tions. Human CETP and PLTP as positive controls.

Lakomy et al LXR Regulation of CETP in Inflammatory Macrophages 3



ment and significant increase in oil red O staining (Figure 4Aand 4B). Lipid loading significantly increased the expressionof CETP without affecting that of PLTP (Figure 4C). Thesynthetic LXR agonist induced a significant increase in theexpression of both CETP and PLTP genes, whether the cellswere pretreated or not with oxidized LDL. Both lipid loadingand the LXR agonist produced a synergistic up to 90-fold risein CETP mRNA levels in macrophages (Figure 4C).

Inflammation Produced Differential Effects onCETP and Other LXR Targets inHuman MacrophagesDifferentiated human macrophages were pretreated for 6hours with inflammatory stimuli (TNF�, LPS or INF�), thentreated for 24 hours with 1 �mol/L of the T0901317 LXRagonist. All the inflammatory stimuli increased the level ofIL1� mRNA (Figure 5A), and a concentration-dependenteffect was demonstrated for TNF (Figure 5B). The LXR-mediated induction of the ABCA1 gene was maintained ininflammatory cells, whereas the major 120-fold increase inCETP mRNA levels observed after the LXR agonist treat-ment was suppressed in inflammatory cells treated withTNF�, LPS, or IFN� (Figure 5A and 5B). The moderate4-fold increase in PLTP mRNA level was also prevented byLPS and IFN�, with no effect of the TNF treatment. Inter-estingly, LXR� expression in macrophages was significantlyinduced by the LXR agonist, and this autoregulatory effectwas magnified in inflammatory cells (Figure 5C). TheT0901317 LXR agonist did no longer increase CETP mRNAand CETP activity levels in LPS-treated macrophages (Figure6). In the meantime, the ability of LXR to upregulate the

ABCG1 and SREBP1c target genes was maintained (Figure6A). As previously described,27 TLR4 gene expression wasupregulated after long-term (48 hours), but not after short-term (16 hours) treatment of human macrophages with theLXR agonist (results not shown). Here, we show that theability of LXR to upregulate ABCG1 and SREBP1c targetgenes is maintained (Figure 6A). Interestingly, and in contrastto the pretreatment with proinflammatory agents (Figure 5A;Figure 6A), the LPS stimulus did no longer modify theLXR-mediated overexpression of CETP when applied afterthe LXR agonist treatment, with concordant observationsafter short-term (16-hour) and long-term (48-hour) treatmentswith the LXR agonist (Figure 6C).

DiscussionThe present study shows that CETP is upregulated by LXR indifferentiated human macrophages but not in human periph-eral blood monocytes. The ability of LXR agonists to upregu-late the CETP gene remained intact when macrophages wereloaded with oxidized lipids. In contrast, it was lost onexposure to TNF� or LPS inflammatory stimuli. While LPSreduced the expression of the LXR target ABCA1 in J774mouse macrophages28 and in RAW cells,29 the present studynow indicates that LXR agonists rescue the expression ofABCA1 in primary macrophages exposed to inflammatorystimuli.

In a first attempt to bring insight into the molecularmechanism implicated in the failure of the LXR agonist toupregulate CETP expression in inflammatory macrophages,we sought for putative alteration in LXR� expression. Thispoint was urged by earlier studies which demonstrated thatLXR� expression is suppressed during the LPS-inducedacute-phase response in the HK-2 human proximal tubularcell line, in human kidney mesangial cells, and in the mousekidney.30,31 In contrast to these observations, it is shown herein macrophages that expression level of LXR� is not onlymaintained, but even enhanced in inflammatory cells. Theseobservations come in new support of the existence of anLXR� autoregulation loop that has been described earlier.32

