Inflammation in Myocardial Ischemia/Reperfusion Injury: Underlying Mechanisms and Therapeutic Potential

Acute myocardial infarction (MI) occurs when blood flow to the myocardium is restricted, leading to cardiac damage and massive loss of viable cardiomyocytes. Timely restoration of coronary flow is considered the gold standard treatment for MI patients and limits infarct size; however, this intervention, known as reperfusion, initiates a complex pathological process that somewhat paradoxically also contributes to cardiac injury. Despite being a sterile environment, ischemia/reperfusion (I/R) injury triggers inflammation, which contributes to infarct expansion and subsequent cardiac remodeling and wound healing. The immune response is comprised of subsets of both myeloid and lymphoid-derived cells that act in concert to modulate the pathogenesis and resolution of I/R injury. Multiple mechanisms, including altered metabolic status, regulate immune cell activation and function in the setting of acute MI, yet our understanding remains incomplete. While numerous studies demonstrated cardiac benefit following strategies that target inflammation in preclinical models, therapeutic attempts to mitigate I/R injury in patients were less successful. Therefore, further investigation leveraging emerging technologies is needed to better characterize this intricate inflammatory response and elucidate its influence on cardiac injury and the progression to heart failure.


Introduction: Myocardial Ischemia/Reperfusion Injury
Heart disease, often a result of myocardial infarction (MI), remains the leading cause of mortality worldwide [1][2][3][4].Acute MI results from obstruction of the coronary arteries that supply the myocardium with blood; therefore, timely reperfusion (typically via percutaneous coronary intervention; PCI) is critical to preserving myocardial integrity and is considered the current gold standard treatment for MI patients [1,5].Paradoxically, therapeutic reperfusion causes additional injury through several mechanisms, including rapid changes in pH, Ca 2+ overload, and hyperoxia, leading to altered metabolism, the reversal of surface ion pumps, mitochondrial dysfunction/ROS production, and opening of the mitochondrial permeability transition pore (mPTP) [3,[5][6][7][8][9][10].This presents clinically as myocardial stunning, arrhythmias, and lethal reperfusion injury, in which salvageable cardiomyocytes in the area at risk undergo necrosis and/or additional forms of regulated cell death [3,[7][8][9]11].Thus, reperfusion directly contributes to infarct expansion and is now believed to account for up to half of the total infarct size [1,3,[7][8][9]12].In addition to MI, heart transplantation, which is the only therapeutic option for end-stage heart failure, is another setting for cardiac ischemia/reperfusion (I/R) injury, and I/R injury limits transplant effectiveness [3,5].Despite its recognition as a significant contributor to myocardial damage following ischemia, reperfusion injury remains without approved therapeutic intervention and novel approaches to ameliorate this disease modality are needed [1,3,[7][8][9]12].Although a sterile environment, I/R initiates a complex inflammatory response that plays an important role in modulating the extent of cardiac injury and repair.The objectives of this review are to provide a detailed analysis of inflammatory cell functions as they relate to our current understanding of the pathology of acute MI, and to highlight studies that target inflammation therapeutically for cardiac I/R injury in preclinical models and patients.
The binding of DAMPs to PRRs increases the expression of proinflammatory cytokines and chemokines that recruit innate immune cells from the bone marrow and spleen to the site of injury in a process referred to as sterile inflammation, or inflammation in the absence of pathogens [1,[5][6][7][8][9][12][13][14][15][16][17][18].The engagement of TLR2 and 4, as well as RAGE, promote the activation of NF-κB to upregulate proinflammatory gene expression and prime the NLRP3 inflammasome, while signaling via TLR3 and 9 activate cGAS-STING and NF-κB to propagate a type 1 interferon response [5,10,27,28].Interestingly, cardiac resident cells uniquely contribute to I/R-induced inflammation.Following injury, cardiomyocytes, cardiac fibroblasts, and resident macrophages release inflammatory and chemoattractant molecules, such as IL-1β, TNFα, IL-6, and CCL2, to generate a chemotactic gradient and recruit inflammatory myeloid cells to the infarct region [3,5,10,29].IL-1β also mediates paracrine effects by upregulating the expression of adhesion molecules needed for immune cell extravasation and collagenases in the fibroblast to delay repair, while decreasing cardiomyocyte contractility through L-type channel uncoupling and increased ROS, worsening cardiac outcomes [19,30].The endothelium weakens its cell junctions and upregulates selectins and cell adhesion molecules to facilitate leukocyte extravasation into the injured tissue [13].Cardiac fibroblasts secrete granulocyte-macrophage stimulating factor (GM-CSF) and chemoattractants including CCL2, CCL7, and CXCL1, which stimulates the recruitment of myeloid cells and initiates their proliferation and differentiation in the bone marrow during emergency hematopoiesis [13,20,[31][32][33][34].Taken together, the release of DAMPs during I/R trigger PRR signaling that initiates the inflammatory response in multiple cell types within the myocardium, generating a chemoattractant gradient to promote leukocyte recruitment to the heart [4,32,33] (Figure 1).5) differentiate into inflammatory macrophages, aiding in the clearance of debris and additional leukocyte recruitment via the production of inflammatory cytokines and chemokines.(6) Dendritic cells begin to activate the adaptive immune response by presenting antigens to lymphocytes in mediastinal lymph nodes.(7) The transition to resolution (shown in yellow), is promoted by macrophage efferocytosis of dying neutrophils, which initiates a phenotypic switch toward repair and resolution.(8) Treg release of anti-inflammatory signaling also facilitates resolution.(9) Further recruitment of neutrophils and macrophages now assume a reparative phenotype (shown in green), releasing anti-inflammatory/reparative mediators to further resolve inflammation, as well as pro-angiogenic and fibrotic factors to stimulate repair processes and scar stabilization.(10) Lymphoid cells infiltrate the injured myocardium and can contribute to fibroblast activation and scar formation.(11) The presentation of autoantigens to lymphocytes elicits an autoimmune response, delaying repair in the chronic phase of stable scar formation.Dysregulation of any phase of the immune response results in defective scar formation and impaired cardiac function.Importantly, this sequence of events is accelerated in reperfused MI (I/R) compared to non-reperfused MI.

Neutrophil Priming
The first myeloid cells recruited to the heart after I/R are neutrophils, which are defined as short-lived myeloid-derived granulocytes and represent over half of all human leukocytes in circulation [35][36][37][38][39].Under homeostatic conditions, neutrophils exist in a quiescent state and follow circadian-mediated release from the bone marrow into circulation via the reciprocal regulation of CXCR2/CXCR4 [36][37][38][39].In response to I/R, neutrophils mobilize within minutes to hours along the chemoattractant gradient of cytokines and  9) Further recruitment of neutrophils and macrophages now assume a reparative phenotype (shown in green), releasing anti-inflammatory/reparative mediators to further resolve inflammation, as well as pro-angiogenic and fibrotic factors to stimulate repair processes and scar stabilization.(10) Lymphoid cells infiltrate the injured myocardium and can contribute to fibroblast activation and scar formation.(11) The presentation of autoantigens to lymphocytes elicits an autoimmune response, delaying repair in the chronic phase of stable scar formation.Dysregulation of any phase of the immune response results in defective scar formation and impaired cardiac function.Importantly, this sequence of events is accelerated in reperfused MI (I/R) compared to non-reperfused MI.

