Reciprocal regulation of inflammation and lipid metabolism by ... - Nature

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Reciprocal regulation of inflammation and lipid metabolism by liver X receptors

© 2003 Nature Publishing Group http://www.nature.com/naturemedicine

SEAN B. JOSEPH1, ANTONIO CASTRILLO1, BRYAN A. LAFFITTE1, DAVID J. MANGELSDORF2 & PETER TONTONOZ1 1

Howard Hughes Medical Institute, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA 2 Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA S.B.J. and A.C. contributed equally to this work. Correspondence should be addressed to P.T.; email: [email protected]

Published online 13 January 2003; doi:10.1038/nm820

Macrophages have important roles in both lipid metabolism and inflammation and are central to the pathogenesis of atherosclerosis. The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression, but their role in inflammation and immunity is unknown. We demonstrate here that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Transcriptional profiling of lipopolysaccharide (LPS)-induced macrophages reveals reciprocal LXR-dependent regulation of genes involved in lipid metabolism and the innate immune response. In vitro, LXR ligands inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2 and interleukin-6 (IL-6) in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune functions in macrophages.

Atherosclerosis is a disorder of lipid metabolism as well as a chronic inflammatory disease. Macrophages have a central role in atherogenesis through the accumulation of oxidized lowdensity lipoprotein (oxLDL) and the production of inflammatory mediators, cytokines and extracellular-matrix–degrading enzymes1,2. Mutations that alter lipid metabolic or immune functions of monocyte/macrophages have been shown to influence the development of atherosclerotic lesions in animal models. Inhibition of the ability of macrophages to accumulate lipid through genetic ablation of SR-A or CD36 reduces atherosclerosis3,4. Mice lacking inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), or bone marrow cyclooxygenase COX-2 expression also show a reduction in lesion formation on an atherosclerotic background5–7. In addition to its well-established role as a substrate for macrophage lipid accumulation, oxLDL exerts complex effects on inflammatory gene expression. On one hand, oxLDL induces the expression of several inflammatory mediators in resting macrophages8. On the other hand, oxLDL antagonizes certain inflammatory gene expression programs initiated after LPS stimulation. For example, pretreatment with oxLDL has been shown to inhibit LPS-induced iNOS activity9. Biochemical studies have determined that oxidized cholesterol, rather than fatty acids or phosphatidylcholine, is responsible for this inhibition10. The molecular mechanisms that mediate the effects of oxidized lipids on inflammatory gene expression are poorly understood. NATURE MEDICINE • VOLUME 9 • NUMBER 2 • FEBRUARY 2003

Previous studies have suggested a role for peroxisome proliferator-activated receptor γ (PPARγ) in control of inflammation. Oxidized lipids derived from oxLDL activate PPARγ and induce expression of CD36 (refs. 11,12). Certain PPARγ ligands, especially 15-deoxy-∆12,14-prostaglandin J2, antagonize the expression of iNOS, tumor necrosis factor (TNF)-α and IL-6 in response to macrophage activation13,14. Studies using PPARγdeficient macrophages, however, have shown that at least some of these effects are independent of PPARγ (refs. 15,16). Clearly, additional uncharacterized signaling pathways contribute to the regulation of inflammatory gene expression by oxidized lipids. The liver X receptors (LXRα, encoded by the gene Nr1h3, and LXRβ, encoded by the gene Nr1h2) constitute a second class of nuclear receptors activated by oxidized lipids. LXRs are ‘cholesterol sensors’ that regulate the expression of genes involved in lipid metabolism in response to specific oxysterol ligands17. In macrophages, these oxysterols may be derived from internalized oxLDL or generated intracellularly through modification of cholesterol18,19. Activation of LXR in macrophages induces expression of several genes involved in lipid metabolism and reverse cholesterol transport, including Abca1, Abcg1 and Apoe (ref. 20). Recent studies have demonstrated that LXRs exert an important atheroprotective effect in macrophages. Systemic administration of an LXR agonist reduced atherosclerosis in Ldlr–/– and Apoe–/– mice21. Conversely, loss of LXR expression from bone marrow increases lesion formation in these same models22. 213