However, although this mechanism may contribute to theenhanced response of the prototypical ABCA1 target genethat was observed under inflammatory conditions, it cannotapply to downregulation of the CETP gene expression. Then,we turned to explore the NFkappaB pathway because it isknown to be activated in an inflammatory context and tointerfere with LXR-mediated effects.29,33 However, involve-ment of this pathway in the inhibition of CETP response toLXR agonists could be excluded as inhibitors such as TPCKor PDTC, which restore ABCA1 in LPS-treated RAW cells,29

did not restore the ability of the LXR agonist to up-regulateCETP expression in inflammatory macrophages (not shown).Finally, the molecular mechanism involved might relate tochanges in the expression/recruitment of transcription factorsand coactivator proteins which might have contributed toalteration of CETP expression under inflammatory condi-tions. Although the latter point will deserve full examinationin a specific and comprehensive study, it can be anticipatedthat the effect of inflammatory stimuli might not simply relateto the selective displacement of LXR coactivators on the CETP

Figure 4. Effect of lipid-loading on CETP mRNA modulation asinduced in human macrophages by the LXR agonist. Humanmacrophages were incubated (Mac�oxLDL) or not (Mac) for 48hours with oxidized LDL. A and B, Oil red O staining. *P�0.05vs (�). C, Macrophages were further incubated for 24 hourswith 1 �mol/L T0901317 or not. Mean�SD of triplicate determi-nations. *P�0.05 vs corresponding control; †P�0.05 vs Mac inthe same conditions; #P�0.05 vs corresponding control and vsMac in the same conditions.




promoter only. Indeed, in the present study, the LXR-mediatedupregulation of CETP gene expression was disrupted by aninflammatory pretreatment, but not an inflammatory posttreat-ment (ie, when the LPS stimulus was applied after a formerincubation with the LXR agonist). Again, results of the presentstudy illustrate the complexity of macrophage LXR signalingand its modulation by inflammation.27

The relative contribution of tissues and cell types to thesystemic pool of CETP has long been a matter of debate.Earlier studies reported that nonparenchymal sinusoidal cellswere the principal source of CETP mRNA in cynomolgusmonkey liver.6 More recently, it was shown in LDLr-KOmice transplanted with bone marrow from CETP transgenic(CETPTg) mice that liver Kupffer cells (hepatic macro-phages) contributed to 50% of the total hepatic CETPexpression.2 The present study demonstrates that the capacityto produce CETP and to upregulate this production inresponse to LXR agonists is turned on when monocytesdifferentiate into macrophages. The differentiation status wasalready shown to affect CETP expression in other cell types(ie, CETP expression decreases during the loss of differenti-

ation of freshly isolated human hepatocytes34) as well asduring the preadipocyte-to-adipocyte differentiation.35 Over-all, observations of the present study come in further supportof macrophages and Kupffer cells as significant contributorsto the circulating pool of CETP, and in addition they suggestthat the loss of LXR-mediated regulation of CETP expressionin inflammatory cells can be of physiological relevance. Ourresults demonstrate that the differentiation status is only oneof the parameters that determine the capability of a cell toproduce CETP as inflammatory stimuli could specificallyabrogate LXR-mediated secretion of CETP by macrophages.It was consistently observed in peritoneal mouse macro-phages activated in situ by thioglycollate and in mouse andhuman macrophages obtained by ex vivo differentiation ofprecursors through M-CSF stimulation and exposed to TNF�or LPS. In support of the pathophysiological relevance ofthese observations, the ability of one single dose of theT0901317 LXR agonist to increase plasma cholesteryl estertransfer activity in CETPTg mice was suppressed whenanimals were intraperitoneally injected with LPS beforereceiving the LXR agonist.

Figure 5. Effect of inflammatory stimuli on CETPmRNA modulation as induced in human macro-phages by the LXR agonist. Human macrophageswere treated for 6 hours with TNF� (20 ng/mL),LPS (1 �g/mL), or INF� (1000 UI/mL; A) or indi-cated concentrations of TNF� (B), and then for 24hours with or without T0901317. C, Macrophageswere treated as in A or B. Mean�SD of triplicatedeterminations. *P�0.05 LXR alone vs control;#P�0.05 vs LXR agonist alone; †P�0.05 vs con-trol/TNF� (0).




