Sophie Colin Giulia Chinetti-Gbaguidi Bart Staels

Macrophage phenotypes in atherosclerosis

Authors’ addresses Sophie Colin1,2,3,4, Giulia Chinetti-Gbaguidi1,2,3,4, Bart Staels1,2,3,4 1 Universit
e Lille 2, Lille, France. 2 Inserm, U1011, Lille, France. 3 Institut Pasteur de Lille, Lille, France. 4 European Genomic Institute for Diabetes (EGID), FR 3508, Lille, France.

Summary: Initiation and progression of atherosclerosis depend on local inflammation and accumulation of lipids in the vascular wall. Although many cells are involved in the development and progression of atherosclerosis, macrophages are fundamental contributors. For nearly a decade, the phenotypic heterogeneity and plasticity of macrophages has been studied. In atherosclerotic lesions, macrophages are submitted to a large variety of micro-environmental signals, such as oxidized lipids and cytokines, which influence the phenotypic polarization and activation of macrophages resulting in a dynamic plasticity. The macrophage phenotype spectrum is characterized, at the extremes, by the classical M1 macrophages induced by T-helper 1 (Th-1) cytokines and by the alternative M2 macrophages induced by Th-2 cytokines. M2 macrophages can be further classified into M2a, M2b, M2c, and M2d subtypes. More recently, additional plaque-specific macrophage phenotypes have been identified, termed as Mox, Mhem, and M4. Understanding the mechanisms and functional consequences of the phenotypic heterogeneity of macrophages will contribute to determine their potential role in lesion development and plaque stability. Furthermore, research on macrophage plasticity could lead to novel therapeutic approaches to counteract cardiovascular diseases such as atherosclerosis. The present review summarizes our current knowledge on macrophage subsets in atherosclerotic plaques and mechanism behind the modulation of the macrophage phenotype.

Correspondence to: Bart Staels Inserm UR 1011, Institut Pasteur de Lille 1, rue du Professeur Calmette BP 245, Lille 59019, France Tel.: +33 3 20 87 73 88 Fax: +33 3 20 87 73 60 e-mail: [email protected] Acknowledgements This work was supported by grants from the Universit
e de Lille 2, the R
egion Nord/Pas-de-Calais, the FEDER and the “Fondation Leducq”. Bart Staels is a member of the Institut Universitaire de France. The authors have no conflicts of interest to declare.

This article is a part of a series of reviews covering Monocytes and Macrophages appearing in Volume 262 of Immunological Reviews.

Immunological Reviews 2014 Vol. 262: 153–166 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Keywords: macrophages, lipids, atherosclerosis, polarization, cytokines, chemokines, inflammation

Introduction Atherosclerosis is a chronic inflammatory disease involving numerous cell types. Perturbation of lipid metabolism and local inflammation appear to be main processes responsible for atherogenesis. The first step of atherosclerosis development is characterized by endothelial activation followed by the accumulation and sequestration of low density lipoproteins (LDLs) in the intima (1). These trapped lipoproteins are modified by various mechanisms such as oxidation by reactive oxygen species (ROS) and enzymatic cleavage, transforming them into pro-inflammatory particles (2). Modified LDLs further stimulates the endothelial cells to produce adhesion molecules leading to the adhesion and recruitment of monocytes into the arterial wall where they

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

153

154

(31, 32)

(28–30, 85, 86)

(19, 28)

(23–28, 82, 90, 93, 104, 105)

(16, 18, 19, 21, 79, 88, 92)

M2d

M2c

M2b

M2a

References Functions in atherosclerosis Regulators

STAT1, NF-kB p65/p50, Microbicidal, tumor IL-1b, IL-6, IL-12, IL-23, TNFa, CXCL9, IL-6, TNFa, IL-23, iNOS IRF5 resistance CXCL10, CXCL11, MHCII, CD86, CD80, MARCO IL-4, IL-13 MR, MHCII, AMAC1, Arg1 IL-10, TGFb, IL-Ra, CCL17, STAT6, NF-kB p50/p50, Wound healing CCL22, CCL18 IRF4, PPARc macrophages IC, TLR, IL-1R ligands MR, MHCII, CD86 IL-1b, IL-6,TNFa, IL-10high Immunoregulation and IL-12low IL-10, Glucocorticoids, MR, CD163 IL-10, TGFb STAT3 Efferocytosis capacity TGFb IL-10high, VEGF, TNFalow, NF-kB Protumoral, proangiogenic TLR+A2R ligands TNFalow, IL-12low, VEGF IL-12low capacity IFNc, LPS, TNF

The classical macrophage activation is a response to products of activated T-helper 1 (Th1) lymphocytes such as interferon-c (IFNc) (17), thus referred to as M1 macrophages to mirror the Th1 nomenclature. IFNc, inflammatory cytokines such as TNF and microbial products such as lipopolysaccharide (LPS) drive, alone or in combination, the classical M1 activation (18). M1 macrophages contribute to an increased and sustained inflammatory response via the secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL1b, TNF (19), as well as IL-12 and IL-23 (20). M1 macrophages also produce toxic agents such as nitric oxide and

M1

Classically activated M1 macrophages

Cytokine and chemokine production

Monocytes exposed to specific micro-environments differentiate into a heterogeneous range of macrophages. The macrophage phenotypic diversity can be assessed by the expression of numerous surface markers as well as by their secretome. The concept of macrophage classification was established in the ‘60s when the term ‘classical activation’ was first used (12). Initially, the classically activated M1 macrophages received most attention (13–15) and several years later alternatively activated M2 macrophages were identified (16). Thereafter, classically activated M1 macrophages and alternatively activated M2 macrophages were extensively described mainly in in vitro experimental settings (Table 1).

Markers and surface molecules

Macrophage polarization and plasticity in vitro

Macrophage phenotype Inducers

differentiated into macrophages that phagocytose modified LDL and form lipid-laden foam cells (3). During atherosclerosis progression, macrophage emigration from atherosclerotic plaques is reduced (4), probably through an increased expression of migration-inhibitory molecules such as netrin1 and semaphorine 3E in foam cells (5, 6), thus maintaining the inflammatory state of the plaque and leading to progression into chronic and more complicated lesions. Macrophages, which are present in various location of the plaque, are sensitive to their complex micro-environment and are facing accumulated cytokines, lipids, iron, and calcium (7–11), factors which can influence macrophage phenotypic polarization and activation. Previous research has shown that all atherosclerosis-related processes (initiation, progression, and regression of lesions) are influenced by the presence of macrophages. However, recent advances suggest that the relative proportion of macrophage sub-populations, rather than the absolute number, is a better indicator of atherosclerotic plaque phenotype and lesion progression.

Table 1. Overview of macrophage subset characteristics described in vitro. The table illustrates the macrophage subsets described in vitro, the principal inducers of macrophage polarization and their characteristics. Macrophage receptor with collagenous structure (MARCO). For the other abbreviations refer to the text

