Review ArticleInnate Immune System Cells in Atherosclerosis
Introduction
The innate immune system functions as the first line of host defense against pathogens. This system is composed of diverse cellular components including granulocytes (basophils, eosinophils and neutrophils), mast cells (MCs), monocytes/macrophages, dendritic cells (DCs) and natural killer cells (NK cells) (1). These cells respond to noxious stimuli and conditions including infections and tissue injuries that can trigger inflammatory responses (2). In several pathologies, the inflammatory response coordinates the various mechanisms characteristic of specific diseases. In this sense, atherosclerosis is regarded as a chronic inflammatory disease as it initiates with endothelial dysfunction that permits the expression of adhesion molecules such as platelet endothelial cell adhesion molecule (PECAM)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM) (3) as well as chemokines such as monocyte chemoattractant protein-1 (MCP-1), which produce interactions among circulating monocytes and endothelial cells. Furthermore, monocytes differentiate into macrophages in the intima in response to colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), an event that, along with the accumulation of lipid deposits such as oxidized low-density lipoprotein (oxLDL) as well as debris, favors the development of a necrotic core. Disease progression induces smooth muscle cells to form a fibrous layer that stabilizes the lesion. However, in advanced disease stages, a rupture of this fibrous cap may expose the necrotic core contents; therefore, platelets and fibrin form a blood clot (thrombus) that results in a partial or total ischemic arterial obstruction 4, 5.
During atherosclerotic plaque development and progression, various types of innate immune cells are essential. MCs express a variety of pattern recognition receptors and Fc receptors, which are susceptible to activation by microorganisms and allergens, respectively. Classical MC activation is caused by the crosslinking of Fc receptors and IgE. However, MCs can also be activated by other mechanisms, leading to the release of various inflammatory mediators that can affect lesion development (6). Neutrophils are another cell type involved in atherosclerosis. These granulocytes are the first to respond to different antigens, and neutrophils initiate the inflammatory response by secreting a wide array of inflammatory mediators such as leukotrienes (7) as well as neutrophil extracellular traps (NETs) that activate the endothelium.
NK cells have been amply demonstrated to play an essential role in immune responses against viruses and tumors (8). However, the role of NK cells in atherosclerosis is not clear. Nonetheless, there is evidence to suggest NK cell involvement in lesions due to the secretion of inflammatory cytokines such as IFN-γ (9).
Monocytes are another type of leukocyte recruited during atherosclerosis; these myeloid lineage cells are of great interest in atherosclerotic disease due to their phenotypic and functional heterogeneity. In mice, two monocyte subsets, Lychigh and Lyclow, have been described. Lychigh monocytes have been found to participate in inflammatory responses, whereas Lyclow monocytes have been associated with inflammation resolution (10). In contrast, three monocyte subsets have been identified in humans according to CD14 and CD16 expression, and evidence suggests that the three monocyte subsets function differently in immune responses (11).
Other key myeloid cells in atherosclerosis are macrophages; these cells secrete pro-inflammatory cytokines and take up oxLDL through CD36-induced foam cell formation, which is considered central to atherosclerosis (12). Macrophages can be classified as classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages produce high levels of IL-12 and IL-23 and low levels of IL-10, are efficient producers of free radicals (ROI) and nitrogen intermediaries and participate in Th1 polarization. In contrast, M2 macrophages produce low levels of IL-12 and IL-23 and high levels of IL-10 and are believed to participate in Th2 polarization (13). Additionally, professional antigen-presenting cells such as DCs are found in atherosclerotic lesions and these cells play a critical role in the differentiation and activation of CD4+ and CD8+ T cells and NK cells (14). In vitro studies have established that immature DCs can mature when exposed to oxLDL and secreted inflammatory cytokines, suggesting a possible involvement of these cells in atherosclerosis 15, 16. In this review, we address the involvement of innate immune cells (MCs, neutrophils, NK cells, monocytes, macrophages and DCs) in atherosclerosis pathogenesis.
MCs are innate immune cells that play a central role in allergy and asthma and are found in all vascularized tissues where they reside in close proximity to blood vessels, nerves, smooth muscle cells, mucus-producing glands and hair follicles 17, 6. Interestingly, MCs are also found in the intima of healthy carotid arteries as well as in early and advanced atherosclerotic lesions and are distributed in the shoulder region, in human. The presence of MCs in an atherosclerotic lesion suggests the possibility of a role for these cells in this disease (18) and the participation of MCs was established in an in vivo study that showed that a specific MC deficiency significantly reduced atherogenesis in mice (19).
