Peptides 36 (2012) 100108

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Peptides
j our nal home page: www. el sevi er . com/ l ocat e/ pept i des
The antimicrobial peptide, epinecidin-1, mediates secretion of cytokines in the
immune response to bacterial infection in mice
Shang-Chun Lee, Chieh-Yu Pan, Jyh-Yih Chen

Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Rd., Jiaushi, Ilan 262, Taiwan
a r t i c l e i n f o
Article history:
Received 13 March 2012
Received in revised form3 April 2012
Accepted 3 April 2012
Available online 10 April 2012
Keywords:
Epinecidin-1
Immune response
Bacterial infection
Cytokines
a b s t r a c t
Epinecidin-1, an antimicrobial peptide which encodes 21 amino acids, was isolated from a marine grouper
(Epinephelus coioides). In this study, we investigated its immunomodulatory functions in mice co-injected
with Pseudomonas aeruginosa. In vivo results showed that the synthetic epinecidin-1 peptide induced
signicant secretion of immunoglobulin G1 (IgG1) in mice co-injected with P. aeruginosa. Moreover,
after injection of 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 28 days. Results showed that there were no signicant differences
in IgM, IgG, or IgG2a between mice injected with epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7
days, and 28 days after an epinecidin-1 (40 g/mouse) injection. Injection of 500 g epinecidin-1/mouse
increased IgG1 to peaks at 2 and 3 days; injection of 100 g epinecidin-1/mouse increased IgG1 to a
peak at 21 days. This supports epinecidin-1 being able to activate the Th2 cell response (enhance IgG1
production) against P. aeruginosa infection. Treatment with different concentrations of epinecidin-1 in
mice elevated plasma interleukin (IL)-10 to initial peaks at 24 and 48 h, and it showed a second peak at
16 days. In RAW264.7 cells, treatment with epinecidin-1 alone did not produce signicant changes in
tumor necrosis factor (TNF)-protein secretion at 1, 6, or 24 h after treatment with 3.75, 7.5, or 15 g/ml
epinecidin-1 compared to the lipopolysaccharide group.
2012 Elsevier Inc. All rights reserved.
1. Introduction
Infectious diseases remain some of the most serious health
threats facing the world [1]. More distressing is the rise of
multidrug-resistant pathogens such as New Delhi metallo--
lactamase-1 (NDM-1)-producing Enterobacteriaceae [4] which has
given rise to an urgent need for novel anti-infective agents. Antimi-
crobial peptides (AMPs) are endogenous antibiotics identied from
plants, shrimp, sh, mice, humans, etc. [2,11,21] that have high
potency and efcacy against a broad spectrum of pathogens,
including multidrug-resistant ones. AMPs are described as evolu-
tionarily ancient weapons [28], and many studies investigated the
hypothesis that AMP cationicity is important for the initial attrac-
tion, selectivity, and electronegative targeting of pathogens. Most
bacteria are signicantly more electronegative than neighboring
host cells due to intrinsic structural and physiologic traits [27].
Many AMPs have hydrophobic surfaces which can permeabilize
bacterial plasma membranes [8]. As described above, these inter-
esting features of AMPs have prompted numerous scientists and

Corresponding author. Tel.: +886 920802111; fax: +886 39871035.

E-mail address: zoocjy@gate.sinica.edu.tw(J.-Y. Chen).
biotechnological companies to begin development of various AMPs
as potential therapeutics [5,10].
Recently, we identied an AMP from a marine grouper
(Epinephelus coioides) named epinecidin-1, which was shown to
be active against gram-negative and -positive bacteria, viruses,
Candida albicans, and Trichomonas vaginalis [1316]. In addition
to direct antibacterial functions, epinecidin-1 has another impor-
tant ability to regulate the innate immune system against Vibrio
vulnicus infection in zebrash [17]. Epinecidin-1 was reported to
modulate bacterial infection-induced cytokines and inhibit TNF-
expression using an improved Tol2 transposon systemto produce
transgenic zebrash with epinecidin-1 which are resistant to bac-
terial infection [20]. Those results suggest epinecidin-1s actions
against bacterial infection need to be more-clearly elucidated, and
epinecidin-1 functional studies of immune-related gene modica-
tions shouldbeassessedusinginvivosystems suchas methods used
to study tilapia hepcidin 23s effects against V. vulnicus infection
in mice [18]. Although epinecidin-1 showed bactericidal features
in most in vitro reports, studying the host response by produc-
ing antibodies specic for antigens produced by bacteria or using
epinecidin-1 in animal (mice) systems has not been done.
