Role of prostaglandins in the immune system of gilthead seabream (Sparus aurata L.)

Laburpena

The immune system of teleost fish has been widely studied for decades but, much is still unknown, especially concerning the role of prostaglandins (PGs) and their signaling pathways. The gilthead seabream (Sparus aurata L.), is a marine teleost fish widely used as a research animal model for better understanding the fish immune response. Despite this, few studies have been focused on the role of PGs in gilthead seabream phagocytes. Within this framework, the aim of this thesis is to advance our knowledge of the role of PGs in the immune response of the gilthead seabream. In the first chapter, we study the molecular characterization of two genes (pgds1 and pgds2) which deduced amino acid sequences show high degree of similarity to mammalian Lipocalin-PGDS involved in PGD2 synthesis. Tissues samples of seabream juveniles challenged or not with the pathogen Vibrio anguillarum or AGs stimulated with PAMPS were analyzed by RT-qPCR. The results showed that both genes were expressed in all tissues and their expression were modulated upon bacterial challenge in almost all tissues examined. However, pgds1, but not pgds2, was detected in AGs which expression was upregulated by stimulation with PAMPs with the same kinetics than PGD2 by this cell type, as was determined by enzyme immunoassay. Finally, to determine the effects of PGD2 in the main functions of AGs, control or stimulated AGs were cultured with PGD2. The results showed that treatment of AGs with PGD2 results in decreased ROS production and cytokine expression. These results provide us evidences that PGD2, probably synthesized by PGDS1, could play a key role in the resolution of inflammation by AGs in gilthead seabream. In the second chapter, we analyzed by RT-qPCR the expression profile of genes involved on PGE2 metabolism in tissue samples of seabream juveniles challenged or not with Vibrio anguillarum and phagocytes activated with genomic DNA from V. anguillarum (VaDNA) were analyzed by RT-qPCR. The results showed that pges (involved in PGE2 synthesis) was expressed in all tissues examined as well as in phagocytes and its expression was up-regulated in all immune organs upon bacterial challenge and in phagocytes stimulated with VaDNA. In contrast, the mRNA levels of hpgd (involved in PGE2 catabolism) were lower in all tissues tested from challenged fishes. In addition, the effect of PGE2 in the main functions of AGs was analyzed. Treatment of stimulated AGs with PGE2 resulted in decreased ROS production and cytokine expression. In contrast, treatment of stimulated macrophage with PGE2 resulted in increased mRNA levels of il10, mrc1 and arg2 and decreased il6 ones, resulting in a M2 phenotype. Furthermore, PGE2 signaled through the cAMP/PKA/CREB pathway in seabream macrophages. These results points to PGE2, could play a key role in the regulation of inflammation in gilthead seabream. Finally, in the third chapter, we determine the effects of 15deoxy-?12,14-PGJ2 (15dPGJ2) and its signaling pathway in seabream AGs. 15dPGJ2 is a cyclopentenone PG derivated from PGD2 that can signal through PPAR?, DP2 receptor or can act by covalent binding to proteins. For this purpose, seabream AGs were stimulated with VaDNA and treated with 15dPGJ2, with 15dPGJ2 analogs, and with PPAR? agonists and antagonist. The results showed that 15dPGJ2 decreased ROS production and cytokine expression in stimulated AGs. Similarly, PG analogs and PPAR? agonist were able to mimic the effects of 15dPGJ2 in ROS production by stimulated AGs. Besides, those effects were reversed by PPAR? antagonists. These results show that, 15dPGJ2 could have an anti-inflammatory role in the resolution of the inflammation in gilthead seabream which signal both, through protein covalent binding and PPAR? activation.