National Cholesterol Awareness Month Article

Integrative Physiology and Experimental Medicine

Orally Administered Eicosapentaenoic Acid Induces Rapid

Regression of Atherosclerosis Via Modulating the Phenotype
of Dendritic Cells in LDL Receptor-Deficient Mice
Kenji Nakajima, Tomoya Yamashita, Tomoyuki Kita, Masafumi Takeda, Naoto Sasaki,
Kazuyuki Kasahara, Masakazu Shinohara, Yoshiyuki Rikitake, Tatsuro Ishida,
Mitsuhiro Yokoyama, Ken-ichi Hirata

ObjectiveEicosapentaenoic acid (EPA) has been shown to have beneficial effects on cardiovascular diseases, although
the precise mechanism is unknown. We investigated the effect of EPA on the regression of atherosclerosis.
Methods and ResultsLDL-receptor deficient mice were fed a high-cholesterol diet for 8 weeks to build up aortic sinus
atherosclerotic lesions and then were fed a normal diet with or without 5% EPA for 4 weeks. Atherosclerotic lesions
were histologically assessed, and immunologic assays were performed. EPA treatment significantly regressed
atherosclerosis (22.7%, P0.05) and decreased the content of macrophages, CD4 T cells, and dendritic cells (DCs)
in atherosclerotic lesions, though only changing the chow never induced the regression. Flow cytometric analysis
revealed that EPA increased immature DCs (CD11c CD80 CD86), increased the indoleamine 2,3-dioxygenase
(IDO) in DCs, and decreased the number of CD4 T cells. In the presence of the IDO inhibitor, the beneficial effects
of EPA on regression were inhibited, suggesting that the effect of EPA was mainly mediated through IDO.
ConclusionIn addition to lowering plasma cholesterol, EPA regressed atherosclerosis probably due to modulation of DC
phenotype and reduction in T cell numbers. The present findings might partly explain the beneficial effects of EPA in
clinics and support clinical evidence. (Arterioscler Thromb Vasc Biol. 2011;31:1963-1972.)
Key Words: Atherosclerosis Immune system dendritic cells lymphocytes regression

E vidence for atherosclerosis regression in humans has

been reported.1,2 Although lowering of plasma lipid
levels is a major driving force, the mechanisms that promote
lesion regression are not clear. Recent clinical evidence
indicated that lowering elevated high-sensitive C-reactive
protein levels in plasma with 3-hydroxy-3-methylglutaryl-
coenzyme A reductase inhibitors (statins) significantly re-
duced the incidence of major cardiovascular events in nor-
molipidemic healthy persons.3 These data indicate that
statins, in addition to their aggressive lipid lowering effect,
may also be a hopeful therapeutic strategy for inhibiting
cardiovascular events via stabilization or regression of ath-
erosclerotic plaques through their antiinflammatory actions.
See accompanying articles on page 1939 and 1943
Eicosapentaenoic acid (EPA), a long-chain n-3 fatty
acid, was reported to have beneficial effects on cardiovas-
cular diseases.4,5 We have also demonstrated that the use of
highly purified EPA, in addition to statins, prevented
cardiovascular events in Japanese hypercholesterolemic
patients.6 On the basis of these findings, we hypothesized
that EPA could regress atherosclerosis. Recent studies
demonstrated that a metabolite of EPA inhibited the
maturation of dendritic cells (DCs), downregulated co-
stimulatory molecules, and induced indoleamine 2,3-diox-
ygenase (IDO).7 IDO, an enzyme involved in tryptophan
catabolism, breaks down tryptophan required for T cell
proliferation, and its induction is associated with T cell
immune suppression.8,9 Here, we investigated the effects
of highly purified EPA in an atherosclerosis regression
mouse model. The present results indicate that the inter-
vention to inflammatory responses, such as the induction
of tolerogenic DCs, could be therapeutic methods for
atherosclerotic plaque regression. A better understanding
of the underlying mechanisms involved in atherosclerosis
regression will allow us to develop novel therapeutic

Received on: January 24, 2011; final version accepted on: May 11, 2011.
From the Division of Cardiovascular Medicine, Department of Internal Medicine (K.N., T.Y., T.K., M.T., N.S., K.K., M.S., Y.R., T.I., M.Y., K.H.),
Division of Signal Transduction, Department of Biochemistry and Molecular Biology (Y.R.), Kobe University Graduate School of Medicine, Kobe, Japan,
Department of Experimental Pathology, Institute for Frontier Medical Science (N.S.), Kyoto University, Kyoto, Japan, and Hyogo Brain and Heart Center
(M.Y.), Himegli, Japan.
Correspondence to Tomoya Yamashita, Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of
Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail tomoya@med.kobe-u.ac.jp
2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.111.229443

