Published online before print May 31, 2007, doi: 10.1161/CIRCRESAHA.106.135814
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Liver X Receptor Activation Potentiates the Lipopolysaccharide Response in Human Macrophages
From the Institut Pasteur de Lille, Département d’Athérosclérose, Lille; Inserm U545, Lille; and Université de Lille 2, Faculté de Pharmacie, Lille, France.
Correspondence to Bart Staels, Inserm U545, Institut Pasteur de Lille, 1, rue du Professeur Calmette, BP 245, Lille 59019, France. E-mail Bart.Staels{at}pasteur-lille.fr
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Abstract |
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Macrophages play a central role in host defense against pathogen microbes by recognizing bacterial components, resulting in the activation of an arsenal of anti-microbial effectors. Toll-like receptor (TLR)-4 mediates the recognition of lipopolysaccharide, a pathogen-associated molecular pattern from Gram-negative bacteria. Activation of the TLR-4 signaling pathway by lipopolysaccharide increases antibacterial effects by inducing secretion of cytokines that activate an immune inflammatory response and by generating bactericidal reactive oxygen species via the NADPH oxidase system. Liver X Receptors (LXRs) are nuclear receptors controlling cholesterol homeostasis and inflammation in macrophages. In addition, LXRs are critical for macrophage survival and play a role in the innate immune response in the mouse. In this study, we investigated whether LXR activation also regulates host defense mechanisms in human macrophages. In primary human macrophages, oxidized LDL and synthetic LXR ligands increased TLR-4 gene expression. Transient transfection assays, gel shift and chromatin immunoprecipitation analysis indicated that LXRs induce human TLR-4 promoter activity by binding to a DR4-type LXR response element. LXR induction of TLR-4 mRNA was followed by an induction of TLR-4 protein expression. Moreover, although short-term pretreatment with LXR agonists significantly reduced the inflammatory response induced by lipopolysaccharide, pretreatment of macrophages for 48 hours with LXR agonists resulted in an enhanced lipopolysaccharide response. Finally, LXR activation increased reactive oxygen species generation by enhancing the expression of NADPH oxidase subunits. These data provide evidence for an immunomodulatory function of LXRs in human macrophages via mechanisms distinct from those previously identified in mouse macrophages.
Key Words: macrophages • nuclear receptors • lipopolysaccharide • reactive oxygen species
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Introduction |
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Macrophages participate in the regulation of innate and adaptive immunity. These cells play a central role in the host defense against pathogen microbes by rapidly recognizing bacterial components and activating an arsenal of antimicrobial effectors.1 The ability of macrophages to recognize bacterial pathogen-associated molecular patterns is conferred in part by the Toll-like receptor (TLR) family of proteins. Ten members of the TLR family have been described that collectively recognize a wide range of microbial components.2 Lipopolysaccharide (LPS) is an integral component of the outer membrane of Gram-negative bacteria. LPS signaling is mediated by TLR-4, because invalidation of this gene in mice leads to LPS-unresponsiveness.3 LPS binding to TLR-4 activates the mitogen-activated protein kinases (MAPK) c-Jun N-terminal kinase (Jnk), p38, and extracellular signal-regulated kinase (Erk) cascades2,4 and induces the expression of genes involved in innate immunity (eg, selectins, NADPH oxidase) and inflammatory response (eg, monocyte chemoattractant protein [MCP]-1 and tumor necrosis factor [TNF]
), which recruit and/or activate neighboring cells to eliminate pathogens. In addition, activation of TLR-4 by LPS triggers NADPH oxidase activation.5 NADPH oxidase is a complex enzyme that consists of 5 major subunits: a plasma membrane spanning cytochrome b558 composed of 2 subunits, gp91phox and p22phox, linked to flavin and cytosolic components such as Rac-2, p67phox, and the subunit p47phox, the latter requiring phosphorylation to be active.6 This enzyme produces superoxide anions that can lead to the generation of toxic reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, all of which can directly cause oxidative damage to bacteria.7 Interestingly, several recent lines of evidence indicate that the TLR-4 signaling pathway is also activated by ROS.8,9
