REVIEWS Macrophage subsets in atherosclerosis Giulia Chinetti-Gbaguidi, Sophie Colin and Bart Staels Abstract | Macrophage accumulation within the vascular wall is a hallmark of atherosclerosis. In atherosclerotic lesions, macrophages respond to various environmental stimuli, such as modified lipids, cytokines, and senescent erythrocytes, which can modify their functional phenotypes. The results of studies on human atherosclerotic plaques demonstrate that the relative proportions of macrophage subsets within a plaque might be a better indicator of plaque phenotype and stability than the total number of macrophages. Understanding the function of specific macrophage subsets and their contribution to the composition and growth of atherosclerotic plaques would aid the identification of novel strategies to delay or halt the development of the disease and its associated pathophysiological consequences. However, most studies aimed at characterizing the phenotypes of human macrophages are performed in vitro and, therefore, their functional relevance to human pathology remains uncertain. In this Review, the diverse range of macrophage phenotypes in atherosclerotic lesions and their potential roles in both plaque progression and stability are discussed, with an emphasis on human pathology. Chinetti-Gbaguidi, G. et al. Nat. Rev. Cardiol. 12, 10–17 (2015); published online 4 November 2014; doi:10.1038/nrcardio.2014.173
Introduction
INSERM U1011, Institut Pasteur de Lille, 1, Rue du Professeur Calmette, BP 245, Lille 59019, France (G.C.‑G., S.C., B.S.). Correspondence to: B.S. bart.staels@ pasteur-lille.fr
The development of atherosclerosis involves activation of various cell types (including endothelial cells, smooth muscle cells, lymphocytes, monocytes, and macro phages) in the intima of the arteries, which results in a local inflammatory response.1 An increase in circulating LDL-cholesterol levels and the subsequent accumulation of oxidized LDL in the subendothelial space triggers the recruitment and retention of monocytes and lymphocytes in the arterial wall. In the intima, monocytes differentiate into macrophages, which scavenge lipoprotein particles, and eventually become foam cells.2 These macrophagederived foam cells secrete inflammatory molecules and factors that further promote lipoprotein retention, degrade the extracellular matrix, and sustain inflammation.3,4 Progression of atherosclerosis is characterized by apop tosis of these resident macrophages in the lipid core of the lesion. The clearance of apoptotic cells is mediated by phagocytes, mostly macrophages, which recognize and internalize dead cells in a process termed efferocytosis.5 In early lesions, phagocytes readily clear apoptotic cells, avoiding further progression of atherosclerosis. In chronic, advanced lesions, however, efferocytosis is no longer suf ficient to engulf all dead cells, and the gradual accumu lation of apoptotic debris results in formation of a necrotic core, which triggers further inflammation, necrosis, and thrombosis.5 Macrophages are crucial in the maintenance of efficient efferocytosis, and thereby contribute to both resolving inflammation and preventing the formation of a necrotic core within the plaque (Figure 1).6 Novel observations have challenged these previously well-established concepts. Whereas atherosclerosis was Competing interests The authors declare no competing interests.
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initially considered a type 1 T helper cell (TH1)-driven inflammatory process, the concept of heterogeneity of macrophages resident within lesions has gradually emerged over the past decade.7,8 Firstly, several studies revealed that both monocytes and macrophages comprise heterogeneous cell populations that adapt their functional phenotype in response to specific microenvironmental signals and molecules.9,10 These different monocyte and macrophage subtypes can be identified based on their dif ferential expression of surface markers and chemokine receptors.9,10 Secondly, the general dogma that tissueresident macrophages are incapable of proliferating was challenged by investigators who observed macrophages in mouse lungs that proliferated independently of monocyte recruitment.11 Both resident and recruited macrophages can be induced to proliferate by IL‑4.11 Moreover, in mice, macrophages in early atherosclerotic lesions are pre dominantly derived from recruited monocytes, whereas macrophage proliferation is a preponderant feature of advanced plaques and is influenced by microenvironment signals.12 Accordingly, differences in resident macrophage phenotypes can also influence their capacity to proliferate locally, which can alter the abundance of macrophages with a given phenotype.11,13 In this Review, we describe and discuss the reported functional phenotypes of macrophages in atherosclerotic plaques, focusing mainly on human pathology. The correlative studies suggesting a link between such macro phage phenotypes and the structure or progression of atherosclerotic lesions will also be discussed.
Macrophage polarization and plasticity
Several classes of macrophages have been described based on their expression of markers, the production of specific
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REVIEWS Key points ■■ Only M1 proinflammatory and M2 anti-inflammatory macrophages have been described in vitro—however, a wide spectrum of intermediate phenotypes has been identified in in vivo studies ■■ Various stimuli (cytokines, lipids and their derivatives, senescent cells, iron) can influence macrophage phenotypes in atherosclerotic lesions ■■ Macrophages with different functional phenotypes are likely to perform different roles in the development of atherosclerosis ■■ M1 macrophages are associated with symptomatic and unstable plaques, whereas M2 macrophages are particularly abundant in stable zones of the plaque and asymptomatic lesions ■■ Modulation of macrophage phenotypes might be a novel strategy for the pharmacological treatment of atherosclerosis
Iron deposit M1 macrophage M2 macrophage Vessel Lipid core M1
Endothelial cell
M2 efferocytic macrophage
Prevention of macrophage necrosis Iron deposits Neoangiogenesis Clearance of apoptotic macrophages
M1 inflammatory macrophage
Apoptosis
Postapoptotic necrosis
Necrotic core Resolution of inflammation
Plaque vulnerability
Figure 1 | Potential role of M2 macrophages in efferocytosis within atherosclerotic plaques. M2 macrophages localized in areas of neovascularization or outside the lipid core can phagocytose apoptotic M1 macrophages, contributing to the resolution of inflammation. If efferocytosis is insufficient, dead M1 macrophages accumulate and undergo postapoptotic necrosis, leading to the formation of a necrotic core, which contributes to plaque instability and rupture.
factors, and their biological functions. Classically acti vated (M1) macrophages are typically induced by TH1 cytokines, such as IFN‑γ and tumour necrosis factor (TNF), or by lipopolysaccharide recognition. These macrophages produce high levels of IL‑12 and IL‑23, low levels of IL‑10,14 and secrete the proinflammatory cytokines TNF, IL‑6, and IL‑1β (Figure 2).15 From a func tional point of view, M1 macrophages participate in the removal of pathogens during infection through activa tion of the NADPH oxidase system, and the subsequent generation of reactive oxygen species (ROS). Chronic M1 macrophage activation can, therefore, mediate ROSinduced tissue damage and impair wound healing.16 To protect against such tissue damage, the inflammatory
response is spatially and temporally counterbalanced by regulatory mechanisms driven by alternatively activated (M2) macrophages (Figure 2).17 Three different subclasses of M2 macrophages have been identified: M2a macrophages are induced by the TH2 cytokines IL‑4 and IL‑13; M2b macrophage subtypes are induced by immune complexes in combination with IL‑1β or lipopolysaccharide; and M2c macrophages are induced by IL‑10, transforming growth factor‑β (TGF‑β), or glucocorticoids. 18 All M2 macrophages have an anti-inflammatory cytokine profile character ized by low production of IL‑12 and high production of both IL‑10 and TGF‑β (which is especially prominent in the M2c subtype).18 M2b macrophages are an exception because they additionally produce high levels of pro inflammatory cytokines, including IL‑1, IL‑6, and TNF.19 Functionally, M2 macrophages can scavenge debris and apoptotic cells20 and promote tissue repair and healing. Furthermore, they possess proangiogenic21 and pro fibrotic22 properties. Monocytes differentiated in the presence of granulocyte–macrophage colony stimulat ing factor (GM‑CSF) or macrophage colony stimulating factor (M‑CSF) display functional properties similar to the M1 and M2 phenotype, respectively.7,14 An alterna tive method of classifying macrophage subpopulations has been proposed on the basis of their role in three macrophage homeostatic activities: host defence, wound healing, and immune regulation.23 This classification distinguishes activated M1 macrophages from woundhealing macrophages (M2a subclass) and regulatory macrophages (M2b/c subclass).23 Although the general properties of macrophages are conserved between species, their cell surface markers are not. For example, in mice (but not in humans), the M2 phenotype is defined by expression of Ym1 (chitinase 3‑like protein 3) and arginase I (Table 1).24 Moreover, only a few markers are selective and specific for a given phenotype. Some markers are shared across different macrophage sub types, suggesting that overlapping phenotypes can exist. Given that the majority of these markers are not exclusive for one phenotype, quantitative rather than qualitative variations should be considered. For this reason, macro phage phenotypes are generally characterized on the basis of cell surface marker expression combined with functional studies, when possible. Interestingly, macrophages are remarkably plastic and can switch from one phenotype to another depending on the environmental cues encountered.25,26 The local cytokine milieu can, therefore, orientate macrophage polariz ation and plasticity. The concept of macro phage heterogeneity has raised much interest in the past decade. In addition to the canonical M1 and M2 macro phage subpopulations, emerging evidence suggests that other specific and distinct macrophage phenotypes are driven by the heterogeneous and complex environment of the atherosclerotic plaque. Macrophage plasticity has also been observed during plaque regression.27 Interestingly, not only does plaque composition influence the pheno type of macrophages, but macrophage subtypes, owing to their intrinsic activities (such as phagocytosis) and
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REVIEWS
Monocyte Stimulus in the plaque Cholesterol crystals Lipopolysaccharide Proinflammatory cytokines Oxidized LDL a
Haemoglobin/ haptoglobin
Haem
IL-4 IL-10
M(Hb) macrophage
Mhem macrophage
M2 macrophage
CD163
LXR-α LXR-β
b M1 macrophage
Proinflammatory
CXCL4
c
TLR-4 NF-κB
TNF IL-6 IL-12
Oxidized phospholipids
M4 macrophage
TLR-2 NFE2L2 LXR-α
IL-10 IL-4
IL-10 HMOX-1
IL-10
d Mox macrophage
Resistant to lipid accumulation Iron-handling capacities Anti-inflammatory
IL-1β COX-2
IL-6 TNF MMP-7
Antioxidant
Reduced phagocytosis Proinflammatory
Figure 2 | Main macrophage subtypes found in atherosclerotic lesions. Stimuli present in atherosclerotic lesions drive the differentiation of monocytes towards different macrophage phenotypes. a | M1 macrophages release proinflammatory cytokines. b | M(Hb), Mhem, and M2 macrophages are resistant to lipid accumulation, possess iron-handling capacities, and have anti-inflammatory effects. c | Mox macrophages display an antioxidant gene expression profile. d | M4 macrophages, like M1 macrophages, are proinflammatory, but lack the capacity for phagocytosis. Abbreviations: COX-2, cyclooxygenase; CXCL4, C-X-C motif chemokine 4; HMOX-1, haem oxygenase (decycling) 1; LDL, low-density lipoprotein; LXR, liver X receptor; MMP-7, matrix metalloproteinase-7; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TLR, toll-like receptor; TNF, tumour necrosis factor.