As far as atherosclerosis is concerned, it should be empha-sized that macrophages are heterogeneous cells with distinctinflammatory potentials and athero/inflammatory gene pat-terns.36–38 Oxidized LDL selectively increased the CETPgene expression in macrophages, suggesting that the basalexpression of CETP in the vascular wall macrophages couldbe actually increased in initial steps of foam cell formation. Inaddition, exposure of lipid-loaded macrophages to theT0901317 synthetic LXR agonist strikingly increased CETPand PLTP gene expression in these cells. Conversely, theCETP gene response to LXR agonists may be selectively lostin macrophages that infiltrate advanced/inflammatory athero-sclerotic lesions. To our knowledge, the antiatherogeniceffect of LXR agonists has been addressed in mice that do notexpress CETP. Thus, the present observations will deservefurther attention in animal models with either systemic ormacrophage-targeted expression of CETP. Because macro-phage-derived CETP was found to worsen the atherogenicityof the lipoprotein profile in the CETPTg mouse model,2 theloss of CETP regulation by LXR agonists in inflammatorymacrophages might not be detrimental. Concerns have beenraised as LXR agonists increase the synthesis and secretion oftriglycerides by the liver39,40 and enhance the cholesteryl esteruptake from HDL to macrophages.41 In addition, LXR ago-nists were also suspected to increase CETP secretion into theblood stream, which may enhance the atherogenicity whenthe clearance of apoB-containing lipoproteins is impaired.42

Our study indicates that, in the inflammatory conditions thatcharacterize the atherosclerotic lesion,12,43 LXR agonists maynot promote any increase in macrophage secretion of CETP.Although the accumulation of inflammatory macrophagesubpopulations should mostly be considered an indicator of ahigh risk of developing atherosclerosis, the present study sug-gests that they would actually respond in an appropriate way toLXR agonists (ie, with a rise in the expression of reversecholesterol transport-promoting genes (ABCA1) but a reductionin the expression of potentially proatherogenic genes [CETP]).

Finally, the LXR-mediated regulation of human CETP expres-sion makes it possible to distinguish between activated andnonactivated macrophage subpopulations.

It is concluded that CETP behaves as a peculiar LXR targetthat displays differential responses to agonists in inflamma-tory versus noninflammatory macrophages. Restriction of themanipulation of the LXR pathway to vascular cells has beenproposed as a promising way to treat atherosclerosis.14 Thepresent study indicates that activated/inflammatory macro-phages in the atherosclerotic plaque are specifically pre-vented from contributing to the circulating CETP pool onLXR agonist treatment.

AcknowledgmentsWe thank Philip Bastable for manuscript editing and the platform ofcytometry (Dijon) for access to the flow cytometer.

Sources of FundingThis work was supported by grants from the Universite deBourgogne, the Centre Hospitalier Universitaire de Dijon, theConseil Regional de Bourgogne, INSERM (Institut National de laSante Et de la Recherche Medicale), the ANR (Agence Nationalede la Recherche), and the Fondation de France. E.S. group issupported by La Ligue Nationale Contre le Cancer and INCa.

DisclosuresNone.

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26. Kohro T, Nakajima T, Wada Y, Sugiyama A, Ishii M, Tsutsumi S,Aburatani H, Imoto I, Inazawa J, Hamakubo T, Kodama T, Emi M.Genomic structure and mapping of human orphan receptor LXR alpha:upregulation of LXRa mRNA during monocyte to macrophage differen-tiation. J Atheroscler Thromb. 2000;7:145–151.

27. Fontaine C, Rigamonti E, Nohara A, Gervois P, Teissier E, Fruchart JC,Staels B, Chinetti-Gbaguidi G. Liver X receptor activation potentiates thelipopolysaccharide response in human macrophages. Circ Res. 2007;101:40–49.

