-
Peptides 36 (2012) 100108
Contents lists available at SciVerse ScienceDirect
Peptides
j our na l ho me p age : www.elsev ier .com
The an ateimmun e
Shang-ChMarine Researc Rd., J
a r t i c l
Article history:Received 13 MReceived in reAccepted 3
ApAvailable onlin
Keywords:Epinecidin-1Immune respoBacterial infecCytokines
whice inve
resuulin G
g e28 dajecte1 (40ays; idin-ction
mice elevated plasma interleukin (IL)-10 to initial peaks at 24
and 48 h, and it showed a second peak at16 days. In RAW264.7 cells,
treatment with epinecidin-1 alone did not produce signicant changes
intumor necrosis factor (TNF)- protein secretion at 1, 6, or 24 h
after treatment with 3.75, 7.5, or 15 g/mlepinecidin-1 compared to
the lipopolysaccharide group.
2012 Elsevier Inc. All rights reserved.
1. Introdu
Infectiouthreats facmultidrug-rlactamase-1given rise tocrobial
peptplants, shripotency anincluding mtionarily anhypothesis tion,
selectibacteria arehost cells dMany AMPbacterial plesting featu
CorresponE-mail add
0196-9781/$ http://dx.doi.oction
s diseases remain some of the most serious healthing the world
[1]. More distressing is the rise ofesistant pathogens such as New
Delhi metallo--
(NDM-1)-producing Enterobacteriaceae [4] which has an urgent
need for novel anti-infective agents. Antimi-ides (AMPs) are
endogenous antibiotics identied frommp, sh, mice, humans, etc.
[2,11,21] that have highd efcacy against a broad spectrum of
pathogens,ultidrug-resistant ones. AMPs are described as
evolu-cient weapons [28], and many studies investigated thethat AMP
cationicity is important for the initial attrac-vity, and
electronegative targeting of pathogens. Most
signicantly more electronegative than neighboringue to intrinsic
structural and physiologic traits [27].s have hydrophobic surfaces
which can permeabilizeasma membranes [8]. As described above, these
inter-res of AMPs have prompted numerous scientists and
ding author. Tel.: +886 920802111; fax: +886 39871035.ress:
[email protected] (J.-Y. Chen).
biotechnological companies to begin development of various
AMPsas potential therapeutics [5,10].
Recently, we identied an AMP from a marine grouper(Epinephelus
coioides) named epinecidin-1, which was shown tobe active against
gram-negative and -positive bacteria, viruses,Candida albicans, and
Trichomonas vaginalis [1316]. In additionto direct antibacterial
functions, epinecidin-1 has another impor-tant ability to regulate
the innate immune system against Vibriovulnicus infection in
zebrash [17]. Epinecidin-1 was reported tomodulate bacterial
infection-induced cytokines and inhibit TNF-expression using an
improved Tol2 transposon system to producetransgenic zebrash with
epinecidin-1 which are resistant to bac-terial infection [20].
Those results suggest epinecidin-1s actionsagainst bacterial
infection need to be more-clearly elucidated, andepinecidin-1
functional studies of immune-related gene modica-tions should be
assessed using in vivo systems such as methods usedto study tilapia
hepcidin 23s effects against V. vulnicus infectionin mice [18].
Although epinecidin-1 showed bactericidal featuresin most in vitro
reports, studying the host response by produc-ing antibodies specic
for antigens produced by bacteria or usingepinecidin-1 in animal
(mice) systems has not been done.
To date, most patients are killed by septic shock within 48 h
aftera Pseudomonas aeruginosa infection [23]. Therapy for P.
aeruginosainfections greatly depends on antibiotic treatment. This
may inducenumerous isolates that illustrate resistance to routinely
applied
see front matter 2012 Elsevier Inc. All rights
reserved.rg/10.1016/j.peptides.2012.04.002timicrobial peptide,
epinecidin-1, medie response to bacterial infection in mic
un Lee, Chieh-Yu Pan, Jyh-Yih Chen
h Station, Institute of Cellular and Organismic Biology,
Academia Sinica, 23-10 Dahuen
e i n f o
arch 2012vised form 3 April 2012ril 2012e 10 April 2012
nsetion
a b s t r a c t
Epinecidin-1, an antimicrobial peptide(Epinephelus coioides). In
this study, wwith Pseudomonas aeruginosa. In vivosignicant
secretion of immunoglobafter injection of 40, 100, 200, or 500mice
treated for 1, 2, 3, 7, 14, 21, and in IgM, IgG, or IgG2a between
mice indays, and 28 days after an epinecidin-increased IgG1 to
peaks at 2 and 3 dpeak at 21 days. This supports epinecproduction)
against P. aeruginosa infe/ locate /pept ides
s secretion of cytokines in the
iaushi, Ilan 262, Taiwan
h encodes 21 amino acids, was isolated from a marine
grouperstigated its immunomodulatory functions in mice
co-injectedlts showed that the synthetic epinecidin-1 peptide
induced1 (IgG1) in mice co-injected with P. aeruginosa.
