Download Monocyte and Macrophage Heterogeneity in the Heart

Review
Monocyte and Macrophage Heterogeneity in the Heart
Matthias Nahrendorf, Filip K. Swirski
Abstract: Monocytes and macrophages are innate immune cells that reside and accumulate in the healthy and
injured heart. The cells and their subsets pursue distinct functions in steady-state and disease, and their tenure
may range between hours and months. Some subsets are highly inflammatory, whereas others support tissue
repair. This review discusses current concepts of lineage relationships and crosstalk of systems, highlights
open questions, and describes tools for studying monocyte and macrophage subsets in the murine and human
heart. (Circ Res. 2013;112:1624-1633.)
Key Words: healing
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heart failure
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macrophages
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M
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monocytes
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myocardial infarction
parenchymal cells in the heart while giving attention to other
organ systems that are involved in cardiovascular disease.
yocardial infarction (MI) inflicts a wound to the heart
that, from the perspective of basic biology, shares
many features with wounds resulting from trauma or surgery.
A parallel between both is that the initial injury, whether
traumatic or ischemic, initiates a general healing response
of the body, which involves many organ systems in the task
of restoring equilibrium and ensuring survival. Unlike many
wounds resulting from trauma, MI is a form of sterile injury.
Another distinction is that the wound in the heart is constantly
subjected to the considerable strain of cardiac contractions. MI
is one of the most frequent wounds in the United States because
coronary syndromes occur once every 25 seconds.1 Certainly, it
is one of the most lethal wounds. If it does not kill immediately,
then MI may result in heart failure, a syndrome with high
mortality.1 Increasingly, we understand that the healing process
in the weeks after ischemia paves the road to either recovery or
left ventricular dilation. Insufficient healing may lead to infarct
expansion and trigger left ventricular remodeling. However, no
therapeutic measures exist in the clinic to propagate healing
and prevent heart failure. Wound healing that occurs after
ischemic injury and that may result in heart failure should
be explored from the perspective of the recent advances in
understanding leukocyte biology. In particular, the improved
knowledge of the role of monocytes, macrophages, and their
subsets in steady-state and disease created new insight into
heart failure evolution post-MI. Macrophages reside in the
healthy heart and, although they may not be as frequent as
myocytes, endothelial cells, and fibroblasts, their numbers are
substantial2 and increase during disease.3,4 Their functions,
especially in the steady-state and the remote zone after MI,
remain elusive. Our review article focuses on these protagonist
immune cells and examines their subsets, source, phenotype,
fate, and function in the steady-state and disease. We discuss
the potential interaction of monocytes and macrophages with
Monocytes and Macrophages
The existence of monocytes has been appreciated since at least
the 1920s.5 The idea that monocytes derive from bone marrow precursors, circulate, and give rise to tissue macrophages
was consolidated in the 1960s and has dominated much of
our thinking since.6 A major characteristic of the model is
that macrophage development follows a linear and spatially
restricted trajectory. The model has been influential not only
for what it proposes but also for what it excludes. Restricting
promonocytes to the bone marrow, monocytes to the blood,
and macrophages to the tissue precludes the existence of promonocytes outside the bone marrow, monocytes outside the
blood, and macrophages outside tissue. Many studies, some
also dating back to the 1960s, have provided evidence against
these locational and ontogenic restrictions.7,8 Precursors can
develop and give rise to monocytes outside the bone marrow in
a process called extramedullary monocytopoiesis.9,10 The phenomenon is rare in the steady-state, increases during inflammation, and illustrates a previously unappreciated dynamic of
cellular relocation. Studies also have shown that monocytes are
not exclusively circulating but can reside in tissue as reservoirs
for mobilization during inflammation.11 Moreover, there are
vast transcriptional differences between classical macrophage
populations in various tissues.12 Therefore, we may need to reexamine, at the functional level, in what ways various macrophages differ and in what ways they are similar.
Recent studies show that many tissue macrophages in the
steady-state do not derive from monocytes but from progenitors that have seeded tissue from the yolk sac before the development of definitive hematopoiesis.13–17 The work not
only builds on previous studies that argued for the dual development of macrophages in the spleen8 but also provides
Original received April 9, 2013; revision received May 7, 2013; accepted May 8, 2013. In April 2013, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 13.5 days.
From the Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Correspondence to Matthias Nahrendorf, Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Simches Research
Building, 185 Cambridge St, Boston, MA 02114 (e-mail [email protected]); or Filip K. Swirski, Center for Systems Biology, Massachusetts
General Hospital, Harvard Medical School, Simches Research Building, 185 Cambridge St, Boston, MA 02114 (e-mail [email protected]).
