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Atherosclerosis Compendium
Circulation Research Compendium on Atherosclerosis
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Atherosclerosis: Successes, Surprises, and Future Challenges
Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease
Triglyceride-Rich Lipoproteins and Atherosclerotic Cardiovascular Disease: New Insights From Epidemiology, Genetics, and Biology
Genetics of Coronary Artery Disease
Surprises From Genetic Analyses of Lipid Risk Factors for Atherosclerosis
From Loci to Biology: Functional Genomics of Genome-Wide Association for Coronary Disease
Are Genetic Tests for Atherosclerosis Ready for Routine Clinical Use?
Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis
Macrophages and Dendritic Cells: Partners in Atherogenesis
Macrophage Phenotype and Function in Different Stages of Atherosclerosis
Adaptive Response of T and B Cells in Atherosclerosis
Microdomains, Inflammation, and Atherosclerosis
Vascular Smooth Muscle Cells in Atherosclerosis
MicroRNA Regulation of Atherosclerosis
The Success Story of LDL Cholesterol Lowering
From Lipids to Inflammation: New Approaches to Reducing Atherosclerotic Risk
Imaging Atherosclerosis
Peter Libby, Karin E. Bornfeldt, and Alan R. Tall, Editors
Macrophages and Dendritic Cells
Partners in Atherogenesis
Myron I. Cybulsky, Cheolho Cheong, Clinton S. Robbins
Abstract: Atherosclerosis is a complex chronic disease. The accumulation of myeloid cells in the arterial intima,
including macrophages and dendritic cells (DCs), is a feature of early stages of disease. For decades, it has
been known that monocyte recruitment to the intima contributes to the burden of lesion macrophages. Yet, this
paradigm may require reevaluation in light of recent advances in understanding of tissue macrophage ontogeny,
their capacity for self-renewal, as well as observations that macrophages proliferate throughout atherogenesis
and that self-renewal is critical for maintenance of macrophages in advanced lesions. The rate of atherosclerotic
lesion formation is profoundly influenced by innate and adaptive immunity, which can be regulated locally
within atherosclerotic lesions, as well as in secondary lymphoid organs, the bone marrow and the blood. DCs are
important modulators of immunity. Advances in the past decade have cemented our understanding of DC subsets,
functions, hematopoietic origin, gene expression patterns, transcription factors critical for differentiation, and
provided new tools for study of DC biology. The functions of macrophages and DCs overlap to some extent, thus
it is important to reassess the contributions of each of these myeloid cells taking into account strict criteria of cell
identification, ontogeny, and determine whether their key roles are within atherosclerotic lesions or secondary
lymphoid organs. This review will highlight key aspect of macrophage and DC biology, summarize how these
cells participate in different stages of atherogenesis and comment on complexities, controversies, and gaps in
knowledge in the field. (Circ Res. 2016;118:637-652. DOI: 10.1161/CIRCRESAHA.115.306542.)
Key Words: atherosclerosis
■
colony-stimulating factors
■
dendritic cells
■
macrophages
■
monocytes
Original received October 23, 2015; revision received December 3, 2015; accepted December 16, 2015.
From the Division of Advanced Diagnostics, Toronto General Research Institute, Peter Munk Cardiac Centre, University Health Network, Toronto, Ontario,
Canada (M.I.C., C.S.R.); Departments of Laboratory Medicine and Pathobiology (M.I.C., C.S.R.) and Immunology (C.S.R.), University of Toronto, Toronto,
Ontario, Canada; and Laboratory of Cellular Physiology and Immunology, Institut de Researches Cliniques de Montréal, Montréal, Québec, Canada (C.C.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
115.306542.
Correspondence to Myron I. Cybulsky, MD, 101 College St, TMDT 3–306, Toronto, Ontario M5G 1L7, Canada. E-mail [email protected]
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.306542
637
638 Circulation Research February 19, 2016
Nonstandard Abbreviations and Acronyms
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Apoe−/−
CSF
CRD
DC
DTR
Flt3L
IFN
LDL
Ldlr−/−
Lyve-1
M-CSF or CSF1
MHC-II
Msr1
oxLDL
Tregs
TLRs
apolipoprotein E deficient
colony-stimulating factor
cholesterol-rich diet
dendritic cell
diphtheria toxin receptor
FMS-like tyrosine kinase 3 ligand
interferon
low-density lipoprotein
LDL receptor deficient
lymphatic vessel endothelial hyaluronan receptor-1
macrophage colony-stimulating factor
major histocompatibility complex class II
type 1 scavenger receptor class A
oxidized LDL
regulatory T cells
toll-like receptors
T
wo Nobel Prizes in Physiology or Medicine awarded
just >100 years apart (1908 and 2011) recognized Elie
Metchnikoff for elucidating a key aspect of innate immunity—phagocytosis by macrophages—and Ralph Steinman
for the discovery of the dendritic cell (DC) and its role in
adaptive immunity. Both macrophages and DCs play a critical role in many diseases including atherosclerosis, but the
relative contributions of these myeloid cells are still debated.
We have known for decades that macrophages are critical to
atherogenesis and that monocytes are recruited to atherosclerotic lesions.1,2 On the basis of the foundations of modern
macrophage biology laid by van Furth and Cohn,3 including
the concept that these professional phagocytes are derived
from circulating blood monocytes, a notion has been firmly
entrenched that chronic inflammation characterized by monocyte recruitment leads to the accumulation of macrophages in
atherosclerotic lesions. More recent studies, however, have
revealed a new paradigm related to the ontogeny of tissueresident macrophages and have conclusively established that
these cells can be derived from embryonic or fetal progenitors
and self-renew through proliferation.4,5
Tissue-Resident Macrophages and Their
Ontogeny
Most tissues of the body contain tissue-resident macrophages.
The diversity of the tissue environments in which macrophages reside has resulted in considerable phenotypic and
functional heterogeneity. Gene expression profiling of macrophage populations from several tissues, for example, has
established that only a small number of transcripts (eg, CD64
and MerTK) associate with all macrophage populations.6 In
addition to maintaining tissue homeostasis and responding
to invading pathogens, macrophages contribute to numerous
pathological processes, making them an attractive potential
target for therapeutic intervention. However, to do so will require a comprehensive understanding of macrophage origins,
the mechanisms that maintain them, and their functional attributes in different tissues and disease contexts.
Macrophage ontology has been debated for decades,7,8 yet
the concept of the mononuclear phagocyte system—which asserts tissue macrophages develop exclusively from circulating
bone marrow–derived monocytes—has prevailed for nearly a
half century.3 However, recent studies using sophisticated fatemapping approaches have determined that some tissue macrophages and their precursors are established embryonically
in the yolk sac and fetal liver before the onset of definitive
hematopoiesis.9–16 Specifically, tissue macrophages arise from
2 distinct developmental programs; early yolk sac–derived
erythro-myeloid progenitors that give rise to macrophages
without monocyte intermediates, and fetal liver monocytes
that derive from late c-Myb+ erythro-myeloid progenitors
generated in the yolk sac and hemogenic endothelium, a specialized subset of endothelial cells found in the dorsal aorta
at the level of the gonad/mesonephros15,16 (Figure 1). These
pathways contribute to macrophage development to varying
degrees in several tissues, including the brain, skin, heart,
liver, and lung.10,13,17–20 Tissue-resident macrophages can also
be derived from recruited monocytes in the postnatal period
(Figure 1), although the mechanisms that determine the balance between prenatal versus postnatal derivation are not fully
understood. Regardless of their origin, resident macrophage
populations in many organs maintain themselves in adulthood through local proliferation, and are largely independent
of blood monocyte recruitment.17,21 Intestinal macrophages
are an exception. Their renewal is constant throughout adult
life and is dependent on circulating monocytes.22 Circulating
monocytes in humans and mice consist of at least 2 subpopulations—classical and nonclassical (patrolling), with distinct
of cell surface marker and chemokine receptor expression patterns and functions during inflammatory responses.23–25
Tissue-resident macrophages densely populate the normal arterial wall, yet their ontogeny, function, and the mechanisms that sustain them are poorly understood. The arterial
wall consists of 3 layers: a thin intima, a thick media, and
an outer adventitia. Arterial macrophages reside largely
within the adventitia. Through gene expression analysis,
we recently demonstrated that arterial macrophages constitute a distinct population among tissue macrophages.26
Using multiple fate-mapping approaches, we showed that
arterial macrophages arise embryonically from CX3CR1+
yolk sac erythro-myeloid progenitors and fetal liver monocytes. Macrophage colonization also associated with a period of recruitment of circulating monocytes immediately
after birth. The postnatal period of monocyte influx was
brief, and corresponded with transient expression of chemokines and cell adhesion molecules implicated in leukocyte
recruitment. In this way, development of the arterial macrophage pool is unique among previously described macrophage populations. Arterial adventitial macrophages are
maintained in adulthood through local proliferation without further meaningful contribution from blood monocytes.
Importantly, proliferation sustains arterial macrophages not
only during steady-state conditions but also mediates their
rebound after severe depletion after sepsis. Maintenance
of the arterial macrophage niche depended on interaction
between macrophage CX3CR1 and CX3CL1 expressed on
PDGFRα+ mesenchymal cells.
Cybulsky et al Myeloid Cells in Atherosclerosis 639
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Figure 1. Macrophage and dendritic cell
ontogeny. Hematopoiesis during embryonic
development occurs in the yolk sac and the fetal
liver. The hemogenic endothelium is a specialized
subset of endothelial cells found in the dorsal
aorta at the level of the gonad/mesonephros
(AGM). During a narrow window of between E9.5
and E14, these endothelial cells can differentiate
into hematopoietic stem and progenitor cells and
translocate to the fetal liver. Similarly, stem and
progenitor cells from the yolk sac translocate to
the fetal liver. Tissue-resident macrophages (Mφ)
are capable of self-renewal. They arise directly
from yolk sac progenitors without monocyte
intermediates and from fetal blood monocytes that
are produced in the fetal liver. Blood monocytes
can also contribute to this population in the
postnatal period. Postnatal hematopoiesis occurs
in the bone marrow. Bone marrow, blood and tissue
cells derived from the common myeloid progenitor
(MP) are shown. cDC indicates classical DC; CDP,
common dendritic cell progenitor; MDP, common
macrophage-dendritic cell-restricted precursor;
Mo-DC, monocyte-derived DC; pDC, plasmacytoid
DC; and pre, percursors.
In addition to expressing core signature macrophage
markers, including CD64, MerTK, F4/80, and CD11b, arterial macrophages uniquely express lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1). Historically, Lyve-1
was thought to be a specific marker of lymphatic endothelium; however, it is also expressed on some macrophages in
the aorta, heart, eye, and tumors. In the arterial adventitia,
Lyve-1 expression is unique to tissue-resident macrophages
because macrophages that arise from recruited blood monocytes do not express the receptor.26 Thus, Lyve-1 expression
distinguishes resident arterial macrophages from monocytederived cells recruited after systemic injection of the bacterial
cell wall component, lipopolysaccharide. Lipopolysaccharide
exposure induces a reduction of Lyve-1+ macrophages, transient replenishment of the arterial compartment by bone
marrow–derived Lyve-1− macrophages and subsequent reestablishment of functional homeostasis by the Lyve-1+-resident
macrophage population. Lyve-1+ macrophages renew locally
through proliferation, but may also partly derive from arterial-resident macrophage progenitor cells. In atherosclerosis,
most macrophages that amass in developing intimal lesions
are derived from blood monocytes. Whether resident arterial
macrophages influence lesion progression, however, remains
unknown. The ability to distinguish resident and monocytederived macrophages may prove a powerful tool for advancing
our understanding of macrophage biology in atherosclerosis
progression.
DC Origin and Classification
DCs are subdivided into classical (cDCs) and plasmacytoid
(pDCs). These cells are derived from a common DC progenitor and precursors of cDCs (pre-cDCs) form in the bone
marrow and disseminate to lymphoid organs (Figure 1). In
addition to cDCs derived from common DC progenitors and
pre-cDCs, DCs can originate from blood monocytes. DC developmental pathways have been studied mostly in lymphoid
tissues, and additional work will be required to map DC development in other tissues, including the normal aorta. Several
excellent reviews provide a comprehensive summary of recent
advances in the DC field27,28 and key facts related to their origin, cell surface markers, gene expression, transcription factors, lineage tracing, in vitro derivation from cultured bone
marrow, important complexities, and gaps in knowledge are
summarized in the Online Data Supplement.
Macrophage and DC Functions
The homeostatic functions of tissue macrophages are common and diverse. Common functions include immune surveillance against invading pathogens, tissue remodeling and
regeneration and support of angiogenesis. For example, tissue
macrophages play a key role in limb regeneration in lower
species such as the salamander29 and regeneration of neonatal
myocardium in mouse injury models.30,31 Diverse macrophage
behavior is largely determined by tissue context. For example,
microglial cells in the brain facilitate neuronal pruning,32,33
macrophages produce catecholamines to sustain adaptive
thermogenesis by adipose tissue and orchestrate the development of beige fat,34,35 and bone marrow macrophages are an
integral component of the hematopoietic stem cell niche.36,37
The steady-state functions of other tissue macrophage populations, including resident arterial macrophages, remain largely
unknown.
DCs induce tolerance against self-antigens and promote antigen-specific immunity on exposure to pathogens.38
Antigen presentation without costimulation leads to T-cell anergy or deletion. A related homeostatic function of DCs is to
640 Circulation Research February 19, 2016
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induce CD4+CD25+Foxp3+ regulatory T cells (Tregs), which
are critical for maintenance of peripheral self-tolerance.38 This
important function is under control of a positive regulatory
feedback loop.39,40 Treg depletion in Foxp3-diphtheria toxin
receptor (DTR) mice induces FMS-like tyrosine kinase 3 ligand (Flt3L) secretion from yet unknown cells, which in turn
initiates the expansion of pre-cDCs and cDCs in lymphoid tissues.39 Ablation of cDCs in CD11c-DTR mice led to a loss of
Tregs and increased interferon (IFN)-γ and interleukin (IL)-17
cytokine production by T cells, whereas increasing DC number by Flt3L treatment resulted in Treg expansion by a mechanism that required major histocompatibility complex (MHC)
class II (MHC-II) expression on cDCs.40
In response to pathogens, DCs in tissues upregulate chemokine receptors such as CCR7, migrate via lymphatics into T
cell–enriched areas of lymph nodes and undergo maturation by
upregulating T-cell costimulatory molecules, including CD40,
CD80/B7-1, CD83, and CD86/B7-2. To fulfill their immunogenic functions, DCs possess machinery to capture, process, and
present antigens to T cells (ie, pattern recognition receptors/tolllike receptors [TLRs], c-type lectin receptors and Fc receptors,
and MHC molecules). Each DC subsets expresses differential
repertoires of c-type lectin receptors and TLRs, which biases
the T-cell response. Splenic CD8α+CD205+ DCs are superior
at cross-presenting antigen by MHC-I and stimulate CD8+ T
cells and CD8α−DCIR2+ DCs are specialized for presentation
by MHC-II and stimulate CD4+ T cells.41 CD8α+ DCs express
CD205, TLR3 and secrete IL-12p70, whereas monocyte-derived
DCs express CD209/DC-SIGN and TLR4.41–44 pDCs express
high levels of the nucleic acid sensors such as TLR7 and TLR9
and secrete type I IFNs during viral infection.45 pDCs can also
prime and cross-prime antigens and induce Th1 polarization.46
Recent studies revealed spatio-temporal complexity in
DC–T cell interactions in lymphoid tissue that is critical to
virus-specific T-cell responses.47,48 Skin CD11b+ DCs are infected early, migrate to the draining lymph node and present
viral antigens to CD4+ T cells. At this early time point, there
is minimal cross-presentation to CD8+ T cells. Later, noninfected chemokine (C motif) receptor 1+ (XCR1+) DCs that
reside in the draining lymph node acquire viral antigen from
infected DCs and cross-present to CD8+ T cells. A prerequisite
for this process is licensing of XCR1+ DCs by CD4+ T cells
that were activated by CD11b+ migratory DCs. This implies
that sequential interactions between DC subsets and T cells
in lymph nodes facilitate diversification of T-cell responses.