Colin et al
Macrophages in atherosclerosis

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

ROS, in an attempt to eradicate bacterial, fungal, and viral infections (16, 21). The different chemokine (C-X-C motif) receptor ligands (CXCL9, CXCL10, and CXCL5) expressed by M1 macrophages further promote the recruitment of Th1 and natural killer cells, which play a crucial role in killing intracellular pathogens (22). Although these effects are beneficial upon acute infection, a chronic induction of M1 macrophage activation can cause tissue damage and impair wound healing, especially under condition of sterile inflammation. Alternatively activated M2 macrophages At the extreme opposite of the macrophage spectrum, the alternatively activated M2 macrophages, mainly referred to as anti-inflammatory macrophages, counteract spatially and temporally the inflammatory response sustained by the M1 macrophages. These M2 macrophages, mainly involved in tissue repair, display phagocytic, pro-angiogenic, and profibrotic capacities (23–25). The term ‘M2 macrophage’ reflects also the Th2 nomenclature, because M2 activation was first thought to be primarily mediated by IL-4 and IL13 cytokines produced by Th2 cells (16, 21). Further in vitro experiments led to an extended classification with a subdivision into four sub-groups: M2a, M2b, M2c, and M2d. M2a macrophages are triggered in response to IL-4 and IL-13, express high levels of the mannose receptor (MR or CD206) and secrete pro-fibrotic factors such as fibronectin, insulinlike growth factor (IGF), and transforming growth factor b (TGFb) contributing to the tissue repair (23–27). Based on their functions, Mosser et al. (28) have proposed a functional classification and refer to these M2a macrophages as ‘wound-healing macrophages’. M2b macrophages are induced upon combined exposure to immune complexes and Toll-like receptor (TLR) ligands or IL-1 receptor agonists (28). M2c macrophages are induced by IL-10 and glucocorticoids. These M2c macrophages, together with M2b macrophages, are also referred to as ‘regulatory macrophages’ (28). They exhibit high expression levels of the Mer receptor tyrosine kinase (MerTK) providing them with high efferocytosis capacity (29, 30). Although M2 macrophages were considered as anti-inflammatory macrophages characterized by high production levels of IL-10 and low levels of IL-12, M2b macrophages appear an exception with simultaneous high production levels of pro-inflammatory cytokines (IL-1b, IL-6 and TNF), IL-10 and low IL-12 expression (19). Finally, M2d macrophages are induced by co-stimulation with TLR and adenosine A2A receptor agonists. These © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

M2d macrophages are characterized by high levels of IL-10 and vascular endothelial growth factor (VEGF) and low levels of TNF and IL-12. However, the high MR expression observed in the other M2 macrophage subtypes is not found in M2d macrophages (31, 32). Although significant progress has been made in characterizing the phenotype and functions of the macrophage subpopulations, several questions are still open and much remains to be discovered. Different markers are used to identify and characterize several populations of M1 or M2 macrophages in vitro and in vivo, creating a confusion on the identity and similarity of the described macrophage subtypes, i.e. one needs to assure that a population identified in one study using one set of markers is phenotypically identical to the one obtained in other studies using different markers. Second, although the general properties of macrophages are conserved between species, some alternative macrophage markers are species specific. Indeed, arginine metabolism in general as well as the expression of arginase1 (Arg1) or the transcription factor found in inflammatory zone 1 (FIZZ1) are specific for mouse M2 macrophages (33). Most usefully, transglutaminase 2 (TGM2) has been recently identified as a novel M2 marker in both species (34). In addition, numerous evidences have shown that the in vitro classification of macrophage phenotypes can be, to certain extend, applied to the macrophage phenotypes in the atherosclerotic plaque, with both M1 and M2 being present in atherosclerotic lesions (9). Macrophage phenotypes in atherosclerotic plaque In atherosclerosis, the heterogeneity of macrophages is a now well-recognized concept. Besides the simplified M1-M2 classification, with M1 and M2 macrophages representing, respectively, 40% and 20% of total atherosclerotic lesion macrophages in mice (35), a multitude of macrophages with different phenotypes co-exist in the atherosclerotic plaque. The main macrophage subsets present in atherosclerotic lesions are shown in Fig. 1. The macrophage phenotype in response to lipids Macrophages adapt their phenotype to the plaque microenvironment which is extremely rich in oxidatively modified lipids and lipoproteins. Cholesterol crystals, found in advanced atherosclerotic lesions, were recently shown to be also present at early stages of atherosclerotic lesions upon appearance of immune cells in the sub-endothelial space (36). In LPS-primed human peripheral blood mononuclear

155

Colin et al
Macrophages in atherosclerosis

Fig. 1. Macrophage subpopulations in atherosclerotic plaques. In atherosclerotic plaques, macrophages adapt their phenotype to different stimuli. The M1 macrophages are driven by IFNc and LPS, as well as by OxLDL and cholesterol crystals, IL-4 induces the M2 macrophages, oxidized phospholipids (OxPL) drive monocytes toward a Mox phenotype, Mhem are induced by heme and M(Hb) are polarized by the Hb/Hp complex and finally, CXCL4 polarize to the M4 phenotype. From a functional point of view, Mhem, M(Hb) and M2 macrophages prevent foam cell formation and play a crucial role in iron handling. M1 macrophages display a pro-inflammatory profile and are found in rupture-prone lesions which suggest that these macrophages are associated to plaque vulnerability. Mox macrophages exhibit reduced phagocytic capacity and express anti-oxidant genes. M4 macrophages also display reduced phagocytic capacity and a pro-inflammatory profile. Some examples of markers or related genes of these macrophages are indicated. Macrophage subpopulations identified in human (H) and/or in mouse (M) atherosclerotic plaques.

cells and in vivo in mice, cholesterol crystals activate the caspase-1-activating-NLRP3 inflammasome thus inducing the cleavage and the release of IL-1 family cytokines (including IL-1b and IL-18) (36). These observations suggest that cholesterol crystals act as a M1 pro-inflammatory stimulus. Likewise, the accumulation of oxidized LDL in M2 macrophages increases the production of pro-inflammatory cytokines (IL6, IL-18, and MCP-1) by a Kr€ uppel-like transcription factor2 (KLF2)-dependent mechanism leading to a polarization toward a M1 phenotype (37, 38). In addition, minimally modified LDL (mmLDL) promote an inflammatory response in human and mouse macrophages through activation of the TLR-4 and TLR-2 pathways (39–41). Oxidized cholesteryl esters are biologically active components of mmLDL present in human atherosclerotic lesions (42). Different forms of oxidized cholesteryl esters drive macrophages to the M1 phenotype through distinct mechanisms. Cholesteryl linoleate, the major cholesteryl ester in atherosclerotic plaque,

156

activates M1 polarization via a MAP-kinase dependent mechanism, whereas 7-keto-cholesteryl-9-carboxinonanoate drives the M1 phenotype through the NF-jB dependent signaling pathway (43–45). In contrast, cholesteryl 9-oxononanoate, the main oxidation product of cholesteryl linoleate, induces the expression and secretion of TGFb1, suggesting a potential role as antiinflammatory mediator (46). Docosahexaenoic acid (DHA), a polyunsaturated omega-3 fatty acid, downregulates the expression of MCP-1 and increases the expression of M2 markers (including IL-10, Arg1, MR) in adipose tissue from high fat diet-induced obese mice (47). Moreover, resolvin D1 (a DHA-derived inflammation resolution mediator) was reported induce the M2 phenotype in mice (47). Sphingosin-1-phosphate, a biologically active sphingolipid, enhances the M2 macrophage phenotype by inducing Arg1 through activation of the sphingosin-1-phosphate-1 receptor (48). Finally, conjugated linoleic acid increases the secretion of © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

IL-10 contributing to a switch to a M2 phenotype in mice (49). However, most of these modulators were studied in mouse models and the relevance for human atherosclerotic lesions requires to be demonstrated. Among the lipids accumulated in the sub-endothelial space, oxidized phospholipids and their derivatives were also found in the atherosclerotic lesions (50). Recently, oxidized phospholipids were reported to induce a different population of macrophages called Mox. Mox polarization is mediated by the transcription factor nuclear erythroid-2 related factor (Nrf2) (35). Mox macrophages exhibit different morphological structure and biological functions from M1 and M2 macrophages. Indeed, Mox display lower chemotactic and phagocytic capacities (35). Furthermore, specific Mox markers were identified as Nrf2-dependent redox-regulating genes such as heme oxygenase-1 (HMOX-1), sulforedoxin-1 (Srx1), thioredoxin reductase 1 (Txnrd1), dual-specificity phosphatase (Dusp)-1 and Dusp-5, thrombospondin (Thsp), glutamate-cystein ligase modifier subunit (Gclm), and glutathione reductase-1 (GSR1) (35). However, some proinflammatory genes, such as IL-1b and COX-2, are upregulated through the TLR-2 pathway (51) in Mox macrophages, similar as in M1 macrophages (35). In established atherosclerotic lesions in mice, Mox macrophages are prominent representing approximately 30% of the total number of macrophages (35). However, the presence of Mox in human atherosclerotic lesions has not been yet reported. The macrophage phenotype in response to hemorrhage and iron Different macrophage sub-populations have been identified in areas of intraplaque hemorrhage in humans. Hemoglobin-stimulated macrophages, referred to as M(Hb) (52), are characterized by high levels of MR and CD163, a scavenger receptor for the hemoglobin/haptoglobin (Hb/Hp) complex involved in hemoglobin clearance after plaque hemorrhage (53). Stimulation of human monocytes by the Hb/Hp complex increases MR and CD163 expression, reproducing the M(Hb) phenotype present in hemorrhagic sites in atherosclerotic plaques. The Hb/Hp complex induces the secretion of anti-inflammatory cytokines, such as IL-10, through a CD163/phosphoinositide 3-kinase phosphatidylinositol-3kinase (PI3K)/phoshoAKT-dependent mechanism in vitro in human macrophages and also ex vivo in tissue macrophages (54, 55). Moreover, M(Hb) exhibit an enhanced cholesterol efflux capacity via an increased activity of the nuclear receptor liver X receptor (LXR)a (NR1H3), thus preventing foam © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

