In the last decade, important advances in the knowledge of macrophage origin have triggered an essential conceptual progress in the mononuclear phagocyte system field. Parabiosed mice and genetic fate-mapping experiments have revealed that the majority of resident macrophages in healthy tissues are established from the yolk sac and fetal liver before birth (2–4). This cellular compartment locally self-maintains throughout life within the tissue and is independent from the hematopoietic input. On the other hand, during adulthood tissue-infiltrating macrophages can develop from circulating monocytes. The recruitment of monocytes is associated with pathological, but also with homeostatic response. Macrophages derived from monocytes display a short lifespan, although exceptions have also been reported. Embryonic- and adult-derived macrophages generally coexist in a given tissue, and their respective number correlates with the origin and records of their tissue of residence. Seminal experiments have demonstrated that embryonic- and monocyte-derived macrophages make different functional contributions in homeostatic conditions or following challenge. In this sense, Levine and coworkers have revealed that embryonic-derived macrophages are clue for cardiac recovery after injury (5). Their results suggest that targeting specific macrophage lineages could have important therapeutic implications in order to improve treatments for heart diseases.

Adult resident macrophage compartments seem to be independent from monocyte recruitment in a given tissue, as was demonstrated by parabiotic experiments. Monocyte compartments of the parabionts reach, with time, considerable chimerism in the joined circulation. However, tissue macrophages failed to equilibrate even after several months of parabiosis, suggesting the absence of an ongoing steady-state contribution of bone marrow-derived cells to adult tissue macrophage compartments (3). Furthermore, recruited monocytes are short-lived effector cells in tissues and assumed different roles that have yet to be better defined; emerging reports suggest, for example, that monocytes can promote angiogenesis and arteriogenesis (6). In addition, it was recently reported that inflammatory Ly6C^high monocytes persist during the steady state without commitment toward macrophage or dendritic cell (DC) fates and might contribute to antigen transport toward lymph nodes (7). Remarkably, macrophage effector function also needs to be tailored to its tissue of residence, an adaptation that is driven by the local microenvironment and by the inflammatory history of a given tissue.

Monocytes/macrophages are very plastic cells and can acquire distinct phenotypes and activation states under the influence of different microenvironments. Although several authors have shown that macrophages treated with different stimuli display altered phenotypes or functional capacities, many of these studies are limited considering that they compare only one particular activation state with non-polarized macrophages. Although macrophage activation was initially seen as a dichotomy between classically and alternatively activated states, it is now clear that the spectrum of macrophage activation states is much more diverse.

Monocytes develop in steady state in the bone marrow from hematopoietic precursors, and they enter the circulation via CCR2 receptor. In mice, circulating monocytes are phenotypically and functionally heterogeneous and can be defined according to Ly6C expression marker (Figure 1). In mice, during steady-state conditions, about 50–60% of circulating monocytes belongs to the Ly6C^high CCR2^high CX3CR1^low CD62L⁺ subset. These inflammatory or classical monocytes have a relatively short-circulating lifespan and are preferentially recruited to injured/inflamed tissues where they maturate to macrophages. The remaining non classical (Ly6C^low CCR2^low CX3CR1^high CD62L⁻) subset patrols blood vessels and accumulates at low numbers in the steady state (8). The number of Ly6C^high monocytes rises during inflammation, at expenses of enhanced monocytopoiesis in the bone marrow and spleen (1, 9–11). Regarding the origin, evidence shows that monocyte subpopulations do not arise from separate progenitors, but rather convert from the Ly6C^high to Ly6C^low subset (12) (Figure 1).

In humans, circulating monocytes can be segregated into three major subsets based on the expression of CD14 and CD16 (13). Over the past decades, human circulating monocytes had been separated into two subpopulations based on CD16 expression, the CD14⁺ CD16⁻ and CD14⁺ CD16⁺ monocyte subsets (hereby designated as CD16⁻ and CD16⁺, respectively). A new nomenclature defines three monocyte populations, where the minor CD16⁺ subset is further separated into two subpopulations (Figure 2). The intermediate subset expresses relatively high levels of CD14 coupled with low CD16 expression (CD14⁺⁺ CD16⁺), while the non-classical subset expresses low levels of CD14 with high expression of CD16 (CD14⁺ CD16⁺⁺). The subset with high CD14 and no CD16 expression, termed classical subset (CD14⁺⁺ CD16⁻), constitute approximately 85–90% of human monocytes in the peripheral blood. Several studies have demonstrated that circulating CD16⁺ monocytes are found in large numbers in patients with inflammation processes (14) and infectious diseases (15, 16).

