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Systemic and Distal Repercussions of Liver-Specific Peroxisome Proliferator-Activated Receptor- Control of the Acute-Phase Response

Institut Pasteur de Lille (R.M,M., E.B., B.S., P.G.), Département d'Athérosclérose, and Institut National de la Santé et de la Recherche Médicale Unité 545, Lille F59019, France; and Faculté des Sciences Pharmaceutiques et Biologiques (R.M.M., B.S., P.G.), Université de Lille 2, Lille F-59006, France

Address all correspondence and requests for reprints to: Philippe Gervois, Laboratoire de biochimie, Faculté des Sciences Pharmaceutiques et Biologiques, 3, rue du professeur Laguesse, BP83, Lille F-59006, France. E-mail: philippe.gervois{at}univ-lille2.fr.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The acute-phase response is characterized by the modulation of liver expression of many proteins involved in a diversity of biological functions. Among them, some are associated with the pathology of atherosclerosis. We previously found that peroxisome proliferator-activated receptor- (PPAR) agonists attenuate the IL-6 induction of acute-phase response gene expression in vitro and in vivo. In the current work, we found a PPAR-dependent regulation of hepatic acute-phase response stimulated by IL-1. We also found that IL-1-stimulated expression of secondary wave cytokines such as IL-6 is prevented upon PPAR activation in liver. Direct involvement of hepatic PPAR was demonstrated using a liver-restricted expression of PPAR in mice. IL-1- or IL-6-mediated acute-phase response was inhibited by fenofibrate treatment in liver-specific PPAR-expressing mice but not in PPAR-deficient mice. In addition, we demonstrated that PPAR exerts a general control of the acute-phase response by using an inflammation/infection model of lipopolysaccharide. In such a context, liver-specific PPAR-expressing mice displayed lower circulating levels of TNF, IL-1, and IL-6 cytokines. We found a distal repercussion of this lowering at the vascular wall level as illustrated by a decreased expression of adhesion molecules in aorta. In conclusion, we demonstrated that through a specific liver action, PPAR behaves as a modulator of systemic inflammation and of the associated vascular response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
EVEN WHEN INFLAMMATION, mediated by varied set of cytokines (1), is crucial to the host defense, chronic inflammation or infection may become deleterious, especially for cardiovascular diseases. Atherosclerosis, especially, is thought too be closely linked to the inflammatory state (2). Prediction of the risk of developing atherosclerosis is based on the identification of risk factors and their modulation. Among them, hemostatic factors such as cytokines and their liver target genes are associated with an increased risk.

The acute-phase response (APR) is an alarm response of the organism stimulated by drastic disturbances including infection, inflammation, trauma, necrosis, and malignant growth. Among a diversity of biological functions, the proinflammatory cytokines TNF, IL-1, and IL-6 are known to induce the APR. The APR corresponds to the gene modulation of acute-phase proteins in liver among which are found fibrinogen, C-reactive protein (CRP), serum amyloid A (SAA), and haptoglobin (HG). Both cytokines and targets of the APR are considered as cardiovascular risk markers of hepatic origin (3, 4, 5). Inflammation and infection are accompanied by the secretion of TNF, IL-1, and IL-6 that activate either separate or common sets of genes (6, 7). TNF and IL-1 belong to the primary wave of cytokines that trigger an immediate modulation of some of the APR target genes. In that context, IL-6 is an acute-phase protein itself and belongs to the secondary wave of cytokines. Bacterial endotoxins such as lipopolysaccharide (LPS) exhibit a broad spectrum of action and provoke a violent response from the entire organism. LPS may induce IL-6 expression and its plasma concentrations and launch TNF/IL-1 signaling, which in turn stimulates and maintains the IL-6 response. Therefore, preventing exacerbation of cytokine stimulation either by controlling concentrations of plasma cytokines or by modulating cytokine signaling pathways might represent a strategy aimed at counteracting deleterious effects of systemic inflammation.