Neutrophil Priming
The first myeloid cells recruited to the heart after I/R are neutrophils, which are defined as short-lived myeloid-derived granulocytes and represent over half of all human leukocytes in circulation [35][36][37][38][39].Under homeostatic conditions, neutrophils exist in a quiescent state and follow circadian-mediated release from the bone marrow into circulation via the reciprocal regulation of CXCR2/CXCR4 [36][37][38][39].In response to I/R, neutrophils mobilize within minutes to hours along the chemoattractant gradient of cytokines and cellular debris to the injured heart [16,31,[39][40][41][42][43][44].This rapid status change in response to environmental cues is essential for neutrophil function as an immune first responder [36,37].This "ready" state, known as priming, was shown to enhance neutrophil effector functions, including generation of ROS, release of neutrophil extracellular traps (NETs), degranulation, chemotaxis and adhesion, phagocytosis, synthesis of inflammatory mediators, and survival [36,37].Neutrophil priming is initiated by exposure to DAMPs induced by I/R (e.g., TNFα, GM-CSF) [36,37], and is marked by the shedding of CD62L and subsequent increased surface expression of CD11b, CD18, CD66, and β2 integrins.Priming also mobilizes secretory granules as well as the NOX2 complex to the plasma membrane to facilitate rapid ROS generation upon activation [45].Enhanced NETosis and phagocytotic capacitance were also observed in primed neutrophils, but the underlying mechanisms are not fully understood [36,37].Although previously thought to be terminally differentiated and transcriptionally inactive, it is now appreciated that neutrophils are highly plastic and modulate gene expression governing key effector functions via engagement and activation of transcription factors NF-κB, C/EBP, CREB, HIF-1α, and MYC [35][36][37]46].Primed neutrophils increase the synthesis and subsequent release of inflammatory mediators including IL-1α, IL-1β, IL-6, TNFα, among others.Interestingly, a slow regression of superoxide burst and CD11b upregulation in primed neutrophils over time was observed ex vivo, suggesting the ability to "de-prime", or revert to a quiescent state, perhaps to minimize nonspecific tissue damage [47].However, whether this aspect of neutrophil function can be leveraged therapeutically to mitigate cardiac damage caused by I/R remains unexplored.

Neutrophil Activation
The ability of neutrophils to transition to varied states of activation in vivo, and their resulting heterogeneity in response to MI, has only recently begun to be defined and appreciated.Elegant studies employing single-cell RNA sequencing observed markers of neutrophil priming and activation that are time-dependent in response to MI in mice, including altered CD62L, activation of C/EBP and HIF-1α, and gene expression signatures indicative of proinflammatory status [46,48].Additional evidence was generated in models of ischemic heart failure, including time-dependent alterations in neutrophil gene expression, enhanced release of ROS, NETs, cytokine secretion, and enhanced neutrophil lifespan in infarcted tissue [35][36][37]39,41,46,[49][50][51].Genetic manipulation of neutrophils can also modulate their priming and activation status.In one such study, neutrophils were engineered to express a common gain-of-function JAK2(V617F) mutation.This resulted in elevated basal priming, as indicated by the constitutive phosphorylation of p47phox (subunit of NOX2) in these cells [36,37,51].In response to acute MI, mice expressing mutant JAK2(V617F) had augmented inflammation and enlarged infarcts compared to WT controls, suggesting that enhanced neutrophil priming and activation exacerbates cardiac injury [49].Reduced effector functions demonstrated by unprimed neutrophils coupled with the potential to de-prime these cells highlight the potential of this response for therapeutic intervention and warrants further investigation in I/R injury [36,37].

Neutrophil Polarization and Function in I/R
Neutrophils rapidly infiltrate the heart within minutes of reperfusion, peaking at ~1 day post MI and lasting up to 3-4 days, with decreases detectable around day 5 and later in mouse models [39,41,50].Recently, neutrophils were shown to polarize to heterogeneous phenotypes and subsets within the heart following MI in a time and context-dependent manner, similar to macrophages [39,41,46,48] (Figure 2).In the infarcted heart, neutrophils are activated by local inflammatory factors via PRR engagement, and polarize to a proinflammatory phenotype (sometimes referred to as "N1"), promoting their anti-microbial functions [39,43,52,53].Polarization can also begin in the peripheral blood and the bone marrow in models of MI, a process that may involve neutrophil reverse transmigration to stimulate granulopoiesis and propagate inflammation [35,46].Distinctively, proinflam-matory "N1" neutrophils produce robust levels of ROS, release granules, and deploy chromatin NETs to neutralize perceived threats.Unlike most cells, neutrophils convert superoxide into secondary oxidants through the neutrophil-specific enzyme myeloperoxidase (MPO), the most abundant protein found in neutrophils, which are then used in microbial killing within the phagosome [54].Importantly, release of extracellular granules and their specific components was shown to modulate the severity of the innate immune response by increasing chemotactic and inflammatory cytokines [55,56].The generation of ROS and activation of NOX2 and MPO during phagosome formation also trigger the release and activation of neutrophil elastase (NE), which degrades the nuclear membrane of neutrophils and allows for formation and expulsion of extracellular traps.NETs can also be activated by citrullination of histones via protein arginine deiminase 4 (PAD4), which decondenses neutrophil chromatin and allows for expulsion.Decondensed chromatin released from the nuclear membrane adsorbs granule components and is released extracellularly to trap and prevent the spreading of pathogens [55].
tively, proinflammatory "N1" neutrophils produce robust levels of ROS, release granules, and deploy chromatin NETs to neutralize perceived threats.Unlike most cells, neutrophils convert superoxide into secondary oxidants through the neutrophil-specific enzyme myeloperoxidase (MPO), the most abundant protein found in neutrophils, which are then used in microbial killing within the phagosome [54].Importantly, release of extracellular granules and their specific components was shown to modulate the severity of the innate immune response by increasing chemotactic and inflammatory cytokines [55,56].The generation of ROS and activation of NOX2 and MPO during phagosome formation also trigger the release and activation of neutrophil elastase (NE), which degrades the nuclear membrane of neutrophils and allows for formation and expulsion of extracellular traps.NETs can also be activated by citrullination of histones via protein arginine deiminase 4 (PAD4), which decondenses neutrophil chromatin and allows for expulsion.Decondensed chromatin released from the nuclear membrane adsorbs granule components and is released extracellularly to trap and prevent the spreading of pathogens [55].

Neutrophil Metabolism
Neutrophils can utilize a variety of energetic substrates depending on their environment, an important feature due to the broad range of oxygen and glucose concentrations experienced during cardiac I/R [3,10,16,75].The impact of metabolic status on neutrophil function is therefore of great interest, yet studies directly examining this interaction remain limited [76].In hypoxic environments, neutrophils favor glycolysis to generate ATP needed not only for survival, but also for NET formation and ROS production [77,78].Studies also demonstrated that activated neutrophils upregulate OXPHOS genes and rely upon mitochondrial respiration to generate sufficient ATP necessary for cell migration and chemotaxis, a process shown to be mediated by mTORC1/2 signaling [79][80][81].However, evidence linking electron transport chain function to neutrophil migration largely derives from bacterial infection studies, and whether this mechanism occurs during sterile inflammation, e.g., I/R, or if additional neutrophil effector functions are dependent upon altered metabolic status requires further investigation.Taken together, neutrophil polarization to inflammatory or reparative phenotypes is a spatially and temporally regulated mechanism during I/R, and their functional heterogeneity modulates the extent of myocardial injury and wound healing.As technological advances allow for more nuanced evaluation of neutrophil transcriptional, functional, and metabolic heterogeneity, more work is required to determine if and how these processes may integrate to regulate cardiac inflammation and injury caused by I/R.

