Modulation of LPS-induced pulmonary neutrophil infiltration and cytokine production by the selective PPARb/d ligand GW0742

Z. Haskova1, B. Hoang2, G. Luo3, L. A. Morgan2, A. N. Billin3, F. C. Barone, B. G. Shearer3, M. E. Barton4,
K. S. Kilgore5
1 Biopharmaceutical Center for Excellence in Drug Discovery, GlaxoSmithKline, King of Prussia, PA USA
2 Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA
3 Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals, Research Triangle Park, NC, USA
4 Clinical Pharmacology and Discovery Medicine, GlaxoSmithKline, Collegeville, PA, USA.
5 ImmunoInflammation Center for Excellence in Drug Discovery, GlaxoSmithKline, 1250 Collegeville Rd., Collegeville, PA 19426, USA, Fax: 610-270-6505, e-mail: [email protected]
Received 15 August 2007; returned for revision 17 September 2007; received from final revision 7 February 2008; accepted by J. Di Battista 15 February 2008
Published Online First 4 July 2008

Abstract. Objective: To define the anti-inflammatory effects of PPARb/d activation by use of the selective PPARb/d lig- and (GW0742) in a model of lipopolysaccharide (LPS)-in- duced pulmonary inflammation.

Methods: Male BALB/c mice were pretreated for three days with the PPARb/d agonist, GW0742, prior to induction of LPS-mediated pulmonary inflammation. Bronchial alveolar lavage fluid (BALF) was analyzed for inflammatory cell in- flux and for levels of pro-inflammatory mediators. BALF- derived inflammatory cells were also collected for mRNA analysis.

Results: Pretreatment with GW0742 resulted in a significant decrease in leukocyte recruitment into the pulmonary space. Protein and mRNA levels of the pro-inflammatory cytokines IL-6, IL-1b and TNFa in BALF were found to be signifi- cantly decreased in GW0742-treated animals (30 mg/kg). A significant decrease in granulocyte macrophage-colony stim- ulating factor (GM-CSF), a major regulator of neutrophil chemotaxis (via its downstream actions on TNFa and other cytokines/chemokines), activation and survival, was also noted in the BALF levels of GW0742-treated animals.

Conclusions: The present study demonstrates that activation of PPARb/d attenuates the degree of inflammation in a model of LPS-induced pulmonary inflammation and may therefore represent a novel therapeutic approach for the treatment of inflammation-mediated pathologies.

Key words: PPARb/d agonism – PPARb/d activators – Inflammation – Lipopolysaccharide-induced inflammation – Pulmonary injury – Neutrophilia – Cytokines


Peroxisome proliferator-activated receptors (PPARs) are members of the steroid hormone nuclear receptor family [1] with three primary isoforms; a, g and d (also known as b/d). Each isoform represents an intracellular ligand activated re- ceptor which, when heterodimerized with a retinoid X recep- tor, functions as a regulator of transcription for a wide variety of genes [2]. PPARa, which is predominantly expressed in liver, heart, kidney and muscles, has been shown to regulate a number of physiological processes including lipid metabo- lism and homeostasis. PPARg is most highly expressed in adipose tissues and is believed to control adipocyte prolif- eration, and lipid/glucose metabolism [3; 4]. PPARb/d has been less well described as compared to the other isoforms. Higashiyama et al. [5] have shown that PPARb/d is ubiqui- tously expressed in a variety of tissues including liver, adi- pose tissue, skeletal muscle, kidney, cerebellum, thalamus, cerebellar cortex and intestine. Physiologically, PPARb/d has been associated with adipocyte precursor cell prolifera- tion [6], oligodendrocyte differentiation [7], and cholesterol homeostasis [8].

PPARs have been implicated in the pathogenesis of a number of diseases including diabetes mellitus, obesity, atherosclerosis and neurological diseases [9], and therefore represent an important pharmacologic target. For example, fibrates such as fenofibrate, clofibrate and gemfibrozil are PPARa ligands utilized for the treatment of dyslipidemia while the PPARg ligands, rosiglitazone and pioglitazone, act as insulin sensitizers, and are therefore marketed for the treatment of type-2 diabetes. Preclinical, in vivo studies us- ing high-affinity PPARb/d agonists have demonstrated ef- ficacy in models of diabetes as well as obesity b-oxidation, suggesting that modulation of the delta isoform may have a role in treating these diseases as well as metabolic syndrome.