Moreover,
pinecidin-1/mouse, we detected IgM, IgG, IgG1, and IgG2a inys.
Results showed that there were no signicant differencesd with
epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7
g/mouse) injection. Injection of 500 g
epinecidin-1/mouseinjection of 100 g epinecidin-1/mouse increased
IgG1 to a1 being able to activate the Th2 cell response (enhance
IgG1. Treatment with different concentrations of epinecidin-1
in
-
S.-C. Lee et al. / Peptides 36 (2012) 100108 101
antibiotics for P. aeruginosa infections, such as imipenem,
lev-ooxacin, and gentamicin [9]. Grouper epinecidin-1
displayedmarked in vivo antiviral and antibacterial activities
against exper-imental infections including Japanese encephalitis
virus (JEV),nervous necrosis virus, V. vulnicus, and Riemerella
anatipestifer inexperimental animals [7,13,16,24,25]. Furthermore,
epinecidin-1 issold by Bachem
(http://shop.bachem.com/ep6sf/prodH7228.html)as a product.
Therefore, epinecidin-1 has emerged as a promis-ing agent,
especially against antibiotic-resistant pathogens. Mostresearch
results described epinecidin-1 as possessing antimicrobialactivity
due to its ability to disrupt bacterial membrane integrityand cause
lysis of microorganisms [15]. Those research resultsmentioned above
sparked our interest in studying host immuneresponses against
bacterial infection in mice using epinecidin-1 infurther clinical
applications of grouper epinecidin-1 as a candidatefor an
antimicrobial drug.
To conrm whether epinecidin-1 is clinically valuable as
acandidate for an antimicrobial drug, we evaluated the effects
ofsynthetic epinecidin-1 on mice, by comparing the
antibacterialneutralization efciency, and measuring serum cytokine
levels ofimmunoglobulin G (IgG), IgM, IgG1, IgG2a, interferon
(IFN)-, inter-leukin (IL)-10, IL-12, and others.
2. Materials and methods
2.1. Mice and the bacterial strain
Adult Balb/C mice were purchased from BioLASCO Taiwan(Taipei,
Taiwan) and housed at the Laboratory Animal House(Jiaushi, Taiwan).
Mice were maintained in pathogen-free sterile
Table 1Primer sequences and Tm values listed in this paper.
Primer Sequence (53) Tm
Murine MCP-1 forward AAC TGC ATC TGC CCT AAG GTC TT 55.3Murine
MCP-1 reverse TGC TTG AGG TGG TTG TGG AA 51.8Murine MCP-3 forward
AAG ATC CCC AAG AGG AAT CTC AAG 55.7Murine MCP-3 reverse CAG ACATG
CCC TTC TTT G 54.8Murine MIP-1 forward TCA GAC ACC AGA AGG ATA C
48.9Murine MIP-1 reverse CTG AGA AGA CTT GGT TGC 48Murine TNF-
forward CAA CGG CAT GGA TCT CA 47.1Murine TNF- reverse GGA CTC CGC
AAA GTC T 45.9Murine GAPDH forward TCA TCC CAG AGC TGA ACG
50.3Murine GAPDH reverse GGG AGT TGC TGT TGA AGT C 51.1
isolators according to animal house guidelines. All
experimentscomplied with relevant Laboratory Animal House
guidelines andinstitutional policies. Food, water, caging, and ller
were steril-ized before use in the experiments. P. aeruginosa
culture followeda previous publication without modication [14].