cell formation (52). The enhanced LXRa activity also induces the expression of ferroportin, an iron exporter, thus reducing cellular iron content leading to a decreased production of ROS by M(Hb) macrophages (52). This reduced ROS production in M(Hb) was confirmed in vivo in the atherosclerotic plaque (52). After endocytosis of the Hb/Hp complex and erythrocytes, the released heme can prime macrophages toward a Mhem macrophage phenotype. Mhem macrophages display an induction of the activating transcription factor (ATF)-1 leading to the induction of LXRb (NR1H2) and HMOX1 expression by distinct mechanisms in humans. Indeed, ATF-1 and LXRb are induced by the phosphorylation of 50 -AMP-activated protein kinase (AMPK), whereas the increased HMOX1 expression is mediated by a Nrf2-dependent pathway (56, 57). This coordinate induction contributes to the upregulation of LXRa and ABCA1 expression, which in turn enhances the cholesterol efflux capacity to prevent foam cell formation and oxidative stress (58). A population of iron-loaded macrophages abundantly present in areas of neo-vascularization in human atherosclerotic plaques was identified as CD68+ MR+ macrophages (10). In vitro, IL-4 induced M2 human macrophages display a higher iron uptake and storage capacity than un-polarized resting macrophages (RM), with an higher expression of iron uptake-associated genes, such as DMT1, TfR1, and LRP1 (10). In line, these M2 macrophages exhibit also a higher capacity to phagocyte senescent erythrocytes than un-polarized macrophages (10). However, after iron exposure, the iron-loaded M2 macrophages change their phenotype and adapt their response to counteract the iron-induced oxidative damages by increasing the iron release capacity via the induction of ferroportin expression by a LXRa/Nrf2 dependent mechanism (10). Furthermore, iron exposure increases LXRa transcriptional activity and thus expression of its target genes ABCA1, ABCG1 and apoE (10). The macrophage phenotype in response to cytokines, chemokines, and growth factors Macrophage subpopulations can also be differently polarized depending on cytokines, chemokines and growth factors present in the atherosclerotic plaques (59). Among these factors, granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) are key players in macrophage differentiation and polarization (28, 60). While GM-CSF-derived human macrophages have a rounded shape, M-CSF macrophages are

157

Colin et al
Macrophages in atherosclerosis

elongated (61). Moreover, M-CSF-derived macrophage have been referred to as CD68+ CD14+, whereas GM-CSF derived macrophages were identified as CD68+ CD14 characterized by a high expression level of genes involved in reverse cholesterol transport (PPARc, LXRa, and ABCG1) and in macrophage emigration (CCR7) (61). Based on in vitro data in human macrophages, GM-CSF is assimilated with a M1polarized macrophage phenotype, whereas M-CSF macrophages are assimilated to M2-like macrophages (62). M-CSF is expressed in human healthy arteries and atherosclerotic lesions, whereas GM-CSF expression is low in smooth muscle cells and endothelial cells in healthy arteries, but increases with atherosclerosis progression (63, 64), suggesting that the GM-CSF:M-CSF balance can play a role in atherosclerosis progression. The predominance of M-CSF in early lesions induces a M2-like phenotype, while the increase of GM-CSF expression upon atherosclerosis development favors a M1-like phenotype. In contrast to the M1 and M2 polarization induced by GM-CSF and M-CSF or Th1 and Th2 cytokines, which are reversible processes (65), a novel human macrophage phenotype induced by the platelet-derived chemokine CXCL4 was reported to be irreversible (66). CXCL4, also known as platelet factor 4 (PF4), is expressed in various cells involved in atherosclerosis including endothelial cells, monocytes, and macrophages (67, 68). Expression of CXCL4 in human carotid atherosclerotic plaques correlates with lesion severity (69). CXCL4-differentiated macrophages, called M4, display specific phenotypic characteristics, with metalloproteinase 7 (MMP7) and the calcium binding protein S100A8 being specific M4 markers in vitro in CXCL4-induced macrophages and in vivo in human atherosclerotic plaques (70). Moreover, a complete loss of CD163 expression occurs in CXCL4-induced macrophages, resulting in the absence of atheroprotective HMOX1 induction after Hb/Hp exposure (71). Thus, CD163 M4 macrophages display a pro-atherogenic profile, whereas CD163+ Mhem macrophages are likely atheroprotective. The fact that these two macrophage sub-populations within the human atherosclerotic plaque can exert opposite effects suggests that an imbalance between macrophage populations may have consequences for atherosclerosis progression. Among the cytokines identified to polarize macrophages in vitro, IL-4 expression has been detected in human atherosclerotic plaques (72). IL-4 expression is enriched in CD68+ MR+ areas, suggesting that IL-4 may be one of the M2-inducers in human atherosclerotic plaques (72). Initially, CD68+ MR+ macrophages were identified in an area

158

overlying the lipid core, a more stable zone of the lesions (9). These CD68+ MR+ macrophages display a lower cholesterol efflux capacity related to a deactivation of the LXRa signaling pathway but have enhanced phagocytic and reparative capacity (72). Distinct localization of macrophage sub-populations in atherosclerotic plaques During atherosclerosis progression, the number of total macrophages as well as the presence of inflammatory cytokines within the human atherosclerotic plaques, such as from the carotid artery, increase (73), a phenomenon associated with increased plaque vulnerability (74, 75). However, besides the total number of macrophages, histological analysis revealed a specific spatial distribution of distinct macrophage subpopulations in human atherosclerotic plaques (73). In the human plaque shoulder, the site most prone to plaque rupture (76), M1 macrophages represent the principal population, while the repartition of M1/M2 macrophages is similar in fibrous cap regions (73). Thus, potential deleterious effects of M1 macrophages may be counteracted by protective pro-fibrotic and tissue repair effects of M2 macrophages in the fibrous cap, while the limited number of M2 macrophages cannot balance the M1mediated effects in the plaque shoulder. Interestingly, M2 macrophages are the predominant population in the adventitia of the human atherosclerotic plaque (73), suggesting that these M2 macrophages may have migrated from the surrounding perivascular adipose tissue. In line, the distribution of macrophage phenotypes in carotid plaques from patients suffering from an acute ischemic attack (symptomatic) versus patients without symptoms (asymptomatic) (77) reveals that M1 macrophages are exclusively found in symptomatic unstable plaques, whereas M2 macrophages are present in both symptomatic and stable asymptomatic plaques and to a higher extent in the latter (77). Likewise, compared to asymptomatic plaques, carotid plaques from patients with acute myocardial infarction (AMI) due to plaque rupture (78), the amount of CD11-c immuno-positive areas, reflecting the M1 phenotype, was found to be higher (78). These findings support the hypothesis that plaque vulnerability is more related to the macrophage phenotype than to the total number of macrophages within the plaque. The observation that, M1 macrophages are the predominant macrophage population in rupture-prone zones and abundant in unstable plaques, identifies the macrophage phenotype as a marker of plaque stability. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