PPAR ligands have also been shown to possess anti-in- flammatory effects. Selective ligand agonists of the a and g isoforms have demonstrated improved outcomes in a va- riety of inflammatory disease models (as reviewed by [10]) including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and pulmonary inflammation [11]). These in vivo systems, as well as in vitro studies, have demonstrat- ed that agonism of the a and g isoforms reduce signaling through the nuclear factor kappa B (NF-kB) and AP1 signal- ing pathways, and ultimately suppress pro-inflammatory cy- tokine expression and cell adhesion [9; 12; 13]. As compared to the a and g isotypes, relatively little is known about the anti-inflammatory properties of PPARb/d. Cell-based stud- ies have show that PPARb/d agonists/activators inhibit the expression of pro-inflammatory cytokines, chemokines and adhesion molecules, likely via modulation of NF-kB [14– 18]. Translating these anti-inflammatory effects of PPARb/d ligands from cell-based systems to the in vivo setting has met with mixed success. Use of either PPARb/d agonists or knockout animals have yielded some encouraging results in the epidermis and models of atherosclerosis [19; 20]. How- ever, GW0742 has recently been shown to be inactive in a model of inflammatory colitis and in two murine models of asthma/airway inflammation (acute (single dose) administra- tion of the PPARb/d agonist GW501516) [21; 22]).

The present study examined the ability of the high affini- ty PPARb/d agonist GW0742, when administered in a multi- ple-dosing paradigm, to modulate the inflammatory response in a murine model of LPS-induced pulmonary inflammation. An emphasis was placed on the early, neutrophil-mediated inflammatory events associated with LPS administration. GW0742 represents a highly specific (murine EC50’s: 28 nM for PPARb/d; 8,900 nM for PPARa and >10,000 nM for PPARg), high-affinity PPARb/d agonist with an acceptable pharmacokinetic profile and activity in vivo, as previously shown [8].

Materials and methods


The procedures outlined in this study were conducted in accordance with NIH regulations of animal care and were approved by GlaxoSmith- Kline Institutional Animal Care and Use Committee. All experiments were performed using eight to ten week old BALB/c male mice (Jack- son Laboratory, Bar Harbor, ME, USA). Animals were maintained under standard conditions consisting of a 12-h light–dark schedule (lights on at 6.00 a. m.) with constant temperature (22 ± 1 °C) and humidity 48 %, and free access to food (Purina rodent chow 5001) and water. Mice were housed in the groups of ten, and a five day acclimatization/ quarantine period was allowed before use.

LPS-induced pulmonary inflammation

Mice were anesthetized by inhalation of isoflurane and LPS 0111:B4 (Sigma-Aldrich Co., St. Louis, MO) was administered intranasally via pipette over a period of 30–45 s as described previously [23]. To determine non-stimulated, baseline values, a separate group of mice received intranasal saline (20 µl/mouse). The concentration of LPS and time-point utilized in the study were selected based on pilot experiments designed to determine the optimal conditions for inflammatory cell influx into the lung. These studies (data not shown) demonstrated that an LPS con- centration of 10 µg/20 µl and a pretreatment time of 4 hours produced maximal inflammatory cell influx as defined by peak neutrophil infiltra- tion. Four hours post-LPS administration, animals were euthanized and bronchoalveolar lavage fluid (BALF) harvested (as described below) and stored (–80 °C) for further evaluation.

Fig. 1. Chemical structure of the selective PPARd agonist GW0742 ([4- [[[2-[3-FLUORO-4-(TRIFLUOROMETHYL)PHENYL]-4-METHYL- 5-THIAZOLYL] METHYL]THIO]-2-METHYLPHENOXY] ACETIC ACID). Molecular Weight- 471.5.

