Interleukin-10 protects against atherosclerosis by modulating multiple atherogenic macrophage function

Abstract

Atherosclerosis is primarily a disorder of lipid metabolism, but there is also a prominent chronic inflammatory component that drives the atherosclerotic lesion progression in the artery wall. During hyperlipidaemic conditions, there is a rapid influx of circulating monocytes into the atherosclerosis-prone areas of the arterial intima. These infiltrated monocytes differentiate into macrophages and take up the atherogenic lipoproteins in the intima of the vessel wall that have been modified within the lesion environment. Interleukin (IL)-10 is a prototypic anti-inflammatory cytokine made primarily by the macrophages and Th2 subtype T lymphocytes. In terms of atherosclerosis its major roles include inhibition of macrophage activation as well as inhibition of matrix metalloproteinase, pro-inflammatory cytokines and cyclooxygenase-2 expression in lipid-loaded and activated macrophage foam cells. Recent discoveries suggest another important role of IL-10 in atherosclerosis: its ability to alter lipid metabolism in macrophages. The current review will highlight the present knowledge on multiple ways in which IL-10 mediates atherosclerosis. As macrophages play a critical role in all stages of atherosclerosis, the review will concentrate on how IL-10 regulates the activities of macrophages that are especially important in the development of atherosclerosis.

Full text

© Schattauer 2015 Thrombosis and Haemostasis 113.3/2015
505Theme Issue Article
Interleukin-10 protects against atherosclerosis by modulating multiple
atherogenic macrophage function
Xinbing Han1; William A. Boisvert2,3
1Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; 2Center for Cardiovascular Research, John A. Burns School of
Medicine, University of Hawaii, Honolulu, Hawaii, USA; 3Kazan Federal University, Kazan, Russia
Summary
Atherosclerosis is primarily a disorder of lipid metabolism, but there is
also a prominent chronic inflammatory component that drives the
atherosclerotic lesion progression in the artery wall. During hyperlipi-
daemic conditions, there is a rapid influx of circulating monocytes into
the atherosclerosis-prone areas of the arterial intima. These infiltrated
monocytes differentiate into macrophages and take up the athero-
genic lipoproteins in the intima of the vessel wall that have been
modified within the lesion environment. Interleukin (IL)-10 is a proto-
typic anti-inflammatory cytokine made primarily by the macrophages
and Th2 subtype T lymphocytes. In terms of atherosclerosis its major
roles include inhibition of macrophage activation as well as inhibition
of matrix metalloproteinase, pro-inflammatory cytokines and cyclo-
oxygenase-2 expression in lipid-loaded and activated macrophage
foam cells. Recent discoveries suggest another important role of IL-10
in atherosclerosis: its ability to alter lipid metabolism in macrophages.
The current review will highlight the present knowledge on multiple
ways in which IL-10 mediates atherosclerosis. As macrophages play a
critical role in all stages of atherosclerosis, the review will concentrate
on how IL-10 regulates the activities of macrophages that are es-
pecially important in the development of atherosclerosis.
Correspondence to:
William A. Boisvert
Center for Cardiovascular Research
John A. Burns School of Medicine
University of Hawaii
651 Ilalo Street
Honolulu, HI 96813, USA
Tel.: +1 808 692 1567, Fax: +1 808 692 1973
E-mail: wab@hawaii.edu
Received: June 11, 2014
Accepted after minor revision: August 22, 2014
Epub ahead of print: November 6, 2014
http://dx.doi.org/10.1160/TH14-06-0509
Thromb Haemost 2015; 113: 505–512
Introduction
Cardiovascular diseases, including coronary artery disease (CAD),
stroke, abdominal aortic aneurysms, and many cases of heart fail-
ure collectively account for the largest rate of mortality in the
Western world (1). Atherosclerosis is the common cause of all of
these diseases, which is well known as a disorder of lipid metab-
olism but with a prominent inflammatory component as well. Per-
sistent chronic inflammation fuels the atherosclerotic lesion pro-
gression in the artery wall throughout the different stages of the
disease (2–5), from early fatty streak to advanced fibro-fatty
plaque formation. During hyperlipidaemic conditions, there is a
rapid influx of circulating monocytes into the atherosclerosis-
prone areas of the arterial intima which differentiate into macro-
phages and take up the atherogenic cholesteryl ester-rich lipopro-
teins in the intima of the vessel wall that have been modified with-
in the lesion environment (6–8). The accumulation of cholesterol-
loaded macrophages in the arterial wall called “foam cells” is a key
feature of early atherosclerotic lesions (9). The importance of these
foam cells is illustrated by their participation in every stage of
atherosclerosis and their ability to trigger an acute thrombotic
event (1). As the most numerous inflammatory cell type in the
plaque, macrophages are the most important source of cytokine
production in the lesion environment (10) and can produce pro-
inflammatory cytokines such as tumour necrosis factor (TNF)-α,
interleukin (IL)-1, IL-6, IL-12, IL-15, IL-18, as well as anti-inflam-
matory cytokines such as IL-10 and transforming growth factor-β
(TGF-β). Many studies have shown that pro-inflammatory cyto-
kines promote the development of atherosclerosis (4) whereas
anti-inflammatory cytokines like TGF-β (11) and IL-10, as will be
discussed in detail below, can have an anti-atherogenic effect.