Modulators of macrophage phenotypes As mentioned above, macrophages can display a continuum of phenotypes illustrated by distinct gene expression profiles with the capacity to switch from one phenotype to another depending on the external stimuli. Transcription factors, post-transcriptional regulators, humoral factors and signaling molecules have been found to play pivotal roles in the control of macrophage polarization. Modulation by transcription factors STAT signaling The balance between activation of the Janus kinase signal transducer and activator of transcription-1 (JAK-STAT) and STAT-3/STAT-6 pathways regulates macrophage polarization. The binding of IFNc to its receptors leads to the phosphorylation of STAT-1 which promotes the transcription of M1-associated genes (79). Moreover, macrophage STAT-1 deficiency in mice reduces atherosclerotic lesions, foam cell formation, and macrophage apoptosis (80, 81). In contrast, IL-4 and IL-13, both M2 phenotype inducers, activate the JAK-STAT-6 pathway (82), which promotes the transcription of M2 phenotype-associated genes, such as Arg1, Fizz1, Ym1, MR (83, 84) contributing to alternative M2 polarization. Likewise, IL-10 upregulates the expression of M2 phenotype genes through the activation of the STAT-3 pathway in mice (85, 86). However, a direct link among STAT-6, STAT-3, and atherosclerosis development remains to be demonstrated. NF-jB signaling The nuclear factor-jB (NF-jB) family members, composed of the p50, p52, p65, RelB, and c-Rel subunits, can form different homodimers and heterodimers which regulate specific target genes and functions. NF-jB is a pivotal regulator of the initiation and resolution of inflammation (87). Indeed, activation of the NF-jB p65 subunit appears a hallmark of M1 activation in human atherosclerotic plaques (88). The activation of NF-jB by TLR ligands produces proinflammatory factors (89). In contrast, activation of p50 NF-jB homodimers appears essential for M2 polarization in vitro in human macrophages and in vivo in mice (90). Furthermore, LDLR/ mice transplanted with p50 subunitdeficient bone marrow display smaller, but more inflammatory lesions linked to reduced foam cell formation (91). However, the role of NF-jB in human macrophages in atherosclerosis has not yet been clarified. Thus, while the © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

literature on NF-jB activity and atherosclerosis is complex, qualitative differences in the activation of the different NF-jB subunits can have opposite effects on the acquisition of M1 and M2 macrophage phenotypes. IRF signaling Interferon regulatory factors (IRFs) are a family of transcription factors expressed in macrophages, playing a role in macrophage polarization either to the M1 or the M2 phenotype, depending on the member of the IRF family. Inflammatory stimuli induce IRF5 expression which in turn upregulates pro-inflammatory genes and downregulates the expression of M2-associated genes; hence, IRF5 acts as a mediator of M1 macrophage polarization in human and murine macrophages (92). By contrast, IRF4 is a modulator of alternative M2 macrophage polarization in response to helminth infection and chitin administration in mice (93). However, the role of IRF members in atherosclerosis remains to be determined. CREB and C/EBP signaling The CCAAT/enhancer-binding protein (C/EBP) transcription factor family, which comprises six members (a-f), and the cAMP-responsive element-binding protein (CREB) have been shown to play roles in macrophage activation (94). C/EBPa and C/EBPb are highly expressed in myeloid progenitors (95). C/EBPb was first identified as a macrophage polarization modulator. Although the expression of Arg1 is induced by IL-4 and IL-13 via the STAT-6 pathway, its expression is regulated in a stimulus-specific manner. In response to TLR signaling, Arg1 induction is mediated by C/EBPb in murine macrophages (96). Deletion of two CREB-binding sites in the cebpb promoter prevents the upregulation of M2-associated genes (Arg1, IL-10, and MR), suggesting that the C/EBPb/CREB cascade is required for M2 macrophage polarization (94). On the other hand, LPS-induced M1 polarization and IL-4 induced M2 polarization are impaired in C/EBPa-deficient macrophages, suggesting that C/EBPa is crucial for both M1 and M2 macrophage phenotype polarization in mice (97). Kr€ uppel-like transcription factor 4 (KLF4), a member of the zinc finger family of transcription factors, induces the expression of M2 markers (Arg1, MR, Fizz1, and PPARc) through STAT-6 signaling and inhibits M1 polarization by sequestering NF-jB cofactors in murine macrophages (98). On the other hand, expression of KLF2 is enhanced by IL-4 stimulation in bone marrow-derived macrophages (BMDM)

159

Colin et al
Macrophages in atherosclerosis

(99). KLF2 inhibits M1 marker gene expression in monocytes and macrophages (100, 101). Finally, KLF6 primes human and mouse macrophages to a M1 phenotype through cooperation with NF-jB and inhibits M2 polarization via a reduced recruitment of PPARc to the Arg1 promoter (102). Modulation by nuclear receptors The role of nuclear receptors in macrophage biology has been extensively studied. Peroxisome Proliferator-Activated Receptor-c (PPARc or NR1C3) is a nuclear receptor highly expressed in macrophages where it controls lipid metabolism and inflammation preventing atherosclerosis development (103). PPARc expression is upregulated in IL-4-stimulated macrophages in a STAT-6 dependent manner (104, 105). PPARc exhibits anti-inflammatory properties by inhibiting NF-jB and AP-1 pathways (106). The first evidence of PPARc acting as a modulator of macrophage polarization was in relation to obesity. In obese mice, PPARc activation decreases the number of M1 macrophages and increases the expression of M2 markers in mouse adipose tissue (107). A macrophage-specific deletion of PPARc impaired alternative activation of mouse macrophages (108). In vitro, PPARc activation promotes the M2 phenotype in human primary macrophages priming macrophages toward an alternative activated M2 phenotype (9). By contrast, the expression of M2-associated genes is not modulated by PPARa (NR1C1) nor PPARb/d (NR1C2) activation in human macrophages (109), whereas PPARb/d activation induces a M2 polarization in adipose tissue and Kupffer cells in mice (110). Other nuclear receptors also participate in the control of macrophage polarization. Among them, Nur77 (NR4A1) is highly expressed in mouse Ly6Clow monocytes and macrophages (111) and suggested to be required for monocyte differentiation into alternative M2 macrophages (112, 113). Macrophage-specific deletion of Nur77 in mice enhances TLR signaling and induces a shift to a pro-inflammatory M1 phenotype (112, 113). However, Chao et al. reported, using Nur77-deficient hematopoietic precursors, that Nur77 is not a crucial modulator neither for M1 nor for M2 macrophage polarization in mice (114). Rev-erba (NR1D1) has been recently reported as a novel modulator of macrophage polarization, expressed in macrophages of human atherosclerotic plaques (115). Rev-erba over-expression in BMDM increased the expression of M2 markers (Arg1, MR, Ym1), suggesting that Rev-erba can promote monocyte differentiation into a M2 macrophage phenotype (115).