Among drugs affecting plasma acute-phase proteins, fibric acid derivatives are reported as repressors. Fibrates are generally effective at normalizing hypertriglyceridemia and hypercholesterolemia and may also lower fibrinogen and CRP plasma levels. Fibrates exert their action at the gene level through activation of the nuclear receptor peroxisome proliferator-activated receptor- (PPAR). The positive regulation of PPAR target genes occurs through the binding of PPAR with its heterodimeric partner retinoid X receptor to a specific response element (8, 9, 10, 11, 12). This pathway is mainly involved in the gene regulation of lipid and lipoprotein metabolism-related genes yielding to the normolipemic action of fibrates (13, 14). Furthermore, PPAR was shown to down-regulate the expression of genes involved in the inflammatory process by interfering with several transcription factors such as signal transducers and activators of transcription, CCAAT box/enhancer-binding proteins, nuclear factor (NF)-B and activator protein (AP)-1 that are stimulated by pro-inflammatory cytokines (15, 16).

A role of PPAR in modulating inflammation came from the initial observation that PPAR-deficient mice displayed a prolonged inflammatory response (17). The involvement of PPAR in the control of inflammatory signaling was first demonstrated in vascular cells (15, 16, 18, 19, 20, 21, 22). Because PPAR is highly expressed in liver, a potent function in the control of the APR was thereafter investigated. We previously found that fibrate-activated PPAR negatively regulates IL-6-stimulated fibrinogen expression (16) and IL-1-stimulated CRP gene expression (19) in isolated primary human hepatocytes. We also reported that chronic activation of PPAR attenuates IL-6-induced APR gene expression in vivo (18). Here, we show that IL-1-mediated stimulation of acute-phase protein expression is counteracted by fibrate treatment in vivo. This effect occurs at the transcriptional level as demonstrated by run-on experiments. In addition, we found that IL-1-stimulated expression of IL-6 in liver is prevented upon PPAR activation. Finally, we demonstrated that PPAR exerts a general control of the APR in inflammation and infection conditions and lowers circulating concentrations of proinflammatory cytokines. This effect had a repercussion at the vascular level as demonstrated by a decreased expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in aorta.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal studies
Animal studies were performed in compliance with European Community specifications regarding the use of laboratory animals. The Nord-Pas de Calais Ethical Committee for animal use approved the animal experiments. C57BL6/J mice were fed for 2 wk with either a standard mouse chow or one containing 0.2% (wt/wt) fenofibrate. Mice were subjected to ip injection of IL-1 (0.5 µg/mouse) or saline buffer 3 h before being killed. Animals were killed by exsanguination under anesthesia. Livers and aortas were removed.

Preparation of nuclei and run-on assay
Nuclei were prepared from fresh livers as described by Gorski et al. (23). Transcription run-on assays were performed as described by Nevins (24). Equivalent amounts of labeled nuclear RNA were hybridized for 48 h at 42 C to 1 µg purified cDNA probes immobilized on Hybond C Extra filters (Amersham, Arlington Heights, IL). The following cDNAs were spotted: an ACO cDNA probe, a glyceraldehyde-3-phosphate dehydrogenase cDNA probe, a serum amyloid cDNA probe, and an HG cDNA probe. After hybridization, filters were washed at room temperature for 10 min in 0.5x standard saline citrate and 0.1% (wt/vol) SDS and twice for 30 min at 65 C and subsequently exposed to x-ray film (X-OMAT-AR; Eastman-Kodak, Rochester, NY).

RNA analysis
RNA from livers, primary mouse hepatocytes, or aorta were extracted with TRIzol reagent (Invitrogen, Cergy Pontoise, France) using the supplier’s instructions. RT was done on 1 µg RNA using random hexameric primers and Superscript reverse transcriptase (Invitrogen). cDNA levels were measured by real-time quantitative PCR using Brilliant SYBR Green Q-PCR Master Mix (Stratagene, La Jolla, CA) on the Mx4000 detection system (Stratagene) using the specific primers. PCR amplification was performed in a total volume of 20 µl containing 100 nM of each primer. The conditions were 95 C for 10 min, followed by 40 cycles of 30 sec at 95 C, 30 sec at 55 C, and 30 sec at 72 C. For each primer pair, the linearity of the reaction was confirmed to have a correlation coefficient of 0.98 and a PCR efficiency of 100% over the detection area by measuring a 10-fold dilution curve with cDNA from mouse liver extract. Samples were analyzed in duplicate and normalized to the cyclophilin values as internal control. PCR was performed with oligonucleotides 5'-TTC TGC TCC CTG CTC CTG-3' and 5'-GTA ATT GGG GTC TTT GCC-3' for SAA, 5'-AAA AAC CTC TTC CTG AAC CAC-3' and 5'-AAC GAC CTT CTC AAT CTC CAC-3' for HG, 5'-GGC TCA GAC TCT GGG AAC TTT AG-3' and 5'-GAA CGA TGT GTG GTG CTT GTG-3' for fibrinogen-, 5'-AGC ACA GAA AGC ATG ATC CG-3' and 5'-CCC GAA GTT CAG TAG ACA GAA GAC-3' for TNF, 5'-CCA GTT GCC TTC TTG GGA CTG-3' and 5'-CAG GTC TGT TGG GAG TGG TAT CC-3' for IL-6, and 5'-GAA TGA CCT GTT CTT TGA AGT T-3' and 5'-TTT TGT TGT TCA TCT CGG AGC C-3' for IL-1, 5'-AAC CGA ATC CCC AAC TTG TGC AG-3' and 5'-TCT CCA GCT TCT CTC AGG AAA TGC C-3' for VCAM-1, and 5'-CCT GGC CTC GGA GAC ATT AGA GAA C-3' and 5'-ACC CCA AGG AGA TCA CAT TCA CGG-3' for ICAM-1. Quantification of mRNA expression was corrected using cyclophilin gene expression as an internal control with the primers 5'-GCA TAC GGG TCC TGG CAT CTT GTC C-3' and 5'-ATG GTG ATC TTC TTG CTG GTC TTG C-3'. Statistical analysis was performed using Student’s t test.