28. Khovidhunkit W, Moser AH, Shigenaga JK, Grunfeld C, Feingold KR.Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver andABCA1 and ABCG1 in J774 murine macrophages: differential role ofLXR. J Lipid Res. 2003;44:1728–1736.

29. Baranova I, Vishnyakova T, Bocharov A, Chen Z, Remaley AT, Stonik J,Eggerman TL, Patterson AP. Lipopolysaccharide down regulates bothscavenger receptor B1 and ATP binding cassette transporter A1 in RAWcells. Infect Immun. 2002;70:2995–3003.

30. Wang Y, Moser AH, Shigenaga JK, Grunfeld C, Feingold KR. Down-regulation of liver X receptor-alpha in mouse kidney and HK-2 proximaltubular cells by LPS and cytokines. J Lipid Res. 2005;46:2377–2387.

31. Ruan XZ, Moorhead JF, Fernando R, Wheeler DC, Powis SH, VargheseZ. Regulation of lipoprotein trafficking in the kidney: role of inflam-matory mediators and transcription factors. Biochem Soc Trans. 2004;32:88–91.

32. Laffitte BA, Joseph SB, Walczak R, Pei L, Wilpitz DC, Collins JL,Tontonoz P. Autoregulation of the human liver X receptor alphapromoter. Mol Cell Biol. 2001;21:7558–7568.

33. Rigamonti E, Chinetti-Gbaguidi G, Staels B. Regulation of macrophagefunctions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men.Arterioscler Thromb Vasc Biol. 2008;28:1050–1059.

34. Agellon LB, Zhang P, Jiang XC, Mendelsohn L, Tall AR. The CCAAT/enhancer-binding protein trans-activates the human cholesteryl estertransfer protein gene promoter. J Biol Chem. 1992;267:22336–22339.

35. Gauthier B, Robb M, McPherson R. Cholesteryl ester transfer proteingene expression during differentiation of human preadipocytes to adi-pocytes in primary culture. Atherosclerosis. 1999;142:301–307.

36. Waldo SW, Li Y, Buono C, Zhao B, Billings EM, Chang J, Kruth HS.Heterogeneity of human macrophages in culture and in atheroscleroticplaques. Am J Pathol. 2008;172:1112–1126.

37. Bouhlel MA, Derudas B, Rigamonti E, Dievart R, Brozek J, Haulon S,Zawadzki C, Jude B, Torpier G, Marx N, Staels B, Chinetti-Gbaguidi G.PPARgamma activation primes human monocytes into alternative M2macrophages with anti-inflammatory properties. Cell Metab. 2007;6:137–143.

38. Gordon S. Macrophage heterogeneity and tissue lipids. J Clin Invest.2007;117:89–93.

39. Li AC, Glass CK. The macrophage foam cell as a target for therapeuticintervention. Nat Med. 2002;8:1235–1242.

40. Tall AR, Costet P, Wang N. Regulation and mechanisms of macrophagecholesterol efflux. J Clin Invest. 2002;110:899–904.

41. Bultel S, Helin L, Clavey V, Chinetti-Gbaguidi G, Rigamonti E, Colin M,Fruchart JC, Staels B, Lestavel S. Liver X receptor activation induces theuptake of cholesteryl esters from high density lipoproteins in primaryhuman macrophages. Arterioscler Thromb Vasc Biol. 2008;28:2288–2295.

42. Plump AS, Masucci-Magoulas L, Bruce C, Bisgaier CL, Breslow JL, TallAR. Increased atherosclerosis in ApoE and LDL receptor gene knock-outmice as a result of human cholesteryl ester transfer protein transgeneexpression. Arterioscler Thromb Vasc Biol. 1999;19:1105–1110.

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Supplemental Material

Liver X receptor-mediated induction of cholesteryl ester transfer protein expression is

selectively impaired in inflammatory macrophages

Daniela Lakomy, Cédric Rébé, Anne-Laure Sberna, David Masson, Thomas Gautier,

Angélique Chevriaux, Magalie Raveneau, Nicolas Ogier, Anh-Thoai NGuyen, Philippe

Gambert, Jacques Grober, Bernard Bonnotte, Eric Solary, and Laurent Lagrost.