Dosing protocol and PPARb/d ligand

Up to four groups of the animals were analyzed (5–8 animals per group) in each study. Animals were randomly selected and placed into one of the following treatment groups: sham injury (saline only, no LPS); LPS-vehicle; LPS-dexamethasone (positive control group); and LPS- GW0742. The selective PPARb/d ligand GW0742 (Figure 1A) and dex- amethasone were dissolved in vehicle (0.5 % Methylcellulose with 0.1 % Tween 80) and administered orally (p. o.) in a volume of 10 ml/kg at the dose indicated. Once-daily dosing (q. d.) of GW0742 was initiated three days prior to LPS with the final dose given 0.5 hour prior to LPS admin- istration. Dexamethasone was dosed as described for GW0742 with the exception that the final dose was given 1.5 hours before LPS.

Collection of BALF and cell count/differentials

Upon sacrifice, mouse lungs were perfused three times with 0.7 ml of saline. Fluid from the first lavage was centrifuged at 4 oC and 1200 RPM for 5 minutes and the supernatant was removed and stored (–80 oC) for assessment of cytokine levels. The cell pellet was resuspended in 200 µl of PBS, and combined with the cell pellets from the two remaining lav- age fluid samples. The final volume of resuspended cells was 500 µl. For determination of cell number, 20 µl of each sample was combined with Tuerk’s stain and counted using a hemocytometer. Cell differential evaluation was conducted by cytospin and use of Quick-Diff stain.

Histological assessment of inflammatory cell localization

Histological analysis of lung tissue was used for localization of inflam- matory cells and was not meant for use as a quantitative tool. Non- lavaged lungs were prepared for histological analysis by instilling the lungs with buffered formalin (~5 ml) via the trachea, followed by rou- tine processing for light microscopy. Samples of the right frontal lobes were embedded in paraffin sectioned, and stained with hematoxylin and eosin. Tissues were examined microscopically, in a blinded fashion, by two board-certified veterinary pathologists (HH, SA).

Assessment of proinflammatory cytokine levels (TNFa, IL-1b and IL-6)

Proinflammatory cytokine levels were determined by use of Meso Scale Discovery platform (Meso Scale Discovery, Gaithersburg, MD). The MSD methodology is an ELISA based methodology in which multiple cytokines may be evaluated simultaneously. In the present study, TNFa, IL-6, and IL-1b were analyzed using MSD methodology. Plates were preincubated for 30 minutes at room temperature with the dilution serum sample (20 µl/well). The experimental samples and serial dilutions of cytokine standards were distributed in duplicates. Following incubation (2 h), plates were washed (3x) (PBS with 2 % Tween 20), incubated for an additional 2 h with the detection antibody, and washed (3x) again (PBS with 2 % Tween 20) before addition of the reading buffer. The fluorescent signal was evaluated using a Meso Scale Discovery reader and cytokine concentrations were determined by a non-linear curve fit using the read- outs from wells that contained serially diluted cytokine standards.

Assessment of KC, MIP-2a, and GM-CSF

In order to ascertain the chemotactic mediators affected by GW0742 treatment, protein levels of KC, MIP-2 and GM-CSF were quantified in bronchoalveolar lavage fluid supernatants. Commercially available ELI- SA kits for the specific chemokine/cytokine were obtained from R&D Systems, Inc., (Minneapolis, MN) and assays conducted as outlined in the manufacturer’s instructions.

RNA isolation and quantitative PCR

Cell pellets from BAL fluid were obtained as described above. RNA was isolated as described previously using Trizol reagent [24]. Real- time quantitative PCR (RT-QPCR) analysis was performed using an ABI PRISM 7700 Sequence Detection System instrument and software (PE Applied Biosystems, Inc., Foster City, CA). Primers and probes (Table 1) were designed using Primer Express Version 2.0.0 (Applied Biosys- tems) and synthesized by Keystone Laboratories (Camarillo, CA). All primers and probes were entered into the NCBI Blast program to ensure specificity. Fold change values were calculated by subtracting the mean threshold cycle number (Ct) for each treatment group from the mean Ct for the vehicle group and raising 2 to the power of this difference.

