Characterization of P2X7R and Its Function in the Macrophages of ayu, Plecoglossus altivelis

School of Marine Sciences, Ningbo University, Ningbo, China.
PLoS ONE (Impact Factor: 3.23). 02/2013; 8(2):e57505. DOI: 10.1371/journal.pone.0057505
Source: PubMed


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.

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Available from: Jiong Chen, Oct 04, 2015
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    • "2.2. Primary culture of ayu head kidney-derived MO/MF Ayu head kidney-derived MO/MF were isolated as previously described [15]. Briefly, fish were sacrificed by anesthetic overdose (50 mg/ml; 0.03% [v/v] ethylene glycol monophenyl ether). "
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    ABSTRACT: C-type lectin-like receptors (CLRs) are important pathogen pattern recognition molecules that recognize carbohydrate structures. However, the functions of these receptors in fish keep less known. In this study, we characterized a novel CLR from a teleost fish, Plecoglossus altivelis (ayu), tentatively named PaCD209L. The cDNA of PaCD209L is 1464 nucleotides (nts) in length, encoding a polypeptide of 281 amino acid residues with a calculated molecular weight of 31.5 kDa. Multiple alignment of the deduced amino acid sequences of PaCD209L and other related fish CLRs revealed that the PaCD209L sequence had typical characteristics of fish CLRs, but without Ca(2+)-binding sites. Sequence comparison and phylogenetic tree analysis showed that PaCD209L shared the highest amino acid identity (44%) with rainbow trout (Oncorhynchus mykiss) CD209aE. PaCD209L transcripts were detected in all of the tissues examined, mainly expressed in the brain and heart. Upon Vibrio anguillarum infection, PaCD209L transcripts were upregulated in all tested tissues and in monocytes/macrophages (MO/MΦ). We prepared recombinant PaCD209L (rPaCD209L) by prokaryotic expression and raised antiserum against PaCD209L. Western blot analysis revealed that native PaCD209L was glycosylated, and its protein expression significantly increased in ayu MO/MΦ upon V. anguillarum infection. In addition, rPaCD209L was able to bind Gram-positive and Gram-negative bacteria in the absence of Ca(2+). After PaCD209L was blocked by anti-PaCD209L IgG, the phagocytosis and bacterial killing activity of MO/MΦ significantly decreased. These results suggest that PaCD209L plays an important role in the regulation of MO/MΦ functions in ayu. Copyright © 2015. Published by Elsevier Ltd.
    Fish &amp Shellfish Immunology 04/2015; 44(2). DOI:10.1016/j.fsi.2015.03.037 · 2.67 Impact Factor
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    • "P2RX7 cDNAs have been found in human, mice, dog and several other vertebrate species since it was cloned from rat macrophages by Surprenant et al. in 1996 [14]–[17]. In teleost, P2RX7 orthologues have been identified from zebrafish [18], seabream [19] and ayu [20]. Available literature has documented that seabream P2RX7 exhibits different agonist (ATP/BzATP)-evoked pharmacological responses from mammalian and zebrafish P2RX7s [19], suggesting species differences of P2RX7 in agonist/antagonist action may exist in teleost. "
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    ABSTRACT: ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60-88% sequence similarity and 45-78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.
    PLoS ONE 05/2014; 9(5):e96625. DOI:10.1371/journal.pone.0096625 · 3.23 Impact Factor
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    ABSTRACT: Oral administration of chicken egg yolk immunoglobulins (IgY) has attracted much attention as a means for controlling infectious diseases caused by microorganisms. This study evaluated the protective effect of IgY against Vibrio anguillarum infection in ayu, Plecoglossus altivelis. IgY was isolated from egg yolks laid by hens initially immunized with formalin-inactivated V. anguillarum. Lower mortality of ayu was observed in groups treated with anti-V. anguillarum IgY (aVIgY), compared with those treated with saline or with nonspecific IgY (nspIgY). All fish in saline-treated groups died within seven days after bacterial inoculation. The bacterial load in blood, liver, and spleen was significantly lower in fish treated with aVIgY than in fish treated with nspIgY. aVIgY treatment significantly reduced tumor necrosis factor-α (PaTNF-α), interleukin-1β (PaIL-1β), transforming growth factor-β (PaTGF-β), and leukocyte cell-derived chemotaxin-2 (PaLECT2) transcript levels in the head kidney, spleen, and liver of ayu challenged by V. anguillarum, compared with nspIgY treatment. The phagocytic activity of macrophages for V. anguillarum in the presence of specific IgY was significantly higher than that seen for nonspecific IgY. These results suggest that passive immunization by oral intubation with pathogen-specific IgY may provide a valuable treatment for V. anguillarum infection in ayu.
    Fish &amp Shellfish Immunology 03/2014; 37(1). DOI:10.1016/j.fsi.2014.01.018 · 2.67 Impact Factor
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