Antigen presentation can also occur in peripheral tissues,
but is stochastic because of a lack of a full T-cell repertoire.
However, re-exposure to a previously encountered antigen
significantly increases the chances of DC antigen presentation
to a memory T cell in peripheral tissues because the initial
exposure will produce long-lived antigen-specific memory T
cells that traffic through tissues.
It is interesting that the abundance of tissue macrophages
rapidly decreases in the early stages of inflammation, that
macrophages express MHC-II and costimulatory molecules,
and in cell culture assays can present antigen to T cells although less efficiently than DCs. Yet, in a diseased tissue
where macrophages outnumber DCs by orders of magnitude,
local antigen presentation by macrophages may be significant.
In vitro studies established that macrophages can be
classified into functionally distinct subsets.49 Functional polarization is also observed in vivo under physiological and
pathological conditions.50 Macrophages undergo either classical M1 or alternative M2 activation, although other subsets
have been described.51–54 In fact, M1 and M2 can be viewed
as the ends of a spectrum and this nomenclature may be misleading.55,56 M1 macrophages express high levels of inflammatory cytokines, increased production of reactive nitrogen
and oxygen intermediates, and promote Th1 responses.49,57 M2
macrophages participate in tissue remodeling, wound healing,
and immune regulation51,58; are highly phagocytic; express
high levels of scavenging molecules, mannose and galactose
receptors; and produce ornithine and polyamines through the
arginase pathway.50 Both M1 and M2 macrophage phenotypes
are found in atherosclerotic lesions. The phenotypic profile of
macrophages at different stages of atherosclerosis is poorly
understood,54,59 but is likely influenced by both lineage commitment and responsiveness to the local microenvironment.
Although macrophages and DCs have distinct origins and
specialized functions, it is important to remember that the
biology of these myeloid cells overlaps and is not fully understood in the context of complex diseases. In light of the
well-documented fact that the phenotype of differentiated
cells can be modulated by environmental cues (eg, plasticity of T-cell subsets and epithelial/endothelial–mesenchymal
transdifferentiation), it is possible and even likely that similar phenotypic modulation may occur in macrophages and
DCs during the pathogenesis of complex diseases, such as
atherosclerosis.
Stages of Atherogenesis
Atherosclerotic lesions in mouse models progress through
stages where intimal myeloid cell biology and molecular
mechanisms may be distinct (Table). Nascent lesions begin
in specific regions of the arterial tree within days of initiating a cholesterol-rich diet (CRD) in low-density lipoprotein
(LDL) receptor–deficient (Ldlr−/−) mice. During this stage,
myeloid cells that normally reside in the intima in regions predisposed to atherosclerosis begin accumulating intracellular
lipid.60 Increased expression of endothelial cell adhesion molecule and chemotactic cytokines, monocyte recruitment and
the proliferation of intimal myeloid cells begin after 1 to 2
weeks of hypercholesterolemia.61 This combined with continued accumulation of intracellular lipid results in early lesions
that are composed almost entirely of myeloid foam cells. The
abundance of myeloid cells in the intima continues to increase
and their numbers stabilize over weeks to months. Advanced
lesions progress through phases that include atheroma with
large necrotic areas, fibro-fatty nodules containing chondrocyte-like cells and calcified acellular regions. Smooth muscle
cells migrate from the media into the intima, deposit matrix,
and contribute to the formation of a fibrous cap.2,62 The biology of this stage is complex. Macrophage abundance in advanced lesions remains stable. They turn over within weeks,
and proliferation, not monocyte recruitment, is the primary
mechanism of macrophage renewal.63 These findings suggest that the local environment in advanced lesions is distinct
from early lesions where monocyte recruitment is a significant
Cybulsky et al Myeloid Cells in Atherosclerosis 641
Table. Stages of Atherosclerotic Lesion Formation in
Mouse Models
Stage
Description
Duration of
CRD (Wk)*
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1
Nascent lesions
Uptake of lipid by resident intimal
myeloid cells
<1
2
Early lesions
(myeloid foam
cell lesions)
Composed predominantly of myeloid
foam cells, with minimal intimal
smooth muscle cells and no fibrous
cap or necrotic regions. Lesions
grow laterally (increased lesion
surface area) and vertically.
2–8
3
Advanced
(complex) lesions
Atheromatous core composed of
macrophage foam cells surrounding
extracellular lipid, cholesterol
clefts, necrotic cell debris, foci of
calcification, and a fibrous cap
composed of intimal smooth muscle
cells and collagenous matrix. Lesion
growth is mainly vertical.
>12
4
Hemorrhagic
complicated
lesions
Complex lesions with hemorrhage
(in the innominate artery of old
Apoe−/− mice)
>40
*Ldlr−/− mice fed a cholesterol-rich diet (CRD).
component of local myeloid cell expansion. Intra-plaque hemorrhages occur in advanced lesions located in the innominate
artery of apolipoprotein E–deficient (Apoe−/−) mice. These lesions exhibit fibrotic conversion of necrotic zones, accompanied by loss of the fibrous cap. Hemorrhages are associated
with fissures through fatty streaks that form adjacent to or
on top of established plaques.64 This suggests that foam cell
lesions can form over established plaques possibly through
monocyte recruitment and macrophage proliferation, and this
process may contribute to plaque destabilization.
Topographical Predisposition to
Atherosclerosis and Intimal Myeloid Cells in the
Normal Intima
In all species, regions of the arterial tree have a distinct predilection to atherosclerosis. Atherosclerotic lesions form in
curvatures, branch points, and bifurcations, whereas straight
segments of arteries are protected. This topographical pattern is related to the unique hemodynamic profile of arterial
regions.65 In straight segments, blood flow is uniform laminar. It has a parabolic vector profile and a forward direction
throughout the majority of the cardiac cycle.66 Disturbed laminar flow (frequently referred to as disturbed flow) is found
in lesser (inner) curvatures, bifurcations, and branch points.
Its direction and velocity during the cardiac cycle is complex,
predictable, and includes directionality that is perpendicular
to the mean bulk flow.67 Furthermore, during systole disturbed
flow is usually in the forward direction and reverses during
diastole, with the location of the stagnation point (the site that
momentarily has no flow) changing during the cardiac cycle.
Turbulent flow has chaotic fluctuations, is irregular and unpredictable. Turbulent flow occurs rarely in humans and very
rarely in mice because flow is readily stabilized by a small
arterial diameter.
Wall shear stress is the frictional force per unit area exerted by flowing blood on endothelial cells, and is dependent
on fluid viscosity and velocity adjacent to the wall.65 Shear
stress varies throughout the cardiac cycle. In straight segments
of arteries, shear stress is high during systole and low during
diastole, with a time-averaged shear stress typically 10 to 20
dynes/cm2. Arterial endothelial cells sense shear stress, and
this is essential for regulating flow in arteries and normalizing shear stress in situation when the requirement for oxygen
changes. For example, a sudden increase in oxygen demand
results in local dilatation of arterioles and increased tissue
blood flow. This in turn increases the blood velocity and shear
stress in the corresponding conduit artery. In response to the
increased shear stress, endothelial cells produce more nitric
oxide, which mediates dilatation of the conduit artery. Arterial
dilatation maintains increased delivery of blood while reducing the velocity, and this normalizes the wall shear stress. If
blood flow is altered for a prolonged period, arteries undergo
outward or inward remodeling that permanently normalizes
wall shear stress. In regions with disturbed flow, the time-averaged shear stress is low; however, the artery does not remodel probably because disturbed flow regions usually encompass
only a portion of the arterial circumference, such as the inner
but not the outer curvatures.
Uniform laminar flow induces the expression of endothelial cell genes that are considered to be anti-inflammatory and
antiatherogenic, such as Krüppel-like factor 2, Krüppel-like
factor 4, and endothelial nitric oxide synthase.65,66 In response
to disturbed laminar flow endothelial cells exhibit unique homeostatic properties. This includes a polygonal or randomly
elongated morphology, as opposed to cells elongated uniformly in the direction of uniformly laminar blood flow. Endothelial
cell gene expression, signal transduction, and production of
matrix and proinflammatory molecules are also unique in regions with disturbed flow.65,68 Several mechanisms contribute
to distinct gene expression patterns in disturbed flow regions,
including gene transcription, chromatin-based/epigenetic
mechanisms, mRNA processing, and post-transcriptional
regulation.69 The lesser curvature of the mouse aortic arch experiences disturbed flow and the biology of this region has
been studied extensively70,71 (Figure 2). Even in the absence of
hypercholesterolemia, unique endothelial properties including
expression of leukocyte adhesion molecules and chemokines
promote low-grade chronic inflammation in the lesser aortic
curvature characterized by continuous recruitment of circulating monocytes.72 Myeloid cells accumulate in the intima of
the lesser curvature and reside immediately below the luminal
endothelium. Their numbers are relatively small in comparison with adventitial macrophages that populate all regions of
the artery wall.26,72,73 Interestingly, myeloid cells also reside in
the intima of the mouse aortic sinus, which is a region highly
predisposed to atherosclerosis.73 Intimal myeloid cells have
also been reported in atherosclerosis-susceptible regions of
the rabbit aorta74 and are found in human arteries.75,76
Myeloid cells in the normal mouse arterial intima express
both myeloid and DC markers such as CD68, CD11b, F4/80,
CD11c, class II MHC, DCIR2/33D1, and αEβ7integrin
(CD103), and exhibit long dendritic processes, a morphological feature typical of DCs.72,73 It is likely that the local arterial
642 Circulation Research February 19, 2016
Figure 2. Biology of the normal arterial intima is unique in regions predisposed to atherosclerosis. A schematic representation of
the mouse thoracic aorta showing the location of the greater curvature (GC) and the lesser curvature (LC) of the ascending aortic arch
and the descending thoracic aorta (DTA). Arrows represent hemodynamic profiles. Intimal myeloid cells (CD103+/Flt3L dependent and
CD11b+/CSF1 dependent) in the LC and in the aortic root are shown. eNOS indicates endothelial nitric oxide synthase.
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intimal microenvironment influences this phenotype. Intimal
cells are as effective as splenic DCs in antigen handling, processing, and presentation to T cells.73 In this study, CD11c
promoter–enhanced yellow fluorescent protein transgenic
mice were injected intravenously with ovalbumin, DCs were
isolated by fluorescence-activated cell sorting from the aorta
and spleen, and antigen presentation to transgenic OT-I and
OT-II T cells was monitored in vitro. T cells are rarely found
in the normal aortic intima,72 although their numbers increase
significantly during atherogenesis. It remains to be determined if antigen presentation is a physiological function of intimal myeloid cells in situ in the normal intima. Recently, we
obtained data showing that they can protect the intima from
infection by intracellular pathogens that are imported within
recruited blood monocytes.
The ontogeny of intimal myeloid cells has not yet been
determined using fate-mapping strategies that have been used
to distinguish fetal from definitive hematopoietic origins, as
is reviewed above for tissue-resident macrophages. This is an
important endeavor because it may provide clues related to
their function and why the local microenvironment can sustain a population of myeloid cells in the absence of disease.
Although classical (Ly6Chi) monocytes are constantly recruited at a low rate61,72 and can differentiate into DCs,77 there is
no evidence that these cells contribute to the population of
myeloid cells that resides in the intima. Monocytes do not
necessarily transform to macrophages after recruitment into
tissues78 and have the capacity to migrate away or undergo
apoptosis. It is also possible that a subpopulation of intimal
myeloid cells is derived from pre-cDCs. Approaches such
as parabiosis could address whether ongoing recruitment of
blood monocytes or pre-cDCs contribute to the population of
intimal myeloid cells.
The abundance of intimal myeloid cells seems to be tightly regulated. In the lesser curvature of the ascending aortic
arch, their abundance increases with age and plateaus in the
range of 450 to 650 cells after 12 to 16 weeks of age. Intimal
myeloid cell abundance in the thoracic aorta may increase
throughout adulthood79; and these cells are located near the
ostia of intercostal arteries72,73 or at a lesser curvature in the
lower thoracic aorta. Both growth factors and chemokines
influence the abundance of intimal myeloid cells. Relevant
growth factors include Flt3L and its receptor Flt3 (also known
as CD135 and fetal liver kinase-2 or Flk2), and macrophage
colony–stimulating factor (M-CSF or CSF1), and its receptor (CSF1R or cFMS).80 A distinct subpopulation of myeloid
cells was regulated by each growth factor. CD103+, Flt3+,
CX3CR1−, CD11b−, and F4/80− cells that exhibit the same
phenotype as cDCs were dependent on Flt3L–Flt3 signaling,
whereas CX3CR1+, CD11b+, F4/80+, DC-SIGN+, and CD103−
cells that exhibit the same phenotype as monocyte-derived
DCs were dependent on CSF1-CSF1R signaling.80 Mice deficient in CX3CR1, the receptor for CX3CL1, a chemokine also
called fractalkine, exhibited reduced numbers of intimal myeloid cells relative to wild-type controls.79 It remains to be determined whether this is because CX3CR1 signaling supports
intimal myeloid cell survival,81 or the recruitment of blood
monocytes to the intima.
In the absence of risk factors for atherosclerosis and especially if the plasma lipoprotein levels are normal, the unique
gene expression pattern, low-grade chronic inflammation and
the accumulation of myeloid cells in regions of disturbed flow
do not lead to any pathology in the arterial intima. However
in the setting of hypercholesterolemia, these regions readily
accumulate intimal lipid and develop atherosclerotic lesions.
In contrast, straight segments of arteries with uniform laminar
flow are protected from atherogenesis. It is likely that the absence of protective effects induced by uniform laminar flow
combined with the direct effect of disturbed laminar flow predisposes an arterial region to atherosclerosis.
Initiation of Atherosclerosis in the Setting of
Hypercholesterolemia and Monocyte Recruitment
to Early Lesion Formation
Transgenic mouse models of atherosclerosis have been developed because mice are resistant to developing diet-induced
hypercholesterolemia. The Apoe−/−82,83 and Ldlr−/−84,85 mice
are used most frequently and develop lesions throughout the
aorta with morphological features that resemble human atherosclerosis.85–87 Apoe−/− mice spontaneously develop hypercholesterolemia (400–500 mg/dL) even during embryological
development, thus the earliest events in the onset of atherosclerosis are difficult to pinpoint. In contrast, Ldlr−/− mice fed
standard chow have only a modest elevation in plasma lipoproteins (primarily intermediate and low-density) relative to
wild-type mice. Switching to a CRD rapidly increases plasma
Cybulsky et al Myeloid Cells in Atherosclerosis 643
Figure 3. Expression of myeloid markers in early
atherosclerotic lesions. Serial frozen sections of
the ascending aortic arch from an Ldlr−/− mouse
fed a cholesterol-rich diet for 8 weeks were
immunostained for CD68, MOMA-2, and CD11c.
Nuclei were counter stained with hematoxylin (blue).
CD11c expression shows a membrane-staining
pattern in the majority of intimal foam cells. CD68
and MOMA-2 are expressed by all foam cells in
the intima and occasional cells in the adventitia (A).
Aortic lumen (L). Bar, 50 μm.
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intermediate, low and very low-density lipoproteins, decreases
high-density lipoprotein,84 and induces lesion formation.88 The
introduction of a CRD to Ldlr−/− mice induces within days the
accumulation of intracellular lipid in intimal myeloid cells and
converts them into foam cells.60 During the first week, intimal
cell proliferation remains low (as in the normal aorta) and
monocyte recruitment is not significantly different from baseline.61 Thus, initial foam cells in nascent lesions are derived
primarily from preexisting intimal myeloid cells rather than
recently recruited blood monocytes.