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Fig. 1 LXR agonists inhibit the macrophage response to bacterial pathogens. a, LXR agonist inhibits nitrite production in response to bacterial infection. b, LXR agonist inhibits induction of iNOS mRNA in response to bacterial infection. c, LXR agonist inhibits induction of iNOS mRNA in response to LPS. d, Dose-dependent inhibition of nitrite production by GW3965. e, Reciprocal dose-dependent regulation of ABCA1 () and iNOS () mRNA

expression by GW3965. Thioglycolate-elicited murine peritoneal macrophages were pretreated with vehicle or 1 µM GW3965 (LXR), rosiglitazone (PPARγ), GW1516 (PPARδ) or GW7647 (PPARα). Cells were then infected with E. coli (a, b) or treated with 100 ng/ml LPS (c, d, e) as indicated. After 18 h, NO production was determined using a colorimetric assay (a, d), or mRNA levels were determined using real-time quantitative PCR assays (b, c, e).

To determine the molecular basis for the atheroprotective effects of LXRs, we explored other roles for these receptors in macrophage biology. Here we outline a previously unrecognized role for LXRs in the control of inflammatory gene expression. These observations identify the LXR pathway as a common regulator of lipid metabolic and immune functions in macrophages and suggest that LXR ligands may have utility in macrophage-dependent inflammatory diseases such as atherosclerosis and rheumatoid arthritis.

macrophages (Fig. 2a). Furthermore, iNOS activity closely correlated with iNOS protein expression in the four genotypes (Fig. 2b). LXR agonists also inhibited COX-2 protein expression. The oxysterol LXR ligand 22(R)-hydroxycholesterol (2 µM), but not the inactive stereoisomer 22(S)-hydroxycholesterol (2 µM), also caused a receptor-dependent inhibition of iNOS mRNA expression (Fig. 2c). Finally, the previously reported ability of oxLDL to inhibit iNOS activity is at least partially mediated by LXRs, as the response to 50 µg/ml (protein) oxLDL was significantly impaired in Nr1h3–/–Nr1h2–/– macrophages (Fig. 2d).

Inhibition of LPS-induced iNOS production by LXR agonists To investigate the role of LXRs in host defense, we tested the influence of LXR agonists on macrophage responses to bacterial pathogens. Thioglycollate-elicited peritoneal macrophages were infected in vitro with Escherichia coli. After 18 hours, expression of iNOS mRNA and nitric oxide (NO) production were assessed. Macrophages pretreated with the LXR agonist GW3965 showed significantly reduced iNOS mRNA and reduced iNOS activity compared with those treated with vehicle alone (Fig. 1a and b). A similar inhibition was observed in response to stimulation with LPS (100 ng/ml; Fig. 1c). The inhibitory effect of LXR agonists on iNOS expression was dose dependent and occurred over the same range of concentration as that required to induce ABCA1 (Fig. 1d and e). In contrast, ligands for PPARγ (rosiglitazone), PPARδ (GW1516) and PPARα (GW7647) had minimal effects on iNOS expression at concentrations of 1 µM (Fig. 1a–c). To confirm that these effects were mediated by LXRs, we compared peritoneal macrophages from wild-type and LXR-null mice. Inhibition of LPS-induced nitrite production by the LXR agonists T1317 and GW3965 (2 µM) was preserved in Nr1h3–/– and Nr1h2–/– macrophages, but abolished in Nr1h3–/–Nr1h2–/– 214

Reciprocal regulation of metabolism and inflammation We used DNA microarrays to further examine the effect of LXR activation on LPS-induced gene transcription. Thioglycolate-elicited peritoneal macrophages from wild-type, Nr1h3–/–, Nr1h2–/– and Nr1h3–/–Nr1h2–/– mice were treated with 2 µM GW3965 for 18 hours before treatment with 100 ng/ml LPS. Cells were harvested 6 hours after LPS stimulation and total RNA from these samples was used to probe Affymetrix U74Av2 arrays. The array data were analyzed using GeneSpring software to identify genes regulated by ligand in LXR-positive, but not Nr1h3–/–Nr1h2–/–, cells. Selected genes fitting these criteria are represented graphically in Fig. 3. As expected, the most highly induced genes were those involved in lipid metabolism, including established LXR target genes such as Abca1. A large cluster of genes involved in the macrophage innate immune response was inhibited by the LXR agonist. These genes encoded iNOS and COX-2, cytokines such as IL-6, IL-1β and granulocyte colony-stimulating factor (G-CSF), chemokines such as monocyte chemoattractant protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1β (MIP-1β) and interferon-inNATURE MEDICINE • VOLUME 9 • NUMBER 2 • FEBRUARY 2003

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Fig. 3 Transcriptional profiling of LXR agonist effects in LPS-activated macrophages. Graphic representation of gene expression changes in macrophages treated with 100 ng/ml LPS and vehicle or synthetic LXR agonist GW3965 (2 µM) as indicated. Genes involved in inflammation or lipid metabolism were selected based on ligand-dependent changes in expression in wild-type, Nr1h3–/– and Nr1h2–/–, but not Nr1h3–/–Nr1h2–/–, cells.