Macrophages 4.1. Cardiac-Resident Macrophages
Macrophages are the most abundant leukocyte found in the heart and play a critical role in cardiac development, homeostasis, and injury responses [10,13,[82][83][84][85][86][87][88][89].Recent advances in sequencing and lineage tracing technologies demonstrated cardiac macrophage heterogeneity at steady state and following injury, revealing distinct ontologies, regenerative capacitance, surface markers, and spatial and functional niches [4,13,82,[84][85][86][87]90].At a basic level, macrophages can be classified as resident or recruited cells [13,82,[84][85][86][87]91].Cardiac-resident macrophages originate from the yolk sac and fetal liver during development, whereas recruited macrophages are derived from peripheral monocytes and are generally denoted as CCR2 + [13,82,[84][85][86][87]91].CCR2 − resident macrophages are maintained through self-renewal and can be further classified by their relative expression of MHCII, TimD4, and Lyve1.These tissue-resident macrophages perform homeostatic functions within the heart.They are important for the proper patterning of coronary and lymphatic vessels during development, they facilitate cardiomyocyte conduction, phagocytosis of dysfunctional ejected mitochondria (exophers) and cellular debris, regulate angiogenesis, and provide defense against pathogens [13,82,[84][85][86][87][90][91][92][93].Importantly, humans and mice demonstrate significant conservation in cardiac-resident macrophage subset markers and function [88].Following acute MI, some resident macrophages die; however, the surviving subset of these cells responds to injury and proliferates (far fewer perish if reperfusion is provided [94]).Importantly, cardiac-resident macrophages also negatively regulate the recruitment of monocytes to the heart, thereby antagonizing the inflammatory process [13,82,85,90,95] (Figure 3).Indeed, depletion of cardiac-resident macrophages through genetic targeting approaches worsened pathological remodeling in response to MI, suggesting cardioprotective effects of these cells in response to heart injury [90,95].Thus, the resident macrophages within the myocardium are a unique cell subpopulation with distinct functional states that are essential to cardiac development, homeostasis, and stress responses.In the developing and homeostatic heart, self-renewing resident macrophages (CCR2 -) contribute to the formation and maintenance of the vasculature, electrical conduction, and phagocytosis of dysfunctional organelles in the myocardium.These macrophages can be subclassified by the expression of MHCII, LYVE1, and TimD4 on their cell surface.In response to injury, monocyte-derived macrophages (CCR2 + ) are recruited to the heart, where they further leukocyte infiltration during inflammation, clear dead cells and debris, and modulate stable scar formation during repair.Polarization and metabolism: DAMPs and inflammatory cytokines polarize recruited monocytes into inflammatory macrophages through activation of PRRs (e.g., TLR2/4).Upregulation of inflammatory genes (HIF-1α) and glucose transporters (Glut1) enhance glycolysis and suppress oxidative phosphorylation (OXPHOS).This results in the accumulation of succinate and activation of the pentose phosphate pathway (PPP), increasing ROS production.Activation of the inflammasome releases inflammatory and chemoattractant molecules, furthering inflammation.MerTK-mediated efferocytosis of dead cells, including neutrophils containing NGAL, as well as the binding of anti-inflammatory cytokines, initiates a phenotypic switch in the macrophage toward reparative function.Enhanced OXPHOS and the subsequent increase in itaconate and NAD + suppresses inflammatory gene expression (HIF-1α) and effector functions (ROS, inflammasome).Reparative macrophages express more CD206, YM-1, and FRP2, and release mediators that contribute to revascularization and stable scar formation.

Cardiac Recruited Macrophages
In response to acute MI, there is a rapid and robust increase in circulating pro-inflammatory (CCR2 + ) monocytes derived from the bone marrow and spleen [4,10,13,82,[84][85][86][87]90,95].Studies in mice demonstrated that monocytes arriving soon after MI differentiate into pro-inflammatory macrophages, whereas monocytes recruited later in the immune response differentiate into macrophages that favor reparative phenotypes [4,10,13,82,86,87,90,95].Recruited CCR2 + macrophages are mobilized to the injured heart during both reperfused (I/R) and non-reperfused MI models; however this process is accelerated by reperfusion, with recruited macrophages present in the heart by 24 h post contribute to the formation and maintenance of the vasculature, electrical conduction, and phagocytosis of dysfunctional organelles in the myocardium.These macrophages can be subclassified by the expression of MHCII, LYVE1, and TimD4 on their cell surface.In response to injury, monocytederived macrophages (CCR2 + ) are recruited to the heart, where they further leukocyte infiltration during inflammation, clear dead cells and debris, and modulate stable scar formation during repair.Polarization and metabolism: DAMPs and inflammatory cytokines polarize recruited monocytes into inflammatory macrophages through activation of PRRs (e.g., TLR2/4).Upregulation of inflammatory genes (HIF-1α) and glucose transporters (Glut1) enhance glycolysis and suppress oxidative phosphorylation (OXPHOS).This results in the accumulation of succinate and activation of the pentose phosphate pathway (PPP), increasing ROS production.Activation of the inflammasome releases inflammatory and chemoattractant molecules, furthering inflammation.MerTK-mediated efferocytosis of dead cells, including neutrophils containing NGAL, as well as the binding of antiinflammatory cytokines, initiates a phenotypic switch in the macrophage toward reparative function.Enhanced OXPHOS and the subsequent increase in itaconate and NAD + suppresses inflammatory gene expression (HIF-1α) and effector functions (ROS, inflammasome).Reparative macrophages express more CD206, YM-1, and FRP2, and release mediators that contribute to revascularization and stable scar formation.

Macrophages in Resolution
As the inflammatory process progresses, the efferocytosis of dead neutrophils, cardiomyocytes, and other parenchymal cells, as well as contribution from the adaptive immune system, induce a phenotypic switch and favor a reparative macrophage state, formerly referred to as alternatively activated macrophages [4,84,86,87,96,98,99].Reparative macrophages can be induced by IL-4 and IL-13, among other factors, which leads to the upregulation of molecules important for wound healing (e.g., Arg1, CD206, VEGF, IGF-1, and YM1) via the activation of transcription factors MYC, PPARγ, STAT3, and others [4,84,86,87,96].In addition, monocyte-derived macrophages that are recruited during the later phase of MI downregulate Ly6C expression and polarize to a reparative phenotype (~4-7 days post I/R) [4,84,86,87,96].The secretion of pro-resolving mediators from these macrophages, such as IL-4, IGF-1, and TGFβ, act in a paracrine fashion to further propagate reparative macrophage polarization, as well as elicit cardiac fibroblast differentiation into myofibroblasts, thereby mediating extracellular matrix remodeling and subsequent scar formation after MI [4,84,86,87,96].Reparative macrophages also produce angiogenic and lymphogenic factors, such as VEGFA and VEGFC, respectively, to promote revascularization and repair of the damaged myocardium after infarction [92].