Analyses based on hierarchical clustering of gene expression profiles of human monocyte subsets have been a matter of debate. Cros et al. reported that the classical and intermediate subpopulations were most closely related among the subsets, while the most distant was the non-classical subset (17). However, other independent groups described that both CD16⁺ subpopulations are more closely related (18, 19). Indeed, the number of genes that were significantly different between both CD16⁺ subsets was the lowest among the three subpopulations. Until now, there is poor agreement on the effector functions of the three monocyte subsets. In this sense, it was reported that TNF is produced mainly by CD16⁺ monocytes (intermediate and non-classical subsets) (20, 21). However, recent results have evidenced that TNF is produced by the three subsets. While Cros et al. (17) described that non-classical monocytes were poor producers of several cytokines [TNF, interleukin (IL)-1β, CCL2, IL-10, IL-8, IL-6, and CCL3] in response to LPS, other reports showed that the stimulation of intermediate subset with LPS produced the most TNF, IL-1β, and IL-6. In agreement, others also showed that intermediate monocytes produced the most TNF and IL-1β upon treatment with LPS and that this subset is the highest producer of TNF when cocultured with pre-activated T cells (22). In addition, Wong et al. (18) showed that non-classical monocytes upon LPS stimulation produced the highest levels of TNF and IL-1β, while equivalent amounts of IL-6 and IL-8 were produced by all three subsets. Other reports also recognized non-classical monocyte population as the greatest producer of TNF (21). Hence, it seems that non-classical monocytes constitute the subset capable of producing inflammatory cytokines in response to TLR ligands. There are also controversies on which monocyte subset is the major producer of the anti-inflammatory cytokine IL-10. In this sense, although it was demonstrated that intermediate monocytes produced the most IL-10 in response to LPS and zymosan (23), some recent reports showed that classical monocytes produced the most IL-10 (17, 18, 24). Further studies are necessary to clarify this relevant question.

Several studies on macrophage biology have focused on the particular functions that these cells acquire after tissue accumulation. Macrophages are highly plastic and can adapt to environmental stimuli, displaying either a classically activated (M1) or alternatively activated (M2) profile, which represent extremes of a spectrum of functional phenotypes (25, 26). M1 macrophages are activated by pro-inflammatory Th1 cytokines and LPS. Besides being antigen-presenting cells, the M1 subset shows an enhanced microbicidal capacity attributable to the production of reactive oxygen species (ROS) (such as hydrogen peroxide, superoxide, nitric oxide (NO), and peroxynitrite) and inflammatory cytokines (TNF, IL-1β, IL-12, and IL-23). On the other extreme, M2 macrophages are activated by Th2 cytokines (IL-4 and IL-13) or by anti-inflammatory mediators (IL-10), enhancing the arginase activity and mannose receptor (CD206) expression, to promote wound healing and reduce Th1 response. However, the M2 subset development can also be detrimental to host tissue, leading to fibrosis when their matrix-enhancing activity is not regulated (27). Although embryonic and monocyte-derived macrophages likely are on a continuum spectrum that lies between (and outside of) the M1 and M2 classifications, the terminology nevertheless has been helpful in order to elucidate macrophage heterogeneity. When Ly6C^high monocytes are recruited to atherosclerotic lesions, they mature to F4/80⁺ macrophages. In a persistent inflammatory environment, these Ly6C^high monocyte-derived macrophages contribute to oxidative stress and are inflammatory by producing IL-1β and TNF (9). In this respect, Ly6C^high monocyte-derived cells are M1 macrophages. In the setting of inflammation resolution, M1 macrophages are replaced by M2 repairing macrophages. Although it has been proposed that M1 to M2 conversion occurs locally (28–30), M2 macrophages also could derive from non-classical and less inflammatory Ly6C^low monocytes (12). Otherwise, M2 macrophages may arise through direct differentiation of Ly6C^high monocytes in a microenvironment that favors wound healing. This option should be further explored because it is enticing for potential therapeutic reasons.

Cardiac resident macrophages coexpress M1 and M2 markers suggesting no specific polarization (31). However, within myocardium macrophages respond to systemic Th2 environment induced by helminth parasites infection, adopting an M2 phenotype associated with enhanced fibrosis. In this model, the increased amount of cardiac macrophages relies on recruitment of Ly6C^highCCR2⁺ monocytes instead of IL-4-induced expansion. In this sense, Jenkins et al. have shown that IL-4 and IL-13 through IL-4Rα not only activate macrophages but also cause proliferative expansion of resident macrophages (32, 33). Moreover, IL-33 also induces macrophages proliferation, but in an IL-4Rα-signaling-independent manner (34), suggesting that the number and activation state of cardiac macrophages largely depend on mediators locally produced.