Isolation of primary hepatocytes
Hepatocytes were isolated from the livers of fed mice by a modification of the collagenase method (25). Hepatocytes were cultured in serum-free William’s E medium (Invitrogen) supplemented with 2 mmol/liter glutamine, 25 µg/ml gentamicin, 100 nmol/liter dexamethasone, 0.1% fatty acid-free BSA, and 2% (vol/vol) Ultroser G (Invitrogen) at 37 C in a humidified atmosphere of 5% CO₂, 95% air. After cell attachment (6 h), medium was changed fresh DMEM (Invitrogen) supplemented with 2 mmol/liter glutamine, 25 µg/ml gentamicin, 100 nmol/liter dexamethasone, and 5 mmol/liter D-glucose, and cells were treated either with fenofibrate (100 µmol/liter) or dimethylsulfoxide for 24 h and IL-1 (10 ng/ml) for 3 h.

Transfection assays
HepG2 cells were grown in MEM containing 10% fetal calf serum (Invitrogen), 2 mM glutamine, 25 µg/ml gentamycine, 1 mM sodium pyruvate, and nonessential amino acids (Invitrogen) in a 5% CO₂ humidified atmosphere at 37 C. For reporter assays, HepG2 cells were transiently transfected with the p1168 h.IL6P-luc+ corresponding to the wild-type human IL-6 promoter construct coupled to the firefly luciferase and the empty vector or the PPAR expression vector using ExGen 500 (Euromedex, Souffelweyersheim, France) according to the manufacturer’s protocol. The total amount of DNA was equalized to 0.5 µg by adding pBSSK⁺ vector. After a 5-h incubation period, cells were treated in MEM containing 0.2% fetal calf serum with IL-1 (10 ng/ml). Cells extracts were prepared using reporter lysis buffer (Promega, Charbonnières, France), and luciferase and β-galactosidase activities were measured as previously described (26). Statistical analysis was performed using Student’s t test.

Hydroporation method
Animal studies were performed on 10-wk-old PPAR-null female mice on a C57BL6/J genetic background. For injection, mice were anesthetized by ip injection with a solution of Domitor (0.68 mg/kg; Orion Pharma, Espoo, Finland) and ketamine (67 mg/kg; Virbac, Carros, France). Rapid tail vein injection (hydroporation) was performed as previously described (27, 28, 29, 30, 31, 32). Briefly, injections were performed in the morning using 20 µg of the control empty vector or PPAR expression vector in 9% saline (wt/vol) solution in a total volume of 1.6 ml per mouse (18–20 g). Mice were allowed to recover on cotton after sc injection of the antidote antisedan at 1.7 mg/kg (Orion Pharma). Fenofibrate treatment was performed by gavage (200 mg/kg). Mice received the first gavage 8 h after hydroporation and the second one 24 h after hydroporation and were subjected to ip injection of murine IL-1 or murine IL-6 or LPS 1 h later. After a total 3-h period, mice were anesthetized and then killed. Each group was composed of five mice. Expression of hydroporated PPAR was analyzed in several tissues including liver, heart, aorta, skeletal muscle, adipose tissue, kidney, and spleen. PPAR expression was exclusively detected in the liver.

