Peroxisome proliferator-activated receptor-alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis

Abstract

The interleukin-12 (IL-12) family of cytokines which includes IL-12, IL-23, and IL-27 play critical roles in T cell differentiation and are important modulators of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Previously, we demonstrated that peroxisome proliferator-activated receptor (PPAR) -alpha agonists suppress the development of EAE. The present studies demonstrated that the PPAR-alpha agonist fenofibrate inhibited the secretion of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 by lipopolysaccharide-stimulated microglia. The cytokines interferon-gamma and tumor necrosis factor-alpha also stimulated IL-12 p40 and IL-27 p28 expression by microglia, which was suppressed by fenofibrate. Furthermore, fenofibrate inhibited microglial expression of CD14 which plays a critical role in TLR signaling, suggesting a mechanism by which this PPAR-alpha agonist regulates the production of these pro-inflammatory molecules. In addition, fenofibrate suppressed the secretion of IL-12p40, IL-23, and IL-27p28 by lipopolysaccharide-stimulated astrocytes. Importantly, fenofibrate suppression of EAE was associated with decreased expression of IL-12 family cytokine mRNAs as well as mRNAs encoding TLR4, CD14, and MyD88 known to play critical roles in MyD88-dependent TLR signaling. These novel observations suggest that PPAR-alpha agonists including fenofibrate may modulate the development of EAE, at least in part, by suppressing the production of IL-12 family cytokines and MyD88-dependent signaling.

Fig. 1

Fenofibrate inhibits lipopolysaccharide (LPS) induction of IL-12 family proteins in primary mouse microglia. Cells were pre-treated for 1 h with the indicated concentrations (µmol/L) of fenofibrate. LPS (0.1 µg/mL) was then added as indicated, and 24 h later, the concentration of IL-12p40 (a), IL-12p70 (p40/p35) (b), IL-23 (p40/p19) (c), and IL-27p28 (d) in the culture medium was determined. Cell viability was determined by MTT assay (e). Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. **p < 0.01 and ***p < 0.001 versus LPS-treated cultures.

Fig. 2

Fenofibrate inhibits interferon-γ (IFN-γ) plus TNF-α (IT) induction of IL-12p40 and IL-27p28 proteins in primary mouse microglia. Cells were pre-treated for 1 h with the indicated concentrations (µmol/L) of fenofibrate. IFN-γ (250 U/mL) and TNF-α (500 U/mL) were added as indicated, and 24 h later, the concentration of IL-12p40 (a) and IL-27p28 (b) in the culture medium was determined. Cell viability was determined by MTT assay (c). Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. **p < 0.01 and ***p < 0.001 versus IFN-γ plus TNF-α-treated cultures.

Fig. 3

Effects of fenofibrate on lipopolysaccharide (LPS) induction of CD14 and MyD88 mRNA expression in primary mouse microglia. Cells were pre-treated for 1 h with fenofibrate (100 µmol/L). LPS (0.1 µg/mL) was added as indicated, and 6 h later, total RNA was isolated. CD14 (a) and MyD88 (b) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to LPS-treated cells and all values were normalized against GAPDH. Values are mean ± SEM from four independent experiments, and duplicate reactions were performed on each sample in each experiment. **p < 0.01 and ***p < 0.001 versus LPS-treated cultures.(c)

Fig. 4

Effects of fenofibrate on lipopoly-saccharide (LPS) induction of CD14 and MyD88 protein expression in mouse primary microglia. Cells were pre-treated for 1 h with fenofibrate (100 µmol/L). LPS (0.1 µg/mL) was added as indicated to cultures, and 24 h later, protein extracts from whole cell lysates were prepared. Protein expression for CD14 and MyD88 was evaluated by western blotting as described in Materials and methods. Western blot analyses are representative of three independent experiments (a). Each CD14, MyD88 and actin band was quantified by densitometry. Results are expressed in arbitrary units as the ratio of CD14 or MyD88 to actin, and LPS was used as reference and assigned the arbitrary value of 1. Values represent the mean ± SEM from three independent experiments. *p < 0.05 versus LPS-treated cultures (b).

Fig. 5

Fenofibrate inhibits lipopolysaccha-ride (LPS) induction of IL-12p40, IL-23 and IL-27p28 in primary mouse astrocytes. Cells were pre-treated for 1 h with the indicated concentrations (µmol/L) of fenofibrate. LPS (2.5 µg/mL) was then added as indicated, and 24 h later, the concentration of IL-12p40 (a), IL-23 (p40/p19) (b) and IL-27p28 (c) in the culture medium was determined. Cell viability was determined by MTT assay (d). Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. **p < 0.01 and ***p < 0.001 versus LPS-treated cultures.

Fig. 6

Oral administration of fenofibrate inhibits IL-12 family subunit mRNA expression in experimental autoimmune encephalomyelitis mice. C57BL/6 mice were fed fenofibrate daily by gavage. Vehicle-treated mice were fed water containing 0.5% carboxymethylcellulose by gavage daily. Spinal cords were harvested on day 18 post-immunization, and total RNA was isolated. IL-12p40 (a), IL-12p35 (b), IL-23p19 (c), IL-27EBI3 (d), and IL-27p28 (e) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold-changes relative to experimental autoimmune encephalomyelitis mice in vehicle group and all values are normalized against GAPDH. Values are mean ± SEM from five independent experiments, and duplicate reactions were performed on each sample in each experiment. *p < 0.05, **p < 0.01, and ***p < 0.001 versus vehicle-treated mice.

Fig. 7

Oral administration of fenofibrate inhibits CD14, MyD88, and TLR4 mRNA expression in experimental autoimmune encephalomyelitis mice. C57BL/6 mice were fed fenofibrate daily by gavage. Vehicle-treated mice were fed water containing 0.5% carboxymethyl-cellulose by gavage daily. Spinal cords were harvested on day 18 post-immunization, and total RNA was isolated. CD14 (a), MyD88 (b), and TLR4 (c) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold-changes relative to experimental autoimmune encephalomyelitis mice in vehicle group and all values are normalized against GAPDH. Values are mean ± SEM from five independent experiments, and duplicate reactions were performed on each sample in each experiment. *p < 0.05 and ***p < 0.001 versus vehicle-treated mice.