Although inflammation is the process in which components of the innate immune system respond to an injury or infection, inflammatory response can also be triggered in absence of infection by a process known as sterile inflammation. This type of inflammation is implicated in the development of a variety of clinical conditions such as atherosclerosis, cardiac injury, neurodegenerative and metabolic disorders. Unlike microbial-induced inflammation, sterile inflammation is activated by endogenous danger-associated molecular patterns (DAMPs) released as a consequence of cellular injury and/or inflammation, such as oxidized low-density lipoprotein (oxLDL), oxidized phospholipids (oxPLs), amyloid β or uric-acid crystals (104, 105). This sterile inflammatory response induces recruitment of neutrophils and monocytes leading to production of pro-inflammatory cytokines and chemokines, mainly IL-18 and IL-1β in a caspase-1-dependent manner. As mentioned above, monocytes subsets (classical, intermediate, and non-classical) extensively participate in the response to infectious disease; however, their role in sterile inflammation in cardiac ischemia-reperfusion injury and atherosclerosis is still not well understood. Myocardial ischemia-reperfusion injury is a condition that involved cardiomyocytes death affecting contractibility and loss of heart function as a result of a vascular occlusion and loss of tissue irrigation. While different pathways contributed to limit the damage, the inflammatory response in the ischemic heart can be detrimental (106).

The knowledge gained on the origin of resident cardiac macrophages has greatly improved the therapeutic potential of macrophage manipulation in the context of sterile inflammation. Considering that adult mammalian heart contains two macrophage pools which include: Ly6C^low MHC-II^low and MHC-II^high (CCR2⁻) representing embryonically established macrophages and the second pool and much less abundant derived from blood Ly6C^high CCR2⁺ monocytes. Altered homeostasis induced by transient depletion (chemically) of monocytes and macrophages population in heart shown proliferation of CCR2⁻ resident macrophages and recruitment of Ly6C^high CCR2⁺ monocytes from blood to contribute to tissue macrophages repopulation (107). Thus, after myocardial injury and knowing that NLRP3 inflammasome activation impair tissue regeneration, blockage of CCR2 abolishes ischemic damage. This concept suggests that blockage of CCR2⁺ monocytes influx or recruitment during cardiac ischemia-reperfusion will be protective, while a strategy of depletion of resident macrophages (CCR2⁻) abolished that protection. In the same way, Marchetti et al. applied a pharmacological inhibitory strategy for NLRP3 inflammasome in a mouse model of myocardial injury. They found that in acute myocardial infarction (MI) with reperfusion and in a model of non-ischemic injury induced with doxorubicin, the inhibition of NLRP3 inflammasome cause a significantly reduction of infarct size and preserved systolic function (108).

The understanding of circulating monocytes and cardiac macrophage biology under physiology and disease condition may help discover new mechanisms to target specific subset population of macrophage that impact in cardiac cytoprotective pathways.

Atherosclerosis is the result of lipid accumulation and chronic inflammation in the arterial wall where macrophage plays a central role in the development and progression of plaque formation (Figure 4). Thus, transmigration of monocytes into the subendothelial space, lipid uptake (oxLDL) and foam cell differentiation are key step in atherogenesis. The maintenance of a healthy endothelium regulates the precise balance in vascular homeostasis between vasoconstriction and vasodilation, trombogenesis and fibrinolysis, cellular migration and proliferation (109). Disturbance of normal vascular physiology is characterized by endothelial activation, which may be caused by infection or by cardiovascular risk factor such as hyperlipidemia (pathophysiologic). Thus, pro-inflammatory mediators (IL-1β, TNF, and MCP-1) induce a deleterious effect on NO bioavailability increasing ROS which stimulate the expression of endothelial adhesion molecules (VCAM-1, ICAM-1) to favor the passage of monocytes to the subendothelium and promoting their differentiation into macrophages (110).

In atherosclerosis, circulating low-density lipoprotein (LDL) molecules undergoes modifications mainly oxidation, acetylation and glycation that alter the normal metabolism of lipoprotein to promote cytotoxic damage on endothelial cells and macrophages. There is a broad spectrum of mediators that induce LDL oxidation from enzymatic (lipoxygenase and cyclooxygenase) to non-enzymatic reactions (free radicals and oxidants). Some of these reactions have differential selectivity for LDL components as occur with ROS that oxidize predominantly the lipid portion of LDL or the neutrophil myeloperoxidase that oxidize the protein portion (Apo B) of LDL by action of hypochlorous acid byproducts (111, 112).

In this inflammatory milieu, other biomolecules, in addition to LDL, such as lipids and proteins are target of oxidation and nitration reactions generating new species that may affect the fate of the plaque development and limit or exacerbate the inflammatory state. Thus, nitro-fatty acids (NO₂-FAs) are the product of the reaction of unsaturated fatty acids and nitrogen-derived species (NO and nitrites) (113). Administration of NO₂-FA in a mice model of atherosclerosis (ApoEKO) exhibited anti-inflammatory and protective actions, decelerating the development of the atheroma plaque, affecting oxLDL uptake and foam cell formation in macrophages (114).