Todate, most patients are killedby septic shockwithin48hafter
a Pseudomonas aeruginosa infection [23]. Therapy for P. aeruginosa
infections greatlydepends onantibiotic treatment. This mayinduce
numerous isolates that illustrate resistance to routinely applied
0196-9781/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.peptides.2012.04.002
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 displayed
marked in vivo antiviral and antibacterial activities against exper-
imental infections including Japanese encephalitis virus (JEV),
nervous necrosis virus, V. vulnicus, and Riemerella anatipestifer in
experimental animals [7,13,16,24,25]. Furthermore, epinecidin-1is
sold 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. Most
researchresults describedepinecidin-1as possessing antimicrobial
activity due to its ability to disrupt bacterial membrane integrity
and cause lysis of microorganisms [15]. Those research results
mentioned above sparked our interest in studying host immune
responses against bacterial infection in mice using epinecidin-1 in
further clinical applications of grouper epinecidin-1 as a candidate
for an antimicrobial drug.
To conrm whether epinecidin-1 is clinically valuable as a
candidate for an antimicrobial drug, we evaluated the effects of
synthetic epinecidin-1 on mice, by comparing the antibacterial
neutralization efciency, and measuring serum cytokine levels of
immunoglobulinG(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 1
Primer sequences and Tmvalues listed in this paper.
Primer Sequence (5

) Tm
Murine MCP-1 forward AAC TGC ATC TGC CCT AAG GTC TT 55.3
Murine MCP-1 reverse TGC TTG AGG TGG TTG TGG AA 51.8
Murine MCP-3 forward AAG ATC CCC AAG AGG AAT CTC AAG 55.7
Murine MCP-3 reverse CAG ACATG CCC TTC TTT G 54.8
Murine MIP-1 forward TCA GAC ACC AGA AGG ATA C 48.9
Murine MIP-1 reverse CTG AGA AGA CTT GGT TGC 48
Murine TNF- forward CAA CGG CAT GGA TCT CA 47.1
Murine TNF- reverse GGA CTC CGC AAA GTC T 45.9
Murine GAPDH forward TCA TCC CAG AGC TGA ACG 50.3
Murine GAPDH reverse GGG AGT TGC TGT TGA AGT C 51.1
isolators according to animal house guidelines. All experiments
complied with relevant Laboratory Animal House guidelines and
institutional policies. Food, water, caging, and ller were steril-
ized before use in the experiments. P. aeruginosa culture followed
a previous publication without modication [14].
2.2. Injection of epinecidin-1 co-treated with bacteria or
epinecidin-1 alone in mice for an immunological assay and
detection of antibody titers
Mice were intraperitoneally injected with0.2ml of P. aeruginosa
alone (0.2ml; 10
6
colony-forming units (cfu)/ml/per mouse),
P. aeruginosa (0.2ml; 10
6
cfu/ml/per mouse) mixed with CpG
(10g/mouse), P. aeruginosa (0.2ml; 10
6
cfu/ml/per mouse) mixed
with epinecidin-1 (40g/mouse), or medium. The day of the
injection was designated day 0. Serum was collected on days
0, 1, 2, 3, 7, 14, 21, and 28. Mice were re-injected with 0.2ml
Fig. 1. Epinecidin-1 induced the production of neutralizing antibodies against an inactivated Pseudomonas aeruginosa antigen. Mice were injected with PBS alone (Medium),
P. aeruginosa alone (B), CpG with P. aeruginosa (B+CpG), or P. aeruginosa with epinecidin-1 (B+AMP). Then, mice were re-challenged with P. aeruginosa alone on day 14.