.

From INSERM UMR866- Université de Bourgogne (D.L., C.R., AL.S., D.M. , T.G., N.O.,

AT.N., P.G., J.G., B.B., E.S., L.L.), IFR Santé-STIC (D.L., C.R., AL.S., D.M. , T.G., A.C.,

M.R., N.O., AT.N, P.G., J.G., B.B., E.S., L.L.), and Centre Georges-François Leclerc (CR)

Dijon Cedex, France

Supplementary Materials and Methods

Chemical reagents

Macrophage-Colony Stimulating Factor (M-CSF) was purchased from Abcys (Paris, France),

Tumor Necrosis Factor-α from PeproTech (Neuilly-sur-Seine, France) and T0901317 from

Merck Chemical Ltd (Nottingham, UK). Lipopolysaccharide (LPS) (Ecoli O128:B12) was

purchased from Sigma-Aldrich.

FITC-conjugated anti-human CD71 antibody was provided by Pharmingen (San Diego, CA)

and FITC-conjugated isotype IgG1 matched control from Immunotech (Marseille, France).

PE-conjugated anti-mouse F4/80 and PE-conjugated isotype IgG matched control were

provided by Ozyme (Saint-Quentin Yvelines, France). The TP2 mouse monoclonal antibody



for CETP (1 µg/ml) (Ottawa Heart Institute Corporation, Canada), the 59 monoclonal

antibody for PLTP (2 µg/ml) (kindly provided by Dr Ehnholm and Dr Jauhiainen) and the

secondary antibody HRP-conjugated polyclonal goat anti-mouse immunoglobulins (Dako,

Glostrup, Denmark) were used in western blotting experiments.

Purification and differentiation of human peripheral blood monocytes

Human peripheral blood monocytes were obtained from buffy coats of healthy donors

(provided by the Etablissement Français du Sang, Dijon, France) with informed consent in

accordance with the Declaration of Helsinki. Briefly, peripheral blood mononuclear cells

(PBMC) were isolated by Ficoll gradient centrifugation and monocyte negative selection was

performed via magnetic activated cell sorting using the Monocyte Isolation Kit II (Miltenyi

Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Briefly,

non-monocytes (i.e. T cells, NK cells, B cells, dendritic cells and basophils) are magnetically

labeled by incubating PBMC with a cocktail of biotin-conjugated antibodies against CD3,

CD7, CD16, CD19, CD56, CD123 and Glycophorin A for 10 min at 4°C in the presence of a

Fc receptor blocking agent. Then, after anti-biotin microbeads had been added for a further 15

mins of incubation at 4°C, the cells were washed and passed through a light scattering (LS)

column attached to a magnet. Unlabeled monocytes (CD14+) eluted from the column were

more than 95% pure, as judged by flow cytometry. Monocytes were grown in suspension in

RPMI 1640 medium with glutamax-I (Invitrogen, Paisley, UK) supplemented with 10% (v/v)

fetal bovine serum (FBS, Invitrogen) in an atmosphere of 95% air and 5% CO2 at 37°C and

exposed to 100 ng/mL M-CSF. Macrophage differentiation was observed by fibroblast-like

shape changes visualized with an Olympus IX70 microscope and assessed by FAM-LETD-



fmk cleavage and CD71 cell surface expression (flow cytometry) and SRA-I transcriptional

levels (PCR).

Animals

C57Bl6 mice expressing the human CETP under the control of its natural flanking regions

(CETPTg) were used in the present study.1 The mice had free access to water and food, and

they were placed on a standard chow diet. All experimental procedures were in accordance

with the local guidelines for animal experimentation.