IL-10 is a prototypic anti-inflammatory cytokine made pri-
marily by the macrophages and T lymphocytes of the Th2 subtype.
In terms of atherosclerosis its major roles include inhibition of
macrophage activation as well as inhibition of matrix metallopro-
teinase (MMP), pro-inflammatory cytokines and cyclooxyge-
nase-2 expression in lipid-loaded and activated macrophage foam
cells (12). More recently, another important role of IL-10 in
atherosclerosis has emerged by identifying its ability to alter lipid
metabolism in macrophages. The current review will highlight the
present knowledge on how IL-10 mediates atherosclerosis. As
macrophages play a critical role in the pathogenesis of athero-
sclerosis, the review will concentrate on how IL-10 regulates the
activities of macrophages that are important in the development of
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506 Han, Boisvert: Athero-protective role of IL-10
atherosclerosis. Identification of the mechanisms that regulate
these responses could be invaluable in the development of new
therapeutic approaches to prevent and/or treat atherosclerosis.
IL-10 modulates multiple atherogenic
macrophage functions
IL-10 exerts its anti-atherogenic effects on plaque development
throughout the different stages of atherosclerosis by influencing
the local inflammatory process within the atherosclerotic lesion.
IL-10 is produced within the atherosclerotic lesion predominantly
by macrophages where it could play a significant role in the modu-
lation of the local inflammatory reaction on both macrophages
and T cells (13).
Macrophages play a central role during all stages of athero-
sclerosis (14). Atherogenesis is initiated with the recruitment of
monocytes to the intima, followed by inflammatory activation
which differentiates the recruited monocytes into macrophages.
The intimal macrophages can then take up modified low-density
lipoproteins (LDL) particles such as oxidised LDL (oxLDL)
through upregulated scavenger receptors, thereby promoting cho-
lesterol loading and foam cell formation in the plaque’s core. Lipid-
loaded macrophages make multiple pro-inflammatory mediators,
reactive oxygen species (ROS), and pro-coagulants that promote
local inflammation as well as thrombotic complications.
An early study on IL-10 and atherosclerosis showed detection
of IL-10 mRNA by RT-PCR in four of five human atherosclerotic
tissues but not in plaque-free aortic specimens (15). The presence
of IL-10 mRNA was subsequently verified in 12 of 17 advanced
human atherosclerotic plaques, mainly in macrophages (13).
These studies suggest that progressive inflammation during
atherosclerosis may induce macrophages to express IL-10.
The mechanisms by which IL-10 may protect against athero-
genesis can be categorised into five general aspects of macrophage
function as follows: 1) anti-inflammatory properties, 2) inhibition
of matrix metalloproteinases (MMPs) and tissue factor (TF), 3)
anti-apoptotic feature, 4) effects on macrophage polarisation, and
5) modulation of lipid metabolism. IL-10’s role in each of these
properties of macrophages is described in detail below.
