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THE PPARγ LIGAND 15d-PGJ2 MODULATES MACROPHAGE ACTIVATION AFTER INJURY IN A MURINE TRAUMA MODEL

Monroy, M. Alexandra*†; Opperman, Kay K.*; Pucciarelli, Margo*; Yerrum, Smitha; Berg, David A.*; Daly, John M.*

doi: 10.1097/shk.0b013e3180310982
Basic Science Aspects
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In macrophages, peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be important for differentiation, and it serves as a negative regulator of activation. Major trauma/injury causes a dramatic host response that disrupts cellular immune homeostasis and initiates an inflammatory cascade that predisposes the injured host to subsequent infections. In prior studies using a murine trauma model consisting of femur fracture and hemorrhage, splenic macrophages from traumatized mice had significantly enhanced LPS-induced cyclooxygenase enzyme (subtype 2) and iNOS production as well as elevated levels of inflammatory cytokines at 1 week after injury compared with uninjured controls. These up-regulated cellular responses corresponded to increased mortality when animals were challenged with LPS or Candida. In the current study, we used the injury model to determine the effect of treatment of injured mice with the endogenous PPARγ ligand 15-deoxy-Δ 12-,14-PGJ2 (15d-PGJ2). It was found that in vivo 15d-PGJ2 treatment significantly reduced the levels of inflammatory mediators produced by splenic macrophages 7 days after injury. The mechanism of inhibition is dependent on PPARγ because concomitant treatment of animals with the PPARγ antagonist GW9662 reversed the inhibitory effect of 15d-PGJ2. Endogenous PPARγ modulated activation of LPS-induced p38 mitogen-activated protein kinase. Furthermore, treatment of injured mice with 15d-PGJ2 conferred a significant survival advantage after infectious challenge induced by cecal ligation and puncture. Thus, this PPARγ ligands significantly attenuate the postinjury inflammatory response and improve survival after infectious challenge.

*Departments of Surgery and Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania

Received 24 Apr 2006; first review completed 11 May 2006; accepted in final form 22 Nov 2006

Address reprint requests to M. Alexandra Monroy, PhD, 3400 North Broad Street, Room 513 OMS, Philadelphia, PA 19140. E-mail: amonroy@temple.edu.

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INTRODUCTION

Alterations in cellular immune function occur after injury or surgical trauma (1). Injury triggers a cascade of systemic proinflammatory reactions. It induces an up-regulated cytokine response, which then initiates downstream changes in cellular immunity, leading to susceptibility to subsequent infections (2). Macrophages contribute significantly to the immune dysfunction after injury (3-5). Data from our laboratory demonstrated that there is altered expression of macrophage products and mediators in both a murine trauma model and in patients after major elective surgery (3, 6).

Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in the regulation of cellular metabolism and has substantial anti-inflammatory effects (7, 8). PPARγ is a member of the nuclear hormone receptor family of ligand-dependent transcription factors that includes retinoid, steroid, and thyroid receptors (9). PPARγ regulates gene expression by binding as a heterodimer with the retinoid X receptor to PPAR response elements in the promoter region of target genes (9). PPARγ can also function in a DNA-binding independent manner to transrepress the transcription of target genes. PPARγ inhibits production of broad subsets of LPS and interferon γ target genes in macrophages such as TNF-α, IL-6, iNOS and cyclooxygenase enzyme (subtype 2; COX-2) (10). PPARγ inhibits gene expression in macrophages without binding to the gene promoters. It is thought that this is accomplished by inhibiting signaling pathways required for inflammatory gene activation (10).

PPARγ is activated by the binding of a variety of lipophilic ligands, including long-chain polyunsaturated fatty acids and several eicosanoids (11). The naturally occurring PPARγ agonist that has been most commonly used experimentally is 15-deoxy-Δ 12-,14-PGJ2 (15d-PGJ2) (11). Several studies have shown that PPARγ ligands have potent anti-inflammatory properties in experimental models of acute and chronic inflammation as well as sepsis (12). Furthermore, PPARγ activation has been shown to be protective in I/R injury of various organs (13-15). The purpose of this study was to determine the effects of treatment with 15d-PGJ2 on macrophage cellular function in a murine trauma model. In this study, we demonstrated that 15d-PGJ2 inhibited inflammatory mediator production in splenic macrophages from injured mice through a PPARγ-dependent mechanism and modulation of p38 mitogen-activated protein (MAP) kinase activation. Additionally, the decrease in inflammatory markers resulted in a significant increase in survival for injured and septic mice.

