Characterization of P2X7R and Its Function in the
Macrophages of ayu, Plecoglossus altivelis
Yu-Qing He, Jiong Chen*, Xin-Jiang Lu, Yu-Hong Shi
School of Marine Sciences, Ningbo University, Ningbo, China
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.
Citation: He Y-Q, Chen J, Lu X-J, Shi Y-H (2013) Characterization of P2X7R and Its Function in the Macrophages of ayu, Plecoglossus altivelis. PLoS ONE 8(2):
Editor: Sebastian D. Fugmann, Chang Gung University, Taiwan
Received November 6, 2012; Accepted January 22, 2013; Published February 21, 2013
Copyright: ? 2013 He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The project was supported by the Program for the National Natural Science Foundation of China (31201970), the Scientific Research Fund of Zhejiang
Provincial Education Department (Y201119291), the Research Fund for the Doctoral Program of Higher Education of China (20113305120001), the Discipline
Project of Ningbo University (szx11069, xkc11004) and the KC Wong Magna Fund in Ningbo University. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The P2X purinoceptor 7 (P2X7R), an ionotropic receptor gated
by adenosine triphosphate (ATP), was first identified in rat . It is
widely distributed in nearly all tissues and organs, with the highest
expression observed in macrophages , . P2X7R possesses
two transmembrane domains, intracellular N- and C-termini, and
a long carboxyl terminus, containing five ATP ligand-binding
motifs in the ectodomain and one conserved LPS-binding motif in
the C-terminus . Activation of P2X7R leads to a variety of
downstream events, including Ca2+influx , nonselective large
pore formation , cell death , interleukin (IL)-1b release ,
membrane permeabilization , and reactive oxygen species
P2X7R expression has been reported to be upregulated upon
pathogen infection . Moreover, P2X7R is involved in the
functional regulation of immune cells, and activation of P2X7R
strongly enhances intracellular bacterial killing in macrophages
and induces macrophage death . Transfection with P2X7R
confers phagocytic abilities on nonphagocytic HEK-293 cells,
while blocking P2X7R expression by siRNA significantly reduces
the phagocytic abilities of THP-1 cells, a monocytic leukemia cell
line . Furthermore, ATP can activate P2X7R to release IL-1b
in human monocytes priming by lipopolysaccharide . Thus,
P2X7R plays important roles in innate immunity in mammals.
Because of the economic and environmental impact of fish and
diseases in fish, many studies conducted over the past decade have
studied the immune system of fish . However, little informa-
tion is available on the functions and characteristics of fish P2X7R,
although the function of P2X7R is known to be important in
mammalian macrophages [7–9]. Therefore, it is necessary to
investigate the function of P2X7R in the fish immune system.
Until recently, only zebrafish (Danio rerio)  and gilthead
seabream (Sparus aurata)  P2X7R genes had been cloned in fish.
P2X7R does not mediate IL-1b release in the gilthead seabream,
which is different from that reported in mammals . However,
the role of P2X7R in regulating the fish immune system still
The ayu (Plecoglossus altivelis) is an economically important fish
species in Asia. Intensive ayu farming has promoted the growth of
many bacterial and viral diseases that have resulted in both
production and animal welfare problems , . Hence,
because of the economic importance of this fish, it seems especially
important to study the immune response of fish against
microbiological pathogens. In this work, we aimed to clone ayu
P2X7R (aP2X7R) cDNA, study the expression and functional
responses of aP2X7R during Listonella anguillarum challenge, and its
potential role in macrophages.
Materials and Methods
About 120 healthy ayu, weighing 40–50 g each, were purchased
from a fishery in Fuxi, Ninghai County, Ningbo City, China.
These fish were maintained and acclimatized in aerated fresh
water at 20–22uC with regular feeding as previously described
PLOS ONE | www.plosone.org1February 2013 | Volume 8 | Issue 2 | e57505
. Only healthy fish, without any pathological signs, were used
in the study. All animal work in this paper was conducted
according to relevant national and international guidelines. All
animal care and experimental procedures were approved by the
Committee on Animal Care and Use and the Committee on the
Ethics of Animal Experiments of Ningbo University.
L. anguillarum challenge in the ayu was performed as described
previously . Briefly, overnight cultures of L. anguillarum were
diluted 1:50 in basic peptone water medium, cultured at 28uC with
shaking, and harvested in the logarithmic growth. Cells were
washed, resuspended, and adjusted to a final concentration of
1.06106colony-forming units (CFU) ml21in sterile normal saline.