160

A role of the Estrogen/Estrogen Receptor (ER) pathway in macrophage polarization has been also outlined. Hematopoietic or myeloid-specific deletion of ERa (ESR1) in female mice has been shown to alter IL-4 responsiveness and ERadeficient macrophages were unable to acquire a M2 phenotype, suggesting that ERa mediates the effects of estrogens to promote alternative M2 polarization (116, 117). Modulation by miRNA Small non-coding RNA called microRNAs (miRNAs) play crucial roles in immune and inflammatory responses (118, 119) and modulate many cellular processes including cell proliferation, apoptosis, and differentiation (120, 121). Upon binding to the 30 -untranslated region (UTR) of target mRNAs, miRNAs negatively regulate gene expression by blocking the translation process or by increasing mRNA degradation (122). Dysregulated expression of miRNAs is associated with the progression of diseases such as cancer, diabetes, and cardiovascular disease (123–125). The demonstration that several miRNAs, including miR-21, miR27b, and miR-130, are expressed in human and murine atherosclerotic plaques (126) and differential expression between M1 and M2 macrophages (127) has provoked a growing interest on the role of miRNAs in the regulation of macrophage polarization. miR-21, miR-147, miR-146a, miR-214, miR-125b, and miR-455 are upregulated in LPS-driven M1 macrophages, whereas miR-143-3p, miR-145-5p, and miR-let7c are more abundant in M2 macrophages (127–130). Although miRNAs play roles in atherosclerosis, only few were studied in macrophage polarization. miR-155 is abundantly expressed in atherosclerotic plaques, especially in inflammatory macrophages (131). Polarization of BMDM into pro-inflammatory M1 macrophages by LPS and IFNc stimulation induces miR-155 expression, whereas miR-155 expression is not modulated upon IL-4 treatment in M2-like macrophages (131). The upregulation of miR-155 in M1 macrophages contributes to the increased production of CCL2 and TNF (both M1 markers) by directly repressing B-cell lymphoma-6 (BCL6) expression (131). Moreover, the expression of miR-155 decreases following M1 to M2 re-polarization, but increases again during the shift toward a M1 phenotype (127). miR-155 deletion in mouse M1 macrophages decreases M1 markers such as TNF, Nos2, IL-12, and increases M2 markers including Arg1, Ym1, Fizz1, thus suggesting a shift toward a M2 phenotype. In contrast, miR-155 overexpression in M2 macrophages © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

leads to a M1 phenotype by inducing M1 markers and repressing canonical M2 markers (127). miR-223 is regulated during macrophage polarization. In BMDMs, miR-223 expression is reduced in LPS-induced M1 macrophages and induced in IL-4 driven M2 macrophages. Moreover, miR-223-deficient mice exhibit a M1-oriented phenotype, and miR-223-deficient macrophages were found to be hypersensitive to LPS stimulation and exhibited a delayed response to IL-4 (132). Altogether these results suggest that miR-223 promotes alternative M2 polarization. Likewise, miR-124, a brain-specific miRNA expressed in microglia (133), drives macrophage polarization toward a M2 phenotype in mice (133). The miR-125 family is composed of miR-125a, miR125b1, and miR125b2, all being expressed in macrophages, in which they are differentially regulated by LPS, with an induction of miR-125a and a reduction in miR-125b expression (134, 135). Using different experimental approaches of over-expression and downregulation in murine macrophages, miR-125a and the miRNA let-7c have been reported to act as potential M2 phenotype inducers (130). Modulation by humoral factors Epidemiological studies have consistently demonstrated an inverse correlation between plasma high density lipoprotein cholesterol (HDL-C) and the risk of coronary artery disease and its thrombotic complications (136, 137). HDL exerts anti-atherosclerotic properties by removing cholesterol from lipid-laden macrophages via ATP-binding cassette transporters, ABCA1 and ABCG1 (138). In addition, HDL exerts direct vasoprotective effects, in particular in endothelial cells, by promoting endothelial repair and enhancing nitric oxide production (139). Several in vitro and in vivo studies have shown potent anti-inflammatory properties of HDL (140, 141). Based on these observations, it has been hypothesized that HDL, as an anti-inflammatory signal, can modulate the macrophage phenotype. In different engineered mouse models of atherosclerosis regression, a normalization of HDL-C levels led to a decrease in CD68+ cell content and to an enrichment of M2 markers in atherosclerotic plaques (142). In primary murine macrophages, HDL increases the expression of M2 markers (Fizz1, Arg1) in a STAT6-dependent manner (143). However, HDL does not modulate the expression of M2 markers in primary human macrophages (144). Moreover, monocytes from subjects with low HDL phenotypes differentiate toward a M2 macro© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

phage phenotype similarly as monocytes from healthy subjects, suggesting that a chronic low exposure to HDL does not alter the alternative polarization potential of monocytes (144). Various other humoral factors with potent antiinflammatory properties, such as apolipoprotein E, adiponectin, IL-33 or angiotensin converting enzyme, enhance the M2 macrophage phenotype (145–148), whereas angiotensin type 1 receptor, activin A, and C-reactive protein (CRP) block the M2 phenotype (149–151). Modulation by irradiation Radiotherapy is known to induce aorta injury in cancer patients (152). Until now this field of research has not attracted much attention, but the continuous rise in patients diagnosed with cancer provoked a growing interest to study the effect of radiotherapy on carotid arteries. It has been reported that thoracic/neck radiotherapy increases cardiovascular events and atherosclerosis development in patients with cancer (153, 154) and induces an inflammatory response in endothelial cells, as well as necrosis and fibrosis in the media and adventitia (155). Irradiation of young (10–16 weeks) hypercholesterolemic apolipoprotein E-deficient (ApoE/) mice accelerates the development of macrophage-rich, inflammatory atherosclerotic lesions (156, 157). Although cancer and atherosclerotic lesions affect people at all ages, both diseases increase with aging. Thus, the effect of local irradiation has been investigated on pre-existing atherosclerotic lesions in aged ApoE/ mice (28 weeks), resulting in smaller lesions with an increased number of apoptotic cells and M1 macrophages and a reduced amount of M2 macrophages (158). In vitro experiments have shown that irradiation reduces the phagocytic capacity of mouse M2 macrophages probably contributing to the increase in apoptotic cells and to the shift of macrophage polarization toward a pro-inflammatory M1 phenotype with increased expression of iNOS, IFN-c, TNF, IL-6, and IL-12b (158). Further studies are needed to fully understand how irradiation affects macrophage polarization in atherosclerotic plaques. Modulation by the mTOR pathway The mammalian target of rapamycin (mTOR), a serine/threonine kinase, forms two complexes: the mTOR complex 1 (mTORC1) which contains the protein raptor and mTORC2 which includes the protein rictor. Rapamycin, an inhibitor of mTOR, is an immunosuppressive agent with potent antiinflammatory and anti-proliferative properties used to

161

Colin et al
Macrophages in atherosclerosis

prevent transplant rejection and vasculopathy (159, 160). Despite increasing plasma LDL levels, rapamycin reduces atherosclerosis in ApoE/ mice (161). The effect of rapamycin on human macrophage polarization was recently studied. Rapamycin induced apoptosis of M2 macrophages without affecting M1 macrophage survival (162). Moreover, rapamycin reduced the expression of M2 markers (CXCR4, MR) and the release of CCL18 and CCL13 in IL-4-induced M2 human macrophages (162). By contrast, in LPS-stimulated human macrophages, rapamycin increases the expression of M1 markers (CCR7, CD86) and the secretion of IL6, TNF, and IL1b, whereas the secretion of M2 factors (IL10, VEGF and CCL18) were reduced, suggesting a shift toward a M1-like phenotype in vitro in rapamycin-treated human macrophages and ex vivo in PBMC from rapamycin-treated patients with type 1 diabetes (162). Around the same time, the role of mTORC1 in macrophage was investigated using a mouse model of constitutive mTORC1 activation. BMDM from these mice display reduced levels of M2 markers (such as Arg1 and Fizz1) and an enhanced pro-inflammatory responsiveness to LPS stimulation, partly due to an attenuation of Akt signaling (163). Moreover, macrophage-specific deletion of mTORC1 protein raptor decreased mTORC1 activity and resulted in a reduction in atherosclerosis and a reduced secretion of the inflammatory chemokine CCL2 in response to mmLDL (164). Altogether these results highlight a role for the mTOR pathway in macrophage polarization to a M1-like phenotype. However, the M1 phenotype appears induced both by the activation of mTORC1 and by the inhibition with

rapamycin. Thus, further investigations are needed to understand the exact roles of mTOR in macrophage biology. Conclusion During atherosclerosis development and progression, macrophages are exposed to many environmental signals, such as lipids and their derivatives, pro- and anti-inflammatory cytokines and heme from senescent erythrocytes, which modulate their functional phenotypes in atherosclerotic lesions. The nature and intensity of these signals vary continuously during plaque progression; thus, the phenotypic polarization of macrophages is a dynamic process which changes over time. Based on their localization and distribution within the plaque, macrophages display distinct functional phenotypes, the M1 macrophages being localized near the lipid core, whereas the M2 macrophages are more enriched in neo-angiogenic areas. Hence, direct targeting of specific macrophage subpopulations at different sites and stages of atherosclerotic plaque formation could be an interesting approach. Nevertheless, the modulation of macrophage phenotypes as therapeutic approach is still difficult to envisage and several questions remain to be addressed. Are the macrophage sub-populations derived from resident macrophages upon environmental stimuli or are they derived from specific monocyte sub-populations recruited in specific sites in atherosclerotic plaque? Further research in this field is required to answer this and other capital and unresolved questions.