Statistical analysis

All results were expressed as the mean ± SEM. One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was used to evaluate the difference between treatment groups and the vehicle group in all experiments comparing one variable. Two-way ANOVA fol- lowed by Bonferroni post-test was used to determine statistical signifi- cance between treatment groups when comparing more than one vari- able in cell differential experiments. Values of p <0.05 were considered to be statistically significant. Fig. 2. Effect of GW0742 (30 mg/kg) on inflammatory cell influx in lungs of LPS-treated animals. Animals were dosed (q. d.) for 3 days prior to intranasal administration of LPS (10 µg). A: Leukocytes were quanti- fied in BALF 4h post-administration of LPS. Dexamethasone (3 mg/kg) was utilized as a positive control. Statistically significant differences were determined using a one-way ANOVA with Dunnett's post-test (**p <0.01, *p <0.05). B-D: Representative photomicrographs of lung tissue from mice receiving (B) vehicle; (C) GW0742; and (D) dexamethasone. Neutrophils (arrows) may be noted in the perivascular and intra-bronchi- olar areas. Few macrophages were noted. Magnification: 20. Statistical differences were determined using a one-way ANOVA with Dunnett’s post-test (**p <0.01, *p <0.05). Results Effect of the PPARb/d ligand GW0742 on BALF cell counts A prophylactic, multi-dose paradigm was chosen for deter- mining the anti-inflammatory properties of the PPARb/d lig- and GW0742. Mice were pretreated with GW0742 (30 mg/ kg, p. o., q. d.) for 3 days prior to administration of LPS with the final dose given 0.5 hour prior to LPS on day 3. Analy- sis of BALF 4 hours following intranasal administration of LPS demonstrated a significant (p <0.01) increase in cell number as compared to vehicle treated animals (Figure 2A). In contrast, GW0742 pretreatment significantly (p <0.05) decreased the number of BALF-associated cells, when com- pared to vehicle. A similar observation was noted with the positive control compound dexamethasone. The significant decrease in the leukocyte population not- ed in the BALF from GW0742- and dexamethasone-treated mice correlated with qualitative morphological changes in lung tissue from a separate set of similarly treated mice. When harvested 4 hours post-LPS administration, histologi- cal analysis of lungs from vehicle-treated mice (Figure 2B) showed infiltration of neutrophils in both the perivascular and intra-bronchiolar areas that was associated with some hemorrhage. Dexamethasone- and GW0742-treated mice (C, D) demonstrated a notable decrease in the number of neu- trophils present when compared to vehicle-treated animals. Few monocytes/macrophages were noted in any of the treat- ment groups. Fig. 3. Differential analysis of BALF inflammatory infiltrates (A). Re- sults are expressed as mean ± SEM for 5 mice per group. Statistical differences among the treatment groups were determined by two-way ANOVA followed by Bonferroni post-test (*p <0.001). B-D: Represent- ative photomicrographs of stained BALF cells from (B) Vehicle; (C), dexamathasone-; and (D) GW0742-treated animals show the relative populations of neutrophils (arrows) and monocyte/macrophages (arrow- heads). Neutrophils as the primary cell type affected by PPARb/d activation: differential analysis of BALF cell infiltrate Cytospin analysis of BALF (Figure 3) revealed that the in- flammatory cell infiltrate in vehicle-treated animals was composed primarily of neutrophils with few associated monocytes and macrophages. In contrast, monocytes/macro- phages constituted the primary cell types in animals treated with either GW0742 (30 mg/kg) or the positive control dex- amethasone (3 mg/kg). In all groups, neutrophils and mac- rophages constituted greater than 98 % of the population with the remainder composed of lymphocytic cells. There was a significant (P <0.001) decrease in BALF neutrophils in both the GW0742- and dexamethasone-treated groups as compared to vehicle whereas the relative number of monocytes /macrophages did not change between groups. These data suggest that neutrophils, as opposed to monocyte-de- rived cell types, were the primary inflammatory cell type that was most affected by PPARb/d activation. GW0742 dose-response relationship GW0742 dose-dependently inhibited the inflammatory cell infiltration post-LPS, as shown in Figure 4. Doses of 30,3.0 and 0.3 mg/kg significantly reduced BALF cell number by 77, 85 and 62 % respectively while the 0.03 mg/kg dose did not significantly inhibit cell infiltration. The minimal ef- fective