Anti-inflammatory properties of IL-10
As a potent anti-inflammatory cytokine, increased IL-10 serum
level is a beneficial prognostic determinant in patients with acute
coronary syndromes (16). A number of studies have shown that
IL-10 expression by plaque macrophages limits the inflammatory
response and promotes plaque healing by inhibiting IL-12 (15) and
iNOS production (13, 17). Attenuation of atherogenesis by IL-10 is
attributed to its anti-inflammatory effects, most notably its ability
to inhibit the release of several pro-inflammatory cytokines (in-
cluding IL-1β, TNF-α, and IL-8) from monocytic cells, and to in-
duce the production of IL-1 receptor antagonist (18). As IL-10 re-
ceptor blockage in macrophages results in significantly higher nu-
clear factor (NF)-kB activation (19), IL-10’s ability to suppress the
expression of inflammatory mediators such as TNF-α, MCP-1
(monocyte chemotactic protein 1) and ICAM-1 (intracellular ad-
hesion molecule 1) is likely attributed to the inhibition of NF-κB
activity (20). IL-10 also suppresses the production of the chemo-
kine, KC/GRO-α (21) which is implicated in intimal macrophage
accumulation and the progression of complex atherosclerotic
lesions in advanced disease (22). Inhibition of both MCP-1 and
ICAM-1 by IL-10 (23–25) is an important property of IL-10 that
may inhibit monocyte influx into the plaque area and thereby curb
the disease development. Recently, microRNA-155 has been
shown to promote atherosclerosis by inhibiting the expression of
BCL6 in macrophages (26). As IL-10 has been shown to suppress
microRNA-155 (27), it is possible that one of the ways that IL-10
mediates its anti-atherogenic role is by inhibiting this microRNA
in macrophages..
Inhibition of matrix metalloproteinases and tissue
factor
Extracellular matrix content of a plaque is intimately associated
with how vulnerable the plaque is to rupture. Clinically unstable
atherosclerosis is associated with the activation of local inflamma-
tory and immune cells with increased expression of MMPs (28)
and TF (29) in the culprit plaque as well as increased systemic pro-
duction of MMPs (30) and thrombin (31). Within atherosclerotic
lesions macrophages are important sources of MMPs, including
MMP-2, MMP-8, MMP-9, MMP-12, MMP-13, and MMP-14 (4,
32). MMPs influence lesion progression by degrading extracellular
matrix proteins which can lead eventually to the development of
unstable, rupture-prone atherosclerotic lesions (4, 33). However, it
appears that not all MMPs promote unstable plaques and there are
conflicting reports of their effects on plaque stability in the litera-
ture. While an early study suggests that remodelling of the neointi-
mal extracellular matrix by MMP-1 is beneficial in the progression
of lesions (34), other studies suggest that MMP-1 and MMP-9 con-
tribute to the weakening of fibrous caps and plaque disruption
leading to the destabilisation of atherosclerotic plaques (35, 36).
Another study highlighting the widely differing effects of MMPs
on atherogenesis shows MMP-3 and MMP-9 to play protective
roles by limiting plaque growth and promoting a stable plaque
phenotype, while MMP-12 supports lesion expansion and desta-
bilisation, and MMP-7 does not have effect on plaque growth or
stability (37).
Some studies suggest that IL-10 may have protective effects
against plaque rupture and thrombus formation. IL-10 can inhibit
the secretion of MMP9 (38, 39), the synthesis of TF (40), and the
production of thrombin (41) from PBMC and macrophages. Low
collagen synthesis and increased activity of macrophage-derived
MMPs are responsible for fibrous cap thinning and fragility.
Therefore, low levels of IL-10 may lead to augmented MMP activ-
ity which may in turn promote plaque instability and acute cardio-
vascular events in certain individuals (38). On the other hand, pro-
inflammatory cytokines like interferon (IFN)-γ may destabilise
plaques by inhibiting collagen production (42) in human vascular
smooth muscle cells, and also by stimulating MMP production in
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507Han, Boisvert: Athero-protective role of IL-10
macrophages (28) and modulating the fibrinolytic response of en-
dothelial cells (43).