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hibition was partially reduced in Nr1h3–/– and Nr1h2–/– macrophages and abrogated in Nr1h3–/–Nr1h2–/– macrophages. A modest receptor-independent induction of MCP-1 and IL-6 was observed in response to both GW3965 and T1317.

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ducible protein-10 (IP-10) , and the metalloproteinase MMP-9. Array analysis also confirmed the specificity of GW3965 (Supplementary Fig. 1 online) and showed that the expression of genes involved in other cellular processes was not significantly altered by LXR ligand (Supplementary Fig. 2). Thus, in activated macrophages, the cholesterol efflux pathway and the innate immune response are reciprocally regulated Genotype: by LXRs. Ligand: - + LPS is an established inducer of the NFLPS: + + κB signaling pathway in macrophages. Several genes inhibited by LXR ligands in 3.0 the array studies above are established targets of NF-κB. To further investigate the 2.5 role of LXRs in NF-κB–dependent gene ex2.0 pression, we analyzed expression of NF-κB target genes in peritoneal macrophages from wild-type, Nr1h3–/–, Nr1h2–/– and 1.5 Nr1h3–/–Nr1h2–/– mice. Northern blot analysis (3 hours and 6 hours after LPS stimula1.2 tion, Fig. 4a) and real-time quantitative PCR analysis (6 hours after LPS stimulation, Fig. 4b) confirmed that LXR agonists were 1.0 effective inhibitors of COX-2, IκBα, IL-1β, MCP-1, IL-6 and IL1-receptor antagonist 0.8 (IL-1RN) expression (Fig. 4a and b). This inExpression


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Fig. 2 LXR-dependent inhibition of iNOS expression and NO production by natural and synthetic LXR ligands. a, Inhibition of LPS-induced nitrate production by the synthetic LXR ligands T1317 () and GW3965 () requires expression of LXRs. b, Inhibition of iNOS and COX-2 protein expression by T1317 (T) and GW3965 (GL) requires expression of LXRs. c, 22(R)-hydroxycholesterol (), but not 22(S)-hydroxycholesterol () inhibits iNOS mRNA expression in an LXR-dependent manner. d, oxLDL (), but not native LDL (), inhibits iNOS mRNA expression in an LXR-dependent manner. , vehicle control (a–d). Thioglycolate-elicited murine peritoneal macrophages from wild-type, Nr1h3–/–, Nr1h2–/– or Nr1h3–/–Nr1h2–/– mice were pretreated for 18 h with 2 µM GW3965, T1317, 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, or 50 µg/ml (protein) LDL or oxLDL. Cells were treated with LPS for an additional 18 h. NO production was determined using a colorimetric assay (a), iNOS and COX-2 protein expression were analyzed by western blot (b, ns denotes non-specific signal), and iNOS mRNA levels were determined using real-time quantitative PCR assays (c, d).

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interleukin-6 MIP-1β G-CSF gelatinase B (MMP-9) MCP-3 COX-2 interferon inducible protein (IP-10) interleukin-1-β nitric oxide synthase cytokine receptor-like molecule (EBl3) suppressor of cytokine signaling -1 suppressor of cytokine signaling -3 MCP-1 TNF-resistance related protein high affinity IgG receptor (Fc-γ RI) IgG high affinity Fc receptor thrombospondin (THBS1) anaphylatoxin C3a receptor protease-activated receptor 3 (PAR3) dendritic cell immunoreceptor similar to platelet-activating factor acetylhydrolase 45 ABCA1 stearoyl-CoA desaturase plasma phospholipid transfer protein fatty acid synthase stearoyl-CoA desaturase 2 (SCD2) Apo CII α-D-galactosidase sphingosine-1-phosphate lyase CTP:phosphocholine cytidylytransferase peroxisome membrane protein (PEX2) long-chain acyl-CoA dehydrogenase fatty acid binding protein (H-FABP) 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)