dose of 0.3 mg/kg suggests that GW0742 is a potent pharmacologic inhibitor of LPS-induced neutrophil pulmo- nary inflammation. Although the 3 mg/kg dose decreased in- flammatory cell infiltration to a similar degree as the 30 mg/ kg dose, the higher dose was chosen for subsequent experi- ments in order to maximize the exposure and subsequent po- tential pharmacodynamic effects of the compound. Fig. 4. GW0742 dose-response relationship of leukocyte levels in the BALF from LPS-stimulated lungs. Statistical differences were deter- mined using a one-way ANOVA with Dunnett’s post-test (*p <0.05, **p <0.01). Results are means ± SEM for 5 mice per group. Effect of GW0742 on archetypal pro-inflammatory cytokines The pro-inflammatory cytokines TNFa, IL-1b and IL-6 are rapidly released (2–4 hours) upon pulmonary administration of LPS and are thought to play an important role in orches- trating subsequent inflammatory events. It was of interest therefore, to determine the effect of PPARb/d activation on mRNA levels and release of these pro-inflammatory media- tors into the BAL compartment. Intranasal administration of LPS resulted in increased protein levels of TNFa (Figure 5A), IL-1b (B) and IL-6 (C) when analyzed 4 hours post-LPS as compared to that noted in the BALF from non-LPS-stimu- lated animals (p <0.01). As compared to vehicle-treated ani- mals, GW0742 (30 mg/kg) significantly (P <0.05) decreased the levels of all three cytokines. Analysis of inflammatory cell mRNA demonstrated similar trend to that observed for BALF cytokines. As shown in Figure 6 (A-B), levels of both TNFa (A), and IL-1b (B) mRNA were significantly decreased in GW0742-treated ani- mals as compared to vehicle (P <0.05). The mRNA encoding IL-6 (Figure 6C) was notably decreased as compared to ve- hicle, but the degree of the reduction did not reach the level of statistical significance. Chemotactic mediators in the BALF from GW0742-treated animals Chemokine levels were measured in the BALF 4 hours fol- lowing LPS stimulation to determine what chemotactic me- diators were affected by PPARb/d activation. As shown in Figure 7, intranasal administration of LPS significantly in- creased BALF KC (A) and MIP-2 (B) as compared to ani- mals receiving saline. Treatment with GW0742 had no effect on either KC or MIP-2 expression. Figure 8 illustrates that LPS significantly (P <0.01) up-regulated the amount of GM-CSF in BALF as compared to the animals receiving saline. Treatment with GW0742 (30 mg/kg) significantly (P <0.01) decreased the levels of GM-CSF protein as compared to ve- hicle-treated animals. Fig. 5. Effect of the PPARd lig- and GW0742 on (A) TNF-a; (B) IL-1b; and (C) IL-6 levels in BALF from vehicle- and GW0742-treated mice 4 hours post-LPS administration. Pro- tein levels were determined by ELISA (pg/ml). Results are expressed as mean ± SEM for 6 mice per group. Statistical differences were determined using one-way ANOVA with Dunnett's post-test (*p <0.05, **p <0.01). Fig. 6. Analysis of pro-inflam- matory cytokine mRNA. Inflam- matory cells were isolated from BAL fluid (4 hours post-LPS) and the total RNA analyzed for (A) TNF-a; (B) IL-1b; and (C) IL-6 mRNA using RT-PCR. Equal loading was shown with 18S bands. Densitometry was performed in relation to 18S (n = 5). Data are representative of 2–3 separate experiments. Dif- ferences among groups were statistically significant with **p <0.01 for saline untreated con- trol (-LPS) vs. vehicle, and *p <0.05 for GW0742 vs. vehicle control, as determined by one- way ANOVA with Dunnett’s post-test. Fig. 7. Protein levels (pg/ml) of the CXC chemokines in the BALF were determined by MSD for (A) KC and ELISA for (B) MIP-2a. The data are expressed as mean +/– SEM (n = 5 mice per group). Statistical differences were determined us- ing one-way ANOVA followed by Dunnett’s posttest (*p <0.01) to evaluate differences between the groups. Fig. 8. BALF protein levels (pg/ml) of GM-CSF were determined by ELISA. Results are expressed as mean+/–SEM for 5 mice per group. Statistical differences were determined using one-way ANOVA fol- lowed by Dunnett’s post-test (*p <0.01). Discussion The primary goals of this study were to elucidate the anti- inflammatory activities of GW0742 and, as such, gain in- sight into the potential role that PPARb/d ligands may play in mediating early, acute inflammatory processes. It was found that GW0742 dose-dependently decreased neutrophil recruitment in LPS-stimulated lungs, but did not alter the number of monocyte/macrophages or lymphocytic cells. It should be noted however, that our study focused on