Atherosclerosis is the leading cause of mortality worldwide and one of its main manifestations is ischemic heart disease, mostly determined by the involvement of the coronary arteries, known as coronary artery disease (CAD). CAD is primarily a result of the buildup of atherosclerosis within the coronary arteries, progressively leading to the narrowing of the vessel lumen, thus reducing the blood flow with consequent ischemia. Upon stimulation by deposited lipids and damaged endothelium, innate and adaptive immune cells are activated and recruited to initiate plaque development [1]. The role of the immune system in CAD has been shown during the last few years to be complex and multifaceted. Immunity plays a key role in several types of pathways. First, inflammation is a key part of atherosclerosis [2]. Both innate and adaptive immune responses are activated to remove dead and apoptotic cells, facilitate scar formation, and promote angiogenesis [3]. Innate immunity includes neutrophils and macrophages, which can directly phagocytose dead cells and debris. They also play a role in releasing cytokines and other molecules that promote an inflammatory response ^[4][5][4,5]. Adaptive immunity involves T cells and B cells, which can recognize and respond to specific antigens. This response can also shape the overall immune response to an injury ^[6][7][6,7]. However, in the context of atherosclerosis, this inflammatory response can become chronic and lead to plaque formation. Immune cells, particularly macrophages, play a pivotal role in the uptake of low-density lipoprotein (LDL), mostly oxidized-LDL (ox-LDL), leading to the formation of foam cells within the atherosclerotic plaque [5]. 

Neutrophils are the most abundant leucocytes and play a significant role in mediating sterile inflammation and injury through a variety of mechanisms ^[4][8][4,9]. Previous experimental works have elucidated their roles in atherosclerotic diseases like CAD and its ensuing complications, i.e., acute coronary syndrome and heart failure ^[8][9]. Early aortic lesions and rupture- or erosion-prone atherosclerotic plaques show a significant presence of neutrophils ^[9][10]. A high peripheral neutrophil count directly relates to the degree of atherosclerosis in coronary arteries ^[10][11], infarct size, and declines in left ventricular ejection fraction ^[11][12][12,13]; the neutrophil-to-lymphocytes ratio raises clinical attention, due to its potential relationship with CAD ^[13][14]. Recent evidence also suggests a role for neutrophils in the activation of reparative processes ^[14][15]. In animal studies, for example, long-term depletion of neutrophils after myocardial infarction (MI) resulted in worsened cardiac function and increased fibrosis ^[14][15]. Neutrophils are paramount for innate immune response as they are the first responders in our defense against invading pathogenic microorganisms [4]. However, in sterile inflammatory conditions, activation of neutrophils may have detrimental effects on host tissues and therefore their homeostasis must be tightly regulated ^[15][16]. Interestingly, most clinically recognized cardiovascular risk factors contribute to enhanced granulopoiesis, e.g., the production of neutrophils in the bone marrow [4]. Once production bursts are triggered by those risk factors, neutrophils play a leading role in the initiation and evolution of unstable atherosclerotic plaques. At sites of disturbed blood flow and increased shear stress, they dysregulate vascular endothelial cells (ECs) and trigger leucocyte arrest ^[16][17], setting the stage for atherosclerosis ^[17][18]. Further release of granule proteins degrades the extracellular matrix (ECM), leading to extra-adhesion of monocytes, vascular hyperpermeability, and transfer of LDL particles ^[18][19]. Through the nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome signaling, activated neutrophils in the atheroma undergo neutrophil extracellular trap (NET) formation (NETosis), a type of cell death ^[19][20] using a network of ECM containing a variety of granule proteins. Their release decreases the stability of atherosclerotic plaques and contributes to thrombus formation through a variety of mechanisms ^{[20][21][22][23]}[21,22,23,24]. Not only do neutrophils play an active role in the genesis of atherosclerotic plaques, they also mediate their consequences to the target organs. Rupture of an atherosclerotic plaque can lead to obstruction of blood circulation resulting in ischaemic death of tissues that immediately triggers an acute inflammatory response, led mostly by neutrophils ^[20][21]. Attracted by cellular debris released by dead cells, neutrophils massively infiltrate the infarct area within hours ^[21][22]. At the site of injury, activated neutrophils generate and release reactive oxygen species, proteases, and NETs ^[22][23] which promote cardiomyocyte apoptosis, degrade ECM ^[23][24], lead to leucocyte infiltration, and prime the NLRP3 inflammasome, which then stimulates granulopoiesis in the bone marrow, leading to a vicious circle of the neutrophil infiltration circle and maladaptive remodeling ^[22][23]. Neutrophils are also involved in the modulation of the healing and remodeling response: the protein S100A8/A9 in NETs activated macrophages to phagocyte dead cells ^[24][25]. The transcriptional profile of neutrophils changes from a pro-inflammatory profile to an anti-inflammatory profile, initiating the reparative process mostly by dedifferentiating cardiomyocytes and promoting the accumulation of reparative macrophages ^[25][26][26,27]. Finally, a subset of pro-angiogenesis neutrophils able to control blood vessel growth, a known mechanism of tissue regeneration after injury, has been recently identified ^[27][28][28,29].

