Reciprocal regulation of inflammation and lipid metabolism by liver X receptors

Previous studies have suggested a role for peroxisome proliferator-activated receptor (PPAR) in control of inflammation. Oxidized lipids derived from oxLDL activate PPAR and induce expression of CD36 (refs. 11,12). Certain PPAR ligands, especially 15-deoxy-12,14-prostaglandin J2, antagonize the expression of iNOS, tumor necrosis factor (TNF)- and IL-6 in response to macrophage activation^{13, 14}. Studies using PPAR-deficient macrophages, however, have shown that at least some of these effects are independent of PPAR (refs. 15,16). Clearly, additional uncharacterized signaling pathways contribute to the regulation of inflammatory gene expression by oxidized lipids.

The liver X receptors (LXR, encoded by the gene Nr1h3, and LXR, 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 cholesterol^{18, 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.

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 PPAR (rosiglitazone), PPAR (GW1516) and PPAR (GW7647) had minimal effects on iNOS expression at concentrations of 1 M (Fig. 1a−c).

Figure 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 M GW3965 (LXR), rosiglitazone (PPAR), GW1516 (PPAR) or GW7647 (PPAR). 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). Full Figure and legend (55K)

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 M) was preserved in Nr1h3^-/- and Nr1h2^-/- macrophages, but abolished in Nr1h3^-/-Nr1h2^-/- 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 M), but not the inactive stereoisomer 22(S)-hydroxycholesterol (2 M), 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 g/ml (protein) oxLDL was significantly impaired in Nr1h3^-/-Nr1h2^-/- macrophages (Fig. 2d).

Figure 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 M GW3965, T1317, 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, or 50 g/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). Full Figure and legend (45K)

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 M 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-1 and granulocyte colony-stimulating factor (G-CSF), chemokines such as monocyte chemoattractant protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1 (MIP-1) and interferon-inducible 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 by LXRs.

Figure 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 M) 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. Full Figure and legend (90K)

LPS is an established inducer of the NF-B signaling pathway in macrophages. Several genes inhibited by LXR ligands in the array studies above are established targets of NF-B. To further investigate the role of LXRs in NF-B−dependent gene expression, we analyzed expression of NF-B target genes in peritoneal macrophages from wild-type, Nr1h3^-/-, Nr1h2^-/- and Nr1h3^-/-Nr1h2^-/- mice. Northern blot analysis (3 hours and 6 hours after LPS stimulation, Fig. 4a) and real-time quantitative PCR analysis (6 hours after LPS stimulation, Fig. 4b) confirmed that LXR agonists were effective inhibitors of COX-2, IB, IL-1, MCP-1, IL-6 and IL1-receptor antagonist (IL-1RN) expression (Fig. 4a and b). This inhibition 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.

Figure 4. Activation of LXR inhibits the expression of multiple NF-B target genes in macrophages. a, Receptor-dependent inhibition of iNOS, COX-2 and IB mRNA expression by LXR ligand (GW3965). b, Receptor-dependent inhibition of IL-1 (), 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-B 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. Full Figure and legend (59K)

Activation of LXR antagonizes NF-B 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-B 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 LXR/RXR or LXR/RXR 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-B 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 NF-B, 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-B activity was blocked by transfection of a dominant-negative IKK or when binding sites for NF-B were mutated (Fig. 5e and f). These observations suggest that inhibition of inflammatory gene expression by LXR ligands involves antagonism of NF-B signaling.

Figure 5. LXRs antagonize the action of NF-B on the iNOS and COX-2 promoters. a and b, Expression and activation of LXR/RXR () or LXR/RXR () inhibits induction of the iNOS (a) and COX-2 (b) promoters by LPS. c and d, Expression and activation of LXR/RXR (L) or LXR/RXR () inhibits expression of a luciferase reporter containing multiple NF-B 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 (), GW3965 (), LPS () or GW3965 and LPS (). f, Expression and activation of LXR/RXR () fails to inhibit expression of a COX-2 luciferase reporter lacking the NF-B binding site. , vector control (a−f). RAW 264.7 cells were transiently transfected with expression vectors for LXR/RXR, LXR/RXR or dominant-negative IKK along with iNOS (a), COX-2 (b, e, f), NF-B (c), AP-1 (d) or COX-2/NF-B mutant (f) luciferase reporters. Following transfection, cells were treated with vehicle or 2 M GW3965 (GW) before the addition of LPS, as indicated. Full Figure and legend (62K)

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- 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-1 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.

Figure 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- and IL-1 expression were determined by real-time quantitative PCR assays. Plasma IL-6 levels were determined by ELISA (P < 0.05 for iNOS, TNF- and IL-6). b, Synthetic LXR ligands reduce inflammation in a model of irritant-induced 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-B target genes in response to IL-1 and TNF-. Thioglycolate-elicited murine peritoneal macrophages were pretreated for 18 h with T1317 () or GW3965 () before the addition of IL-1 or TNF- (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. Full Figure and legend (133K)

Next, we tested the influence of LXR agonists on inflammatory gene expression induced by IL-1 and TNF-, 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-1 or TNF- (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 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.

NF-B is the central transcriptional regulator of the innate immune response. Many of the genes inhibited by LXR are established targets of NF-B signaling. Analysis of the iNOS and COX-2 gene promoters indicates that inhibition of these genes by LXR is accomplished through antagonism of NF-B. Other nuclear receptors, most notably the glucocorticoid receptor (GR), have also been shown to inhibit inflammatory gene expression^{26, 27}. GR has been proposed to antagonize the actions of transcription factors such as NF-B and AP-1 through direct physical interactions^{26, 27, 28}. Preliminary data indicate that LXR agonists do not alter nuclear translocation of NF-B and that these compounds may act downstream of NF-B binding to DNA. More research will be needed to precisely define the mechanism by which LXR activation inhibits the NF-B signaling pathway.

Previous studies have linked the nuclear receptor PPAR to the regulation of macrophage inflammatory gene expression. PPAR ligands also antagonize the expression of inflammatory cytokines and these effects seem to be accomplished through both receptor-dependent and -independent mechanisms^{15, 16, 29, 30}. The previous demonstration that Nr1h3 is itself a target of PPAR raises the possibility that some of the actions of PPAR ligands may be mediated by LXR (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). 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-1 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 rupture^{32, 33, 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 development^{1, 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.

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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 LXR, LXR and IKK-DN (refs. 35,36), and reporter plasmids for NOS-2, (NFB)₃-LUC, (AP-1)₄-LUC and COX-2 have been described³⁷.

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 -galactosidase activity. NO production was determined spectrophotometrically³⁹.

Thioglycolate-elicited peritoneal macrophages were cultured in DMEM/10% LPDS and treated with vehicle or 2 M 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.

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 l 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 l 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.

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