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.113.300890
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Nahrendorf and Swirski Cardiac Macrophages 1625
Nonstandard Abbreviations and Acronyms
HSC
IL
MI
hematopoietic stem cells
interleukin
myocardial infarction
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mechanistic insight, showing the specific involvement of
transcription factors Pu-1 and Myb. The nonhematopoietic
origins of macrophages in tissues, such as brain, liver, lung,
and spleen, raise a plethora of questions. What other locations
contain nonhematopoietic macrophages? Are cardiac macrophages derived from local precursors? How are the various
tissue macrophages similar, and how are they different? What
is the identity of the tissue precursor, and how is it maintained
throughout adult life? Intense investigation of these questions
will no doubt reveal answers in the next few years.
Monocytes are produced in the bone marrow in the
steady-state from hematopoietic precursors. The most restricted monocyte progenitor known is the macrophage dendritic cell progenitor, which is developmentally downstream
of granulocyte and macrophages progenitors and which shares
many phenotypic features with the common dendritic cell progenitor.18,19 Monocytes produced in the bone marrow enter the
blood via CCR2.20,21 In the mouse, circulating monocytes are
phenotypically and functionally heterogeneous and can be
separated according to Ly-6C expression. Between 50% and
60% of mouse monocytes in the steady-state belong to the
Ly-6Chigh CCR2high CX3CR1low CD62L+ subset. These
inflammatory or classical monocytes have a relatively short circulating life span and accumulate preferentially in inflammatory sites where they give rise to macrophages. The remaining
Ly-6Clow CCR2low CX3CR1high CD62L− subset, sometimes
referred to as nonclassical, patrols the vasculature and
accumulates at low numbers in the steady-state.19,22 During
inflammation, the number of Ly-6Chigh monocytes increases
through increased monocytopoiesis in the bone marrow and
spleen, contributing to monocytosis.9,10,23,24 The function
of Ly-6Clow monocytes in tissue, however, requires further
study because relatively few Ly-6Clow monocytes accumulate.
Evidence shows that monocyte subsets do not arise from separate progenitors, but rather convert from the Ly-6Chigh to the
Ly-6Clow subset.25–27 In humans, circulating monocytes also
can be separated into subsets based on the expression of CD14
and CD16.28 Phenotypic profiling indicates that CD16− CD14+
monocytes, which comprise 80% to 90% of human monocytes, are similar to Ly-6Chigh monocytes, whereas CD14dim
monocytes are most similar to Ly-6Clow monocytes.29,30 A third
subset in humans can be identified as CD16+ CD14++.30,31 This
subset, which circulates at a frequency of 5% to 7%, was originally called inflammatory because it produces tumor necrosis
factor-α in response to lipopolysaccharide. The relationship
between human CD16+ CD14++ monocytes and mouse subsets had been the subject of debate. CD16+ CD14++ cells
share many phenotypic features with Ly-6Clow monocytes.30
Accordingly, the expression of CD16 is the division line;
CD16− monocytes are akin to Ly-6Chigh monocytes, whereas
CD16+ monocytes are most similar to Ly-6Clow monocytes.
The opposing view argues that CD16+ CD14++ cells, which
cluster with CD16− CD14++ monocytes, are closely associated with Ly-6Chigh monocytes.29 Here, the division line is
CD14; CD14++ monocytes resemble Ly-6Chigh monocytes,
whereas CD14dim monocytes resemble Ly-6Clow monocytes.
Functionally, the patrolling CD14dim and Ly-6Clow monocytes
are vascular sentinels. In that respect, they are macrophages
of the blood.22
The fate of Ly-6Chigh monocytes on tissue accumulation also
has received attention. A focus of much interest in macrophage
biology is the particular function that macrophages acquire on
tissue accumulation. In vitro, macrophages can be generated
from bone marrow precursors by various means. Macrophages
generated in the presence of interferon-γ or lipopolysaccharide
have been termed M1, or classically activated inflammatory
macrophages. Macrophages generated in the presence of
interleukin (IL)-4 or IL-10, however, have been called
M2, or alternatively activated macrophages, and have a
proresolution profile.32,33 Although tissue and inflammatory
macrophages likely are on a continuum that lies between
(and outside of) the M1 and M2 definitions, the terminology
nevertheless has been helpful in efforts aimed at elucidating
macrophage heterogeneity. When Ly-6Chigh monocytes
infiltrate atherosclerotic lesions, for example, they differentiate
to F4/80+ and Mac3+ macrophages. In lesions in which the
inflammatory stimulus is persistent, these Ly-6Chigh monocyte–
derived macrophages remain inflammatory by expressing
IL-1β and tumor necrosis factor-α and by contributing to
oxidative stress.9 In this respect, Ly-6Chigh monocyte–derived
macrophages are M1 macrophages.
