Multiple Hepcidins in a Teleost Fish,
Dicentrarchus labrax: Different Hepcidins for
Different Roles
This information is current as
of June 17, 2017.
João V. Neves, Carolina Caldas, Inês Vieira, Miguel F.
Ramos and Pedro N. S. Rodrigues
J Immunol 2015; 195:2696-2709; Prepublished online 12
August 2015;
doi: 10.4049/jimmunol.1501153
http://www.jimmunol.org/content/195/6/2696
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References
The Journal of Immunology
Multiple Hepcidins in a Teleost Fish, Dicentrarchus labrax:
Different Hepcidins for Different Roles
João V. Neves,*,† Carolina Caldas,*,† Inês Vieira,*,† Miguel F. Ramos,*,† and
Pedro N. S. Rodrigues*,†,‡
D
ue to the often hostile nature of their surrounding environment, fish have developed several defense mechanisms against waterborne pathogens, with one of the first
lines of defense being a number of relatively small peptides called
antimicrobial peptides. Derived from their diversity, fish possess an
enormous variety of antimicrobial peptides that provoke a great
deal of interest, with many being described (1, 2). Among these
antimicrobial peptides is hepcidin, a small cysteine-rich molecule
discovered at the turn of the twenty-first century, which has been the
focus of recent attention. Hepcidin was originally isolated from
human blood ultrafiltrate and named LEAP-1, for liver-expressed
antimicrobial peptide (3). At the same time, it was also independently isolated by another group from human urine, and named
hepcidin, for its liver origins and antimicrobial properties (4). Aside
from its antimicrobial activity, a much more important role was
*Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135
Porto, Portugal; †Ferro e Imunidade Inata, Instituto de Biologia Molecular e Celular,
Universidade do Porto, 4150-180 Porto, Portugal; and ‡Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
Received for publication May 19, 2015. Accepted for publication July 14, 2015.
This work was supported by Fundo Europeu de Desenvolvimento Regional (FEDER)
Funds through the Operational Competitiveness Programme (COMPETE), and by
National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the
project FCOMP-01-0124-FEDER-029339 (PTDC/MAR-BIO/3204/2012). J.V.N. is
supported by FCT under Grant SFRH/BPD/86380/2012.
The Hamp1 and Hamp2 coding and genomic DNA presented in this article have been
submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) under accession
numbers KJ890391, KJ890392, KJ890393, KJ890394, KJ890395, KJ890396,
KJ890397, KJ890398, KJ890399, and KJ890400.
Address correspondence and reprint requests to Dr. João Neves, Ferro e Imunidade
Inata, Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150180 Porto, Portugal. E-mail address: [email protected]
Abbreviations used in this article: DIF, digoxigenin-11-dUTP; EPO, erythropoietin;
JTT, Jones–Taylor–Thornton (model); SNP, single nucleotide polymorphism; TSB-1,
tryptic soy broth growth medium, supplemented with 1% NaCl.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501153
quickly attributed to hepcidin, as the long-sought key regulator of
iron metabolism, through the inhibition of the iron exporter ferroportin (5, 6). A hepcidin homolog was soon after found in a teleost
fish, the hybrid striped bass (Morone saxatilis 3 Morone chrysops)
(7), and several others followed (8–10). However, contrary to
mammals, in which a single hepcidin gene exists [with the mouse
being the only known exception (11)], the studies performed with
other fish species have shown that many teleosts could possess
a large number of hepcidin genes (1, 12), especially in Perciformes
and Pleuronectiformes. This diversity is generally attributed to genome duplications and positive Darwinian selection (13–15),
influenced by host–pathogen interactions, with variations in gene
copy number suggested to be lineage- or species-specific. These fish
isoforms are usually classified into two groups (12): hamp1-type
isoforms, which usually present a single copy sharing a considerable degree of homology with the mammalian counterparts; and
hamp2-type isoforms, of which many copies may exist, with diversified isoforms that can present unique characteristics, suggesting a singular role in the immune response against a variety of
pathogens. As such, and unlike mammals, in which a single molecule is performing a dual function as an antimicrobial peptide and
iron-regulatory hormone, it is possible that the existence of multiple
hepcidins in fish led to a subfunctionalization. As in mammals, the
highest expression levels of hepcidin in fish are found in the liver
(16–25). However, fish seem to have a wide tissue distribution of the
different hepcidin isoforms, suggesting that each hepcidin isoform
may assume a different biological role, depending on the tissue in
which it is expressed.
Although in mammals the role of hepcidin in the control of iron
metabolism has been the preponderant focus of attention, in fish
mainly its activity as an antimicrobial peptide has been explored,
with fewer studies focusing on its involvement in iron metabolism.
Fish hepcidins have been shown to respond to several infectious
stimuli, such as Gram-negative and Gram-positive bacteria (7, 16, 20,
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Teleost fish rely heavily on their innate immunity for an adequate response against pathogens and environmental challenges, with the
production of antimicrobial peptides being one of their first lines of defense. Among those is hepcidin, a small cysteine-rich antimicrobial peptide that is also the key regulator of iron metabolism. Although most mammals possess a single hepcidin gene, with
a dual role in both iron metabolism regulation and antimicrobial response, many teleost fish present multiple copies of hepcidin,
most likely because of genome duplications and positive Darwinian selection, suggesting that different hepcidins may perform different functions. To study the roles of hepcidin in teleost fish, we have isolated and characterized several genes in the European sea
bass (Dicentrarchus labrax) and evaluated variations in their expression levels in response to different experimental conditions.
Although several hepcidin genes were found, after phylogenetic analysis they could be clustered in two groups: hamp1-like, with
a single isoform similar to mammalian hepcidins, and hamp2-like, with several isoforms. Under experimental conditions, hamp1
was upregulated in response to iron overload and infection and downregulated during anemia and hypoxic conditions. Hamp2 did
not respond to either iron overload or anemia but was highly upregulated during infection and hypoxia. In addition, Hamp2
synthetic peptides exhibited a clear antimicrobial activity against several bacterial strains in vitro. In conclusion, teleost fish that
present two hepcidin types show a degree of subfunctionalization of its functions, with hamp1 more involved in the regulation of
iron metabolism and hamp2 mostly performing an antimicrobial role. The Journal of Immunology, 2015, 195: 2696–2709.
The Journal of Immunology
Materials and Methods
Animals
European sea bass (D. labrax), with an average weight of 50 g, were provided
by a commercial fish farm in the south of Portugal (Piscicultura do Vale da
Lama, Lagos, Portugal). Fish were kept at the fish holding facilities of the
Instituto de Biologia Molecular e Celular, Porto, Portugal, in 500-l recirculating sea water tanks at 21 6 1˚C, with a 12-h light/dark cycle and fed daily to
satiation with commercial fish feed having an iron content of ∼200 mg iron/kg
feed. Before each treatment, fish were anesthetized with ethylene glycol
monophenyl ether (2-phenoxyethanol, 0.3 ml/l; Merck, Algés, Portugal). All
animal experiments were carried out in strict compliance with national and
international animal use ethics guidelines, approved by the animal welfare
and ethic committees of Instituto de Biologia Molecular e Celular and conducted by experienced and trained Federation of European Laboratory Animal Science Associations Category C investigators.