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Fig. 4 Activation of LXR inhibits the expression of multiple NF-κB target genes in macrophages. a, Receptor-dependent inhibition of iNOS, COX2 and IκBα mRNA expression by LXR ligand (GW3965). b, Receptor-dependent inhibition of IL-1β (), MCP-1 (), IL-6 () and IL-1RN () mRNA expression by GW3965. Thioglycolate-elicited murine peritoneal

macrophages from wild-type, Nr1h3–/–, Nr1h2–/– or Nr1h3–/–Nr1h2–/– mice were pretreated for 18 h with GW3965 before the addition of 100 ng/ml LPS. Expression of NF-κB target genes was determined by northern blot (a) or by real-time quantitative PCR assays (b). We used 36B4 as a control for RNA loading and integrity.

Activation of LXR antagonizes NF-κB signaling We next investigated the mechanism by which LXR inhibits iNOS and COX-2 expression. The iNOS and COX-2 promoters contain binding sites for NF-κB that are required for maximal responses to LPS (refs. 23,24), but do not contain LXR binding sites. RAW 264.7 cells were transfected with expression vectors for either LXRα/RXRα or LXRβ/RXRα along with luciferase reporter constructs. The LXR agonist inhibited expression of both iNOS and COX-2 gene promoters in a receptor-dependent fashion (Fig. 5a and b). As activation of iNOS and COX-2 gene promoters by LPS is known to depend

on interactions between AP-1 and NF-κB transcription factors, we evaluated the effect of LXR on minimal promoters containing binding sites for these factors. LXR ligand effectively inhibited the reporter containing binding sites for NFκB, but not that containing binding sites for AP-1 (Fig. 5c and d). Moreover, the inhibitory effect of LXR on the COX-2 promoter was lost when NF-κB activity was blocked by transfection of a dominant-negative IKK or when binding sites for NF-κB were mutated (Fig. 5e and f). These observations suggest that inhibition of inflammatory gene expression by LXR ligands involves antagonism of NF-κB signaling.

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Fig. 5 LXRs antagonize the action of NF-κB on the iNOS and COX-2 promoters. a and b, Expression and activation of LXRα/RXR () or LXRβ/RXR () inhibits induction of the iNOS (a) and COX-2 (b) promoters by LPS. c and d, Expression and activation of LXRα/RXR () or LXRβ/RXR () inhibits expression of a luciferase reporter containing multiple NF-κB binding sites (c), but not a reporter containing multiple AP-1 binding sites (d). e, A dominant-negative IKK expression vector (IKK DN) blocks LXR-dependent repression of the COX-2 promoter. Transfected cells were treated with DMSO (), 216

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GW3965 (), LPS () or GW3965 and LPS (). f, Expression and activation of LXRα/RXR () fails to inhibit expression of a COX-2 luciferase reporter lacking the NF-κB binding site. , vector control (a–f). RAW 264.7 cells were transiently transfected with expression vectors for LXRα/RXRα, LXRβ/RXRα or dominant-negative IKK along with iNOS (a), COX-2 (b, e, f), NF-κB (c), AP-1 (d) or COX-2/NF-κB mutant (f) luciferase reporters. Following transfection, cells were treated with vehicle or 2 µM GW3965 (GW) before the addition of LPS, as indicated. NATURE MEDICINE • VOLUME 9 • NUMBER 2 • FEBRUARY 2003

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Fig. 6 LXRs are negative regulators of inflammation in vivo. a, Enhanced responses to LPS stimulation in LXR-null mice. Wild-type (WT) or Nr1h3–/–Nr1h2–/– (DKO) mice (n = 5 per group) were challenged with vehicle () or LPS () by intraperitoneal injection. Hepatic iNOS, TNF-α and IL1β expression were determined by real-time quantitative PCR assays. Plasma IL-6 levels were determined by ELISA (P < 0.05 for iNOS, TNF-α and IL-6). b, Synthetic LXR ligands reduce inflammation in a model of irritantinduced contact dermatitis. Ears of C57Bl/6J mice (n = 5 per group) were treated topically with TPA (left ear) or with vehicle (right ear). Vehicle (ctrl), GW3965 (GW) or T1317 (T) was applied topically post-TPA treatment. Thickness and weight of 6-mm punch biopsies were determined for each group after 18 h TPA treatment and are presented relative to vehicle-

