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Contents lists available at ScienceDirect

Journal of Cardiology
journal homepage: www.elsevier.com/locate/jjcc

Review

Anti-inammatory and immune-modulatory therapies for preventing

atherosclerotic cardiovascular disease
Tomoya Yamashita (MD, PhD)*, Naoto Sasaki (MD, PhD), Kazuyuki Kasahara (MD, PhD),
Ken-ichi Hirata (MD, PhD)
Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

A R T I C L E I N F O A B S T R A C T

Article history: Atherosclerosis is believed to be a chronic inammation of the arterial wall and various immune cells of
Received 30 January 2015 innate and adaptive immunity involves in the pathogenesis of atherosclerosis. Based on this notion,
Accepted 31 January 2015 several anti-inammatory strategies for prevention of atherosclerosis have been examined mainly using
Available online xxx
animal models. Vaccination or mucosal immunization with athero-antigens comes under candidate
therapeutic methods for antigen-specic prevention of atherosclerosis. Immune suppression mediated
Keywords: by regulatory T cells (Tregs) could be another method to regulate pathogenic chronic inammation in
Inammation
atherogenesis. Inducible Tregs are reported to differentiate peripherally in the intestine and we have
Vaccine
Regulatory T cell
been interested in the oral tolerance, in which not only Tregs but also tolerogenic dendritic cells play
Intestinal immunity crucial roles. We demonstrated that modulation of the intestinal immunity including oral tolerance
could be a novel therapy against atherosclerosis. Further, downregulation of effector T cell response and/
or Treg predominant condition was shown to induce atherosclerosis regression and inhibit the
progression of aneurysm.
In clinical situations, none of the approaches to specically and directly treat inammation to prevent
cardiovascular events or reduce atherosclerosis in human individuals were successful, although high-
sensitive C-reactive protein is shown to have a strong relationship with recurrent events of
cardiovascular diseases in several randomized clinical trials. Now two randomized placebo-controlled
clinical trials evaluating anti-inammatory agents are being conducted in the USA and Canada to clarify
whether targeting the inammation itself will reduce cardiovascular events and risks.
In this review, we present the current understanding of anti-inammatory and immune-modulation
therapies against atherosclerosis and discuss the future perspectives.
2015 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Clinical trials of direct anti-inammatory therapies for cardiovascular disease . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Why does continuous chronic inammation exist in atherosclerotic lesions and how to regulate it? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Anti-atherogenic strategy by enhancing regulatory immune response . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Intervention to the Treg/effector T cells ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Intestine as a therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
Sources of funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

* Corresponding author at: 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. Tel.: +81 78 382 5846; fax: +81 78 382 5859.
E-mail address: tomoya@med.kobe-u.ac.jp (T. Yamashita).

http://dx.doi.org/10.1016/j.jjcc.2015.02.002
0914-5087/ 2015 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.

Please cite this article in press as: Yamashita T, et al. Anti-inammatory and immune-modulatory therapies for preventing
atherosclerotic cardiovascular disease. J Cardiol (2015), http://dx.doi.org/10.1016/j.jjcc.2015.02.002
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Introduction deposition in the arterial wall, which are mediated by coronary