2.2. Injection of epinecidin-1 co-treated with bacteria
orepinecidin-1 alone in mice for an immunological assay
anddetection of antibody titers
Mice were intraperitoneally injected with 0.2 ml of P.
aeruginosaalone (0.2 ml; 106 colony-forming units (cfu)/ml/per
mouse),P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse) mixed with
CpG(10 g/mouse), P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse)
mixedwith epinecidin-1 (40 g/mouse), or medium. The day of
theinjection was designated day 0. Serum was collected on days0, 1,
2, 3, 7, 14, 21, and 28. Mice were re-injected with 0.2 ml
Fig. 1. Epineci domonas aeruginosa antigen. Mice were injected
with PBS alone (Medium),P. aeruginosa a AMP). Then, mice were
re-challenged with P. aeruginosa alone on day 14.Serum was co bulin
M (IgM), IgG, IgG1, and IgG2a antibody titers against the
inactivatedP. aeruginosa a mean value from three determinations,
with the standard error (SE). Data(mean SE) wdin-1 induced the
production of neutralizing antibodies against an inactivated
Pseulone (B), CpG with P. aeruginosa (B + CpG), or P. aeruginosa
with epinecidin-1 (B +llected on days 1, 2, 3, 7, 14, 21, and 28
after the primary injection, and immunoglontigen were determined in
a 96-well plate (n = 3; p < 0.05). Each bar represents theith
different letters signicantly differ (p < 0.05) among
treatments.
-
102 S.-C. Lee et al. / Peptides 36 (2012) 100108
of P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse) on day 14.
Theepinecidin-1 peptide was synthesized by GL Biochem (Shang-hai,
China) using a solid-phase procedure of Fmoc chemistry,the detailed
procedures of which were reported in our previouspublication
[16].
In other trials, mice were injected with 0.2 ml of epinecidin-1
at40, 100, 200, or 500 g/mouse. The day of the injection was
desig-nated day 0, and mice were re-injected with 0.2 ml of each
differentconcentration of epinecidin-1 on day 14. Serum was
collected ondays 0, 1, 2, 3, 7, 14, 21, and 28.
Serum was titrated using inactivated P. aeruginosa
antigen-coated enzyme-linked immunosorbent assay (ELISA) plates
withanti-rabbit IgG-horseradish peroxidase (HRP), IgM-HRP,
IgG2a-HRP, or IgG1-HRP following our previous report with
nomodications [18].
2.3. Detection of cytokine expression levels
To understand cytokine variations after injecting epinecidin-1
co-treated with bacteria or epinecidin-1 alone in mice, wemeasured
the cytokines of IL-4, IL-10, IL-12, IFN-, TNF-, macrophage
inammatory protein (MIP)-1, and monocytechemoattractant protein
(MCP)-1 using ELISA kits (PeproTech, USA,product number: 900-K49,
900-K53, 900-K97, 900-K98, 900-K54,900-K125, and 900-K126,
respectively). To detect variations inMCP-1, MIP-1, and TNF-
transcription (Table 1) and translationlevels after treatment of
RAW264.7 macrophages (ATCC no. TIB-71TM, American Type Culture
Collection, Manassas, VA, USA) withepinecidin-1 in 24-well culture
plates for 1, 6, and 24 h, we applieda real-time polymerase chain
reaction (PCR). Lipopolysaccharide(LPS) (0.1 g/ml) was used to
treat RAW264.7 cells. RNA was
puried using a Qiagen RNeasy Kit (Qiagen, USA).
Reverse-transcription of complementary (c)DNA was performed with
aniScript cDNA Synthesis Kit (Epicentre, USA) according to the
manu-facturers recommendations. A real-time PCR analysis was used
toanalyze gene expressions according to the manufacturers
instruc-tions, and primers are shown in Table 1. SYBR Green
(Toyobo,Japan) and specic primer pairs were used for selected
genes, andprimer pairs for GAPDH were used as the reference gene. A
quanti-tative (q)PCR was performed according to the following
conditions:95 C for 180 s, followed by 45 cycles of 10 s at 95 C,
30 s at 60 C,and 12 s at 72 C. Using 0.5 l cDNA, 2 SYBR Green PCR
buffer,and 500 nM of the forward and reverse primers, the threshold
cyclenumber (Ct) was calculated with ABI software (Applied
Biosystems,USA). Relative transcript quantities were calculated
using the Ctmethod with GAPDH as the reference gene, which was
ampliedfrom the same samples. Ct is the difference in the threshold
cyclesof messenger (m)RNA for selected genes relative to those of
GAPDHmRNA. A real-time PCR was performed in triplicate for each
exper-imental group. Protein secretion were determined by ELISA
kits(PeproTech).