treated ear. c, Histological analysis of inflammation in ears of mice treated with TPA and vehicle or LXR agonists. Representative H&E stained sections from each group are shown. Objective magnification ×10 or ×20 as indicated. d, Activation of LXR inhibits the expression of NF-κB target genes in response to IL-1β and TNF-α. Thioglycolate-elicited murine peritoneal macrophages were pretreated for 18 h with T1317 () or GW3965 () before the addition of IL-1 or TNF-α (50 ng/ml). Gene expression was detected by real-time quantitative PCR assays. e, LXR agonist inhibits the expression of MMP-9 in aortas of atherosclerotic mice. Apoe–/– mice of 8 months of age (n = 5 per group) were treated with vehicle () or GW3965 () for 3 d. Total RNA was isolated from individual aortas and gene expression was measured by real-time quantitative PCR. P < 0.05 for MMP-9 (*) and P < 0.01 (**) for ABCA1.

LXRs have anti-inflammatory activity in vivo To address whether the LXR pathway also functions to modulate inflammatory gene expression in vivo, we compared induction of inflammatory genes by LPS in wild-type and Nr1h3–/–Nr1h2–/– mice (n = 5). Induction of hepatic iNOS and TNF-α expression in response to LPS was enhanced in Nr1h3–/–Nr1h2–/– mice compared with controls (Fig. 6a). Circulating levels of IL-6, as determined by ELISA, were also significantly higher in Nr1h3–/–Nr1h2–/– mice compared with controls. In contrast, IL-1β expression was not significantly different between the two groups, indicating that certain inflammatory genes are more sensitive than others to the loss of LXR signaling in vivo. We also examined the effects of LXR ligands in both acute and chronic inflammatory contexts. First, we studied the effects of LXR agonists in a murine model of irritant contact dermatitis25. Ears were treated topically with tetraphorbol myristyl acetate (TPA) in the presence or absence of LXR ligands and inflammation was assessed after 24 hours (n = 5). Ear thickness and weight

were increased following TPA treatment, and both parameters were reduced by treatment with GW3965 or T1317 (Fig. 6b). Analysis of hematoxylin and eosin (H&E) stained sections of the TPA-treated ears confirmed a reduction in edema and inflammatory infiltrate by LXR agonist (Fig. 6c). Thus, two structurally unrelated LXR agonists have anti-inflammatory activity in a model of cutaneous inflammation. Next, we tested the influence of LXR agonists on inflammatory gene expression induced by IL-1β and TNF-α, which have been implicated in the pathogenesis of atherosclerosis. As observed above for LPS, GW3965 inhibited the induction of iNOS and IL-6 expression by IL-1β or TNF-α (50 ng/ml). Finally, we used real-time quantitative PCR to analyze gene expression in the atherosclerotic aortas of Apoe–/– mice treated with vehicle or GW3965 for 3 days. Consistent with previous work, the LXR ligand induced expression of ABCA1, but did not affect expression of the macrophage-specific marker CD68, indicating that the number of macrophages present was similar in each group (Fig. 6e). Expression of MCP-1 and iNOS