Peritoneal macrophages were harvested from the peritoneal cavity of CETPTg mice 5 days

after a 1 ml injection of 3% thioglycolate. The cells were washed and plated on 24-well plates

in DMEM medium (Invitrogen) and supplemented with 1% Nutridoma-SP (Roche) and

penicillin-streptomycin at a cell density of 5x105 cells/mL.

Mouse bone marrow derived macrophages were obtained as previously described by

incubation of bone marrow cells with M-CSF.2 Femoral bone marrow cells were isolated and

cultured for 4 hours on plastic plates in RPMI 1640 medium with glutamax-I (Invitrogen)

supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen) in an atmosphere of 95%

air and 5% Co2 at 37°C. Subsequently, floating cells were cultured for 3 days in the presence

of 100 ng/mL of M-CSF. The cells were scraped, counted and plated on 24-well plates at a

density of 0.25 x 106 cells/mL in RPMI/FBS/M-CSF for 3 days. Macrophage differentiation

was observed by fibroblast-like shape changes visualized with an Olympus IX70 microscope

and assessed by F4/80 cell surface expression (flow cytometry) and CSF1-R transcriptional

levels (PCR).

In vivo inflammation studies were conducted in male CETPTg mice (6-7 month-old) which

were injected with lipopolysaccharide from E.coli (strain E55:B5, Sigma) dissolved in saline



(0.5 mg/ml). The synthetic LXR agonist T0901317 was solubilized in DMSO (100 mg/ml).

The stock solution was further diluted 1:100 (v/v) with water containing 1% carboxy-methyl-

cellulose (vehicle). Mice were injected intraperitoneally with the LPS solution (5mg LPS / kg

body weight) or saline and left for 6 hours before receiving a single dose of LXR agonist

(10mg/kg) or vehicle by gavage. Four groups of animals were generated: 1) the ‘control’

group (n=5) was treated with saline followed by vehicle, 2) the ‘LPS’ group (n=5) was treated

with LPS followed by vehicle, 3) the ‘LXR’ group (n=5) was treated with saline followed by

LXR agonist, and 4) the ‘LPS + LXR’ group was treated with LPS followed by LXR agonist.

Twenty-four hours after the gavage with LXR agonist or vehicle, mice were anaesthetized

with isoflurane. Blood samples were collected by intracardiac puncture in heparin-containing

tubes. Plasmas were isolated after centrifugation at 5000 rpm for 10 minutes and immediately

assayed for CETP activity as described below. Liver pieces were excised and immediately

snap frozen in liquid nitrogen and stored at -80°C before mRNA isolation prior to qPCR

analysis for the quantification of CETP gene expression (see below).

Preparation of oxidized low density lipoproteins

The different lipoprotein subclasses were isolated from plasma by sequential flotation

ultracentrifugation, according to their density (d) as previously described.3 The LDL

(1.019<d<1.063 g/ml) were prepared in a 70.Ti rotor on an ultracentrifuge (XL-90; Beckman,

Palo Alto, CA, USA) by sequential flotation ultracentrifugation from the fresh citrated plasma

of normolipidaemic donors. Densities of plasma were adjusted by adding potassium bromide.

After a 15-hour run at 184,000 g, the top fraction of the sample containing lipoproteins with

d<1.019 g/ml was removed. Finally, LDL were prepared from the infranatant adjusted to a

density of 1.063 g/ml by a 24-hour run at 149,000 g. The lipoprotein fraction was dialysed

overnight against a 10 mmol/l Tris, 150 mmol/l NaCl, pH 7.4 buffer (TBS). The oxidation of



LDL was performed by incubating freshly prepared LDL, adjusted to a final concentration of

1.2 g/l proteins in TBS, with a copper sulfate solution (final concentration, 5 μmol/l) for 24 h

at 37°C. At the end, oxidation was stopped by adding EDTA (final concentration, 200 μmol/l)

and butylated hydroxytoluene (final concentration, 20 μmol/l) and ox-LDL were kept at 4°C.