The uptake of lipoproteins by intimal myeloid cells seems
to be an efficient process because the majority of intimal lipid
is accumulated in foam cells that contain multiple cytoplasmic cholesteryl ester droplets.60 The response to retention
hypothesis proposes that foam cell formation is dependent
on the retention and modification of proatherogenic plasma
lipoproteins (LDL and very low–density lipoprotein) in the
arterial subendothelial matrix. Potential modifications of the
retained lipoproteins include oxidation, aggregation, glycation, immune complex formation, proteoglycan complex
formation, and conversion to cholesterol-rich liposomes.89,90
Many studies have provided circumstantial evidence in support of the response to retention hypothesis, but perhaps the
most convincing are functional data from transgenic mice
showing that ApoB100 mutations that inhibit proteoglycan
but not LDLR binding result in reduced arterial lipid accumulation.91 Myeloid cells may contribute to LDL modification in
the intima by producing reactive oxygen species or enzymes,
such as 15-lipoxygenase, phospholipases, and myeloperoxidase. Internalization of oxidatively modified lipoproteins is
mediated by scavenger receptor–mediated endocytosis.92
Macropinocytosis is an alternative mechanism for lipoprotein
internalization,93 and intimal myeloid cells may also extend
dendrites through the endothelial monolayer into the artery
lumen to capture lipoproteins directly from the circulation,
analogous to DC in the gut and the respiratory mucosa that extend dendrites through the lining epithelial layer.94–96 The latter may explain how intravenously injected antigens, such as
ovalbumin, accumulate in intimal cells.73 Efficient conditional
deletion of intimal myeloid cells in CD11c-DTR transgenic
mice97 immediately before induction of hypercholesterolemia
in the Ldlr−/− background results in a paucity of foam cells,
significantly reduced intimal lipid accumulation and readily
detectable retention of lipoprotein-like particles in the intimal
extracellular matrix after 5 days.60
Early atherosclerotic lesions in mouse models are composed almost entirely of myeloid foam cells (Table). The
majority of these cells express both macrophage markers and
CD11c (Figure 3). Considering that CD11c is expressed by
most myeloid cells in the normal intima and in early atherosclerotic lesions, CD11c should be viewed as a myeloid cell
marker, analogous to the lungs where alveolar macrophages
express CD11c.98,99 Intimal expression of CD11c does not
necessarily indicate DC origin or differentiation. Additional
markers are required to establish this.
The formation of early lesions is critically dependent on
monocytes and macrophages. This was demonstrated using
osteopetrotic (op/op) mice that are deficient in circulating
monocytes, tissue macrophages, and osteoclasts because of
a point mutation that disrupts M-CSF.100–102 The recruitment
of blood monocytes to the arterial intima has been regarded
for decades as one of the earliest events in the formation of
atherosclerotic lesions.1,2,62 It seems to be most prominent at
the periphery of lesions103 and may contribute to lateral lesion
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expansion. Conditional deletion of CD11b+ monocytes reduced the formation of lesions in mice.104
It requires ≈2 weeks of hypercholesterolemia before
monocyte recruitment to nascent lesions increases relative to
the normal rate of recruitment observed at sites predisposed to
atherosclerosis.61 As early lesions age, monocyte recruitment
increases61 and persists in advanced lesions.105,106 Classical
(Ly6Chi) monocytes are preferentially recruited.105,106 Reversal
of hypercholesterolemia in Apoe−/− mice by viral transduction
of hepatocytes with ApoE resulted in a reduction of intimal
lipid within 1 week and reduced monocyte recruitment at 2
weeks.107 Changes in the rate of monocyte recruitment to atherosclerotic lesions occur 2 weeks after induction or reduction
of hypercholesterolemia, which indicates that the regulation
of this process is not rapid and suggest that the system behaves
like a capacitor that may be dependent on the content of intracellular lipid in intimal myeloid cells.
The molecular regulation of inflammation and intercellular communication during atherogenesis is not fully understood.108 The textbook explanation is that intimal foam cells
produce proinflammatory cytokines that activate luminal
endothelium to express leukocyte adhesion molecules and
chemokines. However, molecular diffusion in lesions may be
complex considering that the flow of water molecules away
from the lumen driven by high hydrostatic pressure in blood
relative to the interstitial fluid. It is possible that close proximity or direct contact between myeloid cells and endothelium
is required. One would expect that the concentration of proinflammatory mediators would be highest in the center of early
lesions directly correlating with the abundance and density of
myeloid foam cells; however, the most prominent expression
of vascular cell adhesion molecule-1109 and monocyte recruitment103 are found primarily at lesion borders.
Macrophage Proliferation and Dynamics in
Atherosclerotic Lesions
As was described above, monocyte-derived macrophages are
essential to the development of atherosclerosis. However, our
recent data indicate that, in addition to monocyte recruitment,
in situ macrophage proliferation also contributes significantly
to lesion growth. Evidence of macrophage proliferation in
atherosclerotic lesions has previously been observed in relatively advanced lesions in humans and rabbits.110–112 Actually,
this process begins in early lesions contemporaneously with
increased monocyte recruitment.61,112 In our experience, macrophage apoptosis detected by terminal deoxynucleotidyl
transferase dUTP nick-end labeling (TUNEL) is not a prominent feature of early lesions, and increased apoptosis is first
detected at the 8-week time point when early lesions begin
evolving into advanced (complex) lesions. Macrophage foam
cell egress into the blood is considered to be a rare event during atherogenesis, and thus is not likely to have a major impact
on macrophage accumulation.113 The production and secretion
of repulsive neuroimmune guidance cues such as netrin-1,
ephrin-B, and semaphorins 3A and 3E may contribute to macrophage retention.114–117 Hypercholesterolemia also impairs
the migration of dermal DCs to regional lymph nodes through
generation of platelet-activating factor.118 Oxidized, but not
native LDL, inhibits TLR4-induced peritoneal macrophage
efflux into lymphatics and in vitro migration through a process that involves CD36, inactivation of Src homology 2-containing phosphotyrosine phosphatase, sustained activation of
focal adhesion kinase, and alteration of cytoskeletal dynamics.119 On reversal of hypercholesterolemia or treatment with
statins, the induction of CCR7 in plaque macrophages may
promote plaque regression.120–125 Collectively these data suggest that monocyte recruitment and macrophage proliferation,
but not macrophage egress, are the key cellular events regulating foam cell lesion progression.
Until recently, the importance of macrophage proliferation
relative to monocyte influx was not known. Using innovative
experimental approaches including parabiosis—a procedure
that allows circulating cells to enter partner tissues—and
continuous in vivo delivery of the thymidine analog BrdU
via subcutaneously implanted osmotic pumps, we demonstrated that in advanced atherosclerosis (lesions characterized
by the presence of lipid-laden macrophages, a necrotic core,
and a developing fibrous cap) turnover of macrophages depended mostly on local macrophage proliferation rather than
recruitment of circulating monocytes.63 In contrast, in early
lesions (comprised mainly of lipid-laden macrophages) both
monocyte recruitment and macrophage proliferation contribute to lesion growth. These data highlight the complexity of
chronic inflammation in atherosclerosis, and suggest there is
a point during atherogenesis where monocytes stop differentiating and macrophage proliferation takes over (Figure 4).
Furthermore, these data suggest that intercellular communication between myeloid cells, endothelium, and stromal cells is
distinct in early versus advanced lesions.
Lesion macrophages could accumulate gradually and be
long-lived or turnover rapidly. We recently addressed this
question through continuous BrdU delivery in osmotic pumps.
BrdU incorporates into newly synthesized DNA and reports
on a cell’s proliferative history. Strikingly, the data revealed
that macrophage turnover in advanced lesions is rapid, and
nearly all macrophages stained for BrdU within 4 weeks.63
Furthermore, despite increases in lesion size over time, the
aortic root macrophage burden in established disease did not
change significantly, suggesting the processes that contribute
to cell loss counterbalance macrophage renewal.
Unlike early lesions where the abundance of intimal macrophages increases over time, in advanced lesions the macrophage burden remains constant or even decreases.63 Advanced
lesions grow primarily as a result of increasing matrix
Figure 4. Role of monocyte recruitment versus macrophage
proliferation in early and advanced atherosclerotic lesions.
Cybulsky et al Myeloid Cells in Atherosclerosis 645
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deposition and acellular regions with necrotic cell debris and
dystrophic calcification. Yet, several studies have documented
conclusively that monocyte recruitment continues to advanced
lesions.105,106,126 This raises an interesting question about the
fate of monocytes recruited to advanced lesions in light of our
recent data that circulating monocytes contribute minimally to
maintaining the macrophage population at this stage of atherosclerosis.63 Possibilities include that intimal monocytes in advanced lesions persist but do not differentiate to macrophages,
undergo apoptotic cell death, or migrate though lesions before reentering back into the circulation. Although they do
not contribute to the macrophage pool, recruited monocytes
may produce soluble mediators that promote matrix deposition and remodeling. Elucidation of molecular mechanisms
for the preferential persistence of macrophages over recruited
monocytes in advanced lesions will be fruitful area for future
investigations.
In vitro studies have shown that macrophages proliferate in response to oxidized LDL (oxLDL).127,128 We showed
that proliferation of macrophages in developing atherosclerotic lesions depends on the type 1 scavenger receptor class A
(Msr1).63 Scavenger receptors promote the uptake of modified
lipoproteins, clear apoptotic cells and debris, activate cellular signal transduction, and induce macrophage apoptosis.129
Msr1 is one of the most important receptors in the uptake of
oxLDL130 and is expressed on monocytes and macrophages,
smooth muscle cells, and endothelial cells. Its role in atherogenesis is multifaceted. In vivo, Msr1 may contribute to lesion complexity by facilitating plaque foam cell formation,
inducing macrophage apoptosis, and promoting inflammatory
gene expression.129 Macrophages with targeted deletion of
Msr1 take up oxLDL poorly in vitro.131 Msr1-mediated internalization of lysophosphatidylcholine, a phospholipid component of oxLDL, induces peritoneal macrophage proliferation
in vitro.132 It remains to be determined whether macrophage
proliferation in lesions is a general feature of scavenger receptor activity or supported by Msr1 alone. Additional scavenger
receptors of interest include macrophage receptor with collagenous structure, a class A scavenger receptor with considerable homology to Msr1 but whose role in atherosclerosis is
not known, and CD36, a class B scavenger receptor that has
been implicated in the development of necrotic lesions in advanced atherosclerosis.
Another important unanswered question is whether macrophage proliferation is mediated directly by intracellular
signaling through the Msr1 or other scavenger receptors or indirectly through uptake of lipids into the cell. Indeed, genetic
deficiencies in the reverse cholesterol transport machinery of
hematopoietic stem and progenitor cells results in increased
plasma membrane lipids and upregulation of the common β
chain of the IL-3/granulocyte/macrophage-CSF receptor.133
Notably, infusion of high-density lipoprotein and liver X
receptor agonists in this setting restores reverse cholesterol
transport and reduces hypercholesterolemia-induced hematopoietic stem and progenitor cell proliferative responses.134
Macrophage proliferation may also depend on different
mechanisms at different stages of disease. Indeed, granulocyte/macrophage-CSF, a growth factor that regulates early
intimal foam cell proliferation in nascent lesions,61 does not
seem to orchestrate macrophage proliferation in established
disease because neither neutralizing antibodies to the growth
factor nor genetic manipulation targeting its receptor affected
macrophage proliferation.63 Future studies will be required
to determine mechanisms of macrophage proliferation at different stages of disease and the contribution of signaling that
promotes cell survival, including CX3CL1 and its receptor
CX3CR1.81
It is clear that both local and systemic factors influence
myeloid cell accumulation in atherosclerosis, although the
interplay between these is poorly understood. Systemic risk
factors for atherosclerosis, such as hyperlipidemia, hypertension, hyperglycemia, smoking, etc, are well established. As
was mentioned above, hypercholesterolemia promotes the
accumulation of lipid in the arterial intima, and this together
with local hemodynamics promotes a local inflammatory response. This local inflammatory response may, in turn, release
systemic mediators, but it is also likely that hypercholesterolemia directly affects myeloid cells in the bone marrow, spleen,
and blood. There is a strong evidence for systemic myeloid
responses to hypercholesterolemia. In mice, hypercholesterolemia induces increased levels of circulating classical (Ly6Chi)
monocytes,105,106 and elevated blood monocyte counts are an
established risk factor for human atherosclerosis.135–137
High levels of hypercholesterolemia in mice induce the
formation of foamy monocytes, with abundant cytoplasmic
lipid droplets and a high side scatter pattern when analyzed
by flow cytometry. Monocyte lipid uptake is dependent
on CD36 and is associated with upregulated expression of
CD11c, chemokine receptors, and activation of CD49d (α4)
integrin, which mediates adhesion to vascular cell adhesion
molecule-1.138–141 Monocytes with cytoplasmic lipid droplets
can also be found in humans after a high-fat high-cholesterol
meal, but the extent of intracellular lipid is much lower relative to mice.139,140,142,143 The systemic effects of hyperlipidemia
on circulating monocytes promote monocyte recruitment to
atherosclerotic lesions. Other systemic effects, such as production of cytokines and growth factors, may influence macrophage proliferation, survival and polarization in lesions.
Functions of DCs in Atherosclerosis
Many studies have shown that adaptive immunity can profoundly affect the progression of atherosclerosis in mouse
models by modulating the accumulation of myeloid cells in
lesions.144 As was mentioned above, DCs are key regulators
of adaptive immunity, and thus, it is not unexpected that they
influence atherogenesis. DCs may also modulate lipoprotein
metabolism,145 although the molecular mechanisms remain
unknown.
Pathogenic stimuli rapidly induce DC maturation that includes upregulation of cell surface costimulatory molecules
CD80/B7-1 and CD86/B7-2 as well as antigen presentation
machinery (MHC-II), required for stimulation of T cells.
Combined deficiency of CD80 and CD86 in Ldlr−/− mice resulted in reduced early and to some extent advanced atherosclerotic lesion formation (8 and 20 weeks of 1.25% CRD), a
reduction in MHC-II+ cells in early lesions and a modest decrease in lesion CD3+ cells.146 T cells isolated from the spleen
and lymph nodes of CD80/CD86-deficient mice after 8 weeks
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of diet showed reduced production of IFNγ in response to stimulation by heat shock protein 60, which suggested that priming
of a Th1 response to a self-antigen was compromised in the
absence of key costimulatory molecules. Similarly, deficiency
of the invariant chain (CD74), a molecule that functions as a
chaperone and participates in the trafficking and transport of
peptide-loaded MHC-II to the cell surface, resulted in reduced
atherosclerosis and activated CD25+ T cells in lesions of Ldlr−/−
mice at 12 to 26 weeks of CRD.147 Cd74−/−Ldlr−/− mice had elevated IgM and IgG3 and decreased IgG1, IgG2b, and IgG2c
antibodies directed to malondialdehyde-modified LDL, and
after immunization with malondialdehyde-modified LDL had
lower levels of all anti–malondialdehyde-modified LDL immunoglobulins. Collectively, these studies suggest that antigen
presentation can influence atherogenesis by modulating both
cellular and humoral immunity, including B-1 cells that produce natural IgM antibodies to oxidized phospholipids found
in oxLDL.148 Because both DCs and macrophages can express
MHC-II, CD80, and CD86, the relative contribution of these
cell types remains unknown. Furthermore, it is not clear where
antigen-presenting cells encounter oxLDL and whether pathogenically relevant antigen presentation occurs in lymphoid
tissues or lesions. Dynamic interactions between antigen-presenting CD11c+ cells and T cells in the artery wall can lead to
local T-cell activation and proinflammatory cytokine production, which promotes macrophage uptake of modified LDL.149
DCs express a repertoire of TLRs that sense diverse
maturation signals and 2 key adaptor molecules, MyD88
and TRIF, integrate intracellular signaling downstream of
TLRs. Germline deficiency of TLR4150 or TLR9151 in Apoe−/−
mice resulted in reduced atherosclerotic burden. However,
Tlr4−/−Apoe−/− mice developed more severe atherosclerosis
when infected with a common oral pathogen, a phenomenon
that was more evident with aging, implying the contribution
of pathogen-specific stimulation of TLR4 in Th1 immunity,
Treg infiltration, and protection from disease.152 Germline
TLR2 deficiency also reduced atherosclerosis in Ldlr−/− mice;
however, this phenotype was not recapitulated by transplantation of TLR2-deficient bone marrow.153 A subsequent study,
showed that TLR2 expression was restricted to endothelial
cells in regions of disturbed flow, was increased by hyperlipidemia and was required for the accumulation of bone marrow–
derived macrophages in early lesions.154 The disease burden in
Ldlr−/− mice was increased by administration of Pam3CSK4, a
synthetic TLR2/TLR1 agonist, and this effect was dependent
on global TLR2 expression or just in bone marrow–derived
cells.153 This indicates that TLR2 signaling in bone marrow–
derived cells in response to an exogenous ligand can accelerate
atherogenesis. Distinguishing TLR signaling in macrophages
versus DCs will be required. To this end, transplantation of
bone marrow from mice with deletion of MyD88 in DCs
using CD11c-Cre into Ldlr−/− mice resulted in inhibited DC
maturation and decreased activation of effector T cells in the
lymphoid tissues, but increased atherosclerosis (10 weeks of
Western-type diet).155 Because CD11c is expressed by macrophages in early lesions, it is possible that TLR signaling in
these cells was inhibited; however, one could expect that this
would decrease lesions, which was not the case in this study.
