Phylogenetic, molecular and functional characterization of prostaglandin receptors in teleost fish

Abstract

The immune system of teleost fish has been studied for decades but, despite the important advances made, much is still unknown, especially concerning the presence and role of prostaglandins, such as PGE2 and PGA2, and their receptors. The gilthead seabream (Sparus aurata L.), a marine teleost fish of great commercial value in Mediterranean aquaculture, has been widely used for better understanding the fish immune response. The aim of this thesis is to advance our knowledge of the role of prostaglandins PGE2 and PGA2 and their receptors in the immune response of the gilthead seabream. In the first chapter, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced the highest mRNA levels ofptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses. In the second chapter we have identified Nr4a3 in gilthead seabream and shown that PGA2 regulates in vitro the main functions of professional phagocytes of this species. The gilthead seabream Nr4a3 revealed highly conserved motifs with all their vertebrate orthologs, particularly the DNA binding domain and sumoylation site. Expression analysis showed that nr4a3 was constitutively expressed in macrophages and was upregulated by PGA2 alone and combined with bacterial DNA. Importantly, PGA2 addition weakly decreased the transcript levels of genes encoding the cytokines interleukin-1 (Il1b), Il6 and Il10, while its combination with VaDNA promoted a synergistic induction of the mRNA levels of these three genes. However, PGA2 failed to regulate the mRNA levels of the gene encoding the M2 polarization marker mannose receptor C-type 1 (Mrc1). In contrast, PGA2 inhibited the respiratory burst of acidophilic granulocytes and induced the transcript levels of the genes encoding Il1b and Il10. Finally, PGA2 was able to signals through gilthead seabream Nr4a3 when ectopically expressed in HEK293 cells. In conclusion, we have shown for the first time that PGA2 regulates the main functions of professional phagocytes of teleosts and provided evidences that it may signals through Nr4a3, paving the way for in vivo studies in order to understand the role of this prostaglandin and the Nr4a3 receptor in fish immune responses.