Modified LDLs are recognized and internalized in macrophages by pattern-recognition receptor (PRR) which includes scavenger receptor (SR) and toll-like receptor (TLR). SRs bind diverse ligands and contribute to the clearance of foreign or modified particles. Regarding to macrophage cholesterol metabolism, CD36 (SR-B) and SR-A1 mediate internalization of oxLDL and transform these cells into cholesterol-laden foam cells in the vasculature intima. Exploration in experimental models evidenced that CD36-deficient macrophages exhibited a reduced uptake of oxLDL; further study in ApoE/CD36 double mutant mice described the reduction of the size of atherosclerotic plaque (115, 116). These results emphasized the role of CD36 as a pro-atherogenic receptor in the vasculature and cardiovascular disease. However, discrepancy on CD36 implication as pro- or anti-atherogenic has been raised by other studies in which described advanced atherogenesis in LDLR/CD36 double mutant mice or less macrophage accumulation but increased aortic lesions in ApoE/CD36 double KO (117). The inconsistency of this result may be in part explained by the different experimental model and animal background employed. Besides the discrepancy on CD36 function in atheroma plaque formation the binding of oxLDL stimulates pro-inflammatory signaling pathways (NF-κB) in macrophage contributing to maintain the chronic inflammatory state generated during the onset of atherosclerosis disease.

In atherosclerosis, the recognition of the subsets of recruited monocytes that differentiate into macrophages in the atheroma plaque may be relevant to design new strategies to treat pathological inflammatory process. During the development of atheroma plaque, the recruitment of monocytes are drawn from the Ly6C^high circulating subset (precursor of M1 macrophages) but after treatment and control of hyperlipidemia a rapid reduction of plaque inflammation and regression was exhibited, characterized by decrease number of macrophages and the relative increase in markers of M2 macrophages state. Rahman et al. described in a model of aortic arch transplantation that plaque regression is characterized by recruitment of Ly6C^high circulating monocytes and the posterior polarization to M2 phenotype (118). This result is contrary to the prevailing paradigm about M2 macrophages recruitment in plaque regression and the authors promote the strategy of M2 macrophage accumulation in atherosclerotic lesions to stimulate plaque regression. Consistent results were obtained in mice treated with IL-13 and IL-4 (promote M2 polarization) which describe protective effects in plaque progression in mice (119).

Summing up, the data obtained clearly illustrate the clue role that mononuclear phagocytic cells play in the development of the atherosclerotic pathology, further molecular mechanisms involved in plaque formation are described in the next chapter.

Macrophages are major contributors to the inflammatory response in the setting of cardiovascular diseases. They secrete diverse pro-inflammatory mediators (including cytokines, chemokines, ROS and nitrogen species, among others), and MMPs and they eventually die by necrosis or apoptosis. Dying macrophages release their lipid contents, which lead to the development of a pro-thrombotic necrotic core. The necrotic core is, in turn, a clue component of unstable plaques and contributes to their rupture and the consequent intravascular blood clot that causes MI and stroke. The main effect of cytokines/chemokines released by macrophages is the recruitment of different innate and adaptive immune cell populations into the atherosclerotic lesions (120, 121), thereby amplifying the inflammatory environment. For example, different inflammatory mediators such as IL-1, TNF and IFN-γ increase the expression of MCP-1 in macrophages (120, 122). Also, under inflammatory conditions macrophages produce different enzymes such as MMPs, cathepsins and serine proteases that degrade the extracellular matrix of the atherosclerotic plaque contributing to the pathogenesis of atherosclerosis (123). In this sense, IL-1 and TNF increase the expression of numerous MMPs, such as MMP-9, and their activity contribute to unstable plaque morphology (123).

While initial reports indicated that IFN-γ prevented macrophage foam cell formation (124), subsequent studies demonstrate that this cytokine promotes foam cell formation through diverse mechanisms: enhancing the uptake of oxLDL/AcLDL through SRs (122, 125), diminishing efflux of cholesterol via inhibition of ABCA-1 transporter and ApoE expression (126, 127) and increasing ACAT-1 levels (126) in macrophage-derived foam cells. For example, it was recently reported that the axis IFN-γ/STAT1 mediates the uptake of acLDL/oxLDL by human macrophages in an extracellular signal-regulated kinase (ERK)-dependent signaling (125). The activity of IFN-γ as a pro-foam cell mediator has been supported by several in vivo studies (128).

Since IFN-γ was identified as a pro-foam cell cytokine, different authors evaluated the effect of other pro-inflammatory cytokines. Studies using a murine macrophage cell line have shown that TNF inhibits the expression of SRs and the uptake of oxLDL (129). Regarding additional mechanisms, more recent evidence has suggested that TNF promotes foam cell formation in vitro by reducing the mRNA expression of ABCA-1/ABCG-1 (130), and intracellular lipid catabolism (131), and by enhancing the expression of ACAT-1 and the accumulation of cholesteryl ester (132). However, the role for TNF remains controversial since other studies have shown that TNF enhances ABCA-1 expression and the efflux of cholesterol in peritoneal macrophages stimulated with ApoA–I (133, 134) and since evidences from studies performed in murine models are also contentious (128).

