Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-?12,14-PGJ2 in fish granulocytes

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-delta(12,14)-PGJ(2) (15dPGJ(2)) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor gamma (PPAR gamma) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ(2), and its biotinylated analog, as well as several PPAR gamma ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ(2) was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1 beta (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPAR gamma agonists were able to mimic the effects of 15dPGJ(2). Conversely, the PPAR gamma antagonist T007097 abolished the effect of 15dPGJ(2) on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ(2) and its biotinylated analog signaled via Ppar alpha and Ppar beta, but not by Ppar gamma. These results were further confirmed by HPLC/MS analysis, where Ppar beta was identified as an interactor of biotin-15dPGJ(2) in naive and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ(2) acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Ppar alpha and Ppar beta in the resolution of inflammation mediated by 15dPGJ(2).

Automated summary

This study revealed that 15dPGJ(2) acts on fish granulocytes by activating PPARs and covalently binding to proteins. Additionally, 15dPGJ(2) decreased ROS production induced by bacterial DNA and the expression of certain cytokines.