ability to release proinflammatory and anti-inflammatory factors, can also influence plaque structure and evolution.
Macrophage phenotypes in plaques Responses to lipids and derivatives Macrophages resident in atherosclerotic lesions are continuously exposed to accumulating lipids and their oxidized derivatives.4 In response to this environment, macrophages adapt their phenotype or initiate specific transcriptional programmes. Cholesterol crystals that accumulate during the early stages of the development of atherosclerotic lesions might be responsible for the inflam matory activation of macrophages (Figure 2).28 Choles terol crystals induce inflammation by stimulating the caspase‑1-activating NLRP3 (also known as cr yopyrin) inflammasome, which results in cleavage and secretion of IL‑1.28 The cholesterol crystals deposited in the arteries of mice can, therefore, act as an M1-polarizing stimulus.28 Accumulation of oxidized lipoproteins can also direct human macrophages towards an M1 proinflammatory phenotype by a mechanism that involves inhibition of the transcription factor Kruppel-like factor 2.29,30 Moreover, in mice, minimally oxidized LDL promotes an inflammatory macrophage phenotype by activating a Toll-like receptor (TLR)‑4-mediated pathway.31 Cholesteryl esters (includ ing linoleate and 7‑ketocholesteryl-9‑carboxynonanoate) induce M1 polarization by activating the TLR‑4 or nuclear factor (NF)‑κB signalling pathways. 32,33 Conversely, 9‑oxononanoyl-cholesterol, a major cholesteryl ester 12 | JANUARY 2015 | VOLUME 12
oxidation product, promotes the development of an anti-inflammatory macrophage phenotype by increasing TGF‑β secretion.33 Sphingolipid metabolites, in particular sphingosine‑1-phosphate (S1P), switches the phenotype of mouse macrophages from M1 to M2 by activating its receptor, S1P1.34 ω3‑Polyunsaturated fatty acid deriva tives, notably resolvin D1, an anti-inflammatory and inflammation-resolving mediator biosynthesized from docosahexaenoic acid,35 switches macrophage polarization towards an M2-like phenotype.36 Conjugated linoleic acid, a group of naturally occurring isomers of linoleic acid,37 induces an anti-inflammatory M2 phenotype in vivo characterized by increased secretion of IL‑10.38 In addition to the M1 and M2 phenotypes, macrophages exposed to oxidized phospholipids can also be driven to express the Mox phenotype, which is triggered by the activation of transcription factor NFE2L2.39 Compared with M1 and M2 macrophages, Mox macrophages display reduced phagocytic and chemotactic capacities. In mice, Mox macrophages typically express NFE2L2-mediated redox-regulatory genes, such as Hmox1, Srxn1, Txnrd1, and Gsr.39 However, in response to oxidized phospholipids, Mox macrophages also express some proinflammatory markers, such as IL‑1β and cyclooxygenase 2, via a TLR-2dependent mechanism.40 In advanced atherosclerotic lesions of mice, Mox macrophages comprise approxi mately 30% of the total number of macrophages. 39 Whether Mox macrophages are also present in human atherosclerotic lesions remains to be determined.
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REVIEWS Table 1 | Characteristics of macrophage subtypes in mice and humans Phenotype
Traditional classification
Polarized by
Markers In mice
In humans
M1
Proinflammatory, classically activated
TH1 cytokines (IFN‑γ, TNF, IL‑1β) or lipopolysaccharide
IL‑1β, TNF, IL‑6, IL‑12, IL‑23, CXCL9, CXCL10, CXCL11, arginase II
IL‑1β, TNF, IL‑6, IL‑12, IL‑23, CXCL9, CXCL10, CXCL11
M2a
Anti-inflammatory, alternatively activated
TH2 cytokines (IL‑4, IL‑13)
Arginase I, resistin-like α, Ym1, Ym2, MMGL, stabilin‑1, CD163
MMR, IL‑1RA, factor XIIIa, CD200R, CCL18 stabilin‑1, CD163
M2b
Alternatively activated
Combinations of immune complexes with IL‑1β or lipopolysaccharide
IL‑10high, IL‑12low
IL‑10high, IL‑12low
M2c
Alternatively activated
IL‑10, TGF‑β, or glucocorticoids
Arginase I
MMR
M4
N/A
CXCL4
MMP‑7, S100‑A8, MMR
MMP‑7, S100‑A8, MMR
Mox
N/A
Oxidized phospholipids
HO‑1, Sulfiredoxin‑1, TR, NFE2L2
HO‑1, sulfiredoxin‑1, TR, NFE2L2
M(Hb)
N/A
Haemoglobin–haptoglobin complexes
CD163, MMR
CD163, MMR
Mhem
N/A
Haem
CD163, ATF-1
CD163, ATF-1
HA-mac
N/A
Haemoglobin–haptoglobin complexes
CD163high, HLA-DRlow
CD163high, HLA-DRlow
Abbreviations: ATF-1, cyclic AMP-dependent transcription factor-1; CCL18, C-C motif chemokine ligand 18; CXCL, C-X-C motif chemokine ligand; HLA, human leukocyte antigen; HO‑1, haem oxygenase 1; MMGL, C‑type lectin domain family 10 member A (also known as MGL‑1); MMP‑7, matrix metalloproteinase 7; MMR, macrophage mannose receptor; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; N/A, not applicable; TGF‑β, transforming growth factor‑β; TH1, type 1 T helper cells; TH2, type 2 T helper cells; TNF, tumour necrosis factor; TR, thioredoxin reductase 1, cytoplasmic.