Regarding anti-inflammatory cytokines, it has been demonstrated that TGF-β decreases CD36/SR-A expression and the uptake of oxLDL in human macrophages (135, 136). Additional data emphasized this anti-foam cell effect by showing that TGF-β downregulates CD36/SR-BI mRNA levels in peritoneal macrophages (137) and increases ApoA–I and HDL-stimulated cholesterol efflux and ABCA-1 gene transcription in foam cells (138). Argmann et al. (139) demonstrated that TGF-β1 decreases LPL expression/activity and cholesteryl ester accumulation with a coincident increase in the expression of ABCG-1 in murine macrophages. Recently, it was reported that TGF-β diminishes LPL gene transcription (140) and increases the expression of ApoE (141). Moreover, other group has shown that CD4⁺ CD25⁺ regulatory T cells use TGF-β1 to inhibit the uptake of oxLDL and the expression of CD36/SR-A mRNA (142). These mechanisms evidence an anti-foam cell role for TGF-β1, although it is important to stress that TGF-β may promote development either of anti-atherosclerotic regulatory T cells or of T-helper 17 (Th17) cells, depending on factors in the local milieu. Therefore, TGF-β signaling in T cells could promote stabilization of atherosclerotic plaques through an IL-17-dependent pathway.

The effect of IL-10 on plaque formation has also been reported. This anti-inflammatory mediator downregulates the CD36 SR expression (143) and, in consequence, decreases the accumulation of cholesterol in human macrophages (144). In addition, two independent groups have reported anti-foam cell activity of IL-10. Indeed, they found that IL-10 enhances the uptake and efflux of cholesterol by human macrophages in vitro and reduces foam cell formation and atherosclerotic plaque progression in vivo (145, 146). Altogether, the results suggest that IL-10 counteract atherogenesis.

Besides M1 and M2 macrophage profiles, additional phenotypes have been described within atherosclerotic lesion and they have become a topic of increasing relevance. In areas of hemorrhage, macrophages respond adaptively to heme acquiring an activation status that was designated hemorrhage-associated macrophages (Mhem). In vitro human blood-derived monocytes are induced to Mhem by stimulation with hemoglobin–haptoglobin complexes and it is dependent on an autocrine action of secreted IL-10 (147, 148). This macrophage profile prevents foam cell formation and has antioxidant functions. Other study showed that oxPL products that accumulate in atherosclerotic lesions induce another phenotype designated as Mox. Data indicate that Mox macrophages are characterized by high expression of heme oxygenase-1 and it comprises approximately 30% of all macrophages in established atherosclerotic lesions in experimental models (149). In addition, CXCL4, a chemokine released by activated platelet, was demonstrated to prevent monocytes/macrophages apoptosis and promote its differentiation into a phenotype called “M4” (MMP7⁺ S100A8⁺ CD68⁺). M4 macrophage was described in human coronary atherosclerotic plaques ex vivo and postmortem (150, 151). The prevalence of this macrophage subset is higher in patients with severe coronary artery disease and there is a significant correlation between the accumulation of M4 macrophages within the intima and plaque destabilization, which is independent of the overall number of macrophages in the vascular wall (150).

Recently, several groups have published that IL-17A-stimulated macrophages are suggested to constitute a new macrophage population. Barin et al. (152) showed that IL-17A induces activation of primary macrophages in a unique profile of cytokines and chemokines, including IL-12p70, GM-CSF, IL-3, IL-9, CCL4 and CCL5, that can be distinguished from previously characterized macrophage activation states. Moreover, another work showed that IL-17A induces a pro-inflammatory transcriptome in human monocytes/macrophages, including cytokines such as IL-1α and IL-6, chemokines like CCL2, CCL8, CCL20, CXCL1, CXCL2 and CXCL6, genes involved in oxidative stress, upregulation of CD14, CD163 levels and downregulation of genes associated with T cell costimulation. By comparing the entire transcriptomes of M1, M2 and M4 macrophages, authors confirm that IL-17A-treated macrophages are a unique polarization subset (153). On the other hand, it was reported that IL-17 also can strongly amplifies human monocytes/macrophages differentiation toward M2c profile, making it resistant to apoptosis and promoting its MerTK-dependent clearance of apoptotic bodies (154). M2c cells (CD14^bright CD16⁺ CD163⁺ MerTK⁺) are a subset of alternatively activated macrophages induced by M-CSF plus IL-10 or glucocorticoids and they are involved in inflammation resolution through phagocytosis of early apoptotic neutrophils and anti-inflammatory cytokines production (155). In contrast with the mentioned effects of IL-17, authors demonstrated that both IFN-γ/Th1 environment and IL-4-chronic exposure impair glucocorticoid and IL-10-induced M2c macrophage differentiation and also alters phagocytosis function of stablished M2c macrophages by reducing MerTK levels and promoting its apoptosis. Thus, this work indicates that Th17 environment orchestrates the resolution of innate inflammation through expansion of M2c regulatory macrophages.