The liver X receptors, LXR and LXRß, are nuclear receptors that regulate genes controlling lipid metabolism and inflammation.10 LXRs are activated by oxysterols as well as by intermediate products of the cholesterol biosynthetic pathway.11,12 LXRs bind as heterodimers with the retinoid X receptor (RXR) to specific response elements, termed LXR response elements (LXREs), located in promoters of target genes.13 LXREs usually consist of a (A/G)GGTCA direct repeat motif spaced by 4 nucleotides (DR4). LXR activation in macrophages induces expression of several genes involved in cholesterol trafficking and efflux including genes encoding the Niemann Pick C (NPC)1/NPC2 proteins,14 the ATP-binding cassette transporters (ABC)A115 and ABCG1/ABCG416 and apolipoprotein (apo)E.17 In addition to their well-established role as cholesterol sensors, LXRs regulate transcriptional programs involved in the inflammatory response. In murine macrophages, LXR activation has been shown to inhibit TLR-4–mediated LPS response18,19 by antagonizing the nuclear factor 
B pathway through a mechanism that is not completely understood. Furthermore, TLR-4 activation can inhibit LXR-induced cholesterol efflux from macrophages, indicating a physiological crosstalk between inflammation and lipid metabolism.20 Although the antiinflammatory properties of LXRs are well documented in murine macrophages, the role of LXRs in the inflammatory response in human monocyte/macrophages is controversial. In human monocytes, LXR agonists suppress tissue factor and TNF expression induced by proinflammatory stimuli.21,22 However, TNF and vascular endothelial growth factor are also direct LXR target genes in human monocyte/macrophages.23,24 In addition, a role for LXRs in the control of the innate immune response, especially macrophage survival, in mice has also emerged: LXR activation prevents bacteria-induced macrophage apoptosis and LXR-null macrophages undergo accelerated apoptosis when challenged with intracellular bacteria.25,26
Although the crosstalk between the LXR and TLR signaling pathways is well established in murine macrophages, data showing whether LXRs could also play a role in the TLR-4/LPS signaling pathways in human macrophages are lacking. We demonstrate that LXR activation leads to an increase in TLR-4 expression in human but not in murine macrophages. Our results show that this regulation occurs at the transcriptional level via a LXRE in the human TLR4 promoter. Induction of TLR-4 by LXR activation enhances signaling pathways in response to LPS, leading to an increased MCP-1 and TNF secretion. Moreover, LXR activation increases ROS generation in both resting and LPS-stimulated macrophages by enhancing the expression of the NADPH oxidase subunits. Our results provide evidence for novel mechanisms through which LXRs could modulate the adaptive immune response in human macrophages.
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Materials and Methods |
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Cell Culture
Mononuclear cells were isolated by Ficoll gradient centrifugation from donor blood. Mature monocyte-derived macrophages were used for experiments after 10 days of culture. Murine bone marrow–derived macrophages were prepared from C57BL/6J mice. For experiments, medium was changed to medium without serum (see the online supplement, available at http://circres.ahajournals.org).
RNA Extraction and Analysis
Total cellular RNA from macrophages was extracted using TRIzol (Life Technologies), and reverse transcription was performed using random hexameric primers and Superscript reverse transcriptase (Life Technologies). cDNA were quantified by quantitative PCR on an MX 4000 apparatus (Stratagene) using specific primers (see the online data supplement).
Electrophoretic Mobility Shift Assays
LXR and RXR were in vitro transcribed and subsequently translated. For blocking experiments, 1 µg of polyclonal anti-LXR antibody (P-20; Santa Cruz Biotechnology) was added to the binding reaction. The radiolabeled probes were added, and the binding reaction was incubated at room temperature. Protein complexes were resolved by 6% nondenaturing polyacrylamide gel electrophoresis (see the online data supplement).
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) experiments were performed using differentiated THP-1 macrophages. Cell lysate was immunoprecipitated using anti-LXR (P-20; Santa Cruz Biotechnology) or anti-IgG antibodies (Santa Cruz Biotechnology) as negative control. The same lysate volume was kept without immunoprecipitation for subsequent purification of input genomic DNA. Immunoprecipitated DNA was PCR amplified using primers covering either the –555 to –349 region of the human TLR-4 promoter containing the (–477/–461) TLR-4-LXRE or part of the ß-actin gene as negative control (see the online data supplement).
Plasmid Cloning and Transient Transfection Experiments
HuH7 cells were transfected with reporter plasmids and with expression vectors using the cationic lipid RPR120535B.27 Cells were subsequently incubated in medium containing 2% Ultroser (BioSepra, Cergy Pontoise, France) and T0901317 (1 µmol/L) or GW3965 (1 µmol/L) for 48 hours and luciferase and ß-galactosidase assays were performed (see the online data supplement).