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MATERIALS AND METHODS

Materials

Roswell Park Memorial Institute (RPMI)-1640 medium and Hanks balanced salt solution (HBSS) were purchased from MediaTech (Herndon, Va). Proteinase inhibitor cocktail and enhanced chemiluminescence reagents were from Roche Applied Science (Indianapolis, Ind). The PPARγ antagonist GW9662 and 15d-PGJ2 were from BIOMOL (Plymouth Meeting, Pa). Phospho-p38 MAP kinase (Thr180/Tyr182) antibody and p38 MAP kinase antibody were from Cell Signaling Technology (Beverly, Mass). The actin antibody was from Abcam (Cambridge, Mass). The PPARγ antibody was from Calbiochem (Merck, Darmstadt, Germany). LPS (Escherichia coli serotype O55:B5) and all other chemicals and biochemicals were from Sigma-Aldrich (St. Louis, Mo).

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Trauma model

Female BALB/c mice (6-8 weeks old) were obtained from Harlan (Indianapolis, Ind). On arrival, all animals were acclimatized for 2 to 4 days in an American Association for the Accreditation of Laboratory Animal Care-accredited veterinary facility. Standard mouse chow and water were allowed ad libitum. After acclimatizing, mice were randomized to a control group uninjured or to a trauma group (femur fracture plus 40% blood volume hemorrhage). After anesthesia (isoflurane) was assured, a small skin incision was made over the right femur. The intermuscular plane was dissected to expose the femur, and sterile scissors were used to create a midshaft femur fracture. After hemostasis was achieved, the wound was closed in a single layer with continuous nylon suture. The mouse was then bled 40% of total blood volume (according to the formula mouse weight [in grams] × 75 mL/1,000 mg × 0.40) into a fire-polished heparinized microcapillary tube through a retro-orbital approach (16). Trauma animals were further subdivided into drug treatment (1 mg/kg 15d-PGJ2), a commonly used dose (17) or vehicle treatment (dimethyl sulfoxide) given daily for 7 days by i.p. injection. All experimental protocols were approved by the Institutional Animal Use and Care Committee of Temple University School of Medicine. The experiments were performed in adherence to the National Institute of Health Guidelines on the use of Laboratory Animals.

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Survival experiments

In separate experiments, mice were randomized to trauma vehicle (n = 22) and trauma 15d-PGJ2-treated mice (1 mg/kg daily i.p. injection) (n = 22). On day 7 after injury, they were given a septic challenge by cecal ligation and puncture (CLP) (18). This procedure was performed via a sterile 0.5-cm midline abdominal incision, after which the bowel was gently mobilized to expose the cecum, and a 4.0 silk ligature was placed around a portion of the ileocecal region. After this, a 23-gauge needle was used to puncture the portion distal to the ligature, and a small amount of expressed stool was visualized. The midline incision was then reapproximated using 4.0 silk sutures, and animals were followed for survival.

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Preparation of splenic macrophages

Seven days after trauma, the mice were killed by carbon dioxide inhalation, and total splenocytes were harvested by the following method. Spleens were collected aseptically and placed in separate tissue culture dishes containing cold (4°C) HBSS without calcium and magnesium. Splenocytes were isolated by gentle mechanical disruption of the spleen with filtration through a cell strainer into HBSS to obtain a single-cell suspension (19). The suspension was centrifuged for 10 min at 500g and 4°C. Red blood cells in the pellet were lysed with an ammonium chloride solution (8.29 g/L NH4Cl, 1 g/L KHCO3, and 0.037 g/L Na2EDTA; pH 7.4) for 4 min, immediately diluted with cold HBSS, and centrifuged as previously described. The remaining pellet was resuspended in RPMI-1640 media containing 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum.

Total splenocytes were stained with trypan blue, counted on a hemocytometer, diluted to a final concentration of 1 × 107 cells per milliliter, and plated. After allowing macrophages to adhere for 2 h at 37°C in a 5% carbon dioxide atmosphere, the nonadherent cells were suctioned off, and the macrophages were cultured in RPMI-1640 complete media as described above.