Cardiovascular disease (CVD), a leading cause of mortality in
many developed and developing countries, is caused mainly by
atherosclerosis. Clinical studies and animal experiments have
established that high plasma concentrations of cholesterol,
mainly transported by low-density lipoprotein (LDL), promote
atherosclerotic CVD and statin-based lipid lowering therapy
reduces CV events. However, some clinical trials revealed residual
cardiovascular risks cannot be ignored even after the aggressive
reduction of LDL cholesterol to target levels. Despite great
advances in treating acute coronary syndrome with catheter-
based therapies and controlling risk factors of CVD, there is still an
enormous need for additional therapies as the recurrent rate of CV
events remains at about 20% within 3 years even with optical
medical treatment [1].
Atherosclerosis is considered not only a disorder of lipid
accumulation in the arterial wall but also a chronic inammatory
disease that contains components of both innate and acquired
immune systems [25]. Several immune responses are critical
factors in the initiation and progression of atherosclerosis (Fig. 1).
The rst step preceding the atherosclerotic lesion formation
is endothelial activation or dysfunction and LDL-cholesterol
risk factors such as dyslipidemia, hypertension, diabetes mellitus,
and smoking. Secondly, the accumulated LDL is oxidized and the
resultant formation of oxidized LDL (OxLDL) has been suggested to
be the critical event in deteriorating inammation in vascular wall.
Thirdly, not only monocytes but also various types of leukocytes
adhere to the activated endothelium, migrate into the arterial wall
via upregulated adhesion molecules, and produce pro-inamma-
tory cytokines or chemokines. Subsequently, monocyte-derived
macrophages take up OxLDL via scavenger receptor leading to the
formation of lipid-laden foam cells. Following such steps, the initial
fatty streaks contain lipids and numerous immune cells such as
macrophages, dendritic cells (DCs), and T lymphocytes. Progressed
atherosclerotic lesions involve the migrated smooth muscle cells
(SMCs), debris, apoptotic cells, and extracellular matrix such as
collagen and proteoglycans [5]. B lymphocytes and their producing
immunoglobulins including IgG and IgM are thought to be
associated with atherogenesis. Finally, such indolent progressed
atherosclerotic plaques may suddenly rupture and induce life-
threatening coronary thrombosis presenting as an acute coronary
syndrome. The notable features of unstable rupture-prone plaque
are inltration of many inammatory cells, large lipid core, and
thin brous cap [6,7].

Fig. 1. Role of inammatory cells and immune responses in atherogenesis. Low-density lipoprotein (LDL) is deposited in the subendothelial space, and the accumulated LDL is
oxidized to OxLDL (oxidized LDL) that activates the endothelium. Coronary risk factors also activate the endothelium and induce the adhesion molecules. Monocytes migrate
into the subendothelial space using the adhesion molecules, differentiate into macrophages, take up OxLDL, and change to foam cells. The protein components of the OxLDL
particle are processed and presented as antigens to T cells by macrophages and dendritic cells (DCs). Other self and foreign antigens may also trigger similar immune
reactions. T cells differentiate into effector T cells (Th1, Th2, and Th17) and release cytokines and chemokines, and stimulate the migration of smooth muscle cell (SMC) and
other inammatory reactions. Migrated SMCs change their phenotype from contractile SMCs to synthesized ones that produce cytokines. Synthesized SMCs and foam cells
contribute to form the atherosclerotic plaques including the lipid core and brous cap formation. Proatherogenic cytokines including IFN-g secreted by Th1, and IL-12
secreted by DCs and macrophages deteriorate the lesion formation, might be associated with destabilizing the plaque, and induce the plaque rupture. Regulatory T cells
(Tregs) suppress effector T cell activation, the differentiation of nave T cell into effector T cells, and downregulate antigen presentation of DCs via secretion of anti-
inammatory cytokines including interleukin (IL)-10 and transforming growth factor (TGF)-b. Tolerogenic DCs, characterized by downregulated expressions of CD80/CD86,
maintain the tolerance to self-antigens by inducing Tregs or by inhibiting effector T cells. Immunoglobulins produced by B cells are also thought to play a role in atherogenesis.
apo B, apolipoprotein B; CRP, C-reactive protein; HSP, heat shock protein; IFN, interferon; Ig, immunoglobulin.