40 fish were intraperitoneally injected with 100 mL of L. anguillarum
per fish, and 40 other fish were injected with 100 mL of saline per
fish as a negative control. Each tank contained 20 bacteria-
infected or healthy control fish. Samples of infected and control
fish were randomly collected at 0, 4, 8, 12, and 24 h postinjection
(hpi), frozen in liquid nitrogen, and stored at 270uC until use.
Determination of the cDNA sequence of aP2X7R
Total RNAs were extracted from ayu head kidney with RNAiso
Reagent (TaKaRa, Dalian, China) following the manufacturer’s
instructions and treated with RNase free DNase I. The mRNA in
1 mg total RNA was reverse transcribed using M-MLV reverse
transcriptase (TaKaRa) following standard protocols. Based on the
partial sequence of aP2X7R, which was obtained from previous
transcriptome sequencing, the full-length cDNA sequence was
determined using the rapid amplification of cDNA ends (RACE)
method . PCR amplification products were sequenced by an
ABI 3730 automated sequencer (Invitrogen, Carlsbad, CA, USA).
The similarity of the obtained aP2X7R sequence (accession
number: HE984576) with known P2X7R sequences, i.e., human
(Homo sapiens), Q99572; small-eared galago (Otolemur garnettii)
XM_003795998; dog (Canis lupus familiaris), NM_001113456;
XM_001926804; cattle (Bos Taurus), NM_001206516; rabbit
(Oryctolagus cuniculus), XM_002719745; mouse (Mus musculus)
AJ489297; rat (Rattus norvegicus), NM_019256; clawed frog (Xenopus
laevis), AJ345114; chicken (Gallus gallus), XM_001235162; green
anole (Anolis carolinensis), XM_003222779; gilthead seabream (S.
aurata), AJ887997; and zebrafish (D. rerio), AY292647, was
analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The cleavage site of signal peptides was predicted by the SignalP
4.0 program (http://www.cbs.dtu.dk/services/SignalP/). The
transmembrane helices were predicted by the ‘‘DAS’’- Trans-
membrane Prediction server (http://www.sbc.su.se/˜miklos/DAS/
). Multiple alignments were analyzed using ClustalW (http://
clustalw.ddbj.nig.ac.jp/). Phylogenetic and molecular evolutionary
analyses were conducted using MEGA version 4 .
Real-time quantitative PCR (RT-qPCR)
Changes in the mRNA expression of aP2X7R following L.
anguillarum infection were analyzed by RT-qPCR as previously
described . RT-qPCR was conducted on an ABI StepOne
Real-Time PCR System (Applied Biosystems, USA) using SYBR
premix Ex Taq (Perfect Real Time) (TaKaRa) in accordance with
the manufacturer’s instructions. To assess PCR efficiency, 10-fold
serial dilutions of both aP2X7R and b-actin plasmid cDNA were
used to generate a standard curve for each assay plate. According
to the standard curve, the PCR efficiency was determined to be
92% and 94% for aP2X7R and b-actin, respectively. After the
amplification, melt curves were obtained by slow heating from
60uC to 95uC at 0.1uC/s, with continuous fluorescence collection,
confirming that only our specific product peaks were detected.
Amplifications with aP-F: 59-TCCCAGTTCAGACGGACAG-39
and aP-R: 59-TTAAGGTGTGGTGTTTGCCA-39 primers were
performed with cDNA from the head kidney, spleen, liver, gill,
intestine, heart, and muscle of infected and control fish. As a
control, a 231-bp fragment of the housekeeping b-actin gene was
amplified from the same cDNA preparations using pActin-F: 59-
CGCACTTCATGATGCTGTTG-39 primers. The mRNA ex-
pression of aP2X7R from macrophages was also detected. Relative
gene expression was analyzed by the comparative Ct method (2-
DDCtmethod). The mRNA expression of aP2X7R was normalized
against the expression of b-actin. Data were expressed as the mean
6 SEM and analyzed by one-way analysis of variance (ANOVA)
with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Four
independent experiments were performed. Differences were
considered significant at P,0.05.
Prokaryotic expression of the ectodomain fragment of
Based on the previously determined sequence, a specific primer
pair was designed that would amplify a 750-bp fragment
comprising amino acids 60–310 of aP2X7R ectodomain peptide
(aEP) and included restriction sites for NdeI and BamHI
CGGATCCTTATCCAAAAGCTTTGTACAG-39 primers, re-
spectively, to facilitate directional cloning into the pSBET vector.