T cell and Treg abundance and transforming growth factor-β
expression were decreased in these atherosclerotic lesions,
whereas CCL2 expression, monocyte recruitment, and myeloid cells were increased.155 This study suggested that the atheroprotective effect of Tregs dominates over effector T cells
in atherogenesis and highlights the requirement of MyD88
signaling in DCs in the induction of Tregs.
Given the importance of feedback regulatory loop between
DCs and Tregs, several other studies investigated the role of
DCs on Treg homeostasis in atherosclerosis. Flt3-dependent
DCs were important in maintaining Treg homeostasis, and lack
of these DCs resulted in more atherosclerosis in Ldlr−/− mice fed
a cholesterol-enriched diet for 12 weeks.80 In line with this, Treg
depletion by anti-CD25 antibody156 and Foxp3-DTR157 also promoted atherosclerosis. Increased plasma cholesterol and very
low–density lipoprotein levels and decreased clearance of very
low–density lipoprotein and chylomicron remnants in Tregdepleted Foxp3-DTR mice contributed to this effect.157 The
molecular basis for hepatic lipoprotein metabolism modulation by Tregs has not been established. In contrast to the above
studies, CCL17-expressing DCs, which are found in advanced
atherosclerotic lesions of Apoe−/− mice but not the healthy artery
wall, restricted Treg expansion through secretion of CCL17.158
CCL17+ DCs are mature (CD40+, CD80+, and CD86+) DCs
that express CD11c, MHC-II, CD11b, and CD205 cell surface markers159; however, further studies such as antigen-presentation capability and allogeneic T-cell stimulatory capacity
(eg, in a mixed leukocyte reaction) are required to determine
if CCL17+ DCs represent a distinct DC subset. Collectively,
it is clear that cDCs play a critical role in Treg homeostasis,
although CCL17+ DCs seem to have an opposing role. Tregs
diminish myeloid cell accumulation in lesions likely through
several different mechanisms. Treg abundance within lesions
seems to be important, but these cells may also function in
lymphoid tissues to modulate systemic factors that, in turn,
influence myeloid cell biology in lesions.
pDCs can also modulate atherogenesis. Several studies
adopted a pDC depletion approach using antibodies targeting
PDCA1/BST2, but reached disparate conclusions. Depletion
of pDCs in Ldlr−/− mice exacerbated the development of lesions in the aortic root and in a carotid artery cuff model,160
whereas reduced atherosclerosis was observed in Apoe−/−
mice.161,162 The depleting antibody, the dose and the interval
of administration may have accounted for different effects
on lesions in these studies. It is also important to remember that expression of PDCA1 is not restricted to pDCs and
can be found other cells including inflamed macrophages.6,45
Therefore, anti-PDCA1 treatment regimens may deplete some
macrophages in lesions and not just pDCs.
Recently, E2-2/TCF4, a transcription factor critical for
pDC development, was deleted using CD11c-Cre and atherosclerosis was studied following bone marrow transplantation
into Ldlr−/− mice.163 In TCF4-depleted chimeras, the neutral
lipid content and T cells were reduced in early (8 weeks of
CRD) lesions located in the aortic root in spite of elevated
total plasma cholesterol levels. A compensatory increase in
cDCs and B220lo CD11c+ MHC-II+ cells was found in lymphoid organs, consistent with previous studies,164 but Treg
levels were comparable in the spleen. The authors attributed
the reduced atherogenesis to defective pDC-driven MHC-II
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antigen presentation to T cells based on both in vitro and in
vivo experiments, and not to altered IFN-α production. Given
the expansion of cDCs in TCF4-depleted chimeras, the known
atheroprotective functions of most cDCs, and the important
role of Tregs in atherosclerosis, further studies may be necessary to fully appreciate the observed atherosclerosis phenotype in E2-2/TCF4-deficient chimeric mice.
Although published studies on the role of pDCs in atherosclerosis are informative, spatio-temporal information about
aortic pDCs during different stages of atherosclerosis is lacking and it is still not known whether aortic pDCs produce type
I IFNs in lesions. Given the potential for nonspecific cell depletion by anti-PDCA1 antibodies and compensatory responses
to constitutive pDC depletion in E2-2/TCF4-deficient mice,
it will be necessary to use more specific and inducible pDC
depletion approaches. Lymphoid tissue pDCs can be deleted
efficiently in BDCA2-DTR mice.46 Once pDC accumulation
in aortic atherosclerotic lesions is evaluated, it remains to be
determined if DT will effectively delete these cells in BDCA2DTR mice and what impact this will have on lesion formation.
DC Therapeutics for Atherosclerosis
To harness DCs as a therapeutic vaccine, bone marrow–derived DCs (cultured with granulocyte/macrophage-CSF) were
loaded with oxLDL and adoptively transferred to Ldlr−/− mice
before inducing dietary hypercholesterolemia.165,166 These
studies resulted in a significant reduction in atherosclerotic lesion development, reduced oxLDL–specific T cells, increased
oxLDL–specific IgG levels, and induced Tregs. In contrast,
DCs loaded with malondialdehyde-modified LDL aggravated
atherosclerosis in Apoe−/− mice.167 Similarly, increased lesion development was found when Apoe−/− mice expressing
β-galactosidase under the transcriptional regulation of the
SM22α promoter received bone marrow–derived DCs loaded
with β-galactosidase–specific peptides.168
The above DC immunization approaches have several limitations. Bone marrow–derived DCs cultured with
granulocyte/macrophage-CSF constitute a heterozygous population of DCs and macrophages.169 Even purification of DCs
using approaches such as microbeads coated with antibodies
to CD11c fails to achieve a uniform population. The maturation status of DCs was not evaluated. It is well established that
steady-state immature DCs promote T-cell tolerance through
deletion of reactive T cells170,171 and induction of Tregs,172
whereas mature DCs (after exposure to TLR ligands) induce
Th1 polarization.38 It would be interesting to assess effects
of adoptively transferred cultured DCs loaded with modified
LDL (eg, oxLDL) and exposed to a maturation stimulus (eg, a
TLR agonist) in an animal model of atherosclerosis.
Given the limitations of immunization with cultured DCs
loaded with an atherosclerosis-related protein/peptide, a new
approach was pioneered by Drs Steinman and Nussenzweig171
to directly target antigens to DCs in vivo through the antibodies
against endocytic surface receptors that are highly expressed in
DC subsets (Figure 5). This DC targeting antibody approach
can be used in all species and does not require lengthy in vitro
manipulation steps, which can be problematic when translated to patients. Targeting receptors include CD205/DEC-205,
which is highly expressed in CD8α+ DCs. Targeting a selfantigen fused to an anti-CD205 antibody resulted in the induction of tolerance.38 In contrast, when a foreign protein/peptide
was combined with an adjuvant (TLR agonist such as poly IC),
it induced strong cellular and humoral immunity against the
foreign protein/peptide.38 Several DC subset-specific antibodies have been identified, such as anti-CD207, anti-Clec9A,
and anti-PDCA1/BST2/CD317 and can be used for targeting
proteins/peptides to DCs.170,173–176 Once appropriate protein/
peptide targets for atherosclerosis are identified, the efficacy
of the DC targeting antibody approach in the prevention of atherosclerosis can be tested in animal models.
Conclusions
The accumulation of myeloid cells in the arterial intima is just
one component of atherogenesis. It is influenced by local and
Figure 5. Dendritic cell (DC) vaccination strategies for atherosclerosis. DCs can be harnessed to prevent infections, modulate
diseases, and eliminate tumors. The initial approach was to educate DCs in vitro with protein, then infuse antigen-bearing DCs into
experimental animals or patients to elicit strong T- and B-cell immunity. This in vitro approach was explored in mouse models of
atherosclerosis. Proteins and other antigens can be targeted for DC uptake via various receptors, including c-type lectin receptors by
using genetically cloned antibodies against these receptors. This in vivo approach enhances antigen presentation to both CD4 and CD8 T
cells in animals and humans. mCκ indicates mouse κ light chain constant region; mIgG1, mouse IgG1 isotype heavy chain constant region;
VH, heavy chain variable region; and Vκ, κlight chain variable region. Linker is a 12 to 14 amino acid peptide for flexible fusion of proteins
or peptides to the targeting antibody.
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systemic factors that include modulation of acquired immunity by DCs. Mouse models provide a means for elucidating
molecular insights into how cellular processes such as monocyte recruitement and macrophage proliferation are regulated
at different stages of lesion formation, what are the functions
of newly recruited monocytes versus established proliferating
macrophage populations, what molecules mediate intercellular communication in lesions, and how these are modulated by
various DC subsets residing in lesions and in lymphoid tissues.
Intimal myeloid cells do not only influence the progression
of atherosclerotic lesions but also ultimately may contribute
to lesion destabilization. With respect to the latter, it will be
important to discern the role of newly recruited monocytes
versus established proliferating macrophages because understanding the relationship between these may yield novel
therapeutic strategies. Modeling of thrombotic complications
will be difficult with mouse models. Furthermore, in contrast
to mouse models, a persistent and extreme hypercholesterolemia is not a usual feature in the majority of patients with atherosclerosis; therefore, disease progression and the interplay
between monocyte recruitment and macrophage proliferation
may be more complex and influenced by multiple systemic
risk factors. Nevertheless, we feel that gaining a thorough
understanding of myeloid cell biology in atherogenesis using
the diverse approaches that are available in association with
mouse models is an important endeavor that will ultimately
affect human health.
Acknowledgments
We acknowledge the assistance of Angela Li and Tae Jin Yun
in preparation of figures and Magar Ghazarian for performing
immunostaining.
Sources of Funding
Our research programs are supported by grants from the Canadian
Institutes of Health Research (CIHR): MOP-84446 (M.I. Cybulsky),
MOP-89740 (M.I. Cybulsky), MOP-106522 (M.I. Cybulsky), MOP125933 (C. Cheong), HIG-133050 (C. Cheong), and MOP-133390
(C.S. Robbins), the National Research Foundation of Korea (GRN2013S1A2A2035348 to C. Cheong), and the Peter Munk Cardiac
Centre at University Health Network. M.I. Cybulsky is the recipient
of a Canada Research Chair; C. Cheong holds a Chercheur-Boursier
Junior of Fonds de recherche du Québec-Santé—Société Québécoise
d’Hypertension Artérielle and a CIHR New Investigator Award, and
C.S. Robbins holds the Peter Munk Chair in Aortic Disease Research
and a CIHR New Investigator Award.
Disclosures
None.
References
1. Munro JM, Cotran RS. The pathogenesis of atherosclerosis: atherogenesis
and inflammation. Lab Invest. 1988;58:249–261.
2.Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med.
1999;340:115–126. doi: 10.1056/NEJM199901143400207.
3. van Furth R, Cohn ZA. The origin and kinetics of mononuclear phagocytes. J Exp Med. 1968;128:415–435.
4. Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol. 2014;14:392–404. doi:
10.1038/nri3671.
5. Epelman S, Lavine KJ, Randolph GJ. Origin and functions of tissue macrophages. Immunity. 2014;41:21–35. doi: 10.1016/j.immuni.2014.06.013.
6. Gautier EL, Shay T, Miller J, et al; Immunological Genome Consortium.
Gene-expression profiles and transcriptional regulatory pathways that
underlie the identity and diversity of mouse tissue macrophages. Nat
Immunol. 2012;13:1118–1128. doi: 10.1038/ni.2419.
7. Akazaki K. A concept of reticuloendothelial system. Tohoku J Exp Med.
1962;76:107–118.
8. Naito M, Yamamura F, Nishikawa S, Takahashi K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures. J
Leukoc Biol. 1989;46:1–10.
9. Samokhvalov IM, Samokhvalova NI, Nishikawa S. Cell tracing shows
the contribution of the yolk sac to adult haematopoiesis. Nature.
2007;446:1056–1061. doi: 10.1038/nature05725.
10. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF,
Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–845. doi: 10.1126/science.1194637.
11.Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard
N, Kierdorf K, Prinz M, Wu B, Jacobsen SE, Pollard JW, Frampton J,
Liu KJ, Geissmann F. A lineage of myeloid cells independent of Myb
and hematopoietic stem cells. Science. 2012;336:86–90. doi: 10.1126/
science.1219179.
12. Hoeffel G, Wang Y, Greter M, et al. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. J Exp Med. 2012;209:1167–1181.
doi: 10.1084/jem.20120340.
13. Epelman S, Lavine KJ, Beaudin AE, et al. Embryonic and adult-derived
resident cardiac macrophages are maintained through distinct mechanisms
at steady state and during inflammation. Immunity. 2014;40:91–104. doi:
10.1016/j.immuni.2013.11.019.
14. Molawi K, Wolf Y, Kandalla PK, et al. Progressive replacement of embryo-derived cardiac macrophages with age. J Exp Med. 2014;211:2151–
2158. doi: 10.1084/jem.20140639.
15. Hoeffel G, Chen J, Lavin Y, et al. C-Myb(+) erythro-myeloid progenitorderived fetal monocytes give rise to adult tissue-resident macrophages.
Immunity. 2015;42:665–678. doi: 10.1016/j.immuni.2015.03.011.
16. Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet
L, Garner H, Trouillet C, de Bruijn MF, Geissmann F, Rodewald HR.
Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 2015;518:547–551. doi: 10.1038/nature13989.
17. Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, Strauss-Ayali
D, Viukov S, Guilliams M, Misharin A, Hume DA, Perlman H, Malissen
B, Zelzer E, Jung S. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38:79–
91. doi: 10.1016/j.immuni.2012.12.001.
18. Kierdorf K, Erny D, Goldmann T, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci.
2013;16:273–280. doi: 10.1038/nn.3318.
19.Guilliams M, De Kleer I, Henri S, Post S, Vanhoutte L, De Prijck S,
Deswarte K, Malissen B, Hammad H, Lambrecht BN. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived
cells in the first week of life via GM-CSF. J Exp Med. 2013;210:1977–
1992. doi: 10.1084/jem.20131199.
20.Chorro L, Sarde A, Li M, Woollard KJ, Chambon P, Malissen B,
Kissenpfennig A, Barbaroux JB, Groves R, Geissmann F. Langerhans
cell (LC) proliferation mediates neonatal development, homeostasis, and
inflammation-associated expansion of the epidermal LC network. J Exp
Med. 2009;206:3089–3100. doi: 10.1084/jem.20091586.
21.Hashimoto D, Chow A, Noizat C, et al. Tissue-resident macrophages
self-maintain locally throughout adult life with minimal contribution
from circulating monocytes. Immunity. 2013;38:792–804. doi: 10.1016/j.
immuni.2013.04.004.
22.Bain CC, Bravo-Blas A, Scott CL, Gomez Perdiguero E, Geissmann F,
Henri S, Malissen B, Osborne LC, Artis D, Mowat AM. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol. 2014;15:929–937. doi: 10.1038/ni.2967.
23. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82.
24. Grage-Griebenow E, Flad HD, Ernst M. Heterogeneity of human peripheral blood monocyte subsets. J Leukoc Biol. 2001;69:11–20.
25. Strauss-Ayali D, Conrad SM, Mosser DM. Monocyte subpopulations and
their differentiation patterns during infection. J Leukoc Biol. 2007;82:244–
252. doi: 10.1189/jlb.0307191.
26. Ensan S, Li A, Besla R, et al. Self-renewing resident arterial macrophages
arise from embryonic CX3CR1+ precursors and circulating monocytes
immediately after birth. Nat Immunol. 2016;17:159–168. doi: 10.1038/
ni.3343.