Isolation of sea bass hepcidin genes
Pairs of oligonucleotide PCR primers were designed according to conserved regions of hepcidin mRNA sequences from sea bass and other fish
species, sea bass expressed sequence tags, and whole-genome shotgun
sequences available in the National Center for Biotechnology Information
nucleotide database (http://www.ncbi.nlm.nih.gov). cDNA preparations
from liver, spleen, and head kidney were used in PCR amplifications. PCR
products were run on 1.2% agarose gels, and then relevant fragments were
purified with the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine,
CA), cloned into pCRII-TOPO vectors, propagated in One Shot Mach1-T1R
Competent Cells (Invitrogen, Life Technologies, Carlsbad, CA), and sent for
sequencing (GATC Biotech, Konstanz, Germany). Both strands were sequenced, and chromatograms were analyzed in FinchTV (Geospiza, Seattle,
WA) and assembled using Multalin (http://bioinfo.genopole-toulouse.prd.fr/
multalin/multalin.html).
Genomic organization
Genomic DNAwas isolated from sea bass RBCs, as described elsewhere (38);
quantification was performed using a NanoDrop 1000 spectrophotometer
(Thermo Scientific, Waltham, MA); and quality was checked by agarose gel
electrophoresis. One microgram of genomic DNA was amplified by RT-PCR
with the primers based on the previously obtained cDNA sequences, with the
following cycling profile: 94˚C for 5 min, 30 cycles of 94˚C for 90 s, 59˚C for
30 s, and 72˚C for 90 s. Several PCR products were purified, cloned, and sent
for sequencing. Comparisons were made between cDNA and genomic DNA
to assess the similarity of the coding regions and to identify intron/exon
boundaries. A comparison between the genomic sequences of sea bass
hepcidins with those of other species was also made, with GenBank and
Ensembl accession numbers for sequences used as follows: Morone chrysops
hamp (AF394245), Sparus aurata hamp1 (EF625901), Paralichthys oliva-
ceus hamp (AY623817), Paralichthys olivaceus hamp2 (AY623818),
Scophthalmus maximus hamp (AY994075), Gadus morhua hamp
(EU334515), Danio rerio hamp1 (AY363452), Salmo salar hamp1 (AF542965),
Ictalurus punctatus hamp (AY834211), Xenopus tropicalis hamp (ENSXETT00000065495), Mus musculus Hamp1 (ENSMUST00000062620), Mus
musculus Hamp2 (ENSMUST00000074671), and Homo sapiens HAMP
(ENST00000222304).
Amplification of 59 and 39 flanking regions
The 59 and 39 RACE were carried out using the 59/39 RACE Kit, 2nd Generation (Roche Applied Science, Amadora, Portugal) according to the
manufacturer’s instructions. Conditions for PCR were as follows: 94˚C for
2 min, 94˚C for 15 s, 59˚C for 30 s, 72˚C for 40 s, for 10 cycles; 94˚C for 15 s,
59˚C for 30 s, 72˚C for 40 s (plus 20 s/cycle), for 25 cycles, with a final
elongation at 72˚C for 7 min. When necessary, a second PCR amplification
was performed using the same conditions for an additional 30 cycles. To
increase sequence coverage and obtain possible promoter regions, amplifications of genomic DNA were performed using the Universal Genome
Walker Kit (Clontech, Mountain View, CA), according to the manufacturer’s
instructions. Amplification products were run on agarose gels; relevant
fragments were purified with the Zymoclean Gel DNA Recovery Kit (Zymo
Research), cloned, and sequenced as described earlier.
Sequence alignment and phylogenetic analysis
Alignments of the amino acid sequences of the hamp-predicted proteins were
performed using MUSCLE from MEGA v6 (39). A phylogenetic tree was
constructed using the Maximum Likelihood method, with the Jones–Taylor–
Thornton (JTT) model, Nearest-Neighbor-Interchange heuristic model,
complete deletion of gaps, and 1000 bootstrap replications. Sequences
used for comparisons and phylogenetic trees and their accession numbers
were as follows: Danio rerio Hamp1 (NP_991146); Danio rerio Hamp2
(NP_001018873); Epinephelus coioides Hamp1 (ADC93804); Epinephelos
coioides Hamp2 (ADC93805); Gadus morhua Hamp (ACA42770);
Ictalurus punctatus Hamp (ABA43709); Lateolabrax japonicus Hamp
(AAS55063); Lates calcarifer Hamp1 (AEO51036); Lates calcarifer Hamp2
(AEO51037); Morone chrysops Hamp (AAM28440); Oncorhynchus mykiss
Hamp (ADU85830); Oplegnathus fasciatus Hamp2 (ACF49394); Oreochromus mossambicus HampTH1-5, TH2-2, and TH2-3 (18); Pagrus major
Hamp (AAS66305); Paralichthys olivaceus Hamp1 (BAE06232); Paralichthys olivaceus Hamp2 (BAE06233); Salmo salar Hamp (AAO85553);
Scopthalmus maximus Hamp1 (AAX92670); Scopthalmus maximus Hamp2
(AFE88622); Sparus aurata Hamp (CAO78619); Columba livia HAMP
(ABV00675); Crocodylus siamensis HAMP (ADA68357); Xenopus tropicalis HAMP1 (NP_001090729); Xenopus tropicalis HAMP2 (ABL75284);
Mus musculus HAMP1 (NP_115930); Mus musculus HAMP2 (NP_899080);
and Homo sapiens HAMP (NP_066998).
Southern blot assay
To determine the number of copies of each hepcidin type in sea bass, 5 mg
genomic DNA was independently digested for 24 h at 37˚C with the restriction enzymes EcoRI, HindIII, or SstI (Roche Applied Science). Digestion products were run on an appropriate electrophoresis gel and blotted
onto a positively charged nylon membrane (Amersham Hybond N+; GE
Healthcare, Buckinghamshire, U.K.). The membrane was subjected to
Southern blot analysis using the digoxigenin-11-dUTP (DIG) System (Roche
Applied Science) according to the manufacturer’s specifications. Briefly,
505 bp (hamp1) and 463 bp (hamp2) DIG labeled probes were prepared with
the DIG Probe Synthesis Kit (Roche Applied Science), using primers
designed on the basis of the obtained sea bass hamp1 and hamp2 sequences;
the membrane was hybridized overnight at 44˚C and washed, and detection
was performed with the chemiluminescent substrate CDP-Star (Roche
Applied Science). Visualization was achieved by exposing the membrane to
x-ray film (Hyperfilm; GE Healthcare) for 1–5 min.