In the context of inflammation resolution, M1 macrophages are replaced by M2 resolution–mediating macrophages. It has been proposed that this occurs through local
M1 to M2 conversion.34–36 M2 macrophages also could derive
from less inflammatory Ly-6Clow monocytes. Alternatively,
M2 macrophages may arise through direct differentiation of
Ly-6Chigh monocytes in a microenvironment that favors resolution (Figure 2). This possibility should be explored experimentally because it is enticing for several reasons. First, the
macrophage turnover kinetics in the infarcted myocardium
are remarkably fast. This suggests that newly recruited cells
are continuously replacing old cells. Second, recent evidence indicates that Ly-6Clow monocytes function predominantly as vascular sentinels and infiltrate tissue at very low
frequencies. Third, in the steady-state, Ly-6Clow monocytes
derive from Ly-6Chigh monocytes, and it is not clear whether
the conversion path involves intermediate stages, that is, Ly6Chigh monocyte to Ly-6Clow monocyte to M2 macrophage.
Sophisticated lineage-tracing studies are therefore required
to elucidate monocyte/macrophage lineage relationships during resolution of inflammation in the ischemic myocardium,
testing the hypothetical options outlined in Figure 2.
Infarct Healing
The typical trigger of MI is plaque rupture in a coronary
artery, which suddenly stops the arterial blood flow feeding
downstream organ areas. Deprived of oxygen, the nonperfused myocardial tissue undergoes necrosis. Accumulation
of leukocytes in the first minutes to hours after MI was
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recently imaged with intravital microscopy for the first
time.37,38 Surprisingly, monocyte recruitment may outpace
neutrophils soon after coronary ligation.38 Nevertheless,
neutrophils are the first leukocyte population to reach a
robust peak of several hundred-thousand cells within the first
day after onset of ischemia.3 The short-lived cells are recruited
through adhesion molecules, such as E-selectin, P-selectin,
and ICAM-1, and the chemokine IL-8.39 The presence of neutrophils is generally seen as detrimental because it contributes to the damage inflicted by reperfusion injury. Their main
function is to defend the organism against invaders such as
microbes. To fulfill this task, they carry an armamentarium of
inflammatory cytokines, are phagocytic, and impose oxidative
stress on the tissue in which they accumulate. Their presence
in the healing infarct wanes quickly, but they may linger longer if inflammation does not resolve. Some of these cells can
be found in the cardiac wound as late as 7 days after MI.
The next major cell classes dominating the infarct are
monocytes and macrophages. These myeloid cells can be
found in high numbers, up to a million cells, in the infarct.
An inflammatory phenotype (Ly-6Chigh monocytes/M1-type
macrophages) dominates initially and is followed by cells
with a lesser inflammatory phenotype promoting tissue repair
(Ly-6Cint/low monocytes/M2-type macrophages). These cells
are discussed in more detail in the following paragraphs.
Other leukocytes invading the infarct include dendritic
cells,40 lymphocytes,41,42 and mast cells.43 Compared with
neutrophils, monocytes, and macrophages, their numbers are
lower. However, they may have an important role in regulating
infarct healing and the myeloid cell response.
When the inflammatory activity resolves, nonleukocyte
cells join the rebuilding activities in the infarct. Solicited by
angiogenic factors, such as vascular endothelial growth factor,
numerous vessels sprout into newly forming granulation tissue. These small capillaries can appear as early as a few days
after injury, but their highest numbers are found weeks later.
In nonreperfused infarcts, the highest density of neovessels is
observed in the border zone, that is, next to noninfarcted tissue. Initially, new vessels only consist of endothelial cells but
acquire a more mature morphology, including a pericyte coat,
over time.44
Myofibroblasts produce collagen, the extracellular matrix
that strengthens the emerging infarct scar. Impaired collagen
deposition or accelerated degradation of collagen attributable
to high matrix metalloproteinase activity45 can lead to infarct
rupture and sudden death. Infarct expansion is a less drastic but
still detrimental phenotype of insufficient matrix generation.46
Hence, collagen-producing cells play a major role in later
stages of wound healing. Because parabosis studies linking
row) is faster than recruitment of GFP+ cells in a LysMGFP/+
wild-type mice after coronary ligation with GFP+ mice have
argued that circulating progenitors only give rise to leukocytes
in the infarct,47 α-smooth muscle actin–positive cells may
derive from local progenitors, for instance the very numerous
resident cardiac fibroblasts.