Anti-apoptotic properties
Macrophages and other cell types in the atherosclerotic plaque
constantly undergo apoptosis and necrosis which may accelerate
atherosclerosis by releasing lipids and inflammatory mediators
from the macrophages to the plaque and thereby contributing to
the formation of the necrotic core. IL-10’s anti-apoptotic proper-
ties have been reported in cultured macrophages (24, 25) and in T
lymphocytes (44). The production of ROS is increased in athero-
sclerotic arteries (45), leading to endothelial damage, oxidation of
lipid components (46), and recruitment of inflammatory cells to
the site of injury. Inflammatory nitric oxide has apoptotic effects
(47) and can induce cell death, at least in part through local per-
oxynitrite formation (48). IL-10 can also activate signal transducer
and activator of transcription 3 (STAT3), which suppresses endo-
plasmic reticulum stress-induced apoptosis in lipid-laden macro-
phages by increasing the expression of anti-apoptotic genes like
Bfl-1 and Mcl-1 (49, 50). As excessive accumulation of free choles-
terol in cells causes apoptosis, one other way in which IL-10 may
exert its anti-apoptotic effects is by stimulating ABCA1/ABCG1
production which increases cholesterol efflux from lipid-laden
foam cells (24, 25, 51).
Effects on macrophage polarisation
When responding to certain stimuli such as inflammatory medi-
ators or microbial products, macrophages have the ability to be
polarised into one of two subtypes: classically activated M1 and al-
ternatively activated M2 form (52). Macrophages are polarised
into M1 subtype by IFN-γ, microbial stimuli (e. g. lipopolysacchar-
ide) or cytokines such as TNF-α and GM-CSF, whereas M2 macro-
phages are induced by IL-4, IL-13, immune complexes, glucocorti-
coid or secosteroid (vitamin D3) hormones (53, 54). M1 macro-
phages express low levels of IL-10 and high levels of IL-12 and
IL-23. By contrast, M2 macrophages express abundant IL-10 and
low levels of IL-12 and IL-23. A recent publication shows that
IL-10 induces macrophage polarisation toward the M2 phenotype
(55).
M1 and M2 macrophages can both be detected in athero-
sclerotic lesions (56, 57). Anti-inflammatory M2 macrophages are
more susceptible to foam cell formation than pro-inflammatory
M1 macrophages, and exposure of macrophages to oxLDL renders
M2 macrophages pro-inflammatory (58). Although it is not en-
tirely clear at this point how macrophage polarisation affects
atherosclerosis, there is an indication that M2 phenotype may
exert an athero-protective action in experimental atherosclerosis
(56). Although M2 macrophages dominate at the initial stages of
atherosclerosis, macrophage phenotypic switch from M2 to M1
occurs with lesion progression (57). Furthermore, M1 macro-
phages dominate over M2 macrophages in the rupture-prone
shoulder regions of the plaque, whereas M2 polarised cells are
found in stable plaques (57). This concept is further supported by a
recent finding that thioredoxin-1, an oxidative stress-limiting pro-
tein with anti-inflammatory and anti-apoptotic properties, pro-
motes M2 macrophage polarisation and antagonises athero-
sclerosis (59). Another study suggests that helminth-derived
antigens reprogram macrophages to M2 phenotype which reduces
murine atherosclerosis (60).
Modulation of lipid metabolism
As macrophages accumulate lipid and become foam cells during
atherosclerosis, their properties can change. OxLDL can promote
immune activation by inducing pro-inflammatory cytokines IL-12
and TNF-α as well as IL-10 production by mononuclear leuko-
cytes from human atherosclerotic plaque (61). Induction of IL-10
in lipid-laden macrophages may be an indication that IL-10 may
be involved in lipid metabolism in these cells. During foam cell
formation, two steps are critical in maintaining lipid homeostasis
in macrophages: cholesterol uptake mediated by scavenger recep-
tors, and cholesterol efflux mediated by ABCA1/ABCG1. Scav-
enger receptors such as CD36 and scavenger receptor A (SR-A) on
macrophages mediate the uptake of modified lipoproteins from
the vessel wall (62). On the other hand, reverse cholesterol trans-
port via ABCA1 and ABCG1 is critical to export the cytotoxic cel-
lular free cholesterol to lipid-poor apoA1 and lapidated high-den-
sity lipoprotein (HDL) particles (63). It is well documented that
cholesterol efflux via ABCA1 and ABCG1 is essential to slow the
development of atherosclerosis by decreasing lipid loading (64,
65). Although the role of scavenger receptors appears confusing
because of conflicting results from gene knockout or transgenic
mouse studies as reviewed by Hansson and Hermansson (66), sev-
eral recent publications demonstrated that these receptors are pro-
tective against atherosclerosis due to their ability to remove modi-
fied LDL from the vessel wall (67, 68).