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1963 by guest on May 17, 2015
1964 Arterioscler Thromb Vasc Biol September 2011
interventions to enhance plaque regression and reduce
cardiovascular events.
Methods
Please see the Online Appendix for a detailed description of
Methods, available at http://atvb.ahajournals.org.
Animals and Experimental Design
Low-density lipoprotein receptor knockout (LDLR/) mice were
kept in a specific pathogen-free animal facility at Kobe University
Institute. All animal experiments were conducted according to the
Guidelines for Animal Experiments at Kobe University School of
Medicine. Six-week-old male LDLR/ mice received a high-fat
and cholesterol diet (21% fat, 1.25% cholesterol, and 0.5% cholate;
Oriental Yeast, Tokyo, Japan) for 8 weeks. At 14 weeks of age, their
diet was changed to fish powder-free diet with or without 5%
ultrapure EPA ethylester (99% purity; Mochida Pharmaceutical
Co, Ltd, Tokyo, Japan) and continued until euthanized. In several
experiments, mice were treated with the IDO inhibitor (1-methyl-
DL-tryptophan, 1-MT; Sigma, St. Louis, MO)10 or its solvent. Mice
were euthanized at 14, 16, 18, and 22 weeks of age, and atheroscle-
rotic lesions were assessed as previously described.11
Cell Isolation and Flow Cytometry Analyses
Purified CD11c DCs and CD4 T cells were isolated from
mesenteric lymph nodes (LNs) and spleens using an AutoMACS
separator (Miltenyi Biotec, Inc, Auburn, CA) according to the
manufacturers instructions and were used for flow cytometry
analyses, cell culture experiments, and total RNA extraction.
Assessment of mRNA Expression
Total RNA was extracted from CD11c DCs and mouse aortic roots
after perfusion with RNAlater (Ambion, Austin, TX) using TRIzol
reagent (Invitrogen, Carlsbad, CA). Quantitative real-time reverse
transcription polymerase chain reaction (RT-PCR) was performed as
described previously.11
Statistical Analysis
Data were expressed as meansSEM. Mann-Whitney U test was
used to detect significant differences between two groups. Kruskal-
Wallis test was used to detect significant differences when 3 groups
were compared. Statistical values of P0.05 were considered
statistically significant.
Results
EPA Induces Regression of Established
Atherosclerotic Lesions
Our original regression mouse model and the design of
experiments presented here are outlined in Figure 1A. In
brief, 6-week-old LDLR/ mice were fed a high-cholesterol
and high-fat diet for 8 weeks to build up atherosclerotic
lesions at the aortic sinus. When mice were 14 weeks old, the
diet was changed to normal food with (EPA) or without 5%
EPA (control) and continued for 4 weeks. Changing the diet
markedly reduced plasma cholesterol levels as shown in
Table. Orally administered EPA further decreased plasma
cholesterol levels as previously observed.12 For baseline
levels, atherosclerosis was assessed when mice were 14
weeks (baseline; 5.500.46105 m2) and 18 weeks old for (TGF)- were increased, but IL-10 levels were decreased in
each group (Figure 1B and 1C). There was no observable
decrease in plaque area in mice who were switched to the
normal diet at 18 weeks of age (control; 5.380.33
105 m2) or at 22 weeks of age (Figure 1C). Addition of EPA
to the normal diet normalized plasma cholesterol and signif-
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icantly regressed established atherosclerosis at 18 weeks of
age (EPA; 4.250.33105 m2; 22.7% versus baseline
and 20.9% versus control, P0.05; Figure 1B and 1C),
although further EPA treatment for 4 weeks showed no
additional effects. As EPA significantly reduced plasma
cholesterol level, we examined EPA-treated group with
adjusted plasma cholesterol level to control group to rule out
the effect of lowering cholesterol on the regression. As shown
in Supplemental Figure I, EPA could induce the regression
compared with control group even under the same plasma
cholesterol condition.
Qualitative Analyses of Atherosclerotic Plaques
Before and After Regression
Immunohistochemical analysis of the aortic root (baseline,
control, and EPA) was performed to determine changes in
atherosclerotic plaques before and after regression. Mac-
rophage accumulation and CD4 T cell content was
markedly decreased with EPA treatment compared with
those in the baseline and control groups (Figure 1D and
1E). In addition, smooth muscle cell content, as deter-
mined by -smooth muscle actin staining and collagen
content assessed by Masson Trichrome staining, was
increased by EPA compared with those in the baseline and
control groups (Figure 1D and 1E).
Next, we performed the assays focusing on DCs in
atherosclerotic lesions. CD86 is recognized as an impor-
tant costimulatory molecule related to maturation of DCs.
We examined the number of CD11c and CD11c CD86
mature DCs using immunohistochemical studies, and
mRNA expression of CD11c and costimulatory molecules
with quantitative RT-PCR in atherosclerotic lesions. Low-
ering cholesterol significantly reduced the number of DCs,
and EPA further markedly decreased both the number of
DCs and the expression of costimulatory molecules (Fig-
ure 2A). The same observation applied to the mRNA
expression of CD11c and costimulatory molecules (Figure
2B). Taken together, these results indicate that EPA
decreased the number of mature DCs in the plaque at
18 weeks.
To assess the mechanism of regression of the atheroscle-
rotic plaque, we examined mRNA expression of cytokines,
chemokines, matrix metalloproteinases (MMPs), and adhe-
sion molecules in atherosclerotic plaques by quantitative
RT-PCR (Figure 2C). We found that proinflammatory cyto-
kines and chemokines, such as interferon (IFN)-, interleukin
(IL)-12p40, monocyte chemoattractant protein (MCP)-1, and
tumor necrosis factor (TNF)-, were significantly reduced in
the EPA group compared with those in the baseline or control
groups. MMPs, which degrade the extracellular matrix, were
also reduced in the EPA group. On the other hand, the levels
of the antiinflammatory cytokine transforming growth factor
the EPA group. Levels of adhesion molecules such as
intracellular adhesion molecule (ICAM)-1 were reduced in
the EPA group compared with those in the baseline or control
groups, but vascular cell adhesion molecule (VCAM)-1 levels
were not affected by EPA treatment.
 Nakajima et al EPA Induces Regression of Atherosclerosis 1965

14 weeks
A 6 weekst (Baseline) 18 weekst

Control High cholesterol and high fat diet Normal chow

EPA High cholesterol and high fat diet Normal chow + EPA

B Baseline C

700 Control

Atherosclerotic lesion size

EPA
600

( 103 m2 )
Control EPA 500
*# *#
400

300
14 16 18 20 22
B a s e lin e w eeks

MOMA-2 positive area (%)

D E 80
Baseline Control EPA
60
MOMA-2

40 *#
20

0
Baseline Control EPA

250
CD4+cells/mm2

200

150
*#
CD4

100

50

0
Baseline Control EPA

*#
-SMA positive area (%)

6
#
-SMA

0
Baseline Control EPA

60
*#
Collagen area (%)

#
Collagen

40

20

0
Baseline Control EPA

Figure 1. Eicosapentaenoic acid (EPA) induces rapid regression of established atherosclerosis in male low-density lipoprotein receptor
knockout (LDLR/) mice. A, Experimental design. B, Representative photomicrographs of aortic sinus atherosclerotic lesions stained
by Oil Red O. A black bar represents 200 m. C, Time course of atherosclerotic lesion size in the aortic sinus of LDLR/ mice at 14
weeks old (baseline; closed square) and after changing to normal chow with (EPA; open squares) or without EPA (control; gray
squares). D, Representative sections of aortic sinus stained with antibodies specific for MOMA-2 for macrophages, CD4 T cells, and
-smooth muscle actin for smooth muscle cells in male LDLR/ mice at 14 weeks (baseline) and 18 weeks (control and EPA) of age.
The fibrous area was stained by Massons trichrome. A white bar represents 200 m. E, Quantitative analyses of the immunohisto-
chemical and Massons trichrome stainings. Data represent meansSEM of n10 at 14 weeks old, n5 at 16 weeks old, n9 at 18
weeks old, and n8 at 22 weeks old in both groups. *P0.05 vs control group, and #P0.05 vs baseline.