References 1. Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011;145:341–355. 2. Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med 2001;11:93–102. 3. Simionescu M. Implications of early structural-functional changes in the endothelium for vascular disease. Arterioscler Thromb Vasc Biol 2007;27:266–274. 4. Bellingan GJ, Caldwell H, Howie SE, Dransfield I, Haslett C. In vivo fate of the inflammatory macrophage during the resolution of inflammation: inflammatory macrophages do not die locally, but emigrate to the draining lymph nodes. J Immunol 1996;157:2577– 2585. 5. van Gils JM, et al. The neuroimmune guidance cue netrin-1 promotes atherosclerosis by

162

6.

7.

8.

9.

inhibiting the emigration of macrophages from plaques. Nat Immunol 2012;13:136–143. Wanschel A, et al. Neuroimmune guidance cue semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention. Arterioscler Thromb Vasc Biol 2013;33:886– 893. Uzui H, et al. Increased expression of membrane type 3-matrix metalloproteinase in human atherosclerotic plaque: role of activated macrophages and inflammatory cytokines. Circulation 2002;106:3024–3030. Bobryshev YV. Intracellular localization of oxidized low-density lipoproteins in atherosclerotic plaque cells revealed by electron microscopy combined with laser capture microdissection. J Histochem Cytochem 2005;53:793–797. Bouhlel MA, et al. PPARg activation primes human monocytes into alternative M2

10.

11.

12. 13.

14.

macrophages with anti-inflammatory properties. Cell Metab 2007;6:137–143. Bories G, et al. Liver X receptor (LXR) activation stimulates iron export in human alternative macrophages. Circ Res 2013;113:1196–1205. Li X, et al. A pattern of disperse plaque microcalcifications identifies a subset of plaques with high inflammatory burden in patients with acute myocardial infarction. Atherosclerosis 2011;218:83–89. Mackaness GB. Cellular resistance to infection. J Exp Med 1962;116:381–406. Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 1983;158:670–689. Pace JL, Russell SW, Schreiber RD, Altman A, Katz DH. Macrophage activation: priming activity from a T-cell hybridoma is attributable to

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

15.

16.

17.

18.

19. 20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

interferon-gamma. Proc Natl Acad Sci USA 1983;80:3782–3786. Celada A, Gray PW, Rinderknecht E, Schreiber RD. Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J Exp Med 1984;16:55–74. Stein M, Keshav S, Harris N, Gordon S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med 1992;176:287–292. Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 1993;259:1739–1742. Mantovani A, Garlanda C, Locati M. Macrophage diversity and polarization in atherosclerosis: a question of balance. Arterioscler Thromb Vasc Biol 2009;29:1419–1423. Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73:209–212. Verreck FA, et al. Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco) bacteria. Proc Natl Acad Sci USA 2004;101:4560–4565. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003;3:23–35. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677–686. Jetten N, Verbruggen S, Gijbels MJ, Post MJ, De Winther MP, Donners MM. Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis 2014;17:109–118. Lee CG, et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001;194:809–821. Sierra-Filardi E, Vega MA, Sanchez-Mateos P, Corbi AL, Puig-Kroger A. Heme oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release. Immunobiology 2010;215:788–795. Spencer M, et al. Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation. Am J Physiol Endocrinol Metab 2010;299:E1016–E1027. Mahdavian Delavary B, van der Veer WM, van Egmond M, Niessen FB, Beelen RH. Macrophages in skin injury and repair. Immunobiology 2011;216:753–762. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008;8:958–969. Zizzo G, Hilliard BA, Monestier M, Cohen PL. Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol 2012;189:3508–3520. Zizzo G, Cohen PL. IL-17 stimulates differentiation of human anti-inflammatory macrophages and phagocytosis of apoptotic

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

neutrophils in response to IL-10 and glucocorticoids. J Immunol 2013;190:5237– 5246. Grinberg S, Hasko G, Wu D, Leibovich SJ. Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype. Am J Pathol 2009;175:2439–2453. Ferrante CJ, et al. The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Ralpha) signaling. Inflammation 2013;36:921–931. Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol 2006;177:7303–7311. Martinez FO, et al. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood 2013;121:e57–e69. Kadl A, et al. Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2. Circ Res 2010;107:737–746. Duewell P, et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 2010;464:1357– 1361. van Tits LJ, Stienstra R, van Lent PL, Netea MG, Joosten LA, Stalenhoef AF. Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: a crucial role for Kruppel-like factor 2. Atherosclerosis 2011;214:345–349. Hirose K, et al. Different responses to oxidized low-density lipoproteins in human polarized macrophages. Lipids Health Dis 2011;10:1. Bae YS, et al. Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2. Circ Res 2009;104:210–218. Chavez-Sanchez L, Garza-Reyes MG, Espinosa-Luna JE, Chavez-Rueda K, Legorreta-Haquet MV, Blanco-Favela F. The role of TLR2, TLR4 and CD36 in macrophage activation and foam cell formation in response to oxLDL in humans. Hum Immunol 2014;75:322– 329. Pasini AF, et al. Enhanced levels of oxidized low-density lipoprotein prime monocytes to cytokine overproduction via upregulation of CD14 and toll-like receptor 4 in unstable angina. Arterioscler Thromb Vasc Biol 2007;27:1991– 1997. Harkewicz R, Hartvigsen K, Almazan F, Dennis EA, Witztum JL, Miller YI. Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein. J Biol Chem 2008;283:10241– 10251. Huber J, et al. Oxidized cholesteryl linoleates stimulate endothelial cells to bind monocytes via the extracellular signal-regulated kinase 1/2

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

pathway. Arterioscler Thromb Vasc Biol 2002;22:581–586. Fang L, et al. Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high cholesterol diet: macrophage binding and activation. J Biol Chem 2010;285:32343–32351. Huang Z, et al. 7-ketocholesteryl-9-carboxynonanoate induced nuclear factor-kappa B activation in J774A.1 macrophages. Life Sci 2010;87:651–657. Sottero B, et al. Expression and synthesis of TGFbeta1 is induced in macrophages by 9-oxononanoyl cholesterol, a major cholesteryl ester oxidation product. BioFactors 2005;24:209–216. 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 2011;187:5408–5418. Hughes JE, Srinivasan S, Lynch KR, Proia RL, Ferdek P, Hedrick CC. Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages. Circ Res 2008;102:950–958. McCarthy C, et al. IL-10 mediates the immunoregulatory response in conjugated linoleic acid-induced regression of atherosclerosis. FASEB J 2013;27:499–510. Subbanagounder G, et al. Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position. Arterioscler Thromb Vasc Biol 2000;20:2248– 2254. Kadl A, et al. Oxidized phospholipid-induced inflammation is mediated by Toll-like receptor 2. Free Radic Biol Med 2011;51:1903–1909. Finn AV, et al. Hemoglobin directs macrophage differentiation and prevents foam cell formation in human atherosclerotic plaques. J Am Coll Cardiol 2012;59:166–177. Nielsen MJ, Moller HJ, Moestrup SK. Hemoglobin and heme scavenger receptors. Antioxid Redox Signal 2010;12:261–273. 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 2004;94:119–126. Landis RC, Philippidis P, Domin J, Boyle JJ, Haskard DO. Haptoglobin genotype-dependent anti-inflammatory signaling in CD163(+) macrophages. Int J Inflam 2013;2013:980327. Wan X, et al. 50 -AMP-activated protein kinase-activating transcription factor 1 cascade modulates human monocyte-derived macrophages to atheroprotective functions in response to heme or metformin. Arterioscler Thromb Vasc Biol 2013;33:2470–2480. Boyle JJ, et al. Heme induces heme oxygenase 1 via Nrf2: role in the homeostatic macrophage response to intraplaque hemorrhage. Arterioscler Thromb Vasc Biol 2011;31:2685–2691. Boyle JJ, et al. Activating transcription factor 1 directs Mhem atheroprotective macrophages