Short-Interfering RNA
Short-interfering (si)RNA specific for human LXR and LXRß (SMARTpool siRNA) and nonsilencing control siRNA were purchased from Dharmacon. Human macrophages were transfected using the transfection reagent DharmaFECT Reagent 4. Forty-eight hours after transfection, cells were incubated in the presence of T0901317 (1 µmol/L) or vehicle (DMSO) and harvested 24 hours later.
Protein Extraction and Western Blot Analysis
Protein lysate was separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with polyclonal anti–TLR-4 (H-80; Santa Cruz Biotechnology); anti-p47phox and anti-p91phox (kindly provided by Dr A. Shah, GKT School of Medicine, King’s College, London, UK); anti–ß-actin (I-19; Santa Cruz Biotechnology); anti-MAPK p38, phospho-p38, Erk, and phospho-Erk (Cell Signaling Technology) antibodies. Immunoreactive bands were quantified using ChemiGenius 2 (Syngene) (see the online data supplement).
Fluorescein Isothiocyanate–Conjugated LPS-Binding Assay
Primary human macrophages treated or not with T0901317 (1 µmol/L) or GW3965 (1 µmol/L) were incubated with fluorescein isothiocyanate (FITC)-conjugated LPS (200 ng/mL). Cellular binding of FITC-LPS was analyzed by flow cytometry.
Cytokine Secretion in Cell Culture Supernatants
MCP-1 and TNF secreted in cell culture supernatants were quantified using ELISA purchased from Peprotech according to the instructions of the manufacturer.
ROS Quantification
Cells were exposed to T0901317 (1 µmol/L) or GW3965 (1 µmol/L) for 48 hours and then incubated with dichlorofluorescein diacetate (10µmol/L) for 30 minutes. Cells were lysed in 1 mL of 1 mol/L NaOH, and dichlorofluorescein fluorescence was read at 
exc=485 nm and em=530 nm (see the online data supplement).
Statistical Analysis
Statistical differences between groups were analyzed by Student t and ANOVA tests and were considered significant when P<0.05.
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Results |
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LXR Activation Increases TLR-4 Gene Expression in Human, but Not in Murine, Macrophages
Quantitative PCR analysis was performed on RNA isolated from primary human differentiated macrophages treated for 24 hours with LXR ligands. Treatment with either T0901317 (1 µmol/L) or GW3965 (1 µmol/L) resulted in the induction of TLR-4 mRNA levels (Figure 1A). Similar results were obtained in 4-phorbol 12-myristate 13-acetate–differentiated THP-1 macrophages (see Figure I in the online data supplement). In addition, treatment with oxidized LDL (oxLDL) (50 µg/mL), which contains natural LXR ligands, strongly induced TLR-4 mRNA levels (Figure 1B). Kinetic studies in primary human macrophages demonstrated that TLR-4 gene induction by LXR agonists appears only after 24 hours of treatment (Figure 1C). Strikingly, LXR activation by T0901317 (2 µmol/L) or GW3965 (2 µmol/L) had no impact on TLR-4 expression in murine bone marrow–derived macrophages, pointing to a species-specific regulation of TLR-4 by LXR (Figure 1D). ABCA1 gene expression was measured as positive control of LXR activation both in human and murine macrophages (data not shown). Together, these results demonstrate a species-specific increase of TLR-4 mRNA levels after LXR activation.