This dichotomous and apparently paradoxical effect of neutrophil activity in atherosclerotic diseases highlights the difficulty of researching beneficial therapeutical strategies involving neutrophils and calls for an expertly tailored approach to the matter. In the short term, stunning inhibition of neutrophils such as through beta-adrenergic antagonists as metoprolol or through inhibition of S100A8/A9 has been shown to reduce infarct size and increase left ventricular ejection fraction ^[29][30][30,31]. However, long-lasting depletion of neutrophils or even long-term inhibition of S100A8/A9 resulted in worse cardiac function and increased fibrosis ^[24][31][25,32]. Identifying the right window to effectively suppress the inflammatory functions of neutrophils while retaining their reparative functions could pave the way for important therapeutical applications.

Just as for neutrophils, the role of macrophages in inflammation, particularly in its cardiovascular aspects, is multifaceted ^[5][32][5,33]. Many studies have shown their ability to trigger and drive robust and damaging inflammatory responses ^[33][34], while others have shown their involvement in tissue repair and even cardiac regeneration ^[5][34][35][5,35,36]. This seems to be because different macrophage populations mediate different responses ^[36][37]. Furthermore, due to their high plasticity, they can adopt different phenotypes in response to varying stimuli and environments, a process called polarization ^[32][33]. Classically, cardiac macrophages have been categorized according to inflammatory states and cell surface markers into pro-inflammatory (M1) and anti-inflammatory (M2) subsets ^[25][26].

Newly discovered subsets of macrophages with mixed M1/M2 cell surface markers have challenged the adequacy of the classification ^[37][38]. In vitro, culture of human macrophages revealed considerable deviation from the M1/M2 spectrum when in contact with a cardiovascular relevant stimulus, e.g., free fatty acids or high-density lipoprotein (HDL) ^[38][39]. Furthermore, in vivo studies of murine atherosclerosis showed how, for example, inflammatory macrophages express cell surface markers like CD206, typically used to define the M2 anti-inflammatory subset ^[39][40]. In other words, the traditional classification of macrophages into M1 and M2 phenotypes does not fully capture the diversity of the population in vivo.

An alternative approach would classify macrophages according to developmental lineage, transcriptional factors, and recruiting dynamic as well as cell surface markers [5]. Through this classification, new functionally distinct cardiac macrophage populations have been elucidated. The adult heart contains three distinct populations of macrophages to the expression of C-C chemokine receptor type 2 (CCR2) and major histocompatibility complex class II (MHC-II): CCR2^-MHC-II^low, CCR2^-MHC-II^high, and CCR2⁺ MHC-II^high.