Monocytes in the Infarct
Monocytes are among the very first cells recruited to ischemic myocardium. Intravital microscopy of the beating mouse
heart37,38 allows the following of leukocyte action in the ischemic heart with high temporal and spatial resolution in vivo.
Monocyte recruitment outpaced neutrophils within the first 30
minutes after the onset of ischemia (Figure 1).38 It is currently
not clear which monocyte subset is recruited during the first
minutes after MI or what the function of these cells entails.
However, one could hypothesize that these are patrolling Ly6Clow monocytes that amplify the initial inflammatory signal.
Discriminating monocyte subsets by imaging will contribute to
a better understanding of their phenotype, source, and function.
Two sequential phases defined by the expression of Ly-6C
on monocytes/macrophages can be identified in the infarcted
myocardium.3 The inflammatory Ly-6Chigh monocyte subset
is recruited during the first days after MI via the chemokine
monocyte chemotactic protein-1.48 Days later, when inflammation resolves in the cardiac wound, the presence of these
inflammatory cells wanes. Starting at approximately day 4 after MI, the infarct tissue accumulates Ly-6Cint/low monocytes/
macrophages (Figure 2). On day 7, Ly-6Cint/low F4/80high cells
outnumber Ly-6Chigh cells in the heart.3 Similar monocyte kinetics can be observed in the blood of mice after coronary
ligation3 and in patients with acute MI.49 This time course corresponds to expression of M1-type markers in the tissue early
after injury and M2-type macrophage markers later.50
Interestingly, the turnover of myeloid cells in the infarct is
very rapid. Even days after injury, monocytes are recruited at
a rather high rate and reside only for an average of 20 hours in
the cardiac wound.10 Thereafter, most of them become apoptotic. Although only between 5% and 13% of the entire accumulating CD11b+ leukocyte population exits the infarct within
24 hours, some of these cells accumulate in the liver, lymph
nodes, and the spleen.10 It is currently unknown whether cells
that exit the site of inflammation do so actively or whether
they merely perish elsewhere. Alternatively, they may have
a specific function, for instance, transmitting information to
sites of monocyte production.
Within the first week of injury, the function of myeloid cells
changes over time. During day 1 to 4 after ischemia, the milieu
is highly inflammatory. Ly-6Chigh monocytes have a high payload
Figure 1. Intravital microscopy of monocyte and
neutrophil recruitment in the first 30 minutes
after myocardial infarction in green fluorescent
protein (GFP) reporter mice. Dedicated image
stabilization and motion compensation techniques
allowed following of this process in vivo.
Interestingly, within the first 30 minutes after injury,
the recruitment of monocytes in a transgenic
mouse in which GFP reports on monocytes (top
mouse (bottom row and graph on the right). Adapted from Jung et al.38
Nahrendorf and Swirski Cardiac Macrophages 1627
Advanced flow cytometry gating strategies, and new surface
markers,12 together with a higher number of channels available
in current flow cytometers, enable better discrimination between
tissue monocytes and macrophages.
Lineage Relationships in the Infarct
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Figure 2. Potential lineage relationships of monocytes and
macrophages after myocardial infarction. In steady-state,
monocyte conversion from Ly-6Chigh to Ly-6Clow may occur in
blood and bone marrow.27 It is generally accepted that Ly-6Chigh
monocytes give rise to inflammatory M1 macrophages in tissue.
Other lineage relationships, indicated by dashed arrows, remain to be
experimentally explored in the setting of acute myocardial infarction.
of inflammatory cytokines, such as tumor necrosis factor-α and
proteases.3 Together with inflammatory macrophages, they form
a demolition crew that digests dead cells and extracellular matrix.
These cells clear the infarct of debris by phagocytosis, a prerequisite for replacing the injured tissue with granulation tissue and
the evolving scar. An unresolved question is whether this debris
is removed from the heart to distant organs or recycled onsite.
During the following rebuilding phase, the inflammatory activity
resolves and gives way to Ly-6Cint/low monocytes/macrophages.
These cells have a lower content of inflammatory molecules and
proteases. Instead, they release vascular endothelial growth factor
and transforming growth factor-β, supporting angiogenesis and
collagen production. Both healing phases are required for sufficient wound healing. If either is ablated, then the scar-building
process is disturbed.3
Monocytes and macrophages may influence tissue regeneration in yet to be discovered ways. Work on the hematopoietic stem cell (HSC) niche illustrates that macrophages can
have close relationships with multipotent progenitor cells.51,52
Recent insight highlights their role in regulating production
of red blood cells.53 These regulatory macrophage actions
are based on crosstalk with HSCs in the bone marrow. It is
tempting to speculate that macrophages could act similarly
in other tissues, for instance, by interacting with cells that
have regenerative capacities in the injured heart. Specifically,
macrophage subsets may differentially influence activity
of proliferating endothelial cells, fibroblasts, endogenous
cardiac progenitors, or stem cells that were transferred into
the heart with the intention to rebuild myocardium after MI.