An important point to note is that in a predominantly Th17 environment, monocytes/macrophages have a key role in the IL-17-induced inflammatory responses. In an experimental murine autoimmune myocarditis (EAM) model, the genetic ablation of IL-17RA alters the recruitment of monocytes/macrophages, which is the most numerous component of the inflammatory infiltrate and it is implicated in the cardiac damage (152). In concordance, other work shows that IL-17A promotes cardiac infiltration of Ly6C^high monocytes by inducing fibroblasts to produce high levels of cytokines and chemokines. Particularly, GM-CSF production by IL-17-stimulated cardiac fibroblasts drives differentiation of infiltrating monocytes into an even more inflammatory phenotype which further intensifies the myocarditis. Experiments performed in IL17RAKO mice or depletion of Ly6C^high monocytes/macrophages demonstrates the involvement of these innate cells in the inflammatory dilated cardiomyopathy (156). On the other hand, in an advanced atherosclerotic model in ApoEKO mice, in vivo IL-17A blocking markedly prevents atherosclerotic lesion progression and improves its stability by reducing inflammatory burden and monocyte infiltration (153). Moreover, the CCL2 and CCL5 production were significantly reduced in those IL-17A mAb-treated mice. In vitro experiments demonstrated that IL-17A induces adhesion of human monocytes, adhesion and rolling of platelets on endothelial cells. Other studies performed in early stages of atherosclerosis demonstrate that IL-17/IL-17RA axis has a pro-atherogenic role by promoting early monocyte recruitment into plaque through CCL5 production, endothelial VCAM-1 expression and increasing CXCL1 expression on the vessel lumen (157–159).

Purinergic system has been recognized as a main pathway to regulate immune response and it is critical to prevent the collateral damage produced by the exacerbation of the immune response in several diseases including cardiac pathologies. In normal conditions, ATP is localized intracellularly where it is the main source of energy for cell functions and it accomplishes indispensable functions in cellular metabolism such as proliferation, migration, motility, biosynthesis and contraction functions within the cardiomyocytes. In addition to its metabolic function, ATP also acts as an important signaling molecule when it is released to the extracellular compartment. After it is released, the extracellular ATP hydrolysis is governed by the activity of two membrane ectoenzymes, the CD39 that catalyzes the phosphohydrolysis of ATP to ADO monophosphate (AMP) and CD73 that hydrolyzes AMP into ADO and inorganic phosphate (45). CD39 and CD73 expression on immune and vascular cells and their enzymatic activities play decisive role in the regulation of the magnitude and duration of purinergic signals that modulate cellular function in an autocrine or paracrine manner (160). These effects are mediated by two types of nucleotide/nucleoside receptors: P1 receptors activated by extracellular ADO and P2 receptors activated by ATP and other nucleotides such as ADP and AMP. The first group includes 4 ADO receptors (ADORA 1, ADORA 2A, ADORA 2B, and ADORA 3), and the second group includes several P2X and P2Y subtypes of receptors (161).

Purinergic signaling is highly described in tumoral context (162) and in autoimmune diseases (163) but poorly studied in the context of cardiac pathologies. Given the high production rate of ATP and the turnover required to maintain its continuous mechanical work, disruption of heart tissue generates the release of high amounts of ATP to the extracellular space. Bonner et al. have reported that cardiomyocytes and erythrocytes do not detectably express the ATP catabolic machinery, but coronary endothelial cells were highly positive for CD39 under basal conditions and a low proportion of these cells express CD73. Furthermore, they stated that resident cardiac APCs and monocytes were the responsible of the first step of ATP degradation in the heart after ischemic injury because they were highly positive for the CD39 while CD73 was absent. Likewise, the dephosphorylating step of AMP to immunosuppressive ADO seems to take place on T lymphocytes and granulocytes because CD73 was mainly expressed in those populations (164).

