Abstract
Uridine 5’-diphosphate (UDP)-activated purinergic receptor P2Y6 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 P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R 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 P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1β, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1β and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.
Highlights
Nucleotides, such as adenosine 5 -triphosphate (ATP), adenosine 5 -diphosphate (ADP), uridine 5 -triphosphate (UTP), and uridine 5 -diphosphate (UDP), perform important roles in modulating inflammatory and cell death responses following their release into the extracellular milieu and activation of purinergic receptors at the plasma membrane surface [1,2,3]
Mammalian cells can express various combinations of the eight known subtypes of P2Y receptor (P2Y1R, P2Y2R, P2Y4R, P2Y6 receptor (P2Y6R), and P2Y11R–P2Y14R) that belong to the G-protein-coupled receptor (GPCR) superfamily, the seven P2X receptors (P2X1R–P2X7R) that act as adenosine -triphosphate (ATP)-gated ion channels and the four different G-protein-coupled adenosine (P1) receptors [4]
A Basic Local Alignment Search Tool (BLAST) search of the National Center for Biotechnology Information (NCBI) database reveals that the P2Y6 receptor is highly conserved
Summary
Nucleotides, such as adenosine 5 -triphosphate (ATP), adenosine 5 -diphosphate (ADP), uridine 5 -triphosphate (UTP), and uridine 5 -diphosphate (UDP), perform important roles in modulating inflammatory and cell death responses following their release into the extracellular milieu and activation of purinergic receptors at the plasma membrane surface [1,2,3]. It has been demonstrated that UDP and lipopolysaccharides (LPS)-induced IL-8 release from human monocytic THP-1 cells is mediated by an autocrine stimulation of the P2Y6 receptor [8]. P2Y6 receptors significantly up-regulated the mRNA levels of IL-8, IP-10, and IL-1β in human monocytic cells stimulated with UDP [9]. Studies demonstrated that the P2Y6 receptor is a novel mediator in up-regulating innate immune response against invading pathogens through recruiting monocytes/macrophages [11]. We previously showed that functional P2Y2 and P2Y12 receptors are expressed in Japanese flounder immune cells [12], while the presence and immune function of other P2YRs in fish is still unknown. We show that in addition to previously demonstrated P2Y2 and P2Y12 receptors, Japanese flounder immune cells express functional P2Y6 receptors. We further reveal the association of the P2Y6 receptor with TLR3/ 4-mediated immune signaling in fish
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