The latter point may guide timing of cell therapy, because
an inflammatory milieu provided by M1 macrophages could
hinder survival, differentiation, and integration of therapeutically transferred cells.
It is currently unclear how monocytes and macrophages
share the burden of work because of the difficulties when trying
to clearly separate the cell populations using surface markers.
Although evidence for lineage relationship in the infarcted
myocardium is rather sparse, there is considerable evidence
in other tissues regarding how monocyte subsets and macrophages correspond to each other developmentally. The
emerging picture is as follows: in the steady-state, the tissue
is populated by resident macrophages, many of which do not
rely on the bone marrow for replenishment but rather selfrenew from local progenitors or through proliferation.16 This
has not yet been explored specifically for macrophages in the
heart. Soon after injury, Ly-6Chigh monocytes infiltrate the infarct in large numbers.3 Many of these monocytes may not
differentiate to macrophages but either exit or die in tissue.
Those that differentiate acquire M1-like properties, continue
to express Ly-6C, and contribute to inflammation (Figure 2).
Over time, as inflammation gives way to resolution, a second
Ly-6Cint/low phase emerges. We need to address experimentally
whether macrophages dominating this regenerative wound
healing phase arise via the differentiation of Ly-6Chigh monocytes (Figure 2) and less via differentiation of Ly-6Clow monocytes or local M1 to M2 macrophage conversion. Over time,
as inflammation completely subsides, a population of F4/80high
macrophages resembling steady-state macrophages returns.
Future studies will determine whether these macrophages
derived from a circulating or local precursor.
Sources of Monocytes After MI
The bone marrow is the primary site of production for blood
cells. The production of leukocytes is tightly regulated by a
team of housekeeping cells forming the hematopoietic niche.
Key constituents include CD169+ macrophages, mesenchymal stem cells, osteoblasts, and endothelial cells. These niche
cells organize HSC retention and provide signals that regulate
proliferation or quiescence of HSCs via adhesion molecules
(eg, selectins) and soluble cytokines (eg, CXCL12, stem cell
factor).51,52 Release of monocytes from the bone marrow into
the blood stream depends on the chemokine receptor CCR2,20
which also regulates recruitment to the site of inflammation.48
The infarct recruits these cells from the blood stream.
Within the first 24 hours after coronary ligation in mice, approximately half of all monocytes recruited to the heart derive
from a splenic reservoir.11 Reservoir monocytes reside in the
subcapsular red pulp of the spleen and closely resemble their
circulating counterparts, which they outnumber significantly.
Upon MI, splenic monocytes increase motility, at least partially,
by signaling of angiotensin-2 through the angiotensin-2 subtype-1 receptor expressed on their cell surface.11,54 The cells then
intravasate into splenic veins and leave the organ (Figure 3) to
accumulate at inflammatory sites. In mice, this leads to a drastic shrinkage of the spleen within the first 24 hours after MI.
The organ weight declines by ≈50%, and so does the number of
splenic monocytes. The bone marrow and blood pool also substantially contribute to the monocytes recruited to the infarct.
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Figure 3. Intravital microscopy of the spleen in a CX3CR1GFP/+
mouse soon after myocardial infarction shows intravasation
and departure of a splenic monocyte. Adapted from Swirski
et al.11
Because the residence time of monocytes and macrophages
is short, and because recruitment to the infarct continues at levels of several hundred-thousand cells per day, the production
of these cells intensifies to meet the high demand after the reservoirs exhaust. We55 and others56 observed that MI increases
the activity of the hematopoietic system. In humans, increased
bone marrow uptake of the positron emission tomography
tracer 18F-fluorodeoxyglucose, a radiolabeled glucose analog,
indicates a higher metabolic activity.56 The human bone marrow releases hematopoietic progenitors after MI.57 In mice
after coronary ligation, these cells home for the spleen, where
they seed the organ depending on very late antigen 4 and stem
cell factor to initiate extramedullary hematopoiesis.55 This
monocyte production is stimulated by signaling through the
IL-1 receptor, replenishes the splenic reservoir by day 6 after
MI, and continues to supply the infarct with a significant number of cells (≈50% of infarct monocytes in mice derive from
the spleen on day 6 after coronary ligation).10
Sympathetic nervous signaling is a trigger of bone marrow
stem cell release after MI. Treatment with a β3-specific adrenoreceptor inhibited the release of HSCs into the blood.55 These
findings raise the question of whether β-blockers in clinical use
with affinity to the β3-receptor subtype inhibit HSC release in patients after MI, and perhaps have an anti-inflammatory effect by
reducing extramedullary production of inflammatory leukocytes.