There is a wealth of evidence that IL-10 can influence cellular
lipid metabolism by facilitating both cholesterol uptake and cho-
lesterol efflux (reverse cholesterol transport). In 2005, it was re-
ported that IL-10 enhances oxLDL-induced formation of macro-
phage foam cells as well as inhibits apoptosis, albeit indirectly, by
increasing the expression of anti-apoptotic genes Bfl-1 and Mcl-1
(49). Soon after, it was demonstrated that IL-10 not only stimulates
ABCA1/ABCG1 function, aiding in cholesterol efflux from lipid-
laden foam cells, but inhibits CD36-mediated oxLDL uptake by
macrophages, both of which would lead to preventing foam cell
formation (51). These seemingly conflicting findings were some-
what clarified by our study in 2009 in which we reported that
IL-10 modulates lipid metabolism in macrophages by facilitating
both cholesterol uptake and efflux (24). Our results showed that
IL-10 can concomitantly up-regulate ABCA1 in a PPAR-γ-de-
pendent mechanism and increase the expression of scavenger re-
ceptors (SR-A and CD36). In support of this another group re-
ported that IL-10 stimulates the expression of scavenger receptors
and enhances foam cell formation (69). Collectively, these findings
support the hypothesis that enhanced cholesterol uptake mediated
by IL-10 may be athero-protective by actively removing the highly
atherogenic modified lipoproteins from the artery wall. At the
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508 Han, Boisvert: Athero-protective role of IL-10
same time, IL-10-mediated increase in ABCA1-dependent choles-
terol efflux is important for the efficient disposal of cytotoxic free
cholesterol through reverse cholesterol transport. Interestingly, it
was suggested recently that anti-inflammatory M2 macrophages
but not pro-inflammatory M1 macrophages rapidly accumulate
oxLDL (58). Because IL-10 is a known promoter of M2 macro-
phage polarisation as mentioned above (55, 70, 71), it is likely that
IL-10’s role in lipid accumulation is predominantly in M2 macro-
phages. Overall, there is a wealth of data to suggest a comprehen-
sive anti-atherogenic role of IL-10 in macrophages, including its
role in lipid homeostasis along with a more traditional role of
IL-10 in inhibiting inflammatory molecules (e. g. TNF-α, iCAM-1,
and MMP9) and reducing apoptosis (24, 25). A cartoon demon-
strating the multi-faceted anti-atherogenic role of IL-10 in macro-
phages is shown in
▶
Figure 1.
Human studies and in vivo animal models
The role of IL-10 in atherosclerosis has been investigated using dif-
ferent animal models as listed in
▶
Table 1.
It was first described in 1996 that IL-10 was present in human
atherosclerotic lesions and that ox-LDL induced IL-10 release
from monocytes in vitro (15). Interestingly, the inhibition of IL-12
by IL-10 observed in this study suggests that the balance between
IL-12 and IL-10 production likely contributes to the level of im-
mune-mediated tissue injury in atherosclerosis. Activated mono-
cytes produce IL-12 and IL-10 that regulate the Th1 and Th2 re-
sponses, respectively. IL-12 can act as a T cell growth factor that
selectively induces the Th1 cytokine pattern. One of the roles of
IL-10 is to inhibit the local production of IL-12 which may po-
tentiate the chronic inflammatory Th1 cell and macrophage re-
sponses leading to tissue injury in atherosclerosis (15). The issue of
athero-regulation by both IL-12 and IL-10 was further compli-
cated by IL-12 being expressed at an earlier stage of atherosclerosis
than IL-10 in apoE-/- mice (81). This suggests that IL-12 and
IL-10 may have distinct roles in regulating the immune response
during different stages of the disease.
The expression and potential effects of IL-10 in advanced
human atherosclerotic lesions was reported in 1999 (13). Immu-
nostaining from this study indicated that the main source of IL-10
in advanced human atherosclerotic plaques is the macrophage.