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1966 Arterioscler Thromb Vasc Biol September 2011

Table. Effect of EPA on Body Weight and Plasma Lipid Profile

N Body Weight (g) Total Cholesterol (mg/dl) HDL Cholesterol (mg/dl) Triglycerides (mg/dl)
Baseline (14 wk old) 10 22.60.4 1809.0139.2 17.04.2 72.016.4
Control 9 27.70.4 263.814.9 61.32.0 64.36.1
EPA 9 27.00.4 160.48.7* 36.92.0* 73.97.1
Control1-MT 7 27.10.6 264.66.5 62.60.4 65.06.2
EPA1-MT 7 27.30.6 160.86.1$ 40.40.4$ 61.06.2
ControlSolvent 7 27.50.8 264.216.5 62.32.9 66.07.8
EPASolvent 7 27.60.5 160.911.0# 38.02.8# 63.58.2
Results are expressed as meansSEM. *P0.05 vs control, $P0.05 vs control1-MT, #P0.05 vs controlsolvent.

Effect of EPA on DCs in Systemic assessed the expression of IDO in CD11c DCs by flow
Lymphatic Organs cytometry analysis. IDO is an enzyme that catabolizes the
We performed flow cytometry analyses using CD11c DCs amino acid tryptophan and plays an important role in T cell
from spleens and LNs after positive selection with anti- proliferation and apoptosis.9 We observed significant increase
CD11c microbeads in each group and found that CD11c in IDO expression in CD11c DCs from EPA-treated mice
DCs in the EPA group expressed lower levels of CD80, compared with those from the baseline and control groups
CD86, CD40, and major histocompatibility complex class II (P0.05; Figure 3D). We next examined whether the DC-
compared with levels in the control groups (Figure 3A, 3B, expressed IDO from EPA-treated mice was functional. IDO
and 3C). We also confirmed another maturation marker CD83 activity assessed by the levels of kynurenine, a metabolite of
was reduced in EPA group (data not shown). These results tryptophan, in the culture supernatant of DCs from spleens
indicate that EPA systemically induces immature DCs char- indicated higher values in EPA-treated group than that in the
acterized by low expression of costimulatory molecules. We baseline or control group (P0.01; Figure 3E). We also

A Baseline Control EPA B

CD11c Nuclei

Figure 2. Phenotype of dendritic cells

CD40 (DCs) in atherosclerotic lesions in the
*##
aortic sinus. A, Representative photomi-
CD80 crographs of CD11c (green), CD86
*## (red), and CD11cCD86 mature DCs
(yellow) in the plaques of the aortic sinus
CD86 from 14-week-old (baseline) and
*#
18-week-old (control and EPA) mice. A
white bar represents 20 m. B, Quanti-
CD86

CD11c ##
*##
tative analyses of mRNA expression of
CD11c and costimulatory molecules
0.0 0.5 1.0 1.5 (CD80, CD86, and CD40) related to mat-
Relative mRNA expression uration of DCs were shown. n6 in
baseline, n9 in control, and n6 in ei-
cosapentaenoic acid (EPA) group. C,
Cytokines and chemokines (interferon
Merge

[IFN]-, interleukin [IL]-10, transforming

growth factor (TGF)-, IL-12p40, mono-
cyte chemoattractant protein [MCP]-1),
activated macrophage and DC-releasing
materials (tumor necrosis factor [TNF]-,
matrix metalloproteinases [MMP]2,
MMP9), and adhesion molecules (intra-
Baseline cellular adhesion molecule [ICAM]-1,
vascular cell adhesion molecule
Control
[VCAM]-1) in atherosclerotic aortas were
EPA quantified by quantitative real-time
reverse transcription polymerase chain
C 1.5 reaction and normalized to glyceralde-
Relative mRNA expression

hyde 3-phosphate dehydrogenase. Total

*
RNA was extracted from aortas of
1.0 14-week-old (baseline) and 18-week-old
*
* ** **
*
(control and EPA) mice. Fold changes
## * # # ## ** # ##
## ## relative to each baseline group are
0.5 ##
## shown. At least n4 in baseline, n7 in
##
control, and n5 in EPA group. *P0.05
0.0
and **P0.01 vs control, #P0.05 and
##P0.01vs baseline.
0

P2

P9

-1

-1

-1

p4

P-
F-

F-
N

AM

AM
M

M
IL
IF

TG

C
TN
2

M
M
-1

IC

VC
IL

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 Nakajima et al EPA Induces Regression of Atherosclerosis 1967

Figure 3. Effect of eicosapentaenoic acid (EPA) on dendritic cells (DCs) in vivo. A, Representative results of CD80, CD86, CD40, and
major histocompatibility complex (MHC) class II expression in CD11c DCs isolated from spleens and lymph nodes (LNs) of 14-week
old (baseline) and 18-week old (control and EPA) mice assessed by flow cytometry. In the representative histograms, isotype control
stains are given as under black line area. Surface marker expression on CD11c DCs of baseline (black shaded area), control (gray
area), and EPA (under blue line area) is shown. B and C, Quantitative analysis of surface expression for each marker was determined
as mean fluorescence intensity (MFI). n5 in baseline, n7 in control, and n7 in EPA group. D, Cells from spleens and LNs of
14-week-old (baseline) and 18-week-old (control and EPA) mice were prepared, stained with fluorescein isothiocyanate-conjugated
anti-CD11c and anti- indoleamine 2,3-dioxygenase (IDO) antibody followed by allophycocyanin-labeled secondary antibody, and
assessed by flow cytometry. The histograms were gated on CD11c DCs. Quantitative analysis of expression for IDO was determined
as MFI. n4 in baseline, n7 in control, and n7 in EPA group. E, The IDO activity was assessed by kynurenine production in the cul-
ture supernatant of CD11c DCs isolated from spleens of each group, which were incubated in Hanks balanced salt solutions for 8
hours. n4 per group. F, Total RNA was extracted from CD11c DCs isolated from spleens of each group. Expressions of interleukin
(IL)-6, IL-12p40, IL-10, transforming growth factor (TGF)- were quantified by quantitative real-time reverse transcription polymerase
chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. Fold changes relative to each baseline group are shown.
n6 per group. *P0.05 and **P0.01 vs control. #P0.05 and ##P0.01 vs baseline.