2.4. Glutamic oxaloacetic transaminase (GOT), glutamic
pyruvictransaminase (GPT), and the cell proliferation assay
To detect GOT and GPT, mice were injected with
epinecidin-1co-treated with bacteria or epinecidin-1 alone as
described above.Serum and urea were collected and sent to the
Taiwan MouseClinic (http://tmc.sinica.edu.tw/;
http://tmc.sinica.edu.tw/sopc chemistry.html; Taipei, Taiwan) for
further analysis. Spleencells were collected and centrifuged with
red blood cell lysisbuffer. Leukocyte pellets were collected and
separated on days
Fig. 2. Effect o lone (P. aeruginosa w e on dprimary inject ted
inrepresents the SE) wf Pseudomonas aeruginosa on serum cytokine
levels. Mice were injected with PBS aith epinecidin-1 (B + AMP).
Then, mice were re-challenged with P. aeruginosa alon
ion. Amounts of interleukin (IL)-12, interferon (IFN)-, IL-10,
and IL-4 were estima mean value from three determinations, with the
standard error (SE). Data (mean Medium), P. aeruginosa alone (B),
CpG with P. aeruginosa (B + CpG), oray 14. Serum was collected on
days 1, 2, 3, 7, 14, 21, and 28 after the
a 96-well plate using respective antibodies (see Section 2).
Each barith different letters signicantly differ (p < 0.05)
among treatments.
-
S.-C. Lee et al. / Peptides 36 (2012) 100108 103
14 and 28 from mice as described in the section of Injection
ofepinecidin-1 co-treated with bacteria or epinecidin-1 alone
inmice for the immunological assay and detection of antibody
titers.One hundred thousand cells per milliliter was mixed with 100
lP. aeruginosa (106 cfu/ml) in 100 l of medium that contained
10%serum. To these cells was added 100 l concanavalin A (ConA;10
g/ml, Sigma, St. Louis MO, USA) as a positive control, andcells
were mixed with 100 l of medium as a negative control.Cells were
cultured in a 37 C incubator (5% CO2) for 72 h andassessed using a
CellTiter 96 Aqueous one solution kit to ana-lyze cell
proliferation (Cat. No. G3582, Promega, Madison, WI,USA).
2.5. Statistical analysis
Students t-test was used to graph and analyze the data. p
valuesof
-
104 S.-C. Lee et al. / Peptides 36 (2012) 100108
3.2. Effects of epinecidin-1 and CpG co-treatment with
P.aeruginosa on GOT and GPT in mice
Mouse plasma GOT and GPT levels were used to reect
liverfunction. GOT increased to a peak at 7 days after P.
aeruginosainfection (p < 0.05; Fig. 4). Compared to the P.
aeruginosa group, P.aeruginosa co-treatment with epinecidin-1
increased plasma GOTat 24 h (Fig. 4). Blood GPT reached a peak at
24 h in the group co-treated with P. aeruginosa and epinecidin-1
(Fig. 4). Compared tothe P. aeruginosa group, P. aeruginosa
co-treated with CpG showedincreased plasma GPT at 28 days (Fig.
4).
3.3. Effects of epinecidin-1 on plasma levels of IgM, IgG, IgG1,
andIgG2a
To understand the cytokine and immune responses after inject-ing
40, 100, 200, or 500 g epinecidin-1/mouse, we detected IgM,IgG,
IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28days.
Results showed that there were no signicant differencesin IgM, IgG,
or IgG2a between mice injected with epinecidin-1alone (Fig. 5).
IgG1 increased to peaks at 24 h, 7 days, and 28 days
after the epinecidin-1 (40 g/mouse) injection. Injection of 500
gepinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days;
injec-tion of 100 g epinecidin-1/mouse increased IgG1 to a peak at
21days (Fig. 5). These results support epinecidin-1 being able to
acti-vate the Th2 cell response (enhance IgG1 production) against
P.aeruginosa infection.