The local anti-inflammatory response of IL-10 and its inhibitory
effects on excessive cell death in the plaque was indirectly shown
by the observation that IL-10 expression was associated with low
levels of inducible nitric oxide synthase (iNOS) expression and cell
death.
IL-10-deficient and IL-10-overexpressing murine models on
either apoE-/- or LDLR-/- background have greatly advanced our
understanding of how IL-10 might modulate atherogenesis. In
1999, two groups independently showed athero-protective proper-
ties of IL-10 (72, 73). Many other groups since then have utilised
various animal models and IL-10 delivery systems to understand
how IL-10 affects atherosclerosis. The first study involving athero-
Figure 1: Cartoon
depicting the diverse
role of IL-10 in macro-
phages during athero-
sclerosis. Upon binding
to its receptor, IL-10 up-
regulates scavenger re-
ceptors, SR-A and CD36,
which facilitates modified
LDL uptake by macro-
phages and promotes
cholesteryl ester accumu-
lation and foam cell
formation. IL-10 also pro-
motes ABCA1-mediated
free cholesterol efflux to
apoAI in a PPAR
γ
-de-
pendent manner. As a
prototypic anti-inflamma-
tory cytokine, IL-10 sup-
presses the expression of
inflammatory mediators
such as TNF-
α
, MCP-1
and iCAM-1, presumably
through the inhibition of
NF-
κ
B activity (20), and
diminishes apoptosis in
the lipid-laden foam cells
(24).
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509Han, Boisvert: Athero-protective role of IL-10
genic diet-fed IL-10-deficient mice showed increased lipid ac-
cumulation, T-cell infiltration, IFN-γ expression, as well as de-
creased collagen in the lesion compared with wild-type mice (72).
Intramuscular electrotransfers of IL-10 plasmid DNA resulted in a
60 % reduction in lesion size in IL-10-deficient mice. Another
group found that diet-induced atherosclerotic lesions were larger
in IL-10-/- mice than in control mice (73). They also observed that
transgenic murine IL-10 expression driven exclusively in T cells by
human IL-2 promoter significantly attenuated atherosclerosis de-
velopment (73).
In 2001, von der Thusen et al. reported that increased plasma
concentrations of IL-10 as a result of adenoviral gene transfer in
LDLR-/- mice led to reduction in atherosclerotic lesion size by in-
hibiting the production of TNF-α (25, 74). The mechanism in-
volves inhibition of anti-inflammatory TNF-α production by IL-10
(25, 74). Concordantly, Pinderski et al. demonstrated that overex-
pression of IL-10 by activated T lymphocytes attenuated lesion
formation by driving the shift to a Th2 phenotype with decreased
IFN-γ production (by peripheral blood lymphocytes, splenocytes,
and circulating monocytes) (75). Alteration of macrophage func-
tion was exhibited by markedly decreased apoptosis in macro-
phage foam cells within the lesions of IL-10 transgenic mice (75).
The athero-protective results obtained with IL-10-deficient
mice on the C57BL/6J background (72, 73) were confirmed in
IL-10 and apoE double knockout mice as demonstrated by Cali-
giuri et al. (31). Several significant findings were revealed by this
study: 1) Th-1 response and lesion size were dramatically in-
creased in double knockout mice compared with apoE-/- controls
at the early phase of lesion development, 2) the proteolytic and
procoagulant activity was elevated in advanced lesions as indicated
by an increase in TF and MMP activities, suggesting that IL-10
may reduce atherogenesis and improve the stability of plaques, and
3) lipid metabolism regulated by IL-10 was implicated in this study
as LDL cholesterol was increased but VLDL was decreased in the
double knockout mice without significant changes in total choles-
terol or triglyceride levels (31).
In an attempt to utilise IL-10 as a therapeutic agent, several
techniques have been used by different groups to deliver the IL-10
gene in vivo. One study showed that intramuscular gene transfer of
IL-10 cDNA reduces atherosclerotic lesion formation in apoE-/-
Table 1: The role of IL-10 in atherosclerosis investigated using different animal models.