Please cite this article in press as: Yamashita T, et al. Anti-inammatory and immune-modulatory therapies for preventing
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Taken together, we might prevent atherosclerotic CVD by
intervening at each step or factor that is associated with the
inammatory processes in atherogenesis, although none of the
approaches treating specic inammation to prevent the CV
events in humans were successful to date. Several anti-inamma-
tory drugs are being examined for their potential to reduce the
residual CV risks in CVD patients. In this review, we focus on the
anti-inammatory therapies of clinical trials and animal experi-
ments and consider the future perspectives of ghting against
atherosclerotic CVD.
Clinical trials of direct anti-inammatory therapies for
cardiovascular disease
To date, no clinical trial has addressed whether targeting the
inammation itself will reduce CV events and risks. Now two
randomized placebo-controlled clinical trials evaluating anti-
inammatory agents are being conducted in the USA and Canada
(Fig. 2) [8,9]. High-sensitive (hs) C-reactive protein (CRP) has been
shown to have a strong relationship with recurrent events of CVD
in several randomized clinical trials [10]. HsCRP is produced in the
liver stimulated by interleukin-1b (IL-1b), tumor necrosis factor-a
(TNF-a), and interleukin-6 (IL-6). Based on the basic animal
studies, inhibition of such cytokines and chemokines could reduce
atherosclerosis.
The Canakinumab Anti-inammatory Thrombosis Outcomes
Study [CANTOS] is the rst large randomized controlled clinical
study testing whether the use of canakinumab, human anti-IL-1b
monoclonal antibody, can prevent secondary CV events in subjects
with stable coronary artery disease considered at high inamma-
tory risk despite usual therapy [8]. Results of this CANTOS trial are
expected in 2016. If successful, this trial would provide a new
cytokine-based therapy for secondary prevention of CVD.
Another one is the National Heart Lung and Blood Institute
initiated Cardiovascular Inammation Reduction Trial [CIRT], in
which patients with chronic atherosclerosis and either diabetes
mellitus or metabolic syndrome will be randomized to usual care
or usual care plus low-dose methotrexate (1520 mg/week)
[9]. This low-dose methotrexate is routinely and safely used as
an anti-inammatory regimen for the treatment of rheumatoid
arthritis and the primary endpoint is major cardiovascular events.
Results of this clinical trial can be expected in 2018.
Fig. 2. Ongoing clinical trials evaluating anti-inammatory therapies. Two large
clinical trials (CANTOS and CIRT) are examining whether cytokine-based therapies
can accomplish the secondary prevention of cardiovascular events and death.
CANTOS, Canakinumab Anti-inammatory Thrombosis Outcomes Study;
Canakinumab, anti-interleukin-1b antibody; CIRT, Cardiovascular Inammation
Reduction Trial; hsCRP, high-sensitivity C-reactive protein; IL, interleukin; TNF,
tumor necrosis factor.
Please cite this article in press as: Yamashita T, et al. Anti-inammatory and immune-modulatory therapies for preventing
atherosclerotic cardiovascular disease. J Cardiol (2015), http://dx.doi.org/10.1016/j.jjcc.2015.02.002
3
If both or either of the two clinical trials are successful, they
would support the inammatory hypothesis of atherosclerosis and
provide a novel IL-1b-, IL-6-, and CRP-associated cytokine-based
therapy for inhibiting CV events and we could have new anti-
inammatory therapies for secondary prevention of CVD.
Why does continuous chronic inammation exist in
atherosclerotic lesions and how to regulate it?
Numerous studies of human atherosclerotic lesions and
genetically altered mouse strains including the apolipoprotein E
knockout (apoE / ) and LDL receptor knockout (LDLR / ) mice
have provided many important clues on the link between immune
cells and the pathogenesis of atherosclerosis [5]. Following antigen