Responses to haemorrhage Neovascularization often occurs in atherosclerotic plaques and can result in intraplaque haemorrhage after vessel rupture.41 The rupture of microvessels releases erythro cytes, which can be phagocytosed by macrophages, leading to an increase in iron content and release of haem asso ciated with oxidized lipid deposition.42 Accumulation of iron by M1 macrophages is a defence mechanism against microbial infection and bacterial proliferation.43 In the haemorrhagic zones of human atherosclerotic lesions, macrophages present the M(Hb) phenotype.44 M(Hb) macrophages are a subpopulation of CD68+ macro phages expressing both macrophage mannose receptor 1 (MMR, also known as CD206) and scavenger receptor cysteine-rich type 1 protein M130 (CD163). CD163 is involved in clearance and endocytosis of haemoglobin– haptoglobincomplexes, and is necessary for effective haemoglobin clearance after plaque haemorrhage.45 The M(Hb) macrophage phenotype is induced in vitro by haemoglobin–haptoglobin complexes, and is characterized by the production of anti-inflammatory factors. Owing to increased activity of the transcription factor oxysterols receptor LXR‑α (also known as liver X receptor‑α) and induction of cholesterol efflux, these macrophages are pro tected against lipid accumulation.44 M(Hb) macrophages also exhibit reduced intracellular iron accumulation owing to increased ferroportin expression, and thereby produce less ROS than other macrophage subsets.44 In response to haemoglobin–haptoglobin complex ligation via CD163, these macrophages produce IL‑10 by a mechanism involv ing the phosphoinositide 3-kinase (PI3K)–Akt pathway.46,47 Interestingly, in areas of neovascularization, a population of CD68+MMR+ macrophages that colocalize with iron deposits and oxidized lipids have been identified in human
atherosclerotic lesions.48 In vitro, these macrophages sense the cellular iron content and modify their gene expression profile in response.48 Indeed, IL‑4-polarized M2 macro phages have a phenotype that favours iron accumulation, which activates both LXR‑α and expression of its target genes.48 Upon exposure to iron, however, M2 macrophages favour iron release by increasing the expression of ferro portin through LXR‑α-dependent and NFE2L2-dependent mechanisms.48–50 Interestingly, these M2 macrophages also avidly phagocytose both senescent erythrocytes and apoptotic cells.48,51 Haem directs macrophage polarization towards the Mhem phenotype.52 This pathway is driven by the induc tion of activating cyclic AMP-dependent transcription factor ATF‑1 and oxysterols receptor LXR‑β, through phosphorylation of 5'-AMP-activated protein kinase (AMPK).53 LXR‑β subsequently induces the expression of both LXR‑α and ATP-binding cassette subfamily A member 1 (ABCA1).52 Mhem macrophages are character ized by increased expression of haem oxygenase 1 (HO‑1), which in mice is induced by CD163 via a mechanism involving the NFE2L2 pathway,54 and are protected from oxidative stress and lipid accumulation. Functionally, all these macrophages described above seem to be resistant to lipid accumulation and foam cell formation; their oxidative state, however, differs between subtypes. Mhem and M(Hb) macrophages have reduced levels of oxidative stress,41,52 whereas iron-loaded M2 macrophages have increased oxidative capacity.48 These differences might reflect their sterol oxidation activi ties, which results in the generation of potential antiinflammatory LXR ligands. 55 All these macrophage phenotypes might coexist in areas of neovascularization or haemorrhage in human atherosclerotic plaques, or
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REVIEWS might represent different stages of differentiation of the same cell population. Moreover, the spectrum of macro phage phenotypes can change during atherosclerotic plaque progression. Macrophages constitute the first line of defence against senescent erythrocytes and iron-induced damage. However, iron sequestration by M1 macrophages leads to an unrestrained proinflammatory phenotype that impairs wound healing.56 Given their increased capac ity to scavenge iron from the microenvironment and to phagocytose senescent erythrocytes, these macrophages might have a role in the recycling of potentially detrimen tal iron. Furthermore, although direct in vivo evidence is not yet available, M2 macrophages in human athero sclerotic lesions are highly competent in the phagocytosis and scavenging of cellular debris.51 They might, therefore, contribute to both maintenance of efficient efferocytosis within the plaque and resolution of inflammation.6
Responses to cytokines and growth factors Cytokines, chemokines and growth factors present in atherosclerotic lesions can contribute to macrophage phenotype determination.57 IL‑4, a potent inducer of the M2a phenotype,17 is expressed in human atherosclerotic plaques and could be one of the factors responsible for inducing polarization of a population of CD68+MMR+ M2 macrophages localized not only in zones of neo vascularization, but also in stable lesion areas away from the lipid core.8,51 These human CD68+MMR+ macrophages are smaller than CD68+MMR– macrophages and contain several small lipid droplets in their cytoplasm. Although CD68+MMR+ macrophages display reduced lipoprotein accumulation and decreased cholesterol efflux capacity (owing to reduced expression of LXR‑α and ABCA1), they are highly competent for phagocytosis.51 Growth factors such as GM‑CSF and M‑CSF are important regulators of macrophage differentiation and polarization. In vitro treatment of human macrophages with M‑CSF induced the expression of genes such as SEPP1, STAB1, and CD163L1 (encoding selenoprotein P, stabilin‑1, and CD163 antigen-like 1, respectively), whereas treatment with GM‑CSF induced the expression of PPBP (encoding platelet basic protein, also known as C‑X‑C motif chemokine 7).58 Interestingly, these genes are also expressed in human atherosclerotic lesions in vivo, which suggests that both GM‑CSF-induced and M‑CSF‑induced macrophage populations are present.58 Two additional macrophage populations, CD68+CD14+ and CD68 + CD14 – , have been also studied. 7 The CD68+CD14+ subpopulation, prevalent in human lesions, resembles M‑CSF-induced macrophages and expresses many proinflammatory genes. Conversely, CD68+CD14– cells (which resemble GM‑CSF-induced macrophages) express several genes involved in reverse cholesterol transport (PPARG, NR1H3, and ABCG1) and macro phage migration through the vessel wall (CCR7).7 The GM‑CSF:M-CSF ratio fluctuates in vivo during athero sclerosis progression.58,59 M‑CSF is expressed in both healthy arteries and atherosclerotic lesions,58 whereas GM‑CSF expression increases in smooth muscle cells and 14 | JANUARY 2015 | VOLUME 12
endothelial cells upon atherosclerosis development and macrophage accumulation.59 This change in the balance between GM‑CSF and M‑CSF might contribute to the increased prevalence of M1 macrophages observed in advanced plaques. Platelet factor 4 (also known as C‑X‑C motif chemo kine 4, or CXCL4) is abundantly expressed in macro phages and the neovascular endothelium of human atherosclerotic plaques.60,61 In humans, CXCL4 induces the M4 macrophage phenotype, which, despite display ing some characteristics of the M1 and M2 phenotypes, completely lacks the capacity for phagocytosis (Figure 2).62 CXCL4-induced M4 macrophages do not express CD163, and are consequently unable to induce expression of the atheroprotective enzyme HO‑1 in response to the haemoglobin–haptoglobin complexes.63 M4 macrophages might, therefore, prevent the development of macro phages displaying the Mhem phenotype. Interestingly, although macrophages can switch between the M1, M2, and Mox phenotypes (at least in vitro),39 polarization to the M4 phenotype seems to be irreversible.62 M4 macrophages have been found in human atherosclerotic plaques, where they stain positive for MMP‑7 and the calcium-binding protein S100‑A8,64 suggesting a potential role for this macrophage phenotype in human atherosclerosis.
Macrophage roles in plaque stability
The total number of macrophages gradually increases with plaque progression and severity, and is higher in symptomatic than asymptomatic plaques.65–67 Several research groups have examined the spatial distribution of different macrophage subsets within human athero sclerotic lesions. Macrophages located in the lesion shoulder, which is considered one of the most unstable areas within the plaque,68 mainly express M1 polariza tion markers, whereas macrophages present in the fibrous cap surrounding the necrotic core express both M1 and M2 markers (Figure 3).65 Consequently, the potentially deleterious proinflammatory effects of M1 macrophages within the fibrous cap might be counterbalanced by poten tially beneficial (that is, profibrotic and plaque-stabilizing) effects of M2 macrophages.22 In the adventitia, M2 macro phages are twofold to threefold more abundant than M1 macrophages.65 The observation that some markers of specific macrophage subsets are expressed in overlap ping regions, whereas others are only present in distinct zones of human plaques, suggests three possibilities: first, that these macrophage subpopulations arise from distinct monocyte origins; secondly, that sequential spatiotemporal recruitment of M1 and M2 macrophages occurs within the plaque; thirdly, that macrophage phenotype switch ing occurs in response to the local microenvironment. Evidence of phenotype switching in resident macrophages has been reported in mice.69 Arginase II, an M1 marker, has been detected alongside arginase I, an M2 marker, in macrophages within advanced atherosclerotic plaques. Furthermore, immunohistochemical staining for argi nase I and arginase II was equally distributed across advanced lesions.69 These findings are probably related to changes in the cytokine microenvironment during plaque
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REVIEWS Adventitia Media
Lumen
Fibrous cap
Plaque shoulder
Intima
Lipid core
Vessel Neovascularization in iron-rich areas
Iron deposit M1 macrophage M2 macrophage Smooth muscle cell
Figure 3 | Localization of macrophage subsets in human atherosclerotic lesions. M1 macrophages are predominantly found in the plaque shoulder and lipid core, whereas M2 macrophages are most abundant in the adventitia and areas of neovascularization, which also contain iron deposits. Fibrous caps contain similar amounts of both macrophage subsets.