3.4. Effects of epinecidin-1 on plasma levels of TNF-, IL-4,
IL-10,IL-12, and IFN-
Epinecidin-1 (200 g/mouse) elevated plasma TNF- at 1and 3 days
(Fig. 6). Then, the level had returned to the basalvalue by 15
days. Epinecidin-1 (200 g/mouse) increased plasmaIL-12 at 3 and 16
days (Fig. 6). Injection of 100 g epinecidin-1/mouse increased
plasma IL-12 at 3 days, and injection of 500 gepinecidin-1/mouse
increased plasma IL-12 at 2 days (Fig. 6).Epinecidin-1 (200
g/mouse) signicantly increased plasma IL-4at 3, 15, 17, and 21 days
(Fig. 6). The other dosage of 40 g/mouseincreased plasma IL-4 at 3,
15, and 16 days (Fig. 6). Then, thelevel had returned to the basal
value by day 21. Treatment withdifferent concentrations of
epinecidin-1 elevated plasma IL-10
Fig. 5. Epinec ent cocollected on d (IgM)plate (n = 3; p <
ndard(p < 0.05) amoidin-1 induced the production of neutralizing
antibodies after injection with differays 1, 2, 3, 7, 14, 21, and
28 after the primary injection, and immunoglobulin M
0.05). Each bar represents the mean value from three
determinations, with the stang treatments.ncentrations (40, 100,
200, or 500 g/mouse) into mice. Serum was, IgG, IgG1, and IgG2a
antibody titers were determined in a 96-well
error (SE). Data (mean SE) with different letters signicantly
differ
-
S.-C. Lee et al. / Peptides 36 (2012) 100108 105
Fig. 6. Effects on days 1, 2, 3estimated in a(mean SE) w
to initial p(Fig. 6).
3.5. Effects expressions
In RAWmacrophagexpressions24 h) and Tment with (15 g/ml) TNF-
mRNepinecidin-6 and 24 hand 7.5 gepinecidin-(Fig. 7b). Coproduce
sigtreated witof epinecidin-1 on serum cytokine levels. Mice were
injected with different concentrations, 7, 15, 16, 17, 21, and 28
after the primary injection. Amounts of interleukin (IL)-12,
int
96-well plate using respective antibodies (see Section 2). Each
bar represents the meanith different letters signicantly differ (p
< 0.05) among treatments.
eaks at 24 and 48 h, and a second peak on day 16
of epinecidin-1 on MCP-1, MIP-1, and TNF-
264.7 cells (morphologically monocytes andes), LPS greatly
elevated MCP-1 mRNA and protein
at 6 and 24 h (Fig. 7a). LPS elevated MIP-1 (6 andNF- (1 and 6
h) mRNA expressions (Fig. 7a). Treat-epinecidin-1 from a low (3.75
g/ml) to a high doseshowed dose-dependent effects on MCP-1, MIP-1,
andA expressions (Fig. 7a). Compared to the LPS group,1 treatment
increased MCP-1 and MIP-1 secretion at
(Fig. 7b). Compared to the group treated with 3.75/ml
epinecidin-1, the group treated with 15 g/ml1 showed signicant
changes in MCP-1 at 1 and 6 hmpared to the LPS group, epinecidin-1
alone did notnicant TNF- changes at 1, 6, or 24 h after being
h 3.75, 7.5, or 15 g/ml epinecidin-1.
4. Discussi
The majepinecidin-quent deteshowed thaThe inductiaeruginosa
epinecidin-CpG or P. splenocytesCpG, AMP, ment was P. aeruginowith
epineon establistreatment win mice indlevels [7]. Aepinecidin- of
epinecidin-1 (40, 100, 200, or 500 g/mouse). Serum was
collectederferon (IFN)-, IL-10, IL-4, and tumor necrosis factor
(TNF)- were
value from three determinations, with the standard error (SE).
Data
on
or ndings of the present study are that synthesized1 co-treated
with P. aeruginosa in mice with subse-ction of antibody titers of
IgG, IgM, IgG1, and IgG2at epinecidin-1 activated Th2 cells against
P. aeruginosa.on level was low possibly because of the low dose of
P.(0.2 ml; 106 cfu/ml/per mouse) in the treatments. Anti-1 IgG was
produced in mice challenged with eitheraeruginosa. Marked cell
proliferation occurred when
from bacterially infected mice were stimulated withor ConA in
vitro. Cell proliferation with ConA treat-higher compared to
stimulation with P. aeruginosa,sa co-treated with CpG, or P.
aeruginosa co-treatedcidin-1. Thus, in agreement with our previous
studyhing an epinecidin-1-based inactivated vaccine, co-ith
epinecidin-1 and Japanese encephalitis virus (JEV)uced higher Th2
cytokine levels than Th1 cytokinentibody isotyping revealed that
induction of IgG1 by1 was through Th2 cells, so it was a humoral
response.