CCR2^-MHC-II^low and CCR2^-MHC-II^high, are long-lived, derive from embryonic progenitors maintained through the proliferation of local macrophages in the heart, and represent the vast majority at steady state [5]. They show an enhanced capacity to phagocyte death cardiomyocytes and exhibit a low inflammatory profile ^[40][41][41,42], while CCR2^-MHC-II^high can, in vitro, elicit an important inflammation response, for example through antigen-presenting cells (APC) to T-cells ^[41][42][42,43]. CCR2⁺ MHC-II^high are relatively short-lived and derive exclusively from circulating monocytes [5]. At a steady state, their function is still unclear. Following an MI, however, the presence of mitochondrial deoxyribonucleic acid (DNA) and alarmins from dying cardiomyocytes activates a vast array of proinflammatory genes in this subpopulation of macrophages, for example in the NLRP3 pathway, involved in neutrophil-associated inflammatory response, as previously stated ^[43][44]. In the first acute response to ischemic injury (first 4–7 days) CCR2^-MHC-II^low and CCR2^-MHC-II^high continue to show their classical phagocytic, non-inflammatory function in the lesion area ^[25][44][26,45]. This triggers apoptosis of all resident macrophages and by 24 h post-MI they are almost completely absent ^[33][34]. At the same time, abundant blood monocytes infiltrate the lesion area and differentiate into pro-inflammatory CCR2⁺ MHC-II^{high [33]}[34]. The fact that inhibition of monocyte extravasation into the cardiac tissue decreases macrophage numbers and improves cardiac physiology, highlights the importance of this population of macrophages in the adverse post-MI response ^[45][46]. In the reparative phase (days 5–14), monocytes differentiate into CCR2^-MHC-II^high macrophages as opposed to CCR2⁺ MHC-II^{high [46]}[47]. This switch in polarisation seems to be determined by changes in the local ischemic region: the infarct microenvironment is initially filled with early pro-M1 mediators, like interferon-γ (IFN-γ) and the granulocyte-macrophage colony-stimulating factor (GM-CSF), which trigger the initial differentiation into CCR2⁺ MHC-II^high macrophages and later with pro-M2 factors, like interleukin (IL) 10 and transform growth factor- β (TGF-β), stimulating differentiation into CCR2^-MHC-II^high macrophages ^[47][48][48,49]. These promote angiogenesis and scar formation and regulate the ECM microenvironment ^[46][49][50][47,50,51], orchestrating the fine mechanisms leading to tissue modelling and healing.

The mechanistic aspects of this flexible macrophage polarization are still poorly understood ^[51][52]. In the past 5 years, several studies have suggested an extensive epigenetic and transcriptional crosstalk between pro-inflammatory and anti-inflammatory signaling ^[52][53][53,54]. Responding to local stimuli, macrophages not only react at a transcriptional level ^[54][55], mounting the real-time response but they also adopt unique and permissive epigenetic changes, creating a cellular memory ^[55][56]. This memory enables the cells to launch a faster response upon reactivation, changing the macrophage activation state ^[51][52]. This allows a potentially more efficient response to pathological stimuli ^[56][57][57,58] but makes the system also prone to the dysregulation responsible for the clinical disease ^[51][58][52,59]. The tight control by transcription factors and epigenetic modifiers makes these pathways in macrophages a promising therapeutic target for inflammation-driven diseases.

Natural Killer T (NKT) cells have been the subject of increasing research about their role in the immune response to atherosclerosis and CAD ^[59][60]. NKT cells can be broadly categorized into two main subsets: Type I (invariant) and Type II NKT cells; type I NKT cells are the most well-studied subset and are characterized by their invariant T-cell receptor (TCR) alpha chain combined with a limited set of beta chains. These cells recognize glycolipid antigens such as α-galactosylceramide; they can rapidly produce a wide spectrum of cytokines, making them versatile regulators of immune responses. Type II NKT cells are less well-defined than Type I NKT cells and exhibit more distinct TCRs. They recognize a broader range of lipid antigens, including sulfatides, phospholipids, and glycolipids. Their functions are less clear, but they may also influence immune responses in various contexts ^[60][61][61,62]. The activation of these lipid-reactive NKT cells involves the interaction between lipid antigens, both endogenous and exogenous, and the nonclassical major histocompatibility complex class I (MHC-I) molecules of the cluster of differentiation (CD) 1 family on APC ^[62][63]. These lipid antigens bind to specific TCRs on T-cell subsets, including NKT cells. The structures of molecules in the CD1 family have been studied to understand how these lipid antigens associate with them. Group I CD1 molecules present lipid antigens from microbes and self-lipids to T cells, while group II CD1 molecules, specifically CD1d, present lipids to NKT cells. These CD1-presenting molecules are found on APC-like dendritic cells (DC), macrophages, and B cells, all of which play a role in the development of atherosclerotic lesions ^[63][64][64,65].