Chromosomal localization of hepcidin by fluorescence in situ
hybridization
To stimulate cellular mitosis, healthy sea bass were i.p. injected twice with
PHA (20 mg/g body weight in 0.2 ml distilled water solution), with a 24-h
interval. At 12 h after the second injection, mitosis was inhibited by injecting
colchicine (10 mg/g body weight). At 4–5 h after colchicine injection, fish
were sacrificed and the head kidneys were removed for leukocyte isolation.
Tissues were then squeezed through a 70-mM nylon cell strainer and the
volume adjusted to 5 ml with PBS. Cells were then centrifuged at 400 3 g for
5 min, 4˚C; the pellet was resuspended in 5 ml 13 PBS, overlaid on 5 ml
Lymphoprep, and centrifuged at 800 3 g for 30 min at room temperature.
Cells were collected from the interface and washed twice with 13 PBS at
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
21, 23–29), virus (16, 24), parasites (30), and LPS (17, 18, 22, 31–
33), in some cases with different isoforms presenting differential
expressions (16–18, 24, 29, 33). Other studies have examined the
effects of iron overload in hepcidin (16–18, 21, 24, 28, 32), showing
its involvement in the control of iron levels in fish. However, studies
concerning other conditions known to affect hepcidin regulation,
such as anemia (28, 34–36) or hypoxia (37), are scarce. Therefore,
currently no comprehensive study addresses the functions of the
two hepcidin types in a single fish species and gives equal attention
to their role in both iron metabolism and immune response.
In this article, we describe several hepcidin genes for the European
sea bass (Dicentrarchus labrax), one hamp1-type and four hamp2type; thoroughly characterize them at the genomic and protein
levels; evaluate variations in their expression when fish are subjected
to different conditions known to affect hepcidin regulation, including iron overload, anemia, infection and hypoxia; and test their
antimicrobial potential.
With this study, we provide an integrative insight into the functions of the different hepcidin genes in a teleost fish, showing that
although the functions described for mammals also persist in teleosts, they are clearly partitioned, with hamp1-type hepcidin mostly
performing the role of iron regulatory hormone and the various
hamp2-type hepcidins being involved in host antimicrobial defense.
2697
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FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
RNA isolation and cDNA synthesis
Total RNA was isolated from tissues with the PureLink RNA Mini Kit protocol
for animal tissues (Invitrogen) with the optional on-column PureLink DNase
treatment (Invitrogen), according to the manufacturer’s instructions. Total
RNA quantification was performed using a NanoDrop 1000 spectrophotometer (Thermo Scientific), and quality was assessed by running the samples in an Experion Automated Electrophoresis Station (Bio-Rad, Hercules,
CA). For all samples, 1.25 mg of each was converted to cDNA by Thermoscript and an oligo(dT) 20 primer (Invitrogen), for 40 min at 50˚C, according
to the manufacturer’s protocol.
Constitutive expression of sea bass hepcidin
Five healthy sea bass were sacrificed, and several tissues were collected for
RNA isolation and cDNA synthesis, as previously described. Relative levels of
hamp1 and all hamp2 mRNAs were quantified by real-time PCR analysis
using an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). A
total of 1 ml of each cDNA sample was added to a reaction mix containing 10
ml iQ SYBR Green Supermix (Bio-Rad), 8.5 ml double distilled H2O, and 250
nM of each primer, making a total volume of 20 ml per reaction. A nontemplate control was included for each set of primers. The cycling profile was
the following: 94˚C for 3.5 min, 40 cycles of 94˚C for 30 s, 59˚C for 30 s, and
72˚C for 30 s. Samples were prepared in duplicates, a melting curve was
generated for every PCR product to confirm the specificity of the assays, and
a dilution series was prepared to check the efficiency of the reactions. b-Actin
was used as the housekeeping gene. The comparative CT method (22DDCT
method) based on cycle threshold values was used to analyze gene expression
levels.
Hepcidin expression in in vivo models of iron overload,
anemia, infection, and hypoxia
To evaluate hepcidin transcriptional regulation in response to different
conditions, several experimental models were created, RNA was isolated
and converted into cDNA, and gene expression analysis was conducted as
previously described for the liver, spleen, and head kidney.
Iron overload. To induce iron overload, fish were i.p. injected with 200 ml iron
dextran (Sigma-Aldrich, St. Louis, MO) diluted in sterile PBS to a final
concentration of 10 mg/ml, as previously reported (40). Control fish were
injected with 200 ml sterile PBS. At 4, 7, and 14 d after treatment, five fish
from each of the experimental groups were anesthetized, and blood was
drawn from the caudal vessels for evaluation of hematological parameters.
Subsequently, fish were euthanized with an overdose of anesthetic and dissected; their tissues were excised, snap frozen in liquid nitrogen, and stored
at 280˚C until further use.
Anemia. To induce the experimental anemia, fish were individually weighted
and bled from the caudal vessels the equivalent v/w of 2% body mass. Control
fish were subjected to the same manipulation (anesthesia, weighting,
pinching), but no blood was removed. At 1, 4, 7, and 14 d after treatment, four
fish from each of the experimental groups were anesthetized, and blood was
drawn from the caudal vessels for evaluation of hematological parameters.
Subsequently, fish were euthanized with an overdose of anesthetic and dissected; their tissues were excised, snap frozen in liquid nitrogen, and stored at
280˚C until further use.
Infection. Photobacterium damselae spp. piscicida, strain PP3, known to be
pathogenic in sea bass, was used for the experimental infections. P. damselae
was cultured to midlogarithmic growth in tryptic soy broth growth medium,
supplemented with 1% NaCl (TSB-1). After measuring absorbance at 600
nm, bacteria were resuspended in TSB-1 to a final concentration of 5.0 3 105
CFUs ml21.
For the experimental infection, fish were anesthetized and i.p. injected with
200 ml (1.0 3 105 CFU) bacterial suspension. For the control group, fish were
injected with 200 ml TSB-1. At 1, 2, 3, 4, and 9 d post infection, six fish from
each group were anesthetized, and blood was drawn from the caudal vessels
for evaluation of hematological parameters. Fish were then euthanized with
an overdose of anesthetic and dissected; their tissues were excised, snap
frozen in liquid nitrogen, and stored at 280˚C until further use. Mortality was
assessed every 12 h during the experimental infection.
Acute hypoxia. To induce experimental hypoxia, fish were maintained in
water with an oxygen concentration of 4.0 mg/l (hypoxia). Oxygen levels in the
water were decreased using an oxygen depletion system, controlled by nitrogen
flow. Surface gas exchange was limited by setting the water inflow under
the water’s surface. Control animals were maintained at 7.4 mg/l (normoxia),
and oxygen levels were maintained by setting water inflow above the water’s
surface. The oxygen levels in each tank were checked every 10 min using an
oxygen sensor, and adjusted when necessary. At 4 h later, four fish from each
group were euthanized with an overdose of anesthetic and dissected; several
tissues were excised, snap frozen in liquid nitrogen, and stored at 280˚C until
further use.
Antimicrobial activity of sea bass hepcidins
The biological activity of sea bass Hamp1 and two different Hamp2 mature
peptides was studied by determining their antimicrobial properties.