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LXRs Bind to the Human TLR-4 Promoter In Vitro and In Vivo
To determine whether TLR-4 is directly responsive to LXRs, the human TLR-4 proximal promoter was examined by bioinformatics analysis to determine the presence of potential LXRE sites. A potential LXRE very similar to the consensus sequence was found between nucleotides –477 and –461 upstream of the transcription initiation site (Figure 2A). Electrophoretic mobility shift assays were performed to test the ability of the human LXR/RXR heterodimer to bind to this putative (–477/–461) TLR-4–LXRE. A specific DNA–protein complex was formed when in vitro synthesized LXR and RXR proteins were incubated with the P32-labeled probe covering the TLR-4–LXRE (Figure 2B, lane 7). RXR or LXR alone did not bind to this site, confirming that these receptors cannot bind as homo- or monomers. The binding of LXR/RXR to the probe is specific, because the complex was competed by an excess of the unlabeled TLR-4–LXRE oligonucleotide (Figure 2B, lanes 8 to 10) but not by an excess of the unlabeled mutated TLR-4–LXRE oligonucleotide (Figure 2B, lanes 11 to 13). The specificity of LXR/RXR binding was verified by the addition of a polyclonal anti-LXR antibody, which inhibited the formation of the LXR/RXR complex (lane 6). Furthermore, no binding was observed on the mutated (–477/–461) TLR-4–LXRE (Figure 2B, lane 15). Similar results were obtained for the LXRß/RXR heterodimer (data not shown). These data demonstrate that LXR and LXRß bind as a heterodimer with RXR to the (–477/–461) TLR-4–LXRE present in the promoter of the human TLR-4 gene. Alignment of the human and murine TLR-4 5'-promoter region sequence28 indicated that the LXRE site is not conserved between the human and the murine promoters, probably explaining the species-specific regulation of TLR-4 by LXR (Figure 2A).
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To validate the binding of LXR to the native TLR-4 promoter in vivo, a ChIP assay was performed in human THP-1 macrophages, in which TLR-4 mRNA is also strongly induced by treatment with LXR agonists (see the online data supplement). The genomic DNA region encompassing the LXRE of the TLR-4 gene was immunoprecipitated with a polyclonal anti-LXR antibody (Figure 2C). PCR amplification with primers specific for the ß-actin gene did not result in any significant signal, thus demonstrating the specificity of immunoprecipitation and PCR amplification reactions. These results provide qualitative evidence that in human macrophages, LXR binds to the LXRE sequence of the TLR-4 gene.
LXR/RXR Heterodimers Induce TLR-4 Promoter Activity
To test whether LXRs/RXR activate transcription from the (–477/–461) TLR-4–LXRE site, 3 copies of this element were cloned in front of the herpes simplex virus thymidine kinase promoter to obtain the (TLR-4–LXRE)3x-Tk-Luc luciferase reporter vector. In HuH7 cells, cotransfection of pCMX-hLXR and pSG5-hRXR expression vectors on the (TLR-4–LXREwt)3x reporter vector led to significant induction of transcriptional activity compared with empty pCMX and pSG5 vectors. This effect was enhanced by T0901317 (1 µmol/L) or GW3965 (1 µmol/L) treatment (Figure 3A). Induction of transcriptional activity after LXR/RXR transfection and activation was abolished when the mutated LXRE site was tested (Figure 3A).
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To further investigate whether LXR activates the TLR-4 promoter, transfection assays were performed using luciferase reporter constructs driven by the natural TLR-4 promoter29 of 620 bp or by the TLR-4 promoter fragment that contains 480 bp and lacks the TLR-4–LXRE site. Activity of the 620-bp TLR-4 promoter was induced by LXR/RXR cotransfection and enhanced by the presence of T0901317 (1 µmol/L) (Figure 3B). By contrast, the truncated 480-bp TLR-4 promoter was unaffected by either LXR/RXR cotransfection or T0901317 treatment. In addition, introduction of specific mutations of the LXRE in the context of the 620-bp TLR-4 promoter abolished induction of the TLR-4 promoter by either LXR/RXR cotransfection in the absence or in the presence of T0901317 (1 µmol/L) (Figure 3B).
Similar results on both TLR-4–LXRE site and natural human TLR-4 promoter were obtained when LXRß and RXR were transfected under the same experimental conditions (data not shown). These results demonstrate that the human TLR-4 promoter contains a functional LXRE that confers responsiveness to LXRs.
LXR Expression Is Necessary for the Induction of TLR-4 Gene Expression by LXR Agonists
To address whether the stimulatory effect of LXR ligands on TLR-4 gene expression was mediated by LXR, a siRNA approach was used to reduce LXR and LXRß expression. Quantitative PCR analysis indicated that transfection of a specific double LXR/LXRß siRNA pool significantly suppressed LXR and LXRß gene expression by 
70% and 80%, respectively, in comparison with control siRNA-transfected cells (Figure 4A and 4B). Inhibition of LXR/LXRß expression drastically abolished the ability of T0901317 (1 µmol/L) to induce TLR-4 gene expression (Figure 4C). ABCA-1 gene induction, measured as positive control, was also significantly reduced (Figure 4D). Taken together, these data demonstrate that LXR ligands induce TLR-4 gene expression through a receptor-dependent mechanism.