MC activation by allergens or microorganisms induces the release of preformed inflammatory mediators, which are localized in specialized granules and the de novo synthesis and secretion of cytokines, chemokines and eicosanoids (17). Classical MC activation occurs through crosslinking of the high-affinity FcεRI receptor, which binds to IgE (20). However, MCs are also susceptible to activation by other mechanisms such as IgG or immune complexes (21). In this context, it was recently discovered that FcεR1α deficiency resulted in a marked reduction in lipid deposition in the aortic arch intima in ApoE−/− mice. This reduction might be due to a lack of FcεR1α-mediated MC activation, leading to a drastic reduction in the release of pro-inflammatory mediators that could reduce the amounts of inflammatory cells such as macrophages and T cells in atherosclerotic lesions in ApoE−/−/FcεR1α−/− mice (22). Notably, immune complexes formed by oxLDL-IgG have been found in plaques in animal models, suggesting a role of oxLDL-IgG complexes in MC activation (23). In vitro data have reinforced this idea, as oxLDL-IgG immune complexes have been shown to induce the release of pro-inflammatory cytokines such as IL-8 and TNF-α from human MCs, along with other powerful mediators such as histamine and tryptase (24). Another MCs activation pathway is triggered by the Toll-like receptors (TLR). In this respect, the administration of TLR4 antagonists in mice ApoE−/− was shown to have no effect on the recruitment of MCs to plaques but instead to reduce the activation of lesion-derived MCs, suggesting that the MCs are susceptible to activation via TLR4. Moreover, MCs-specific activation induced apoptosis in vascular smooth muscle cells present within atherosclerotic plaques, a process that could be counteracted by a TLR4 antagonist, suggesting that TLR4-mediated MCs activation is involved in the induction of vascular smooth muscle cell apoptosis (25). Additionally, MCs in lesions could be activated through the receptor for the complement system components C3a and C5a 26, 27. A study showed that ApoE−/− mice overexpressed the complement 3a receptor and complement 5a receptor (C5aR) in atherosclerotic plaques in response to exogenous C5a administration, suggesting that C5a acts as a potent chemoattractant for MCs as well as a ligand for C5aR, which triggers MCs activation. (26). Collectively, these studies suggest that MCs are susceptible to activation in response to soluble mediators such as C5a and receptors such as IgE, IgG and TLR, and this activation is likely to induce the release of inflammatory mediators, leading to the recruitment of leukocytes toward the lesion and promoting disease development (Figure 1).
MCs can initiate atherosclerosis through various mediators such as histamine, which augments vascular permeability and alters vascular tone (27) and can also produce cytokines; for example, specific IL-6 or IFN-γ deficiency in the MCs of mice prone atherosclerotic development leads to reductions in lesion degrees and intimal size. Furthermore, the reconstitution of these mice with MCs from wild-type mice results in an increase in lesion degree, implying that IL-6- and IFN-γ-derived MCs promote atherosclerosis pathogenesis (19). Additionally, the in vitro release of these pro-inflammatory mediators by MCs of mouse stimulates the expression of VCAM-1, ICAM-1, P-selectin and E-selectin in endothelial cells which, in turn, facilitates the adhesion of leukocytes to the endothelium (Figure 1). This finding suggests that MCs are an important source of inflammatory mediators that enhance leukocyte adhesion and that this MCs function is partly associated with mast cell cytokines (28). MCs also secrete tryptase and chymase; in vivo experiments in ApoE−/− mice have shown that tryptase overexpression significantly increased the areas of carotid plaques and the degree of carotid artery stenosis, whereas the carotid plaque area remains small when tryptase expression is disrupted (29). A possible role for tryptase might include promoting leukocyte recruitment. In this regard, tryptase increases the in vitro production of MCP-1 and IL-8, which are potent chemoattractants for monocytes and neutrophils in endothelial cells in ApoE−/− mice (30). Moreover, in human, tryptase promotes foam cell formation by suppressing activation of the nuclear receptor liver x receptor (LXR)-α (31) and inhibiting reverse cholesterol transport (32). Additionally, tryptase degrades pericellular fibronectin, vitronectin and collagen type IV, which promotes angiogenesis and tissue remodeling (33) and can contribute to atherosclerotic plaque hemorrhage by regulating the balance between plasminogen activator inhibitor-1 and tissue plasminogen activator levels in mice (30). In MCs, chymase is another serine peptidase associated with atherosclerosis. Inhibition of chymase in ApoE−/− mice reduced plaque progression and necrotic core size, which affected plaque stability by increasing collagen levels (34). On the other hand, mouse chymase destabilizes plaques by inducing smooth muscle cell (SMC) apoptosis and degrading fibronectin and vitronectin (35). Interestingly, in animal models and humans, MC-produced tryptase stimulates microvessel tube formation and enhances the growth of microvessel endothelial cells, whereas chymase promotes angiogenesis through the effects of angiotensin-II 36, 37. These angiogenic effects can contribute to lesion growth and induce plaque destabilization, leading to rupture (Figure 1).