Publication
Mallat et al. 1999, Circ Res (72)
Pinderski Oslund et al. 1999, ATVB (73)
Von der Thusen et al. 2001, FASEB J (74)
Pinderski et al. 2002, Circ Res (75)
Caligiuri G et al. 2003,
Mol Med (31)
Yoshioka et al. 2004, Gene Ther (77)
Liu et al. 2006, Atherosclerosis (78)
Namiki et al. 2004, Atherosclerosis (76)
Han X, et al. 2010, FASEB J (25)
Du L, et al. 2011, Human Gen Therapy (79)
Sun J, et al. 2011, PloS One (80)
Approach
IL-10-encoding plasmid transferred to muscle
cells using electrotransfer procedures
Murine IL-10 transgene under the control of
human IL2 promoter (overexpression of IL-10
by T cells)
Systemic adenovirus-mediated transfer of IL-10
Systemic overexpression of IL-10 by T cells,
bone marrow transplantation
IL-10 deficiency
Systemic delivery of adeno-associated virus
vector (tibial muscle injection)
Systemic delivery (tail vein injection)
Transfer of murine IL-10 cDNA plasmid to
femoral muscle with Hemagglutinin virus of
Japan (HVJ)-liposome
Overexpression of IL-10 by macrophages, bone
marrow transplantation
Expression of IL-10 in carotid arteries achieved
with helper-dependent adenoviral vector
Magnetic resonance imaging bone marrow cells
transduced by IL-10 /lentivirus, bone marrow
transplantation
Animal model
C57BL/6 mice
C57BL/6J mice
LDLR-/- mice
LDLR -/- mice
ApoE -/- mice
ApoE-/- mice
LDLR-/- mice
ApoE-/- mice
LDLR-/- mice
Rabbit
ApoE-/- mice
Underlying mechanism
Inhibit inflammation, plaque collagen content
and stability
Block monocyte adhesion to human aortic
endothelial cells
Monocyte deactivation by inhibition of TNF-
α
and lowering of serum cholesterol levels
Polarisation to Th2 phenotype; lowered
activation of monocytes; decreased apoptosis
of macrophage foam cells within lesion
Increased Th1 response; increased TF and
MMP activity; increase in LDL and decrease in
vLDL in IL-10-/-ApoE-/- mice
Inhibition of inflammation and oxidative stress
Anti-inflammatory (MCP-1) and cholesterol-
lowering effects
Reduced macrophage infiltration and altered
Th1 response
Inhibition of inflammation and apoptosis;
modulation of lipid metabolism in foam cells
(both lipid uptake and cholesterol efflux)
No athero-protective effect
Bone marrow cells transduced by IL10
entivirus were recruited to atherosclerotic
lesions and prevented the progression of
atherosclerosis.
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510 Han, Boisvert: Athero-protective role of IL-10
mice (76). IL-10 gene transfer quelled the Th1 response by in-
hibiting IL-12 and IFN-γ expression in transgenic mice (76).
These results were confirmed in another study in which adeno-as-
sociated virus vector-mediated IL-10 gene transfer via intramus-
cular injection inhibited atherosclerosis in apoE-/- mice (77) by
lowering MCP-1 expression in both the vascular wall of the as-
cending aorta and serum. Similar results were observed in apoE-/-
mice transplanted with bone marrow cells transduced by IL-
10/lentivirus (80). In agreement with these results, a systemic de-
livery of adeno-associated virus type 2-hIL-10 inhibited athero-
genesis in LDLR-/- mice by combating inflammation and oxidative
stress (78). Similar effects of IL-10 overexpression on neointima
formation were seen in the hypercholesterolaemic apoE*3-Leiden
mice as well (82).
Anti-atherosclerotic properties of IL-10 were further displayed
in high fat diet-fed LDLR-/- mice in which IL-10 was overexpressed
in macrophages by utilising a macrophage-specific retroviral vec-
tor that allows long-term in vivo expression of IL-10 in macro-
phages through transplantation of retrovirally transduced bone
marrow cells (BMCs) (25). The IL-10 expressed by macrophages
in the plaques derived from transduced BMCs inhibited athero-
sclerosis in these mice, at least in part by reducing inflammation
and apoptosis in IL-10-overexpressing macrophages. These results
are consistent with previous findings (24) and provided evidence
that IL-10 production in macrophages is protective against athero-
sclerosis. Their results also highlight a novel therapeutic technique
against atherosclerosis using an effective stem cell transduction
system that allows prolonged production of IL-10 from macro-
phages.