Plasma cytokine concentrations
Plasma levels of TNF, IL-1, or IL-6 were determined by ELISA (R&D Systems Europe, Abingdon, UK) following manufacturer’s instructions. Statistical analysis was performed using Student’s t test.

Reagents
Fenofibric acid (Laboratories Fournier, Dijon, France) was dissolved in dimethylsulfoxide (Me₂SO). Fenofibrate suspended in 1% carboxymethylcellulose (low viscosity; Sigma Chemical Co., St. Louis, MO) was administered by gavage at a dose of 200 mg/kg. Control animals received equivalent volumes (100 µl) of 1% carboxymethylcellulose in similar conditions. Murine recombinant IL-1 or IL-6 (0.5 µg per mouse) (Tebu Le Perray-en-Yvelines, France) and LPS (Escherichia coli, serotype 055:B5; Sigma) prepared in 9 g/liter saline were administered by ip injection at the dose of 50 µg per mouse.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PPAR activation prevents IL-1-mediated APR gene expression in vivo
To study the effect of PPAR activation on IL-1-mediated APR gene expression in vivo, we examined mRNA levels of APR genes in liver such as SAA and HG in mice untreated or treated with fenofibrate and subsequently stimulated or not by IL-1 (Fig. 1). The APR genes were analyzed together with the well-characterized PPAR target gene acyl-coenzyme A oxidase (ACO). As expected, ACO mRNA levels were increased in fibrate-treated mice (data not shown). Moreover, mice stimulated with IL-1 responded with a strong induction of the mRNA expression of SAA (Fig. 1A) and a lower but obvious increase of HG mRNA expression (Fig. 1B). Interestingly, this IL-1 effect was strongly impaired by fenofibrate treatment. Next, we analyzed the effect of fibrate-activated PPAR on APR gene transcription in vivo by performing nuclear run-on transcription assays on nuclei prepared from livers of mice untreated or treated with fenofibrate and stimulated or not by IL-1 (Fig. 2). Mice treated with fenofibrate showed an increased transcription rate of ACO. Remarkably, mice stimulated with IL-1 exhibited an increased transcription rate for SAA and HG genes, which was strongly attenuated once PPAR was activated by its agonist. Together, these results demonstrate that PPAR controls the APR gene expression stimulated by IL-1 at a transcriptional level in vivo.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Fenofibrate attenuates APR gene expression induced by IL-1. Mice were treated with 0.2% (wt/wt) fenofibrate (FF) or not (Cont) for 2 wk and subsequently stimulated or not for 3 h with IL-1 as indicated (four mice per group). Total RNA extracted from liver was analyzed by quantitative RT-PCR to measure SAA (A) and HG (B) mRNA expression levels. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Effect of fenofibrate treatment on IL-1-induced transcription of APR genes. Mice were treated with 0.2% (wt/wt) fenofibrate (FF) or not (Cont) for 2 wk and stimulated or not for 3 h with IL-1 as indicated. Nuclear run-on assays were performed on nuclei isolated from liver. The autoradiogram shows glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control, membrane background (Blank), ACO, SAA, and HG mRNA expression levels.

PPAR controls IL-1-induced APR gene expression through a direct action within the hepatocyte

View larger version (11K): [in this window] [in a new window]   FIG. 3. Fenofibrate attenuates APR gene expression induced by IL-1 through a direct effect within the hepatocyte. Primary hepatocytes were isolated from mice liver and were in vitro treated with fenofibrate (100 µM) (FF) or vehicle (0.1% Me2SO) (Cont) for 24 h and stimulated with IL-1 (10 ng/ml) for an additional 6-h period. Total RNA was extracted and subjected to quantitative RT-PCR analysis to measure SAA (A) and HG (B) mRNA expression levels. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

PPAR activation prevents IL-1-induced secondary wave IL-6 cytokine both in vitro and in vivo