Serumwas collected on days 1, 2, 3, 7, 14, 21, and 28 after the primary injection, and immunoglobulin M(IgM), IgG, IgG1, and IgG2a antibody titers against the inactivated
P. aeruginosa antigen were determined in a 96-well plate (n=3; p<0.05). Each bar represents the mean value fromthree determinations, with the standard error (SE). Data
(mean SE) with different letters signicantly differ (p<0.05) among treatments.
102 S.-C. Lee et al. / Peptides 36 (2012) 100108
of P. aeruginosa (0.2ml; 10
6
cfu/ml/per mouse) on day 14. The
epinecidin-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 previous
publication [16].
In other trials, mice were injected with 0.2ml of epinecidin-1 at
40, 100, 200, or 500g/mouse. The day of the injection was desig-
nated day 0, and mice were re-injected with0.2ml of eachdifferent
concentration of epinecidin-1 on day 14. Serum was collected on
days 0, 1, 2, 3, 7, 14, 21, and 28.
Serum was titrated using inactivated P. aeruginosa antigen-
coated enzyme-linked immunosorbent assay (ELISA) plates with
anti-rabbit IgG-horseradish peroxidase (HRP), IgM-HRP, IgG2a-
HRP, or IgG1-HRP following our previous report with no
modications [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, we
measured the cytokines of IL-4, IL-10, IL-12, IFN-, TNF-
, macrophage inammatory protein (MIP)-1, and monocyte
chemoattractant protein(MCP)-1using ELISAkits (PeproTech, USA,
product number: 900-K49, 900-K53, 900-K97, 900-K98, 900-K54,
900-K125, and 900-K126, respectively). To detect variations in
MCP-1, MIP-1, and TNF- transcription (Table 1) and translation
levels after treatment of RAW264.7 macrophages (ATCC no. TIB-
71
TM
, American Type Culture Collection, Manassas, VA, USA) with
epinecidin-1 in 24-well culture plates for 1, 6, and 24h, we applied
a real-time polymerase chain reaction (PCR). Lipopolysaccharide
(LPS) (0.1g/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 an
iScript cDNA Synthesis Kit (Epicentre, USA) according to the manu-
facturers recommendations. A real-time PCR analysis was used to
analyze 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, and
primer pairs for GAPDHwere used as the reference gene. A quanti-
tative (q)PCRwas performedaccording to the following conditions:
95

C for 180s, followed by 45 cycles of 10s at 95

C, 30s at 60

C,
and 12s at 72

C. Using 0.5l cDNA, 2 SYBR Green PCR buffer,

and 500nMof the forward and reverse primers, the threshold cycle
number (Ct) was calculatedwithABI software (AppliedBiosystems,
USA). Relative transcript quantities were calculated using the Ct
method with GAPDH as the reference gene, which was amplied
fromthe same samples. Ct is the difference inthe thresholdcycles
of messenger (m)RNAfor selected genes relative to those of GAPDH
mRNA. 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 pyruvic
transaminase (GPT), and the cell proliferation assay
To detect GOT and GPT, mice were injected with epinecidin-1
co-treated with bacteria or epinecidin-1 alone as described above.
Serum and urea were collected and sent to the Taiwan Mouse
Clinic (http://tmc.sinica.edu.tw/; http://tmc.sinica.edu.tw/sop
c chemistry.html; Taipei, Taiwan) for further analysis. Spleen
cells were collected and centrifuged with red blood cell lysis
buffer. Leukocyte pellets were collected and separated on days
Fig. 2. Effect of Pseudomonas aeruginosa on serumcytokine levels. Mice were injected with PBS alone (Medium), P. aeruginosa alone (B), CpG with P. aeruginosa (B+CpG), or
P. aeruginosa with epinecidin-1 (B+AMP). Then, mice were re-challenged with P. aeruginosa alone on day 14. Serumwas collected on days 1, 2, 3, 7, 14, 21, and 28 after the
primary injection. Amounts of interleukin (IL)-12, interferon (IFN)-, IL-10, and IL-4 were estimated in a 96-well plate using respective antibodies (see Section 2). Each bar
represents the mean value fromthree determinations, with the standard error (SE). Data (meanSE) with 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 of
epinecidin-1 co-treated with bacteria or epinecidin-1 alone in
mice for the immunological assay and detection of antibody titers.