163

Colin et al
Macrophages in atherosclerosis

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

through coordinated iron handling and foam cell protection. Circ Res 2012;110:20–33. Wolfs IM, Donners MM, de Winther MP. Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation. Thromb Haemost 2011;106:763–771. Fleetwood AJ, Cook AD, Hamilton JA. Functions of granulocyte-macrophage colony-stimulating factor. Crit Rev Immunol 2005;25:405–428. Waldo SW, et al. Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am J Pathol 2008;172:1112–1126. Xu W, Schlagwein N, Roos A, van den Berg TK, Daha MR, van Kooten C. Human peritoneal macrophages show functional characteristics of M-CSF-driven anti-inflammatory type 2 macrophages. Eur J Immunol 2007;37:1594– 1599. Brocheriou I, et al. Antagonistic regulation of macrophage phenotype by M-CSF and GM-CSF: implication in atherosclerosis. Atherosclerosis 2011;214:316–324. Plenz G, et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates the expression of type VIII collagen mRNA in vascular smooth muscle cells and both are codistributed during atherogenesis. Arterioscler Thromb Vasc Biol 1999;19:1658–1668. Porcheray F, et al. Macrophage activation switching: an asset for the resolution of inflammation. Clin Exp Immunol 2005;142:481– 489. Gleissner CA, Shaked I, Little KM, Ley K. CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages. J Immunol 2010;184:4810–4818. Gleissner CA, Ley K. CXCL4 in atherosclerosis: possible roles in monocyte arrest and macrophage foam cell formation. Thromb Haemost 2007;98:917–918. Gleissner CA, von Hundelshausen P, Ley K. Platelet chemokines in vascular disease. Arterioscler Thromb Vasc Biol 2008;28:1920– 1927. Pitsilos S, et al. Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters. Thromb Haemost 2003;90:1112–1120. 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 2014. In Press. Gleissner CA, Shaked I, Erbel C, Bockler D, Katus HA, Ley K. CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages. Circ Res 2010;106:203– 211. Chinetti-Gbaguidi G, et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARgamma and LXRalpha pathways. Circ Res 2011;108:985– 995. Stoger JL, et al. Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis 2012;225:461–468.

164

74. Koutouzis M, et al. Serum interleukin-6 is elevated in symptomatic carotid bifurcation disease. Acta Neurol Scand 2009;119:119–125. 75. Cheuk BL, Cheng SW. Annexin A1 expression in atherosclerotic carotid plaques and its relationship with plaque characteristics. Eur J Vasc Endovasc Surg 2011;41:364–371. 76. Barlis P, Serruys PW, Devries A, Regar E. Optical coherence tomography assessment of vulnerable plaque rupture: predilection for the plaque ‘shoulder’. Eur Heart J 2008;29:2023. 77. Cho KY, et al. The phenotype of infiltrating macrophages influences arteriosclerotic plaque vulnerability in the carotid artery. J Stroke Cerebrovasc Dis 2013;22:910–918. 78. Lee CW, et al. Macrophage heterogeneity of culprit coronary plaques in patients with acute myocardial infarction or stable angina. Am J Clin Pathol 2013;139:317–322. 79. Darnell JE, Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415–1421. 80. Agrawal S, Febbraio M, Podrez E, Cathcart MK, Stark GR, Chisolm GM. Signal transducer and activator of transcription 1 is required for optimal foam cell formation and atherosclerotic lesion development. Circulation 2007;115:2939– 2947. 81. Lim WS, et al. Signal transducer and activator of transcription-1 is critical for apoptosis in macrophages subjected to endoplasmic reticulum stress in vitro and in advanced atherosclerotic lesions in vivo. Circulation 2008;117:940–951. 82. Pauleau AL, Rutschman R, Lang R, Pernis A, Watowich SS, Murray PJ. Enhancer-mediated control of macrophage-specific arginase I expression. J Immunol 2004;172:7565–7573. 83. Sica A, Bronte V. Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 2007;117:1155– 1166. 84. Goenka S, Kaplan MH. Transcriptional regulation by STAT6. Immunol Res 2011;50:87–96. 85. Lang R, Patel D, Morris JJ, Rutschman RL, Murray PJ. Shaping gene expression in activated and resting primary macrophages by IL-10. J Immunol 2002;169:2253–2263. 86. Gao S, et al. Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-kappaB and signal transducer and activator of transcription 3 pathways. Exp Biol Med (Maywood) 2014;239:366–375. 87. Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible new role for NF-kappaB in the resolution of inflammation. Nat Med 2001;7:1291–1297. 88. Brand K, et al. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest 1996;97:1715–1722. 89. Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004;25:280–288.

90. Porta C, et al. Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB. Proc Natl Acad Sci USA 2009;106:14978–14983. 91. Kanters E, et al. Hematopoietic NF-kappaB1 deficiency results in small atherosclerotic lesions with an inflammatory phenotype. Blood 2004;103:934–940. 92. Krausgruber T, et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat Immunol 2011;12:231–238. 93. Satoh T, et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat Immunol 2010;11:936–944. 94. Ruffell D, et al. A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair. Proc Natl Acad Sci USA 2009;106:17475–17480. 95. Friedman AD. Transcriptional control of granulocyte and monocyte development. Oncogene 2007;26:6816–6828. 96. El Kasmi KC, et al. Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens. Nat Immunol 2008;9:1399–1406. 97. Lee B, et al. C/EBPalpha regulates macrophage activation and systemic metabolism. Am J Physiol Endocrinol Metab 2014;306:E1144–E1154. 98. Liao X, et al. Kruppel-like factor 4 regulates macrophage polarization. J Clin Invest 2011;121:2736–2749. 99. Lingrel JB, et al. Myeloid-specific Kruppel-like factor 2 inactivation increases macrophage and neutrophil adhesion and promotes atherosclerosis. Circ Res 2012;110:1294–1302. 100. Das H, et al. Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes. Proc Natl Acad Sci USA 2006;103:6653–6658. 101. Mahabeleshwar GH, et al. The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock. Immunity 2011;34:715–728. 102. Date D, Das R, Narla G, Simon DI, Jain MK, Mahabeleshwar GH. Kruppel-like Transcription Factor 6 Regulates Inflammatory Macrophage Polarization. J Biol Chem 2014;289:10318– 10329. 103. Babaev VR, et al. Conditional knockout of macrophage PPARgamma increases atherosclerosis in C57BL/6 and low-density lipoprotein receptor-deficient mice. Arterioscler Thromb Vasc Biol 2005;25:1647–1653. 104. Huang JT, et al. Interleukin-4-dependent production of PPAR-g ligands in macrophages by 12/15 lipoxygenase. Nature 1999;400:378–382. 105. Szanto A, et al. STAT6 transcription factor is a facilitator of the nuclear receptor PPARgamma-regulated gene expression in macrophages and dendritic cells. Immunity 2010;33:699–712. 106. Ricote M, Li AC, Willsson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-g is a negative regulator of macrophage activation. Nature 1998;391:79–82.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