Synthetic peptides coding for the predicted mature peptides of Hamp1
(QSHLSLCRWCCNCCRGNKGCGFCCKF), Hamp2.1 (HSSPGGCRFCCNCCPNMSGCGVCCTF), and Hamp2.2 (HSSPGGCRFCCNCCPNMSGCGVCCRF) were commercially produced (Bachem AG, Bubendorf,
Switzerland) and incubated in serial dilutions with four bacterial strains:
P. damselae spp. piscicida (PP3), Vibrio anguillarum (VCO.06.05), Lactococcus
garviae (TPV19.04), and Streptococcus parauberis (CM408.04). In short, 1 3
107 bacteria per milliliter were incubated in optimal growth conditions with the
peptide in flat-bottom 96-well plates and OD read at 600 nm in a plate reader after
24 h of incubation. Wells with no added peptide were used as controls, and wells
without bacteria were used as blanks.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 6 (GraphPad
Software, La Jolla, CA). Data normality was checked by performing the
Kolmogorof–Smirnoff test, and the Student t test was used for estimating
statistical significance. Multiple comparisons were performed with one-way
ANOVA and the post hoc Tukey test. A p value , 0.05 was considered statistically significant.
Results
Molecular characterization of sea bass hepcidins
Several different sea bass hepcidin transcripts were obtained by
PCR amplification and 59/39 RACE using liver, spleen, and head
kidney cDNA. One of the transcripts was identified as being of the
HAMP1-type, with all others being of the HAMP2-type.
Hamp1 coding DNA (deposited in GenBank under accession
number KJ890396) consists of an open reading frame of 273 bp
(Fig. 1), with flanking 59 and 39 untranslated regions of 195 bp and
126 bp, respectively, and encodes a 91 aa preprohepcidin (Fig. 2). A
potential cleavage site for the signal peptide was predicted between
Ala24 and Val25 (SSA/VP) and a motif for the propeptide cleavage
was identified between Arg65 and Gln66 (KR/QS). Thus, Hamp1
prepropeptide consists of a 24-aa signal peptide, a 41-aa prodomain,
and a 26-aa mature peptide. The mature peptide has a predicted m.w.
of 2957 Da and an isoelectric point of 8.32.
All hamp2 coding DNAs (deposited in GenBank under accession
numbers KJ890397, KJ890398, KJ890399, KJ890400) consist of an
open reading frame of 255 bp (Fig. 1), with variable size flanking 59
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800 3 g for 5 min at room temperature. The cell pellet was then resuspended in
0.075 M KCl and incubated for 45 min at room temperature. After hypotonization, cell suspensions were centrifuged at 1500 3 g for 5 min and fixed
with cold, fresh Carnoy’s fluid (3:1 v/v methanol and glacial acetic acid).
After 4 min, the old fixative was replaced with a fresh one, centrifuged as
above, and placed at 220˚C for 10 min, three times. The cells were finally
fixed in 200 ml fresh Carnoy’s fluid and kept at 4˚C for 24 h. Fixed cells were
transferred to a cold glass slide by dropping two to three drops of cell suspension at a distance of ∼70 cm. Glass slides were then air dried at room
temperature for 24 h and used for fluorescence in situ hybridization. Probes
were labeled with DIG. Labeled nucleotides were incorporated in PCR
products using the PCR DIG Probe Synthesis Kit (Roche Applied Science),
with T7 and SP6 primers. For each slide, 30 ml hybridization solution
(containing 2.5 mg each labeled probe, 50% formamide, 10% dextran sulfate,
23 SSC) was added, denatured for 5 min at 73˚C, and allowed to hybridize
for 16–18 h at 37˚C. Posthybridization washes were in 23 SSC/0.05% Tween
20 for 2 min at room temperature, 0.43 SSC for 5 min at 73˚C, and again in
23 SSC/0.05% Tween 20 for 2 min at room temperature, blocked in 5% BSA/
13 PBS for 45 min at room temperature, and incubated with FITCconjugated anti-DIG Ab (Roche Applied Science). Following washes in
33 PBST (PBS, 0.1% Tween 20) for 10 min, 13 PBST for 10 min, and 13
PBS for 10 min, slides were mounted with Mounting Medium with DAPI
(Vectashield; Vector Laboratories, Burlingame, CA). Images were captured
with an Axiocam MR Rev3.0 (Zeiss, Thornwood, NY) camera on an Axio
Imager Z1 epifluorescence microscope (Zeiss) and optimized using AutoQuant 33 (Media Cybernetics, Bethesda, MD).
The Journal of Immunology
2699
and 39 untranslated regions, ranging between 67 and 97 bp and 73 and
143 bp, respectively, and encode 85 aa preprohepcidins (Fig. 2). A
potential cleavage site for the signal peptide was predicted between
Ala24 and Val25 (SSA/VP) and a motif for the propeptide cleavage
was identified between Arg59 and His60 (KR/HS). Thus, Hamp2
prepropeptides consist of a 24 aa signal peptide, a 35 aa prodomain
and a 26 aa mature peptide. The mature peptides have predicted
molecular weights ranging between 2617 and 2729 Da and isoelectric points ranging between 7.50 and 7.85. Alignment between
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. DNA and predicted
amino acid sequence of sea bass
hamp1 and hamp2. Nucleotides are
indicated in the upper row, and amino
acids are indicated in italic in the
lower row. Signal peptide is underlined and mature peptide is boxed.
Intron positions are indicated by
arrows. Polymorphic nucleotides and
amino acids in hamp2 are in bold.
both sea bass hepcidin types revealed several amino acid differences
between Hamp1 and Hamp2, but all mature peptides retain the characteristic eight cysteine residues at conserved positions (Fig. 2).
Identity scores between Hamp1 and Hamp2 ranged from 60.0 to
62.4% and between the different Hamp2 from 95.3 to 98.8%. Furthermore, comparison shows that Hamp1 presents a hypothetical iron
regulatory sequence Q-S/I-H-L/I-S/A-L in the N terminus region of
the mature peptide, whereas the same is lacking from all Hamp2
mature peptides (Fig. 2).
FIGURE 2. Alignment of sea bass hepcidins. Identical amino acids are shaded gray. Signal peptide (SSA/VP) and propeptide (KR/QS and KR/HS) predicted
cleavage sites are underlined and double underlined, respectively. Conserved cysteine residues are indicated by hashes.
2700
FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
Southern blot
To determine the number of copies of each hepcidin in the sea bass
genome, a southern blot analysis was performed. After 5 ug of genomic DNA were independently digested with predicted zero cutter
(EcoRI and HindIII) or single cutter (SstI) restriction enzymes, and
hybridized with hamp1 or hamp2 specific DIG-labeled probes,
different hybridization bands were visible (Fig. 3). For hamp1,
a single band was observed for EcoRI and HindIII, and two bands for
SstI. For hamp2, 4 bands were observed for EcoRI and HindIII, and 6
bands for SstI.