One hundred thousand cells per milliliter was mixed with 100l
P. aeruginosa (10
6
cfu/ml) in 100l of mediumthat contained 10%
serum. To these cells was added 100l concanavalin A (ConA;
10g/ml, Sigma, St. Louis MO, USA) as a positive control, and
cells were mixed with 100l of medium as a negative control.
Cells were cultured in a 37

C incubator (5% CO
2
) for 72h and
assessed 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 values
of <0.05 were considered to indicate statistical signicance.
3. Results
3.1. Effects of epinecidin-1 on the immune response against P.
aeruginosa infection
To understand cytokine and immune responses against P.
aeruginosa infection and in re-challenge P. aeruginosa experiments,
Fig. 3. Effect of epinecidin-1 on Pseudomonas aeruginosa infection-mediated induc-
tion of splenic antigen-presenting cells. Mice were injected with PBS alone
(Medium), P. aeruginosa alone (B), CpG with P. aeruginosa (B+CpG), or P. aeruginosa
withepinecidin-1(B+AMP). Then, mice were re-challengedwithP. aeruginosa alone
on day 14. The spleen was collected on days 14 (co-culture) and 28 (re-challenge),
and (A) lysed with RBC lysis buffer, and leukocyte pellets were cultured in RPMI
medium. Cell proliferation was estimated after 24h. (B) Monocyte chemoattrac-
tant protein (MCP)-1 secretion in the cell culture supernatant was measured by an
ELISA. Eachbar represents the meanvalue fromthree determinations, withthe stan-
dard error (SE). Data (meanSE) with different letters signicantly differ (p<0.05)
among treatments.
we detected IgM, IgG, IgG1, and IgG2a in mice treated for 1, 2, 3,
7, 14, 21, and 28 days. Results showed no signicant difference
between mice injected with P. aeruginosa alone and those injected
with both P. aeruginosa and epinecidin-1 (Fig. 1). On day 3, mice co-
treated with P. aeruginosa and epinecidin-1 showed a signicant
difference in IgG2a compared to the other experimental groups.
These data showthat the induction of bacterium-neutralizing anti-
bodies was similar between these two conditions (P. aeruginosa
and epinecidin-1 vs. P. aeruginosa and CpG). Next, to understand
Th1 and Th2 cell activation, neutralization against IgG1 and IgG2a
isotypes was separately determined. Compared to IgG2a, IgG1 acti-
vation in serum after epinecidin-1 co-injected with P. aeruginosa
was higher than that in P. aeruginosa-injected mice on days 14 and
28 after infection (Fig. 1). This shows that epinecidin-1 can activate
the Th2cell response against P. aeruginosa infection. The level of the
Th1-activated cytokine, IL-12, was also induced in serum after an
epinecidin-1 plus P. aeruginosa injectioncomparedto mice injected
with P. aeruginosa alone on day 21 (Fig. 2). In contrast, neither
the Th1 cell-associated cytokine, IFN-, nor the Th2 cell-associated
cytokine, IL-4, showed signicant elevation (Fig. 2). These data
showthat epinecidin-1 can activate Th2 cells against P. aeruginosa
infection in mice. Spleen cells were cultured, and the survival rate
of spleen cells was calculated by an MTS assay. There was no differ-
ence in cell survival rates between mice injected with P. aeruginosa
and those injected with both epinecidin-1 and P. aeruginosa after
the primary infection (Fig. 3). To determine whether monocytes,
T-lymphocytes, and macrophages were attracted to the spleen, we
studied the chemokine, MCP-1. Surprisingly, there was little dif-
ference in levels of MCP-1 between cells of mice injected with
P. aeruginosa and those injected with both epinecidin-1 and P.
aeruginosa (Fig. 3). This shows that epinecidin-1 only induced a
weak immune response against P. aeruginosa.