presentation by antigen-presenting cells such as macrophages or
DCs to nave T cells, adaptive T cell-mediated immune responses
are initiated, and many activated CD4+ T cells are observed in
atherosclerotic lesions in both humans and mice [11,12]. CD4+ T
cell clones which respond to some specic antigens derived from
atherosclerotic plaques have been shown to recognize suspected
self-antigens important for atherosclerosis. To keep the chronic
inammation in the atherosclerotic lesions, several continuous
antigenic stimuli might be needed, although sterile inammation
(inammasome) was also reported to play critical roles in
atherogenesis [13]. The candidate antigens related with athero-
sclerosis are OxLDL, heat shock proteins (HSP), and several foreign
antigens including bacteria such as Chlamydia pneumonia and
Porphyromonas gingivalis [14]. However, the true antigens respon-
sible for driving atherosclerosis remain unidentied.
The elimination of foreign antigens, immunization of autoanti-
gens, or tolerance induction to antigens might alleviate inamma-
tory disease. To eliminate the bacterial antigens, several human
clinical trials using antibiotics for prevention of atherosclerotic
CVD were performed. Unfortunately, no antibiotic clinical trials
were effective for preventing CV events in humans and antibiotics
cannot be considered as anti-atherogenic agents [15]. The
question, whether this is the proof that infections and their
related foreign antigens have no role in atherogenesis or whether
the treatment was too late to start for preventing CV events, is still
unanswered.
The immunization study with OxLDL or modied LDL
demonstrated a remarkable protective effect on atherosclerosis,
reducing lesion area in hypercholesterolemic rabbits and mice by
3050% [1618] (Fig. 3). Since LDL particle contains apoproteins,
mainly apolipoprotein B (apoB), and many lipid species, the key
antigens associated with atherosclerosis could not be identied to
date. In any case, this series of experiments were the rst trial of
immunization (vaccine) therapies against atherosclerosis and they
proposed a new notion that we might prevent CVD via vaccination
with some specic antigens. Immunization with some specic
apoB peptides also reduced atherosclerotic lesion area in mice
[19,20]. Further, whole atherosclerotic plaque homogenates were
used as antigens for immunization in mice [21]. Their immuniza-
tion could induce antibodies against modied LDL and inhibited
atherosclerotic lesion formation. Several reports indicated the
atheroprotective role of anti-oxLDL IgM, but the functional role of
anti-oxLDL IgG remains elusive [18]. We had already reported that
xenogenic macrophage homogenates immunization could reduce
atherosclerosis in mice via inducing anti-macrophage antibodies
that inhibited macrophage functions [22].
The cytosolic chaperon Hsp60 has a high degree of homology to
mycobacterial Hsp65. Different from the case of OxLDL, the
immunization against Hsp60 or Hsp65 aggravate atherosclerosis in
rabbits and mice [23] (Fig. 3). On the other hand, oral administra-
tion or mucosal immunization with Hsp60 could reduce the
atherosclerotic lesions [24,25]. Taken together, immunization with
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Fig. 3. Vaccine therapies against atherosclerosis. Putative athero-antigen candidates are injected or mucosally administered to atherosclerosis mouse models. Parenteral
immunization with OxLDL and its related molecules inhibit atherosclerotic lesion formation. Injection with whole atherosclerotic lesion or xenogenic macrophages also
inhibits atherosclerosis. Oral or nasal administration of Hsp60 or Hsp65 can reduce the atherosclerotic lesion formation, whereas parenteral immunization with them
deteriorates atherosclerosis. OxLDL, oxidized low-density lipoprotein; apoB, apolipoprotein B; Ig, immunoglobulin; Treg, regulatory T cells; Hsp, heat shock protein.