The activation of NKT cells can occur through various pathways. When an antigen is presented by CD1d molecules, a subset of NKT cells called invariant NKT cells (iNKT) respond rapidly by releasing cytokines, like helper T cells ^[65][66][66,67]. These cytokines have the potential to influence the development of atherosclerotic lesions in multiple ways ^[67][68][68,69]. The cytokines secreted by activated iNKT cells within the lesion may affect the response of other cells involved in the immune system, both innate and adaptive. In addition to antigen presentation by CD1d molecules, NKT cells can also be activated through a CD1d-independent pathway: DC or macrophages can be activated by Toll-like receptor (TLR) ligands, which then produce cytokines like IL-12, IL-18, or type I interferons. These cytokines can activate NKT cells without the involvement of CD1d molecules ^[69][70]. Moreover, TLR2 and TLR4 activation have been linked with atherosclerosis ^[70][71][71,72]. The effects of iNKT cells have been observed in studies conducted on mice. These studies administer different diets to mice to examine the impact of varying levels of iNKT cell activation or quantity. For instance, in mouse models treated with a Western-type diet, increased iNKT cell activity leads to increased plaque formation on the aortic root ^[72][73]. Conversely, in mice with genetically iNKT-deficient cells, an opposite effect on atherosclerotic lesions was shown ^[73][74].

NKT cells therefore play a crucial role in the immune response to CAD and atherosclerosis. The activation of these cells can occur through various pathways, including antigen presentation by CD1d molecules and CD1d-independent pathways. The cytokines released by activated NKT cells can influence the development of atherosclerotic lesions and modulate the response of other immune cells. Further research is required to fully understand the complex mechanisms by which NKT cells contribute to these diseases.

B cells play a multifaceted role in atherosclerosis, depending on cellular differentiation: this process leads to subtypes B1 and B2 cell formation ^[74][75][76][75,76,77]. When naive B cells are exposed to a complex set of stimuli, they undergo differentiation and become antibody-secreting cells, specifically plasma blasts and plasma cells. In dyslipidemia, activated endothelium lining atherosclerotic plaques allow different immunoglobulins to enter the plaque area ^[77][78]. B1 cells are associated with producing antibodies, including IgM antibodies, with anti-inflammatory properties ^[78][79]. B1 cells may have a protective role in atherosclerosis by reducing inflammation and promoting plaque stability through their antibody production ^[79][80][81][80,81,82]. Contrary, B2 cells were initially viewed to be proatherogenic after preferential B2-cell depletion using CD20-targeted antibodies ^[82][83][83,84]. However, recent studies provide marginal zone B cells can conduct protective effects potentially secreting IgM ^[84][85].

In atherosclerosis, B cells produce antibodies directed against specific antigens present within the plaques. The most well-studied antibody target is ox-LDL. When LDL cholesterol particles become oxidized, they produce Oxidation-Specific Epitopes (OSEs) that can be recognized by the immune system. Anti-ox-LDL antibodies can promote inflammation and contribute to plaque formation by facilitating the uptake of ox-LDL by macrophages, leading to the formation of foam cells ^[85][86][87][86,87,88]. Advanced stages of plaque formation give rise to artery tertiary lymphoid organs, such as those found in the adventitia, where plasma cells are formed within the plaque. This leads to the production of immunoglobulins in the adventitia. To support this, atherosclerotic plaques contain immunoglobulins specific to different OSEs ^[88][89]. A substantial number of IgM antibodies in our immune system can identify OSEs ^[89][90]. These epitopes can be found on ox-LDL, apoptotic cells, and microvesicles. They also inhibit the pro-inflammatory responses of macrophages triggered by microvesicles ^[90][91]. Additionally, when macrophages are triggered by microvesicles, the IgM antibodies also play a role in reducing the pro-inflammatory responses of the macrophages.

In contrast, IgG antibodies form immune complexes with ox-LDL, promoting inflammatory responses by macrophages ^[91][92]. IgE antibodies are known to have proatherogenic properties, as they stimulate macrophages and mast cells in both the plaque and perivascular area. Hamze et al. found that atherosclerotic plaques are rich in IgA and IgG, secreted by B cells during the inflammation process ^[92][93]. The involvement of IgA antibodies in atherosclerosis is still not well understood: a positive association between IgA and cardiovascular (CV) outcomes is reported, but functional roles have yet to be investigated ^[93][94].