progression; a local increase in the expression of IFN‑γ occurs at the expense of IL‑4 expression within advanced atherosclerotic lesions, at least in mice.69 However, these suggestions are mainly derived from correlation studies, and no direct experimental evidence is available as yet. Interestingly, polarization of macrophages resident in epicardial adipose tissue can influence atherosclerosis development in the coronary arteries. Macrophage infiltration and cytokine expression are both elevated in the epicardial adipose tissue of patients with coro nary artery dise ase compared with patients without coronary artery disease.70,71 The M1:M2 macrophage ratio in epicardial adipose tissue is positively correlated with the severity of coronary artery disease, and a shift towards a proinflammatory macrophage phenotype is evident in patients with coronary artery disease.71 These observations strongly suggest that macrophage polariza tion, not only within the plaque, but also in surrounding epicardial adipose tissue, could influence the progression of coronary atherosclerosis in humans. Experiments in animals will be useful to determine whether the polariza tion of macrophages resident in perivascular fat is affected because of the proximity of these adipose deposits to the inflamed vessel, or whether it contributes to coronary
inflammation. M2 macrophages in the adventitia might also have originated in the surrounding perivascular adipose tissue, given the strong migratory capacity of this macrophage subset.65,72 At present, however, no evidence exists to support this theory. The phenotype of macrophages resident in the carotid plaques of symptomatic patients during an acute ischae mic attack has been compared with those from asymp tomatic patients. A majority of the macrophages in the plaques of symptomatic patients display an M1 pheno type, whereas those from asymptomatic patients pre dominantly express M2 polarization markers.66 Consistent with this observation, coronary atherosclerotic lesions from patients with acute myocardial infarction (which often results from inflammation and plaque rupture) are enriched with CD11c+ M1 macrophages, whereas the number of MMR+ M2 macrophages was no differ ent from that in coronary plaques isolated from patients with stable angina pectoris.73 The relative proportions of M1 and M2 macrophages have also been investigated in plaques of different location and composition, including symptomatic carotid artery and femoral artery plaques.67 The proportion of M1 macrophages (positive for iNOS [inducible nitric oxide synthase], SOCS‑3 [suppressor of cytokine signalling-3], and MHC‑II) was higher in carotid than femoral plaques, whereas expression of M2 markers (CD163, dectin‑1) was more abundant in macrophages from femoral than carotid plaques.67 Several metalloproteinases that are abundantly expres sed in vitro by proinflammatory macrophages colocalize with M1 macrophage markers in human atherosclerotic lesions.74 Reciprocally, in experimental mouse models of atherosclerotic plaque regression (in which an athero sclerotic aortic arch from a hyperlipidaemic donor is transplanted into a normolipidaemic recipient mouse), resident macrophages express fewer M1 markers and more M2 markers after transplantation, a switch that is thought to contribute to suppression of inflammation.75,76 However, whether these results can be translated to the human situation, in which regression studies are not feasible and prospective studies virtually impossible, is still unclear. The results of correlation studies indicate that M1 macrophages predominate in plaques with an unstable phenotype, suggesting that plaque instability might be the consequence of an imbalance between M1 and M2 pheno types. However, whether the abundance of M1 macro phages in unstable plaques is the cause or consequence of plaque rupture remains unclear.
Conclusions
Macrophages are important in the development of athero sclerotic lesions because they participate in all stages of plaque formation and progression.77 During their life time, macrophages are exposed to a plethora of micro environmental signals and stimuli (including cytokines modified lipids, senescent erythorocytes, and iron) that influence their transcriptional programme and func tional phenotype. The intensity of these signals changes during plaque progression, and varies between plaque
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REVIEWS regions. Therefore, macrophages adapt their phenotype both over time and in response to their physical location, contributing to and modulating plaque progression and composition. The local accumulation of macrophage subpopulations that are highly competent in clearing apoptotic cells (such as M2 macrophages) can sustain efferocytosis, thereby contributing to the resolution of inflammation and prevention of necrotic core formation within plaques. Whether the distinct macrophage phenotypes represent different stages of differentiation of a single population of phenotypically and functionally plastic resident macro phages, or are consequences of the recruitment and dif ferentiation of specific monocyte subpopulations in the lesion, is still a matter of debate. However, despite rapid advances in this field, novel studies and research methods need to be developed to determine whether monocyte sub populations give rise to specific macrophage phenotypes. 1.
2. 3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Moore, K. J. & Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 145, 341–355 (2011). Lusis, A. J. Atherosclerosis. Nature 407, 233–241 (2000). Libby, P. Inflammation in atherosclerosis. Nature 420, 868–874 (2002). Libby, P., Aikawa, M. & Schonbeck, U. Cholesterol and atherosclerosis. Biochim. Biophys. Acta 1529, 299–309 (2000). Tabas, I. Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency. Arterioscler. Thromb. Vasc. Biol. 25, 2255–2264 (2005). Tabas, I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat. Rev. Immunol. 10, 36–46 (2010). Waldo, S. W. et al. Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am. J. Pathol. 172, 1112–1126 (2008). Bouhlel, M. A. et al. PPARγ activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. 6, 137–143 (2007). Geissmann, F., Jung, S. & Littman, D. R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71–82 (2003). Gordon, S. & Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953–964 (2005). Jenkins, S. J. et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332, 1284–1288 (2011). Robbins, C. S. et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat. Med. 19, 1166–1172 (2013). Jenkins, S. J. et al. IL‑4 directly signals tissueresident macrophages to proliferate beyond homeostatic levels controlled by CSF‑1. J. Exp. Med. 210, 2477–2491 (2013). Verreck, F. A. et al. Human IL‑23‑producing type 1 macrophages promote but IL‑10‑producing type 2 macrophages subvert immunity to (myco) acteria. Proc. Natl Acad. Sci. USA 101, 4560–4565 (2004). Mosser, D. M. The many faces of macrophage activation. J. Leukoc. Biol. 73, 209–212 (2003).
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Our current knowledge suggests that M2 macrophages, owing to their localization within human plaques and their intrinsic anti-inflammatory properties, are primarily associated with plaque stability. However, one of the most important challenges in this field will be to demonstrate a direct causative link between macrophage phenotype and the structure or progression of atherosclerotic lesions. Given that only correlation studies are feasible in humans, specific mouse models need to be developed. Further identification of novel phenotypic and func tional markers, and the use of novel large-scale expression profiling approaches and imaging technology, is expected to lead to an improved definition and understanding of the specific functions of the different macrophage sub types identified within the plaque. The identification of biological stimuli that can modulate macrophage pheno types could lead to the development of novel therapeutic approaches for the treatment of atherosclerosis.
16. Murray, P. J. & Wynn, T. A. Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 11, 723–737 (2011). 17. Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 3, 23–35 (2003). 18. Mantovani, A. et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25, 677–686 (2004). 19. Anderson, C. F., Gerber, J. S. & Mosser, D. M. Modulating macrophage function with IgG immune complexes. J. Endotoxin. Res. 8, 477–481 (2002). 20. Zizzo, G., Hilliard, B. A., Monestier, M. & Cohen, P. L. Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J. Immunol. 189, 3508–3520 (2012). 21. Jetten, N. et al. Anti-inflammatory M2, but not proinflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis 17, 109–118 (2014). 22. Lee, C. G. et al. Interleukin‑13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor β1. J. Exp. Med. 194, 809–821 (2001). 23. Mosser, D. M. & Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008). 24. Martinez, F. O., 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. 177, 7303–7311 (2006). 25. Porcheray, F. et al. Macrophage activation switching: an asset for the resolution of inflammation. Clin. Exp. Immunol. 142, 481–489 (2005). 26. Lee, S. et al. Distinct macrophage phenotypes contribute to kidney injury and repair. J. Am. Soc. Nephrol. 22, 317–326 (2011). 27. Feig, J. E. et al. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocytederived cells. Proc. Natl Acad. Sci. USA 108, 7166–7171 (2011). 28. Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010). 29. van Tits, L. J. et al. Oxidized LDL enhances pro‑inflammatory responses of alternatively
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activated M2 macrophages: a crucial role for Kruppel-like factor 2. Atherosclerosis 214, 345–349 (2011). Hirose, K. et al. Different responses to oxidized low-density lipoproteins in human polarized macrophages. Lipids Health Dis. 10, 1 (2011). Bae, Y. S. et al. Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: Toll-like receptor 4‑ and spleen tyrosine kinasedependent activation of NADPH oxidase 2. Circ. Res. 104, 210–218 (2009). Fang, L. et al. Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high cholesterol diet: macrophage binding and activation. J. Biol. Chem. 285, 32343–32351 (2010). Sottero, B. et al. Expression and synthesis of TGFβ1 is induced in macrophages by 9‑oxononanoyl cholesterol, a major cholesteryl ester oxidation product. Biofactors 24, 209–216 (2005). Hughes, J. E. et al. Sphingosine‑1‑phosphate induces an antiinflammatory phenotype in macrophages. Circ. Res. 102, 950–958 (2008). Serhan, C. N. et al. Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions. J. Exp. Med. 206, 15–23 (2009). Titos, E. et al. Resolvin D1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an M2-like phenotype. J. Immunol. 187, 5408–5418 (2011). Mitchell, P. L. & McLeod, R. S. Conjugated linoleic acid and atherosclerosis: studies in animal models. Biochem. Cell Biol. 86, 293–301 (2008). McCarthy, C. et al. IL‑10 mediates the immunoregulatory response in conjugated linoleic acid-induced regression of atherosclerosis. FASEB J. 27, 499–510 (2013). Kadl, A. et al. Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2. Circ. Res. 107, 737–746 (2010). Kadl, A. et al. Oxidized phospholipid-induced inflammation is mediated by Toll-like receptor 2. Free Radic. Biol. Med. 51, 1903–1909 (2011). Kolodgie, F. D. et al. Intraplaque hemorrhage and progression of coronary atheroma. N. Engl. J. Med. 349, 2316–2325 (2003).