Polymorphonuclear (PMN) cells or neutrophils play an important role in inflammation due to their ability to perform a variety of effector functions that collectively represent one of the major mechanisms of innate immunity. Neutrophils are among the first cells to respond to invading microorganisms or tissue damage by phagocytosis and the production of reactive oxygen species, myeloperoxidase and various proteolytic enzymes, which together function to eliminate microbial pathogens but can also contribute to tissue destruction (38). Many neutrophil functions originate from signals mediated through receptors such as TLRs, NOD-like receptors, C-type lectin receptors and RIG-I-like receptors 39, 40.
Neutrophils are produced in and released from the bone marrow, survive in the circulation, and home to sites where they clear senescent neutrophils (41). Neutrophil production is enhanced under hypercholesterolemia. There is a direct correlation between peripheral neutrophil counts and atherosclerotic lesion sizes in mice, suggesting that factors that regulate neutrophil homeostasis also influence atherosclerosis development by altering the peripheral neutrophil counts, especially under hyperlipidemic conditions. In turn, neutropenic mice display reduced atherosclerotic lesion formation. Evidence suggests that hyperlipidemia-triggered neutrophilic conditions promote early atherosclerosis (42). In humans, epidemiological studies have shown that elevated numbers of circulating neutrophils are predictive of cardiovascular events, independent of serum cholesterol levels (43).
During inflammatory processes, neutrophils increase in the circulation and are recruited by IL-8 to the lesion foci, where they exert their effector functions (38). Interestingly, mice fed a high-fat diet had significantly increased numbers of circulating neutrophils (44), even though the atherosclerotic plaques still expressed IL-8, suggesting that neutrophils might be recruited to the lesions. Several studies have established that neutrophils are recruited to lesions where they are distributed mainly in the shoulder regions in African green monkeys (45) and are consistently located in the unstable layers of human atherosclerotic lesions; fewer neutrophils are found in the centers of atherosclerotic plaques (7).
This distribution of neutrophils in lesion areas with high inflammatory activity suggests that different neutrophil mechanisms may influence atherogenesis (Figure 2). These mechanisms include a wide variety of mediators (pentraxin 3, neutrophil elastase, neutrophil extracellular traps, reactive oxygen species, myeloperoxidase, leukotriene B4, connexin, matrix metalloproteinases and IFN-γ), most of which can contribute to plaque formation, progression and rupture (46). One of these mediators is pentraxin 3 (PTX3) which, in humans, is produced by neutrophils in atherosclerotic lesions in response to inflammatory signals, including bacterial products, IL-1 and TNF-α (47).
In vitro human studies have demonstrated that PTX3 binds to the first subcomponent of the classical complement pathway, C1q, suggesting that PTX3, when produced by neutrophils in the atherosclerotic plaque, may interact with C1q and induce complete complement activation. This activation could increase inflammation, chemotaxis, phagocytosis, or tissue damage, thus influencing atherogenesis development in human 48, 49. Additionally, PMNs are the most abundant source of myeloperoxidase (MPO) (50). Recently, the molecule INV-315 has been reported to inhibit MPO in mice, resulting in a considerable reduction in atherosclerotic plaques due to improved vascular function via attenuated inflammation and oxidative stress and enhanced cholesterol efflux (51). On the other hand, MPO is involved in hypochlorous acid generation, which is crucial to lipoprotein oxidation (52) and MPO-modified high-density lipoprotein (HDL) impairs HDL ability to partake in reverse cholesterol transport in mice (53). These results suggest that MPO promotes macrophage cholesterylester accumulation and foam cell formation, both of which are essential atherosclerotic processes. New functions could be attributed to MPO. Recently, in vitro studies have shown that MPO induces a robust extension of PMN locomotion in comparison with other molecules such as IL-8. Interestingly, this PMN phenomenon remains even when MPO is inactivated or when PMNs are stimulated with MPO variants that lack total enzyme activity, suggesting a mechanism independent of the enzyme’s catalytic activity. Furthermore, the in vivo administration of active MPO or MPO variants without catalytic activity into the carotid arteries of wild-type or Mpo−po mice induced the rapid adhesion of PMNs to the muscular postcapillary veins (54), suggesting that MPO may act as an additional mechanism in neutrophil recruitment to lesions (Figure 2). Various efforts have been made in the search for cardiovascular disease biomarkers. To this end, studies have reported increased levels of circulating PTX3 and MPO and have denoted these as prognostic biomarkers of heart failure, both acute and chronic, that may be an associated risk factor for cardiovascular disease in human 55, 56, 57.