For determination of inflammatory cytokines TNF-α and IL-6, splenic macrophages from control or traumatized mice were cultured and stimulated with 100 ng/mL LPS for 24 h. Supernatants were harvested and stored at −70°C until assayed. For detection of NO, cells were stimulated with 100 ng/mL LPS and 100 U/mL interferon γ. Cytokines were measured by using an enzyme-linked immunosorbent assay kit according to the manufacturer's specifications (BD Biosciences, La Jolla, Calif). Enzyme-linked immunosorbent assay plates were analyzed using a VersaMax microplate reader and SOFTmax PRO software (Molecular Devices, Sunnyvale, Calif). NO production was measured from stimulated supernatants by a Greiss reagent using a kit from Promega (Madison, Wis).

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Western blot analysis

Western blots were performed as described previously (19). Splenic macrophages were washed twice and homogenized in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitors (10 μg/mL leupeptin, 10 μg/mL pepstatin A, 10 μg/mL aprotinin, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride) and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). Proteins (30 μg per lane) were separated on a denaturing 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. For detection of hsp70, 65 μg of protein was loaded per lane. Membranes were blocked in 4% nonfat dry milk in phosphate-buffered saline and subsequently incubated overnight at 4°C with primary antibody. After washing, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Chemiluminescence was detected using the enhanced chemiluminescence reagent according to the manufacturer's protocol. For detection of β-actin, the same membranes were incubated with rabbit polyclonal anti-β-actin antibody overnight at 4°C and processed as described.

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RNA extraction and reverse transcriptase-polymerase chain reaction analysis

Total cellular RNA was extracted from control or traumatized splenic macrophage cells using the RNeasy Mini Kit (Qiagen Inc, Valencia, Calif) according to the manufacturer's instructions. cDNA was reverse-transcribed from 200 ng total cellular RNA using Oligo(dT)12-18 primers and superscript II reverse transcriptase. One to three microliters of the cDNA reaction was used for amplification using 2× SYBR green master mix (Applied Biosystems, Foster City, Calif). Reactions were assayed on an ABI7500 sequence detection system using default parameters. Each condition was plated in triplicate and normalized to β-actin as the housekeeping control gene. Quantitation was performed using cycle threshold and ΔΔCT using ABI software. The PPARγ gene-specific (GenBank accession no. U01841) primers used were 5′-GGA GAT CTC CAG TGA TAT CGA CCA-3′ (sense) and 5′-ACG GCT TCT ACG GAT CGA AAC T-3′ (antisense). The same amount of cDNA was amplified using β-actin (X03672)-specific primers 5′-AGA GGG AAA TCG TGC GTG AC-3′ (sense) and 5′-CAA TAG TGA TGA CCT GGC CGT-3′ (antisense). Melt curve analysis was done to ensure a single polymerase chain reaction product was obtained using SYBR green.

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Nuclear extract preparation

Splenic macrophages were stimulated with LPS (100 ng/mL). Cells were harvested, and nuclear extracts were prepared by modification of the mini extract method (20). Cells were washed once with cold phosphate-buffered saline and centrifuged. Cell pellets were resuspended in buffer I (10 mM HEPES, pH 7.8; 10mM KCl, and protease inhibitor cocktail from Roche) and incubated for 15 min. Subsequently, 5 μL of 10% NP40 /200 μL was added, and tubes were vortexed. The nuclei were isolated by centrifugation, and the cytoplasmic supernatants were saved. The nuclear pellets were gently resuspended in 80 μL of buffer II (20 mM HEPES, pH 7.8; 420 mM NaCl, 25% glycerol, and protease inhibitor cocktail from Roche). After 30 min of incubation, nuclear extracts were cleared by centrifugation for 5 min. The supernatants were transferred to new tubes, and protein concentration was determined using a commercially available Bradford assay (Bio-Rad Protein Assay, Hercules, Calif).

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Electrophoretic mobility shift assay

The consensus nuclear factor (NF)-κB and peroxisome proliferator response element oligonucleotides for gel shift assays were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). The oligonucleotides were end-labeled with γ-32P adenosine triphosphate (New England Nuclear, PerkinElmer, Wellesley, Mass) using T4 polynucleotide kinase (Invitrogen, Carlsbad, Calif). The binding reaction was performed by incubating 5 μg of nuclear protein in 20 mM HEPES (pH 7.9), 10% glycerol, 5 mM dithiothreitol, and 1 μg of poly (dI-dC) in a final volume of 20 μL for 30 min at 25°C (21). To detect an antibody supershift, 2 μL of antibody to PPARγ was added to the reaction mixture for 30 min at 25°C. The protein/DNA complexes were then fractionated on 6% polyacrylamide gels in 0.25× Tris, boric acid, EDTA, and water. Gels were transferred to Whatman filter paper, dried for 1 h, and then subjected to autoradiography at −80°C.