all antigens including atherosclerotic lesions is not always
atheroprotective. The ultimate purpose of this research including
ours is development of anti-atherosclerogenic vaccine to inhibit
atherosclerosis or reduce the incidence of CV events [26]. ApoB
peptides and other antigens as atherosclerosis vaccines are now
clinically tested to develop new immune-therapies against
atherosclerotic CVD [20]. We hope some of the antigens can be
used for anti-atherogenic vaccines, although we should be careful
to apply the experimental results in mice to humans.
Anti-atherogenic strategy by enhancing regulatory immune
response
Recent compelling data suggest that several subsets of Tregs,
responsible for maintaining immunological tolerance and sup-
pressing excessive immune responses [27], inhibit atherosclerosis
development or progression by dampening effector T cell
responses (Fig. 1). Natural Tregs are dened as a population of T
cells expressing CD4, CD25 (IL-2 receptor), and the major
transcription factor forkhead box P3 (Foxp3). These T cells are
differentiated mainly in the thymus and have unique immuno-
suppressive activities without antigen exposure in the periphery.
Deciency of Treg function or development has been shown to
deteriorate atherosclerotic lesion formation [2830]. Other Tregs
are inducible Tregs (iTregs) including type 1 regulatory T cells (Tr1)
and transforming growth factor-b (TGF-b) producing Tregs (Th3).
iTregs are derived from nave or effector T cells and differentiated
in the periphery such as intestine. They are also CD4 CD25-positive
T cells, but do not require Foxp3 to be functional. Tr1 secrete IL-10
and Th3 produce TGF-b. iTregs maintain immunologic homeosta-
sis mainly through secreting immunosuppressive cytokines and
inhibit atherosclerotic lesion formation in mice [3133]. Both
natural and iTregs can prevent autoimmunity and redundant
immune responses by suppressing proliferation of nave and
effector T cells, their differentiation into Th1, Th2, and Th17
lineage, and the function of other types of lymphoid cells including
B cells, macrophages, and DCs. Since Tregs exert suppressive
functions regardless of their antigen specicity after activation by
antigen presentation [27], we speculate that induction of
polyclonal activated Tregs as well as antigen-specic Tregs could
be a possible therapeutic approach to atherosclerosis.
Recently, our clinical study demonstrated that the number of
circulating natural Tregs and the Treg/effector T cell ratio was
decreased in coronary artery disease patients compared with
controls [34]. Other previous papers also indicated that low levels
of circulating Foxp3-positive cells were associated with an
increased risk of coronary events. In consideration of previous
studies, we believe that increasing the Treg/effector T cell ratio, by
suppressing effector T cell responses and increasing the number of
Tregs or promoting Treg responses, could be a promising
therapeutic approach for atherosclerotic CVD [35].
IL-2 has long been thought to be a critical cytokine for T cell
proliferation and differentiation. In particular, it is crucial for the
maintenance of Tregs, because the Treg marker CD25 is a
component of the high-afnity IL-2 receptor and is functionally
essential for the development of Treg by binding IL-2. Low doses of
a recombinant mouse IL-2/anti-IL-2 monoclonal antibody complex
(IL-2 complex) administration induced selective expansion of
CD4+CD25+Foxp3+ Tregs [36]. IL-2 complex therapy was shown to
attenuate atherosclerosis and to inhibit aneurysm formation via
selective Tregs expansion (Fig. 4) [37,38].
Intervention to the Treg/effector T cells ratio
Parental administration of FcR-non-binding anti-CD3 mono-
clonal antibody has been shown to suppress effector T cell immune
responses and be effective for treating autoimmune diabetes in
mice and humans [39], and acute transplant rejection in humans
[40]. Notably, this therapy also inhibits atherosclerosis develop-
ment and progression in atherosclerosis prone mice (Fig. 4)
[41]. Previous studies demonstrated induction of CD4+CD25+
Tregs, along with reduced number of effector T cells, from CD3-Ab