Pfu DNA Polymerase (Fermentas, Vilnius, Lithuania) was used for
gene amplification according to the manufacturer’s protocol. BL21
(DE3) Escherichia coli transformed with pSBET-aEP plasmid were
used for overexpression of aEP. After a 4-h induction of protein
expression by 1 mM isopropyl-bD-thiogalactopyranoside (IPTG),
the bacterial pellets were collected and detected by 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The recombinant peptide was extracted from inclusion bodies and
purified by molecular sieve filtration. Protein concentration was
determined by the Coomassie light blue method with bovine
serum albumin as the standard.
Antibody production and purification
The purified aEP of aP2X7R was used as an antigen to
immunize mice to produce antiserum . ICR mice (20–22 g)
were intraperitoneally immunized with 0.5 ml purified aEP (1 mg
ml21) emulsified with an equal volume of Freund’s complete
adjuvant. Thereafter, the mice were injected intraperitoneally with
the same amount of aEP emulsified with Freund’s incomplete
adjuvant on days 14 and 28 post-primary immunization. One day
after the final injection, the mice were fasted overnight, and the
blood was collected from the caudal vein. After standing at 4uC for
8 h, sera were collected by centrifuging at 140006g for 10 min at
4uC and stored at 270uC until use. Control mice were injected
with the same volume complete Freund’s adjuvant. The specificity
of anti-aEP serum was determined by Western blot with
macrophage lysates and recombinant aEP protein. Anti-aEP
polyclonal antibody (aEPAb) from the generated antisera and
control isotype immunoglobulin G (IgG) from control mice were
purified by protein G chromatography media (Bio-Rad, Shanghai,
China) according to the manufacturer’s protocol. Western blotting
was performed to detect the purified recombinant aEP of
The Role of ayu P2X7R on Macrophages
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aP2X7R. The same recombinant protein sample was sequenced to
detect the specificity of aEPAb. The protein sample was resolved
by SDS-PAGE and subsequently transferred onto a polyvinylidene
fluoride (PVDF) membrane. The N-terminal amino acid sequence
of the purified aEP of aP2X7R was determined by automated
Edman degradation on a PE/ABD Model 470A protein sequencer
(Foster City, CA) operated with gas-phase delivery of trifluor-
Figure 1. Multiple alignment of vertebrate P2X7R. Threshold for shading was . 60% of similarity. Similar residues are shadowed gray and
identical residues are shadowed black. Two predicted transmembrane domains (yellow), the P2X family signature (blue), five residues important for
nucleotide binding (red), and the LPS/lipid-binding domain (pink) are shown. The accession numbers of P2X7R sequences are HE984576 for ayu (P.
altivelis), AJ887997 for gilthead seabream (S. aurata), AY292647 for zebrafish (D. rerio), AJ345114 for clawed frog (X. laevis), Q99572 for human (H.
sapiens), and AJ489297 for mouse (M. musculus).
The Role of ayu P2X7R on Macrophages
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Ayu macrophages were isolated as previously described with
some modifications . Briefly, head kidneys were aseptically
extracted, collected, and meshed in RPMI 1640 (Invitrogen,
Shanghai, China) supplemented with 2% fetal bovine serum (FBS)
(Invitrogen), penicillin (100 U ml21), streptomycin (100 mg ml21),
and heparin (20 U ml21). The cell suspension was centrifuged at
4006g for 20 min on Ficoll (GE Healthcare Life Sciences, New
Jersey, USA) at a suspension:Ficoll ratio of 2:1. Cells were
collected from the interphase, washed, and dissolved in RPMI
1640 supplemented with 0.1% FBS and antibiotics. The cells were
then seeded into 35-mm well plates at a density of 26106cells
well21and allowed to adhere overnight at 24uC in an atmosphere
with 5% CO2. The medium was changed to complete medium
(4% ayu serum, 6% FBS, 100 U ml21penicillin, 100 mg ml21
streptomycin), and cells were kept in the incubator under the same
Infection of macrophages with L. anguillarum
Before infection, the medium was changed to antibiotic-free
medium, and cells were incubated for another 12 h. Macrophages
were infected with live L. anguillarum at a multiplicity of infection
(MOI) of 10. Infected and uninfected cells were harvested at 0, 4,
8, 12, and 24 hpi. Cell RNA was extracted using RNAiso Reagent
(TaKaRa). Simultaneously, cells were also lysed in buffer
containing protease inhibitors (20 mM Tris-HCl, 1 mM EDTA,
1% Triton X-100, 1 mM PMSF, 10 mg ml21aprotinin, 10 mg
ml21leupeptin, and 10 mg ml21pepstatin-A, pH 8.0), and total
proteins were prepared as described previously .