Chromosomal localization of hamp1 and hamp2
Hybridization with a hamp1-specific DIG-labeled probe showed
only two enriched signals in sister chromatids, with similar signal
strength (Fig. 4A–C), whereas with hamp2, twelve enriched signals
were observed, with varying signal strengths (Fig. 4D–F).
Genomic organization
Analysis of hepcidin promoter regions
Several putative cis-regulatory elements were identified in the
flanking 59 regions of the genomic DNA of hamp1 and each hamp2
(Fig. 6), using Possum web server (http://zlab.bu.edu/∼mfrith/
possum/), with additional matrices obtained from the TRANSFAC
database (http://www.gene-regulation.com/pub/databases.html).
Transcription factor binding sites found included TATA-boxes,
GATA, STAT1, STAT4, STAT6, C/EBP a, C/EBP b, AP1, AP4, NF-Y,
and HNF4. All promoters presented almost all transcription factor
binding sites identified, with NF-Y absent from the promoter of
hamp2.3, STAT4 from the promoter of hamp2.4, and GATA from the
promoter of hamp1. In addition, transcription factor binding sites
EF2 and SP1 were found only in the promoter for hamp1.
Sequence comparison and phylogenetic analysis
Sequence comparison with other vertebrate species hepcidins
showed a high degree of conservation (Fig. 7), particularly of the
mature peptide and its characteristic cysteine residues, with Hamp1
sharing an identity with other vertebrate hepcidins between 28 and
94%, Hamp2.1 between 25 and 98%, Hamp2.2 between 25 and
100%, Hamp2.3 between 24 and 95%, and Hamp2.4 between 25 and
98%. Phylogenetic analysis clusters fish hepcidins separated from
other vertebrate hepcidins (Fig. 8), and among them, two separate
clusters are clear. One of the clusters includes type 2 hepcidins from
fish of the Perciformes and Pleuronectiformes orders. The other
cluster includes type 1 hepcidins, and can be divided in two
subclusters, one including fish of the Perciformes and Pleuronectiformes orders, and the other, fish of the Gadiformes, Salmoniformes, Siluriformes, and Cypriniformes orders. D. labrax
Hamp1 clusters with other fish Hamp1 and is closer to S. maximus
and P. olivaceus Hamp, whereas all Hamp2 cluster with other
Hamp2 hepcidins and are closer to M. chrysops Hamp, especially
D. labrax Hamp2.2.
Constitutive expression of sea bass hepcidins
Hepcidin constitutive expression was evaluated in several healthy sea
bass tissues, namely, liver, spleen, head and trunk kidney, pyloric
ceca, stomach, several sections of the intestine, gill, brain, and
leukocytes (Fig. 9). Both hepcidins were detected in all tested tissues, with an overall predominance of hamp1. Hamp1 was found to
have high expression in the liver and leukocytes; moderate in the
pyloric ceca, stomach, midintestine, posterior intestine, gill, and
brain; and low in the spleen, head kidney, trunk kidney, and anterior
intestine. Hamp2 was also found to present the highest expression in
the liver and leukocytes (although not as high as hamp1); moderate
in pyloric ceca, stomach, midintestine, gill, and brain; and low in the
spleen, head kidney, trunk kidney, anterior intestine, and posterior
intestine.
Hepcidin expression in in vivo models
FIGURE 3. Southern blot. To determine the number of hepcidin copies in
the sea bass genome, 5 mg of genomic DNAwas independently digested with
EcoRI, HindIII, or SstI, and hybridized with hamp1– or hamp2–specific
probes. Molecular weights are indicated on the left margin.
Hamp1 and hamp2 mRNA expression levels were evaluated in
response to an assortment of experimental conditions known to
influence hepcidin expression, such as iron overload, anemia, infection with the Gram-negative bacteria P. damselae spp. piscicida
strain PP3, and acute hypoxia (Fig. 10).
Iron overload. In the experimental model of iron overload, hepcidin
expression was evaluated in the liver, spleen, and head kidney 4, 7, and
14 d after overload with i.p. injected iron dextran (Fig. 10A). Hamp1
expression was found to be increased in all tested tissues. In the liver,
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The genomic structure of hamp1 consisted of exons of 87, 78, and
108 bp and introns of 100 and 163 bp (deposited in GenBank under accession number KJ890391) (Fig. 5A). All hamp2 genomic
structures presented same-sized exons of 87, 63, and 105 bp, the first
intron with 99 bp but a variable-sized second intron, ranging
between 173 and 191 bp (deposited in GenBank under accession numbers KJ890392, KJ890393, KJ890394, and KJ890395)
(Fig. 5A). For all hepcidins, exon 1 encodes the signal peptide and
the 59 terminus of the propeptide, exon 2 encodes the middle region
of the propeptide, and exon 3 encodes the 39 terminus of the propeptide and the mature peptide. The introns have high identity
scores, even higher than those for exons, with identity percentages
between hamp1 and hamp2 ranging from 70.0 to 74.5% and between
the different hamp2 from 97.8 to 99.6%.
Genomic organization of sea bass hamp1 and hamp2 was compared with that of other vertebrates and found to be very similar
(Fig. 5B), with the usual organization in three exons and two introns.
Similarities in exon sizes were also observed, particularly with other
perciform fish, but significant variations occur in intron sizes.
Generally, mammalian introns are much larger than fish introns,
and the first intron is larger than the second, but there are some
exceptions.
The Journal of Immunology
2701
hamp1 upregulation peaked at day 4 and gradually decreased toward
control levels up to day 14. Similar patterns of expression were
observed for the spleen and head kidney, with an increase starting
at day 4, reaching maximum levels at day 7, and decreasing or
returning to control levels at day 14, respectively. No significant
changes were observed for hamp2 expression in any of the tested
organs.
Anemia. Hepcidin expression was evaluated in the liver, spleen, and
head kidney of anemic fish, 1, 4, 7, and 14 d after blood was drawn
from the caudal vessels (Fig. 10B). Similar patterns of hamp1
downregulation were shared by the liver and head kidney, with
significant decreases at day 1 and a gradual recovery to control levels
toward day 14. In the spleen, decreased expression was observed at
day 7. No significant changes were observed for hamp2 expression
in any of the tested organs.
Infection. In the experimental model of infection with P. damselae,
hepcidin expression was evaluated in the liver, spleen, and head
kidney at 1, 2, 3, 4, and 9 d post infection (Fig. 10C). Similar patterns
of hamp1 increase were observed in all tissues, although to a much
higher degree in the liver, with significant increases at day 1 and
recovery to control levels at day 2. For hamp2, a massive increase
in expression was observed in the liver at day 1 (.12,000-fold increase), followed by a steady decline but still over 100-fold higher
than control levels at day 9. In the spleen and head kidney, hamp2
expression started to increase at day 1, reached maximum values at
day 2, and recovered to control levels at day 3 in the spleen and more
gradually toward day 9 in the head kidney.