Fig. 4. Changes in serumaspartate transferase (GOT) and alanine transferase (GPT)
after an injection with PBS alone (Medium), Pseudomonas aeruginosa alone (B), CpG
with P. aeruginosa (B+CpG), or P. aeruginosa with epinecidin-1 (B+AMP) into mice.
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 liver
function. GOT increased to a peak at 7 days after P. aeruginosa
infection (p<0.05; Fig. 4). Compared to the P. aeruginosa group, P.
aeruginosa co-treatment with epinecidin-1 increased plasma GOT
at 24h (Fig. 4). Blood GPT reached a peak at 24h in the group co-
treated with P. aeruginosa and epinecidin-1 (Fig. 4). Compared to
the P. aeruginosa group, P. aeruginosa co-treated with CpG showed
increased plasma GPT at 28 days (Fig. 4).
3.3. Effects of epinecidin-1 on plasma levels of IgM, IgG, IgG1, and
IgG2a
To understand the cytokine and immune responses after inject-
ing 40, 100, 200, or 500g epinecidin-1/mouse, we detected IgM,
IgG, IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28
days. Results showed that there were no signicant differences
in IgM, IgG, or IgG2a between mice injected with epinecidin-1
alone (Fig. 5). IgG1 increased to peaks at 24h, 7 days, and 28 days
after the epinecidin-1 (40g/mouse) injection. Injection of 500g
epinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days; injec-
tion of 100g epinecidin-1/mouse increased IgG1 to a peak at 21
days (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 (200g/mouse) elevated plasma TNF- at 1
and 3 days (Fig. 6). Then, the level had returned to the basal
value by 15 days. Epinecidin-1 (200g/mouse) increased plasma
IL-12 at 3 and 16 days (Fig. 6). Injection of 100g epinecidin-
1/mouse increased plasma IL-12 at 3 days, and injection of 500g
epinecidin-1/mouse increased plasma IL-12 at 2 days (Fig. 6).
Epinecidin-1 (200g/mouse) signicantly increased plasma IL-4
at 3, 15, 17, and 21 days (Fig. 6). The other dosage of 40g/mouse
increased plasma IL-4 at 3, 15, and 16 days (Fig. 6). Then, the
level had returned to the basal value by day 21. Treatment with
different concentrations of epinecidin-1 elevated plasma IL-10
Fig. 5. Epinecidin-1 induced the production of neutralizing antibodies after injection with different concentrations (40, 100, 200, or 500g/mouse) into mice. Serum was
collected on days 1, 2, 3, 7, 14, 21, and 28 after the primary injection, and immunoglobulin M (IgM), IgG, IgG1, and IgG2a antibody titers were determined in a 96-well
plate (n=3; p<0.05). Each bar represents the mean value fromthree determinations, with the standard error (SE). Data (meanSE) with different letters signicantly differ
(p <0.05) among treatments.
S.-C. Lee et al. / Peptides 36 (2012) 100108 105
Fig. 6. Effects of epinecidin-1onserumcytokine levels. Mice were injectedwithdifferent concentrations of epinecidin-1(40, 100, 200, or 500g/mouse). Serumwas collected
on days 1, 2, 3, 7, 15, 16, 17, 21, and 28 after the primary injection. Amounts of interleukin (IL)-12, interferon (IFN)-, IL-10, IL-4, and tumor necrosis factor (TNF)- were
estimated in a 96-well plate using respective antibodies (see Section 2). Each bar represents the mean value fromthree determinations, with the standard error (SE). Data
(mean SE) with different letters signicantly differ (p<0.05) among treatments.
to initial peaks at 24 and 48h, and a second peak on day 16
(Fig. 6).