Cybulsky et al Myeloid Cells in Atherosclerosis 649
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
27. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage:
ontogeny and function of dendritic cells and their subsets in the steady
state and the inflamed setting. Annu Rev Immunol. 2013;31:563–604. doi:
10.1146/annurev-immunol-020711-074950.
28. Swiecki M, Colonna M. The multifaceted biology of plasmacytoid dendritic cells. Nat Rev Immunol. 2015;15:471–485. doi: 10.1038/nri3865.
29. Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult
salamander limb regeneration. Proc Natl Acad Sci U S A. 2013;110:9415–
9420. doi: 10.1073/pnas.1300290110.
30. Lavine KJ, Epelman S, Uchida K, Weber KJ, Nichols CG, Schilling JD,
Ornitz DM, Randolph GJ, Mann DL. Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart. Proc Natl Acad Sci U S A. 2014;111:16029–16034.
doi: 10.1073/pnas.1406508111.
31. Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R,
Sadek HA, Olson EN. Macrophages are required for neonatal heart regeneration. J Clin Invest. 2014;124:1382–1392. doi: 10.1172/JCI72181.
32. Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P,
Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, Gross CT.
Synaptic pruning by microglia is necessary for normal brain development.
Science. 2011;333:1456–1458. doi: 10.1126/science.1202529.
33.Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR,
Yamasaki R, Ransohoff RM, Greenberg ME, Barres BA, Stevens B.
Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron. 2012;74:691–705. doi: 10.1016/j.
neuron.2012.03.026.
34. Nguyen KD, Qiu Y, Cui X, Goh YP, Mwangi J, David T, Mukundan L,
Brombacher F, Locksley RM, Chawla A. Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis.
Nature. 2011;480:104–108. doi: 10.1038/nature10653.
35. Qiu Y, Nguyen KD, Odegaard JI, Cui X, Tian X, Locksley RM, Palmiter
RD, Chawla A. Eosinophils and type 2 cytokine signaling in macrophages
orchestrate development of functional beige fat. Cell. 2014;157:1292–
1308. doi: 10.1016/j.cell.2014.03.066.
36. Chow A, Huggins M, Ahmed J, Hashimoto D, Lucas D, Kunisaki Y, Pinho
S, Leboeuf M, Noizat C, van Rooijen N, Tanaka M, Zhao ZJ, Bergman A,
Merad M, Frenette PS. CD169⁺ macrophages provide a niche promoting
erythropoiesis under homeostasis and stress. Nat Med. 2013;19:429–436.
doi: 10.1038/nm.3057.
37. Dutta P, Hoyer FF, Grigoryeva LS, et al. Macrophages retain hematopoietic stem cells in the spleen via VCAM-1. J Exp Med. 2015;212:497–512.
doi: 10.1084/jem.20141642
38.
Steinman RM. Decisions about dendritic cells: past, present, and future. Annu Rev Immunol. 2012;30:1–22. doi: 10.1146/
annurev-immunol-100311-102839.
39. Liu K, Victora GD, Schwickert TA, Guermonprez P, Meredith MM, Yao
K, Chu FF, Randolph GJ, Rudensky AY, Nussenzweig M. In vivo analysis
of dendritic cell development and homeostasis. Science. 2009;324:392–
397. doi: 10.1126/science.1170540.
40. Darrasse-Jèze G, Deroubaix S, Mouquet H, Victora GD, Eisenreich T, Yao
KH, Masilamani RF, Dustin ML, Rudensky A, Liu K, Nussenzweig MC.
Feedback control of regulatory T cell homeostasis by dendritic cells in
vivo. J Exp Med. 2009;206:1853–1862. doi: 10.1084/jem.20090746.
41. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller
C, Yamazaki S, Cheong C, Liu K, Lee HW, Park CG, Steinman RM,
Nussenzweig MC. Differential antigen processing by dendritic cell subsets in vivo. Science. 2007;315:107–111. doi: 10.1126/science.1136080.
42. del Rio ML, Bernhardt G, Rodriguez-Barbosa JI, Förster R. Development
and functional specialization of CD103+ dendritic cells. Immunol Rev.
2010;234:268–281. doi: 10.1111/j.0105-2896.2009.00874.x.
43. den Haan JM, Bevan MJ. Constitutive versus activation-dependent crosspresentation of immune complexes by CD8(+) and CD8(-) dendritic cells
in vivo. J Exp Med. 2002;196:817–827.
44. Edelson BT, KC W, Juang R, et al. Peripheral CD103+ dendritic cells form
a unified subset developmentally related to CD8alpha+ conventional dendritic cells. J Exp Med. 2010;207:823–836. doi: 10.1084/jem.20091627.
45. Reizis B, Colonna M, Trinchieri G, Barrat F, Gilliet M. Plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system? Nat
Rev Immunol. 2011;11:558–565. doi: 10.1038/nri3027.
46. Swiecki M, Gilfillan S, Vermi W, Wang Y, Colonna M. Plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral NK
and CD8(+) T cell accrual. Immunity. 2010;33:955–966. doi: 10.1016/j.
immuni.2010.11.020.
47.Hor JL, Whitney PG, Zaid A, Brooks AG, Heath WR, Mueller SN.
Spatiotemporally distinct interactions with dendritic cell subsets facilitates
CD4+ and CD8+ T cell activation to localized viral infection. Immunity.
2015;43:554–565. doi: 10.1016/j.immuni.2015.07.020.
48. Eickhoff S, Brewitz A, Gerner MY, Klauschen F, Komander K, Hemmi
H, Garbi N, Kaisho T, Germain RN, Kastenmüller W. Robust anti-viral
immunity requires multiple distinct t cell-dendritic cell interactions. Cell.
2015;162:1322–1337. doi: 10.1016/j.cell.2015.08.004.
49. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8:958–969. doi: 10.1038/nri2448.
50. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–795. doi: 10.1172/JCI59643.
51. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164:6166–6173.
52.Biswas SK, Mantovani A. Macrophage plasticity and interaction with
lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010;11:889–
896. doi: 10.1038/ni.1937.
53. Kadl A, Meher AK, Sharma PR, Lee MY, Doran AC, Johnstone SR, Elliott
MR, Gruber F, Han J, Chen W, Kensler T, Ravichandran KS, Isakson BE,
Wamhoff BR, Leitinger N. Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2. Circ
Res. 2010;107:737–746. doi: 10.1161/CIRCRESAHA.109.215715.
54. Ley K, Miller YI, Hedrick CC. Monocyte and macrophage dynamics during atherogenesis. Arterioscler Thromb Vasc Biol. 2011;31:1506–1516.
doi: 10.1161/ATVBAHA.110.221127.
55.Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and
polarization: nomenclature and experimental guidelines. Immunity.
2014;41:14–20. doi: 10.1016/j.immuni.2014.06.008.
56. Xue J, Schmidt SV, Sander J, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity.
2014;40:274–288. doi: 10.1016/j.immuni.2014.01.006.
57.Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol.
2006;177:7303–7311.
58. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2
mononuclear phagocytes. Trends Immunol. 2002;23:549–555.
59. Mantovani A, Garlanda C, Locati M. Macrophage diversity and polarization in atherosclerosis: a question of balance. Arterioscler Thromb Vasc
Biol. 2009;29:1419–1423. doi: 10.1161/ATVBAHA.108.180497.
60. Paulson KE, Zhu SN, Chen M, Nurmohamed S, Jongstra-Bilen J, Cybulsky
MI. Resident intimal dendritic cells accumulate lipid and contribute to the
initiation of atherosclerosis. Circ Res. 2010;106:383–390. doi: 10.1161/
CIRCRESAHA.109.210781.
61.Zhu SN, Chen M, Jongstra-Bilen J, Cybulsky MI. GM-CSF regulates
intimal cell proliferation in nascent atherosclerotic lesions. J Exp Med.
2009;206:2141–2149. doi: 10.1084/jem.20090866.
62.Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis.
Circulation. 2002;105:1135–1143.
63.Robbins CS, Hilgendorf I, Weber GF, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med.
2013;19:1166–1172. doi: 10.1038/nm.3258.
64. Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz
SM. Advanced atherosclerotic lesions in the innominate artery of the ApoE
knockout mouse. Arterioscler Thromb Vasc Biol. 2000;20:2587–2592.
65. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6:16–26.
doi: 10.1038/ncpcardio1397.
66.Gimbrone MA Jr, García-Cardeña G. Vascular endothelium, hemodynamics, and the pathobiology of atherosclerosis. Cardiovasc Pathol.
2013;22:9–15. doi: 10.1016/j.carpath.2012.06.006.
67.Mohamied Y, Rowland EM, Bailey EL, Sherwin SJ, Schwartz MA,
Weinberg PD. Change of direction in the biomechanics of atherosclerosis.
Ann Biomed Eng. 2015;43:16–25. doi: 10.1007/s10439-014-1095-4.
68. Conway DE, Schwartz MA. Flow-dependent cellular mechanotransduction in atherosclerosis. J Cell Sci. 2013;126:5101–5109. doi: 10.1242/
jcs.138313.
69.Cybulsky MI, Marsden PA. Effect of disturbed blood flow on endothelial cell gene expression: a role for changes in RNA processing.
Arterioscler Thromb Vasc Biol. 2014;34:1806–1808. doi: 10.1161/
ATVBAHA.114.304099.
70. Hajra L, Evans AI, Chen M, Hyduk SJ, Collins T, Cybulsky MI. The NFkappa B signal transduction pathway in aortic endothelial cells is primed
for activation in regions predisposed to atherosclerotic lesion formation.
Proc Natl Acad Sci U S A. 2000;97:9052–9057.
650 Circulation Research February 19, 2016
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
71. Cuhlmann S, Van der Heiden K, Saliba D, Tremoleda JL, Khalil M, Zakkar
M, Chaudhury H, Luong le A, Mason JC, Udalova I, Gsell W, Jones H,
Haskard DO, Krams R, Evans PC. Disturbed blood flow induces RelA
expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation. Circ Res. 2011;108:950–959.
doi: 10.1161/CIRCRESAHA.110.233841.
72. Jongstra-Bilen J, Haidari M, Zhu SN, Chen M, Guha D, Cybulsky MI.
Low-grade chronic inflammation in regions of the normal mouse arterial
intima predisposed to atherosclerosis. J Exp Med. 2006;203:2073–2083.
doi: 10.1084/jem.20060245.
73. Choi JH, Do Y, Cheong C, Koh H, Boscardin SB, Oh YS, Bozzacco L,
Trumpfheller C, Park CG, Steinman RM. Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves. J Exp Med.
2009;206:497–505. doi: 10.1084/jem.20082129.
74.Malinauskas RA, Herrmann RA, Truskey GA. The distribution of intimal white blood cells in the normal rabbit aorta. Atherosclerosis.
1995;115:147–163.
75. Bobryshev YV, Lord RS. Ultrastructural recognition of cells with dendritic
cell morphology in human aortic intima. Contacting interactions of vascular dendritic cells in athero-resistant and athero-prone areas of the normal
aorta. Arch Histol Cytol. 1995;58:307–322.
76. Millonig G, Niederegger H, Rabl W, Hochleitner BW, Hoefer D, Romani N,
Wick G. Network of vascular-associated dendritic cells in intima of healthy
young individuals. Arterioscler Thromb Vasc Biol. 2001;21:503–508.
77. Cheong C, Matos I, Choi JH, et al. Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell
areas. Cell. 2010;143:416–429. doi: 10.1016/j.cell.2010.09.039.
78.Jakubzick C, Gautier EL, Gibbings SL, et al. Minimal differentiation
of classical monocytes as they survey steady-state tissues and transport
antigen to lymph nodes. Immunity. 2013;39:599–610. doi: 10.1016/j.
immuni.2013.08.007.
79. Liu P, Yu YR, Spencer JA, Johnson AE, Vallanat CT, Fong AM, Patterson
C, Patel DD. CX3CR1 deficiency impairs dendritic cell accumulation in
arterial intima and reduces atherosclerotic burden. Arterioscler Thromb
Vasc Biol. 2008;28:243–250. doi: 10.1161/ATVBAHA.107.158675.
80. Choi JH, Cheong C, Dandamudi DB, Park CG, Rodriguez A, Mehandru
S, Velinzon K, Jung IH, Yoo JY, Oh GT, Steinman RM. Flt3 signalingdependent dendritic cells protect against atherosclerosis. Immunity.
2011;35:819–831. doi: 10.1016/j.immuni.2011.09.014.
81.Landsman L, Bar-On L, Zernecke A, Kim KW, Krauthgamer R,
Shagdarsuren E, Lira SA, Weissman IL, Weber C, Jung S. CX3CR1 is
required for monocyte homeostasis and atherogenesis by promoting cell
survival. Blood. 2009;113:963–972. doi: 10.1182/blood-2008-07-170787.
82. Plump AS, Smith JD, Hayek T, Aalto-Setälä K, Walsh A, Verstuyft JG,
Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis
in apolipoprotein E-deficient mice created by homologous recombination
in ES cells. Cell. 1992;71:343–353.
83. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science.
1992;258:468–471.
84. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J.
Hypercholesterolemia in low density lipoprotein receptor knockout mice
and its reversal by adenovirus-mediated gene delivery. J Clin Invest.
1993;92:883–893. doi: 10.1172/JCI116663.
85. Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein
receptor-negative mice. J Clin Invest. 1994;93:1885–1893. doi: 10.1172/
JCI117179.
86. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient
mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994;14:133–140.
87. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E.
Evaluation of lesional development and progression. Arterioscler Thromb.
1994;14:141–147.
88. Lichtman AH, Clinton SK, Iiyama K, Connelly PW, Libby P, Cybulsky
MI. Hyperlipidemia and atherosclerotic lesion development in LDL receptor-deficient mice fed defined semipurified diets with and without cholate.
Arterioscler Thromb Vasc Biol. 1999;19:1938–1944.
89. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–561.
90.Tabas I. Nonoxidative modifications of lipoproteins in atherogenesis.
Annu Rev Nutr. 1999;19:123–139. doi: 10.1146/annurev.nutr.19.1.123.
91. Skålén K, Gustafsson M, Rydberg EK, Hultén LM, Wiklund O, Innerarity
TL, Borén J. Subendothelial retention of atherogenic lipoproteins in early
atherosclerosis. Nature. 2002;417:750–754. doi: 10.1038/nature00804.
92.Horiuchi S, Sakamoto Y, Sakai M. Scavenger receptors for oxidized
and glycated proteins. Amino Acids. 2003;25:283–292. doi: 10.1007/
s00726-003-0029-5.
93. Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang J, Combs CA,
Malide D, Zhang WY. Macropinocytosis is the endocytic pathway
that mediates macrophage foam cell formation with native low density lipoprotein. J Biol Chem. 2005;280:2352–2360. doi: 10.1074/jbc.
M407167200.
94. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio
R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P. Dendritic cells
express tight junction proteins and penetrate gut epithelial monolayers to
sample bacteria. Nat Immunol. 2001;2:361–367. doi: 10.1038/86373.
95. Schon-Hegrad MA, Oliver J, McMenamin PG, Holt PG. Studies on the
density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC)
in the conducting airways. J Exp Med. 1991;173:1345–1356.
96. Sertl K, Takemura T, Tschachler E, Ferrans VJ, Kaliner MA, Shevach
EM. Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. J Exp Med.
1986;163:436–451.
97. Jung S, Unutmaz D, Wong P, Sano G, De los Santos K, Sparwasser T, Wu
S, Vuthoori S, Ko K, Zavala F, Pamer EG, Littman DR, Lang RA. In vivo
depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells
by exogenous cell-associated antigens. Immunity. 2002;17:211–220.
98. Guth AM, Janssen WJ, Bosio CM, Crouch EC, Henson PM, Dow SW.
Lung environment determines unique phenotype of alveolar macrophages. Am J Physiol Lung Cell Mol Physiol. 2009;296:L936–L946. doi:
10.1152/ajplung.90625.2008.
