Functional characterization of purinergic P2Y2 and P2Y12 receptors involved in Japanese flounder (Paralichthys olivaceus) innate immune responses

Abstract

G-protein-coupled P2Y receptors activated by extracellular nucleotides play important roles under different physiological and pathophysiological conditions in mammals. To investigate the immunological relevance of P2Y receptors in fish, we identified and characterized the P2Y₂ and P2Y₁₂ receptors in Japanese flounder Paralichthys olivaceus. The P. olivaceus P2Y₂ and P2Y₁₂ receptors harbor seven transmembrane domains but share only 24% sequence identity. Real-time PCR analysis revealed the constitutive but unequal mRNA expression pattern of P2Y₂R and P2Y₁₂R in normal Japanese flounder tissues with the dominant expression of P2Y₂R in head kidney and blood and P2Y₁₂R in hepatopancreas. In addition, the expression of P2Y₂ and P2Y₁₂ receptors was markedly modulated by PAMPs stimulation and Edwardsiella tarda infection. Furthermore, blockage of P2Y₁₂R potently increased ADP-activated pro-inflammatory cytokine IL-1beta gene expression in the head kidney macrophages (HKMs). Moreover, inhibition of P2Y₂ and P2Y₁₂ receptor activity with their respective potent antagonists significantly altered some of the LPS-induced pro-inflammatory cytokine gene expression in the HKMs. However, blockade of P2Y₁₂R did not affect the poly(I:C)-induced pro-inflammatory cytokine gene expression examined in the HKMs. Collectively, we have for the first time reported the role of purinergic P2Y₂ and P2Y₁₂ receptors in fish innate immunity. Our findings have also addressed the importance of extracellular ATP and its metabolites in fish innate immune responses.

Keywords: Expression analysis; Innate immunity; P2Y receptor; Paralichthys olivaceus; Teleost fish.