Neutrophils have been widely documented to use different strategies against microorganisms. Recently, a new strategy described in neutrophils is the NETs, which can trap microorganisms and could thus be important in immune defense. NETs are composed of nuclear components, DNA and histones and are decorated with proteins from primary granules (e.g., MPO and neutrophil elastase), secondary granules (e.g., lactoferrin and PTX3) and tertiary granules (e.g., matrix metalloproteinase 9) and short peptidoglycan recognition proteins (38). Interestingly, inflammatory signals induce the release of NETs from neutrophils in human 58, 59, and these conditions are present in atherosclerosis (5). In vitro studies in humans reported that neutrophil-derived NETs accumulate and adhere strongly to platelets and induce a filopod in these cells, suggesting that NET-adherent platelets are activated and that platelet stimulation by purified histones is sufficient for aggregation. Another interesting mechanism involving NETs is thrombus formation (Figure 2), which is generated by fibrin deposition and induces a red thrombus (NETs associated with red blood cells) similar to that observed in arteries (60). Moreover, NETs induce cytotoxicity in endothelial cells, suggesting involvement of NETs in plaque destabilization through the induction of endothelial cell apoptosis in human (61).
NK cells are innate immune cells that sense pathological changes in tissues through the balanced cognate activities of inhibitory and activating receptors, which respectively recognize the reduced expression of major histocompatibility complex (MHC) class I molecules and the increased expression of MHC class I homologues such as MICA and MICB on affected cells 62, 63. When a NK cell encounters an affected cell, the NK cell can be activated by activating receptors, which can induce NK cell activation and can increase NK cell perforin-mediated cytotoxicity and the secretion of pro-inflammatory cytokines such as IL-1β, TNF and IFN-γ, leading to the direct elimination of the affected cell, induction of inflammation and polarization of adaptive immune responses 62, 63, 64. NK cells are present in atherosclerotic lesions in both humans and mice 65, 66, 67, 68 where they are distributed near the luminal endothelium, which covers the fibrous layer of the subendothelial space in the plaque shoulders and are absent in the necrotic lesion nucleus (18). Identification of the in vivo role of NK cells in atherosclerosis has been complicated. A previous study analyzed the role of NK cells in mice that harbored the beige mutation on the LDLR−mu background (these mice have a defect in NK cell cytolytic activity). This study showed an increase in atherosclerosis in “beige” LDLR−L mice compared with wild-type LDLR−mi animals, suggesting that the cytolytic activity of NK cells does not play a significant role in atherosclerosis (67). Many physiological functions of NK cells are likely mediated by NK cell cytokine production (69). In vivo, anti-NK1.1-treated wild-type mice were shown to produce slightly lower levels of IFN-γ in response to lipopolysaccharide (LPS). These results indicated that NK cells are the predominant source of acute IFN-γ production in response to LPS (70). Moreover, IFN-γ deficiency dramatically reduces atherosclerotic lesion development in ApoE−/− mice. In contrast, restoration of IFN-γ in the same mouse model induces lesion development as well as an increase in T cell infiltration (71). This evidence is supported by the functional depletion of NK cells in mice prone to atherosclerosis development. This depletion led to a significant reduction in atherosclerotic plaque development, suggesting a strong NK cell intervention. It is noteworthy that functional depletion does not affect the trafficking of NK cells, T lymphocytes or macrophages to lesions in LDLr−/− mice (9).