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T. Yamashita et al. / Journal of Cardiology xxx (2015) xxxxxx 5

Fig. 4. Intervention to the effector T cells and/or Tregs for prevention of atherosclerosis. Parenteral anti-CD3 antibody treatment decreases atherosclerosis and regresses
(reduces) atherosclerotic lesions in mice. IL-2 complex (recombinant mouse IL-2/anti-IL-2 monoclonal antibody complex) administration decreases atherosclerosis and
prevents aneurysm formation in mice. Combination treatment with both anti-CD3 antibody and IL-2 complex further decreases atherosclerotic lesion formation compared
with either therapy. IL, interleukin; Treg, regulatory T cells.

treatment, explaining the long-term protective effects observed in
mouse models of autoimmune diseases [42], although this increase
in CD4+CD25+ Treg number was not observed in atherosclerosis-
prone mice [41].
To test the hypothesis that under inammatory conditions such
as hypercholesterolemia, suppression of effector T cell immune
responses before Treg expansion is important in augmenting
regulatory immune responses for efcient reduction in athero-
sclerosis, we employed the combination of anti-CD3 antibody and
IL-2/anti-IL-2 monoclonal antibody complex in apoE / mice. We
demonstrated that this novel combination therapy could effec-
tively reduce atherosclerotic lesion formation and plaque inam-
mation in mice by strikingly enhancing a regulatory immune
response (Fig. 4) [43]. We believe that our ndings are highly
relevant for shaping future clinical strategies for preventing
atherosclerotic diseases.
Recently, we examined the impact of T cell modulation by anti-
CD3 antibody on atherosclerosis regression in a mouse model
previously reported [44], in which LDLR / mice were fed a high-
cholesterol diet for 8 weeks to form atherosclerotic lesions and
were then changed to a standard diet to lower plasma cholesterol.
We demonstrated for the rst time that intravenous administra-
tion of anti-CD3 antibody, in addition to lowering plasma
cholesterol, could induce rapid regression of atherosclerosis by
reducing CD4+ T cells and increasing the proportion of Foxp3+
Tregs in both lymphoid organs and atherosclerotic plaques
[45]. Depletion of Tregs by anti-CD25 antibody injection abolished
the anti-CD3 antibody-mediated atherosclerosis regression, indi-
cating the essential role for Tregs in this process.
Intestine as a therapeutic target
Based on the experimental results that oral administration or
mucosal immunization of Hsp60 or Hsp65 inhibited atheroscle-
rotic lesion formation in animal models [24,25], we have been
interested in the intestinal mucosal immunity and we thought that
the intestine could be a therapeutic target for preventing
atherosclerosis. Further, we focused on the mechanism of oral
tolerance to apply its mechanism for prevention of atherosclerosis
[46,47]. Accumulating evidence indicated that DCs and Tregs play
critical roles in maintaining the tolerance (Fig. 5).
Tregs differentiate from nave T cells in the periphery under
certain conditions, as well as in the thymus [48]. Gut-associated
lymphoid tissue (GALT) is known to be the main site for generation
of peripheral iTregs, where there are multiple preferential stimuli
such as commensal microbiota and food antigens for the induction
of Tregs [46]. It was demonstrated that iTreg might play a crucial
role in the maintenance of mucosal immune tolerance and in the
suppression of allergic inammation in the intestine. However, the
differences between iTregs and natural Tregs or functional stability
of iTregs in vivo remain to be determined, and further studies are
needed.
The intestinal immune system must protect our body from
invading pathogens, but never attacks food antigens and com-
mensal bacteria. Recent studies have revealed that DCs in the small
intestine have a crucial role in determining inammatory or
tolerogenic immune responses, but distinct subtypes have not
been clearly dened. In the small intestine, DCs are identied by
their CD11c expression and can be divided into two subsets
depending on their CD80 and CD86, or CD103 expressions
[46,47]. CD80+CD86+ DCs are thought to be active and mature
DCs and work as immune activated APCs. On the other hand,
CD80 CD86 DCs are immature or tolerogenic DCs and induce
tolerance in the intestine together with Tregs [46]. CD103+ DCs can
be divided into two subsets that do or do not express CD11b.
Lamina propria CD103+CD11b+ DCs capture antigens and migrate
into mesenteric lymph nodes. Although the precise function of
CD103+CD11b DCs in GALT for regulating Tregs remains to be
determined, lamina propria CD103+CD11b+ DCs promote the
generation of Tregs through the production of TGF-b and vitamin A