Hypoxia. Hepcidin expression in response to low oxygen levels
was measured in the liver, spleen, and head kidney of fish subjected
to 4 h of acute hypoxia (4.0 mg O2/l) (Fig. 10D). Significant decreases
in hamp1 expression were observed in the liver and head kidney,
with no changes observed in the spleen. In contrast, an opposite
response of hamp2 was observed in the liver, with a significant in-
crease in expression, with no changes observed for the spleen and
head kidney.
Biological activity of synthetic sea bass hepcidins
The antimicrobial activities of sea bass Hamp1, Hamp2.1, and
Hamp2.2 were tested using Gram-positive and Gram-negative
bacteria (Fig. 11).
The synthetic Hamp1 mature peptide did not significantly inhibit
the growth of any of the tested bacteria, up to concentrations of
100 mM. Both synthetic Hamp2.1 and Hamp2.2 mature peptides
significantly affected all strains, with the Gram-positive bacteria
strains being much more susceptible, even at the lowest peptide
concentration tested (0.78125 mM). Furthermore, Hamp2.2 showed
a stronger antimicrobial activity than did Hamp2.1, particularly
against the Gram-negative P. damsela and V. anguillarum. Differences were also observed in the concentrations at which each Hamp2
started to significantly inhibit bacterial growth, with V. anguillarum
being the more resistant strain.
Discussion
In mammals, the small cysteine-rich peptide hepcidin was originally
identified as an antimicrobial peptide (3, 4), but more importantly, it
was later found to be the long-sought key regulator of iron homeostasis (5). Most mammals present only one copy of the hepcidin
gene, with the exception of the mouse, for which recent genome
duplication after the mouse-rat ancestor divergence has been proposed (11, 41). Thus, a single molecule is effectively performing
a dual role, being involved in both the immune response and the
regulation of iron metabolism. However, the same does not apply to
fish, in which multiple copies of the hepcidin gene have been described (16–18, 21, 24, 31, 33), and these can be divided into two
types, hamp1 and hamp2, presumably with different functions (12).
As such, the study of hepcidin in fish can bring new insights into its
evolution and roles.
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FIGURE 4. Chromosomal localization of sea bass hepcidins determined by fluorescent in situ hybridization, using DIG-labeled probes. (A) hamp1 DAPI
channel, (B) hamp1 FITC channel, (C) hamp1 overlapped DAPI/FITC, (D) hamp2 DAPI channel, (E) hamp2 FITC channel, (F) hamp2 overlapped DAPI/FITC.
Chromosomes were blue fluorescence counterstained with DAPI to observe morphology. Arrows indicate hepcidin-enriched areas of the chromosomes.
2702
FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
In this study, we cloned and characterized five hepcidin genes in the
European sea bass D. labrax. Putative protein sequence alignment
and phylogenetic analysis showed that the identified hepcidin genes
could be divided into two distinct groups, with one gene in one group
(HAMP1-type) and the other four in another group (HAMP2-type),
suggesting the possibility of different functions for each hepcidin
type. The fact that a hypothetical iron regulatory sequence Q-S/I-HL/I-S/A-L (42) is present in the N terminus region of the Hamp1
putative mature peptide, but not in any Hamp2, further reinforces the
possibility of different roles for Hamp1 and Hamp2. In addition, it is
clear from the Southern blot analysis that the sea bass genome
presents a single hamp1 gene, contrary to hamp2, for which several
genes are found. Similar results were observed with chromosomal
localization by FISH, where for hamp1 only a pair of signals is
detected in sister chromatids, indicating a single copy, but multiple
signals spread across the karyotype were observed for hamp2,
pointing toward multiple gene copies in different chromosomes,
with varying signal strengths, suggesting the existence of hamp2
gene clusters. This plethora of hepcidin genes in sea bass, of the
HAMP2-type, is likely the result of genome duplications, cumulative mutations, and positive Darwinian selection, previously reported for these antimicrobial peptides and in particular in
Perciformes and Pleuronectiformes fish (13–15). The fact that sea
bass hamp2 genes show a much higher number of single nucleotide
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FIGURE 5. Genomic organization of sea bass hepcidin genes. (A) Exon–
intron diagram of sea bass hamp1 and hamp2 genomic structure. (B) Comparative view with other vertebrate species. Exons are shown as boxes,
introns as solid lines, and untranslated regions as dashed lines, with sizes in
base pairs indicated below.
polymorphisms (SNP) in the mature peptide (7 SNPs) than in the rest
of the prepropeptide (1 SNP), further indicates the functional relevance of the mature peptide active region, as previously discussed
(43).
The genomic organization of both hamp1 and the various hamp2
genes is highly conserved when compared with other fish, amphibian, and mammalian species, sharing their tripartite organization. As with many other teleost fish (25), intron sizes are smaller
than those of other vertebrates, especially mammals, and the second
intron is also generally larger than the first. Furthermore, whereas the
first intron has a conserved size between the different hamp2, the
second intron presents some size variability and is larger than its
hamp1 equivalent. It is possible that during the evolutionary process,
different degrees of insertion or deletion in the intron regions may
have occurred, along with mutations introducing several SNPs;
however, they are still highly conserved not only between the different hamp2 but also between hamp1 and hamp2, probably owing to
a common ancestrality.
Analysis of the genomic sequence upstream of the coding regions
revealed different putative promoter regions for hamp1 and the
various hamp2. Several known mammalian transcription factor
binding sites were also found in sea bass, with the more relevant
being discussed in this article. Binding sites for C/EBPs, STATs, and
HNF4 were found in the promoters of all hepcidins. These are all
known to be required for an effective hepcidin expression and to be
critical for BMP, SMAD1, and HJV responsiveness (44). C/EBPa
and C/EBPb are both potent and weak activators, respectively, of
human and mouse hepcidin promoters (45). Furthermore, it has also
been recently proposed that in conditions of anemia or hypoxia,
erythropoietin (EPO) could act directly to repress hepcidin in hepatocytes through EPO receptor–mediated regulation of the transcription factor C/EBPa (46). STAT has recently been identified as
one of the most important regulators of hepcidin, being essential for
hepcidin expression during inflammatory conditions (47, 48).
Two transcription factors, E2F and SP1, were found only in the
promoter of hamp1. Hepcidin mRNA induction is known to be TLR4
dependent, and, in turn, E2F is recruited by NF-kB upon TLR4
activation (49, 50). SP1 is a common motif in gene promoters, being
involved in many cellular processes, including immune responses,
but its involvement with hepcidin is still mostly unknown. Of interest, no GATA transcription binding factor family members were
found in the hamp1 promoter. One member of this family, GATA-4,
is known to be a likely participant in the control of hepcidin expression, and it is suggested that alterations of its expression could
contribute to the development of iron-related disorders (51). As
such, this variability in the transcription factor binding sites suggests
the possibility of a differential expression in basal conditions and
in response to different stimuli, reinforcing the idea of a subfunctionalization of sea bass hepcidins.