99. Bradford BM, Sester DP, Hume DA, Mabbott NA. Defining the anatomical localisation of subsets of the murine mononuclear phagocyte
system using integrin alpha X (Itgax, CD11c) and colony stimulating
factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages. Immunobiology. 2011;216:1228–1237.
doi: 10.1016/j.imbio.2011.08.006.
100.Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J, Miyata M.
Decreased atherosclerosis in mice deficient in both macrophage colonystimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci U S A.
1995;92:8264–8268.
101. Qiao JH, Tripathi J, Mishra NK, Cai Y, Tripathi S, Wang XP, Imes S,
Fishbein MC, Clinton SK, Libby P, Lusis AJ, Rajavashisth TB. Role of
macrophage colony-stimulating factor in atherosclerosis: studies of osteopetrotic mice. Am J Pathol. 1997;150:1687–1699.
102. de Villiers WJ, Smith JD, Miyata M, Dansky HM, Darley E, Gordon S.
Macrophage phenotype in mice deficient in both macrophage-colonystimulating factor (op) and apolipoprotein E. Arterioscler Thromb Vasc
Biol. 1998;18:631–640.
103. Walker LN, Reidy MA, Bowyer DE. Morphology and cell kinetics of
fatty streak lesion formation in the hypercholesterolemic rabbit. Am J
Pathol. 1986;125:450–459.
104.Stoneman V, Braganza D, Figg N, Mercer J, Lang R, Goddard M,
Bennett M. Monocyte/macrophage suppression in CD11b diphtheria
toxin receptor transgenic mice differentially affects atherogenesis and
established plaques. Circ Res. 2007;100:884–893. doi: 10.1161/01.
RES.0000260802.75766.00.
105. Swirski FK, Libby P, Aikawa E, Alcaide P, Luscinskas FW, Weissleder
R, Pittet MJ. Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata. J Clin
Invest. 2007;117:195–205. doi: 10.1172/JCI29950.
106.Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra
J, Garin A, Liu J, Mack M, van Rooijen N, Lira SA, Habenicht AJ,
Randolph GJ. Monocyte subsets differentially employ CCR2, CCR5,
and CX3CR1 to accumulate within atherosclerotic plaques. J Clin Invest.
2007;117:185–194. doi: 10.1172/JCI28549.
107.Potteaux S, Gautier EL, Hutchison SB, van Rooijen N, Rader DJ,
Thomas MJ, Sorci-Thomas MG, Randolph GJ. Suppressed monocyte
recruitment drives macrophage removal from atherosclerotic plaques of
Apoe-/- mice during disease regression. J Clin Invest. 2011;121:2025–
2036. doi: 10.1172/JCI43802.
108.Randolph GJ. Mechanisms that regulate macrophage burden in
atherosclerosis. Circ Res. 2014;114:1757–1771. doi: 10.1161/
CIRCRESAHA.114.301174.
109. Iiyama K, Hajra L, Iiyama M, Li H, DiChiara M, Medoff BD, Cybulsky
MI. Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions
and at sites predisposed to lesion formation. Circ Res. 1999;85:199–207.
Cybulsky et al Myeloid Cells in Atherosclerosis 651
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
110.Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell prolif
eration in human coronary arteries. Proc Natl Acad Sci U S A.
1990;87:4600–4604.
111. Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1990;10:680–687.
112. Lutgens E, de Muinck ED, Kitslaar PJ, Tordoir JH, Wellens HJ, Daemen
MJ. Biphasic pattern of cell turnover characterizes the progression from
fatty streaks to ruptured human atherosclerotic plaques. Cardiovasc Res.
1999;41:473–479.
113. Llodrá J, Angeli V, Liu J, Trogan E, Fisher EA, Randolph GJ. Emigration
of monocyte-derived cells from atherosclerotic lesions characterizes
regressive, but not progressive, plaques. Proc Natl Acad Sci U S A.
2004;101:11779–11784. doi: 10.1073/pnas.0403259101.
114. van Gils JM, Derby MC, Fernandes LR, et al. The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration
of macrophages from plaques. Nat Immunol. 2012;13:136–143. doi:
10.1038/ni.2205.
115. Ramkhelawon B, Yang Y, van Gils JM, Hewing B, Rayner KJ, Parathath
S, Guo L, Oldebeken S, Feig JL, Fisher EA, Moore KJ. Hypoxia induces netrin-1 and Unc5b in atherosclerotic plaques: mechanism for
macrophage retention and survival. Arterioscler Thromb Vasc Biol.
2013;33:1180–1188. doi: 10.1161/ATVBAHA.112.301008.
116.Wanschel A, Seibert T, Hewing B, Ramkhelawon B, Ray TD,
van Gils JM, Rayner KJ, Feig JE, O’Brien ER, Fisher EA, Moore
KJ. Neuroimmune guidance cue Semaphorin 3E is expressed
in atherosclerotic plaques and regulates macrophage retention.
Arterioscler Thromb Vasc Biol. 2013;33:886–893. doi: 10.1161/
ATVBAHA.112.300941.
117. van Gils JM, Ramkhelawon B, Fernandes L, Stewart MC, Guo L, Seibert
T, Menezes GB, Cara DC, Chow C, Kinane TB, Fisher EA, Balcells M,
Alvarez-Leite J, Lacy-Hulbert A, Moore KJ. Endothelial expression of
guidance cues in vessel wall homeostasis dysregulation under proatherosclerotic conditions. Arterioscler Thromb Vasc Biol. 2013;33:911–919.
doi: 10.1161/ATVBAHA.112.301155.
118. Angeli V, Llodrá J, Rong JX, Satoh K, Ishii S, Shimizu T, Fisher EA,
Randolph GJ. Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization. Immunity. 2004;21:561–574.
doi: 10.1016/j.immuni.2004.09.003.
119. Park YM, Febbraio M, Silverstein RL. CD36 modulates migration of
mouse and human macrophages in response to oxidized LDL and may
contribute to macrophage trapping in the arterial intima. J Clin Invest.
2009;119:136–145. doi: 10.1172/JCI35535.
120. Feig JE, Pineda-Torra I, Sanson M, et al. LXR promotes the maximal
egress of monocyte-derived cells from mouse aortic plaques during
atherosclerosis regression. J Clin Invest. 2010;120:4415–4424. doi:
10.1172/JCI38911.
121. Feig JE, Rong JX, Shamir R, Sanson M, Vengrenyuk Y, Liu J, Rayner
K, Moore K, Garabedian M, Fisher EA. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque
monocyte-derived cells. Proc Natl Acad Sci U S A. 2011;108:7166–7171.
doi: 10.1073/pnas.1016086108.
122. Feig JE, Shang Y, Rotllan N, Vengrenyuk Y, Wu C, Shamir R, Torra IP,
Fernandez-Hernando C, Fisher EA, Garabedian MJ. Statins promote
the regression of atherosclerosis via activation of the CCR7-dependent
emigration pathway in macrophages. PLoS One. 2011;6:e28534. doi:
10.1371/journal.pone.0028534.
123. Hewing B, Parathath S, Barrett T, et al. Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and
atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2014;34:779–
789. doi: 10.1161/ATVBAHA.113.303044.
124. Trogan E, Feig JE, Dogan S, Rothblat GH, Angeli V, Tacke F, Randolph
GJ, Fisher EA. Gene expression changes in foam cells and the role of
chemokine receptor CCR7 during atherosclerosis regression in ApoEdeficient mice. Proc Natl Acad Sci U S A. 2006;103:3781–3786. doi:
10.1073/pnas.0511043103.
125.Wu C, Hussein MA, Shrestha E, Leone S, Aiyegbo MS, Lambert
WM, Pourcet B, Cardozo T, Gustafson JA, Fisher EA, Pineda-Torra I,
Garabedian MJ. Modulation of macrophage gene expression via liver X
receptor α serine 198 phosphorylation. Mol Cell Biol. 2015;35:2024–
2034. doi: 10.1128/MCB.00985-14.
126.Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P,
Weissleder R. Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc Natl Acad Sci U S A.
2006;103:10340–10345. doi: 10.1073/pnas.0604260103.
127. Martens JS, Reiner NE, Herrera-Velit P, Steinbrecher UP. Phosphatidylinositol
3-kinase is involved in the induction of macrophage growth by oxidized low
density lipoprotein. J Biol Chem. 1998;273:4915–4920.
128. Hamilton JA, Myers D, Jessup W, Cochrane F, Byrne R, Whitty G, Moss
S. Oxidized LDL can induce macrophage survival, DNA synthesis, and
enhanced proliferative response to CSF-1 and GM-CSF. Arterioscler
Thromb Vasc Biol. 1999;19:98–105.
129. Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis: beyond
lipid uptake. Arterioscler Thromb Vasc Biol. 2006;26:1702–1711. doi:
10.1161/01.ATV.0000229218.97976.43.
130. Hazen SL. Oxidized phospholipids as endogenous pattern recognition
ligands in innate immunity. J Biol Chem. 2008;283:15527–15531. doi:
10.1074/jbc.R700054200.
131. Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S,
Rhee JS, Silverstein R, Hoff HF, Freeman MW. Scavenger receptors class
A-I/II and CD36 are the principal receptors responsible for the uptake of
modified low density lipoprotein leading to lipid loading in macrophages.
J Biol Chem. 2002;277:49982–49988. doi: 10.1074/jbc.M209649200.
132.Sakai M, Miyazaki A, Hakamata H, Kodama T, Suzuki H, Kobori
S, Shichiri M, Horiuchi S. The scavenger receptor serves as a route
for internalization of lysophosphatidylcholine in oxidized low density lipoprotein-induced macrophage proliferation. J Biol Chem.
1996;271:27346–27352.
133.Yvan-Charvet L, Pagler T, Gautier EL, Avagyan S, Siry RL, Han
S, Welch CL, Wang N, Randolph GJ, Snoeck HW, Tall AR. ATPbinding cassette transporters and HDL suppress hematopoietic stem
cell proliferation. Science. 2010;328:1689–1693. doi: 10.1126/
science.1189731.
134. Murphy AJ, Akhtari M, Tolani S, Pagler T, Bijl N, Kuo CL, Wang M,
Sanson M, Abramowicz S, Welch C, Bochem AE, Kuivenhoven JA,
Yvan-Charvet L, Tall AR. ApoE regulates hematopoietic stem cell
proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice. J Clin Invest. 2011;121:4138–4149. doi: 10.1172/
JCI57559.
135.Chapman CM, Beilby JP, McQuillan BM, Thompson PL, Hung J.
Monocyte count, but not C-reactive protein or interleukin-6, is an independent risk marker for subclinical carotid atherosclerosis. Stroke.
2004;35:1619–1624. doi: 10.1161/01.STR.0000130857.19423.ad.
136. Nasir K, Guallar E, Navas-Acien A, Criqui MH, Lima JA. Relationship
of monocyte count and peripheral arterial disease: results from the
National Health and Nutrition Examination Survey 1999-2002.
Arterioscler Thromb Vasc Biol. 2005;25:1966–1971. doi: 10.1161/01.
ATV.0000175296.02550.e4.
137. Johnsen SH, Fosse E, Joakimsen O, Mathiesen EB, Stensland-Bugge E,
Njølstad I, Arnesen E. Monocyte count is a predictor of novel plaque formation: a 7-year follow-up study of 2610 persons without carotid plaque
at baseline the Tromsø Study. Stroke. 2005;36:715–719. doi: 10.1161/01.
STR.0000158909.07634.83.
138.Wu H, Gower RM, Wang H, Perrard XY, Ma R, Bullard DC, Burns
AR, Paul A, Smith CW, Simon SI, Ballantyne CM. Functional role
of CD11c+ monocytes in atherogenesis associated with hypercholesterolemia. Circulation. 2009;119:2708–2717. doi: 10.1161/
CIRCULATIONAHA.108.823740.
139.Gower RM, Wu H, Foster GA, Devaraj S, Jialal I, Ballantyne CM,
Knowlton AA, Simon SI. CD11c/CD18 expression is upregulated on
blood monocytes during hypertriglyceridemia and enhances adhesion
to vascular cell adhesion molecule-1. Arterioscler Thromb Vasc Biol.
2011;31:160–166. doi: 10.1161/ATVBAHA.110.215434.
140. Foster GA, Gower RM, Stanhope KL, Havel PJ, Simon SI, Armstrong
EJ. On-chip phenotypic analysis of inflammatory monocytes in atherogenesis and myocardial infarction. Proc Natl Acad Sci U S A.
2013;110:13944–13949. doi: 10.1073/pnas.1300651110.
141. Xu L, Dai Perrard X, Perrard JL, Yang D, Xiao X, Teng BB, Simon
SI, Ballantyne CM, Wu H. Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia.
Arterioscler Thromb Vasc Biol. 2015;35:1787–1797. doi: 10.1161/
ATVBAHA.115.305609.
142.den Hartigh LJ, Connolly-Rohrbach JE, Fore S, Huser TR, Rutledge
JC. Fatty acids from very low-density lipoprotein lipolysis products
induce lipid droplet accumulation in human monocytes. J Immunol.
2010;184:3927–3936. doi: 10.4049/jimmunol.0903475.
143. Varela LM, Ortega A, Bermudez B, Lopez S, Pacheco YM, Villar J, Abia
R, Muriana FJ. A high-fat meal promotes lipid-load and apolipoprotein
B-48 receptor transcriptional activity in circulating monocytes. Am J
Clin Nutr. 2011;93:918–925. doi: 10.3945/ajcn.110.007765.
652 Circulation Research February 19, 2016
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
144. Lichtman AH, Binder CJ, Tsimikas S, Witztum JL. Adaptive immunity
in atherogenesis: new insights and therapeutic approaches. J Clin Invest.
2013;123:27–36. doi: 10.1172/JCI63108.
145. Gautier EL, Huby T, Saint-Charles F, Ouzilleau B, Pirault J, Deswaerte V,
Ginhoux F, Miller ER, Witztum JL, Chapman MJ, Lesnik P. Conventional
dendritic cells at the crossroads between immunity and cholesterol homeostasis in atherosclerosis. Circulation. 2009;119:2367–2375. doi:
10.1161/CIRCULATIONAHA.108.807537.
146.Buono C, Pang H, Uchida Y, Libby P, Sharpe AH, Lichtman AH.
B7-1/B7-2 costimulation regulates plaque antigen-specific T-cell
responses and atherogenesis in low-density lipoprotein receptordeficient mice. Circulation. 2004;109:2009–2015. doi: 10.1161/01.
CIR.0000127121.16815.F1.
147. Sun J, Hartvigsen K, Chou MY, Zhang Y, Sukhova GK, Zhang J, LopezIlasaca M, Diehl CJ, Yakov N, Harats D, George J, Witztum JL, Libby P,
Ploegh H, Shi GP. Deficiency of antigen-presenting cell invariant chain
reduces atherosclerosis in mice. Circulation. 2010;122:808–820. doi:
10.1161/CIRCULATIONAHA.109.891887.
148. Tsiantoulas D, Diehl CJ, Witztum JL, Binder CJ. B cells and humoral immunity in atherosclerosis. Circ Res. 2014;114:1743–1756. doi: 10.1161/
CIRCRESAHA.113.301145.
149. Koltsova EK, Garcia Z, Chodaczek G, Landau M, McArdle S, Scott SR,
von Vietinghoff S, Galkina E, Miller YI, Acton ST, Ley K. Dynamic T
cell-APC interactions sustain chronic inflammation in atherosclerosis. J
Clin Invest. 2012;122:3114–3126. doi: 10.1172/JCI61758.
150. Yin YW, Liao SQ, Zhang MJ, Liu Y, Li BH, Zhou Y, Chen L, Gao CY, Li
JC, Zhang LL. TLR4-mediated inflammation promotes foam cell formation of vascular smooth muscle cell by upregulating ACAT1 expression.
Cell Death Dis. 2014;5:e1574. doi: 10.1038/cddis.2014.535.
151. Ma C, Ouyang Q, Huang Z, Chen X, Lin Y, Hu W, Lin L. Toll-like receptor 9 inactivation alleviated atherosclerotic progression and inhibited
macrophage polarized to M1 phenotype in ApoE-/- mice. Dis Markers.
2015;2015:909572. doi: 10.1155/2015/909572.