In vitro studies have reinforced the possible role of NK cells in atherosclerosis. Human DCs stimulated with anti-phosphorylcholine (anti-PC)-opsonized, minimally modified LDL (mmLDL) demonstrated enhanced maturation and rapid IL-12p70 production. NK cells produced IFN-γ upon interacting with IL-12-producing DCs that had been activated by anti-PC-opsonized mmLDL. Moreover, IFN-γ promoted DC IL-12 responses that were further augmented when mmLDL was opsonized with anti-PC (16). Similar results in mouse have been observed in cultures of NK cells and dendritic cells with oxLDL (70). This observation suggests that the interaction between DCs and NK cells induces the secretion of IL-12p70 and IFN-γ, which may affect atherosclerosis development (Figure 3).
Monocytes originate from a myeloid precursor in the bone marrow (71) and are released into the circulation, from which they enter tissues. The half-life of monocytes in the blood is believed to be relatively short (∼1 day in mice and 1–3 days in humans) 72, 73. This short half-life in the blood has fostered the concept that blood monocytes may continuously repopulate macrophage or DC populations to maintain homeostasis and, during inflammation, may fulfill critical roles in innate and adaptive immunity 74, 75.
Monocytes are currently understood to be a heterogeneous population, and monocyte subsets have different phenotypes in mice and humans 72, 73. For example, murine blood monocytes are identified primarily by the expression of CD115, CD11b, F4/80, CCR2 and CX3CR1 76, 77; this classification has permitted the identification of two monocyte subpopulations in mice (Figure 4). The first has the phenotype Gr1+Ly6ChighCCR2+ CX3CR1low (78) and the second has the phenotype Gr1−Ly6ClowCCR2−CX3CR1high (11). Ly6Chi monocytes (inflammatory) extravasate into the blood in a CCR2–CCL2-dependent manner and are essential in inflammation. Ly6Clow monocytes (patrolling) emigrate into the blood via CX3CR1–CX3CL1 signaling and monitor the luminal face of the small blood vessel endothelium under homeostatic and inflammatory conditions 78, 79, 80.
During atherogenesis, hypercholesterolemia is a key factor that induces the progressive and selective expansion of Ly-6Chigh monocytes. Interestingly, Ly-6Chigh monocytes preferentially adhered to activated endothelium and accumulated in lesions. Furthermore, in mice this monocyte subpopulation increased in the first days after acute myocardial infarction 78, 81. Meanwhile, Ly6Clow monocytes expand and prevail at 5 days after acute myocardial infarction and are recruited to sites of vascular damage to facilitate wound healing and revascularization (81). These data suggest that monocyte subpopulations can be mobilized from the bone marrow into the lesions in response to high circulating levels of cholesterol. Furthermore, after an acute disease event, monocyte subpopulations expand in a biphasic manner, suggesting that these cells may have distinct functions in damaged tissues.
The arrival of monocytes to the lesion depends on several molecules, among which chemokines and their receptors are fundamental. In mice, CCR2 deficiency dramatically reduces atherosclerosis development (78), and in ApoE−/− mice, treatment with a CCR2 antagonist reduces monocyte/macrophage infiltration in lesions (81). Within the same pathway, Apoe−/−/CCL2−/−/CX3CR1−/− mice showed considerable reductions in Gr1−Ly6Clow monocyte infiltration in lesions (82). Furthermore, a deficiency of P-selectin glycoprotein ligand-1, the common ligand of P- and E-selectin on leukocytes, contributed to the preferential homing of Ly6Chigh monocytes to atherosclerotic lesions of mice (83). In contrast, P-selectin, VCAM-1, ICAM-1 or PECAM-1 deficiency or blocking reduced monocyte adhesion and rolling as well as atherosclerosis development in mice 84, 85, 86, 87. The recruitment of distinct monocyte subsets to plaques suggests that these cells can differentiate into different types of macrophages. Ly6Chigh and Ly6Clow monocytes differentiate into M1 and M2 macrophages, respectively (88) and both monocyte subsets may be crucial to disease development, making them potential therapeutic targets.