GAUU-39) and a scrambled siRNA (59-GAGACACAGGCUC-
GUUAAUAGGAGU-39) were purchased from Invitrogen. Trans-
fection of cells with siRNA was performed using Lipofectamine
2000 transfection reagent (Invitrogen) according to the manufac-
ture’s protocol. Briefly, 5 ml of Lipofectamine 2000 in 250 ml of
Opti-MEM (Invitrogen) was mixed with either 100 pmol aP2X7R
siRNA or 100 pmol scrambled siRNA in 250 ml of Opti-MEM.
The mixture was then incubated for 20 min at room temperature
and was added to macrophages with a final siRNA concentration
of 40 nM. After a 5.5-h incubation, media were changed to
complete media, and cells were cultured for another 48 h before
collection for cell death, phagocytosis, and bacterial killing assays.
RT-qPCR and Western blotting were used to confirm aP2X7R
Western blot analysis
Protein samples from macrophages subjected to bacterial
infection or siRNA blocking were analyzed by SDS–PAGE and
Western blotting, as previously reported . aEPAb was used as
the primary antibody at 6 mg ml21, and the bound primary
antibody was evaluated using the relevant HRP-labeled goat anti-
mouse IgG at 160 ng ml21. The proteins were visualized using an
enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA). Changes in relative band intensity
were analyzed by the NIH ImageJ program. Three biological
replicates were used for each treatment.
Figure 2. Phylogenetic analysis of aP2X7R amino acid
sequences using the neighbor-joining method. The values at
the forks indicate the percentage of trees in which this grouping
occurred after bootstrapping (1000 replicates). The scale bar shows the
number of substitutions per base. The accession numbers of sequences
used are human (H. sapiens), Q99572; small-eared galago (O. garnettii)
XM_003795998; dog (C. familiaris), NM_001113456; horse (E. caballus),
XM_001495572; pig (S. scrofa), XM_001926804; cattle (B. Taurus),
NM_001206516; rabbit (O. cuniculus), XM_002719745; mouse (M.
musculus), AJ489297; rat (R. norvegicus), NM_019256; clawed frog (X.
laevis), AJ345114; chicken (G. gallus), XM_001235162; green anole (A.
carolinensis), XM_003222779; gilthead seabream (S. aurata), AJ887997;
ayu (P. altivelis), HE984576; and zebrafish (D. rerio), AY292647.
Figure 3. Bacterial expression of aEP and preparation of
antiserum. (A) SDS-PAGE analysis of recombinant aEP. Lane M: protein
marker; 1 and 2: protein from BL21 E. coli transformed with the pSBET-
aEP plasmid before and after IPTG induction; 3: purified recombinant
protein. (B) Western blot analysis of recombinant aEP and native
aP2X7R. Lane 4: purified recombinant protein; 5: ayu head kidney-
derived macrophages; NC: negative control, BL21 lysate before IPTG
The Role of ayu P2X7R on Macrophages
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Measurement of cell death
Ayu head kidney-derived macrophages were seeded in 96-well
plates (16104cell well21) and treated with various concentrations
of ATP (0.01, 0.1, 0.5, 1, 1.5, 2.5, 5, and 10 mM) for 30 min at
24uC in an atmosphere with 5% CO2. The media were then
removed and replaced with complete media. The cells were
incubated for an additional 6 h, and cytoplasmic histone-
associated DNA fragments were quantified using a Cell Death
Detection ELISAPLUS(Roche Applied Science, Indianapolis,
USA) according to the manufacturer’s protocol. To assess the
effects of aP2X7R on ATP-induced cell death, cells were
transfected with aP2X7R siRNA for 48 h, pre-incubated with
different concentrations of aEPAb (1, 5, 10, 15, 25, 50, 100, 200,
and 500 mg ml21) for 30 min, or pre-treated with various
concentrations of oxidized ATP (oATP; 1, 10, 100, 150, 300,
500, and 1000 mM; Sigma, Shanghai, China) for 2 h. oATP is a
small Schiff base molecule that has been used as an antagonist of
P2X7R . Scrambled siRNA, mouse isotype IgG, and PBS
were added as controls. Cells were treated with 5 mM ATP for
30 min, followed by a 6-h incubation in the absence of ATP. Cell
death was determined as described above.