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was not different between the two groups; however, expression of MMP-9 was substantially reduced in mice treated with LXR agonist. Thus, LXR agonist reduces expression of an established inflammatory mediator in the vessel wall. Discussion Macrophages have a central role in innate immunity. They function to scavenge pathogens and apoptotic cells as well as to coordinate the inflammatory response to such stimuli through the production of cytokines and other mediators. Previous studies have outlined the importance of LXRs in the macrophage cholesterol efflux pathway. Here, we have explored the possibility that cellular lipid accumulation might affect the production of inflammatory mediators by activating LXR. Global analysis of gene expression in activated macrophages revealed that LXRs inhibit genes involved in the innate immune response while simultaneously inducing those involved in lipid metabolism. In vivo, activation of the LXR pathway was shown to antagonize inflammatory gene expression and to reduce inflammation in a model of contact dermatitis. These observations identify LXR as a molecular link between lipid metabolism and inflammation. The function of LXRs as cholesterol sensors in macrophages is likely to be important for the scavenger function of these cells. Internalization of apoptotic cells and cellular debris leads to the accumulation of large amounts of fatty acid and cholesterol. In this context, the role of LXR is to activate the cholesterol efflux pathway to protect the cell from lipid overload. Our results indicate that another consequence of LXR activation in cholesterol-loaded cells is to limit the production of inflammatory mediators. When apoptotic cells are being scavenged, an inflammatory response is not appropriate. In fact, the absence of inflammation is a hallmark of apoptotic cell death. In contrast, phagocytosis of pathogens, such as Gram-negative bacteria, elicits a marked immune response. Not only do pathogens contain molecules such as LPS that are recognized by the Toll family of receptors, they also lack cholesterol and therefore would not be expected to activate the LXR pathway. Thus, LXRs may function to integrate metabolic and immune signaling. NF-κB is the central transcriptional regulator of the innate immune response. Many of the genes inhibited by LXR are established targets of NF-κB signaling. Analysis of the iNOS and COX-2 gene promoters indicates that inhibition of these genes by LXR is accomplished through antagonism of NF-κB. Other nuclear receptors, most notably the glucocorticoid receptor (GR), have also been shown to inhibit inflammatory gene expression26,27. GR has been proposed to antagonize the actions of transcription factors such as NF-κB and AP-1 through direct physical interactions26–28. Preliminary data indicate that LXR agonists do not alter nuclear translocation of NF-κB and that these compounds may act downstream of NF-κB binding to DNA. More research will be needed to precisely define the mechanism by which LXR activation inhibits the NF-κB signaling pathway. Previous studies have linked the nuclear receptor PPARγ to the regulation of macrophage inflammatory gene expression. PPARγ ligands also antagonize the expression of inflammatory cytokines and these effects seem to be accomplished through both receptordependent and -independent mechanisms15,16,29,30. The previous demonstration that Nr1h3 is itself a target of PPARγ raises the possibility that some of the actions of PPAR ligands may be mediated by LXRα (ref. 31). The ability of GW3965 and T1317 to inhibit inflammatory gene expression is strictly receptor-dependent; however, both compounds were observed to induce expression of certain inflammatory genes in LXR-null cells (Figs. 3 and 4b). 218

Thus, these compounds are also likely to have LXR-independent effects that should be taken into account. The ability of LXRs to regulate both lipid homeostatic and innate immune responses is likely to be particularly relevant in the context of atherogenesis. The importance of inflammation in atherosclerosis is well established. Inflammatory mediators such as MCP-1, IL-6 and IL-1β promote monocyte recruitment, stimulate smooth muscle cell proliferation and increase extracellular matrix production8. Metalloproteinases such as MMP-9 have been implicated in both lesion remodeling and plaque rupture32–34. Oxidized lipids can be either inducers or inhibitors of inflammatory gene expression, depending on the context9. Our results indicate that the ability of oxysterols to antagonize LPS responses is mediated by LXRs. In contrast, the stimulatory effects of oxidized lipids on inflammatory gene expression in resting macrophages are likely to be mediated by different pathways, as LXR agonists did not alter expression of inflammatory targets in the absence of LPS. Thus, ligand activation of LXRs in lesion macrophages might be predicted to limit production of inflammatory molecules that exacerbate lesion development1,2. In support of this idea, we found that treatment with LXR ligand reduced the expression of MMP-9 in the aortas of 8-month-old Apoe–/– mice. Although we did not detect a change in acute inflammatory mediators in these animals, this may reflect the presence of long-standing and relatively advanced disease. In the future, it will be interesting to examine the effects of LXR agonists in more acute models of vessel wall injury. Our findings may be directly related to recent studies demonstrating that activation of LXR inhibits the development of atherosclerosis in murine models. We previously reported that GW3965 reduces lesion formation in both Ldlr–/– and Apoe–/– mice21. The modest changes in plasma lipid levels observed in these studies indicate that local actions of LXR ligands on macrophages within the artery wall are likely to contribute to their pharmacological effects. The recent observation that loss of LXR expression in bone marrow cells increases atherosclerosis further supports the hypothesis that the macrophage is a primary target of the anti-atherogenic effects of LXR (ref. 22). Our results indicate that LXR agonists may reduce atherosclerosis not only by promoting cholesterol efflux, but also by acting to limit the production of inflammatory mediators in the artery wall. Finally, these results raise the possibility that LXR agonists may have utility in the treatment of other chronic inflammatory diseases in which activated macrophages play an important role. Methods Reagents and plasmids. GW3965, GW1516, GW7647 and T0901317 were from GlaxoSmithKline (Research Triangle Park, North Carolina). Oxysterols, LDL and ox-LDL were from Sigma and Intracell (St. Louis, Missouri; Fredrick, Maryland), respectively. LPS from Salmonella typhimurium was from Sigma. Expression plasmids for LXRα, LXRβ and IKKDN (refs. 35,36), and reporter plasmids for NOS-2, (NFκB)3-LUC, (AP-1)4-LUC and COX-2 have been described37. Cell culture and transfections. Thioglycolate-elicited peritoneal macrophages were cultured in RPMI containing 10% fetal bovine serum (FBS). RAW 264.7 cells were cultured in DMEM containing 10% FBS. For ligand treatments, cells were in cultured medium supplemented with 5% lipoprotein-deficient serum (LPDS) and ligands for 18 h before stimulation. Bacterial infection in vitro was performed as described38. Transient transfections of RAW 264.7 cells were performed in triplicate in 48-well plates using Superfect reagent (Life Technologies). After transfection, cells were incubated in medium containing 10% LPDS and the indicated ligands for 18 h before stimulation with LPS for an additional 18 h. Luciferase activity was normalized to β-galactosidase activity. NO production was determined spectrophotometrically39. NATURE MEDICINE • VOLUME 9 • NUMBER 2 • FEBRUARY 2003