B cells also produce various cytokines, including proatherogenic tumor necrosis factor -α (TNF-α) and antiatherogenic interleukin-10. Several studies on mouse models have described the protective role of B cells, remodeling the atheromatic plaque and increasing the lesion in case of B cell depletion ^[94][95][95,96]. However, there are different functional subsets of B cells, recognizing the heterogeneous population: both the proatherogenic and the antiatherogenic activities of various subsets are described ^[96][97][97,98]. While traditionally thought of as primarily involved in the production of antibodies, B cells also have antibody-independent pathways that influence the development and progression of atherosclerosis: the presence of B cells was characterized in the adventitia of atherosclerotic aortas but not in the atheromatous plaque ^[98][99]; this suggests a local immune response, associated with T cells, DC, and macrophages ^[99][100].

T cells have been found in the blood vessel walls near various CV diseases. They can contribute to immune responses in two ways: directly, by producing cytokines and molecules that promote inflammation, and indirectly through the activation of B cells. The different subsets of T cells have distinct functions in CV diseases, depending on whether they produce pro-inflammatory or anti-inflammatory molecules. CD4+ T cells, when in a naive state, can be differentiated into several subsets: T helper 1 (TH1), TH2, TH17, or regulatory T (Treg) cells. TH1 cells are pro-atherogenic and act through the production of IFN-γ and TNF-α ^[100][101][101,102].

On the other hand, Treg cells have an anti-atherosclerotic effect by secreting IL-10 and transforming growth factor-β (TGF-β). In fact, studies have shown that IL-10 produced by Treg cells can slow down the progression of abdominal aortic aneurysm and the formation of artery blockages following angioplasty ^{[101][102][103][104]}[102,103,104,105].

TH2 cells secrete molecules such as IL-4, IL-5, and IL-13. While TH2 cells and IL-4 may be associated with advanced atherosclerosis in mice lacking the apolipoprotein E (ApoE) gene ^[105][106], atherosclerosis decreased in mice lacking both the IL4 and Ldlr or IL4 and ApoE genes ^[106][107][107,108].

Lastly, there is inconsistent contrasting evidence on the impact of TH17 cells in atherosclerosis. TH17 cells produce cytokines like IL-17A and IL-17F. Blocking or inhibiting IL-17 in mice lacking the ApoE gene was found to promote the development of atherosclerosis ^{[108][109][110]}[109,110,111]. However, mice lacking the ILl17a gene actually showed an accelerated formation of unstable atherosclerotic lesions compared to mice lacking only the ApoE gene ^[111][112].

Assorted studies suggest that CD4+ T-cells are crucially involved in left-ventricular (LV) remodeling during both ischemic ^[112][113] and non-ischemic ^[113][114] heart failure. Mice studies show activation of CD4+ T-cells post-MI is a controlled response designed to subside rapidly with scar formation to achieve complete immune resolution within 2 weeks post-MI. HF, on the other hand, is associated with a second wave of CD4+ T-cell activation, and their transmigration into the heart promotes LV remodeling, end-diastolic volume and end-systolic volume increasing, ejection fraction reduction, and progressive cardiac dysfunction ^[114][115][115,116].

CD8+ T cells play a role in the development of atherosclerosis. When these cells are activated, they release cytotoxins, perforin, and granzymes. The cytotoxins can induce programmed cell death, or apoptosis, in macrophages, vascular smooth muscle cells (VSMCs), and ECs. This contributes to the formation of vulnerable atherosclerotic lesions, which are areas of plaque that can rupture and lead to complications ^[116][117]. Furthermore, the absence of programmed cell death ligand (PDL)-1 and PDL-2 in mice has been shown to increase the development of atherosclerotic lesions in the aorta. It also leads to an increase in the numbers of CD4+ T cells and CD8+ T cells, suggesting that these cells are more involved in atherosclerosis when these molecules are lacking ^[117][118].