3.5. Effects of epinecidin-1 on MCP-1, MIP-1, and TNF-
expressions
In RAW264.7 cells (morphologically monocytes and
macrophages), LPS greatly elevated MCP-1 mRNA and protein
expressions at 6 and 24h (Fig. 7a). LPS elevated MIP-1 (6 and
24h) and TNF- (1 and 6h) mRNA expressions (Fig. 7a). Treat-
ment with epinecidin-1 from a low (3.75g/ml) to a high dose
(15g/ml) showed dose-dependent effects on MCP-1, MIP-1, and
TNF- mRNA expressions (Fig. 7a). Compared to the LPS group,
epinecidin-1 treatment increased MCP-1 and MIP-1 secretion at
6 and 24h (Fig. 7b). Compared to the group treated with 3.75
and 7.5g/ml epinecidin-1, the group treated with 15g/ml
epinecidin-1 showed signicant changes in MCP-1 at 1 and 6h
(Fig. 7b). Compared to the LPS group, epinecidin-1 alone did not
produce signicant TNF- changes at 1, 6, or 24h after being
treated with 3.75, 7.5, or 15g/ml epinecidin-1.
4. Discussion
The major ndings of the present study are that synthesized
epinecidin-1 co-treated with P. aeruginosa in mice with subse-
quent detection of antibody titers of IgG, IgM, IgG1, and IgG2a
showed that epinecidin-1 activated Th2 cells against P. aeruginosa.
The induction level was low possibly because of the low dose of P.
aeruginosa (0.2ml; 10
6
cfu/ml/per mouse) in the treatments. Anti-
epinecidin-1 IgG was produced in mice challenged with either
CpG or P. aeruginosa. Marked cell proliferation occurred when
splenocytes from bacterially infected mice were stimulated with
CpG, AMP, or ConA in vitro. Cell proliferation with ConA treat-
ment was higher compared to stimulation with P. aeruginosa,
P. aeruginosa co-treated with CpG, or P. aeruginosa co-treated
with epinecidin-1. Thus, in agreement with our previous study
on establishing an epinecidin-1-based inactivated vaccine, co-
treatment with epinecidin-1 and Japanese encephalitis virus (JEV)
in mice induced higher Th2 cytokine levels than Th1 cytokine
levels [7]. Antibody isotyping revealed that induction of IgG1 by
epinecidin-1 was through Th2 cells, so it was a humoral response.
106 S.-C. Lee et al. / Peptides 36 (2012) 100108
Fig. 7. Changes in mRNA expressions (a) and serum secretion (b) of monocyte chemoattractant protein (MCP)-1, macrophage inammatory protein (MIP)-1, and tumor
necrosis factor (TNF)- after epinecidin-1 treatment of RAW264.7 cells for 1, 6, and 24h. Concentrations of epinecidin-1 used to treat RAW264.7 cells were 0, 3.75, 7.5, and
15 g/ml. The concentration of lipopolysaccharide (LPS) used to treat RAW264.7 cells was 0.1g/ml. Each bar represents the mean value from three determinations, with
the standard error (SE). Data (meanSE) with different letters signicantly differ (p<0.05) among treatments. *p<0.05, **p <0.01, and ***p<0.001.
Antibody titers of IgG, IgM, IgG1, andIgG2a against aninactivatedP.
aeruginosa antigen showed that epinecidin-1 can activate Th2 cells
against that pathogen. The Th1 cell-responsive cytokine, IL-12, also
increased in mice injected with both epinecidin-1 and P. aeruginosa
at 7 days after bacterial re-challenge (Fig. 2). From results of
serum-neutralizing antibody levels of IL-10, leukocyte prolif-
eration from the spleen of MCP-1 showed slight induction,
but MCP-1 induction was not seen after re-challenge exper-
iments (Fig. 3). Recently, enhancement of resistance against
bacterial strains was studied in transgenic sh overexpress-
ing the epinecidin-1 peptide [20]. Those results suggested that
epinecidin-1 has direct antimicrobial and bacteriostatic activities
against bacterial infection and also possesses immunomodulatory
functions.