Western blot analysis

Culture cell supernatants were applied on 8-12% polyacrylamide gels and then electroblotted

to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). After incubation for 1

hour at room temperature with 3% BSA in Tris-buffered saline (TBS) – 0.1% Tween-20,

membranes were incubated overnight at 4°C with the primary antibody diluted in TBS-BSA-

Tween, washed three times in TBS-Tween incubated with the secondary antibody for 30 min.

at RT and washed three times Finally, the blots were revealed using a chemiluminescent

substrate (ECLTM; Amersham-Pharmacia Biotech).

RNA isolation and real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen). One hundred to 500 ng of RNA

were reverse-transcribed into cDNA using M-MLV reverse transcriptase, Random Primers

and RNAseOUT inhibitor (Invitrogen). cDNA were quantified by real time PCR using either

a SYBR® Green Real-time PCR kit (Invitrogen) on a LightCycler 2.0 detection system

(Roche Diagnostics, Meylan, France). Relative mRNA levels were determined using the ΔCt

method. Values were expressed relative to cyclophilin A levels for experiments on humans or

GAPDH levels for experiments on mice. The sequences of the oligonucleotides used are

shown in Table 1.



Measurement of CETP activity

Cholesteryl ester (CE) transfer activity was measured with a commercially available

fluorescence assay using synthetic liposomes enriched with nitrobenz-oxadiazol (NBD)-

labeled CEs as donors and VLDLs as acceptors (Roar Biomedical, New York, NY). Briefly,

samples (50 µl), fluorescent-CE-labeled liposomes (4 µl), and unlabeled VLDL acceptors (4

µl) were incubated at 37°C in a final volume of 200 µl Tris-buffered saline in 96-well

microplates. Changes in fluorescence were monitored for a 3 h period using a Victor2 TM

fluorescent counter (Perkin Elmer Life Sciences) with 465 nm excitation and 535 nm emission

wavelengths.

Measurement of PLTP activity

Phospholipid transfer activity was measured using a commercially available fluorescence

activity assay (Cardiovascular targets, New York, NY, USA) following the instructions

provided by the manufacturer. Briefly, samples (50 µl), fluorescent-labeled donors (3 µl) and

unlabeled acceptors (50 µl), were incubated at 37°C in a final volume of 150 µl of TBS in 96

well microplates. Changes in fluorescence were monitored every minute using a Victor2–TM

fluorescent counter (PerkinElmer Life Sciences) for a 30 min period, with a 465 nm excitation

and a 535 nm emission wavelength.

Flow cytometry



To detect caspase activity, we used FAM-LETD-fmk (caspase-8) from Serotec (Oxford, UK)

according to the manufacturer’s instructions. Briefly cells were incubated with the fluorescent

substrate for 1 hour at 37°C, then detached and washed with 1X washing buffer and analyzed

with an LSRII flow cytometer (BD Biosciences, Erembodegem, Belgium).

To detect membrane expression of CD71 or F4/80, cells were washed with 1X PBS-0.1%

azide, incubated with IgG matched control or specific antibody for human CD71 (BD

Biosciences) or mouse F4/80 (Biolegend) diluted in PBS-azide-1% BSA for 1 hour at 4°C,

then washed in PBS-azide and analyzed by flow cytometry as described above.

Oil Red O staining

Lipid level analysis was performed by using Oil Red O (Sigma-Aldrich). Oil Red O was

resuspended at 0.5% (wt/vol) in 98% propane-2-ol and stored at 4°C protected from light.

Human macrophages were seeded into 6-well plates, treated or not with oxidized LDL

(50µg/mL) for 48 h and fixed in 4% (v/v) formaldehyde for 15 min. The cells were then

washed in 1x PBS and in 60% propane-2-ol, and then stained with freshly diluted (60% stock

solution in water) and filtered Oil Red O for another 15 min. The cells were rinsed in 60%

propane-2-ol, and lipid droplets were detected by light microscopy with an Olympus IX70

microscope and quantified with Photoshop-Pro.