In humans, three monocyte subpopulations have been described (Figure 4): classical (CD14++CD16−), intermediate (CD14++CD16+) and nonclassical (CD14+CD16++). Phenotypically, CD14+CD16− monocytes express high levels of CCR1, CCR2 and CXCR2 and low levels of CX3CR1, HLA-DR, CD80 and CD86, whereas CD14++CD16+ monocytes express high levels of CX3CR1, HLA-DR, CD80 and CD86 (89). Functionality analyses have shown that CD14++CD16− monocytes produce high levels of IL-10 and low levels of TNF, whereas CD14++CD16+ monocytes produce high levels of pro-inflammatory cytokines such as TNF and low levels of anti-inflammatory cytokines such as IL-10 (90); CD14+CD16++ monocytes exhibit a cytokine secretion pattern similar to that of CD14+CD16+ monocytes (91). Human monocyte subsets were recently demonstrated to produce pro- and anti-inflammatory cytokines when stimulated by specific ligands through TLR2 and TLR4 (90). Furthermore, these TLR are activated by oxidatively modified LDL to induce the secretion of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α 92, 93. Notably, CD14+CD16+ and CD14+CD16++ monocyte subpopulations are differentially recruited by MCP-1 and fractalkine, respectively 5, 11. Together these data demonstrate that monocyte subsets are phenotypically and functionally different and are also recruited differentially, suggesting that these subsets may have different roles in atherosclerosis.
Several clinical studies have yielded interesting data regarding the roles of these monocyte subsets. One study of patients with arterial disease and diabetes showed an increase in circulating CD14+CD16+ monocytes compared with the levels in healthy subjects and suggested a possible role for CD14+CD16+ monocytes in the inflammatory responses in these patients (94). Interestingly, acute myocardial infarction increases the numbers of CD14+CD16+ monocytes in the early days after the event; these cells decrease on the seventh day after the event, after which CD14+CD16− monocytes prevail 95, 81. The magnitude of CD14+CD16− monocyte mobilization is associated with cardiac recovery, whereas the numbers of CD14+CD16+ monocytes do not appear to have any impact on cardiac recovery, suggesting a dynamic specific to monocyte subpopulations after myocardial infarction (95). Additionally, future research should be targeted to the investigation of biomarkers (CD86 or HLA-DR, among others) in human monocyte populations that reflect patients’ disease states. This discovery would be potentially useful in clinical practice during decision making for disease treatment.
Macrophages are derived from monocytes and are found in all tissues where they display great anatomic and functional diversity. Macrophages have roles in almost every aspect of an organism’s biology including development, homeostasis, repair and immune responses (96). Currently, macrophages are known to be highly heterogeneous cells that can rapidly change their functions in response to local microenvironmental signals (97). In this sense, IFN-γ, in combination with LPS or GM-CSF, induces the classically activated or pro-inflammatory M1 macrophages. In turn, IL-4 and IL-13 induce the alternatively activated or regulatory M2 macrophages 97, 98. M1 macrophages have an IL-12high, IL-23high and IL-10low phenotype, are efficient producers of ROI, nitrogen intermediaries and pro-inflammatory cytokines (IL-1β and TNF) and participate in Th1 polarization. M2 macrophages have an IL-12low, IL-23low and IL-10high phenotype and are believed to participate in Th2 responses; additionally, M2 macrophages express scavenger and mannose receptors, among others 13, 99, 100. Macrophages are essential during all phases of atherosclerosis (12). In mice, early lesions feature a high density of M2 macrophages, and M1 macrophages predominate in advanced lesions 100, 101. In humans, M1 macrophages are distributed in the rupture-prone shoulder regions of the plaque. In contrast, most M2 macrophages are located in the vascular adventitial tissue, as well as in areas of intraplaque hemorrhage and in stable plaques (102).
During early atherosclerotic lesion development in mice, there is a predominant infiltration of M2 macrophages causing that the atherosclerotic lesions are small, suggesting that M2 macrophages may favor a state atheroprotective. In contrast, in advanced stages of atherosclerosis in mice, M1 macrophages predominate and express higher levels of inducible NO synthase (iNOS) that contribute to plaque development (101).
Moreover, M1 macrophages in advanced lesions (101) could cause plaque vulnerability that originates with lesion shoulder thinning, which occurs when macrophages degrade the fibrous layer matrix via metalloproteinases (MMPs) (5). For example in human, the collagenase (MMP1) and the stromelysin (MMP-10) are both expressed by M1 macrophages and can cause plaque rupture and, subsequently, myocardial infarction (103).
Interestingly, human M2 macrophages accumulate low levels of cytoplasmic lipid droplets. This accumulation is because macrophages display a reduced capacity to handle and efflux cellular cholesterol due to low expression levels of LXR-α and its target genes ATP binding cassette transporter A1 and apolipoprotein E. Additionally, in M2 macrophages, peroxisome proliferator-activated receptor-γ activation enhances the phagocytic but not the cholesterol trafficking pathways (104). These, human macrophages have a protective mechanism for controlling free cholesterol toxicity in conditions in which the cholesterol efflux pathway is defective (105) and are therefore less susceptible to foam cell transformation (106).