152.Hayashi C, Papadopoulos G, Gudino CV, Weinberg EO, Barth KR,
Madrigal AG, Chen Y, Ning H, LaValley M, Gibson FC III, Hamilton JA,
Genco CA. Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen. J Immunol.
2012;189:3681–3688. doi: 10.4049/jimmunol.1201541.
153. Mullick AE, Tobias PS, Curtiss LK. Modulation of atherosclerosis in
mice by Toll-like receptor 2. J Clin Invest. 2005;115:3149–3156. doi:
10.1172/JCI25482.
154.Mullick AE, Soldau K, Kiosses WB, Bell TA III, Tobias PS, Curtiss
LK. Increased endothelial expression of Toll-like receptor 2 at sites of
disturbed blood flow exacerbates early atherogenic events. J Exp Med.
2008;205:373–383. doi: 10.1084/jem.20071096.
155. Subramanian M, Thorp E, Hansson GK, Tabas I. Treg-mediated suppression of atherosclerosis requires MYD88 signaling in DCs. J Clin Invest.
2013;123:179–188. doi: 10.1172/JCI64617.
156. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, Zoll
J, Merval R, Esposito B, Cohen JL, Fisson S, Flavell RA, Hansson GK,
Klatzmann D, Tedgui A, Mallat Z. Natural regulatory T cells control the
development of atherosclerosis in mice. Nat Med. 2006;12:178–180. doi:
10.1038/nm1343.
157. Klingenberg R, Gerdes N, Badeau RM, et al. Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J Clin
Invest. 2013;123:1323–1334. doi: 10.1172/JCI63891.
158. Weber C, Meiler S, Döring Y, et al. CCL17-expressing dendritic cells
drive atherosclerosis by restraining regulatory T cell homeostasis in
mice. J Clin Invest. 2011;121:2898–2910. doi: 10.1172/JCI44925.
159. Alferink J, Lieberam I, Reindl W, Behrens A, Weiss S, Hüser N, Gerauer
K, Ross R, Reske-Kunz AB, Ahmad-Nejad P, Wagner H, Förster I.
Compartmentalized production of CCL17 in vivo: strong inducibility in
peripheral dendritic cells contrasts selective absence from the spleen. J
Exp Med. 2003;197:585–599.
160.Daissormont IT, Christ A, Temmerman L, et al. Plasmacytoid den
dritic cells protect against atherosclerosis by tuning T-cell proliferation and activity. Circ Res. 2011;109:1387–1395. doi: 10.1161/
CIRCRESAHA.111.256529.
161.Döring Y, Manthey HD, Drechsler M, et al. Auto-antigenic proteinDNA complexes stimulate plasmacytoid dendritic cells to promote
atherosclerosis. Circulation. 2012;125:1673–1683. doi: 10.1161/
CIRCULATIONAHA.111.046755.
162.Macritchie N, Grassia G, Sabir SR, Maddaluno M, Welsh P, Sattar
N, Ialenti A, Kurowska-Stolarska M, McInnes IB, Brewer JM,
Garside P, Maffia P. Plasmacytoid dendritic cells play a key role
in promoting atherosclerosis in apolipoprotein E-deficient mice.
Arterioscler Thromb Vasc Biol. 2012;32:2569–2579. doi: 10.1161/
ATVBAHA.112.251314.
163. Sage AP, Murphy D, Maffia P, et al. MHC Class II-restricted antigen
presentation by plasmacytoid dendritic cells drives proatherogenic
T cell immunity. Circulation. 2014;130:1363–1373. doi: 10.1161/
CIRCULATIONAHA.114.011090.
164. Ghosh HS, Cisse B, Bunin A, Lewis KL, Reizis B. Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells. Immunity. 2010;33:905–916. doi: 10.1016/j.
immuni.2010.11.023.
165. Habets KL, van Puijvelde GH, van Duivenvoorde LM, van Wanrooij EJ,
de Vos P, Tervaert JW, van Berkel TJ, Toes RE, Kuiper J. Vaccination
using oxidized low-density lipoprotein-pulsed dendritic cells reduces atherosclerosis in LDL receptor-deficient mice. Cardiovasc Res.
2010;85:622–630. doi: 10.1093/cvr/cvp338.
166.Hermansson A, Johansson DK, Ketelhuth DF, Andersson J, Zhou
X, Hansson GK. Immunotherapy with tolerogenic apolipoprotein
B-100-loaded dendritic cells attenuates atherosclerosis in hypercholesterolemic mice. Circulation. 2011;123:1083–1091. doi: 10.1161/
CIRCULATIONAHA.110.973222.
167.Hjerpe C, Johansson D, Hermansson A, Hansson GK, Zhou X.
Dendritic cells pulsed with malondialdehyde modified low density lipoprotein aggravate atherosclerosis in Apoe(-/-) mice. Atherosclerosis.
2010;209:436–441. doi: 10.1016/j.atherosclerosis.2009.10.003.
168. Ludewig B, Freigang S, Jäggi M, Kurrer MO, Pei YC, Vlk L, Odermatt
B, Zinkernagel RM, Hengartner H. Linking immune-mediated arterial
inflammation and cholesterol-induced atherosclerosis in a transgenic
mouse model. Proc Natl Acad Sci U S A. 2000;97:12752–12757. doi:
10.1073/pnas.220427097.
169. Helft J, Böttcher J, Chakravarty P, Zelenay S, Huotari J, Schraml BU,
Goubau D, Reis e Sousa C. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages
and dendritic cells. Immunity. 2015;42:1197–1211. doi: 10.1016/j.
immuni.2015.05.018.
170. Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, Ravetch
JV, Steinman RM, Nussenzweig MC. Dendritic cells induce peripheral T
cell unresponsiveness under steady state conditions in vivo. J Exp Med.
2001;194:769–779.
171. Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad
Sci U S A. 2002;99:351–358. doi: 10.1073/pnas.231606698.
172. Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC,
von Boehmer H. Inducing and expanding regulatory T cell populations
by foreign antigen. Nat Immunol. 2005;6:1219–1227. doi: 10.1038/
ni1265.
173. Caminschi I, Proietto AI, Ahmet F, et al. The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement. Blood.
2008;112:3264–3273. doi: 10.1182/blood-2008-05-155176.
174. Cheong C, Choi JH, Vitale L, et al. Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic
cells within human anti-human DEC205 monoclonal antibody. Blood.
2010;116:3828–3838. doi: 10.1182/blood-2010-06-288068.
175.Idoyaga J, Cheong C, Suda K, Suda N, Kim JY, Lee H, Park CG,
Steinman RM. Cutting edge: langerin/CD207 receptor on dendritic cells
mediates efficient antigen presentation on MHC I and II products in vivo.
J Immunol. 2008;180:3647–3650.
176. Loschko J, Schlitzer A, Dudziak D, Drexler I, Sandholzer N, Bourquin
C, Reindl W, Krug AB. Antigen delivery to plasmacytoid dendritic cells
via BST2 induces protective T cell-mediated immunity. J Immunol.
2011;186:6718–6725. doi: 10.4049/jimmunol.1004029.
Macrophages and Dendritic Cells: Partners in Atherogenesis
Myron I. Cybulsky, Cheolho Cheong and Clinton S. Robbins
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Circ Res. 2016;118:637-652
doi: 10.1161/CIRCRESAHA.115.306542
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CIRCRES/2015/306542/R1 Suppl. Page 1
Dendritic cell origin and classification
DCs are subdivided into classical (cDCs) and plasmacytoid (pDCs). Murine cDCs were
discovered in lymphoid tissues and defined based on functional and morphological features that
distinguish them from monocytes and macrophages. These include: (a) nonadherent cells with
probing morphology (continuously forming and retracting processes), (b) preferential localization
to T cell areas thereby facilitating clonal section of antigen-specific T cells, (c) strong stimulators
of the mixed leukocyte reaction that measures the capacity for stimulating allogeneic T cells, (d)
superior antigen presentation to CD4 and CD8 T cells relative to other myeloid cells in the
context of major histocompatibility complex (MHC) class II (MHC-II) and MHC-I, (e) lower
phagocytic activity compared with monocytes and macrophages, and (f) loss of monocyte and
macrophage markers.1-5 pDCs were identified subsequently. Their morphology is distinct from
cDCs, and upon stimulation by foreign nucleic acids they have the unique capacity to produce
abundant type I interferon (IFN) and prime T cells against viral antigens.4,5 In mice, both cDCs
and pDCs reside in lymphoid and most nonlymphoid tissues, but only pDCs are detected in the
blood.6
DC subsets are based on their origins, yet within tissue DC populations are though to acquire
distinct phenotypes based on local environmental cues. Both cDCs and pDCs are derived from
bone marrow precursors, although the mapping of DC developmental pathway is still being
debated. Previously, a common macrophage-DC-restricted precursor (MDP) was proposed7-9;
however, the existence of this precursor was recently challenged10, and additional studies will
be necessary to resolve this controversy. Nonetheless, has been firmly established that a
common DC progenitor (CDP) can generate cDCs and pDCs in lymphoid organs, but lacks the
potential to generate monocytes and macrophages11,12 (Figure 1). Precursors of cDCs (precDCs) form in the bone marrow and disseminate to lymphoid tissues.8,13,14 In addition to cDCs
derived from CDPs and pre-cDCs, DCs can originate from blood monocytes (Mo-DCs).2,15-18
Mo-DCs are as capable as cDCs at antigen presentation, including cross-presentation of
proteins and live gram-negative bacteria.2 Mo-DCs can be distinguished from CDP-derived
cDCs by cell surface markers (see below). Although DCs and macrophages constitute the bulk
of tissue myeloid cells, it should be noted that DC developmental pathways have been studied
mostly in lymphoid tissues, and additional work will be required to map DC development in other
tissues, including the aorta. For example, pre-cDCs have not been identified in the normal or
diseased aorta.
Epidermal Langerhans cells, which express Langerin (CD207), constitute a subset of cDCs.
Interestingly, these cells are maintained by local proliferation19 and are radio-resistant in bone
marrow chimera experiments where they remain as host-derived cells for at least 18 months.20
In contrast, Langerin+ dermal DCs are rapidly replaced by donor cells in bone marrow chimera
experiments, implying differential origin of dermal and epidermal Langerin+ DCs.21 Epidermal
Langerhans cells are derived predominantly from embryonic fetal liver monocytes with a minor
contribution of yolk sac-derived cells,22 but in inflamed skin they are derived from blood
monocytes.23
Cell surface markers are commonly used for identification of DCs. Mouse DCs including dermal
and epidermal Langerin+ DCs express CD11c (alpha-X integrin) and high levels of MHC-II, but
lack T, B, natural killer cell, granulocyte and erythroid markers. Other markers include
chemokine receptors, and c-type lectin receptors. Mouse pDCs express CD4, Siglec-H,
mPDCA1/BST2/CD317, B220/CD45RA, and low levels of CD11c and MHC-II (Figure 1). CDPderived cDCs express CD8α in lymphoid tissue and CD103 (alpha-E integrin) in nonlymphoid
tissues, and may express CD11b (alpha-M integrin), but do not express macrophage markers
such as F4/80 and CD206 (mannose receptor). It is presumed that lymphoid CD8α+CD103cDCs are equivalent to nonlymphoid CD103+CD8α- cDCs.24 Mo-DCs express macrophage
markers CD11b, F4/80, CD14, CD206 and CD209a/DC-SIGN, but do not express CD103 or
CIRCRES/2015/306542/R1 Suppl. Page 2
CD8α. It is important to note that lineage tracing approaches were not used to establish the
above paradigm, and it is possible that in some tissues local environmental cues may dominate
over origin (CDP versus monocyte) in establishing the local cDC phenotype.
Surface markers are insufficient for identification of distinct myeloid cell subsets across tissues.
Several markers previously considered to be DC-specific are actually expressed by other cell
types. For example, some macrophages25 and T cells26 can also express CD11c. Thus, when
using the CD11c promoter to regulate the expression of Cre or DTR, it is important to remember
that other cell types may be targeted in addition to DCs. The pDC-specific markers
mPDCA1/BST2/CD317 and Siglec-H can be expressed by many cell types including inflamed
macrophages.4,27 CD11b is not a reliable marker for CD8α- DC, and CD11b+ DCs may be a
mixed population of DCs and macrophages. Given the promiscuous expression of CD11c and
higher autofluorescence of macrophages, it is prudent to use CD64 and MerTK in combination
with CD11b and F4/80 to identify macrophages and to exclude these cells from analysis of DC
populations.28,29 It should also be noted that many markers that are used successfully to identify
DC subsets in lymphoid organs are difficult to use in tissues such as the aorta that require a
combination of collagenases for cell isolation. In this case, intracellular staining can be
employed to visualize some markers. For example, CD4 and CD206/MR expression can be
rescued by intracellular staining.2,30 Thus, in addition to cell surface markers, multiple
parameters including intracellular staining and transcription factors (see below) should be used
to identify DC subset equivalents to lymphoid counterparts in the aorta. Recently, a
classification of DC subsets mainly in the bone marrow and lymphoid tissues was proposed
based on surface markers, growth factor requirements, and subset-specific transcription
factors.31-33
While traditional flow cytometry forms the foundations for defining myeloid cell populations, it
cannot elucidate the full complexity of the myeloid system. To overcome this, a recent advance
was to combine flow cytometry and mass spectrometry34. This technology, known as CyTOF,
enables determination of more than 30 different cell surface or intracellular markers per cell due
to minimum spectral overlap between transition element isotopes conjugated to antibodies.
Interestingly, this approach identified more than 28 clusters of myeloid cells across eight tissues
35
. Analysis of the DC transcriptome by transcription profiling has also contributed to defining
the ontogeny and subtypes of myeloid cells across the entire mouse hematopietic lineage.27,36,37
Recent studies through the Immunological Genome (ImmGen) Project have defined a core-DC
gene signature compared with macrophages36. The above approaches may help to identify
aortic DC subsets. The only problem is the small number of aortic myeloid cells, especially
cDCs and pDCs. Recent advance in single cell trancriptome-analysis using RNAseq38 may
overcome this limitation.
Bone marrow culture systems have been developed to obtain large numbers of DCs for in vitro
studies. This approach also contributed to studies of DC immunizing properties.39-42
Unfortunately, bone marrow culture does not fully recapitulate the properties of tissue-resident
cDCs in vivo. Moreover, DCs derived from bone marrow cultured with
granulocyte/macrophage-colony stimulating factor (GM-CSF or CSF2) were recently shown to
be a heterogeneous population consisting of DCs and macrophages.43 Bone marrow cells
cultured with FMS-like tyrosine kinase 3 ligand (Flt3L) also generate multiple subsets of DCs,
including pDCs (CD11cloB220+) and two types of cDCs (CD24+ and CD11b+CD172a+). These
cDC subsets are considered equivalent to lymphoid CD8α+ and CD8α- DCs, respectively.
However, in vitro-derived cDCs do not express cell surface CD8α, CD103, CD205/DEC-205 and
DCIR2/33D1, suggesting the acquisition of cell surface markers is subject to tissue or
environment context or appropriate activation signals.44
Mature cDCs derived from pre-DCs or monocytes, but not pDCs, monocytes and macrophages,
express the transcription factor Zbtb46/BTBD4.45 Zbtb46 is not required for early cDC
CIRCRES/2015/306542/R1 Suppl. Page 3
development, but Zbtb46-deficiency resulted in the alteration of the proportion of CD8α+ and
CD8α- DCs in the spleen and a small increase in DCs that migrated from the skin to draining
lymph nodes.46 Zbtb46 is a useful tool for visualization and deletion of cDC in vivo. Coupled to
a fluorescent reporter transgenic mice with the Zbtb46 promoter enable cDCs visualization, or
their selective depletion when coupled to the diphtheria toxin receptor (DTR).45,46 Zbtb46
expression is also a useful tool to determine if a population of immune cells has cDC features.