Macrophage Metabolism and Functional Regulation
As alluded to above, the infarcted heart is a hostile environment with extreme ranges of available oxygen, nutrients, and metabolites.Inflammatory monocytes mobilized acutely during MI are highly dependent upon glycolysis, and increased glycolytic flux in monocytes is correlated with increased secretion of inflammatory cytokines, increased infarct size, and decreased systolic function in human MI patients [100,101].Glucose uptake and greater dependence on glycolysis relative to oxidative respiration provide the energy necessary for proliferation, inflammatory cytokine production, generation of ROS, and adhesion and transmigration of monocytes into the heart following MI.This reliance on glycolysis continues as inflammatory monocytes differentiate into CCR2 + macrophages.Hypoxic conditions that result from ischemia activate macrophage HIF-1α, which upregulates glucose transporters (e.g., Glut1), as well as glycolytic enzymes (e.g., PDK1, hexokinase, 6-PFK), all of which favor glycolysis [13,84,87,95,[100][101][102].This also activates the pentose phosphate pathway (PPP), which provides NADPH needed for ROS generation through NADPH oxidase.Moreover, HIF-1α positively regulates the production of IL-1β and stimulates the proteolysis and inhibition of the phagocytic receptor MerTK, which is needed for reparative macrophage function [103].Preventing HIF-1α-mediated glycolysis attenuated these responses [104,105].Inflammatory CCR2 + macrophages also exhibit impaired TCA cycle and oxidative phosphorylation, which causes the accumulation of TCA metabolites that can influence cell behavior.In particular, succinate is oxidized during reperfusion, leading to the reversal of electron transport chain complex I and increased ROS, further stabilizing HIF-1α and exacerbating inflammatory conditions [106,107].
In contrast to proinflammatory CCR2 + cardiac macrophages, reparative macrophages are associated with mitochondrial OXPHOS, although this is an oversimplification and both oxidative respiration and glycolysis can influence pro-and anti-inflammatory mechanisms [108].Following MI, genes related to mitochondrial respiration are upregulated and several mitochondrial-related metabolic intermediates were shown to promote macrophagereparative functions.For example, TCA cycle-derived itaconate antagonizes the oxidation of succinate and blunts complex I-generated ROS to suppress inflammation.Itaconate was also shown to attenuate inflammasome function, and itaconate supplementation in vivo was demonstrated to reduce infarct size and subsequent adverse cardiac remodeling [109][110][111].The TCA cycle also generates NADH, which when oxidized, produces NAD + , a potent anti-inflammatory metabolite.Indeed, NAD + levels are reduced in heart failure and supplementation of NAD + or precursors that increase NAD + levels were shown to protect against cardiac I/R injury and increase reparative macrophage populations [112][113][114][115]. NAD + is inhibitory against HIF-1α and can promote its degradation, thereby favoring macrophage reparative polarization [116].
In response to efferocytosis, macrophages direct excess lipids to mitochondria for increased fatty acid oxidation and mitochondrial respiration, which activates PGC-1β and elevates NAD + levels, leading to a reparative phenotype [117].Consistent with these findings, disruption of the electron transport chain complex I function in the myeloid compartment resulted in an augmented inflammatory response and worsened cardiac injury after MI [118].The process of removing dying cells also induces the synthesis of IL-10, another pro-reparative cytokine that plays an important role in resolution and wound healing [73].Efferocytosis can also stimulate glycolysis and cause increased lactate in the macrophage.Interestingly, lactate can be secreted to elicit paracrine effects in neighboring cells that promote macrophage anti-inflammatory polarization [119].Together, these findings support the concept that selective activation of metabolic pathways and the relative enrichment of individual metabolites have the capacity to alter macrophage polarization and function, thus impacting cardiac outcomes following injury.How this regulation might be impacted by cardiovascular comorbidities, such as aging and obesity/metabolic syndrome, and whether macrophage metabolism can be harnessed for therapeutic benefit, remains unclear and warrants future investigation.

Dendritic Cells
Cardiac resident myeloid cells also include dendritic cells (DCs), which coordinate both the innate and adaptive immune responses via antigen presentation and cytokine release [88,120].Similar to macrophages, DCs are heterogeneous and consist of two major subsets within the heart, conventional (cDC) and plasmacytoid (pDC), which can be selectively targeted in vivo [120][121][122][123].In the context of acute MI, both cDCs and pDCs expand and activate within the myocardium, and global depletion of DCs worsened MI-induced remodeling and dysfunction, suggesting that a component of DC function contributes to wound healing [122].However, the selective individual inhibition of either subtype was shown to be cardioprotective, indicating a deleterious role for DCs, although particular subtype contributions may differ in the absence of reperfusion [120,121,123].pDCs were shown to release type I interferons early after I/R, and their depletion afforded cardiac benefit [120].cDCs contribute to pathological remodeling post MI, presumably through alterations in macrophage and Treg recruitment, as demonstrated by selective cDC depletion [121,122,124].Importantly, dendritic cells specialize in antigen presentation and further modulate MI injury through auto-reactive T cell activation [125][126][127].For example, necrotic cardiomyocytes release α-myosin heavy chain, which is presented to T cells by DCs in the mediastinal lymph nodes or circulating blood, resulting in heart-specific autoimmune responses that contribute to prolonged inflammation and exacerbate I/R injury [125,128] (Figure 4).specific autoimmune responses that contribute to prolonged inflammation and exacerbate I/R injury [125,128] (Figure 4).During steady state, DC metabolism relies mainly on fatty acid oxidation and OXPHOS; however, following TLR-mediated activation, DC metabolism shifts to glycolysis [129,130].This transition promotes inflammatory cytokine secretion and antigen presentation [129].Intriguingly, DC subsets display distinct metabolic profiles upon activation, as cDCs favor glycolysis and pDCs favor oxidative phosphorylation [131].How these differences in energetics might modulate inflammatory behaviors in DC subsets, and whether this contributes to functional differences in MI injury and remodeling remains largely unknown.However, given the implied importance of DCs in heart disease During steady state, DC metabolism relies mainly on fatty acid oxidation and OX-PHOS; however, following TLR-mediated activation, DC metabolism shifts to glycolysis [129,130].This transition promotes inflammatory cytokine secretion and antigen presentation [129].Intriguingly, DC subsets display distinct metabolic profiles upon activation, as cDCs favor glycolysis and pDCs favor oxidative phosphorylation [131].How these differences in energetics might modulate inflammatory behaviors in DC subsets, and whether this contributes to functional differences in MI injury and remodeling remains largely unknown.However, given the implied importance of DCs in heart disease after injury, further elucidation of mechanisms underlying their activity and interactions with other immune cell types may uncover novel therapeutic targets to improve cardiac outcomes after I/R.