-
106 S.-C. Lee et al. / Peptides 36 (2012) 100108
Fig. 7. Changenecrosis factor15 g/ml. Thethe standard e
Antibody titaeruginosa aagainst thatincreased inat 7 days
serum-neuteration fros in mRNA expressions (a) and serum secretion
(b) of monocyte chemoattractant prot (TNF)- after epinecidin-1
treatment of RAW264.7 cells for 1, 6, and 24 h. Concentration
concentration of lipopolysaccharide (LPS) used to treat RAW264.7
cells was 0.1 g/ml. Error (SE). Data (mean SE) with different
letters signicantly differ (p < 0.05) among trea
ers of IgG, IgM, IgG1, and IgG2a against an inactivated P.ntigen
showed that epinecidin-1 can activate Th2 cells
pathogen. The Th1 cell-responsive cytokine, IL-12, also mice
injected with both epinecidin-1 and P. aeruginosaafter bacterial
re-challenge (Fig. 2). From results ofralizing antibody levels of
IL-10, leukocyte prolif-m the spleen of MCP-1 showed slight
induction,
but MCP-1iments (Fibacterial sing the epiepinecidin-against
bacfunctions.ein (MCP)-1, macrophage inammatory protein (MIP)-1,
and tumors of epinecidin-1 used to treat RAW264.7 cells were 0,
3.75, 7.5, andach bar represents the mean value from three
determinations, withtments. *p < 0.05, **p < 0.01, and ***p
< 0.001.
induction was not seen after re-challenge exper-g. 3). Recently,
enhancement of resistance againsttrains was studied in transgenic
sh overexpress-necidin-1 peptide [20]. Those results suggested
that1 has direct antimicrobial and bacteriostatic activitiesterial
infection and also possesses immunomodulatory
-
S.-C. Lee et al. / Peptides 36 (2012) 100108 107
Endotoxic shock is generally associated with the
systemicinammatory response and causes multiple organ failure
[22].Pretreatment with epinecidin-1 decreased TNF- gene
expressionafter bacterial infection [14]. In this study,
co-treatment with P.aeruginosa GOT and Gconcentratiicant
differantibiotics (Epinecidin-after virus 1 attenuateIgG1 after
organ failurels of GPT ainjection ofsignicant rthat epinecas
post-treain plasma Gmean arterThus, epineliver againsseveral
othe
The imming a host viruses, partant early refcacy of Ias adjunct
tIgM and Igand IgG2a aresults suggtion by moepinecidin-temic immuan
importantory processmight modinjection ofproduction tion. We
fotreating theinfection-ining CpG- orcould strenas IL-12. Re4 and
IL-10might induof both Th1epinecidin-wagandha,
In suma signicanour study epinecidin-tory effects.epinecidin-our
results epinecidin-
Acknowled
Researchtion, InstituJiaushi, Ilan
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in
the online version, at http://dx.doi.org/10.1016/
des.2
nces
cher s, no erica.valhoiologi1;17:plin 0;125obo-Prel L,
pneumherichcock i-infec
BG, Leliora6;79:ang H
of im esta1;32:tsuzaks Actaura S
tors fopenemokherulator7;64:aan bial
p2;33:rby-T-enrielatio6;260
CY, n and inephe. DNA
CY, naeus udom700
CY, s derpionib9;30:
CY, s of a
letha15.
CY, imminst 1;31:
CY, L immnicusdey Rune
g KC, roducacteranbabspectidemanulatio202ugott ombiherapng
YDctingimicro
Sheland epinecidin-1 attenuated the increases in plasmaPT after
7 days in mice (Fig. 4). Injection of differentons of epinecidin-1
alone in mice presented no signif-ences in GOT or GPT values
compared to injection of4 and 10 mg) from days 1 to 15
(Supplementary Fig. S2).1 also alleviated histopathologic changes
in the braininfection [7]. Accordingly, treatment with epinecidin-d
the LPS-induced organ damage with an increase inendotoxic shock. In
addition, the criteria for deninge in patients with septic shock
are increased serum lev-nd GOT, which suggest liver cell injury.