DH5a E. coli in the logarithmic phase of growth were labeled
with fluorescein isothiocyanate (FITC; cells were hereafter
designated as FITC-DH5a; Sigma) according to the manufactur-
er’s protocol. Ayu macrophages grown on cover slips in 6-well
plates (26106cell well21) were transfected with aP2X7R siRNA
for 48 h, pre-incubated with 200 mg ml21aEPAb for 30 min or
300 mM oATP for 2 h. As a control, scrambled siRNA, mouse
isotype IgG, and PBS were added. FITC-DH5a were added at an
MOI of 10, and cells were further incubated for 30 min. Then,
cells were washed extensively with sterile PBS to remove
extracellular particles. Trypan blue (0.4%) was used to quench
the fluorescence that resulted from particles, which were outside of
the cells or sticking to the surface of the cells. The engulfed
bacteria were examined by fluorescence microscopy (6006
magnification; Nikon Eclipse Ti-U, Tokyo, Japan). The mean
fluorescence intensity (MFI) of bacteria engulfed by cells among
siRNA, aEPAb, and oATP treatments was analyzed by the NIH
ImageJ program, and at least 400 macrophages were counted for
each independent assay. The results were expressed as the percent
MFI of the control and were shown as the mean 6 SEM of a
typical example from at least three independent experiments.
Bacterial killing assay
Ayu macrophages were transfected with aP2X7R siRNA for
48 h, pre-incubated with 200 mg ml21aEPAb for 30 min or
300 mM oATP for 2 h, and then infected with live L. anguillarum at
an MOI of 10 as described above. As a control, scrambled siRNA,
mouse isotype IgG, and PBS were added. Bacterial phagocytosis
was allowed to proceed for 30 min at 24uC in an atmosphere of
5% CO2, and the noninternalized L. anguillarum were removed by
washing with sterile PBS. One set of samples (the uptake group)
was lysed in 1% Triton X-100 solution and plated onto solid
thiosulfate citrate bile salts sucrose (TCBS) agar medium to
provide bacterial uptake values. The remaining set (the kill group)
was incubated with 5 mM ATP (Sigma) for a 30 min pulse. The
Figure 4. RT-qPCR analysis of aP2X7R mRNA expression
following bacterial infection in various tissues and macro-
phages. Fish or macrophages were infected with L. anguillarum for 4,
8, 12, or 24 h. aP2X7R transcript levels were normalized by the b-actin
content at the same time point. Each bar represents the mean 6 SEM of
the results from four independent experiments. *P,0.05; **P,0.01;
Figure 5. Western blot analysis of aP2X7R protein expression
in macrophages upon L. anguillarum treatment. (A) Protein
collected from L. anguillarum-infected macrophages at 0, 4, 8, 12 and
24 hpi was assayed by Western blotting using an antiserum specific to
aP2X7R. (B) Histogram displaying the changes in relative band intensity
of aP2X7R protein in L. anguillarum-infected macrophages at 0, 4, 8, 12,
and 24 h. Data are representative of three independent experiments.
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ATP was then removed and replaced with an equal volume of
fresh medium, and the cells were further incubated for 1.5 h to
allow bacterial killing to occur. Cell lysate-bacterial samples were
collected and pelletted at 14000 rpm for 20 min, lysed in 1%
Triton X-100 solution, and then plated onto TCBS agar medium.
After incubation at 30uC for 18 h, we counted the CFU in the
plates. After aEPAb or isotype IgG incubation, cells were treated
with a 30-min ATP plus, then cultured in the absence of ATP and
harvested at different time intervals (0.5, 1, 2, and 4 h) to
investigate whether the bacteria were killed or could not replicate
. Bacterial survival was determined by dividing the CFU in the
kill group by the CFU in the uptake group. Three independent
experiments were performed.
AP2X7R gene analysis
The full-length cDNA of aP2X7R, measuring 2046 nucleotides
(nts) long, was obtained and deposited into GenBank with the
accession number HE984576. The 1725-nt open reading frame of
aP2X7R encoded a polypeptide of 574 amino acids corresponding
to a calculated molecular weight (MW) of 65.0 kDa. The deduced
protein contained no putative signal peptide predicted by the
SignalP 4.0 program, suggesting that it was not a secretory protein.
Multiple alignment revealed that a high level of conservation of
the P2X receptor family signature, the two transmembrane
domains and a long C-terminal domain, were present in aP2X7R
(Fig. 1). Five residues important for nucleotide binding in
mammalian P2X7R molecules ,  were intact in aP2X7R
(Fig. 1). Sequence analysis showed that aP2X7R had the highest
amino acid identity to P2X7R from the gilthead seabream (67%).
Phylogenetic tree analysis showed that P2X7R proteins from the
ayu and other fish were grouped together, forming a fish cluster
distinct from the mammalian cluster (Fig. 2).