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RNA and protein analysis. Total RNA was extracted using Trizol reagent (Life Technologies, Inc., Rockville, Maryland). Northern blot analysis was carried out as described using radiolabeled cDNA probes12. Real-time quantitative PCR (TaqMan) analysis was performed using an Applied Biosystems 7700 sequence detector. Two-step real-time PCR was performed as described40. Primer and probe sequences are available upon request. Reactions were performed in duplicate and normalized to 36B4 expression. Western blot analysis was performed as described35. Antibodies against NOS-2 (SC-651) and COX-2 (160106) were from Santa Cruz Biotechnology and Cayman Chemicals (Santa Cruz, California; Ann Arbor, Michigan), respectively. DNA microarray analysis. Thioglycolate-elicited peritoneal macrophages were cultured in DMEM/10% LPDS and treated with vehicle or 2 µM GW3965 for 18 h before stimulation with 100 ng/ml LPS for 6 h. Total RNA was reverse transcribed using a T7-(dT)24 primer (Genset Corp., La Jolla, California) and Superscript Choice (Life Technologies). Biotin-labeled cRNA was generated using the Bioarray High Yield Transcript Labeling Kit (ENZO). Samples were hybridized to single Affymetrix Murine U74Av2 microarrays and visualized by the PAN Facility at Stanford University. Data was analyzed using GeneSpring and GeneChip Analysis Suite software (Affymetrix). Only those results determined by the software to represent significant expression differences in the presence or absence of ligand are presented. Positive results were defined as ligand-dependent changes in expression in wild-type, Nr1h3–/– and Nr1h2–/– cells, but not in Nr1h3–/–Nr1h2–/– cells. Animals. Nr1h3+/+Nr1h2+/+, Nr1h3–/–, Nr1h2–/– and Nr1h3–/–Nr1h2–/– mice on a mixed background (C57Bl/6 and 129Sv) and Apoe–/– mice on a C57Bl/6 background were maintained on standard chow. Where indicated, diets were supplemented with GW3965 at a level sufficient to provide a 20 mg/kg body weight dose per day. Irritant contact dermatitis was induced as described25. Briefly, 10 µl of 0.03% TPA in acetone was applied to the left ears of male mice. The right ears were treated with vehicle alone. We applied 20 µl of 10 mM T1317, 10 mM GW3965, or vehicle to both left and right ears at 45 min and 4 h post-TPA treatment. After 18 h of TPA treatment, the thickness and weight of 6-mm punch biopsies were determined. Statistical analysis was performed using Student’s t-test. Experiments were conducted in accordance of the Animal Research Committee of the University of California, Los Angeles.

Supplementary information is available on the Nature Medicine website.

Acknowledgments We thank T.M. Willson and J. Collins for GW3965 and T1317, and L. Bosca and P. Martin for reagents and comments. P.T. is an Assistant Investigator of the Howard Hughes Medical Institute at the University of California, Los Angeles. This work was supported by grants from the NIH (HL 660881) the Human Frontier Science Project (RGY-021) to P.T. Competing interests statement The authors declare that they have no competing financial interests.

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