Sequence alignment of the putative proteins with hepcidins from
other vertebrates showed that sea bass hepcidins also have the
characteristic features of hepcidin, such as conserved cleavage sites
for the processing of the propeptide and the mature peptide and eight
cysteine residues in the mature peptide (12). These residues are involved in the formation of disulfide bonds, which give hepcidin the
characteristic hairpin-like structure, believed to be essential for its
activity, but there are reports of some fish hepcidins with as little as
four cysteine residues (24, 52) and still likely to be fully functional.
Phylogenetic analysis clearly shows two different clusters of
hepcidins: fish hepcidins and those of other vertebrates, including
reptiles, amphibians, birds, and mammals. Fish hepcidins can be
further subdivided into two clusters, hamp1-type and hamp2-type
(12), that do not seem to be divided by species of origin (with several fish presenting both hamp1- and hamp2-type hepcidins) and, as
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The Journal of Immunology
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2704
FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
FIGURE 7. Amino acid alignment of sea bass hepcidins with peptides from other vertebrates. Signal peptide (SSA/VP) and propeptide (KR/QS and KR/HS)
predicted cleavage sites are represented by x and #, respectively. Conserved cysteine residues are shaded gray and numbered above. Gaps are indicated by dashes.
The transcriptional response to experimental iron overload of
hamp1 alone, and not hamp2, places the first in the center of iron
regulation and suggests other roles for the latter. The increase in
hamp1 expression is likely to be a response to the elevated iron levels,
to achieve homeostasis and avoid iron toxicity. The liver is known to
be the major contributor to the control of systemic iron metabolism,
producing and releasing vast amounts of hepcidin that will directly
inhibit ferroportin-mediated iron release from hepatocytes, macrophages, and enterocytes (58–60), and, indirectly, due to increased
ferritin levels, reduce or suppress slc11a2-mediated iron uptake by the
enterocytes (61–63). The increased expressions in the spleen and head
kidney are likely to have a low impact on systemic control of iron
levels, rather being more involved in the control of local iron fluxes
because these are the two major organs involved in hematopoiesis in
fish. When compared with other reports of hepcidin expression in
conditions of iron overload, we observe both similar and contradictory
results. Similar results were observed for S. maximus (21), in which
iron overload induced the expression of hamp1 mostly in the liver, but
also somewhat in the head kidney. In P. olivaceus (17), hepcidin JF2 is
also more expressed in response to iron overload in the liver and head
kidney, with some increase in hepcidin JF1 in the liver. Despite their
naming, phylogenetic analysis places hepcidin JF2 in the hamp1
cluster of fish hepcidins and JF1 in the hamp2 cluster.
Hepcidin expression in the experimentally anemic animals mirrors
what is observed in iron overload. A decrease in hepcidin expression
was to be expected (34), but, once again, only hamp1 is found to
respond, with no significant changes in hamp2 expression. Again,
the hamp1 gene alone seems to take up role of iron regulator. The
decreased hepcidin levels would lead to decreased ferroportin
degradation and increased iron release from hepatocytes, macrophages, and enterocytes (58–60), and increased iron uptake by the
enterocytes mediated by slc11a2 (61–64), to allow sufficient iron to
reach the hematopoietic organs for an increased erythropoiesis,
necessary to quickly replenish the lost RBCs and avoid possible
damage caused by hypoxia. Studies on the impact of anemia on
hepcidin expression in fish are scarce, with the exception of our own
previous studies (28, 34) and in Ictalurus punctatus (36). Never-
FIGURE 6. Promoter regions analysis. Putative transcription factor binding sites for the hamp1 promoter and the four different hamp2 promoters were
predicted using the POSSUM Web server, with additional matrices obtained from the TRANSFAC database. The horizontal axis represents upstream distance
from the start codon; the vertical axis represents probability score. Direction is indicated by positioning above (forward) or below (reverse) the horizontal axis.
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such, suggesting that although they share a common ancestor, different hepcidins are likely to have distinct functions. Furthermore,
hamp2-type hepcidins seem to be confined to fish from the Acanthopterygii superorder, whereas the hamp1-type is present in fish of
the Acanthopterygii, Protacanthopterygii, Paracanthopterygii, and
Ostariophysi superorders.
As in many fish species and other vertebrates (3, 4, 16, 17, 19–
21, 24, 25, 27, 28, 53), both sea bass hamp1 and hamp2 are
abundantly expressed in the liver. Of interest, although expression is very low in the spleen, hepcidin is also abundantly
expressed in spleen-derived leukocytes. Further analysis will be
required to determine the exact cells responsible for this expression,
but hepcidin expression has been reported to have an important
role in macrophages, monocytes, and lymphocytes (54–57). Comparatively, hamp1 is more expressed in the majority of the different
tissues, with the exception of the gill and brain, where hamp2 is
predominant. Assuming the higher antimicrobial potential of
Hamp2, it is not surprising to find it highly expressed in the gill,
a tissue in constant contact with the surrounding environment,
many times a doorway to microbial infection. As for the brain,
because the CNS possesses little immunological vigilance, the
bypass of the blood–brain barrier makes it particularly susceptible to infection, and, as such, the ability to rapidly produce
antimicrobial peptides could complement the existing protective
mechanisms.
To better understand the possible roles of each sea bass hepcidin,
expression of both hamp1 and hamp2 was evaluated in several
experimental models, including in vivo models of iron overload,
anemia, hypoxia, and infection.
In the iron-overloaded animals, an increase of hepcidin expression
was observed in all tested tissues, but only for hamp1, with no significant changes for hamp2 genes. We had previously shown an increase in hepcidin expression in response to iron overload (28, 34);
however, in the mentioned studies, the specific upregulated hepcidin
genes were not discriminated. The differential expression between
sea bass hamp1 and hamp2 is presented for the first time, to our
knowledge, in this work.
The Journal of Immunology
2705
theless, it seems clear that hepcidin, and in particular hamp1,
assumes a preponderant role during alterations in the iron status
of sea bass, and most certainly of other teleost fish.
FIGURE 9. Constitutive expression of sea bass
hamp1 and hamp2. Expression was measured
by real-time PCR in several organs and leukocytes of healthy sea bass specimens (n = 5).
A significant induction of both sea bass hepcidin types was observed after experimental bacterial challenge with P. damselae spp.
piscicida, with hamp2 being the most responsive, particularly in the
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FIGURE 8. Phylogenetic analysis. The evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix–based model.
The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding
to partitions reproduced in ,50% bootstrap replicates are collapsed. The initial tree or trees for the heuristic search were obtained by applying the NeighborJoining method to a matrix of pairwise distances estimated using a JTT model. The analysis involved 34-aa sequences. All positions containing gaps and
missing data were eliminated. There were a total of 49 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.
