Peroxisome proliferator-activated receptor {delta} regulates inflammation via NF-{kappa}B signaling in polymicrobial sepsis

Abstract

The nuclear peroxisome proliferator-activated receptor δ (PPARδ) is an important regulator of lipid metabolism. In contrast to its known effects on energy homeostasis, its biological role on inflammation is not well understood. We investigated the role of PPARδ in the modulation of the nuclear factor-κB (NF-κB)-driven inflammatory response to polymicrobial sepsis in vivo and in macrophages in vitro. We demonstrated that administration of GW0742, a specific PPARδ ligand, provided beneficial effects to rats subjected to cecal ligation and puncture, as shown by reduced systemic release of pro-inflammatory cytokines and neutrophil infiltration in lung, liver, and cecum, when compared with vehicle treatment. Molecular analysis revealed that treatment with GW0742 reduced NF-κB binding to DNA in lung and liver. In parallel experiments, heterozygous PPARδ-deficient mice suffered exaggerated lethality when subjected to cecal ligation and puncture and exhibited severe lung injury and higher levels of circulating tumor necrosis factor-α (TNFα) and keratinocyte-derived chemokine than wild-type mice. Furthermore, in lipopolysaccharide-stimulated J774.A1 macrophages, GW0742 reduced TNFα production by inhibiting NF-κB activation. RNA silencing of PPARδ abrogated the inhibitory effects of GW0742 on TNFα production. Chromatin immunoprecipitation assays revealed that PPARδ displaced the NF-κB p65 subunit from the κB elements of the TNFα promoter, while recruiting the co-repressor BCL6. These data suggest that PPARδ is a crucial anti-inflammatory regulator, providing a basis for novel sepsis therapies.

Figure 1

A: Effect of in vivo treatment with GW0742 on mean arterial blood pressure in rats subjected to cecal ligation and puncture (CLP). Data represent the mean ± SEM of 3 to 17 animals for each time point. *P < 0.05 versus time 0 in the same treatment group subjected to CLP. B: Effect of in vivo treatment with GW0742 on survival in rats (n = 15 for each group) subjected to cecal ligation and triple puncture (CL3P). *P < 0.05 versus vehicle-treated group at Gehan-Breslow analysis. GW0742 (0.5 mg/kg i.p.) was administered at the time of CLP or CL3P procedure and at 6 and 12 hours thereafter.

Figure 2

Effect of in vivo treatment with GW0742 (0.5 mg/kg i.p.) on myeloperoxidase (MPO) activity in the lung (A), liver (B), and cecum (C) in rats at 18 hours after cecal ligation and puncture (CLP). Each data point represents the mean ± SEM of 4 to 10 animals for each group. *P < 0.05 versus sham rats. **P < 0.05 versus vehicle-treated rats. Treated rats received GW0742 at the time of CLP procedure and at 6 and 12 hours thereafter.

Figure 3

Effect of in vivo treatment with GW0742 (0.5 mg/kg i.p.) on plasma levels of TNFα (A), IL-6 (B), IL-1β (C), leptin (D), and MCP-1 (E) in rats at 18 hours after cecal ligation and puncture (CLP). Each data point represents the mean ± SEM of 3 to 10 animals for each group. *P < 0.05 versus sham rats. **P < 0.05 versus vehicle-treated rats. Treated rats received GW0742 at the time of CLP procedure and at 6 and 12 hours thereafter.

Figure 4

Effect of in vivo treatment with GW0742 (0.5 mg/kg i.p.) on PPARδ expression and production of TNFα in ex vivo rat peritoneal macrophages. A: Western blot analysis of PPARδ and β-actin (used as loading control protein) in nuclear extracts. Blot is representative of three independent experiments. B: Supernatant levels of TNFα production. Data are mean ± SEM of three independent experiments. *P < 0.05 versus sham rats. **P < 0.05 versus vehicle-treated rats. Peritoneal macrophages were harvested at 6 hours after cecal ligation and puncture (CLP). Treated rats received GW0742 at the time of CLP procedure.

Figure 5

Sepsis induces down-regulation of PPARδ expression and up-regulation of NF-κB activity. A: Western blot analysis of PPARδ and β-actin (used as loading control protein) in nuclear extracts of livers. B: Image analysis of IκBα expression relative to β-actin expression in cytosolic extracts of livers determined by densitometry of Western blots (n = 3 time-course experiments). C and D: Representative autoradiographs of EMSA for NF-κB DNA binding in liver (C) and lung (D); DNA binding complexes are indicated with a left bracket. E and F: Image analyses determined by densitometry of three independent EMSAs for DNA binding of NF-κB in liver (E) and lung (F). Fold increase was calculated versus respective sham value (time 0) set to 1.0. Tissue samples were obtained from vehicle-treated and GW0742-treated rats at 0, 1, 3, 6, and 18 hours after cecal ligation and puncture (CLP). Treatment with vehicle or GW0742 (0.5 mg/kg i.p.) was given at the time of CLP procedure and at 6 and 12 hours thereafter. * P < 0.05 versus vehicle-treated group.

Figure 6

Mortality was significantly higher in heterozygous PPARδ^+/− than wild-type PPARδ^+/+ mice subjected to polymicrobial sepsis (P = 0.007 at log-rank analysis). Wild-type PPARδ^+/+ (n = 12) and heterozygous PPARδ^+/− (n = 12) mice underwent cecal ligation and puncture (CLP), and were monitored for survival for five days.