In contrast to the pro-inflammatory action of ATP, ADO exhibits diverse immunoregulatory roles. Bonner and coworkers found that macrophages stimulated through ADORA 2A and ADORA 2B receptors with ADO undergo M2 phenotype in infarcted mice. In addition, in CD73KO mice there was augmented expression of microbicidal M1 genes, while the expression of M2 genes like arginase-1, IL-10, and TGF-β decreased. This monocyte imbalance toward inflammatory Ly6C^high-expressing monocytes in mice lacking CD73 had been related to adverse myocardial healing and ventricular dilatation after MI (165). Furthermore, Haskó et al. reported that ADO diminish the oxidative burst by downregulating NO production and inhibit TNF release from monocytes and macrophages (166). A parallel mechanism has also been reported by Cain group in murine isquemia/reperfusion model where ADO pretreatment decreased myocardial TNF production (167). There is strong evidence that ADO can modulate leukocyte adhesion to vascular endothelium in vivo. In this sense, Koszalka et al. demonstrated that ADO is an important endogenous pathway to modulate the inflammatory vascular response. After a period of ischemia-reperfusion, they observed significant increase of leukocyte adherence to the vascular endothelium only in the CD73 mutant (168). It has also been suggested that purinergic signals play a role in cellular migration. Analysis of atherosclerotic lesions of P2Y6 KO mice revealed fewer macrophages, diminished RNA expression of IL-6 and VCAM-1, suggesting that deficiency in this ATP receptor limits atherosclerosis and plaque inflammation (169). Recently, it was reported that lack of P2X7 receptor resolute plaque destabilization by inhibiting inflammasome activation and consequently ameliorates experimental atherosclerosis. ATP binding to P2 receptors is crucial to NLRP3 assembly and the subsequent IL-1β release. P2X7 null mice had decreased amount of macrophages in the wound and, in consequence, smaller and less inflamed atherosclerotic lesions than respective control animals (170). Altogether, the data indicate that purinergic signaling can modulate macrophage polarization and infiltration; and that ADO is a key metabolite in suppressing inflammation following injury or infection.

In addition, ADO induces the production of vascular endothelial growth factor (VEGF) in human macrophages, associated with increased hypoxia inducible factor-1α (HIF-1α) expression, the main transcriptional inducer of VEGF in hypoxic milieu. VEGF promotes blood vessel formation, induces cell proliferation and initiates immune cell migration to stimulate vasculogenesis and angiogenesis (171). On the other hand, extracellular ATP contributes to tissue repair by the stimulation of P2X7 receptor and release of the pro-angiogenic factor VEGF (172). Summing up, activation of purinergic receptors in macrophages can promote wound healing and may be targeted to improve cardiac repair mechanisms.

The heart changes its substrate preferences throughout the life, under physiological or pathological conditions. This metabolic flexibility allows it to adapt to environmental changes. However, shifts in fuel selectivity can become detrimental in the pathological heart (173).

Pathological cardiac hypertrophy is a common consequence of several cardiovascular diseases and is a maladaptive response to chronic stress, sometimes resulting in cardiac failure. Pathological hypertrophy involves a shift in fuel metabolism from fatty-acid oxidation to enhanced reliance on glucose. Increased glucose utilization is characterized by upregulation of glucose uptake and glycolysis concomitant with no change or a decrease in glucose oxidation, resulting in uncoupling of glucose uptake/oxidation metabolism. Nowadays, it is accepted that changes in mitochondrial function and cardiac metabolism precede heart dysfunction, indicating that metabolic remodeling is an early event in the development of cardiac diseases.

Recent interest in understanding the impact of these fundamental metabolic changes on immune cell differentiation and function has yielded numerous studies examining the pathways and metabolites involved in driving protective immune responses. A key question that has emerged is whether differential metabolic activity (i.e., use of glycolysis versus mitochondrial respiration) is coincident with differentiation or if it is a direct driver of alterations in immune cell differentiation and function. Classically activated macrophages that promote inflammation utilize glycolysis and glutamine metabolism to generate large quantities of succinate which enhances inflammation and IL-1β production (174). In contrast, macrophages that play a role in tissue healing and inflammatory resolution increase mitochondrial lipid oxidation (175). A key differentiator between these metabolic phenotypes may arise in the mitochondria, where rather than engaging in conventional electron transport, altered electron flow through the succinate dehydrogenase complex promotes ROS generation and inflammation (176). Enhanced glycolysis in coronary artery disease patients drives mitochondrial ROS production, resulting in pyruvate kinase M2 (PKM2) assembly and translocation to the nucleus. It then phosphorylates and activates STAT3 to promote the production of the cytokines IL-1β and IL-6 (177). Indeed, the increased activation of glycolytic pathway was associated with epigenetic changes in monocyte-derived macrophages describing a new mechanism to explain trained immunity in human innate immune cells (178).

Diabetes is a major risk factor for cardiovascular diseases (179). As such it may lead to heart failure indirectly, by promoting the development of coronary artery disease. However, it is now known that diabetes may cause heart failure also by eliciting a direct detrimental impact on the myocardium leading to the development of cardiac hypertrophy, diastolic and systolic dysfunction (180). The clinical condition associated with the spectrum of cardiac abnormalities induced by diabetes is termed diabetic cardiomyopathy. High glucose levels and dyslipidemia directly induce the upregulation and secretion of cytokines, chemokines and adhesion molecules in immune cells by modulating multiple signaling pathways that converge toward NF-κB signaling (181–183). Activation of the renin–angiotensin–aldosterone system and accumulation of advanced glycation end-products (AGE) and DAMPs molecules like extracellular high-mobility group box-1 protein (HMGB1), also represent key mechanisms that mediate inflammatory response in the diabetic heart primarily by acting TLRs (184–186). Following these initial molecular events, leukocytes infiltrate the myocardium and perpetuate the inflammatory process through secretion of cytokines and pro-fibrotic factors and by increasing the production of ROS. Mediators resulting from this inflammatory cascade, in turn, modulate specific intracellular signaling mechanisms in cardiac cells causing hypertrophy, mitochondrial dysfunction, ER stress and cell death fibroblast proliferation and collagen production. In addition, inflammatory factors may affect myocardial metabolic processes and interfere with cardiomyocyte contractile properties. These abnormalities together promote the development of diabetic cardiomyopathy.