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REVIEWS 42. Kockx, M. M. et al. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 23, 440–446 (2003). 43. Ganz, T. Macrophages and systemic iron homeostasis. J. Innate. Immun. 4, 446–453 (2012). 44. Finn, A. V. et al. Hemoglobin directs macrophage differentiation and prevents foam cell formation in human atherosclerotic plaques. J. Am. Coll. Cardiol. 59, 166–177 (2012). 45. Nielsen, M. J., Moller, H. J. & Moestrup, S. K. Hemoglobin and heme scavenger receptors. Antioxid. Redox Signal. 12, 261–273 (2010). 46. Philippidis, P. et al. Hemoglobin scavenger receptor CD163 mediates interleukin‑10 release and heme oxygenase‑1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circ. Res. 94, 119–126 (2004). 47. Landis, R. C., Philippidis, P., Domin, J., Boyle, J. J. & Haskard, D. O. Haptoglobin genotypedependent anti-inflammatory signaling in CD163+ Macrophages. Int. J. Inflam. 2013, 980327 (2013). 48. Bories, G. et al. Liver X receptor (LXR) activation stimulates iron export in human alternative macrophages. Circ. Res. 113, 1196–1205 (2013). 49. Recalcati, S. et al. Differential regulation of iron homeostasis during human macrophage polarized activation. Eur. J. Immunol. 40, 824–835 (2010). 50. Corna, G. et al. Polarization dictates iron handling by inflammatory and alternatively activated macrophages. Haematologica 95, 1814–1822 (2010). 51. Chinetti-Gbaguidi, G. et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways. Circ. Res. 108, 985–995 (2011). 52. Boyle, J. J. et al. Activating transcription factor 1 directs Mhem atheroprotective macrophages through coordinated iron handling and foam cell protection. Circ. Res. 110, 20–33 (2012). 53. Wan, X. et al. 5'‑AMP‑activated protein kinaseactivating transcription factor 1 cascade modulates human monocyte-derived macrophages to atheroprotective functions in response to heme or metformin. Arterioscler. Thromb. Vasc. Biol. 33, 2470–2480 (2013). 54. Boyle, J. J. et al. Heme induces heme oxygenase 1 via Nrf2: role in the homeostatic macrophage response to intraplaque
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hemorrhage. Arterioscler. Thromb. Vasc. Biol. 31, 2685–2691 (2011). Spann, N. J. et al. Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses. Cell 151, 138–152 (2012). Sindrilaru, A. et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J. Clin. Invest. 121, 985–997 (2012). Wolfs, I. M., Donners, M. M. & de Winther, M. P. Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation. Thromb. Haemost. 106, 763–771 (2011). Brocheriou, I. et al. Antagonistic regulation of macrophage phenotype by M‑CSF and GM‑CSF: implication in atherosclerosis. Atherosclerosis 214, 316–324 (2011). Plenz, G., Koenig, C., Severs, N. J. & Robenek, H. Smooth muscle cells express granulocytemacrophage colony-stimulating factor in the undiseased and atherosclerotic human coronary artery. Arterioscler. Thromb. Vasc. Biol. 17, 2489–2499 (1997). Pitsilos, S. et al. Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters. Thromb. Haemost. 90, 1112–1120 (2003). Gleissner, C. A. & Ley, K. CXCL4 in atherosclerosis: possible roles in monocyte arrest and macrophage foam cell formation. Thromb. Haemost. 98, 917–918 (2007). Gleissner, C. A., Shaked, I., Little, K. M. & Ley, K. CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages. J. Immunol. 184, 4810–4818 (2010). Gleissner, C. A. et al. CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages. Circ. Res. 106, 203–211 (2010). Erbel, C. et al. CXCL4-induced plaque macrophages can be specifically identified by co‑expression of MMP7+S100A8+ in vitro and in vivo. Innate Immun. (in press). Stoger, J. L. et al. Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis 225, 461–468 (2012). Cho, K. Y. et al. The phenotype of infiltrating macrophages influences arteriosclerotic plaque vulnerability in the carotid artery. J. Stroke Cerebrovasc. Dis. 22, 910–918 (2013). Shaikh, S. et al. Macrophage subtypes in symptomatic carotid artery and femoral artery plaques. Eur. J. Vasc. Endovasc. Surg. 44, 491–497 (2012).
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68. Barlis, P., Serruys, P. W., Devries, A. & Regar, E. Optical coherence tomography assessment of vulnerable plaque rupture: predilection for the plaque ‘shoulder’. Eur. Heart J. 29, 2023 (2008). 69. Khallou-Laschet, J. et al. Macrophage plasticity in experimental atherosclerosis. PLoS ONE 5, e8852 (2010). 70. Hirata, Y. et al. Enhanced inflammation in epicardial fat in patients with coronary artery disease. Int. Heart J. 52, 139–142 (2011). 71. Hirata, Y. et al. Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue. J. Am. Coll. Cardiol. 58, 248–255 (2011). 72. Cougoule, C. et al. Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments. Eur. J. Cell Biol. 91, 938–949 (2012). 73. Lee, C. W. et al. Macrophage heterogeneity of culprit coronary plaques in patients with acute myocardial infarction or stable angina. Am. J. Clin. Pathol. 139, 317–322 (2013). 74. Huang, W. C., Sala-Newby, G. B., Susana, A., Johnson, J. L. & Newby, A. C. Classical macrophage activation up-regulates several matrix metalloproteinases through mitogen activated protein kinases and nuclear factor-κB. PLoS ONE 7, e42507 (2012). 75. Feig, J. E. et al. Regression of atherosclerosis is characterized by broad changes in the plaque macrophage transcriptome. PLoS ONE 7, e39790 (2012). 76. Feig, J. E. et al. Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophages in atherosclerotic plaques. Circulation 123, 989–998 (2011). 77. Gui, T., Shimokado, A., Sun, Y., Akasaka, T. & Muragaki, Y. Diverse roles of macrophages in atherosclerosis: from inflammatory biology to biomarker discovery. Mediators Inflamm. 2012, 693083 (2012). Acknowledgements The authors acknowledge grants from the Fondation de France (G.C.‑G.), the Fondation pour la Recherche Médicale (G.C.‑G.), and the Transatlantic Leducq HDL Network (B.S.). B.S. is a member of the Institut Universitaire de France. Author contributions G.C.‑G. and S.C. researched data for the article. S.C. and B.S. substantially contributed to the discussion of content. G.C.‑G. wrote the manuscript. B.S. reviewed and edited the manuscript before submission.
VOLUME 12 | JANUARY 2015 | 17 © 2015 Macmillan Publishers Limited. All rights reserved
Introduction
INSERM U1011, Institut Pasteur de Lille, 1, Rue du Professeur Calmette, BP 245, Lille 59019, France (G.C.‑G., S.C., B.S.). Correspondence to: B.S. bart.staels@ pasteur-lille.fr
The development of atherosclerosis involves activation of various cell types (including endothelial cells, smooth muscle cells, lymphocytes, monocytes, and macro phages) in the intima of the arteries, which results in a local inflammatory response.1 An increase in circulating LDL-cholesterol levels and the subsequent accumulation of oxidized LDL in the subendothelial space triggers the recruitment and retention of monocytes and lymphocytes in the arterial wall. In the intima, monocytes differentiate into macrophages, which scavenge lipoprotein particles, and eventually become foam cells.2 These macrophagederived foam cells secrete inflammatory molecules and factors that further promote lipoprotein retention, degrade the extracellular matrix, and sustain inflammation.3,4 Progression of atherosclerosis is characterized by apop tosis of these resident macrophages in the lipid core of the lesion. The clearance of apoptotic cells is mediated by phagocytes, mostly macrophages, which recognize and internalize dead cells in a process termed efferocytosis.5 In early lesions, phagocytes readily clear apoptotic cells, avoiding further progression of atherosclerosis. In chronic, advanced lesions, however, efferocytosis is no longer suf ficient to engulf all dead cells, and the gradual accumu lation of apoptotic debris results in formation of a necrotic core, which triggers further inflammation, necrosis, and thrombosis.5 Macrophages are crucial in the maintenance of efficient efferocytosis, and thereby contribute to both resolving inflammation and preventing the formation of a necrotic core within the plaque (Figure 1).6 Novel observations have challenged these previously well-established concepts. Whereas atherosclerosis was Competing interests The authors declare no competing interests.
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initially considered a type 1 T helper cell (TH1)-driven inflammatory process, the concept of heterogeneity of macrophages resident within lesions has gradually emerged over the past decade.7,8 Firstly, several studies revealed that both monocytes and macrophages comprise heterogeneous cell populations that adapt their functional phenotype in response to specific microenvironmental signals and molecules.9,10 These different monocyte and macrophage subtypes can be identified based on their dif ferential expression of surface markers and chemokine receptors.9,10 Secondly, the general dogma that tissueresident macrophages are incapable of proliferating was challenged by investigators who observed macrophages in mouse lungs that proliferated independently of monocyte recruitment.11 Both resident and recruited macrophages can be induced to proliferate by IL‑4.11 Moreover, in mice, macrophages in early atherosclerotic lesions are pre dominantly derived from recruited monocytes, whereas macrophage proliferation is a preponderant feature of advanced plaques and is influenced by microenvironment signals.12 Accordingly, differences in resident macrophage phenotypes can also influence their capacity to proliferate locally, which can alter the abundance of macrophages with a given phenotype.11,13 In this Review, we describe and discuss the reported functional phenotypes of macrophages in atherosclerotic plaques, focusing mainly on human pathology. The correlative studies suggesting a link between such macro phage phenotypes and the structure or progression of atherosclerotic lesions will also be discussed.
