Characterization Of Udp-Activated Purinergic Receptor P2y(6) Involved In Japanese Flounder Paralichthys Olivaceus Innate Immunity

Uridine 5'-diphosphate (UDP)-activated purinergic receptor P2Y(6) is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y(6) receptor (P2Y(6)R) in fish immunity has not been investigated. In this report, we characterized a P2Y(6)R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y(6)R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y(6)R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y(6)R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I: C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y(6) receptor activity by the potently selective P2Y(6)R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1 beta, IL-6 and TNF-alpha gene expression in PBL cells treated with UDP. Moreover, LPS-and poly(I: C)-induced gene expression of IL-1 beta and TNF-alpha in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y(6)R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y(6)R in fish innate immunity.