Figure 6. RT-qPCR and Western blot analysis of aP2X7R
expression following siRNA transfection. aP2X7R siRNA was
transfected into macrophages for 48 or 72 h. Scrambled siRNA was
transfected as a control. (A) Histogram displaying the aP2X7R mRNA
expression following siRNA transfection by RT-qPCR analysis. (B) The
effects of aP2X7R siRNA transfection on knockdown of aP2X7R protein
was confirmed by Western blot analysis. Histogram displays the
changes in relative band intensity of aP2X7R protein upon siRNA
treatment. Data are representative of three independent experiments.
NC: negative control without any siRNA.
Figure 7. ATP induced cell death through activation of aP2X7R.
(A) Cell death induced by different concentrations of ATP. Macrophages
were incubated with PBS (control) or various concentrations of ATP for
30 min and cultured for an additional 6 h without ATP. (B) The effects of
aEPAb on ATP-induced cell death. Cells were pre-incubated with
different concentrations of aEPAb for 30 min before ATP treatment. (C)
The effects of oATP on ATP-induced cell death. The cells were
pretreated with various concentrations of oATP for 2 h before ATP
treatment. (D) The effects of aP2X7R siRNA on ATP-induced cell death.
The cells were transfected with siRNA for 48 h before ATP treatment.
Mouse isotype IgG, PBS, and scrambled siRNA were added as controls.
Cells were treated with ATP for 30 min, followed by a 6-h incubation in
the absence of ATP. Data are representative of three independent
experiments. *P,0.05; **P,0.01.
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Preparation and purification of an antibody against aEP
aEP (comprising amino acids 60–310 of the aP2X7R ectodo-
main peptide) was overexpressed in BL21 (DE3) E. coli
transformed with the pSBET-aEP plasmid (Fig. 3A, lane 2).
Protein from BL21 (DE3) E. coli transformed with the pSBET-aEP
plasmid before and after IPTG induction was shown in Fig. 3A
(lanes 1 and 2, respectively). The recombinant peptide was
extracted from inclusion bodies and purified by molecular sieve
filtration. As shown by SDS-PAGE analysis, the recombinant aEP
was highly pure (Fig. 3A, lane 3). Using our anti-aEP polyclonal
antibody, which was generated by immunizing mice, we detected
aEP by Western blotting. The MW of recombinant aEP was
30.0 kDa, while that of the full-length aP2X7R protein from
macrophages was 65.0 kDa (Fig. 3B). The specificity of the
antibody was verified by sequencing the band from the
recombinant protein detected by Western blotting. The partial
MTKVKGVALTNLPGVGNIVW, which was indeed the N-
terminal sequence of the aEP of aP2X7R. Antibody was
subsequently purified by protein G chromatography media and
stored at 270uC before the following study.
Alteration of tissue and macrophage aP2X7R mRNA
expression upon L. anguillarum infection
aP2X7R transcripts were detected in macrophages and tissues,
including the spleen, head kidney, gill, liver, muscle, intestine and
heart, by RT-qPCR (Fig. 4). The expression levels of the receptor
were higher in macrophages, as well as in the head kidney, liver,
and spleen, as compared to the other studied tissues. After
challenge with L. anguillarum, the aP2X7R transcripts in these
tissues showed a time-dependent increase in expression pattern.
The bacterial infection increased the expression of aP2X7R
mRNA in all examined tissues at 4 hpi. The most dramatic
upregulation in aP2X7R mRNA expression was observed in the
head kidneys (up to 19.99-fold) at 12 hpi (Fig. 4).
Expression of macrophage aP2X7R upon L. anguillarum
To further analyze the protein levels of aP2X7R in ayu
macrophages upon L. anguillarum infection, protein was analyzed
by Western blotting using specific aEPAb. After L. anguillarum
challenge, aP2X7R protein was increased at 8 hpi, peaked (5.05-
fold increase) at 12 hpi, and remained at a significantly higher
level at 24 hpi (3.38-fold increase), as compared to that at 0 hpi
AP2X7R regulated ATP-induced macrophage cell death
An RNAi assay was performed to knockdown aP2X7R
expression. When the cells were transfected with aP2X7R siRNA,
the mRNA and protein levels of aP2X7R were significantly
downregulated as compared to corresponding mRNA and protein
levels in cells transfected with scrambled siRNA and negative
control cells (Fig. 6A and B). We measured cell death in terms of
cytoplasmic histone-associated DNA fragment formation. Treat-
ment with ATP induced cell death in a dose-dependent manner
(Fig. 7A); ATP concentrations below 1 mM had little effect, and
the ATP concentration evoking half-maximal cell death effect
(EC50) was 1.5 mM (Fig. 7A). The blocking activity of aEPAb and
oATP on ATP-induced cell death also showed a dose-dependent
effect (Fig. 7B and C). Knockdown of aP2X7R by siRNA inhibited
ATP-induced cell death (Fig. 7D). These results confirmed that
ATP-induced cell death was mediated by aP2X7R.