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Statistical analysis

Each experiment was conducted at least three times with consistent results. All values in the figures are expressed as mean value ± SD. The data were analyzed using Student t test and ANOVA, with significance determined as P < 0.05.

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RESULTS

Effect of 15d-PGJ2 in vivo treatment on inflammatory mediator production in trauma-derived splenic macrophages

We measured the effects of in vivo treatment with the endogenous PPARγ ligand 15d-PGJ2 on splenic macrophage activation in our murine trauma model consisting of femur fracture and hemorrhage. Splenic macrophages from control and injured mice were isolated 7 days posttrauma, and LPS-induced activation was assessed. As seen in Figure 1, treatment of injured mice with the PPARγ ligand 15d-PGJ2 significantly inhibited production of the proinflammatory cytokines TNF-α and IL-6 as well as the production of NO. The inhibition was mediated by a PPARγ-dependent mechanism because treatment with the PPARγ antagonist GW9662 abrogated the inhibition of mediator production by 15d-PGJ2.

Fig. 1

Fig. 1

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Treatment with 15d-PGJ2 increases expression and activity of PPARγ in splenic macrophages of injured mice

To determine whether trauma induced the expression of PPARγ in splenic macrophages, we measured PPARγ mRNA levels from control and trauma-derived macrophages at 7 days. Peroxisome proliferator-activated receptor γ mRNA expression was decreased in macrophages from injured mice. However, in vivo agonist treatment restored expression of PPARγ mRNA to normal in trauma mice (Fig. 2A). Furthermore, to determine the activation of PPARγ, we assessed DNA binding activity from splenic macrophages using electrophoretic mobility assay. As shown in Figure 2B, macrophages from traumatized mice treated with 15d-PGJ2 displayed higher DNA binding activity to the consensus peroxisome proliferator response element site than macrophages from control mice or trauma vehicle-treated mice.

Fig. 2

Fig. 2

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Treatment with PPARγ agonist reduces activation of the p38 MAP kinase in splenic macrophages

Mitogen-activated protein kinases have been implicated in a number of signaling events that are potentially important in the inflammatory response (22). We have recently shown that p38 activation is altered in splenic macrophages from traumatized mice (19). Therefore, the effects of PPARγ activation on LPS-induced p38 activity from trauma-derived macrophages were measured. Administration of 15d-PGJ2 was associated with significantly reduced levels of phosphorylated p38 in splenic macrophages from injured mice, as shown in Figure 3. The lack of p38 phosphorylation was not due to reduced levels of p38 because the overall levels of p38 did not change with trauma or 15d-PGJ2 treatment (middle panel).

Fig. 3

Fig. 3

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15d-PGJ2 treatment decreases activity of NF-κB

One of the proposed mechanisms of PPARγ anti-inflammatory effect is through inhibition of the NF-κB pathway. Therefore, we determined the DNA binding activity of NF-κB from nuclear extracts of splenic macrophages as a measure of its activation. As shown in Figure 4, administration of 15d-PGJ2 results in a subtle but noticeable decrease in DNA binding activity in macrophages from traumatized mice compared with results in vehicle-treated trauma mice.

Fig. 4

Fig. 4

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15d-PGJ2 treatment does not increase expression of the cytoprotective proteins

Several studies have suggested that PPARγ agonists and, in particular, 15-dPGJ2 may mediate anti-inflammatory mechanisms independent of PPARγ. These mechanisms involve induction of cytoprotective proteins of the heat shock response such as hsp70 and of heme oxygenase (HO) 1 (23, 24). Western blot analysis indicated that in vivo treatment with 15d-PGJ2 did not increase expression of hsp70 and in fact reduced expression of HO-1 in splenic macrophages of injured mice, as shown in Figure 5.