the early, acute phase of LPS-induced inflammation, where the neutrophil is the primary cell type recruited to the lungs and would therefore be expected to be the cell type most affected. The more chronic effects of PPARb/d activation under these conditions remain to be evaluated. The expression of the pro-inflammatory cytokines IL-1b, TNFa and IL-6, all of which have been previously shown to be up-regulated in lungs following administration of LPS [25], were found to be significantly decreased in the BALF of GW0742-treated animals as compared to vehicle. Inter- estingly, transcript analysis of post-lavage lung tissue by RT-PCR showed increased levels mRNAs encoding these cytokines upon administration of LPS, but did not demon- strate a reduction for any of the aforementioned cytokine mRNAs in lung tissue from GW0742-treated animals (data not shown). It is hypothesized that this may be due to an insufficient number of PPARb/d receptors in lung tissue. Indeed, immunohistochemical analysis demonstrated only weak PPARb/d receptor immunoreactivity in lung alveolar type II cells and bronchial epithelium [5]. It was therefore of interest to examine RNA from BALF-derived inflammatory cells. RT-PCR analysis demonstrated a significant decrease in the mRNAs encoding IL-1b, TNFa and IL-6 in GW0742- treated animals as compared to vehicle control group. It is apparent therefore, that, in the setting of LPS-induced pul- monary inflammation, the primary cell types affected by PPARb/d ligands are the newly recruited inflammatory cells as opposed to the resident lung cell types. Interestingly, the cell count data demonstrate a change in the type and number of cells contained in the BAL fluid of GW0742/LPS treated animals as compared to the LPS treated animals. It is ap- parent, based on cell number and differential analysis of the cells that neutrophils constituted the primary cell type analyzed in BAL fluid samples from vehicle-treated animals, while monocyte/macrophages were the predominant type obtained from GW0742-treated animals. Thus, the altera- tions we observed in the expression of cytokine mRNAs and proteins is influenced by a shift in the immune cells resident in the lungs of the drug treated animals. Thus, GW0742 may cause a key change in the immune reaction to LPS that alters the quality and quantity of the cellular infiltrate into the lung tissue. Further experimentation will be required to determine the nature of this cellular mechanism. In order to elucidate the chemotactic mediators affected by the selective PPARb/d ligand GW0742, BALF was ini- tially analyzed for the protein levels of both MIP-2 and KC. These neutrophil chemotactic factors are members of the CXC chemokine family, and are expressed by alveolar mac- rophages, fibroblasts and inflammatory cell types. Both have been previously shown to play a role in mediating neutrophil recruitment in LPS-induced lung inflammation [26; 27]. It was hypothesized that the inhibition of neutrophil influx noted after PPARb/d activation, may be due to decreased ex- pression of one or more of these chemokines in that TNFa has been shown to promote both MIP-2 and KC release. Interestingly, while the expression of both KC and MIP- 2 were up-regulated in the BALF following LPS treatment, neither of the chemokines was decreased to a statistically significant degree in GW0742-treated animals as compared to the vehicle. While these data do not exclude the possibil- ity that other CXC or CC chemokines may be inhibited by PPARb/d, it does suggest that other direct or indirect mecha- nisms may be involved in the inhibition of neutrophil recruitment. A number of reports have suggested that granulocyte macrophage colony stimulating factor (GM-CSF) is an im- portant regulator of neutrophil recruitment (via its down- stream actions on TNFa and other modulators) [28], acti- vation/survival in models of pulmonary inflammation [29] and bleomycin-induced fibrosis [30]. Use of neutralizing anti-GM-CSF antibodies have been shown to significantly decrease neutrophil accumulation following LPS, likely via modulation of Akt, AP-1 and NF-kB [31]. Furthermore, Bozinovski et al. have demonstrated that the expression of TLR-4 is reduced in LPS-stimulated lungs following treat- ment with anti-GM-CSF. More recently, neutralization of GM-CSF in the setting of LPS-induced lung injury was as- sociated with a decrease in both TNFa and MIP-2, but not KC levels [32]. Taken together, these studies suggest that GM-CSF is rapidly expressed following LPS administration and likely plays an important role in modulating the