T Cells
T cells are lymphoid derived and make up ~25% of the non-myeloid resident leukocytes in the myocardium [13,100].T cells are broadly categorized as CD4+ T helper and CD8+ cytotoxic T, with subset classification into multiple effectors, including regulatory T cells (Tregs) [127].Similar numbers of CD4+ and CD8+ T cells were observed in the healthy myocardium [132], although their relative contribution to cardiac homeostasis remains elusive.Following acute MI, a subset of T cells infiltrates the heart prior to antigen activation [133], and contributes to injury by secreting inflammatory cytokines, enhancing leukocyte recruitment, and expanding the infarct [4,134,135].Interestingly, antibody depletion of CD4+, but not CD8+, T cells immediately after I/R improved cardiac outcomes, which was attributed to suppressed interferon gamma secretion and reduced neutrophil recruitment [135].However, in non-reperfused MI, CD8+ antibody depletion was shown to be cardioprotective by suppressing granzyme B-mediated cardiomyocyte apoptosis [136].As mentioned above, dendritic cell-mediated antigen presentation to T cells elicits activation of the adaptive immune response and initiates autoimmunity against the heart, prolonging the inflammatory response and causing additional cardiac damage [4,127,[137][138][139].On the other hand, Tregs demonstrated cardioprotective effects that include restraining CD4+ and CD8+ inflammatory effector functions during acute MI and the subsequent remodeling process [137,[140][141][142]. Tregs, similar to other T cells, accumulate in the heart post MI, yet have distinct transcriptional profiles acquired by activation in the inflamed myocardium that favor a reparative phenotype.After ischemic insult, Tregs were shown to suppress myeloid cell recruitment, as Treg depletion increased inflammatory monocytes and neutrophils in the infarct zone [143].Upon activation by DCs, Tregs also facilitate wound healing and scar formation, and prevent rupture, via the activation of resident cardiac fibroblasts and increased reparative macrophage polarization after MI [142][143][144].Furthermore, depletion of FoxP3 + Tregs enhanced cardiac autoimmunity after I/R, indicating that Tregs normally protect the heart by actively suppressing autoreactivity [141].Finally, Tregs may also provide cardioprotection during ischemic heart injury by promoting cardiomyocyte proliferation, demonstrated by Treg supplementation post MI [145,146] (Figure 4).
T cell metabolism and potential functional implications, in the context of ischemic heart disease, is currently an active area of investigation, and thus far appears to play an important regulatory role in T cell differentiation and effector functions [100].Similar to macrophages, an upregulation of glucose transporters and a metabolic preference for glycolysis promotes CD4+ T cell expansion and production of interferon gamma, as well as the expression of granzyme B in CD8+ T cells [147,148].Conversely, Tregs express lower levels of Glut1 and exhibit greater fatty acid oxidative and mitochondrial respiration, suggesting that reduced reliance on glycolysis may underly the anti-inflammatory function in these cells [100,148,149].Given the distinct transcriptional and metabolic profiles exhibited by cardiac T cells during ischemic injury, it will be important to determine whether metabolic pathway preferences modulate T cell differentiation, function, and potentiate T cell-mediated autoimmunity following I/R.

B Cells
B cells are one of the most abundant leukocyte populations in the healthy myocardium, yet our understanding of B cell function in the heart, during both homeostatic conditions and following injury, remains relatively limited [127,132,150].For example, the role of cardiac resident B cells in maintaining cardiac homeostasis is largely unexplored; however, prior work suggests that B cells can regulate MHCII expression on resident macrophages in the heart [151].B cell numbers expand after MI [127,150,152,153] and are thought to play a role in acute injury and inflammation, as well as autoimmune responses during chronic remodeling [127,150,152,153].Depletion studies demonstrated that the B cell inhibition attenuated inflammatory myeloid cell infiltration and subsequent pathological cardiac remodeling due to repressed CCL7 secretion after MI [153].Additionally, the production of autoantibodies by B cells after I/R was shown to contribute to enhanced cardiomyocyte loss and neutrophil infiltration [154].In contrast, studies in mice that overproduce B cells also demonstrated preserved cardiac function post MI [155], and adipose-derived pericardial B cells were shown to secrete cardioprotective IL-10 [156].These findings demonstrate the pleiotropic nature of these lymphocytes and highlight the need to further define the complex mechanisms underlying the role of B cell function during cardiac injury (Figure 4).Recent work found that metabolic status can influence B cell function.For example, activation of the B cell receptor increases glycolysis, and subsequent autoantibody and cytokine production in B cells appears to be energetically supported by glycolysis [100,157], whereas production of IL-10 relies primarily on fatty acid oxidation [158].Moreover, differentiated B cells (plasma cells) were shown to favor fatty acid oxidation relative to glucose utilization for enhanced survival; however, whether this mechanism is conserved in ischemic heart disease remains unknown [159].

Translational Potential of Targeting Inflammation during I/R Injury
Inflammation plays a fundamental role in modulating I/R-induced injury and wound healing and is therefore of great interest for potential therapeutic intervention.Multiple lines of reasoning provide a compelling rationale for modulating post-I/R inflammatory cascades for cardiac benefit.These include reduced leukocyte-mediated death of vulnerable cardiomyocytes at the infarct border, reduced ECM remodeling and strengthened scar formation, enhanced angiogenic effects, potential reduction in arrhythmogenic incidence, and protection against future coronary events and recurrent MI in humans.Moreover, some standard treatments for MI patients, e.g., β-adrenergic receptor antagonists, may have inherent anti-inflammatory functions that contribute to their therapeutic efficacy [160].Many preclinical studies leveraged our understanding to target inflammatory processes, and showed promise for ameliorating infarction and subsequent pathological remodeling and cardiac dysfunction (Table 1).However, interventions aimed at depleting entire populations of cells (e.g., neutrophils or macrophages [53,161]) or employing broad inhibitors of inflammatory processes had limited success-probably because these cells and processes have pleiotropic effects in the inflamed myocardium.Implementing strategies to effectively target inflammation clinically remains challenging due to multiple translational barriers and will likely require enhanced precision and better understanding of patient heterogeneity.
Table 1.Studies targeting inflammation in preclinical models of I/R.A representative list of relevant small animal studies that modulated inflammatory pathways and assessed cardiac injury and/or function following I/R.Findings from most preclinical studies suggest that attenuating pro-inflammatory responses generally provides cardiac benefit during I/R.↓, reduced compared to control.↑, increased compared to control.

Broad Approaches to Inflammatory Inhibition
Glucocorticoids have wide-ranging effects on many cell types, and were shown to both promote and antagonize inflammatory function depending on the cellular target and status of the injury environment [179].Early studies noted a benefit of glucocorticoid administration for reducing acute MI injury in preclinical animal models; however, subsequent studies reported mixed results, in some cases worsening cardiac outcomes [180][181][182].This may be the result of the ability of glucocorticoids to signal via both glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs), thereby affecting a broad range of cell types to elicit systemic effects [183].Glucocorticoids were also shown to signal through MRs to enhance inflammatory mediators [184,185], and high dose treatment led to dysregulation of macrophage clearance of cellular debris from the infarct site and impaired fibroblast function, leading to compromised scar formation [182].Importantly, results from the clinical application of glucocorticoids for MI patients did not demonstrate convincing effectiveness overall, and several studies raised safety concerns, making this broad approach to limit inflammation largely unattractive [186].
Nonsteroidal anti-inflammatory drugs (NSAIDs) also act to broadly repress inflammatory responses through the inhibition of COX enzymes.Similar to findings employing glucocorticoids, NSAID administration showed mixed results in preclinical studies, with some reports of protection against MI-induced cardiomyocyte death and adverse remodeling, and others reporting worsening cardiac dysfunction, scar thinning, and a protective effect of COX-2 during I/R injury [187][188][189][190][191]. Attempts to translate this approach to patients were largely unsuccessful, with no clear benefit for MI reduction observed, and an increased risk of death and recurrent MI [192,193].These adverse outcomes may be due to local and/or systemic effects including altered blood pressure, atherogenic predisposition, attenuated repair processes, or enhancement of arrhythmogenic events [194].
Cyclosporine A (CsA) is an immunosuppressant that targets and inhibits the function of cyclophilin D, thereby preventing the opening of the mitochondrial PTP and cell death [195].In preclinical animal models of I/R, CsA was somewhat effective at reducing injury and potentially cardiac inflammation, although results were inconsistent [196,197].Early pilot results in MI patients administered CsA showed promise [198]; however, followup investigation did not find a significant benefit when combined with PCI therapy [199].
Ischemic conditioning (IC) is a mechanical intervention for producing multiple cycles of non-lethal ischemia followed by reperfusion.IC can be applied directly to the coronary artery (in the case of MI) to elicit endogenous cardiac benefit, or to vasculature/organs remote from the heart, referred to as remote ischemic conditioning (RIC).Since its discovery more than three decades ago [200], IC has been shown to effectively protect the myocardium against I/R injury in a variety of preclinical models [201].The underlying mechanisms that afford cardioprotection are thought to consist of the reperfusion injury salvage kinase (RISK) pathway, the survivor activating factor enhancement (SAFE) pathway, and the PKC-NO-PKG pathway [202].Recent work also implicated RIC as a modulator of inflammatory signaling in response to I/R, which may facilitate additional myocardial protection.Preclinical evidence from studies in mice, rats, and rabbits demonstrate attenuated pro-inflammatory cytokines (e.g., TNFα, IL-1β, and IL-6) and inflammatory mediators (e.g., TLR4, HMGB1, and ICAM-1) when animals are subjected to RIC compared to I/R without treatment [203][204][205][206][207]. Unfortunately, the robust protective effect of IC is not consistently observed in MI patients, and large randomized controlled trials have been largely inconclusive, with no improvement in clinical outcomes after one year [208][209][210].Discrepancies in clinical observations could be due to limitations inherent to animal models of acute MI [211], as well as our limited understanding of mechanisms that convey the cardiac benefits of RIC.In this regard, results from the clinical administration of RIC in coronary artery bypass graft (CABG) patients did not demonstrate a clear effect on inflammatory mediators, and whether this approach modulates inflammation caused by I/R in humans requires additional investigation [212][213][214][215].