Fig. 5

Fig. 5

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Administration of 15d-PGJ2 confers a survival advantage to injured mice subjected to septic challenge

We next determined whether PPARγ activation plays a protective role in injured mice subjected to polymicrobial sepsis induced by CLP. Injured mice (n = 22) that were treated with vehicle had an overall survival rate of 25%. Injured mice (n = 22) that were treated in vivo with 15d-PGJ2 before being subjected to CLP had a significantly improved survival rate of 70% (P < 0.003). Therefore, 15d-PGJ2 treatment of injured mice had a beneficial effect, as shown in Figure 6.

Fig. 6

Fig. 6

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DISCUSSION

Traumatic injury has a deleterious effect on cellular immune regulation that may result in systemic inflammatory response syndrome when infection supervenes (2). Macrophages play a central role in mediating the host response to injury. Previous studies from our laboratory have shown that at 7 days posttrauma, macrophages have a significantly increased response to LPS. This is characterized by an overproduction of proinflammatory cytokines PGE2 and NO (4). We have also shown that blocking PGE2 production with NS-398, a selective COX-2 inhibitor during the first 24 h posttrauma modifies the cellular immune response and protects the host from a subsequent septic challenge (25). Thus, inhibiting the primed macrophage responses after injury reduces trauma induced cellular immune dysfunction and has a beneficial outcome for the injured host.

It has been suggested that PPARγ agonists might have therapeutic potential in the treatment of inflammatory conditions (12). In addition, multiple studies have demonstrated an anti-inflammatory effect on macrophages specifically (8, 26, 27). Results of the current study demonstrate that in vivo treatment of injured mice with the PPARγ ligand 15d-PGJ2 reduces significantly the macrophage hyperinflammatory response, specifically, the production of proinflammatory cytokines such as TNF-α and the mediator NO. Significantly improved survival of injured mice made septic by CLP was also noted. Peroxisome proliferator-activated receptor γ activation has a protective role in different models of inflammation, injury, and septic shock (14, 28-31). The beneficial effect observed with 15d-PGJ2 treatment is mediated at least in part through PPARγ because administration with the PPARγ antagonist GW9662 reversed the inhibition of inflammatory mediator production (Fig. 1). Trauma decreased expression of PPARγ in splenic macrophages at 7 days posttrauma, and in vivo 15d-PGJ2 treatment restored expression to normal levels (Fig. 2A). This is consistent with other studies. For example, in a study in rats, the expression of PPARγ in lung tissue was significantly reduced in response to polymicrobial sepsis induced by CLP and was restored with administration of PPARγ agonists (31). However, other studies have shown induction of PPARγ in response to inflammation. In a model of allergen-induced bronchial inflammation, ovalbumin inhalation increased expression of PPARγ in the lung (32). Therefore, expression of PPARγ in a particular tissue may depend on the type of injury or inducer. In vivo administration of PPARγ agonists appears to enhance activity of PPARγ in splenic macrophages from traumatized mice because enhanced DNA binding of PPARγ occurred in macrophages derived from 15d-PGJ2-treated mice (Fig. 2B).

Splenic macrophages from traumatized mice have enhanced activation of MAP kinases (19). In the current study, treatment with 15d-PGJ2 inhibits the LPS-induced activation of p38 MAP kinase. This finding is of significance because p38 is involved in the induction of TNF-α and COX_2 (33, 34). The decreased activation of p38 may explain the observed reduction of these inflammatory mediators in the injured 15d-PGJ2-treated mice because it has been proposed that p38 inhibition may be a therapeutic target in inflammatory processes (35). The NF-κB signaling pathway appears to be a major target of 15d-PGJ2 anti-inflammatory effects. The observed inhibition of NF-κB activation by 15d-PGJ2 is in agreement with reports of PPARγ-dependent and PPARγ-independent mechanisms of anti-inflammatory action (36, 37). We also tested the possibility that 15d-PGJ2 treatment may mediate anti-inflammatory effects through the induction of cytoprotective proteins. Our results indicated that 15d-PGJ2 did not increase expression of hsp70 or HO-1. The difference between our results and other reported studies may reflect the difference in the cell type studied and the type of injury studied.

Our studies indicate that 15d-PGJ2 counteracts the macrophage hyperinflammatory response induced by injury. Treatment of injured mice with this agonist suppressed inflammatory mediator production and conferred a significant survival advantage in injured mice. This beneficial effect was mediated at least in part through activation of PPARγ.

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Keywords:

Inflammation; TNF-α; IL-6; p38; NF-κB; macrophages

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