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1968 Arterioscler Thromb Vasc Biol September 2011

A 6
*
# #
CD4+ T cell ( 106 )

4 #
* Figure 4. Effect of eicosapentaenoic
# Baseline acid (EPA) on the number and function
Control of CD4 T cells in vivo and ex vivo. A,
2
# CD4 lymphocytes from spleens and
# EPA lymph nodes (LNs) of 14-week-old
(baseline) or 16- and 18-week old (con-
trol and EPA) mice were prepared,
0
16 weeks 18 weeks 16 weeks 18 weeks stained with FITC-conjugated anti-CD4,
and counted by flow cytometry. n4 at
Spleen LNs 14 weeks old and n5 at 18 weeks old
in both groups. B, Proliferation of CD4
B C 15
T cells from spleens of Balb/c mice
[ H] Thymidine (10 cpm)
4
* cocultured and stimulated by CD11c
[3H] Thymidine (104cpm)

3
dendritic cells from spleens of 14-week-
3 10
old (baseline) and 18-week-old (control
and EPA) mice assessed by [3H]-
2 # * thymidine incorporation; n3 per group.
5 C, Proliferation of CD4 T cells from
spleens of 14-week-old (baseline) or
1 #
18-week-old (control and EPA) mice in
3

0 response to anti-CD3/anti-CD28 anti-

0 (-) (+) body were assessed by [3H]-thymidine
10 : 1 20 : 1
T ce ll / DC ratio Anti-CD3 / CD28 antibody incorporation; n4 per group. D, The
graph represents the percentage of
D E CD25 Foxp3 cells within the CD4
CD25+ Foxp3+cells / CD4+ cells (%)

15 population in spleens and LNs of

3 #
# 14-week-old (baseline) or 18-week-old
# # (control and EPA) mice. n4 per group.
* # E, Cells from spleens and LNs of
Apoptosis %

10
2
14-week-old (baseline) or 18-week-old
(control and EPA) mice were immediately
#
5 prepared, stained with annexin V and
1
propidium iodide. The percentage of
apoptotic cells was counted by flow
cytometry. n4 per group. F, The graph
0 0
Spleen LNs Spleen LNs
represents the percentage of CD44high or
CD62Llow cells within the CD4 popula-
F G 5 tion in spleens of 14-week-old (baseline)
or 18-week-old (control and EPA) mice.
n3 per group. G, The graph represents
CD62Llow cells/CD4+ cells (%)
CD44high cells/CD4+ cells (%)

4
cytokine staining

25 50
# the frequencies of interferon-, interleu-
( % C D 4 + cells)

# # kin (IL)-4, IL-10, and IL-17 CD4 T

20 40
3 cells in spleens of 14-week-old (baseline)
15 30 or 18-week-old (control and EPA) mice.
2 n3 per group. *P0.05 vs control,
10 20 #P0.05 vs baseline.
5 10
1

0 0 0
IFN- IL-4 IL-10 IL-17

assessed mRNA levels of DC-associated cytokines in pressive properties have less capacity to induce T cell
CD11c DCs from spleens and found a significant decrease proliferation. We performed in vitro proliferation assays of
in mRNA levels of proinflammatory cytokines IL-6 and CD4 T cells from Balb/c mouse spleens cocultured with
IL-12p40 compared with control, whereas mRNA levels of and stimulated by CD11c DCs from spleens of each
antiinflammatory cytokines IL-10 and TGF- were upregu- group (Figure 4B). CD4 T cell proliferation was sup-
lated in the EPA group (Figure 3F). Therefore, we could say pressed when cocultured with DCs from the EPA group
that oral administration of EPA makes the phenotype of DCs compared with DCs from the baseline or control groups
immature and tolerogenic in vivo. (P0.05), suggesting that DCs from EPA-treated mice had
less proliferation activity of T lymphocytes. To determine
Effect of EPA on T Lymphocytes in Systemic whether EPA directly contributes to the suppressive po-
Lymphatic Organs tential of lymphoid cells, we performed an in vitro prolif-
Next, we investigated the effect of EPA on T cells. Orally eration assay of CD4 T cells from spleens after positive
administered EPA decreased the number of CD4 T cells selection with anti-CD4 antibody in each group in response
in spleens and LNs (Figure 4A). DCs with immunosup- to anti-CD3/anti-CD28 antibodies. As shown in Figure 4C,
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 Nakajima et al
we observed no reduction in CD4 T cell proliferation in
the EPA group compared with that in the control group. It
is unlikely that EPA directly suppresses T cell prolifera-
tion. Tolerogenic DCs have been previously shown to
inhibit T cell proliferation through the induction of
Tregs.13 Most Tregs are characterized by CD25 and Foxp3
expression and, for some subsets, by increased IL-10 or
TGF- production.14 We observed no increase in CD25 or
Foxp3 expression in the CD4 T cell population in spleens
and LNs of EPA-treated mice at 18 weeks (Figure 4D). To
assess whether EPA induces the apoptosis in T cells or not,
we investigated the annexin V assays. There was no
difference in the expression of annexin V in splenocytes
and LN cells between the two groups (Figure 4E). Previous
studies have shown that DCs generated in the presence of
EPA metabolites are poor stimulators of activated T cells
but do not induce CD4 Foxp3 T cells.7 As such, our data
imply that tolerogenic DCs induced by EPA treatment may
systemically suppress activated immune reactions without
inducing Tregs. Further, to determine the effects of EPA
on the activation of T lymphocytes in vivo, we examined
the surface expression of the activation marker CD44high
and CD62Llow by flow cytometry analysis. These activa-
tion markers on T cells were not changed or slightly
increased in EPA-treated mice compared with controls
(Figure 4F). Next, to determine whether EPA-treatment
affects the cytokine production from T cells, we analyzed
IFN-, IL-4, IL-10, and IL-17 production or expression by
intracellular cytokine staining. We could not detect any
differences in the ex vivo cytokine production from T cells
of spleens among the three groups (Figure 4G). Taken
together, EPA might not directly affect the function of T
cells in vivo but inhibited T cell proliferation via DC-
dependent manner and resulted in decreasing the number
of T cells.
Role of IDO in the Beneficial Action of EPA
To determine whether IDO expressed in DCs is directly
involved in regression of established atherosclerotic
plaques after EPA treatment, we treated mice with an IDO
inhibitor, 1-MT, which blocks the function of IDO.10
Administration of 1-MT significantly increased atheroscle-
rotic lesions (Figure 5A and 5B), macrophage contents,
and CD4 T cell contents (Supplemental Figure IIA and
IIB) in the EPA-treated group compared with the solvent-
treated EPA group, although 1-MT never affected plasma
cholesterol levels (Table). In the presence of 1-MT, there
were no significant differences in atherosclerotic lesions
between the EPA and control groups. 1-MT treatment did
not significantly affect the IDO expression in DCs (Figure
5C). The IDO activity was assessed by the kynurenine
production in the culture supernatant of DCs from spleens
of each group. 1-MT treatment significantly decreased the
kynurenine level in DCs only in EPA group (Figure 5D).
We found that the number of CD4 T cells was signifi-
cantly increased in spleens and LNs in the presence of
1-MT (Figure 5E). There were no differences in DC-
mediated T cell proliferation between 1-MTtreated con-
trol and 1-MTtreated EPA groups (Figure 5F), suggesting
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EPA Induces Regression of Atherosclerosis 1969
1-MT treatment could partly cancel the effect of EPA on
DC-mediated T cell proliferation. These results suggest
that the beneficial effects of EPA on the regression of
atherosclerosis in this model were mediated at least in part
by IDO. Moreover, inhibition of T cell proliferation was
mainly dependent on the activity of IDO likely expressed
in DCs.
Discussion
The major findings of the present study are as follows: (1)
EPA administration to mice with normalized plasma choles-
terol levels induced regression of atherosclerosis in LDLR/
mice over a 4-week period, and (2) we demonstrated a
possible mechanism associated with the regression of athero-
sclerosis. Increasing IDO expression in DCs is highly asso-
ciated with regression, and decreasing the number of CD4 T
lymphocytes is another therapeutic candidate to induce ath-
erosclerosis regression.
Epidemiological and clinical evidence suggests an in-
creased intake of EPA protects against death from coro-
nary artery disease.4,5 We demonstrated that additive EPA
treatment with statins reduced the frequency of major
coronary events without affecting plasma lipid values.6 To
provide a molecular mechanism for the epidemiological
evidence, several animal studies have demonstrated the
antiatherogenic effects of EPA. Matsumoto et al demon-
strated that EPA reduced progression of atherosclerotic
lesions in apolipoprotein E-deficient mice through its
antiinflammatory effects, including attenuation of some
adhesion molecules and chemokines.12 Another article
indicated that a high content of dietary long-chain n-3 fatty
acids minimizes atherosclerosis and inflammatory re-
sponses in LDLR/ mice.15 On the basis of these find-
ings, we investigated the effect of EPA treatment with
normalized plasma cholesterol levels on atherosclerotic
plaques in LDLR/ mice. We demonstrated for the first
time that EPA treatment results in a rapid regression of
atherosclerosis, supporting the clinical beneficial effects of
EPA on secondary prevention of cardiovascular diseases in
patients. Transfer of high-fat and cholesterol-fed LDLR/
mice to a normal diet stopped further progression of
atherosclerosis and led to a less inflammatory plaque
phenotype, but it was not sufficient to induce the regres-
sion of aortic sinus lesions. In contrast, a marked reduction
in aortic plaque area was found in EPA-treated mice,
suggesting that EPA facilitated the net removal of inflam-
matory cells and lipids probably due to antiinflammatory
actions and increase in reverse cholesterol transfer. In the
present study, we demonstrated that the number of infil-
trated cells and mRNA expression of several chemokines,
cytokines, and adhesion molecules were significantly re-
duced in atherosclerotic lesions in EPA-treated mice.
An in vitro study by Wang et al demonstrated that EPA
inhibited LPS-induced DC maturation, decreased IL-12
and TNF- levels, and reduced T cell stimulatory capac-
ity.16 IL-12 induces IFN- production and is involved in
the differentiation of T cells into potent type 1 helper T
cells.17 In this study, we found that EPA systemically
reduced the maturation of DCs (expression of CD80,
1970 Arterioscler Thromb Vasc Biol September 2011