AP2X7R mediated the phagocytosis and bacterial killing
of ayu macrophages
The phagocytosis of FITC-DH5a was significantly downregu-
lated to approximately 50.33% of cells transfected with the
scrambled siRNA control (Fig. 8A). Moreover, aP2X7R blockage
by aEPAb and oATP also altered the phagocytosis of ayu
macrophages (Fig. 8B and C). Next, bacteria survival was
determined by the CFU counting method to assess the bacterial
killing of ayu macrophages (Fig. 9). aP2X7R siRNA transfection
significantly inhibited ATP-induced bacterial killing in ayu
macrophages (Fig. 9A). Blockage with aEPAb and oATP also
suppressed the effects of ATP on the bacterial killing of ayu
macrophages (Fig. 9B and C). Kinetic evaluation of bacterial
killing by ATP-pulsed macrophages was performed. At 1 h after
ATP stimulation, there was a significant reduction in bacterial
Figure 8. The phagocytosis of ayu macrophages after aP2X7R
ablation. Fluorescence images of phagocytosis of FITC-DH5a in
macrophages treated with siRNA (A), aEPAb (B), and oATP (C). After
incubation with siRNA for 48 h, aEPAb for 30 min, or oATP for 2 h,
macrophages were incubated with FITC-DH5a at an MOI of 10 for an
additional 30 min. Scrambled siRNA, mouse isotype IgG, and PBS were
added as controls. Histogram represents the percent mean fluorescence
intensity (MFI) of bacteria engulfed by cells treated with siRNA, aEPAb,
or oATP. Data are representative of at least three independent
experiments. Scale bar, 10 mm. *P,0.05; **P,0.01.
The Role of ayu P2X7R on Macrophages
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Figure 9. ATP-induced bacterial killing activity was inhibited after aP2X7R ablation. Plates displayed the survival L. anguillarum from
macrophages treated with siRNA (A), aEPAb (B), or oATP (C). The histogram demonstrated the effects of siRNA, aEPAb, or oATP on bacterial killing.
Macrophages were infected with live L. anguillarum after siRNA, aEPAb, or oATP treatment. L. anguillarum viability was examined through CFU assay
after ATP treatment. Mouse IgG, scrambled siRNA, and PBS were added as controls for the respective treatments. (D) Reduction of L. anguillarum
viability induced by ATP. After treatment with aEPAb or isotype IgG, cells were treated with ATP. Subsequently, cells were harvested at different time
intervals, and bacterial viability was monitored through CFU assays. Data are representative of at least three independent experiments. *P,0.05;
The Role of ayu P2X7R on Macrophages
PLOS ONE | www.plosone.org8 February 2013 | Volume 8 | Issue 2 | e57505
viability of the control, further decreasing at 2 and 4 h after ATP
stimulation, which could be inhibited by aEPAb (Fig. 9D). These
results further confirmed that aP2X7R could mediate phagocytosis
and bacterial killing of ayu macrophages.
In this study, we provided the full-length sequence of the
aP2X7R gene. The deduced protein possessed several common
features shared by other P2X7R homologues . Sequence
comparison and phylogenetic tree analysis also confirmed
aP2X7R as a distinct member of the fish purinergic subtype
receptor. These data indicated that aP2X7R is homologue to
mammalian P2X7R and may play a role in ayu innate immunity.
Multiple alignment of fish, amphibian (clawed frog), and
mammalian P2X7R sequences revealed a high level of conserva-
tion of the P2X7R family signature, the two transmembrane
domains and the long C-terminal domain. Five residues important
for nucleotide binding in mammalian P2X7R were also present in
a conserved position in the fish and amphibian proteins. The
conserved structures of P2X7R in evolution indicated that P2X7R
may play important roles in both fish and mammals.