Macrophage polarization and plasticity
Several classes of macrophages have been described based on their expression of markers, the production of specific
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REVIEWS Key points ■■ Only M1 proinflammatory and M2 anti-inflammatory macrophages have been described in vitro—however, a wide spectrum of intermediate phenotypes has been identified in in vivo studies ■■ Various stimuli (cytokines, lipids and their derivatives, senescent cells, iron) can influence macrophage phenotypes in atherosclerotic lesions ■■ Macrophages with different functional phenotypes are likely to perform different roles in the development of atherosclerosis ■■ M1 macrophages are associated with symptomatic and unstable plaques, whereas M2 macrophages are particularly abundant in stable zones of the plaque and asymptomatic lesions ■■ Modulation of macrophage phenotypes might be a novel strategy for the pharmacological treatment of atherosclerosis
Iron deposit M1 macrophage M2 macrophage Vessel Lipid core M1
Endothelial cell
M2 efferocytic macrophage
Prevention of macrophage necrosis Iron deposits Neoangiogenesis Clearance of apoptotic macrophages
M1 inflammatory macrophage
Apoptosis
Postapoptotic necrosis
Necrotic core Resolution of inflammation
Plaque vulnerability
Figure 1 | Potential role of M2 macrophages in efferocytosis within atherosclerotic plaques. M2 macrophages localized in areas of neovascularization or outside the lipid core can phagocytose apoptotic M1 macrophages, contributing to the resolution of inflammation. If efferocytosis is insufficient, dead M1 macrophages accumulate and undergo postapoptotic necrosis, leading to the formation of a necrotic core, which contributes to plaque instability and rupture.
factors, and their biological functions. Classically acti vated (M1) macrophages are typically induced by TH1 cytokines, such as IFN‑γ and tumour necrosis factor (TNF), or by lipopolysaccharide recognition. These macrophages produce high levels of IL‑12 and IL‑23, low levels of IL‑10,14 and secrete the proinflammatory cytokines TNF, IL‑6, and IL‑1β (Figure 2).15 From a func tional point of view, M1 macrophages participate in the removal of pathogens during infection through activa tion of the NADPH oxidase system, and the subsequent generation of reactive oxygen species (ROS). Chronic M1 macrophage activation can, therefore, mediate ROSinduced tissue damage and impair wound healing.16 To protect against such tissue damage, the inflammatory
response is spatially and temporally counterbalanced by regulatory mechanisms driven by alternatively activated (M2) macrophages (Figure 2).17 Three different subclasses of M2 macrophages have been identified: M2a macrophages are induced by the TH2 cytokines IL‑4 and IL‑13; M2b macrophage subtypes are induced by immune complexes in combination with IL‑1β or lipopolysaccharide; and M2c macrophages are induced by IL‑10, transforming growth factor‑β (TGF‑β), or glucocorticoids. 18 All M2 macrophages have an anti-inflammatory cytokine profile character ized by low production of IL‑12 and high production of both IL‑10 and TGF‑β (which is especially prominent in the M2c subtype).18 M2b macrophages are an exception because they additionally produce high levels of pro inflammatory cytokines, including IL‑1, IL‑6, and TNF.19 Functionally, M2 macrophages can scavenge debris and apoptotic cells20 and promote tissue repair and healing. Furthermore, they possess proangiogenic21 and pro fibrotic22 properties. Monocytes differentiated in the presence of granulocyte–macrophage colony stimulat ing factor (GM‑CSF) or macrophage colony stimulating factor (M‑CSF) display functional properties similar to the M1 and M2 phenotype, respectively.7,14 An alterna tive method of classifying macrophage subpopulations has been proposed on the basis of their role in three macrophage homeostatic activities: host defence, wound healing, and immune regulation.23 This classification distinguishes activated M1 macrophages from woundhealing macrophages (M2a subclass) and regulatory macrophages (M2b/c subclass).23 Although the general properties of macrophages are conserved between species, their cell surface markers are not. For example, in mice (but not in humans), the M2 phenotype is defined by expression of Ym1 (chitinase 3‑like protein 3) and arginase I (Table 1).24 Moreover, only a few markers are selective and specific for a given phenotype. Some markers are shared across different macrophage sub types, suggesting that overlapping phenotypes can exist. Given that the majority of these markers are not exclusive for one phenotype, quantitative rather than qualitative variations should be considered. For this reason, macro phage phenotypes are generally characterized on the basis of cell surface marker expression combined with functional studies, when possible. Interestingly, macrophages are remarkably plastic and can switch from one phenotype to another depending on the environmental cues encountered.25,26 The local cytokine milieu can, therefore, orientate macrophage polariz ation and plasticity. The concept of macro phage heterogeneity has raised much interest in the past decade. In addition to the canonical M1 and M2 macro phage subpopulations, emerging evidence suggests that other specific and distinct macrophage phenotypes are driven by the heterogeneous and complex environment of the atherosclerotic plaque. Macrophage plasticity has also been observed during plaque regression.27 Interestingly, not only does plaque composition influence the pheno type of macrophages, but macrophage subtypes, owing to their intrinsic activities (such as phagocytosis) and
NATURE REVIEWS | CARDIOLOGY
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REVIEWS
Monocyte Stimulus in the plaque Cholesterol crystals Lipopolysaccharide Proinflammatory cytokines Oxidized LDL a
Haemoglobin/ haptoglobin
Haem
IL-4 IL-10
M(Hb) macrophage
Mhem macrophage
M2 macrophage
CD163
LXR-α LXR-β
b M1 macrophage
Proinflammatory
CXCL4
c
TLR-4 NF-κB
TNF IL-6 IL-12
Oxidized phospholipids
M4 macrophage
TLR-2 NFE2L2 LXR-α
IL-10 IL-4
IL-10 HMOX-1
IL-10
d Mox macrophage
Resistant to lipid accumulation Iron-handling capacities Anti-inflammatory
IL-1β COX-2
IL-6 TNF MMP-7
Antioxidant
Reduced phagocytosis Proinflammatory
Figure 2 | Main macrophage subtypes found in atherosclerotic lesions. Stimuli present in atherosclerotic lesions drive the differentiation of monocytes towards different macrophage phenotypes. a | M1 macrophages release proinflammatory cytokines. b | M(Hb), Mhem, and M2 macrophages are resistant to lipid accumulation, possess iron-handling capacities, and have anti-inflammatory effects. c | Mox macrophages display an antioxidant gene expression profile. d | M4 macrophages, like M1 macrophages, are proinflammatory, but lack the capacity for phagocytosis. Abbreviations: COX-2, cyclooxygenase; CXCL4, C-X-C motif chemokine 4; HMOX-1, haem oxygenase (decycling) 1; LDL, low-density lipoprotein; LXR, liver X receptor; MMP-7, matrix metalloproteinase-7; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TLR, toll-like receptor; TNF, tumour necrosis factor.
ability to release proinflammatory and anti-inflammatory factors, can also influence plaque structure and evolution.
Macrophage phenotypes in plaques Responses to lipids and derivatives Macrophages resident in atherosclerotic lesions are continuously exposed to accumulating lipids and their oxidized derivatives.4 In response to this environment, macrophages adapt their phenotype or initiate specific transcriptional programmes. Cholesterol crystals that accumulate during the early stages of the development of atherosclerotic lesions might be responsible for the inflam matory activation of macrophages (Figure 2).28 Choles terol crystals induce inflammation by stimulating the caspase‑1-activating NLRP3 (also known as cr yopyrin) inflammasome, which results in cleavage and secretion of IL‑1.28 The cholesterol crystals deposited in the arteries of mice can, therefore, act as an M1-polarizing stimulus.28 Accumulation of oxidized lipoproteins can also direct human macrophages towards an M1 proinflammatory phenotype by a mechanism that involves inhibition of the transcription factor Kruppel-like factor 2.29,30 Moreover, in mice, minimally oxidized LDL promotes an inflammatory macrophage phenotype by activating a Toll-like receptor (TLR)‑4-mediated pathway.31 Cholesteryl esters (includ ing linoleate and 7‑ketocholesteryl-9‑carboxynonanoate) induce M1 polarization by activating the TLR‑4 or nuclear factor (NF)‑κB signalling pathways. 32,33 Conversely, 9‑oxononanoyl-cholesterol, a major cholesteryl ester 12 | JANUARY 2015 | VOLUME 12
oxidation product, promotes the development of an anti-inflammatory macrophage phenotype by increasing TGF‑β secretion.33 Sphingolipid metabolites, in particular sphingosine‑1-phosphate (S1P), switches the phenotype of mouse macrophages from M1 to M2 by activating its receptor, S1P1.34 ω3‑Polyunsaturated fatty acid deriva tives, notably resolvin D1, an anti-inflammatory and inflammation-resolving mediator biosynthesized from docosahexaenoic acid,35 switches macrophage polarization towards an M2-like phenotype.36 Conjugated linoleic acid, a group of naturally occurring isomers of linoleic acid,37 induces an anti-inflammatory M2 phenotype in vivo characterized by increased secretion of IL‑10.38 In addition to the M1 and M2 phenotypes, macrophages exposed to oxidized phospholipids can also be driven to express the Mox phenotype, which is triggered by the activation of transcription factor NFE2L2.39 Compared with M1 and M2 macrophages, Mox macrophages display reduced phagocytic and chemotactic capacities. In mice, Mox macrophages typically express NFE2L2-mediated redox-regulatory genes, such as Hmox1, Srxn1, Txnrd1, and Gsr.39 However, in response to oxidized phospholipids, Mox macrophages also express some proinflammatory markers, such as IL‑1β and cyclooxygenase 2, via a TLR-2dependent mechanism.40 In advanced atherosclerotic lesions of mice, Mox macrophages comprise approxi mately 30% of the total number of macrophages. 39 Whether Mox macrophages are also present in human atherosclerotic lesions remains to be determined.