A Control+Solvent Control+1-MT EPA+Solvent EPA+1-MT

Figure 5. Indoleamine 2,3-dioxygenase

(IDO) inhibitor canceled the beneficial
effects of eicosapentaenoic acid (EPA) on
the regression of atherosclerosis. A, Male
low-density lipoprotein receptor knockout
(LDLR/) mice were treated with an IDO
inhibitor, 1-methyl-DL-tryptophan (1-MT) or
B 900
** its solvent, from 14 weeks of age when
Atherosclerotic lesion size

the diet was changed to normal chow

with or without 5% EPA and continued for
800
4 weeks. Representative photomicro-
( 103 m2 )

700 graphs of Oil Red O staining were shown.

A black bar represents 200 m. B, Quan-
600 # Control+Solvent titative analyses of atherosclerotic lesion
size in the aortic sinus of LDLR/ mice
500 Control+1-MT at 18 weeks of age. n7 per group. C,
400 EPA+Solvent Cells from spleens and lymph nodes (LNs)
of 18-week-old (control and EPA with
EPA+1-MT 1-MT or its solvent) mice were prepared,
300
stained with FITC-conjugated anti-CD11c
C Spleen LNs D and anti-IDO antibody followed by APC-
# ** labeled secondary antibody, and
250 300 20 assessed by flow cytometry. Quantitative
#
Kynurenine (uM / 105 cell)
MFI of IDO expression
MFI of IDO expression

## analysis of expression for IDO was deter-

200
15 mined as MFI. n6 in control with sol-
200 vent, n5 in control with 1-MT, n6 in
150
EPA with solvent, n5 in EPA with 1-MT.
10
100 D, The IDO activity was assessed by
100 kynurenine production in the culture
50
5 supernatant of CD11c dendritic cells
(DCs) isolated from spleens of each
0 0 0 group, which were incubated in HBSS for
8 hours. n4 per group. E, CD4 lympho-
Control + Solvent cytes from spleens and LNs of 18-week-
old (control and EPA with 1-MT or its sol-
Control + 1-MT vent) mice were prepared, stained with
EPA + Solvent FITC-conjugated anti-CD4, and counted
E 8 * ** F by flow cytometry. F, Proliferation of
20 EPA + 1-MT CD4 T cells from spleens of Balb/c mice
[3H] Thymidine (104cpm)

cocultured and stimulated by CD11c

CD4+ T cell ( 10 6 )

6 DCs from spleens of 18-week-old (control

15
and EPA with 1-MT or its solvent) mice
# assessed by [3H]-thymidine incorporation;
4 * 10 *
# n3 per group. *P0.05 and **P0.01 vs
corresponding controls (control and EPA
2 ##
# 5 with solvent), #P0.05 and ##P0.01 vs
control with solvent.
0 0
Spleen LNs 10 : 1 20 : 1
T cell / DC ratio