Monocytes and Macrophages in Normal and
Failing Myocardium
Macrophages reside in many normal tissues during
steady-state. The heart is no exception.2,3 Improved
detection strategies, including fluorescent reporter proteins,
visualize these cells nestled within the normal myocardium
in direct contact with endothelial cells and myocytes.2
During steady-state, their gene expression profile resembles
noninflammatory M2 macrophages.2 The number of
extravasated monocytes in the healthy heart is low.
However, monocytes can be found patrolling the myocardial
vasculature.38 The patrolling behavior was previously
described for Ly-6Clow monocytes in other tissues.22,58
The precise function of patrolling monocytes and tissue
macrophages in the heart is currently unclear.
What is the role of myocardial monocytes and macrophages
in disease, ie, in the failing heart? Their increased presence
in the myocardium has recently been described in mice with
heart failure due to pressure overload59 and in the remote
zone after MI in mice and patients (Figure 4).60,61 It is wellknown that myocyte hypertrophy, myocyte death, changes in
the balance of extracellular matrix production and protease
activity are central in the evolution of heart failure, but the
role of monocytes and macrophages within this system is not
well-understood. Specifically, we lack knowledge about their
origin, phenotype, and function in failing hearts. Leukocyte
numbers in the remote myocardial tissue are orders of magnitude lower compared with the acutely ischemic infarct.
Nevertheless, an interesting hypothesis is that monocytes and
macrophages may instruct and interact with parenchymal cells
in the myocardium (Figure 4). The importance of macrophages
in many other diseases is well-recognized.62,63 For instance,
myeloid cells digest extracellular matrix and promote tissue
destruction in atherosclerosis4,64,65 and rheumatoid arthritis,66,67
and they instigate fibrosis in liver disease.68 Hence, it is conceivable that these cells have an active role in the evolution
of heart failure by supplying profibrotic signals or enhancing
protease activity and ventricular dilation. Inflammatory macrophages may even accelerate myocyte apoptosis in failing
hearts. Alternatively, these cells may be innocent bystanders or merely dispose of apoptotic myocytes. Clinical signs
of inflammation in heart failure patients, such as increased
C-reactive protein, high cytokine levels,69,70 and the predictive
value of the white blood count71,72 underscore the likelihood
of an inflammatory component in left ventricular remodeling. Future research should address the mode of recruitment,
whether there is local macrophage proliferation in the failing
heart, the function of leukocytes in the remote myocardium,
and their interaction with endothelial cells, myocytes, and
fibroblasts (Figure 4).
Crosstalk Between Organ Systems
The heart is intimately linked to all organ systems. On the obvious side, it supplies the entire body with oxygenated blood.
A decline of this function compromises the function of other
organs, possibly enhancing heart failure in a vicious cycle.
Renal function is a prominent example, because decreased
perfusion of the kidneys causes fluid retention and increases
stress on the heart due to increased loading conditions. Similar
connections may exist between the cardiovascular and the
metabolic, endocrine, immune, and hematopoietic systems,
and these interactions may enhance heart failure.
Monocytes and macrophages closely link atherosclerosis and MI with the immune and hematopoietic systems.
Derailed defense functions of macrophages, which fail to
remove lipid deposits from the diseased arterial wall, lead
Nahrendorf and Swirski Cardiac Macrophages 1629
Tools to Study Monocytes and Macrophages in
the Heart
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Figure 4. Open questions about the phenotype, behavior, and
function of monocytes and macrophages in the remote zone
after myocardial infarction (MI). The lower right inset shows
immunoreactive staining for CD68+ macrophages in the remote
zone of a patient who died soon after MI. Adapted from Lee et
al.61
to inflammatory complications of atherosclerotic plaque.
