Effect of peroxisome proliferator-activated receptor alpha activators on tumor necrosis factor expression in mice during endotoxemia

Abstract

Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARalpha) as a potential target to modulate regulation of the immune response. Since PPARalpha activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARalpha activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARalpha wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARalpha. PPARalpha WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARalpha KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARalpha in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARalpha in vivo can be masked by other systemic effects of PPARalpha activators.

FIG. 1

Time course of fenofibrate (A) and Wy 14,643 (B) on TNF levels in plasma from mice during endotoxemia. CD-1 mice were fed rodent chow containing no drug, fenofibrate (0.5%), or Wy-14,643 (0.1%) for 2 weeks. The animals were then challenged with 0.5 ml of saline or LPS (12 mg/kg), and plasma samples were collected at the indicated times. The data are taken from a representative experiment (n = 4 animals per group), and the values are expressed as the means ± standard errors of the means. Asterisks denote values that are significantly different (P < 0.05), as determined by Student’s t test, from those of LPS-treated control-fed animals. The values for saline-treated animals were not plotted, since no TNF was detected in the plasma samples.

FIG. 2

Time course of fenofibrate on glucose levels in plasma from mice during endotoxemia. CD-1 mice were fed rodent chow containing no drug or fenofibrate (0.5%) for 2 weeks. The animals were then challenged with 0.5 ml of saline or LPS (12 mg/kg), and plasma samples were collected at the indicated times. The data are taken from a representative experiment (n = four animals per group), and the values are expressed as means ± standard errors of the means. Asterisks denote values that are significantly different (P < 0.05), as determined by Student’s t test, from LPS-treated control-fed animals.

FIG. 3

Time course of Wy-14,643 on LPS-induced TNF expression in primary macrophages from PPARα wild-type (WT) versus knockout (KO) mice. Culture supernatant fractions were collected from LPS-treated (10 ng/ml) primary peritoneal macrophages at the times indicated. The data presented are from a representative experiment (n = 4 wells per group). Asterisks denote values that are significantly different (P < 0.05), as determined by Student’s t test, from LPS-treated cells in the absence of Wy-14,643.

FIG. 4

Effect of overexpression of PPARs and RXRα on hTNF-luciferase activity. 293T cells were cotransfected, by using the calcium phosphate method, with a TNF promoter (−615 to +90)-luciferase construct plus PPARα, PPARβ, or PPARγ with or without RXRα. The medium was changed 24 h after transfection, and cell extracts were harvested 24 h later. Luciferase activity and protein concentration were determined, and the results are expressed as the percentage of activity of hTNF-luciferase alone. The results represent four independent experiments (n = 3 wells/group/experiment).