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Fig. 5. Intestinal immune system as a possible therapeutic target for controlling inammatory diseases including atherosclerosis. Several types of Tregs and tolerogenic DCs
play critical roles in maintaining oral tolerance in the intestine. Oral administration of anti-CD3 antibody and active vitamin D3 induces Tregs and tolerogenic DCs in
mesenteric lymph nodes or gut-associated lymphoid tissues (GALT). Some DCs in the intestine have a crucial role in determining tolerogenic immune responses by prompting
the generation of Tregs. Inducible Treg (iTreg) is reported to differentiate mainly in the intestine. Foxp3+ Tregs inhibit antigen presentation through a cell-contact dependent
manner or production of anti-inammatory cytokines (IL-10 or TGF-b). This phenomenon looks like oral tolerance and results in inhibition of atherosclerotic lesion formation
in atherosclerosis-prone mice. Enhancing Treg-mediated immune responses by modulating intestinal immunity could be a promising therapeutic approach for preventing
atherosclerosis. LAP, latency-associated peptide; Treg, regulatory T cell; DC, dendritic cell; IL, interleukin; TGF, transforming growth factor.

metabolite retinoic acid. CD103+ DCs regulate the balance between
immunity and tolerance in the intestine. Elucidation of the
mechanisms of how different subtypes of intestinal DCs respond
to various antigens and determination of the response toward
either immunity or tolerance will contribute to effective tolerance
induction therapy.
Studies have shown that oral or nasal anti-CD3 antibody is
biologically active and induces TGF-b producing CD4+LAP+
(latency-associated peptide) Tregs = Th3 that suppress experimen-
tal autoimmune encephalitis (EAE) [49] and autoimmune diabetes
[50] in a TGF-b dependent fashion. Autoimmune diseases are
shown to be suppressed by only low doses of oral anti-CD3
antibody in association with an increase in LAP+ Tregs, but not by
high doses, although there is no evidence of antigen specicity
with oral anti-CD3 antibody [49]. As observed in mucosal tolerance
induction, only low doses of oral anti-CD3 antibody administration
may result in the induction of Th3 by delivering a weak signal to T
cells, although further elucidation of the cellular and molecular
mechanisms underlying induction of various Tregs will be needed.
We applied this method for the treatment of atherosclerosis in
apoE / mice and demonstrated that oral anti-CD3 antibody
treatment induced Th3 and CD4+Foxp3+ Tregs, which suppressed
pathogenic immune processes pivotal for atherogenesis through a
TGF-b-dependent mechanism and consequently inhibited athero-
sclerotic plaque formation [33]. Further, we examined the effect of
oral anti-CD3 antibody treatment on the phenotypes of DCs in the
mesenteric lymph nodes in mice and conrmed the CD80 and
CD86 expressions were reduced in anti-CD3 antibody-treated mice
compared to controls (Fig. 5).
Some immature DCs acquire tolerogenic properties by inducing
Tregs and inhibiting nave T cell activation. Epidemiological
studies have highlighted the increasing prevalence of vitamin
D3 deciency and its association with increased risks of CVDs and
mortality [51]. However, the effect of vitamin D3 supplementation
on prevention of CVDs remains controversial in clinical trials and
no reports have been published about the direct effects of orally
administrated calcitriol on atherosclerosis in animal models. We
for the rst time demonstrated that oral administration of active
form of vitamin D3 (calcitriol) decreases atherosclerosis by
promoting induction of tolerogenic DCs and Foxp3+ Tregs
[52]. A cell-therapy strategy, using tolerogenic DCs, revealed that
apoB100-pulsed tolerogenic DCs inhibit the proliferative and
proinammatory T cell response to apoB100, promoted Treg
induction, and reduced atherosclerotic lesion formation [53]. Oral
administration of Hsp60 might also induce Tregs in gastrointesti-
nal tract and affect atherogenesis [25,54].
Although further studies are needed to clarify the precise role of
several types of vascular DCs in atherogenesis, effective methods to
induce atheroprotective DCs could be novel therapies for preven-
tion of atherosclerosis.
Concluding remarks
Since the rst evidence of involvement of immunity in
atherogenesis was discovered more than 30 years ago, a large
number of basic and clinical studies have clearly established that
immune responses play crucial roles in the disease process.
However, none of the approaches to specically and directly treat
inammation to prevent cardiovascular events or reduce athero-
sclerosis in human individuals were successful to date, although
hsCRP has been shown to have a strong relationship with recurrent
events of CVD, and statin treatment could reduce hsCRP and

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prevent CV events independent of lowering LDL cholesterol in
several randomized clinical trials such as the JUPITER study
[10]. Two large clinical trials of direct anti-inammatory therapies
for CVD [8,9] are now being conducted and we might use the
results in our patients for preventing CV events in the near future.
Vaccination against atherosclerosis is a hopeful method for
prevention of CVD and resultingly reducing medical expenses.
Vaccines targeting autoimmunity including atherosclerosis are
tolerance induction against self-antigens and amplication of the
regulatory responses such as Tregs and tolerogenic DCs described
in this review. Since the tolerance induction can yield an
immunosuppressed individual with impaired tumor surveillance
and increased susceptibility to infections, we should be careful to
apply these results of animal research to human clinical situations.
A hopeful method to resolve this problem may be antigen-specic
Treg induction that many researchers including us wish to achieve.
The intestinal immune system has been attracting much
attention as a novel therapeutic target to treat atherosclerosis.
Gut-associated immune tolerance induction by oral administra-
tion of drugs or therapeutic agents possessing immunoregulatory
activities is simple, easy, and a hopeful way to regulate
inammatory diseases, although the detail of mechanisms of
how intestinal immunity affects the systemic immune system
remains to be claried. In association with intestinal immunity in
atherogenesis, we have been interested in the gut bacteria that
might involve the pathogenesis of atherosclerotic CVD. Now the
gut ora types susceptible to CVD are under investigation.
The immune system represents a novel and promising target for
prevention or treatment of CVD. The time when this concept is
ready for clinical testing is coming, but it will be critical not to
underestimate the difculties that will be encountered in
transferring the promising results from experimental animals to
humans.
Conict of interest
The authors report no conict of interest.
Sources of funding
This work was supported by Japan Society for the Promotion of
Science KAKENHI Grant Nos. 24591114 (T.Y.), 23790849,
25860601 (N.S.), Suzuken Memorial Foundation (T.Y. and N.S.),
ONO Medical Research Foundation (N.S.), Takeda Scientic
Foundation (T.Y. and N.S.), Kanae Medical Foundation (N.S.),
Mochida Memorial Foundation (T.Y.), Senshin Medical Research
Foundation (T.Y. and N.S.), Mitsui Life Social Welfare Foundation
(T.Y.), Yakult Bioscience Research Foundation (T.Y.), Uehara
Memorial Foundation (K.H. and N.S.), Japan Heart Foundation/
Novartis Grant for Research Award on Molecular and Cellular
Cardiology 2012 (N.S.), Banyu Life Science Foundation Interna-
tional (N.S.), and The Japan Circulation Society Translational
Research Foundation (K.H.).
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