Figure 7

Representative histology photomicrographs of lung sections of heterozygous PPARδ^+/− and wild-type PPARδ^+/+ mice subjected to polymicrobial sepsis. Lungs were harvested 18 hours after CLP and were stained with H&E. A and B: Normal lung architecture in a sham PPARδ^+/+ mouse (A) and a sham PPARδ^+/− mouse (B) at magnification ×100; C and D: Normal lung architecture in a sham PPARδ^+/+ mouse (C) and a sham PPARδ^+/− mouse (D) at magnification ×400. E and F: Lung derangement in a PPARδ^+/+ septic mouse (E) and a PPARδ^+/− septic mouse (F) at magnification ×100. G and H: Lung derangement in a PPARδ^+/+ septic mouse (G) and a PPARδ^+/− septic mouse (H) at magnification ×400. A similar pattern was seen in n = 3 to 6 different tissue sections in each experimental group.

Figure 8

Inflammatory response is enhanced in heterozygous PPARδ^+/− mice after polymicrobial sepsis. A: Myeloperoxidase (MPO) activity in the lung, (B) plasma levels of keratinocyte-derived chemokine (KC), and (C) TNFα in heterozygous PPARδ^+/− and wild-type PPARδ^+/+ mice 18 hours after cecal ligation and puncture (CLP). Each data point represents the mean ± SEM of 3 to 6 animals for each group. *P < 0.05 versus sham mice of the same genotype. **P < 0.05 versus wild-type mice.

Figure 9

PPARδ deficiency is associated with increased activity of NF-κB in the lung after polymicrobial sepsis. A: Representative autoradiographs of EMSA for PPARs (first panel) and NF-κB (second panel) DNA binding in the lung of heterozygous PPARδ^+/− and wild-type PPARδ^+/+ mice 18 hours after cecal ligation and puncture (CLP). B: Specificity of DNA binding was confirmed by incubation with cold oligonucleotide for PPARs. Supershift assay was also performed in samples incubated with antibodies against PPARα, PPARδ, or PPARγ. C and D: Image analyses of DNA binding of PPAR (C) and NF-κB (D) in the lung determined by densitometry. Fold increase was calculated versus respective sham value set to 1.0. Data represent the mean ± SEM of 3 to 5 animals for each group. *P < 0.05 versus sham mice of the same genotype. **P < 0.05 versus wild-type mice.

Figure 10

PPARδ deficiency is associated with increased activity of NF-κB in the liver after polymicrobial sepsis. A and B: Image analyses of DNA binding of PPAR (A) and NF-κB (B) in the liver determined by densitometry. Fold increase was calculated versus respective sham value set to 1.0. Data represent the mean ± SEM of 3 to 5 animals for each group. *P < 0.05 versus sham mice of the same genotype. **P < 0.05 versus wild-type mice. C–F: Image analysis of PPARδ (C), PPARγ (D), PPARα (E), and RXRα (F) expression relative to β-actin expression in nuclear extracts of livers determined by densitometry of Western blots (n = 3 to 5 animals for each group).

Figure 11

PPARδ activation reduced TNFα production in LPS-stimulated J774.A1 macrophages. A: Western blot analysis of PPARδ and β-actin (used as loading control protein) in nuclear extracts of macrophages stimulated with LPS (100 ng/ml) up to 24 hours. Blot is representative of three independent experiments. B: Effect of the PPARδ ligand GW0742 (10–1000 nmol/L) on TNFα production at 24 hours after LPS stimulation. C: Effect of GW0742 (100 nmol/L) on TNFα production at 24 hours after LPS stimulation in macrophages transfected with scramble siRNA or PPARδ-siRNA. Data are mean ± SEM of three independent experiments performed in quadruplicate. *P < 0.05 versus control cells with media alone. **P < 0.05 versus vehicle treatment.

Figure 12

PPARδ inhibits TNFα production by direct inhibition of NF-κB transactivation. A: Western blot analysis of IκBα expression in cytosol extracts of macrophages stimulated with LPS (100 ng/ml) up to 24 hours with or without GW0742 (100 nmol/L). Blot is representative of three independent experiments. Equal loading was confirmed by Ponceau staining after transfer of proteins on nitrocellulose membrane (data not shown). B: Effect of GW0742 on NF-κB promoter activity 4 hours after LPS stimulation. Cells were transfected with a 3xNF-κB promoter-luciferase plasmid and a Renilla luciferase vector (for normalization). Promoter activity of NF-κB was evaluated by luciferase assay and was expressed as fold increase of activity of control basal cells set to 1. *P < 0.05 versus cells with media alone (basal). C: Representative PCR gels from chromatin immunoprecipitation assays demonstrating binding of p65 subunit of NF-κB, PPARδ, and BCL6 to TNFα promoter. Chromatin was immunoprecipitated using an antibody recognizing the p65 subunit of NF-κB, PPARδ, or BCL6. Positive control (Input) was chromatin recovered without immunoprecipitation. Cells were stimulated with LPS (100 ng/ml) for 1 hour with or without GW0742 (10 to 100 nmol/L), which was added to the cells 30 minutes prior LPS. PCR was performed with primers specific for the murine TNFα promoter region spanning the most proximal NF-κB binding site. D: Image analysis of promoter binding relative to Input recovery. Data are mean ± SEM of three independent experiments. *P < 0.05 versus cells with media alone (basal). **P < 0.05 versus vehicle treatment.