CD86, CD40, and major histocompatibility complex class Vassiliou et al demonstrated that a metabolite of EPA
II) and induced tolerogenic DCs, which was characterized inhibited T cell proliferation through increasing IDO expres-
by low expression of costimulatory molecules, less T cell sion in DCs.7 IDO is an enzyme involved in tryptophan
stimulatory capacity, and minimal secretion of IL-12 in catabolism. IDO breaks down tryptophan, which is necessary
vivo. We also demonstrated that EPA markedly decreased for T cell growth, resulting in the inhibition of T cell
the number of mature DCs in atherosclerotic plaques and proliferation by inducing cell cycle arrest.8,9 Intriguingly, we
DC-releasing material of plaques quantified by real-time detected an increase in IDO expression in DCs from the
RT-PCR as compared with the control. Although several
spleen and LNs of EPA-treated mice in vivo and demon-
research groups examined and reported the role of DCs in
strated that the IDO enzyme activity, assessed by the produc-
atherogenesis, it has been undefined yet.18 Some investi-
gators showed the role of DCs in the cholesterol metabo- tion of kynurenine (a metabolite of tryptophan), was also
lism19 or in antigen presentation in the pathogenesis of increasing. In particular, IDO expression in DCs was indis-
atherosclerosis.20 We also demonstrated the possibility that pensable for atherosclerotic plaque regression because ad-
the inhibition of DC maturation reduced the formation of ministration of an IDO inhibitor canceled the beneficial
atherosclerosis, previously.21 Taken together, we hypoth- effects of EPA and increased inflammatory cell infiltration
esized that EPA may change the phenotype and function of and plaque formation to the extent of control with IDO
DCs, and induce the regression of atherosclerosis. inhibitor. We showed that EPA reduces the maturation of
Downloaded from http://atvb.ahajournals.org/ by guest on May 17, 2015
 Nakajima et al
DCs and increases IDO expression in DCs resulting in
decreased numbers of CD4 T lymphocytes in vivo.
Recent work has demonstrated that natural Tregs, which
show high expression of CD25 on their surface and express
the transcription factor Foxp3, play a protective role in
atherogenesis in mice.22,23 Several articles demonstrated
the relationship between IDO expression in DCs and the
generation of Tregs.8,13 In this study, we found that CD4
CD25 Foxp3 Tregs were not increased in spleens and
LNs of EPA-treated mice, although IDO expression in DCs
was increased. EPA was shown to inhibit T cell activation
through a reduction in IL-12 secretion, which is critical for
T cell proliferation and modification of the membrane lipid
profile.24 It is possible that Tregs are not involved in rapid
atherosclerotic plaque regression, at least in this model.
Moreover, it is unlikely that the apoptosis is a cause of the
reduced number of CD4 T cells in EPA group. We
demonstrated that T cell activation assessed by anti-CD3/
anti-CD28 antibodies induced proliferation, surface mark-
ers, and cytokine production was not inhibited in EPA
group. On the other hand, we also verified that DC-
mediated T cell proliferation was significantly inhibited in
EPA-treated group compared with that of control group,
suggesting the inhibitory effects on the T cell proliferation
was dependent on changes in phenotypes of DCs. Taken
together, based on our experiments associated with T
lymphocytes, EPA might not directly change the function
of T cells but reduced the number of T cells possibly
through the functional changes in DCs.
Systemic reduction of CD4 T cells via induction of
tolerogenic DCs is proposed to be involved in rapid plaque
regression. An IDO-inhibitor administration canceled the
beneficial effects of EPA, rather significantly progressed
atherosclerotic lesion with increasing the number of CD4 T
cells compared with the baseline group. The results of our
experimental study clearly indicate the critical role of CD4
T cells in the pathogenesis of atherosclerotic plaque regres-
sion. Systemic changes in immune responses are crucial
determinants of atherosclerotic plaque progression.25 Acti-
vated DCs might migrate from the vessel wall to secondary
lymphatic tissues, where they interact with other immune
cells.26 On the basis of our observed findings, rapid systemic
changes to antiinflammatory responses involved in tolero-
genic DCs are also crucial determinants of atherosclerotic
plaque regression. In the therapeutic harnessing of their
inherent tolerogenicity on transplantation, considerable in-
sight has been gained into the role of DC subsets in central
and peripheral tolerance, and into the molecular pathways
that regulate the outcome of DC-T cell interactions.13 Results
from our data are the first to demonstrate a possible role of a
decrease in CD4 T cells via induction of tolerogenic DCs in
rapid atherosclerotic regression, although further studies are
required to provide more direct evidence for the interaction
between T cells and DCs in plaques.
Recent vigorous examination using a mouse regression
model revealed that several candidate molecules and markers
are associated with lesion regression. Fisher et al demon-
strated that elimination of foam cells, in addition to normal-
izing the plasma cholesterol levels, is critical to achieve
Downloaded from http://atvb.ahajournals.org/ by guest on May 17, 2015
EPA Induces Regression of Atherosclerosis 1971
atherosclerosis regression.27 They raised the intriguing pos-
sibility that during regression, macrophage foam cells acquire
DC characteristics that permit them to migrate to lymph
nodes. In addition, it has been reported that lesion macro-
phages require intact chemokine receptor 7 (CCR7) signaling,
an essential factor for DC migration, to migrate from athero-
sclerotic plaques under regressive conditions.28 They also
showed that mRNA levels of cholesterol efflux factors such
as adenosine triphosphate-binding cassette transporter A1
(ABCA-1) were increased, and expression levels of VCAM-1
and MCP-1 were reduced in the plaque during regression.
Unlike previous studies, we did not observe higher expression
levels of CCR7 or ABCA-1 in the atherosclerotic plaques of
the EPA group compared to the control group (data not
shown), which may be due to the differences in the model,
mouse background, the type of lesion, or timing of assess-
ment, although further studies are required.
In conclusion, we showed that EPA treatment with nor-
malizing plasma cholesterol can induce a rapid and substan-
tial regression of atherosclerotic lesions, possibly mediated
by changing the function of DCs and decreasing the number
of T-lymphocytes. Our studies also imply the possibility that
intervention of immune systems could be a novel therapeutic
target for regression of atherosclerosis. EPA has been shown
to reduce the incidence of cardiovascular events in humans,
and the present findings might partly explain the beneficial
effects of EPA in clinics and support the clinical evidence,
although care should be taken when interpreting experimental
results in mice. Atherosclerotic plaque regression should
represent a therapeutic goal in the management of ischemic
heart disease. Further studies are needed to clarify the
molecular mechanisms of atherosclerosis regression.
Sources of Funding
This work was supported by a Grant-in-Aid for Scientific Re-
search in Japan and by research grants from Medical Research
Fund of Hyogo Medical Association, Cardiovascular Research
Fund, Hyogo Science and Technology Association, and Takeda
Science Foundation.
Disclosures
None.
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SUPPLEMENTAL MATERIAL

1
Supplemental Methods

Administration of 1-MT

To prepare 1-MT for administration in drinking water, 1 g of 1-MT, 97% (Sigma)

was added to a 15 ml conical tube with 7.8 ml Methocel/Tween [0.5% Tween 80/0.5%

Methylcellulose (v/v in water; both from Sigma)]. The mixture was bead milled

overnight by adding 1-2 ml by volume of 3 mm glass beads and mixing by inversion.

The next day, the 1-MT concentration was adjusted to 85mg/ml by adding an additional

4 ml Methocel/Tween and mixing again briefly. For administration in drinking water,

1-MT was prepared at 2 mg/ml in water as described above, supplemented with a small

amount of aspartame (1 g/L) to improve acceptance by the mice, and filter sterilized.