It is worth emphasising that most strategies mentioned above
had systemic effects on multiple cells including T cells, monocytes
and endothelium resulting from overexpression of IL-10 in circu-
lation. For example, overexpression of IL-10 in activated T lymp-
hocytes inhibited monocyte activation and led to a shift to either
Th2 phenotype (75) or Th1 phenotype (83). As a cytokine with di-
verse effects on most haematopoietic cell types, IL-10 can inhibit
the activation and effector function of T cells, monocytes, and
macrophages (84). In addition, IL-10 can regulate the growth and/
or differentiation of B cells, NK cells, cytotoxic and helper T cells,
mast cells, granulocytes, dendritic cells, keratinocytes, and en-
dothelial cells (84). In particular, several studies support the con-
cept that IL-10 exerts inhibitory effects on vascular smooth muscle
cells (VSMC). There is evidence that athero-protective role of
IL-10 is mediated in part by regulating vessel wall remodelling
through inhibition of VSMC proliferation following vascular in-
jury (85). IL-10 may play an essential role in the maintenance of
normal vasculature, as IL-10 inhibits VSMC activation (86) and
IL-10 deficiency results in vascular damage and remodelling (87).
Interestingly, deficiency in CCR5 has been shown to protect
against neointima formation by up-regulating IL-10 in the neointi-
mal VSMC in atherosclerosis-prone mice (88).
Collectively, these studies indicate alterations in circulating
IL-10 levels can influence the function of other immune cells
which may in turn influence atherosclerosis. In the study by Han
et al. there was no detectable IL-10 in circulating plasma at any
time point during the atherogenic diet feeding whereas IL-10 was
readily detected in IL-10-overexpressing macrophages in athero-
sclerotic lesions (25). This suggests that IL-10 was expressed in dif-
ferentiated macrophages but not in circulating monocytes. There-
fore, their technique of overexpressing IL-10 only in differentiated
macrophages is useful to evaluate the unique role of locally-pro-
duced IL-10 in atherogenesis, and clearly shows that IL-10 acting
in the vessel wall can decrease the development of atherosclerosis
despite ongoing hyperlipidaemia.
Although most studies so far support the protective mechanism
by IL-10, the exact role of IL-10 in attenuating atherosclerosis re-
mains controversial, and is dependent on the animal model in
question. For example, a recent study using a rabbit model showed
that prolonged and stable expression of IL-10 in rabbit carotid ar-
teries achieved with a helper-dependent adenoviral vector had
neither an atheroprotective effect nor any effect on adhesion mol-
ecules or any other atherogenic cytokines (79). This study suggests
that gene therapy involving IL-10 delivery may bring about differ-
ent results in different species.
Therapeutic considerations
In light of the findings that systemic and intralesional delivery of
IL-10 can be anti-atherogenic, it is tempting to speculate that IL-10
treatment may have the potential to be a novel therapeutic agent
against atherosclerosis in the future. IL-10 expression after intra-
muscular DNA electrotransfer or other techniques leads to a per-
sistent expression of this protective cytokine in circulation and in
local lesion (25, 75, 89). It is likely that systemic delivery of IL-10
will result in suppression of immune response and increase the op-
portunity of infection, particularly involving intracellular pa-
thogens such as Chlamydia and Listeria monocytogenes (18).
Compared with systemic delivery of IL-10, local expression of
IL-10 in atherosclerotic lesions may have much less impact on the
general immune response. On the other hand, a robust local ex-
pression driven by retrovirus or adenovirus makes it difficult to
regulate IL-10 expression in a temporally and spatially controllable
manner as desired. Accordingly, the safety and effectiveness of ex-
ogenous IL-10 administration utilizing these techniques will need
to be evaluated in the future before they are adopted in human pa-
tients for the treatment of atherosclerosis.
Acknowledgements
The authors thank Dr. Y. Baumer for her editorial help. The work
pertaining to the authors contained herein was performed accord-
ing to the Russian Government Program of Competitive Growth
of Kazan Federal University.
Conflicts of interest
None declared.
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511Han, Boisvert: Athero-protective role of IL-10
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