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FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
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FIGURE 10. Sea bass hamp1 and hamp2 expression in in vivo experimental conditions. Expression of hamp1 and hamp2 mRNA was evaluated by
real-time PCR in the liver, spleen, and head kidney of fish subjected to (A) iron overload, in which fish were injected with 2 mg of iron dextran (n = 5); (B) anemia,
in which 2% v/w body mass of blood was removed through the caudal vessels (n = 4); (C) infection with 105 CFU of P. damselae spp. piscicida PP3 (n = 6);
(D) acute hypoxia, in which fish were subjected to low-oxygenated water (4.0 mg O2/l) (n = 4). Values are expressed as mean fold change 6 SD. b-Actin was used
as the housekeeping gene. Differences from the control groups were considered significant at *p , 0.05, **p , 0.01, and ***p , 0.001.
liver, with more modest increases in the spleen and head kidney.
Hamp1 expression was increased only at 24 h post infection,
returning to normal levels at 48 h, in all tested tissues. This shortterm response is most likely related to the need to limit iron availability for pathogen growth in the early stages of infection, as it
occurs in the liver, the major organ of iron storage and release, and
the spleen and head kidney, both organs involved in erythropoiesis,
thus leading in turn to the so-called anemia of inflammation. On the
basis of expression results previously discussed for the iron modulation experiments, no antimicrobial activity or, at the very least, an
extremely reduced one is expected for Hamp1, at least against the
pathogen used for these experiments, but further experiments will
have to be performed to confirm this. Hamp2 expression was highly
increased in the liver in the first days of infection and kept at high
levels throughout the course of the experiment. Once again, when
taking into account the expression results from the iron modulation
experiments, this increase in expression suggests an exclusive antimicrobial role for Hamp2. Modest increases in the spleen and in
head kidney were also observed, but they are not expected to significantly contribute to the overall production of Hamp2 as an
The Journal of Immunology
2707
antimicrobial peptide; rather, they are likely involved in local
mechanisms of antimicrobial protection. This increase in hepcidin expression in the liver, spleen, and head kidney after bacterial exposure by i.p. injection is mostly corroborated (17, 18,
20–22, 25, 27, 31–33, 65, 66) but sometimes contrasted (18, 19,
22, 27) by what was observed in other fish species, but two
findings must be pointed out: although sharing high similarities,
fish hepcidins have nevertheless subtle differences among them
and stimulation of hepcidin is known to be tissue specific,
depending on the fish species; and different pathogens can and most
likely will induce different responses in the expression of each
hepcidin type. There is, however, a shared opinion in all these
studies, which is that each hepcidin type is likely to perform a specific function or functions, depending on the stimuli.
During hypoxic conditions, we observed opposite responses of sea
bass hepcidins, with a decrease in hamp1 expression in the liver and
kidney but, at the same time, an increase in hamp2 expression in the
liver. The full extent of the mechanisms by which hypoxia influences
hepcidin expression in fish is yet unknown, but in mammals, during
hypoxic conditions, there is a need to increase the erythropoietic
drive, and, as such, hepcidin synthesis is inhibited to increase iron
release and absorption. One of the factors involved in erythropoiesis
is EPO, synthesized by the kidney in hypoxic conditions and known
to inhibit hepcidin expression. Pinto et al. (46) have proposed that
EPO mediates hepcidin expression in hepatocytes through EPO
receptor–mediated regulation of the transcription factor C/EBPa,
which is present in the promoters of sea bass hepcidins.
It was recently proposed that EPO can indirectly inhibit hepcidin
expression through the suppression of STAT3 and SMAD4 signaling
(67), two pathways already confirmed by us to be present in sea
bass (34).
However, the major hypoxia-mediated pathway that influences
hepcidin expression involves the HIF1 (hypoxia inducible factor 1),
which has been shown to negatively regulate hepcidin levels by
binding to its promoter (68, 69). In addition, hepcidin repression
due to changes in C/EBPa and STAT-3 activities has also been described in response to the increased levels of reactive oxygen species
that occur in hypoxic conditions (70).
The results in sea bass hepcidin expression may have a plausible
explanation. On the one hand, with hamp1 being more involved in
iron metabolism regulation, its decrease allows for an increased
iron release from hepatocytes and iron absorption in enterocytes,
needed for the additional erythropoietic activity required to counterbalance the effects of hypoxia. On the other hand, with Hamp2
exerting primarily an antimicrobial function, a preventive increase
in hamp2 levels may be beneficial because exposure to hypoxic
conditions is bound to introduce a potential debilitative state in fish,
making them more susceptible to pathogens.
We also investigated the antimicrobial potential of Hamp1 and
two Hamp2 synthetic putative mature peptides against both Gram-
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FIGURE 11. Antimicrobial activity of sea bass synthetic hepcidins. Growth inhibition of P. damselae spp. piscicida, V. anguillarum, S. parauberis, and
L. garviae, incubated for 24 h with serial dilutions of the synthetic Hamp1, Hamp2.1, and Hamp2.2 putative mature peptides. The percentage of CFU was defined
relative to CFU obtained in the control wells. Results are representative of two separate experiments.
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FISH HEPCIDINS: ANTIMICROBIAL PEPTIDES AND IRON REGULATION
Disclosures
The authors have no financial conflicts of interest.
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negative and Gram-positive bacteria. We observed that Hamp1 does
not exhibit significant antimicrobial activity, similarly to what was
reported for P. olivaceus (17). However, a recent study shows that
S. maximus Hamp1 may be able to exert some antimicrobial activity
when the initial pathogen density is very low (,4 3 103 CFU/ml)
(71). Contrary to Hamp1, both Hamp2 peptides exhibited strong
antimicrobial activities, especially against Gram-positive bacteria,
even at the lowest tested dosages. Furthermore, although differing in
only a single amino acid, Hamp2.2 showed a more potent activity
than did Hamp2.1. It is highly likely that other Hamp2 mature
peptides may present differential antimicrobial activity. Similar
Hamp2 antimicrobial activities have been reported for P. olivaceus
(17), M. chrysops 3 M. saxatilis (72), Oreochromis mossambicus
(18), Cynoglossus semilaevis (65), Epinephelus coioides (24),
Pseudosciaena crocea (22), and Acanthopagrus schlegelii (23), with
varying spectrums of activity.
In conclusion, we have characterized two types of hepcidin genes
in the European sea bass (D. labrax). Expression results obtained
from experimental iron modulation, infection, and hypoxia clearly
indicate that the two types of hepcidin have distinct roles, with the
hamp1-type being the major regulator of iron metabolism and the
hamp2-type having a preponderant antimicrobial role. In addition,
only the synthetic Hamp2 putative mature peptides have shown
antimicrobial activity against several bacterial strains. As such,
contrary to its mammalian counterparts, in which hepcidin performs
a dual role in iron regulation and innate immunity, in some teleost
fish these functions are performed by different hepcidin types.
Furthermore, although the importance of hepcidin antimicrobial
function in mammals is limited, the presence of a single iron
metabolism–related type 1 hepcidin and multiple type 2 hepcidins in
sea bass strongly suggests a more significant role as antimicrobial
peptides in fish. In fact, in ectothermic animals such as fish, the innate immune system has an increased importance and the various
type 2 hepcidins could significantly contribute to the early immune
response. Further studies will be required to better understand the
relevance of each type 2 hepcidin and to gain insight into their antimicrobial properties.
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