View larger version (8K): [in this window] [in a new window]   FIG. 4. Fenofibrate prevents IL-1-mediated hepatic IL-6 expression in vitro and in vivo. A, Mice were treated with 0.2% (wt/wt) fenofibrate (FF) or not (Cont) for 2 wk and stimulated or not for 3 h with IL-1 as indicated (four mice per group). Total RNA extracted from liver was analyzed by quantitative RT-PCR to measure IL-6 mRNA expression levels. B, Primary hepatocytes were isolated from liver and were subsequently treated in vitro with fenofibrate (100 µM) or vehicle (0.1% Me2SO) for 24 h and stimulated with IL-1 (10 ng/ml) for an additional 6-h period. Total RNA was extracted and subjected to quantitative RT-PCR analysis to measure IL-6 mRNA expression levels. C, HepG2 cells were transiently transfected with the control empty vector (e.v.) or PPAR-expressing vector and the IL-6-promoter-driven reporter gene (p1168 h.IL6P-luc+ containing AP-1 and NF-B response elements) and treated or not by human IL-1 (10 ng/ml) for 4 h or vehicle (water). At 24 h after transfection, cells were harvested, and luciferase and β-galactosidase activities were measured. Results are presented as relative light units/β-galactosidase activities. Results are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effect of fenofibrate occurs via a liver-specific activation of PPAR

View larger version (18K): [in this window] [in a new window]   FIG. 5. Liver-restricted activation of PPAR mediates fenofibrate attenuation of IL-1, IL-6, and LPS stimulation of the APR. PPAR expression was restored in PPAR-deficient mice by rapid tail vein injection (hydroporation) as described in Materials and Methods. Mice received empty vector (e.v.) or PPAR expression vector (PPAR). Mice were treated with 200 mg/kg fenofibrate (FF) and were subsequently stimulated for 3 h with IL-1 (0.5 µg/mouse) (A and B), IL-6 (0.5 µg/mouse) (C and D), or LPS (50 µg/mouse) (E and F) as indicated. Total RNA was extracted from liver. SAA, IL-6, and fibrinogen mRNA expression levels were measured by quantitative RT-PCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Infection-induced APR gene expression is impaired by fenofibrate-activated PPAR in vivo

Effect of liver-specific PPAR on systemic inflammation
LPS triggers signal transduction pathways to release various cellular mediators, including the proinflammatory cytokines TNF, IL-1, and IL-6 (6, 7). Thus, we tested the ability of hepatic PPAR to influence circulating cytokine levels in mice by measuring TNF, IL-1, and IL-6 concentrations after LPS-induced acute inflammation (Fig. 6). As previously, liver-restricted PPAR expressing mice were treated with fenofibrate and LPS or not. As expected, LPS treatment of PPAR-deficient mice resulted in a strong elevation of TNF, IL-1, and IL-6 plasma concentrations (Fig. 6, A–C). This effect was not prevented by fenofibrate treatment in PPAR-deficient mice. Interestingly, the enhancement of these circulating proinflammatory cytokine concentrations was drastically prevented in liver-restricted PPAR-expressing mice treated with fenofibrate (Fig. 6, A–C). Furthermore, the lowering of circulating levels of TNF, IL-1, and IL-6 was correlated with the decrease of their respective liver mRNA levels, (Fig. 6, D, E, and F respectively). Taken together, these results strongly demonstrate that liver-specific PPAR activation lowers concentrations of circulating level of proinflammatory cytokines. These results suggest that through a liver action, PPAR may modulate the propagation of inflammation that may have important consequences in cells targeted by proinflammatory cytokines.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Liver-restricted PPAR activation lowers proinflammatory cytokine plasma levels. PPAR expression was restored in PPAR-deficient mice by rapid tail vein injection as described in Materials and Methods. Control mice received empty vector (e.v.) or PPAR expression vector (PPAR). Mice were treated with 200 mg/kg fenofibrate (FF) and subsequently stimulated for 3 h with LPS (50 µg/mouse). Plasma levels of TNF (A), IL-1 (B), and IL-6 (C) were determined by ELISA. D–H, Real-time PCR analysis of liver (D–F) or aorta (G and H) gene expression for indicated genes. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Repercussion of liver-specific PPAR at the vascular wall level

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Modulation of the APR by PPAR has been studied in human cells (16, 19). We also reported that PPAR behaves as a modulator of the APR mediated by IL-6 in vivo (18, 36). In the present work, we investigated the potential overall action of PPAR on the APR in vivo. First, we demonstrated that PPAR prevents IL-1-induced transcription of the APR gene expression in vivo. Moreover, we also found that the activation of PPAR prevents IL-1-stimulated IL-6 expression, which may be considered as an APR gene, in liver. Furthermore, we demonstrated that PPAR reduces the stimulation of the APR mediated by LPS that was used to mimic infection. Importantly, we also demonstrate that PPAR controls liver expression of proinflammatory mediators TNF, IL-1, and IL-6. Finally, we demonstrate the attenuation of circulating levels of cytokines through a liver-restricted action of PPAR. Our data suggest that PPAR may control circulating risk factors of atherosclerosis through a distal mechanism operating in liver.