It is evident that macrophages are highly plastic in response to microenvironmental changes (107). IL-4 induces the in vitro polarization of M2 macrophages (arginase I) from ApoE−/− mice; in the presence of LPS/IFN, these cells switched to an M1 macrophage phenotype (arginase II) and produced IL-6 and iNOS (101). Interestingly, M1 macrophage polarization requires TLR activation and IFN-γ, and a similar mechanism can be induced by oxLDL, which is known to be activated by TLR4 and to induce TNF secretion in human macrophages 92, 107. This situation leads to the hypothesis that the inflammatory environment in atherosclerotic plaques could influence the conversion of M2 macrophages to M1 macrophages, which then assume the roles of inducing, maintaining and perpetuating the inflammatory response through the secretion of pro-inflammatory mediators. In this context, C-reactive protein (CRP) stimulation in vitro induces human M1 macrophage polarization, accompanied by increased levels of TNF-α, IL-1β, IL-6 and MCP-1 (Figure 5). Surprisingly, human M2 macrophages, which are also characterized by the expression of CD206, CD163 and IL-10, also secrete pro-inflammatory cytokines in the presence of CRP, indicating a conversion from M2 macrophages to M1-like producers of TNF, IL-12 and IL-23; in vivo, CRP similarly induces polarization toward the M1 macrophage phenotype in Wistar rats (106). These data suggest that CRP could induce the conversion of M2 macrophages to M1 macrophages, thus promoting atherosclerotic plaque development to more advanced stages. Additionally, oxLDL has been shown to induce a significant increase in IL-8 expression in human M1 macrophages in vitro (108). Furthermore, oxLDL exerts a cytotoxic effect on human M2 macrophages unlike M1 macrophages, which are more resistant to oxLDL lipotoxicity (109), suggesting that oxLDL could contribute to M1 macrophage activation and M2 macrophage killing, thus favoring other macrophage subpopulations (Figure 5). Despite this evidence, the roles of M1 and M2 macrophages in atherosclerosis remain uncertain and complex. Therefore, future studies are needed to understand the functional consequences of macrophage subpopulations in atherosclerotic disease.
DCs represent a small population of cells that are derived from a common CD34+ progenitor in the bone marrow and are located in different tissues (110). DCs are crucial in both the innate and adaptive immune systems as well as being essential in the maintenance of immunologic tolerance to self-tissues (111). DCs express different receptors (Fc receptors, integrins, C-type lectins, TLR and scavenger receptors) that take up both exogenous and endogenous antigens. After these antigens are processed, they are loaded onto MHC molecules for presentation to T cells. DCs can also process antigens via cross-presentation for presentation to CD8+ T cells (112). During maturation, DCs increase the expression of costimulatory molecules, MHC molecules and chemokine receptors as well as cytokines (IL-12 and TNF) that induce the different subtypes of CD4+ T cells such as T helper 1 (Th1), Th2 and Th17 cells, as well as naïve and memory B cells and NK cells 110, 111, 112.
Mouse DCs are recruited to lesions by receptors for chemokines such as CCL12 and CCL5 113, 114 where they adhere through the P- and E-selectins as well as integrins such as ICAM-1 and PECAM-1 (115). In the 1990s, DCs were identified morphologically and by the expression of S100 protein in the intima of arteries and in human atherosclerotic plaques 116, 117. DCs were subsequently found to infiltrate atherosclerotic lesions in ApoE−/− mice (118).