Moreover, T Lymphocytes crosstalk with other molecules, such as cyclophilins, have a new role in CAD: these proteins are released into the extracellular space in response to inflammatory stimuli. Gegunde et al. described the involvement of a cell surface receptor for extracellular cyclophilins in CAD, the CD147 receptor: patients with CAD had considerably higher levels of membrane expression of CD147, cyclophilin A, B, and C in T lymphocytes purified from these subjects, as well as pro-inflammatory interleukins ^[118][119].

When briefly exposed to certain stimuli, cells of the innate immune system such as monocytes, macrophages, DC, and NKT cells can develop a phenotype resembling immunologic memory, termed trained immunity ^[119][120]. Upon restimulation, trained cells manifest a long-term proinflammatory phenotype with an increased cytokine release, nonspecific with respect to the original stimulus. The persistent overactivation of these trained cells could contribute to the incessant vascular wall inflammation, a peculiar characteristic of atherosclerosis ^[120][121].

Previous works on trained immunity involved microorganisms and microbial products including the Bacillus Calmette–Guerin (BCG) vaccine, Candida albicansi, and its cell wall component β-glucan ^[121][122].

Exposure to these pathogens provokes an increased production of proinflammatory cytokines and chemokines in trained cells as a response to a secondary insult, even if different from the initial one.

It was later recognized that also endogenous, self-derived molecules such as ox-LDL, catecholamines, uric acid, and aldosterone can induce a persistent functional reprogramming of innate immune cells ^{[122][123][124][125][126]}[123,124,125,126,127].

From an evolutionary perspective, trained immunity confers protection to the host against subsequent infection, although it can also be responsible for a maladaptive state. An exogenous stimulus such as BCG vaccination protects against lethal systemic C albicans infection in immunodeficient mice that lack adaptive immunity ^[127][128], as well as administration of β-glucan in mice confers protection against recurrent infections ^[128][129][129,130]. Similar evidence also exists in humans, with the profound decrease in infant mortality rates following BCG vaccination, not solely explained by the protection against tuberculosis ^[130][131].

Diversely from the protective effect against infections, trained immunity might be maladaptive in chronic inflammatory diseases in which innate immunity cells play a pivotal role in the pathophysiology. The detrimental effects of trained immunity are implicated in atherosclerosis, gout, neurodegenerative disorders, and transplant rejection ^{[131][132][133]}[132,133,134], and in other inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus ^[134][135][135,136].

Trained immunity could also be one of the mechanisms contributing to the epidemiological association between infectious burden and atherosclerotic CV diseases ^[136][137].

Different endogenous, nonmicrobial atherogenic stimuli have been recognized to induce trained immunity, such as ox-LDL and lipoprotein(a) (Lp(a)), but also catecholamines and high glucose concentration ^{[123][137][138][139]}[124,138,139,140]. ox-LDL has a key role in atherogenic plaque formation thanks to its ability to activate immune cells and trigger foam cell formation. When exposed to a low concentration of ox-LDL and restimulated with a TLR agonist, macrophages produce higher quantities of atherogenic cytokines and chemokines, such as IL-6, MCP1 (monocyte chemoattractant protein 1), and TNF-α. Foam cell formation is similarly enhanced after exposure to ox-LDL, due to the overexpression of scavenger receptor-A (SR-A) and CD36 and downregulation of cholesterol efflux transporters adenosine triphosphate-binding cassette transporter-A1 and G1 ^[123][140][124,141].

Lp(a) is the main circulating carrier of oxidized phospholipids and plays a key role in atherogenesis ^[141][142][142,143]. Monocytes incubated with Lp(a) for 24 h show an increased proinflammatory cytokine production compared to untrained controls ^[143][144]. Monocytes isolated from patients with elevated Lp(a) levels also show a stronger trans-endothelial migration.

A pivotal role in diabetes and CV diseases is played by diet, and atherosclerosis-prone knock-out for LDL receptor mice display characteristics of trained immunity when fed a Western-type diet ^[144][145].

The characteristic increase in cytokine production in trained immunity has also been observed in patients with already established coronary atherosclerosis, familial hypercholesterolemia, and in patients with cerebral small vessel disease ^[145][146][146,147]. Similarly, hyperuricemia can induce long-term proinflammatory activation of innate immune cells ^[122][125][123,126].