The suppression of the IL-1 response in liver is in line with our previous observations demonstrating that PPAR controls the APR stimulated by IL-6 (18) and underscores the broad inhibition of APR gene expression by PPAR activation. The APR is initiated by the release of soluble mediators, mainly cytokines, secreted by monocytes, among which is the TNF/IL-1 family of cytokines that belong to the primary wave of proinflammatory cytokines and are able to stimulate a second wave of cytokines such as IL-6. TNF and IL-1 are main directors of the inflammatory process and induce a large panel of cytokines and other mediators acting in a signaling cascade on target cells as well as within an autocrine loop (1, 37, 38). Taking into account that IL-1 up-regulates IL-6 expression, the action of PPAR on this link constitutes a complementary mechanism aimed at controlling proinflammatory cytokine signaling. This hypothesis was demonstrated in cells involved in the pathology of atherosclerosis. For instance, ligand activation of PPAR prevents IL-1-induced expression of IL-6 in smooth muscle cells (20). Here, we found that in addition to the inhibition of IL-1-stimulated APR, PPAR prevents the IL-1-mediated induction of hepatic IL-6 expression in vivo. Our findings indicate a potential role of PPAR agonists in the regulation of inflammatory cytokine signaling in liver. The inhibition of the IL-1-stimulated expression of multiple APR genes by PPAR agonists in association with the reported interference between PPAR and AP-1 and NF-B signaling strongly argue in favor of an important role of PPAR in the control of inflammation at the hepatic and the vascular wall level. Therefore, PPAR modulates hepatic inflammatory risk markers of cardiovascular diseases directly via repression of APR protein expression and reduction of a secondary wave of proinflammatory cytokines.

The well-characterized model of infection mimicked by endotoxin treatment such as LPS allowed us to investigate the action of PPAR on a broader spectrum of proinflammatory cytokines secreted by liver. In the present study, we found, in the broader context of inflammation stimulated by LPS, that PPAR is capable of blocking APR gene expression as well as proinflammatory cytokine expression in liver. Of note, LPS is known to down-regulate the expression of several nuclear receptors in liver among which PPAR and some of its well-characterized target genes such as those of lipid-metabolizing enzyme (39, 40). This may explain how APR induced by LPS is associated with metabolic disturbance. Although LPS could attenuate PPAR activity, the remaining expression levels of PPAR in mice (expression decreased to 50% of basal levels) is still sufficient to attenuate deleterious effect of LPS.

Several mechanisms could be evoked to explain the broad control of APR by PPAR. Many studies have reported the involvement of PPAR at different steps of the IL-1 signaling pathway to explain mechanism of inhibition. Indeed, it has been previously reported that soluble IL-1 receptor antagonist (an inhibitor of cytokine signaling) is a direct target gene of PPAR. By inhibiting binding of IL-1 to its receptor via increased expression of its natural antagonist, PPAR might prevent or counteract the activation of the IL-1-signaling cascade (41). Furthermore, previous works from our group identified that PPAR inhibits inflammatory gene expression, such as those of IL-6, at the transcriptional level upon physical interaction with c-Jun and p65 (15). Proinflammatory cytokines (TNF and IL-1) and endotoxin LPS all induce the NF-B-activating signaling. Whereas all these inducer signals act through different receptors, they all converge toward the activation of NF-B. We can therefore speculate that the interaction between PPAR and NF-B might partially explain the inhibitory action of PPAR on LPS-induced APR. In a previous work, we studied PPAR modulation of IL-6-mediated APR (18). IL-6 actions are mediated by a specific cell surface IL-6 receptor, glycoprotein gp80, and a signal-transducing molecule, glycoprotein gp130. We found that a 3-d PPAR activation results in the down-regulation of the IL-6 receptor components gp80 and gp130 in liver explaining the attenuation of the IL-6 response. In the present study, the shorter period of treatment revealed that complementary mechanisms might exist such as sequestration of cofactors (16) for IL-6-induced APR attenuation.