Multiple in vivo studies have demonstrated the role of DCs in atherosclerosis. The depletion of resident intimal DCs with the CD11c-DTR model resulted in a notable reduction in the number of DCs in the intima and marked reductions in lipid accumulation in the intima and the number of foam cells in nascent lesions, leading to a reduction in plaque size in mice (119). Similarly, plasmacytoid DC (pDC) depletion in ApoE−/− mice led to reduced atherosclerotic plaque formation (120). Additionally, in vivo pDC depletion reduced T-cell activation as well as interleukin-12, chemokine (C-X-C motif) ligand 1, monokine induced by IFN-γ, interferon γ-induced protein 10 and vascular endothelium growth factor serum levels in ApoE−/− mice (121). These findings clearly demonstrate that during atherosclerotic plaque formation, DCs trigger lipid ingestion and thus initiate nascent foam cell lesion formation as well as the secretion of cytokines that influence the atherosclerotic microenvironment. DCs are widely considered to be professional antigen-presenting cells. Thus, their contributions at various stages of atherosclerosis are key. In vitro experiments have shown that oxLDL can induce the maturation and activation of immature human DCs as demonstrated by increases in the expression of HLA-DR and costimulatory molecules such as CD83, CD86, CD36 and CD205 122, 123 and can induce the downregulation of CCR7 and CCL21 in human DC, leading to a marked reduction in DC migration (124). Similarly, oxidized lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses in ApoE−/− mice (120). This evidence suggests that DCs can respond to classical atherosclerosis inducers such as oxLDL, which induces the maturation and activation of DCs that arrest the lesion (Figure 6). In vivo experiments in mice showed a high frequency of DCs in atherosclerotic plaques as well as high expression levels of MHCII, CD80 and CD86 on these DCs. Interestingly, DCs interact with CD4+ cells in atherosclerotic plaques, and these interactions induce IFN-γ and TNF-α production by T cells (125).
DCs are crucial to the activation of other cells (Figure 6). In vitro assays of mouse and human cells have shown that oxLDL-activated DCs interact with NK cells to promote IL-12 and IFN-γ production. Moreover, in mice, CD48-2B4 pathway contact-dependent mechanisms are implicated in these interactions (70). These results may have important implications for our understanding of the mechanisms that initiate crosstalk between NK cells and DCs to eventually lead to atherosclerosis generation (70). On the other hand, ex vivo activation of pDCs from human atherosclerotic plaques with CpG ODN (TLR9 ligand) induces a 2.5-fold increase in IFN-α mRNA levels compared with unstimulated control tissues. Additionally in human, IFN-α induces increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on the surfaces of CD4+ T cells (Figure 6). This ligand enables the killing of vascular smooth muscle cells, suggesting that pDCs can respond to pathogen-associated molecular patterns and secreted cytokines such as IFN-α to activate T cells through the induction of TRAIL, a powerful apoptotic mediator implicated in plaque vulnerability-associated cell death (126).
Clinical studies have explored the frequencies of circulating DCs in patients with cardiovascular disease and have found reduced frequencies of myeloid DCs (mDCs) and pDCs in the peripheral blood of patients with acute coronary syndrome compared with healthy controls. This reduction tended to be more pronounced in patients with unstable coronary syndromes and extensive coronary artery disease. Meanwhile, patients who suffered acute myocardial infarction exhibited a further temporary reduction in mDCs that partially normalized after 1 week. This reduction in the frequency of circulating DCs, perhaps due to the increments of DCs in advanced atherosclerotic plaques, has been reported previously and suggests a possible role for dendritic cells in plaque progression and rupture in human 127, 128, 129.
Due to their potent capacity to stimulate T cells, DCs are being investigated in the context of therapeutic and vaccine approaches (130). Interestingly, LDLr−/− mice that are injected with oxLDL-pulsed mDCs induce oxLDL-specific T cells, a reduced Th1-response characterized by a 75% reduction in IFN-γ production and an increased production of oxLDL-specific antibodies (IgG1 and IgG2). This response leads to a reduction in lesion size (131). Similarly, LDLr−/− mice that were injected with apolipoprotein B 100-pulsed tolerogenic DCs had attenuated systemic inflammation and reduced their atherosclerotic plaque burdens (132). On the other hand, in ApoE−/− mice, peptide-loaded DC immunization significantly increased Treg numbers as well as IL-10 secretion (133). These findings indicate a promising role for DCs as possible therapeutics in atherosclerosis. However, future studies are needed to better characterize these mechanisms.
Section snippets
Conclusions
In this review we have analyzed how immune system cells affect atherosclerosis. MCs are innate immune cells that play a central role in allergy and are found in atherosclerotic lesions in mice and human. These cells can be activated by oxLDL or IgG-oxLDL complexes to induce the release of several inflammatory mediators such as IL-6 and IFN-γ in models animals and human, as well as tryptase and chymase, which trigger inflammatory responses in the lesion. Interestingly, MC-specific deficiencies
Acknowledgments
This work was supported in part by the Consejo Nacional de Ciencia y Tecnología (CONACyT) grant no. 177669 and the Instituto Mexicano del Seguro Social (IMSS) project of the Fondo de Investigación en Salud (FIS) project no. FIS/IMSS/PROT/G11-2/1022.
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