The solution was delivered in standard autoclaved drinking-water bottles.1 Male

LDLR-/- mice of control and EPA groups were fed 1-MT or its solvent from 14 weeks of

age when the diet was changed to fish powder free diet with or without 5% EPA and

continued for 4 weeks.

Blood Analysis

After overnight fasting, blood was collected by the cardiac puncture under

anesthesia using pentobarbital sodium (80 mg/kg intraperitoneal injection). Plasma was

obtained through centrifugation and stored at -80C. Concentrations of plasma total

cholesterol, high density lipoprotein, low density lipoprotein and triglyceride were

determined enzymatically using an automated chemistry analyzer.

2
Atherosclerotic Lesions Assessments

Mice were anesthetized and the aorta was perfused with saline. For aortic root

lesion analysis, the samples were cut in the ascending aorta, and the proximal samples

containing the aortic sinus were embedded in OCT compounds (Tissue-Tek; Sakura

Finetek, Tokyo, Japan). Five consecutive sections (10 m thickness), spanning 550 m

of the aortic sinus, were collected from each mouse and stained with Oil Red O (Sigma,

St Louis, MO). For quantitative analysis of atherosclerosis, the total lesion area of 5

separate sections from each mouse was obtained with the use of the Image J (National

Institutes of Health) as previously described.2, 3

We assessed the aortic sinus mean

plaque area in comparison among the groups. Lesion analysis was conducted by a single

observer blinded to the group of the mice.

Immunohistochemical Analyses of Atherosclerotic Lesions

Immunohistochemistry was performed on acetone-fixed cryosections (10 m) of

mouse aortic roots using antibodies to identify macrophages (MOMA-2, 1:400, BMA

Biomedicals, Augst, Switzerland), and T cells (CD4, 1:100, BD Biosciences, San Jose,

CA), followed by detection with biotinylated secondary antibodies and

streptavidin-horseradish peroxidase. For natural Tregs, acetone-fixed cryosections of

mouse aortic roots were incubated with rat anti-Foxp3 antibody (clone FJK-16s, 1:100,

eBioscience, San Diego, CA), followed by Alexa Fluor 588 anti-rat secondary antibody

(1:500, Molecular Probes, Eugene, OR) as described.2 Smooth muscle cells were

identified by immunostaining with fluorescein isothiocyanate (FITC)-conjugated

primary antibody against -smooth muscle actin (clone 1A4, 1:500; Sigma). For mature

DCs, slides were stained by using TSATM- kit (PerkinElmer LAS, Inc, Boston, MA)

3
according to the manufacturers instructions. In brief, endogenous peroxidase activity

was quenched 0.5% H2O2 for 30 minnutes. Sections were blocked with TNB buffer

(TSATM- kit) and hamster anti-CD11c antibody (clone N418, 1:200, BioLegend, San

Diego, CA) and rat anti-CD86 (clone PO3.1, 1:200, eBioscience, San Diego, CA) of

primary antibodies were applied for 1 hour at room temperature. Slides were washed

and incubated with HRP-labeled goat-anti-rat IgG (American Qualex, San Clemente,

CA) for 30 minutes. CD86 were detected with Cy3-Tyramide. Next, primary HRP was

deactivated by treatment with 0.5% H2O2 for 30 minutes, and the sections were then

incubated with biotinylated anti-hamster IgG (Vector Laboratories, Inc, Burlingame,

CA). Slides were washed and incubated with streptavidine-HRP (TSATM- kit). Staining

by hamster anti-CD11c was visualized by amplification of the signal with

FITC-Tyramide. Nuclei were counterstained with DAPI (Molecular Probes). The

appropriate fixation reagent depending on the primary antibodies was used. Negative

controls were prepared with substitution with an isotype control antibody. Staining with

Massons trichrome was used to delineate the fibrous area as previously described.2

Sections were observed under an All-in-one Type Fluorescence Microscope (BZ-8000;

Keyence, Osaka, Japan) using BZ Analyzer Software (Keyence). Stained sections were

digitally captured, and the percentage of the stained area (the stained area per total

atherosclerotic lesion area) was calculated. Quantification of CD4+ T cells in

atherosclerotic lesions was done by counting positively stained cells, which was divided

by total plaque area.

Cell Isolation of CD11c+ DCs and CD4+ T cells Subsets

Purified CD11c+ DCs and CD4+ T cells were isolated from mesenteric lymph

4
nodes or spleens. These cells were positively selected with anti-CD11c microbeads

(Miltenyi Biotec, Inc. Auburn, CA) or anti-CD4 (L3T4) microbeads (Miltenyi Biotec)

using an AutoMACS separator (Miltenyi Biotec) according to the manufacturers

instructions. Purified CD11c+ DCs were used for real-time reverse transcriptional

polymerase chain reaction (RT-PCR), flow cytometry analyses and cell proliferation

assays. Purified CD4+ T cells were used for cell proliferation assays.

Flow Cytometry Analyses

For fluorescent-activated cell sorter (FACS) analyses of lymphoid organs, lymph

nodes (LNs) cells and splenocytes were isolated at 14 weeks, 16weeks, 18 weeks of age.

Cells were stained in PBS containing 2% FCS. FACS analysis was performed by

FACSCalibur using CellQuest Pro software (BD Bioscience). The antibodies used were

as follows; anti-CD16/CD32 (clone 2.4G2; BD Bioscience), FITC-conjugated anti-CD4

(clone H129.19; BD Bioscience), phycoerythrin (PE)-conjugated anti-CD25 (clone

PC61; BD Bioscience), PE-anti-CD44 (clone IM7; BD Bioscience), PE-anti-CD62L

(clone MEL-14; BD Bioscience), allophycocyanin (APC)-conjugated Foxp3 (clone

FJK-16s; eBioscience), APC-conjugated CD40 (clone 1C10; eBioscience),

APC-conjugated CD80 (clone 16-10A1; eBioscience), PE-conjugated CD86 (clone

PO3.1; eBioscience), FITC-conjugated MHC class II (clone M5/114.15.2; Miltenyi

Biotec), and isotype matched control antibodies. For CD40, CD80, CD86 and MHC

class II staining, CD11c+ DCs were positively selected with anti-CD11c microbeads

described above and were stained. Intracellular staining of Foxp3 was performed using

the Foxp3 staining buffer set (eBioscience) and APC-conjugated Foxp3 antibody

described above according to the manufacturers instructions. All staining procedures

5
were performed after blocking Fc receptor with anti-CD16/CD32 antibody. Surface

stainings were performed according to standard procedures at a density of 1x106 cells

per 100 l, and volumes were scaled up accordingly. For analysis of IDO in CD11c+

DCs, cells were stained with PE-conjugated CD11c (clone N418; Miltenyi Biotec),

permeabilized, and were stained further with rat anti-IDO antibody (clone mIDO-48;

BioLegend), followed by FITC-conjugated donkey anti-rat immunoglobulin.

