RESUMO
P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.
Assuntos
Doenças dos Peixes , Proteínas de Peixes/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/imunologia , Osmeriformes/imunologia , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/farmacologia , Morte Celular/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/microbiologia , Listonella/crescimento & desenvolvimento , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Dados de Sequência Molecular , Osmeriformes/genética , Osmeriformes/microbiologia , Fagocitose/efeitos dos fármacos , Filogenia , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Receptores Purinérgicos P2X7/imunologia , Alinhamento de SequênciaRESUMO
The teleost warm temperature acclimation related 65 kDa protein (Wap65) is a plasma glycoprotein with the potential roles in heat adaptation, heme recycling, immune response and copper metabolism. It is most homologous to the mammalian hemopexin, which is the plasma transporter of heme. A full-length cDNA clone of the Wap65 gene, 1,534 bp in size, was isolated from the fish ayu (Plecoglossus altivelis). Its deduced amino acid sequence of 439 residues had 60.4-65.4% and 38.3-47.3% identical to fish Wap65-2-type and Wap65-1-type sequences, respectively. In phylogenetic analysis, aWap65 grouped tightly with those fish Wap65-2-type sequences. In healthy control fish, the highest mRNA signal for aWap65 was from the liver, moderately high in brain and gill, and but weaker in spleen, kidney, muscle, heart and intestine. In Listonella anguillarum-infected fish, aWap65 transcripts were significantly increased in liver, while no obvious changes in other tissues at 12 hpi. However, aWap65 transcripts were significantly increased in various tissues at 24 hpi when hemolysis developing, suggesting that aWap65 might be involved in the immune response of ayu.