Diabetes-associated metabolic derangements can directly induce cytokine expression and release from cardiac cells. It was previously shown that hyperglycemia augmented the expression of HMGB1 in isolated cardiomyocytes, macrophages and cardiac fibroblasts, thereby activating the MAPK and NF-κB pathways and by inducing TNF and IL-6 secretion (187). Inhibition of HMGB1 decreased myocardial inflammation and fibrosis in a murine model of type 1 diabetes and protected diabetic animals in response to postinfarction remodeling (187, 188).

An increase in circulating lipids may also contribute to cardiac inflammation in diabetes. Fatty acids activate TLR4 that strongly promotes inflammation through the NF-κB pathway (189). Mice with a TLR4 gene deletion demonstrate improved cardiac function and reduced cardiac intracellular lipid accumulation in response to streptozotocin-induced diabetes (190). Fatty acids induce the subsequent expression of IL-6, TNF and IL-1, which can be reversed by peroxisome proliferator-activated receptor-γ, β/δ (PPAR-b/d) (191). Previous work showed that hyperglycemia and high circulating levels of lipids promote inflammation through the activation of protein kinase C (PKC), which activates MAPK pathway thereby inactivating IkB (192).

The challenge to improve cardiovascular disorders therapy by modulating metabolic pathways must take into account potential off-target effects on other cells and tissues. In these sense, by exploiting the intrinsic capacity of monocytes and macrophages to take up foreign particles, the use of nanoparticle formulations might be one option to achieve targeting specificity (193).

Oxygen is the molecular source of the production of several molecules that damage vital tissues. Reactive oxygen and nitrogen species (RONS) are continuously produced as by-products of the reaction leading to energy production through the mitochondrial and microsomal electron-transport chains. In phagocytes, the oxidative bursts and enzyme systems such as xanthine oxidase and cytochrome P-450 oxidase are the endogenous sources of RONS. While physiological levels of RONS are crucial for cell function, excessive RONS levels trigger oxidative stress. Oxidative stress may damage clues molecules (proteins, lipids, DNA) and could, eventually, conducted to cell death. To prevent oxidative stress, antioxidants have evolved to protect biological systems against RONS-induced damage. Oxidative stress resulting from uncontrolled production of RONS that exceeds the antioxidant capacity, exacerbate atherosclerosis; since it induces endothelial dysfunction by impairing the bioactivity of endothelial NO and promotes leukocyte adhesion, enhances inflammation and thrombosis.

Numerous reports demonstrated that increased oxidative stress is associated with disturbances in cardiovascular diseases. In this sense, it was reported an overproduction of superoxide anion by cardiac dysfunctional mitochondria or increased production of RONS from non-mitochondrial sources in experimental models of type 1 diabetes (194, 195). The augmented production of RONS induces maladaptive cardiac response causing cardiac cells death contributing to cardiovascular disease (196). An excessive oxidative stress has been associated with increased cardiac cell apoptosis, as was evidenced by TUNEL⁺ cells and caspase 3 activation in T. cruzi-infected cardiac tissue (44). In this sense, high oxidative state has been associated with amplified lipid and DNA damage and the consequent cardiac cell injury and death. Therapeutic treatment that promotes RONS regulation have been shown to be effective in reducing cardiovascular dysfunction.

It is known that increased oxidative stress induces modifications of LDL, generating DAMPs that are detected by TLRs on different immune cell populations, mainly monocytes/macrophages. A variety of mechanisms mediated by enzymatic (such as 12/15-lipoxygenase and myeloperoxidase) or no-enzymatic redox reaction (such as RONS) boost LDL oxidation in the artery wall. The level of LDL oxidation modulates the immune system, when cells were treated with low oxLDL, the expression level of CD86, a marker for M1 macrophages, increases compared with that in cells treated with high oxLDL. In contrast, the expression level of the marker for M2 macrophages, CD206, significantly increases in cells treated with high oxLDL compared with that in cells treated with native LDL or low oxLDL. These results indicate that the degree of LDL oxidation affects the differentiation of monocytes into different subtypes of macrophages (197). Further research is needed to assess the functional consequences of oxidation processes on human macrophage behavior.