Colin et al
Macrophages in atherosclerosis

107. Stienstra R, Duval C, Keshtkar S, van der Laak J, Kersten S, Muller M. Peroxisome proliferator-activated receptor gamma activation promotes infiltration of alternatively activated macrophages into adipose tissue. J Biol Chem 2008;283:22620–22627. 108. Odegaard JI, et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature 2007;447:1116–1120. 109. Bouhlel MA, et al. Unlike PPARgamma, PPARalpha or PPARbeta/delta activation does not promote human monocyte differentiation toward alternative macrophages. Biochem Biophys Res Commun 2009;386:459–462. 110. Kang K, et al. Adipocyte-derived Th2 cytokines and myeloid PPARdelta regulate macrophage polarization and insulin sensitivity. Cell Metab 2008;7:485–495. 111. Hanna RN, et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat Immunol 2011;12:778–785. 112. Hanna RN, et al. NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis. Circ Res 2012;110:416–427. 113. Hamers AA, et al. Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis. Circ Res 2012;110:428– 438. 114. Chao LC, et al. Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in mice. J Lipid Res 2013;54:806–815. 115. Ma H, et al. Increased atherosclerotic lesions in LDL receptor deficient mice with hematopoietic nuclear receptor Rev-erbalpha knock- down. J Am Heart Assoc 2013;2:e000235. 116. Ribas V, et al. Myeloid-specific estrogen receptor alpha deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development. Proc Natl Acad Sci USA 2011;108:16457–16462. 117. Campbell L, et al. Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair. J Invest Dermatol 2014;134:2447–2457. 118. O’Connell RM, Rao DS, Baltimore D. microRNA regulation of inflammatory responses. Annu Rev Immunol 2012;30:295–312. 119. Xiao C, Rajewsky K. MicroRNA control in the immune system: basic principles. Cell 2009;136:26–36. 120. Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004;303:83–86. 121. O’Connell RM, Zhao JL, Rao DS. MicroRNA function in myeloid biology. Blood 2011;118:2960–2969. 122. Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 2008;9:219–230. 123. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009;10:704–714.

124. Pandey AK, Agarwal P, Kaur K, Datta M. MicroRNAs in diabetes: tiny players in big disease. Cell Physiol Biochem 2009;23:221–232. 125. Latronico MV, Condorelli G. MicroRNAs and cardiac pathology. Nat Rev Cardiol 2009;6:419– 429. 126. Raitoharju E, Oksala N, Lehtimaki T. MicroRNAs in the atherosclerotic plaque. Clin Chem 2013;59:1708–1721. 127. Cai X, et al. Re-polarization of tumor-associated macrophages to pro-inflammatory M1 macrophages by microRNA-155. J Mol Cell Biol 2012;4:341–343. 128. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA 2006;103:12481–12486. 129. Zhang Y, Zhang M, Zhong M, Suo Q, Lv K. Expression profiles of miRNAs in polarized macrophages. Int J Mol Med 2013;31:797–802. 130. Banerjee S, et al. miR-125a-5p regulates differential activation of macrophages and inflammation. J Biol Chem 2013;288:35428– 35436. 131. Nazari-Jahantigh M, et al. MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages. J Clin Invest 2012;122:4190– 4202. 132. Zhuang G, et al. A novel regulator of macrophage activation: miR-223 in obesity-associated adipose tissue inflammation. Circulation 2012;125:2892–2903. 133. Ponomarev ED, Veremeyko T, Barteneva N, Krichevsky AM, Weiner HL. MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/ EBP-alpha-PU.1 pathway. Nat Med 2011;17:64– 70. 134. Graff JW, Dickson AM, Clay G, McCaffrey AP, Wilson ME. Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem 2012;287:21816–21825. 135. Tili E, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/ TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007;179:5082–5089. 136. Gordon DJ, Rifkind BM. High-density lipoprotein–the clinical implications of recent studies. N Engl J Med 1989;321:1311–1316. 137. Di Angelantonio E, et al. Major lipids, apolipoproteins, and risk of vascular disease. JAMA 2009;302:1993–2000. 138. Rader DJ. Molecular regulation of HDL metabolism and function: implications for novel therapies. J Clin Invest 2006;116:3090–3100. 139. Besler C, et al. Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease. J Clin Invest 2011;121:2693–2708. 140. Park SH, Park JH, Kang JS, Kang YH. Involvement of transcription factors in plasma HDL protection against TNF-alpha-induced vascular cell adhesion molecule-1 expression. Int J Biochem Cell Biol 2003;35:168–182.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014

141. Suzuki M, et al. High-density lipoprotein suppresses the type I interferon response, a family of potent antiviral immunoregulators, in macrophages challenged with lipopolysaccharide. Circulation 2010;122:1919–1927. 142. Feig JE, et al. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells. Proc Natl Acad Sci USA 2011;108:7166–7171. 143. Sanson M, Distel E, Fisher EA. HDL induces the expression of the M2 macrophage markers arginase 1 and Fizz-1 in a STAT6-dependent process. PLoS ONE 2013;8:e74676. 144. Colin S, et al. HDL does not influence the polarization of human monocytes toward an alternative phenotype. Int J Cardiol 2014;172:179–184. 145. Baitsch D, et al. Apolipoprotein E induces antiinflammatory phenotype in macrophages. Arterioscler Thromb Vasc Biol 2011;31:1160– 1168. 146. Lovren F, et al. Adiponectin primes human monocytes into alternative anti-inflammatory M2 macrophages. Am J Physiol Heart Circ Physiol 2010;299:H656–H663. 147. Miller AM, et al. Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice. Circ Res 2010;107:650–658. 148. Kohlstedt K, Trouvain C, Namgaladze D, Fleming I. Adipocyte-derived lipids increase angiotensin-converting enzyme (ACE) expression and modulate macrophage phenotype. Basic Res Cardiol 2011;106:205–215. 149. Ma LJ, et al. Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obesity. Am J Physiol Renal Physiol 2011;300:F1203–F1213. 150. Sierra-Filardi E, et al. Activin A skews macrophage polarization by promoting a pro-inflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers. Blood 2011;117:5092–5101. 151. Devaraj S, Jialal I. C-reactive protein polarizes human macrophages to an M1 phenotype and inhibits transformation to the M2 phenotype. Arterioscler Thromb Vasc Biol 2011;31:1397– 1402. 152. Thomas E, Forbus WD. Irradiation injury to the aorta and the lung. AMA Arch Pathol 1959;67:256–263. 153. Dorresteijn LD, et al. Increased risk of ischemic stroke after radiotherapy on the neck in patients younger than 60 years. J Clin Oncol 2002;20:282–288. 154. Russell NS, et al. Novel insights into pathological changes in muscular arteries of radiotherapy patients. Radiother Oncol 2009;92:477–483. 155. Plummer C, Henderson RD, O’Sullivan JD, Read SJ. Ischemic stroke and transient ischemic attack after head and neck radiotherapy: a review. Stroke 2011;42:2410–2418. 156. Hoving S, et al. Single-dose and fractionated irradiation promote initiation and progression of atherosclerosis and induce an inflammatory plaque phenotype in ApoE(-/-) mice. Int J Radiat Oncol Biol Phys 2008;71:848–857.

165

Colin et al
Macrophages in atherosclerosis

157. Stewart FA, et al. Ionizing radiation accelerates the development of atherosclerotic lesions in ApoE-/- mice and predisposes to an inflammatory plaque phenotype prone to hemorrhage. Am J Pathol 2006;168:649–658. 158. Gabriels K, et al. Irradiation of existing atherosclerotic lesions increased inflammation by favoring pro-inflammatory macrophages. Radiother Oncol 2014;11:455–460. 159. Snell GI, et al. Rescue therapy: a role for sirolimus in lung and heart transplant

166

recipients. Transplant Proc 2001;33:1084– 1085. 160. Mancini D, et al. Use of rapamycin slows progression of cardiac transplantation vasculopathy. Circulation 2003;108:48–53. 161. Castro C, Campistol JM, Sancho D, Sanchez-Madrid F, Casals E, Andres V. Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27 Kip1 -independent pathway. Atherosclerosis 2004;172:31–38.

162. Mercalli A, et al. Rapamycin unbalances the polarization of human macrophages to M1. Immunology 2013;140:179–190. 163. Byles V, et al. The TSC-mTOR pathway regulates macrophage polarization. Nat Commun 2013;4:2834. 164. Ai D, et al. Disruption of Mammalian target of rapamycin complex 1 in macrophages decreases chemokine gene expression and atherosclerosis. Circ Res 2014;114:1576–1584.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 262/2014