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REVIEWS Table 1 | Characteristics of macrophage subtypes in mice and humans Phenotype
Traditional classification
Polarized by
Markers In mice
In humans
M1
Proinflammatory, classically activated
TH1 cytokines (IFN‑γ, TNF, IL‑1β) or lipopolysaccharide
IL‑1β, TNF, IL‑6, IL‑12, IL‑23, CXCL9, CXCL10, CXCL11, arginase II
IL‑1β, TNF, IL‑6, IL‑12, IL‑23, CXCL9, CXCL10, CXCL11
M2a
Anti-inflammatory, alternatively activated
TH2 cytokines (IL‑4, IL‑13)
Arginase I, resistin-like α, Ym1, Ym2, MMGL, stabilin‑1, CD163
MMR, IL‑1RA, factor XIIIa, CD200R, CCL18 stabilin‑1, CD163
M2b
Alternatively activated
Combinations of immune complexes with IL‑1β or lipopolysaccharide
IL‑10high, IL‑12low
IL‑10high, IL‑12low
M2c
Alternatively activated
IL‑10, TGF‑β, or glucocorticoids
Arginase I
MMR
M4
N/A
CXCL4
MMP‑7, S100‑A8, MMR
MMP‑7, S100‑A8, MMR
Mox
N/A
Oxidized phospholipids
HO‑1, Sulfiredoxin‑1, TR, NFE2L2
HO‑1, sulfiredoxin‑1, TR, NFE2L2
M(Hb)
N/A
Haemoglobin–haptoglobin complexes
CD163, MMR
CD163, MMR
Mhem
N/A
Haem
CD163, ATF-1
CD163, ATF-1
HA-mac
N/A
Haemoglobin–haptoglobin complexes
CD163high, HLA-DRlow
CD163high, HLA-DRlow
Abbreviations: ATF-1, cyclic AMP-dependent transcription factor-1; CCL18, C-C motif chemokine ligand 18; CXCL, C-X-C motif chemokine ligand; HLA, human leukocyte antigen; HO‑1, haem oxygenase 1; MMGL, C‑type lectin domain family 10 member A (also known as MGL‑1); MMP‑7, matrix metalloproteinase 7; MMR, macrophage mannose receptor; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; N/A, not applicable; TGF‑β, transforming growth factor‑β; TH1, type 1 T helper cells; TH2, type 2 T helper cells; TNF, tumour necrosis factor; TR, thioredoxin reductase 1, cytoplasmic.
Responses to haemorrhage Neovascularization often occurs in atherosclerotic plaques and can result in intraplaque haemorrhage after vessel rupture.41 The rupture of microvessels releases erythro cytes, which can be phagocytosed by macrophages, leading to an increase in iron content and release of haem asso ciated with oxidized lipid deposition.42 Accumulation of iron by M1 macrophages is a defence mechanism against microbial infection and bacterial proliferation.43 In the haemorrhagic zones of human atherosclerotic lesions, macrophages present the M(Hb) phenotype.44 M(Hb) macrophages are a subpopulation of CD68+ macro phages expressing both macrophage mannose receptor 1 (MMR, also known as CD206) and scavenger receptor cysteine-rich type 1 protein M130 (CD163). CD163 is involved in clearance and endocytosis of haemoglobin– haptoglobincomplexes, and is necessary for effective haemoglobin clearance after plaque haemorrhage.45 The M(Hb) macrophage phenotype is induced in vitro by haemoglobin–haptoglobin complexes, and is characterized by the production of anti-inflammatory factors. Owing to increased activity of the transcription factor oxysterols receptor LXR‑α (also known as liver X receptor‑α) and induction of cholesterol efflux, these macrophages are pro tected against lipid accumulation.44 M(Hb) macrophages also exhibit reduced intracellular iron accumulation owing to increased ferroportin expression, and thereby produce less ROS than other macrophage subsets.44 In response to haemoglobin–haptoglobin complex ligation via CD163, these macrophages produce IL‑10 by a mechanism involv ing the phosphoinositide 3-kinase (PI3K)–Akt pathway.46,47 Interestingly, in areas of neovascularization, a population of CD68+MMR+ macrophages that colocalize with iron deposits and oxidized lipids have been identified in human
atherosclerotic lesions.48 In vitro, these macrophages sense the cellular iron content and modify their gene expression profile in response.48 Indeed, IL‑4-polarized M2 macro phages have a phenotype that favours iron accumulation, which activates both LXR‑α and expression of its target genes.48 Upon exposure to iron, however, M2 macrophages favour iron release by increasing the expression of ferro portin through LXR‑α-dependent and NFE2L2-dependent mechanisms.48–50 Interestingly, these M2 macrophages also avidly phagocytose both senescent erythrocytes and apoptotic cells.48,51 Haem directs macrophage polarization towards the Mhem phenotype.52 This pathway is driven by the induc tion of activating cyclic AMP-dependent transcription factor ATF‑1 and oxysterols receptor LXR‑β, through phosphorylation of 5'-AMP-activated protein kinase (AMPK).53 LXR‑β subsequently induces the expression of both LXR‑α and ATP-binding cassette subfamily A member 1 (ABCA1).52 Mhem macrophages are character ized by increased expression of haem oxygenase 1 (HO‑1), which in mice is induced by CD163 via a mechanism involving the NFE2L2 pathway,54 and are protected from oxidative stress and lipid accumulation. Functionally, all these macrophages described above seem to be resistant to lipid accumulation and foam cell formation; their oxidative state, however, differs between subtypes. Mhem and M(Hb) macrophages have reduced levels of oxidative stress,41,52 whereas iron-loaded M2 macrophages have increased oxidative capacity.48 These differences might reflect their sterol oxidation activi ties, which results in the generation of potential antiinflammatory LXR ligands. 55 All these macrophage phenotypes might coexist in areas of neovascularization or haemorrhage in human atherosclerotic plaques, or
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REVIEWS might represent different stages of differentiation of the same cell population. Moreover, the spectrum of macro phage phenotypes can change during atherosclerotic plaque progression. Macrophages constitute the first line of defence against senescent erythrocytes and iron-induced damage. However, iron sequestration by M1 macrophages leads to an unrestrained proinflammatory phenotype that impairs wound healing.56 Given their increased capac ity to scavenge iron from the microenvironment and to phagocytose senescent erythrocytes, these macrophages might have a role in the recycling of potentially detrimen tal iron. Furthermore, although direct in vivo evidence is not yet available, M2 macrophages in human athero sclerotic lesions are highly competent in the phagocytosis and scavenging of cellular debris.51 They might, therefore, contribute to both maintenance of efficient efferocytosis within the plaque and resolution of inflammation.6
Responses to cytokines and growth factors Cytokines, chemokines and growth factors present in atherosclerotic lesions can contribute to macrophage phenotype determination.57 IL‑4, a potent inducer of the M2a phenotype,17 is expressed in human atherosclerotic plaques and could be one of the factors responsible for inducing polarization of a population of CD68+MMR+ M2 macrophages localized not only in zones of neo vascularization, but also in stable lesion areas away from the lipid core.8,51 These human CD68+MMR+ macrophages are smaller than CD68+MMR– macrophages and contain several small lipid droplets in their cytoplasm. Although CD68+MMR+ macrophages display reduced lipoprotein accumulation and decreased cholesterol efflux capacity (owing to reduced expression of LXR‑α and ABCA1), they are highly competent for phagocytosis.51 Growth factors such as GM‑CSF and M‑CSF are important regulators of macrophage differentiation and polarization. In vitro treatment of human macrophages with M‑CSF induced the expression of genes such as SEPP1, STAB1, and CD163L1 (encoding selenoprotein P, stabilin‑1, and CD163 antigen-like 1, respectively), whereas treatment with GM‑CSF induced the expression of PPBP (encoding platelet basic protein, also known as C‑X‑C motif chemokine 7).58 Interestingly, these genes are also expressed in human atherosclerotic lesions in vivo, which suggests that both GM‑CSF-induced and M‑CSF‑induced macrophage populations are present.58 Two additional macrophage populations, CD68+CD14+ and CD68 + CD14 – , have been also studied. 7 The CD68+CD14+ subpopulation, prevalent in human lesions, resembles M‑CSF-induced macrophages and expresses many proinflammatory genes. Conversely, CD68+CD14– cells (which resemble GM‑CSF-induced macrophages) express several genes involved in reverse cholesterol transport (PPARG, NR1H3, and ABCG1) and macro phage migration through the vessel wall (CCR7).7 The GM‑CSF:M-CSF ratio fluctuates in vivo during athero sclerosis progression.58,59 M‑CSF is expressed in both healthy arteries and atherosclerotic lesions,58 whereas GM‑CSF expression increases in smooth muscle cells and 14 | JANUARY 2015 | VOLUME 12
endothelial cells upon atherosclerosis development and macrophage accumulation.59 This change in the balance between GM‑CSF and M‑CSF might contribute to the increased prevalence of M1 macrophages observed in advanced plaques. Platelet factor 4 (also known as C‑X‑C motif chemo kine 4, or CXCL4) is abundantly expressed in macro phages and the neovascular endothelium of human atherosclerotic plaques.60,61 In humans, CXCL4 induces the M4 macrophage phenotype, which, despite display ing some characteristics of the M1 and M2 phenotypes, completely lacks the capacity for phagocytosis (Figure 2).62 CXCL4-induced M4 macrophages do not express CD163, and are consequently unable to induce expression of the atheroprotective enzyme HO‑1 in response to the haemoglobin–haptoglobin complexes.63 M4 macrophages might, therefore, prevent the development of macro phages displaying the Mhem phenotype. Interestingly, although macrophages can switch between the M1, M2, and Mox phenotypes (at least in vitro),39 polarization to the M4 phenotype seems to be irreversible.62 M4 macrophages have been found in human atherosclerotic plaques, where they stain positive for MMP‑7 and the calcium-binding protein S100‑A8,64 suggesting a potential role for this macrophage phenotype in human atherosclerosis.