Focused Targeting of Inflammatory Cells 8.2.1. The Complement Pathway
The complement cascade is activated by DAMPs following I/R and plays a role in modulating both the extent of injury and the inflammatory response in the infarcted heart [26,216].Inhibition of the complement pathway in both small and large animal preclinical studies demonstrated cardioprotection against acute MI [163,164,217].Attempts to translate these findings to the clinic, however, have not been successful.For example, treatment with pexelizumab to target complement inhibition in STEMI patients did not improve infarct size, mortality, or heart failure development [218][219][220].

Targeting Immune Cell Recruitment and Adhesion
As discussed above, there are several mechanisms that modulate the recruitment of neutrophils, monocytes/macrophages, and lymphocytes to the injured myocardium post I/R.Attempts have been made to target these mediators and disrupt normal migration, adhesion, and extravasation, in an effort to reduce inflammatory damage and increase cardioprotection.Inhibition of certain chemokine function demonstrated limited cardiac benefit in small animal studies.Blockade of CCL2 or CCL5 improved post-MI remodeling and cardiac function, presumably through attenuation of proinflammatory cell recruitment to the injured heart [221,222].Similar results were reported using an RNAi approach to deplete CCR2, which impaired recruitment of inflammatory monocytes and reduced infarct size in mice [223].Of course, manipulating CC chemokines may also interfere with recruitment of immune cells that provide salutary effects.Indeed, CCR5 knockout mice demonstrated worsened cardiac remodeling after I/R [224], which may be a result of impaired inflammatory resolution, and highlights the delicate balance that should be maintained to limit injury and maximize wound healing.
Integrins and selectins are important for leukocyte adhesion to, and extravasation through, the endothelium following I/R.Strategies that leveraged antibodies to neutralize these mediators demonstrated cardioprotection and reduced infarct size post I/R in preclinical models [225][226][227].Moreover, concomitant depletion of multiple cell adhesion molecules using nanoparticle-mediated administration of siRNA was effective at improving cardiac function following MI in mice [228], indicating the potential to target adhesion therapeutically.Despite these findings, however, clinical trial results of anti-adhesion molecule treatments (anti-CD11/CD18) in MI patients have been underwhelming and did not afford infarct size reduction [229][230][231].Administration of the P-selectin antagonist inclacumab for MI showed more promise and may provide modest protection against cardiac damage; however, no difference in adverse events was observed between treatment regimens [232].

Targeting Immune Cell Function and Inflammatory Mediators
Recent preclinical and clinical data suggest that targeting the bioactive molecules that are produced by inflammatory immune cells can provide benefit to the injured heart.The IL-1β/IL-1R pathway has emerged as an intriguing therapeutic target for treatment of I/R injury.Studies in mice demonstrated that either inhibiting IL-1β activity via treatment with anti-IL-1β neutralizing antibodies, or preventing IL-1R signaling using the receptor antagonist anakinra, can attenuate post-MI remodeling and improve heart function [233,234].Importantly, this benefit may extend to patients, as administration of anakinra demonstrated significant reductions in C-reactive protein (CRP), death, and hospitalization [235][236][237][238]. Results from the CANTOS trial, which leveraged IL-1β neutralization by canakinumab in patients with previous MI, are also positive and demonstrate reduced markers of inflammation and reduced cardiovascular events and hospitalization [239][240][241].However, risk of infection was increased, and the long-term safety profile of this approach requires continued study.The production of mature IL-1β is dependent upon the inflammasome, a multi-protein complex that is highly expressed in leukocytes, as well as in fibroblasts and to a lesser extent, cardiomyocytes [242].Small molecule inhibitors that target inflammasome function have been used in preclinical MI models and shown to protect against I/R injury [170][171][172]233,243], yet examination of the therapeutic potential of this approach in patients has only recently begun [244] and warrants further investigation.
Colchicine is an anti-inflammatory drug that was shown to dampen inflammation in patients with MI [245].Colchicine disrupts microtubule networks and negatively regulates the migration and infiltration of neutrophils following injury, which is thought to be a prominent mechanism providing immunosuppression [246].Additional evidence implicates colchicine as an inhibitor of inflammasome function, which may also contribute to the attenuation of inflammatory burden [247].Pilot evaluation of colchicine for treatment of acute MI in patients found a reduction in cardiac injury [248].Results from the subsequent larger COLCOT study, which also administered colchicine to patients after acute MI, demonstrated a reduction in serious adverse events [245].These data also indicate that colchicine may delay progression of heart failure; however, no reduction in infarct size was observed in this patient population, suggesting colchicine may provide benefit by modulating maladaptive remodeling post I/R.
Inhibitory targeting of IL-6, a prominent pro-inflammatory cytokine involved in reperfusion injury and heart failure [249], is an active area of research for the treatment of acute MI.Preclinical studies have demonstrated that blocking IL-6 signaling affords cardioprotection against I/R injury, including infarct reduction and preservation of cardiac function [250].These promising results, however, are yet to be fully realized clinically.Administration of the IL-6 receptor antagonist tocilizumab reduced markers of cardiac injury in patients with acute MI, including troponin and CRP, as well as leukocyte counts, yet failed to reduce infarct size [251][252][253].While these initial studies demonstrate promise for this approach, additional evaluation is needed in larger patient cohorts.