Interestingly,
epinecidin-1 alone in mice (this study) did not result inises in
serum levels of GPT or GOT. Our ndings suggestidin-1 has a function
similar to that of N-acetylcysteine,tment with N-acetylcysteine
attenuated the increasesOT and GPT after an LPS injection and
increased the
ial pressure and heart rate after endotoxic shock [6].cidin-1
treatment of mice might involve protecting thet LPS-induced
dysfunction in and preventing damage tor organs in septic mice.une
system is an important defensive system protect-from various
pathogenic infections such as bacteria,asites, and so on [3]. In
particular, IgM plays an impor-ole in the course of an infection.
In most trials, thegM-enriched intravenous polyclonal
immunoglobulinherapy in sepsis was reported [12]. We observed thatG
levels were signicantly higher compared to IgG1fter injecting
epinecidin-1 alone in mice (Fig. 5). Ourest that epinecidin-1
stimulates IgM and IgG produc-use splenic lymphocytes in the
absence of LPS. Thus,1 might alleviate inammatory reactions in the
sys-ne system of the mouse. The cytokine network playst role in
immune responses and subsequent inamma-es [19]. We demonstrated
that injection of epinecidin-1ulate cytokine production in splenic
lymphocytes. An
epinecidin-1 (Fig. 6) stimulated IL-12, IL-4, and IL-10to a
great extent, but did not affect TNF- produc-und increased cytokine
production by Th2 cells afterm with epinecidin-1, even in the
presence of bacterialduced inammation. These data suggest that
treat-
P. aeruginosa-induced lymphocytes with epinecidin-1gthen the
immune system by regulating cytokine suchgulation of Th2-related
cytokine production, such as IL-, by injecting epinecidin-1
suggests that epinecidin-1ce an immune-related balance toward the
production
and Th2 cytokines by splenic lymphocytes, so that1 possesses a
balancing function such as Echinacea, Ash-and Brahmi [26].mary, the
administration of epinecidin-1 inducedt increase in the serum IgG
level. Furthermoresuggests the possibility of clinical applications
of1 in mammalian systems based on its immunoregula-
Although the mechanism through which amino acids in1 modify
immune indices by receptors is not yet known,point to a new
possibility for a pharmacological role of1.
gment
funding was obtained from the Marine Research Sta-te of Cellular
and Organismic Biology, Academia Sinica,, Taiwan.
j.pepti
Refere
[1] BoubugAm
[2] Car b201
[3] Cha201
[4] DocPoital Esc
[5] Hanant
[6] Hsuam200
[7] Hutionand201
[8] Maphy
[9] Mikfacimi
[10] Moreg200
[11] Najcro201
[12] NorIgMin r200
[13] Pansio(Eption
[14] Pan(PePse687
[15] PantidePro200
[16] Pantidethe806
[17] Panandaga201
[18] Panandvul
[19] Panimm
[20] Pento pto b
[21] Rajper
[22] RieReg193
[23] Traor ccot
[24] WainfeantFish012.04.002.
HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et
al. Baddrugs: no ESKAPE! An update from the Infectious Diseases
Society of
Clin Infect Dis 2009;48:112. Ade O, Gomes VM. Plant defensins
and defensin-like peptidescal activities and biotechnological
applications. Curr Pharm Des427093.DD. Overview of the immune
response. J Allergy Clin Immunol:S323.rez F, Nordmann P,
Domnguez-Herrera J, Lpez-Rojas R, Smani Y,et al. Efcacies of
colistin and tigecycline in mice with experimen-onia due to
NDM-1-producing strains of Klebsiella pneumoniae and
ia coli. Int J Antimicrob Agents 2012;39:2514.RE, Sahl HG.
Antimicrobial and host-defense peptides as newtive therapeutic
strategies. Nat Biotechnol 2006;24:15517.ee RP, Yang FL, Harn HJ,
Chen HI. Post-treatment with N-acetylcysteinetes endotoxin
shock-induced organ damage in conscious rats. Life Sci20106.N, Pan
CY, Rajanbabu V, Chan YL, Wu CJ, Chen JY. Modula-mune responses by
the antimicrobial peptide, epinecidin (Epi)-1,
blishment of an Epi-1-based inactivated vaccine.