Intracellular Cytokine Staining

Splenocytes were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate

(Sigma) and 1 mmol/L ionomycin (Sigma) for 5 hours in the presence of a Golgi

transport inhibitor (BD Bioscience). After staining with FITC-anti-CD4, intracellular

staining of cytokines was performed using intracellular cytokine staining kit (BD

Bioscience) and PE-anti-IL-4, PE-anti-IL-10, Alexa Fluor 647-anti-IL-17, and

PE-anti-IFN- (all from BD Bioscience) according to the manufacturers instructions.

Cell Proliferation and Apoptosis Assays

In all cell culture experiments, we used RPMI 1640 medium (Sigma)

supplemented with 10% FCS, 10 mmol/L Hepes, 50 mol/L 2-mercaptoethanol and

antibiotics. For analysis of in vitro suppressive function of CD11c+ DCs, CD11c+ cells

from spleens of baseline, EPA-treated and control mice were cultured with 1x105 of

CD4+ T cells from spleens of Balb/c mice at various ratio (total volume, 200 l/well).

These cells were cultured at various ratios in flat-bottomed 96-well plates at 37 C with

5 % CO2 for 72 hours. In these experiments, CD11c+ DC were irradiation with 18.5 Gy

before co-culture. CD4+ T cells after positive selection with anti-CD4 (L3T4)

6
microbeads described above in each group were cultured at 5x105 cells/well (total

volume, 200 L/well) in flat-bottomed 96-well plates and stimulated with

anti-CD3/anti-CD28-coated beads. (0.5 per cell; Dynabeads Mouse T-Activator

CD3/CD28; Invitrogen Dynal AS, Oslo, Norway) at 37 C with 5 % CO2 for 72 hours.

The cells were pulsed with 1 Ci of [3H]-thymidine (GE Healthcare, Buckinghamshire,

UK) for the last 16 hours, and thymidine incorporation was assessed with a LS 6500

liquid scintillation counter (Beckman Coulter, Inc, Brea, CA). To assess whether EPA

induces the apoptosis in T cells, cells from spleens and LNs of 14-week old (Baseline)

or 18-week old (Control and EPA) mice were immediately prepared, stained with

Annexin V and propidium iodide using Annexin V-Fluorescein Staining kit (Wako,

Osaka, Japan). Stained cells were analyzed by FACS.

Measurement of IDO enzymatic activity

The IDO enzyme assay was performed as previously reported.4 In brief, CD11c+

cells from spleens were washed, resuspended in sterile HBSS (Sigma) containing 500

mol/L tryptophan (Sigma), and incubated for 8 hours. The supernatants were then

harvested and assayed for kynurenine. For the assay, 30 l of 30% trichloroacetic acid

was added to 60 l of culture supernatant and the mixture was vortexed and centrifuged

at 10,000 g (12,000 rpm) for 5 minutes. Then, the supernatant was added into an equal

volume of Ehrlich reagent (5 l of glacial acetic acid and

p-dimethylaminobenzaldehyde). The OD was measured at 492 nm using a microplate

reader. Purified L-kynurenine (0500 mol/L; Sigma) was used as a standard.

RT-PCR Analysis

7
 At 18 weeks old, mice were anesthetized and the aortic roots were excised as

described above. Total RNA was extracted from CD11c+ DCs, or from the aortas after

perfusion with RNA later (Ambion, Austin, TX) using TRIzol reagent (Invitrogen,

Carlsbad, CA). Quantitative PCR was performed using One Step SYBR PrimeScript

RT-PCR Kit (Takara, Shiga, Japan) and an ABI PRISM 7500 Sequence Detection

system (Applied Biosystems, Foster City, CA) according to the manufacturers protocol

as described previously.2 The following primers were used to amplify CD11c, CD80,

CD86, CD40, MMP2, MMP9, TNF-, IL-6, IL-12p40, IL-10, TGF-, and GAPDH:

CD11c, 5-AGA CGT GCC AGT CAG CAT CAA C-3 and 5-CTA TTC CGA TAG

CAT TGG GTG AGT G-3'; CD80, 5-AGT TTC CAT GTC CAA GGC TCA TTC-3

and 5-TTG TAA CGG CAA GGC AGC AAT A-3; CD86, 5-TGG CAT ATG ACC

GTT GTG TGT G-3 and 5-ACG TTT GAG CAG ATG GAA ACT CTT G-3; CD40,

5-AAA GGT GGT CAA GAA ACC AAA GGA T-3 and 5-GCA CTG GAG CAG

CGG TGT TA-3; MMP2, 5-GAT AAC CTG GAT GCC GTC GTG-3 and 5-CTT

CAC GCT CTT GAG ACT TTG GTT C-3; MMP9, 5-GCC CTG GAA CTC ACA

CGA CA-3 and 5-TTG GAA ACT CAC ACG CCA GAA G-3; TNF-, 5-CAT GTG

CCA CTG AGA ACC TTG AA-3 and 5-CAG GTC CCA GCG CAA TGT AAC-3

IL-6, 5-CCA CTT CAC AAG TCG GAG GCT TA-3 and 5-GCA AGT GCA TCA

TCG TTG TTC ATA C-3; IL12p40, 5-GCT CGC AGC AAA GCA AGG TAA-3 and

5-CCA TGA GTG GAG ACA CCA GCA-3; IL-10, 5-GAC CAG CTG GAC AAC

ATA CTG CTA A-3 and 5-GAT AAG GCT TGG CAA CCC AAG TAA-3; TGF-,

5-GTG TGG AGC AAC ATG TGG AAC TCT A-3 and 5-TTG GTT CAG CCA CTG

CCG TA-3; IFN-, 5-CGG CAC AGT CAT TGA AAG CCT A-3 and 5-GTT GCT

GAT GGC CTG ATT GTC-3; GAPDH, 5-TGT GTC CGT CGT GGA TCT GA-3 and

8
5-TTG CTG TTG AAG TCG CAG GAG-3. Amplification reactions were performed

in duplicate and fluorescence curves were analyzed with included software. GAPDH

was used as an endogenous control reference.

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9
Orally Administered Eicosapentaenoic Acid Induces Rapid Regression of Atherosclerosis
Via Modulating the Phenotype of Dendritic Cells in LDL Receptor-Deficient Mice
Kenji Nakajima, Tomoya Yamashita, Tomoyuki Kita, Masafumi Takeda, Naoto Sasaki,
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Arterioscler Thromb Vasc Biol. 2011;31:1963-1972; originally published online August 4, 2011;
doi: 10.1161/ATVBAHA.111.229443
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