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LXR Activation Increases TLR-4 Protein Expression
To determine whether TLR-4 gene regulation by LXR ligands is followed by an increase at the protein level, total proteins were isolated from primary human macrophages incubated with T0901317 (1 µmol/L) or GW3965 (1 µmol/L) at different time points. TLR-4 protein levels were subsequently measured by Western blot analysis. Treatment with both LXR agonists increased significantly TLR-4 protein levels after 48 hours of treatment (Figure 5A and 5B). Secondly, binding of FITC-conjugated LPS was examined by flow cytometry in primary human macrophages treated with T0901317 (1 µmol/L) or GW3965 (1 µmol/L) for 48 hours. T0901317 (Figure 5C) and GW3965 (Figure 5D) increased the binding of FITC-LPS to primary human macrophages by 22±5% (P<0.01) and 37±11% (P<0.01) compared with untreated cells, respectively.
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LXR Activation Regulates the Response of TLR-4 Signaling Pathways to LPS in Primary Human Macrophages
To determine the functional consequences of TLR-4 upregulation by LXRs, MAPK activation was examined by measuring Jnk, Erk, and p38 phosphorylation using specific antibodies.30 Interestingly, Jnk, Erk, and p38 phosphorylation in response to LPS was enhanced in primary human macrophages pretreated for 48 hours with LXR agonists (Figure 6A and 6B). In addition, secretion of MCP-1 and TNF in response to LPS was significantly enhanced in macrophages pretreated with the LXR agonists for 48 hours, whereas short-term pretreatment with LXR agonists inhibited the LPS response as previously reported by Walcher et al22 (Figure 6C and 6D). Moreover, 48-hour pretreatment with the LXR agonists strongly enhanced the LPS-induced expression of the MCP-1, TNF, and COX-2 genes in primary human macrophages as well as in THP-1 differentiated macrophages (see supplemental Figure II).
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LXR Activation Increases ROS Production in Human Macrophages
LPS is known to induce ROS production in macrophages, most likely by activating membrane associated NADPH oxidase.5 Therefore, ROS was measured after LPS stimulation of primary human differentiated macrophages incubated with the LXR ligand T0901317 (1 µmol/L) for 48 hours. As reported previously, ROS production was induced by LPS in macrophages.5 Interestingly, LXR activation increased ROS release in both resting and LPS-stimulated macrophages (Figure 7A), suggesting a direct regulation of ROS production by LXRs. The increase in ROS production in the presence of LXR agonists was associated with the induction of membrane NADPH oxidase activity, because the effect was abolished by diphenyliodonium, a specific flavin inhibitor (Figure 7B). Similar results were obtained using THP-1 differentiated macrophages (see supplemental Figure III).
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It was next investigated whether LXRs regulate the expression of genes encoding NADPH oxidase subunits in macrophages. p47phox and gp91phox mRNA levels were increased in human macrophages treated for 24 hours with T0901317 or GW3965, an effect dependent on the expression of LXR/LXRß, as demonstrated by the loss of gene induction by T0901317 in cells transfected with an LXR/LXRß siRNA (see supplemental Figure IV). By contrast, p22phox and p67phox transcripts were not affected by LXR activation (data not shown). Treatment with LXR agonists for 24 hours also led to an induction of p47phox and gp91phox protein expression in human macrophages (see supplemental Figure IV).
Strikingly, LXR activation had no impact on NADPH oxidase subunit gene expression nor on ROS production in murine bone marrow–derived macrophages, pointing again to a species-specific regulation of NADPH oxidase by LXR agonists (see supplemental Figure V).
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Discussion |
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Our results demonstrate that LXR agonists induce the expression of the LPS receptor TLR-4 in human but not in murine macrophages. A LXRE site was identified in the human TLR-4 promoter that mediates the transactivation by LXRs/RXR
. The mouse and human TLR-4 genes are highly conserved.31 However, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by different stimuli.28,31 The LXRE site is not conserved between the human and mouse TLR-4 promoters, likely explaining the species-specific regulation of TLR-4 by LXRs. Increased expression of TLR-4 resulted in an enhanced responsiveness of human macrophages to LPS. Indeed, LXR agonist pretreatment enhanced MAPK activation as well as MCP-1 and TNF secretion in LPS-stimulated human macrophages. Moreover, we show that LXR activation increases ROS production in LPS-activated human macrophages. This effect was also observed in resting macrophages. Enhanced ROS production in LXR-activated macrophages was associated with an increase in the expression of the NADPH oxidase subunits p47phox and gp91phox.