S.-C. Lee et al. / Peptides 36 (2012) 100108 107
Endotoxic shock is generally associated with the systemic
inammatory response and causes multiple organ failure [22].
Pretreatment with epinecidin-1 decreased TNF- gene expression
after bacterial infection [14]. In this study, co-treatment with P.
aeruginosa and epinecidin-1 attenuated the increases in plasma
GOT and GPT after 7 days in mice (Fig. 4). Injection of different
concentrations of epinecidin-1 alone in mice presented no signif-
icant differences in GOT or GPT values compared to injection of
antibiotics (4 and10mg) fromdays 1 to 15 (Supplementary Fig. S2).
Epinecidin-1 also alleviated histopathologic changes in the brain
after virus infection [7]. Accordingly, treatment with epinecidin-
1 attenuated the LPS-induced organ damage with an increase in
IgG1 after endotoxic shock. In addition, the criteria for dening
organ failure in patients with septic shock are increased serumlev-
els of GPT and GOT, which suggest liver cell injury. Interestingly,
injection of epinecidin-1 alone in mice (this study) did not result in
signicant rises in serumlevels of GPT or GOT. Our ndings suggest
that epinecidin-1 has a function similar to that of N-acetylcysteine,
as post-treatment with N-acetylcysteine attenuated the increases
in plasma GOT and GPT after an LPS injection and increased the
mean arterial pressure and heart rate after endotoxic shock [6].
Thus, epinecidin-1 treatment of mice might involve protecting the
liver against LPS-induced dysfunction in and preventing damage to
several other organs in septic mice.
The immune systemis an important defensive systemprotect-
ing a host from various pathogenic infections such as bacteria,
viruses, parasites, and so on [3]. In particular, IgMplays an impor-
tant early role in the course of an infection. In most trials, the
efcacy of IgM-enriched intravenous polyclonal immunoglobulin
as adjunct therapy in sepsis was reported [12]. We observed that
IgM and IgG levels were signicantly higher compared to IgG1
and IgG2a after injecting epinecidin-1 alone in mice (Fig. 5). Our
results suggest that epinecidin-1 stimulates IgM and IgG produc-
tion by mouse splenic lymphocytes in the absence of LPS. Thus,
epinecidin-1 might alleviate inammatory reactions in the sys-
temic immune system of the mouse. The cytokine network plays
an important role in immune responses and subsequent inamma-
toryprocesses [19]. We demonstratedthat injectionof epinecidin-1
might modulate cytokine production in splenic lymphocytes. An
injection of epinecidin-1 (Fig. 6) stimulated IL-12, IL-4, and IL-10
production to a great extent, but did not affect TNF- produc-
tion. We found increased cytokine production by Th2 cells after
treating themwith epinecidin-1, even in the presence of bacterial
infection-induced inammation. These data suggest that treat-
ing CpG- or P. aeruginosa-induced lymphocytes with epinecidin-1
could strengthen the immune systemby regulating cytokine such
as IL-12. Regulation of Th2-related cytokine production, such as IL-
4 and IL-10, by injecting epinecidin-1 suggests that epinecidin-1
might induce an immune-related balance toward the production
of both Th1 and Th2 cytokines by splenic lymphocytes, so that
epinecidin-1possesses abalancingfunctionsuchas Echinacea, Ash-
wagandha, and Brahmi [26].
In summary, the administration of epinecidin-1 induced
a signicant increase in the serum IgG level. Furthermore
our study suggests the possibility of clinical applications of
epinecidin-1 in mammalian systems based on its immunoregula-
toryeffects. Althoughthemechanismthroughwhichaminoacids in
epinecidin-1modify immune indices by receptors is not yet known,
our results point to a new possibility for a pharmacological role of
epinecidin-1.
Acknowledgment
Research funding was obtained from the Marine Research Sta-
tion, Institute of Cellular and Organismic Biology, Academia Sinica,
Jiaushi, Ilan, Taiwan.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.peptides.2012.04.002.
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