Macrophage roles in plaque stability
The total number of macrophages gradually increases with plaque progression and severity, and is higher in symptomatic than asymptomatic plaques.65–67 Several research groups have examined the spatial distribution of different macrophage subsets within human athero sclerotic lesions. Macrophages located in the lesion shoulder, which is considered one of the most unstable areas within the plaque,68 mainly express M1 polariza tion markers, whereas macrophages present in the fibrous cap surrounding the necrotic core express both M1 and M2 markers (Figure 3).65 Consequently, the potentially deleterious proinflammatory effects of M1 macrophages within the fibrous cap might be counterbalanced by poten tially beneficial (that is, profibrotic and plaque-stabilizing) effects of M2 macrophages.22 In the adventitia, M2 macro phages are twofold to threefold more abundant than M1 macrophages.65 The observation that some markers of specific macrophage subsets are expressed in overlap ping regions, whereas others are only present in distinct zones of human plaques, suggests three possibilities: first, that these macrophage subpopulations arise from distinct monocyte origins; secondly, that sequential spatiotemporal recruitment of M1 and M2 macrophages occurs within the plaque; thirdly, that macrophage phenotype switch ing occurs in response to the local microenvironment. Evidence of phenotype switching in resident macrophages has been reported in mice.69 Arginase II, an M1 marker, has been detected alongside arginase I, an M2 marker, in macrophages within advanced atherosclerotic plaques. Furthermore, immunohistochemical staining for argi nase I and arginase II was equally distributed across advanced lesions.69 These findings are probably related to changes in the cytokine microenvironment during plaque
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REVIEWS Adventitia Media
Lumen
Fibrous cap
Plaque shoulder
Intima
Lipid core
Vessel Neovascularization in iron-rich areas
Iron deposit M1 macrophage M2 macrophage Smooth muscle cell
Figure 3 | Localization of macrophage subsets in human atherosclerotic lesions. M1 macrophages are predominantly found in the plaque shoulder and lipid core, whereas M2 macrophages are most abundant in the adventitia and areas of neovascularization, which also contain iron deposits. Fibrous caps contain similar amounts of both macrophage subsets.
progression; a local increase in the expression of IFN‑γ occurs at the expense of IL‑4 expression within advanced atherosclerotic lesions, at least in mice.69 However, these suggestions are mainly derived from correlation studies, and no direct experimental evidence is available as yet. Interestingly, polarization of macrophages resident in epicardial adipose tissue can influence atherosclerosis development in the coronary arteries. Macrophage infiltration and cytokine expression are both elevated in the epicardial adipose tissue of patients with coro nary artery dise ase compared with patients without coronary artery disease.70,71 The M1:M2 macrophage ratio in epicardial adipose tissue is positively correlated with the severity of coronary artery disease, and a shift towards a proinflammatory macrophage phenotype is evident in patients with coronary artery disease.71 These observations strongly suggest that macrophage polariza tion, not only within the plaque, but also in surrounding epicardial adipose tissue, could influence the progression of coronary atherosclerosis in humans. Experiments in animals will be useful to determine whether the polariza tion of macrophages resident in perivascular fat is affected because of the proximity of these adipose deposits to the inflamed vessel, or whether it contributes to coronary
inflammation. M2 macrophages in the adventitia might also have originated in the surrounding perivascular adipose tissue, given the strong migratory capacity of this macrophage subset.65,72 At present, however, no evidence exists to support this theory. The phenotype of macrophages resident in the carotid plaques of symptomatic patients during an acute ischae mic attack has been compared with those from asymp tomatic patients. A majority of the macrophages in the plaques of symptomatic patients display an M1 pheno type, whereas those from asymptomatic patients pre dominantly express M2 polarization markers.66 Consistent with this observation, coronary atherosclerotic lesions from patients with acute myocardial infarction (which often results from inflammation and plaque rupture) are enriched with CD11c+ M1 macrophages, whereas the number of MMR+ M2 macrophages was no differ ent from that in coronary plaques isolated from patients with stable angina pectoris.73 The relative proportions of M1 and M2 macrophages have also been investigated in plaques of different location and composition, including symptomatic carotid artery and femoral artery plaques.67 The proportion of M1 macrophages (positive for iNOS [inducible nitric oxide synthase], SOCS‑3 [suppressor of cytokine signalling-3], and MHC‑II) was higher in carotid than femoral plaques, whereas expression of M2 markers (CD163, dectin‑1) was more abundant in macrophages from femoral than carotid plaques.67 Several metalloproteinases that are abundantly expres sed in vitro by proinflammatory macrophages colocalize with M1 macrophage markers in human atherosclerotic lesions.74 Reciprocally, in experimental mouse models of atherosclerotic plaque regression (in which an athero sclerotic aortic arch from a hyperlipidaemic donor is transplanted into a normolipidaemic recipient mouse), resident macrophages express fewer M1 markers and more M2 markers after transplantation, a switch that is thought to contribute to suppression of inflammation.75,76 However, whether these results can be translated to the human situation, in which regression studies are not feasible and prospective studies virtually impossible, is still unclear. The results of correlation studies indicate that M1 macrophages predominate in plaques with an unstable phenotype, suggesting that plaque instability might be the consequence of an imbalance between M1 and M2 pheno types. However, whether the abundance of M1 macro phages in unstable plaques is the cause or consequence of plaque rupture remains unclear.
Conclusions
Macrophages are important in the development of athero sclerotic lesions because they participate in all stages of plaque formation and progression.77 During their life time, macrophages are exposed to a plethora of micro environmental signals and stimuli (including cytokines modified lipids, senescent erythorocytes, and iron) that influence their transcriptional programme and func tional phenotype. The intensity of these signals changes during plaque progression, and varies between plaque
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VOLUME 12 | JANUARY 2015 | 15 © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS regions. Therefore, macrophages adapt their phenotype both over time and in response to their physical location, contributing to and modulating plaque progression and composition. The local accumulation of macrophage subpopulations that are highly competent in clearing apoptotic cells (such as M2 macrophages) can sustain efferocytosis, thereby contributing to the resolution of inflammation and prevention of necrotic core formation within plaques. Whether the distinct macrophage phenotypes represent different stages of differentiation of a single population of phenotypically and functionally plastic resident macro phages, or are consequences of the recruitment and dif ferentiation of specific monocyte subpopulations in the lesion, is still a matter of debate. However, despite rapid advances in this field, novel studies and research methods need to be developed to determine whether monocyte sub populations give rise to specific macrophage phenotypes. 1.
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Our current knowledge suggests that M2 macrophages, owing to their localization within human plaques and their intrinsic anti-inflammatory properties, are primarily associated with plaque stability. However, one of the most important challenges in this field will be to demonstrate a direct causative link between macrophage phenotype and the structure or progression of atherosclerotic lesions. Given that only correlation studies are feasible in humans, specific mouse models need to be developed. Further identification of novel phenotypic and func tional markers, and the use of novel large-scale expression profiling approaches and imaging technology, is expected to lead to an improved definition and understanding of the specific functions of the different macrophage sub types identified within the plaque. The identification of biological stimuli that can modulate macrophage pheno types could lead to the development of novel therapeutic approaches for the treatment of atherosclerosis.
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