The definition of atherosclerosis as an inflammatory disease came from the intensive investigations showing the role of inflammation in atherogenesis at different steps of the lesion appearance and progression (1). TNF, IL-1, and IL-6, considered as early cytokines, have pleiotropic activity and act both locally and distally (6). Interestingly, the hydroporation method allowed us here to analyze the specific liver-restricted action of PPAR. Importantly, we here demonstrated that PPAR, once activated by its ligand, might prevent the exacerbation and propagation of inflammation by decreasing the concentration of the proinflammatory mediators TNF, IL-1, and IL-6 from liver to blood. An important finding of our study is the observed impact of the lowering of plasma concentrations of the latter cytokines at the vascular wall level. Atherosclerosis is a chronic inflammatory disease in which early atherogenic events include increased expression of vascular adhesion molecules and chemoattractants followed by increased adhesion of monocytes and lymphocytes (42). In our model of liver-specific PPAR-expressing mice treated with LPS, the lowered circulating levels of proinflammatory cytokines were correlated with a decreased inflammatory signaling at the vascular wall level. Indeed, ICAM-1 and VCAM-1 expression in aorta was significantly lower in liver-specific PPAR-expressing mice. Increased ICAM-1 and VCAM-1 expression has been suggested to be associated with or predict atherosclerotic lesions (43, 44). Reduced expression of these adhesion molecules have been shown to protect against atherosclerosis in mice (45, 46). Therefore, our data indicate that PPAR attenuates systemic and vascular inflammatory response via a liver-specific control of proinflammatory cytokine gene expression. This identifies a main role of PPAR in liver in limiting the propagation of inflammation to the whole body. The acute phase can be prolonged and converted to a chronic phase of inflammation. Although inflammatory processes are important for the initiation of defense mechanisms, they can become deleterious under situations of chronic activation. Thus, PPAR activators appear an interesting therapeutic option for the attenuation of inflammatory processes associated with atherosclerosis beyond their effects on lipid levels. Our data suggest that activation of hepatic PPAR could be sufficient to attenuate the circulation of inflammatory factors, allowing protection or at least attenuation of cardiovascular diseases such as atherosclerosis.

In conclusion, this study demonstrates the implication of hepatic PPAR in the control of inflammation and infection. Indeed, our results demonstrate that PPAR controls the primary and secondary wave of proinflammatory production at the hepatic level (Fig. 7). Moreover, PPAR controls the APR by blocking the synthesis of acute-phase reactants. Together, these regulations demonstrate that PPAR controls the exacerbation of inflammation at the hepatic level. Interestingly, the hepatic control of inflammation by PPAR activation leads to decreased circulating cytokine concentrations. Thus, PPAR might prevent the propagation of inflammation, which suggests a beneficial impact of liver-restricted PPAR activation at a distal level as, for example, the vascular wall. It could be of interest to engineer liver-specific pharmacological agonists. Further investigations are required to identify the physiological benefits of the distal response of PPAR activation. Taking into account its properties at the vascular wall level, PPAR can be considered as a factor playing a determining role in the control of inflammation at the molecular level and as an attractive target to attenuate the deleterious effect of inflammation on cardiovascular diseases.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Scheme illustrating the different level of control of the APR by PPAR. Activation of PPAR controls the elevation of circulating TNF/IL-1 or IL-6 cytokines stimulated by endotoxin. PPAR may modulate both TNF/IL-1 and IL-6 signaling through a direct action on the pathway or via the down-regulation of IL-1-mediated stimulation of IL-6 expression. The overall action of PPAR on the APR triggered by infection or inflammation leads to the limitation of atherosclerosis risk factor of hepatic origin. Triangle, PPAR ligands; gray square, TNF/IL-1; white square, IL-6.

	Footnotes

 
R.M. was supported by a grant from Région-Nord-Pas de Calais/Institut Pasteur de Lille and a grant from the Nouvelle Société Française d'Athérosclérose. This work was supported by grants from Agence Nationale de la Recherche and Genfit SA (project acronym COMAX), the Région Nord-Pas de Calais/FEDER, the Fondation Coeur et Artères, and the European Vascular Genomics Network.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online March 6, 2008

Abbreviations: ACO, Acyl-coenzyme A oxidase; AP, activator protein; APR, acute-phase response; CRP, C-reactive protein; HG, haptoglobin; ICAM, intercellular adhesion molecule; LPS, lipopolysaccharide; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor-; SAA, serum amyloid A; VCAM, vascular cell adhesion molecule.

Received October 1, 2007.

Accepted for publication February 26, 2008.

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