Conclusions and Future Perspectives
Advancements in treatments have significantly improved the survival of patients experiencing acute MI, yet there remains a great need for more effective therapies to further reduce cardiac ischemia and reperfusion injury and prevent adverse remodeling and accompanying heart failure.Inflammation plays a critical role in modulating cardiac injury and wound healing, and is therefore an attractive process for targeting novel therapies.However, the inflammatory response within the injured myocardium is complex, involving multiple cell types that exhibit pleiotropic effects and interact in multifaceted ways to dictate the timing and extent of damage and wound healing.Due to the intricate nature of these cellular and molecular responses, future studies will likely benefit from our growing detailed understanding of immune cell subpopulations during I/R injury and technological advances allowing for more precise targeting of therapeutic interventions for selective cell types and/or molecules at specific time points.
While the majority of interventions aim to suppress the pro-inflammatory aspects of cardiac inflammation after MI, it has also been implied that the ability to resolve inflammation, i.e., negatively regulate pro-inflammatory function, may be impaired in some patients and could contribute to pathogenesis of I/R injury and subsequent remodeling [254].In line with this approach, preclinical studies have investigated the therapeutic potential of promoting reparative properties of immune cells post injury to confer cardiac benefit [255].As mentioned above, SPMs signal through the FRP2 receptor to polarize macrophages and neutrophils toward a resolving phenotype and stimulate wound healing.A recent study found that treatment of rats with BMS-986235, a FRP2 agonist, increased phagocytosis and neutrophil clearance, and improved post-MI remodeling and heart function [256].It is possible that future therapies will incorporate multiple aspects of immunomodulation in conjunction for added benefit, yet must also consider additional challenges involving inflammatory targeting for patients with MI.The timing of cellular actions and production of inflammatory mediators is critical, and interventions that disrupt early processes could unintentionally impact later responses and worsen outcomes.Therefore, further elucidating the time course of myocardial inflammation is likely to focus the effective window for therapeutic intervention.Moreover, patients are highly diverse and their susceptibility to inflammation and responsiveness to treatments can vary depending on age, gender, medical co-morbidities, genetic background, as well as other factors.In addition, preclinical work is performed largely in rodent models, which are typically young, healthy, and lack co-morbidities often present in humans.New treatments are usually not tested in combination with standard care regimens that patients with cardiovascular disease commonly rely on, which may also impact responses.Despite these challenges, results from some recent trials targeting inflammation for acute MI show promise and reaffirm the potential of manipulating the immune environment for myocardial salvage and improved patient prognosis.

Figure 1 .
Figure 1.Overview of the immune response to ischemic heart injury (1) After acute MI, damageassociated molecular patterns (DAMPs) are released from the infarcted myocardium and initiate the immune response.(2) Neutrophils are rapidly recruited from the circulation, and upon arrival to the injured myocardium, polarize to an inflammatory phenotype, initiating the inflammatory phase (shown in red).(3) Inflammatory neutrophils release ROS, NETs, inflammatory cytokines/chemoattractants via degranulation, and begin clearing debris.(4) Recruited monocytes arrive at the site of injury and (5) differentiate into inflammatory macrophages, aiding in the clearance of debris and additional leukocyte recruitment via the production of inflammatory cytokines and chemokines.(6) Dendritic cells begin to activate the adaptive immune response by presenting antigens to lymphocytes in mediastinal lymph nodes.(7) The transition to resolution (shown in yellow), is promoted by macrophage efferocytosis of dying neutrophils, which initiates a phenotypic switch toward repair and resolution.(8) Treg release of anti-inflammatory signaling also facilitates resolution.(9) Further recruitment of neutrophils and macrophages now assume a reparative phenotype (shown in green), releasing anti-inflammatory/reparative mediators to further resolve inflammation, as well as pro-angiogenic and fibrotic factors to stimulate repair processes and scar stabilization.(10) Lymphoid cells infiltrate the injured myocardium and can contribute to fibroblast activation and scar formation.(11)The presentation of autoantigens to lymphocytes elicits an autoimmune response, delaying repair in the chronic phase of stable scar formation.Dysregulation of any phase of the immune response results in defective scar formation and impaired cardiac function.Importantly, this sequence of events is accelerated in reperfused MI (I/R) compared to non-reperfused MI.

Figure 1 .
Figure 1.Overview of the immune response to ischemic heart injury (1) After acute MI, damageassociated molecular patterns (DAMPs) are released from the infarcted myocardium and initiate the immune response.(2) Neutrophils are rapidly recruited from the circulation, and upon arrival to the injured myocardium, polarize to an inflammatory phenotype, initiating the inflammatory phase (shown in red).(3) Inflammatory neutrophils release ROS, NETs, inflammatory cytokines/chemoattractants via degranulation, and begin clearing debris.(4) Recruited monocytes arrive at the site of injury and (5) differentiate into inflammatory macrophages, aiding in the clearance of debris and additional leukocyte recruitment via the production of inflammatory cytokines and chemokines.(6) Dendritic cells begin to activate the adaptive immune response by presenting antigens to lymphocytes in mediastinal lymph nodes.(7) The transition to resolution (shown in yellow), is promoted by macrophage efferocytosis of dying neutrophils, which initiates a phenotypic switch toward repair and resolution.(8) Treg release of anti-inflammatory signaling also facilitates resolution.(9) Further recruitment of neutrophils and macrophages now assume a reparative phenotype (shown in green), releasing anti-inflammatory/reparative mediators to further resolve inflammation, as well as pro-angiogenic and fibrotic factors to stimulate repair processes and scar stabilization.(10)Lymphoid cells infiltrate the injured myocardium and can contribute to fibroblast activation and scar formation.(11)The presentation of autoantigens to lymphocytes elicits an autoimmune response, delaying repair in the chronic phase of stable scar formation.Dysregulation of any phase of the immune response results in defective scar formation and impaired cardiac function.Importantly, this sequence of events is accelerated in reperfused MI (I/R) compared to non-reperfused MI.

Figure 2 .
Figure 2. Neutrophil activation and polarization in myocardial I/R injury.Priming and activation: Under homeostatic conditions, quiescent neutrophils are released from the bone marrow into circulation and return to the bone marrow via reciprocal expression of CXCR2 and CXCR4, respectively, under circadian control.During injury, DAMPs prime neutrophils is in circulation, resulting in (1) the shedding of L-selectin, (2) the movement of NOX2 and integrins to the plasma membrane, and (3) the upregulation of inflammatory gene expression.At the site of injury, fully activated neutrophils elicit effector functions, including the release of ROS, NETs, granules/cytokines, and phagocytosis of pathogens, or in the case of I/R, cellular debris.Polarization: During I/R, DAMPs activate PRRs and promote neutrophil polarization toward an inflammatory phenotype.PRR signaling stimulates activation of the inflammasome and release of granules and inflammatory cytokines.Myeloperoxidase (MPO) is also activated, which in turn upregulates NOX2 and NE function, increasing ROS generation both within phagosomes and extracellularly.NE and PAD4 mediate the formation and release of neutrophil extracellular traps (NETs), which contribute to microvascular dysfunction