acute phase of the inflammatory response. In the present study, pretreatment with GW0742 was associated with a significant decrease in BALF-associated GM-CSF protein, suggesting that the noted anti-inflammatory effects may be due to the modulation of GM-CSF and its effects on down-stream in- flammatory regulators. Moreover, the ability of GM-CSF to act as a neutrophil chemoattractant suggests that the lack of neutrophilia noted in the lungs of GW0742-treated animals may also be due to the inhibition of the direct, chemotactic effects of this protein. While we demonstrate that a number of important inflammatory mediators, including TNFa, IL-1b, IL-6 and GM- CSF, are affected by PPARb/d activation, the cellular/molec- ular basis of this anti-inflammatory effect has yet to be fully elucidated. Recent studies however, have begun to offer in- sight as to potential mechanism(s) for this effect. Ding et al., [14] have recently shown that treatment of cardiomyocytes with GW0742 significantly decreased TNFa production fol- lowing LPS stimulation. It was noted that the presence of GW0742 was associated with decreased degradation of both IkBa and IkBb, thereby implicating inhibition of NF-kB as a potential mechanism for the observed decrease in TNFa expression. This hypothesis is supported by additional stud- ies examining the ability of PPARb/d ligands to impact the NF-kB pathway [15;16;18]. The ability of PPARb/d ligands to impair NF-kB activity presents a plausible, direct mecha- nism for the results noted in the present study. The results of this study are in line with multiple reports which indicate that agonists against other PPAR subtypes,specifically PPARa and g, also act to reduce LPS-induced pulmonary inflammation [33]. For example, Delayre-Or- thez et al. [34] have used PPAR knockout mice and spe- cific PPAR agonists to delineate the role of this molecule in modulating LPS-induced pulmonary inflammation. Specifi- cally, cell infiltration, chemoattractant production and MMP activity were significantly decreased in agonist-treated and PPARa knockout mice [34]. This anti-inflammatory effect is not limited to PPAR however, as multiple studies suggest that activation of the PPAR receptor attenuates pulmonary inflammation in a manner similar to that noted for both a and b/d. For example, pretreatment with the PPAR agonist ros- iglitazone significantly decreased pulmonary injury, MPO activity and the overproduction of TNF-alpha and CINC-1 as well as expression of ICAM-1 following LPS [35]. Fur- thermore, studies have shown that rosiglitazone pretreatment reduced body weight loss, mortality, and neutrophil infiltra- tion in the model of bleomycin-induced lung-injury [36] and LPS-induced airway inflammation [37]. In light of the aforementioned studies, it is clear that PPARb/d has a simi- lar anti-inflammatory profile as that noted for both the a and g subtypes. The results of this study would seem to contradict that of Trifilieff et al. [22] however, who demonstrated that, in contrast to PPARa and g, PPARb/d agonists do not inhibit allergen-induced airway inflammation. There are however, a number of important differences between the present inves- tigation and that of Trifilieff et al., [22] including the differ- ing disease pathologies investigated in the respective studies. The authors of the aforementioned study utilized a model of allergen-induced airway disease in which the pathophysiol- ogy is antigen-specific and carried out by effector T lym- phocytes. In contrast, the model of LPS-induced pulmonary inflammation utilized in the present study is characterized by the rapid infiltration of neutrophils into the alveolar space. Another important difference between these studies was the employed dosing paradigm. Trifilieff et al. [22] utilized an acute dosing protocol where a single dose of the com- pound was administered one hour before allergen challenge. In contrast, the present study used a previously optimized prophylactic dosing regimen consisting of three days of pre- treatment with the last dose administered half an hour before LPS challenge. The results of this study are the first to directly demonstrate that treatment with a PPARb/d agonist (GW0742) at- tenuates LPS-induced pulmonary inflammation evidenced by a significant decrease in inflammatory cell influx as well as a decrease in pro-inflammatory cytokine message and pro- tein. Our findings lend further support for the role of PPARs in modulating the inflammatory response and extend these data to encompass PPARb/d. 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