Aberrant cholesterol handling enhances the production
of leukocytes in the bone marrow73 and, at least in mice,
initiates extramedullary myelopoiesis in the spleen.9,74 In
humans, increased activity of macrophages gives rise to
vulnerable plaques, plaque rupture, and MI. Thus, MI occurs in a setting of chronic inflammation, with elevated
activity of the hematopoietic and immune systems. The
increased availability of inflammatory leukocytes attributable to preexisting atherosclerosis changes the inflammatory response to acute infarction and modulates myocardial
healing. In apolipoprotein E−/− mice with atherosclerosis,
inflammatory leukocytes are recruited for a longer time
and in higher numbers to the cardiac wound, disturbing the
resolution of inflammation in the heart and enhancing postMI remodeling.75
There is another perspective to this interaction of chronic
inflammation in the artery wall and acute inflammation in the
infarct. We have long known that the first year after an ischemic event is particularly dangerous, and that many patients
experience reinfarction.76,77 A systemic flare-up of inflammation in the vessel wall after MI may cause these secondary
ischemic events.78 Increased activity of the sympathetic nervous system translocates leukocyte progenitors to the spleen
and accelerates overall production of inflammatory leukocytes.55 These cells not only are available for recruitment to
the infarct but also may increase inflammatory activity in
atherosclerotic plaque. Hence, it seems reasonable to target
the nervous, immune, or hematopoietic systems to either prevent reinfarction or ameliorate the evolution of post-MI heart
failure. This concept is already implemented to some degree,
ie, by using beta-blockers, lowering blood cholesterol levels,
controlling obesity, and treating diabetes mellitus. However,
current clinical therapeutics do not specifically target inflammatory feedback loops.
Although the first histochemical studies reported on leukocyte
and macrophage presence in acute infarcts,79 a number of
newer tools enabled analysis of leukocyte subsets, increased
sensitivity, resulted in quantitative data, and facilitated in vivo
measurements. Flow cytometric analysis of digested infarcts
was a major advance, because this approach enumerates the
total cell number per unit of tissue. Multichannel capabilities
allow staining for surface marker combinations, hence the
identification and isolation of monocyte subsets. The use of
CX3CR1GFP reporter mice improved the sensitivity of ex vivo
histology.2,61 In vivo microscopy of monocytes reported on the
distinct migration paths and patterns of the cell. For instance, it
revealed the increased motility of splenic monocytes after MI
(Figure 3). Imaging the heart with microscopic resolution has
only recently been established (Figure 1), because the cardiac
and respiratory motions represent major hurdles. These were
overcome by using tissue stabilization in conjunction with
ECG and respiratory triggering of image acquisition.37,38 Organlevel imaging modalities, including MRI,80 positron emission
tomography,61 fluorescence molecular tomography,81 and hybrid
approaches,82 can now follow monocyte and macrophages
and their molecular function in murine infarcts noninvasively
(Figure 5). Noninvasive leukocyte imaging answered questions
Figure 5. Noninvasive imaging of monocytes and
macrophages. Left, top, Hybrid fluorescence molecular
tomography/X-ray computed tomography imaging of protease
activity in an apolipoprotein E−/− mice after coronary ligation
(adapted from Panizzi et al75). Right, top, Positron emission
tomography/MRI in a mouse with acute myocardial infarction
using a macrophage-avid, copper-64–labeled cross-linked iron
oxide nanoparticle (64Cu-CLIO; adapted from Majmudar and
Nahrendorf95). Bottom, left, T2*-weighted MRI after injection
of macrophage-avid iron oxide nanoparticles (adapted from
Nahrendorf et al81). Bottom, right, Imaging of macrophages in a
patient with acute MI (adapted from Yilmaz et al87).
1630 Circulation Research June 7, 2013
about how monocyte and macrophage content in the infarct
influences left ventricular remodeling and the evolution of
heart failure. Monocyte and macrophage imaging may rely
on nanoparticles that are taken-up by these cells and that can
be detected noninvasively. These include positron emission
tomography isotope–labeled nanoparticles,83 fluorochromelabeled nanoparticles,81 gadolinium84–labeled or fluorine-1985–
labeled liposomes, and iron oxide nanoparticles that are detectable
with T2*-weighted MRI.80 First pioneered for this application
in the mouse,80 iron oxide nanoparticles were recently used in
2 clinical trials focusing on macrophages in acute infarcts.86,87
These imaging tools will likely enable clinical studies that aim
to translate basic findings and will follow emerging therapeutics
that target monocyte and macrophage biology.
Clinical Translatability
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What is the evidence that discoveries in the mouse translate
to patients with MI? A large number of clinical studies support concepts of cardiac monocyte and macrophage heterogeneity recently described in mice. Studies have shown, for
example, that CD14++CD16+ monocytes predict cardiovascular events.88–90 However, seemingly conflicting data91 as well
as lack of consensus regarding how human and mouse subsets correspond impede translation of mechanistic insights.