Biomaterials362736.i K. Control of cell selectivity of
antimicrobial peptides. Biochim Bio-
2009;1788:168792., Wada H, Okazaki M, Nakamura M, Honda K,
Yasutake T, et al. Riskr bacteraemia attributable to Pseudomonas
aeruginosa resistant to, levooxacin, or gentamicin. J Hosp Infect
2011;79:2678.
jee N, Hancock RE. Cationic host defence peptides: innate
immuney peptides as a novel approach for treating infections. Cell
Mol Life Sci92233.L, Babji AS. A review of sh-derived antioxidant
and antimi-eptides: their production, assessment, and applications.
Peptides17885.eglund A, Haque KN, Hammarstrm L. Intravenous
polyclonalched immunoglobulin therapy in sepsis: a review of
clinical efcacyn to microbiological aetiology and severity of
sepsis. J Intern Med:50916.Chen JY, Cheng YS, Chen CY, Ni IH, Sheen
JF, et al. Gene expres-localization of the epinecidin-1
antimicrobial peptide in the grouperlus coioides), and its role in
protecting sh against pathogenic infec-
Cell Biol 2007;26:40313.Chao TT, Chen JC, Chen JY, Liu WC, Lin
CH, et al. Shrimpmonodon) anti-lipopolysaccharide factor reduces
the lethality ofonas aeruginosa sepsis in mice. Int Immunopharmacol
2007;7:.Chen JY, Lin TL, Lin CH. In vitro activities of three
synthetic pep-ived from epinecidin-1 and an anti-lipopolysaccharide
factor againstacterium acnes, Candida albicans, and Trichomonas
vaginalis. Peptides105868.Chow TY, Yu CY, Yu CY, Chen JC, Chen JY.
Antimicrobial pep-n anti-lipopolysaccharide factor, epinecidin-1,
and hepcidin reducelity of Riemerella anatipestifer sepsis in
ducks. Peptides 2010;31:
Wu JL, Hui CF, Lin CH, Chen JY. Insights into the
antibacterialunomodulatory functions of the antimicrobial peptide,
epinecidin-1,Vibrio vulnicus infection in zebrash. Fish Shellsh
Immunol101925.ee SC, Rajanbabu V, Lin CH, Chen JY. Insights into
the antibacterialunomodulatory functions of tilapia hepcidin (TH)23
against Vibrio
infection in mice. Dev Comp Immunol 2012;36:16673., Mousawy K,
Nagarkatti M, Nagarkatti P. Endocannabinoids andregulation.
Pharmacol Res 2009;60:8592.Pan CY, Chou HN, Chen JY. Using an
improved Tol2 transposon systeme transgenic zebrash with
epinecidin-1 which enhanced resistanceial infection. Fish Shellsh
Immunol 2010;28:90517.u V, Chen JY. Applications of antimicrobial
peptides from sh andves for the future. Peptides 2011;32:41520.n
NC, Guo RF, Bernacki KD, Reuben JS, Laudes IJ, Neff TA, et al.n by
C5a of neutrophil activation during sepsis. Immunity 2003;19:.KA,
Echevarria K, Maxwell P, Green K, Lewis 2nd JS. Monotherapynation
therapy? The Pseudomonas aeruginosa conundrum. Pharma-y
2011;31:598608., Kung CW, Chi SC, Chen JY. Inactivation of nervous
necrosis virus
grouper (Epinephelus coioides) by epinecidin-1 and hepcidin
15bial peptides, and downregulation of Mx2 and Mx3 gene
expressions.
lsh Immunol 2010;28:11320.
-
108 S.-C. Lee et al. / Peptides 36 (2012) 100108
[25] Wang YD, Kung CW, Chen JY. Antiviral activity by sh
antimicrobial peptidesof epinecidin-1 and hepcidin 15 against
nervous necrosis virus in medaka.Peptides 2010;31:102633.
[26] Yamada K, Hung P, Park TK, Park PJ, Lim BO. A comparison of
the immunostim-ulatory effects of the medicinal herbs Echinacea,
Ashwagandha and Brahmi. JEthnopharmacol 2011;137:2315.
[27] Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide
action and resis-tance. Pharmacol Rev 2003;55(1):2755.
[28] Zasloff M. Antimicrobial peptides of multicellular
organisms. Nature2002;415:38995.