TLR-4 is critical for the recognition of LPS, a chief pathogen-associated molecular pattern from Gram-negative bacteria. TLR-4 activation by LPS engages multiple mechanisms that control the initiation of the adaptive immune response4 involved in the surveillance, attack, containment, and clearance of pathogens.32 Cell migration from peripheral blood into the inflamed tissue involves tightly controlled sequences of events. Activation of TLR-4 results in the induction of cytokine expression, which regulates cell migration and activation to the sites of inflammation.32 In addition, LPS-activated macrophages produce ROS, which could directly kill bacteria within the phagosome.33 Because MCP-1 and TNF secretion in response to LPS as well as ROS production are induced in LXR-activated macrophages, it appears that LXRs may play a role in the macrophage response against bacteria. Indeed, LXRs may contribute to bacterial elimination through recruitment and/or activation of neighboring cells as well as through the production of antibacterial ROS.
Progression of the innate immune response is also regulated at the level of macrophage survival. Of note, certain bacterial pathogens target TLR-4–initiated antiapoptotic mechanisms to induce the death of activated macrophages and thereby evade detection and destruction by the host immune response.34 Interestingly, LXR-deficient mice are highly susceptible to infection by the intracellular bacteria Listeria monocytogenes.26 Bone marrow transplant studies pointed to altered macrophage function as the major determinant of this susceptibility. LXR activation antagonized pathogen-induced apoptosis of murine macrophages through upregulation of the antiapoptotic factors AIM and Bcl-xl as well as inhibition of proapoptotic gene expression.25,26 However, we were not able to detect AIM and Bcl-xl expression in primary human macrophages under our experimental conditions (data not shown), thus excluding the possibility that this mechanism is operational in this human model. Nevertheless, silencing of LXR gene expression in human peripheral blood mononuclear cells resulted in significant upregulation of the expression of the proapoptotic c-myc gene.35 In addition, in the antiapoptotic branch of TLR-4 signaling, nuclear factor B and p38 MAPK cooperate to induce transcription of 2 antiapoptotic genes, Pai-2 and Bfl-1/A1, the products of which block the concurrent activation of the proapoptotic pathways.36,37 Thus, it cannot be excluded that LXR agonists, via induction of TLR-4, also modulate the apoptotic response of human macrophages.
Recently, Walcher et al have identified an anti-inflammatory action of LXRs in human cells.22 These authors showed that short-term, acute cotreatment of Th-1 primed human monocytes with LXR agonists, together with interferon 
, led to a slight decrease in TNF secretion. In line with these observations, we observed that pretreatment of primary human macrophages with LXR agonists for 0 to 6 hours before stimulation with LPS resulted in a reduction of LPS-induced MCP-1 and TNF secretion. However, when macrophages were pretreated for 48 hours with LXR agonists, an increase of the LPS/TLR-4 signaling pathway was observed. Thus, it appears that LXR activation prepares macrophages to allow an enhanced antibacterial response via induction of the TLR-4 signaling pathway, whereas, once the inflammatory stimulus is present, LXRs exert antiinflammatory actions. This combination of chronic and acute effects suggests that the LXR pathway may have evolved to potentiate the role of the macrophage in the response to and resolution of inflammation. Clearly, the impact of LXR signaling on macrophage gene expression is complex and context dependent.
In conclusion, our results identify a novel role for LXRs in the preparation of macrophages to exert antibacterial activities via induction of TLR-4 expression and NADPH oxidase activity.
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Acknowledgments |
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We thank C. Duhem and S. Lecher for technical help, K. Bertrand (Genfit, Loos, France) for providing the T0901317 and GW3965 compounds, and Dr A. Shah (London, UK) for providing p47phox and gp91phox antibodies.
Sources of Funding
We acknowledge grant support from the Region Nord-Pas de Calais (to C.F) and the European Vascular Genomics Network (LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received June 30, 2006; revision received April 10, 2007; accepted May 17, 2007.
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