For example, monocytes infected with Listeria monocytogenes produce TNF and iNOS and
have been called TNF/iNOS-producing DCs (TIP-DCs).47 However, TIP-DCs do not express
Zbtb46,45,46 which suggests that they are more closely related to activated monocytes than to
DCs. Other transcription factors are also required for cDC development and differentiation. For
example, BATF3-deficient mice lack splenic CD8α and nonlymphoid CD103+ DCs. Inteferon
+
regulatory factor 8 (IRF8)-deficient mice lack CD8α CD103+ DCs, but also have reduced
+
48
+
pDCs. ID2-deficient mice lack CD8α CD103 DCs.49 In contrast, IRF4 and several additional
transcriptional factors such as RelB, RBP-J, and IRF2 are required for CD8α CD11b+ DC
31,32
differentiation.
Transcription factors E2-2 and IRF8 are critical for differentiation of pDCs.50-52 Interestingly, loss
of E2-2 expression in mature pDCs resulted in their spontaneous conversion to cDCs.50 It is
important to take this into consideration when using E2-2 deficient mice to dissect pDC
functions in pathophysiological settings. While IRF8 is critical for normal development of CD8α+
DCs and pDCs, BXH-2 mice that harbor a point mutation in IRF8 (R294C) do not exhibit an
impairment of pDC development.52,53 This suggests that different transcription factors interact
with IRF8 and influence CD8α+ DC or pDC development.
DC identification and subtyping is still evolving. It is prudent to rely on a combination of
approaches, including surface markers, transcription factor expression and transcriptional
profiling to define tissue-resident DC subsets. Approaches such as parabiosis can distinguish
self-renewing DC populations versus blood-derived replenishment both in steady-state and
inflammatory conditions.54 Sophisticated lineage tracing tools are lacking for DCs, and hopefully
will be developed in the near future. The exception is genetic tracing of Clec9a/DNGR-1
expression, a receptor for dead cells that is expressed by CD115+ CDPs and pre-cDCs, but not
MDPs.55 This lineage tracing approach revealed that some CD11b+ DC populations in
nonlymphoid tissues are in fact derived from CDP and pre-cDC precursors, and is potentially
applicable to DC populations in arteries.
CIRCRES/2015/306542/R1 Suppl. Page 4
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of
mice. I. Morphology, quantitation, tissue distribution. J Exp Med. 1973; 137:1142-1162.
Cheong C, Matos I, Choi JH, Dandamudi DB, Shrestha E, Longhi MP, Jeffrey KL, Anthony
RM, Kluger C, Nchinda G, Koh H, Rodriguez A, Idoyaga J, Pack M, Velinzon K, Park CG,
Steinman RM. Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+)
dendritic cells for immune T cell areas. Cell. 2010; 143:416-429.
Choi JH, Cheong C, Dandamudi DB, Park CG, Rodriguez A, Mehandru S, Velinzon K,
Jung IH, Yoo JY, Oh GT, Steinman RM. Flt3 signaling-dependent dendritic cells protect
against atherosclerosis. Immunity. 2011; 35:819-831.
Reizis B, Colonna M, Trinchieri G, Barrat F, Gilliet M. Plasmacytoid dendritic cells: onetrick ponies or workhorses of the immune system? Nat Rev Immunol. 2011; 11:558-565.
Steinman RM. Decisions about dendritic cells: past, present, and future. Annu Rev
Immunol. 2012; 30:1-22.
O'Keeffe M, Hochrein H, Vremec D, Scott B, Hertzog P, Tatarczuch L, Shortman K.
Dendritic cell precursor populations of mouse blood: identification of the murine
homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors. Blood.
2003; 101:1453-1459.
Fogg DK, Sibon C, Miled C, Jung S, Aucouturier P, Littman DR, Cumano A, Geissmann F.
A clonogenic bone marrow progenitor specific for macrophages and dendritic cells.
Science. 2006; 311:83-87.
Liu K, Victora GD, Schwickert TA, Guermonprez P, Meredith MM, Yao K, Chu FF,
Randolph GJ, Rudensky AY, Nussenzweig M. In vivo analysis of dendritic cell
development and homeostasis. Science. 2009; 324:392-397.
Waskow C, Liu K, Darrasse-Jeze G, Guermonprez P, Ginhoux F, Merad M, Shengelia T,
Yao K, Nussenzweig M. The receptor tyrosine kinase Flt3 is required for dendritic cell
development in peripheral lymphoid tissues. Nat Immunol. 2008; 9:676-683.
Sathe P, Metcalf D, Vremec D, Naik SH, Langdon WY, Huntington ND, Wu L, Shortman
K. Lymphoid tissue and plasmacytoid dendritic cells and macrophages do not share a
common macrophage-dendritic cell-restricted progenitor. Immunity. 2014; 41:104-115.
Naik SH, Sathe P, Park HY, Metcalf D, Proietto AI, Dakic A, Carotta S, O'Keeffe M, Bahlo
M, Papenfuss A, Kwak JY, Wu L, Shortman K. Development of plasmacytoid and
conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo.
Nat Immunol. 2007; 8:1217-1226.
Onai N, Obata-Onai A, Schmid MA, Ohteki T, Jarrossay D, Manz MG. Identification of
clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell
progenitors in mouse bone marrow. Nat Immunol. 2007; 8:1207-1216.
Diao J, Winter E, Cantin C, Chen W, Xu L, Kelvin D, Phillips J, Cattral MS. In situ
replication of immediate dendritic cell (DC) precursors contributes to conventional DC
homeostasis in lymphoid tissue. J Immunol. 2006; 176:7196-7206.
Naik SH, Metcalf D, van Nieuwenhuijze A, Wicks I, Wu L, O'Keeffe M, Shortman K.
Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. Nat
Immunol. 2006; 7:663-671.
Leon B, Lopez-Bravo M, Ardavin C. Monocyte-derived dendritic cells formed at the
infection site control the induction of protective T helper 1 responses against Leishmania.
Immunity. 2007; 26:519-531.
Nakano H, Lin KL, Yanagita M, Charbonneau C, Cook DN, Kakiuchi T, Gunn MD. Bloodderived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1
immune responses. Nat Immunol. 2009; 10:394-402.
Kool M, Soullie T, van Nimwegen M, Willart MA, Muskens F, Jung S, Hoogsteden HC,
CIRCRES/2015/306542/R1 Suppl. Page 5
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Hammad H, Lambrecht BN. Alum adjuvant boosts adaptive immunity by inducing uric acid
and activating inflammatory dendritic cells. J Exp Med. 2008; 205:869-882.
Siddiqui KR, Laffont S, Powrie F. E-cadherin marks a subset of inflammatory dendritic
cells that promote T cell-mediated colitis. Immunity. 2010; 32:557-567.
Chorro L, Sarde A, Li M, Woollard KJ, Chambon P, Malissen B, Kissenpfennig A,
Barbaroux JB, Groves R, Geissmann F. Langerhans cell (LC) proliferation mediates
neonatal development, homeostasis, and inflammation-associated expansion of the
epidermal LC network. J Exp Med. 2009; 206:3089-3100.
Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG,
Engleman EG. Langerhans cells renew in the skin throughout life under steady-state
conditions. Nat Immunol. 2002; 3:1135-1141.
Ginhoux F, Collin MP, Bogunovic M, Abel M, Leboeuf M, Helft J, Ochando J,
Kissenpfennig A, Malissen B, Grisotto M, Snoeck H, Randolph G, Merad M. Blood-derived
dermal langerin+ dendritic cells survey the skin in the steady state. J Exp Med. 2007;
204:3133-3146.
Hoeffel G, Wang Y, Greter M, See P, Teo P, Malleret B, Leboeuf M, Low D, Oller G,
Almeida F, Choy SH, Grisotto M, Renia L, Conway SJ, Stanley ER, Chan JK, Ng LG,
Samokhvalov IM, Merad M, Ginhoux F. Adult Langerhans cells derive predominantly from
embryonic fetal liver monocytes with a minor contribution of yolk sac-derived
macrophages. J Exp Med. 2012; 209:1167-1181.
Ginhoux F, Tacke F, Angeli V, Bogunovic M, Loubeau M, Dai XM, Stanley ER, Randolph
GJ, Merad M. Langerhans cells arise from monocytes in vivo. Nat Immunol. 2006; 7:265273.
Edelson BT, Kc W, Juang R, Kohyama M, Benoit LA, Klekotka PA, Moon C, Albring JC,
Ise W, Michael DG, Bhattacharya D, Stappenbeck TS, Holtzman MJ, Sung SS, Murphy
TL, Hildner K, Murphy KM. Peripheral CD103+ dendritic cells form a unified subset
developmentally related to CD8alpha+ conventional dendritic cells. J Exp Med. 2010;
207:823-836.
Bradford BM, Sester DP, Hume DA, Mabbott NA. Defining the anatomical localisation of
subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax,
CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to
discriminate dendritic cells from macrophages. Immunobiology. 2011; 216:1228-1237.
Huleatt JW, Lefrancois L. Antigen-driven induction of CD11c on intestinal intraepithelial
lymphocytes and CD8+ T cells in vivo. J Immunol. 1995; 154:5684-5693.
Heng TS, Painter MW, Immunological Genome Project C. The Immunological Genome
Project: networks of gene expression in immune cells. Nat Immunol. 2008; 9:1091-1094.
Jakubzick C, Gautier EL, Gibbings SL, Sojka DK, Schlitzer A, Johnson TE, Ivanov S,
Duan Q, Bala S, Condon T, van Rooijen N, Grainger JR, Belkaid Y, Ma'ayan A, Riches
DW, Yokoyama WM, Ginhoux F, Henson PM, Randolph GJ. Minimal differentiation of
classical monocytes as they survey steady-state tissues and transport antigen to lymph
nodes. Immunity. 2013; 39:599-610.
Epelman S, Lavine KJ, Beaudin AE, Sojka DK, Carrero JA, Calderon B, Brija T, Gautier
EL, Ivanov S, Satpathy AT, Schilling JD, Schwendener R, Sergin I, Razani B, Forsberg
EC, Yokoyama WM, Unanue ER, Colonna M, Randolph GJ, Mann DL. Embryonic and
adult-derived resident cardiac macrophages are maintained through distinct mechanisms
at steady state and during inflammation. Immunity. 2014; 40:91-104.
Cheong C, Choi JH. Dendritic cells and regulatory T cells in atherosclerosis. Mol Cells.
2012; 34:341-347.
Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of
monocytes, macrophages, and dendritic cells. Science. 2010; 327:656-661.
Hashimoto D, Miller J, Merad M. Dendritic cell and macrophage heterogeneity in vivo.
CIRCRES/2015/306542/R1 Suppl. Page 6
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Immunity. 2011; 35:323-335.
Murphy KM. Transcriptional control of dendritic cell development. Adv Immunol. 2013;
120:239-267.
Bendall SC, Simonds EF, Qiu P, Amir el AD, Krutzik PO, Finck R, Bruggner RV, Melamed
R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe'er D, Tanner SD, Nolan
GP. Single-cell mass cytometry of differential immune and drug responses across a
human hematopoietic continuum. Science. 2011; 332:687-696.
Becher B, Schlitzer A, Chen J, Mair F, Sumatoh HR, Teng KW, Low D, Ruedl C, RiccardiCastagnoli P, Poidinger M, Greter M, Ginhoux F, Newell EW. High-dimensional analysis of
the murine myeloid cell system. Nat Immunol. 2014; 15:1181-1189.
Miller JC, Brown BD, Shay T, Gautier EL, Jojic V, Cohain A, Pandey G, Leboeuf M, Elpek
KG, Helft J, Hashimoto D, Chow A, Price J, Greter M, Bogunovic M, Bellemare-Pelletier A,
Frenette PS, Randolph GJ, Turley SJ, Merad M, Immunological Genome C. Deciphering
the transcriptional network of the dendritic cell lineage. Nat Immunol. 2012; 13:888-899.
Gautier EL, Shay T, Miller J, Greter M, Jakubzick C, Ivanov S, Helft J, Chow A, Elpek KG,
Gordonov S, Mazloom AR, Ma'ayan A, Chua WJ, Hansen TH, Turley SJ, Merad M,
Randolph GJ, Immunological Genome C. Gene-expression profiles and transcriptional
regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.
Nat Immunol. 2012; 13:1118-1128.
Shalek AK, Satija R, Adiconis X, Gertner RS, Gaublomme JT, Raychowdhury R, Schwartz
S, Yosef N, Malboeuf C, Lu D, Trombetta JJ, Gennert D, Gnirke A, Goren A, Hacohen N,
Levin JZ, Park H, Regev A. Single-cell transcriptomics reveals bimodality in expression
and splicing in immune cells. Nature. 2013; 498:236-240.
Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J. GM-CSF and TNF-alpha
cooperate in the generation of dendritic Langerhans cells. Nature. 1992; 360:258-261.
Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM.
Generation of large numbers of dendritic cells from mouse bone marrow cultures
supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;
176:1693-1702.
Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human
dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus
interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 1994;
179:1109-1118.
Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G,
Fritsch PO, Steinman RM, Schuler G. Proliferating dendritic cell progenitors in human
blood. J Exp Med. 1994; 180:83-93.
Helft J, Bottcher J, Chakravarty P, Zelenay S, Huotari J, Schraml BU, Goubau D, Reis e
Sousa C. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of
CD11c(+)MHCII(+) macrophages and dendritic cells. Immunity. 2015; 42:1197-1211.
Brasel K, De Smedt T, Smith JL, Maliszewski CR. Generation of murine dendritic cells
from flt3-ligand-supplemented bone marrow cultures. Blood. 2000; 96:3029-3039.
Satpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, Murphy TL,
Murphy KM. Zbtb46 expression distinguishes classical dendritic cells and their committed
progenitors from other immune lineages. J Exp Med. 2012; 209:1135-1152.
Meredith MM, Liu K, Kamphorst AO, Idoyaga J, Yamane A, Guermonprez P, Rihn S, Yao
KH, Silva IT, Oliveira TY, Skokos D, Casellas R, Nussenzweig MC. Zinc finger
transcription factor zDC is a negative regulator required to prevent activation of classical
dendritic cells in the steady state. J Exp Med. 2012; 209:1583-1593.
Serbina NV, Salazar-Mather TP, Biron CA, Kuziel WA, Pamer EG. TNF/iNOS-producing
dendritic cells mediate innate immune defense against bacterial infection. Immunity. 2003;
19:59-70.
CIRCRES/2015/306542/R1 Suppl. Page 7
48.
49.
50.
51.
52.
53.
54.
55.
Aliberti J, Schulz O, Pennington DJ, Tsujimura H, Reis e Sousa C, Ozato K, Sher A.
Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells.
Blood. 2003; 101:305-310.
Ginhoux F, Liu K, Helft J, Bogunovic M, Greter M, Hashimoto D, Price J, Yin N, Bromberg
J, Lira SA, Stanley ER, Nussenzweig M, Merad M. The origin and development of
nonlymphoid tissue CD103+ DCs. J Exp Med. 2009; 206:3115-3130.
Cisse B, Caton ML, Lehner M, Maeda T, Scheu S, Locksley R, Holmberg D, Zweier C,
den Hollander NS, Kant SG, Holter W, Rauch A, Zhuang Y, Reizis B. Transcription factor
E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development.
Cell. 2008; 135:37-48.
Tsujimura H, Tamura T, Ozato K. Cutting edge: IFN consensus sequence binding
protein/IFN regulatory factor 8 drives the development of type I IFN-producing
plasmacytoid dendritic cells. J Immunol. 2003; 170:1131-1135.
Schiavoni G, Mattei F, Sestili P, Borghi P, Venditti M, Morse HC, 3rd, Belardelli F,
Gabriele L. ICSBP is essential for the development of mouse type I interferon-producing
cells and for the generation and activation of CD8alpha(+) dendritic cells. J Exp Med.
2002; 196:1415-1425.
Tailor P, Tamura T, Morse HC, 3rd, Ozato K. The BXH2 mutation in IRF8 differentially
impairs dendritic cell subset development in the mouse. Blood. 2008; 111:1942-1945.
Waskow C. Generation of parabiotic mice for the study of DC and DC precursor
circulation. Methods Mol Biol. 2010; 595:413-428.
Schraml BU, van Blijswijk J, Zelenay S, Whitney PG, Filby A, Acton SE, Rogers NC,
Moncaut N, Carvajal JJ, Reis e Sousa C. Genetic tracing via DNGR-1 expression history
defines dendritic cells as a hematopoietic lineage. Cell. 2013; 154:843-858.