P2X7R has a ubiquitous tissue distribution, and its expression
levels may vary over orders of magnitude in mammals . It is
predominantly expressed in immune cells from myeloid lineages,
such as macrophages, monocytes, and dendritic cells , . The
head kidney, thymus, spleen, and mucosa-associated lymphoid
tissues are known to be the major lymphoid tissues in teleost fish
. In this study, aP2X7R transcripts were widely distributed in
ayu tissues and were especially abundant in the head kidney,
spleen, and liver. Similarly, aP2X7R mRNA was abundant in ayu
head kidney-derived macrophages. These results suggest that
aP2X7R is also mainly expressed in immune organs and cells.
Mammalian P2X7R has been found upregulated after infection
, , . A significant increase in the expression of P2X7R
has been observed on human macrophages infected with M.
tuberculosis  or mouse macrophages infected with Leishmania
amazonensis . A significant release of ATP has also been
detected by M. tuberculosis-infected macrophages . In our study,
aP2X7R was upregulated in all examined ayu tissues after
bacterial infection, suggesting that aP2X7R is implicated in ayu
In mammals, it has been demonstrated that prolonged
stimulation of macrophages using high concentrations of ATP
causes cell death via P2X7R-induced apoptosis , . Cell
death by ATP stimulation proceeds through apoptotic nuclear
alterations, such as chromatin condensation and DNA fragmen-
tation, but not cytolytic or membrane damage and occurs
concomitant with a decrease in bacterial viability , , .
However, it is still unclear whether ATP activates P2X7R to
induce cell death in fish. In the current study, ayu macrophage
death was observed following treatment with ATP. The EC50
concentration of ATP needed to induce aP2X7R-dependent cell
death was 1.5 mM, which seemed higher than the concentration
of ATP required in their mammalian counterparts . We also
found that ATP-induced cell death was suppressed after aP2X7R
blockage via RNAi, aEPAb, and oATP. Our data suggest ATP
activates aP2X7R to induce the macrophage cell death in ayu, a
P2X7R has also been reported to be involved in phagocytosis
and clearance of bacteria by macrophages , . Phagocytosis
is inhibited after ATP dissociates myosin IIA from P2X7R
complex , , . However, in the absence of ATP,
P2X7R may function in phagocytosis . Furthermore, the
mechanisms underlying P2X7R and phagocytosis in the absence
of ATP have also been defined . A peptide mimicking the
extracellular domain of P2X7R can bind phagocytosed particles,
suggesting that P2X7R mediates phagocytosis via direct recogni-
tion of the particles . In the present study, we observed that
siRNA, specially designed to knockdown the expression of
aP2X7R, aEPAb (the anti-aP2X7R extracellular domain anti-
body), and oATP (a P2X7R antagonist), could significantly
attenuate the phagocytic activity of ayu macrophages. Therefore,
aP2X7R may mediate phagocytosis as a scavenger receptor in ayu
After infection, intracellular bacterial viability is reduced after
ATP is released into the extracellular media . Here, we found
that the survival of bacteria was downregulated in ATP induced
macrophages compared with negative control. Moreover, the
number of CFUs was confirmed to be reduced after the cells were
treated with ATP for different times. This result suggests that
bacteria may actually be killed in macrophages treated with ATP
rather than just exhibit halted replication. As shown in multiple
studies, P2X7R mediates this process of ATP-induced bacterial
killing of macrophages , , _ENREF_30. Many studies
have also suggested that killing of bacteria and other pathogens via
the P2X7R-mediate pathway is independent of nitric oxide (NO)
, . The death of host cells may explain this method of
bacterial killing , , , . We also found that ATP
induced cell death in ayu macrophages, suggesting that ATP-
induced bacterial killing may also result from cell death.
Furthermore, ATP-induced bacterial killing was inhibited by
aP2X7R knockdown by siRNA or blockage by aEPAb and oATP.
Thus, we speculate that aP2X7R may be involved in bacterial
killing in ayu macrophages.
In conclusion, we found that aP2X7R was mainly distributed in
immune tissues of the ayu. Moreover, upon bacterial challenge,
aP2X7R mRNA was significantly upregulated in all tested tissues.
The mRNA and protein levels of aP2X7R were also significantly
increased in macrophages in response to bacterial infection. In the
seabream, a teleost fish, activation of P2X7R regulates phospha-
tidylserine externalization and cell permeabilization, but fails to
induce IL-1b release in leukocytes . However, the function of
P2X7R in other fish species remains poorly understood. Our data
further demonstrate that aP2X7R may regulate cell death,
phagocytosis, and bacterial killing in ayu macrophages in response
to bacterial infection, suggesting a conserved function for P2X7R
in macrophage modulation.
Conceived and designed the experiments: JC XJL. Performed the
experiments: YQH XJL YHS. Analyzed the data: YQH. Wrote the
paper: YQH JC XJL.
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