Figure 2 .
Figure 2. Neutrophil activation and polarization in myocardial I/R injury.Priming and activation: Under homeostatic conditions, quiescent neutrophils are released from the bone marrow into circulation and return to the bone marrow via reciprocal expression of CXCR2 and CXCR4, respectively, under circadian control.During injury, DAMPs prime neutrophils is in circulation, resulting in (1) the shedding of L-selectin, (2) the movement of NOX2 and integrins to the plasma membrane, and (3) the upregulation of inflammatory gene expression.At the site of injury, fully activated neutrophils elicit effector functions, including the release of ROS, NETs, granules/cytokines, and phagocytosis of pathogens, or in the case of I/R, cellular debris.Polarization: During I/R, DAMPs activate PRRs and promote neutrophil polarization toward an inflammatory phenotype.PRR signaling stimulates activation of the inflammasome and release of granules and inflammatory cytokines.Myeloperoxidase (MPO) is also activated, which in turn upregulates NOX2 and NE function, increasing ROS generation both within phagosomes and extracellularly.NE and PAD4 mediate the formation and release of neutrophil extracellular traps (NETs), which contribute to microvascular dysfunction during reperfusion ("no reflow").During resolution, SPMs and anti-inflammatory cytokines signal through pro-resolution receptors (e.g., FPR2), to upregulate ALOX15 and facilitate a phenotypic switch toward repair.Reparative neutrophils undergo lipid class switching, increase expression of CD206, Arg1, and release resolving factors (e.g., IL-10), pro-angiogenic factors (e.g., MMP9), and profibrotic factors (e.g., TGFβ) that contribute to the resolution of inflammation and wound healing.Metabolically, neutrophils rely on glycolysis for most effector functions, but may require oxidative phosphorylation (OXPHOS) for proper chemotaxis.

Figure 3 .
Figure 3. Macrophage regulation and function during myocardial I/R injury.Resident versus recruited:In the developing and homeostatic heart, self-renewing resident macrophages (CCR2 -) contribute to the formation and maintenance of the vasculature, electrical conduction, and phagocytosis of dysfunctional organelles in the myocardium.These macrophages can be subclassified by the expression of MHCII, LYVE1, and TimD4 on their cell surface.In response to injury, monocyte-derived macrophages (CCR2 + ) are recruited to the heart, where they further leukocyte infiltration during inflammation, clear dead cells and debris, and modulate stable scar formation during repair.Polarization and metabolism: DAMPs and inflammatory cytokines polarize recruited monocytes into inflammatory macrophages through activation of PRRs (e.g., TLR2/4).Upregulation of inflammatory genes (HIF-1α) and glucose transporters (Glut1) enhance glycolysis and suppress oxidative phosphorylation (OXPHOS).This results in the accumulation of succinate and activation of the pentose phosphate pathway (PPP), increasing ROS production.Activation of the inflammasome releases inflammatory and chemoattractant molecules, furthering inflammation.MerTK-mediated efferocytosis of dead cells, including neutrophils containing NGAL, as well as the binding of anti-inflammatory cytokines, initiates a phenotypic switch in the macrophage toward reparative function.Enhanced OXPHOS and the subsequent increase in itaconate and NAD + suppresses inflammatory gene expression (HIF-1α) and effector functions (ROS, inflammasome).Reparative macrophages express more CD206, YM-1, and FRP2, and release mediators that contribute to revascularization and stable scar formation.

Figure 3 .
Figure 3. Macrophage regulation and function during myocardial I/R injury.Resident versus recruited: In the developing and homeostatic heart, self-renewing resident macrophages (CCR2 -)contribute to the formation and maintenance of the vasculature, electrical conduction, and phagocytosis of dysfunctional organelles in the myocardium.These macrophages can be subclassified by the expression of MHCII, LYVE1, and TimD4 on their cell surface.In response to injury, monocytederived macrophages (CCR2 + ) are recruited to the heart, where they further leukocyte infiltration during inflammation, clear dead cells and debris, and modulate stable scar formation during repair.Polarization and metabolism: DAMPs and inflammatory cytokines polarize recruited monocytes into inflammatory macrophages through activation of PRRs (e.g., TLR2/4).Upregulation of inflammatory genes (HIF-1α) and glucose transporters (Glut1) enhance glycolysis and suppress oxidative phosphorylation (OXPHOS).This results in the accumulation of succinate and activation of the pentose phosphate pathway (PPP), increasing ROS production.Activation of the inflammasome releases inflammatory and chemoattractant molecules, furthering inflammation.MerTK-mediated efferocytosis of dead cells, including neutrophils containing NGAL, as well as the binding of antiinflammatory cytokines, initiates a phenotypic switch in the macrophage toward reparative function.Enhanced OXPHOS and the subsequent increase in itaconate and NAD + suppresses inflammatory gene expression (HIF-1α) and effector functions (ROS, inflammasome).Reparative macrophages express more CD206, YM-1, and FRP2, and release mediators that contribute to revascularization and stable scar formation.

Figure 4 .
Figure 4.The adaptive immune response during myocardial I/R injury.Dendritic cells: Cardiac resident dendritic cells (DCs) can be subclassified into plasmacytoid (pDC) and conventional (cDC) subtypes.During I/R, pDCs increase production and release type I interferons and contribute to the pro-inflammatory response.Conversely, cDCs were shown to facilitate the recruitment of Tregs and may favor resolution and repair processes.T cells: T cells are both resident and recruited to the heart during I/R, and can be classified into three main subtypes: cytotoxic T (CD8+), T helper (CD4+), and T regulatory (Treg).CD8+ cells release granzyme B (GZMB), promoting cardiomyocyte apoptosis and inflammation, while CD4+ cells release interferon gamma, which also promotes inflammation.In contrast, Tregs promote resolution by suppressing myeloid recruitment and stimulating fibroblast activation and repair after I/R.B cells: Similar to T cells, B cells are both resident and recruited to the heart after I/R.B cell activation and contribution to I/R injury remains unclear and likely has pleiotropic effects.B cells can positively regulate CCL7, which promotes myeloid cell recruitment and cardiac injury, yet B cells also produce IL-10 that can stimulate resolution and repair following I/R.Autoimmunity: The massive loss of cardiomyocytes elicited to I/R can trigger autoimmune responses in the heart.Resident DCs can present myocardial debris as autoantigens to T and B cells.B cells differentiate into plasma cells (PCs) and produce autoantibodies against the heart, while CD8+ T cells target healthy cardiomyocytes, contributing to infarct expansion and delayed repair.

Figure 4 .
Figure 4.The adaptive immune response during myocardial I/R injury.Dendritic cells: Cardiac resident dendritic cells (DCs) can be subclassified into plasmacytoid (pDC) and conventional (cDC) subtypes.During I/R, pDCs increase production and release type I interferons and contribute to the pro-inflammatory response.Conversely, cDCs were shown to facilitate the recruitment of Tregs and may favor resolution and repair processes.T cells: T cells are both resident and recruited to the heart during I/R, and can be classified into three main subtypes: cytotoxic T (CD8+), T helper (CD4+), and T regulatory (Treg).CD8+ cells release granzyme B (GZMB), promoting cardiomyocyte apoptosis and inflammation, while CD4+ cells release interferon gamma, which also promotes inflammation.In contrast, Tregs promote resolution by suppressing myeloid recruitment and stimulating fibroblast activation and repair after I/R.B cells: Similar to T cells, B cells are both resident and recruited to the heart after I/R.B cell activation and contribution to I/R injury remains unclear and likely has pleiotropic effects.B cells can positively regulate CCL7, which promotes myeloid cell recruitment and cardiac injury, yet B cells also produce IL-10 that can stimulate resolution and repair following I/R.Autoimmunity: The massive loss of cardiomyocytes elicited to I/R can trigger autoimmune responses in the heart.Resident DCs can present myocardial debris as autoantigens to T and B cells.B cells differentiate into plasma cells (PCs) and produce autoantibodies against the heart, while CD8+ T cells target healthy cardiomyocytes, contributing to infarct expansion and delayed repair.