Although tissue data in the heart are still lacking, a 2-phase
monocyte response has been observed in the blood of patients
after MI.49 Inflammatory CD14+CD16− monocytes peaked
on day 3, whereas CD14+CD16+ monocytes peaked on day
5 after MI, recapitulating the temporal pattern observed for
Ly-6Chigh and Ly-6Clow monocytes in mice.3 Patients with
a higher blood level of inflammatory monocytes had worse
myocardial salvage and lower ejection fraction 6 months after
MI, measured by MRI.49 This intriguing study does not yet answer the question of causality in patients. Possibly, increased
inflammatory activity leads to worse healing and outcome.
Alternatively, a more extensive injury may have caused higher
blood monocyte levels and lower ejection fraction 6 months
later. A number of clinical studies confirm the correlation of
blood leukocyte levels in heart failure with prognosis.71,72
Although many open questions persist, there are also some
intriguing clues regarding the response of the hematopoietic
system in patients after MI. An increased level of hematopoietic progenitors was found in the blood,57 and the metabolic
activity of the bone marrow increased.56 There is also evidence
for an increased progenitor activity in the spleen of patients
after MI.55 Future studies will show whether there is splenic
monocyte release in patients with MI, and whether biphasic
monocyte recruitment can be observed in infarct tissue.
The presence of macrophages in the remote myocardium
after MI has been confirmed in patients by histology61 and in a
macrophage imaging study.56 However, their function remains
to be elucidated.
Open Questions
We are beginning to better understand the role of monocytes,
macrophages, and their subsets in the heart, but there are more
questions than answers. The role of these cells in the healthy
heart is unclear. After MI, we do not yet understand what
signals initiate the resolution of inflammation in the cardiac
wound. Newer microscopic imaging techniques may soon
better elucidate how monocytes are recruited to the injured
heart, especially when the endothelium is disrupted. We have
never seen a monocyte or macrophage move from the border
zone into the core of an infarct, although we have found these
cells there. What are their migration patterns, how do the cells
move, and how fast?
The high turnover of leukocytes in the infarct obviates
the importance of leukocyte supply and production. Only
few studies have investigated how the hematopoietic system, including the bone marrow, reacts to MI. Given the
distance of organs such as the spleen and the bone marrow
from the heart, it would be interesting, also as a possible
way of interacting therapeutically with the inflammatory
response, to learn more about the signals that regulate
monocyte supply. What are the danger signals that report
cardiac injury to other organ systems, including the spleen
and the bone marrow?
Although macrophages are present in the remote zone after
MI and in mice with pressure overload, it is unclear whether
they are important and what functions they pursue. Finally,
from a perspective of monocyte and macrophage heterogeneity, precise lineage relationships between these cells and their
subsets have yet to be determined in the steady-state as well as
in the diseased heart.
Conclusion
The motivation behind the questions is the need for new treatment concepts in heart failure. We should also explore how
our current therapeutics interfere with monocyte and macrophage function. For example, monocyte progenitor release
from the bone marrow after coronary ligation in mice depends
on β-adrenergic signaling. Furthermore, angiotensin-converting enzyme inhibition decreases monocyte and macrophage
numbers in the infarct of mice by inhibiting the mobilization of splenic monocytes. Are similar mechanisms active in
patients?
Newer therapeutic avenues may target the inflammatory
activity after MI directly. There are 2 major clinical atherosclerosis trials underway exploring the role of the antiproliferative drug methotrexate92 and the action of neutralizing
IL-1β,93 a cytokine that likely supports post-MI monocyte
production in the spleen.10 These trials may provide important cues regarding the role of monocyte and macrophage
activity in patients.
Finally, understanding the signaling pathways and the fate
of monocytes and their progenitors after MI will support the
search for specific therapies. One example is RNAi silencing
of the chemokine receptor CCR2, which inhibits the recruitment of inflammatory monocyte subset in mice with MI,82,94
possibly sparing noninflammatory monocyte and macrophage
subsets. Any potential immune-targeted therapy will face
scrutiny about unwanted effects. However, targeting a culprit subset, motivated by increased knowledge of monocyte
and macrophage heterogeneity, may spare important immune
functions, such as repair and defense against infection.
Nahrendorf and Swirski Cardiac Macrophages 1631
Acknowledgments
The authors acknowledge Servier Medical Art for help in producing
figures.
Sources of Funding
This work has been funded in whole or in part with federal funds
from the National Heart, Lung, and Blood Institute, the National
Institutes of Health, and the Department of Health and Human
Services (R01HL095612, R01HL095629, R01HL096576, Contract
No. HHSN268201000044C).
Disclosures
None.
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Monocyte and Macrophage Heterogeneity in the Heart
Matthias Nahrendorf and Filip K. Swirski
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Circ Res. 2013;112:1624-1633
doi: 10.1161/CIRCRESAHA.113.300890
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