Statistical analysis

Unpaired, two-tailed Student's t test and one way ANOVA with Dunnett's test were used for

two-group and multiple group comparisons, respectively. P values below 0.05 were

considered statistically significant.



8

Supplementary References

1 Jiang XC, Agellon LB, Walsh A, Breslow JL, Tall A. Dietary cholesterol increases

transcription of the human cholesteryl ester transfer protein gene in transgenic mice.

Dependence on natural flanking sequences. J Clin Invest. 1992;90:1290-1295.

2. Kitaura H, Nagata N, Fujimura Y, Hotokezaka H, Tatamiya M, Nakao N, Yoshida N,

Nakayama K. Interleukin-4 directly inhibits tumor necrosis factor-alpha-mediated

osteoclast formation in mouse bone marrow macrophages. Immunol Lett.

2003;88:193-198.

3. Deckert V, Persegol L, Viens L, Lizard G, Athias A, Lallemant C, Gambert P, Lagrost

L. Inhibitors of arterial relaxation among components of human oxidized low-density

lipoproteins. Cholesterol derivatives oxidized in position 7 are potent inhibitors of

endothelium-dependent relaxation. Circulation. 1997;95:723-731.

Supplementary Figure

Supplementary figure I: Characterization of macrophage differentiation of CETPTg

mouse Bone Marrow Cells. Bone Marrow Cells (BMC) from CETPTg mice were incubated

for 6 days with M-CSF (100ng/mL), and macrophage differentiation (BMC-Mac) was

assessed by morphological features in light microscopy (A), flow cytometry detection of

F4/80 cell surface expression (F4/80 = grey histogram; control Ig = white histograms - B),

and Real Time PCR expression of CSF1-R (C). CSFR1-R mRNA expression normalized to

GAPDH mRNA and expressed relative to the level in untreated cells. Mean ± S.D. of

triplicate determinations; *: P< 0.05 vs BMC.



Supplement Material

TABLE 1. Sequences of the primers used to measure gene expression

Forward Reverse

Human primers

ABCA1

ABCG1

CETP

Cyclophilin A

IL-1β

LXRα

PLTP

SRA-1

SREBP1c

Mouse primers

ABCA1

CSF1-R

GAPDH

MCP1

PLTP

5’-gcactgaggaagatgctgaaa-3’

5'-catctcactgtgcagga-3'

5’-cagctgtgtgttgatctgga-3’

5’-gcatacgggtcctggcatcttgtcc-3’

5’-atgaaggctgcatggatcaa-3’

5’-gaagaaactgaagcggcaaga-3’

5’-acgagcttgttggcattgacta-3’

5’-gatgctcgctcaatgacagc-3’

5'-gacatcgaagacatgcttcagct-3'

5’-gttcctgcagaaacagtagca-3’

5’-cagcaatacctacgtgtgcaa-3’

5’-cctgcaccaccaactgctta-3’

5’-tcccaatgagtaggctggagagc-3’

5’-tgggacggtgttgctcaa-3’

5’-agttcctggaaggtcttgttcac-3’

5'-gttgttcaccagctcca-3'

5’-cagatcagccacttgtcctat-3’

5’-atggtgatcttcttgctggtcttgc-3’

5’-tcatcatcagtgatggattgg-3’

5’-actcgaagccggtcagaaaa-3’

5’-ccatggcagagtcgaagaagaa-3’

5’-ccatgtccctggactgagg-3’

5'-aggcttcaagagaggagctcaat-3'

5’-atgaggttggagatagcagaga-3’

5’-aatcccacttcggcgttagt-3’

5’-tcatgagcccttccacaatg-3’

5’-agaagtgcttgaggtggttgtg-3’

5’-tcgatgcccacgagatca-3’



CETP Tg Mouse BMC CETP Tg Mouse BMC-MacA

CB

FI

F4/80

Eve

nts

BMC

BMC-Mac

BMC0

2

4

6

8

BMC-Mac

Rel

ativ

em

RN

A le

vels CSF1-R *

Figure I

Supplement Material



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