The effect of immune cell‐derived exosomes in the cardiac tissue repair after myocardial infarction: Molecular mechanisms and pre‐clinical evidence

Abstract After a myocardial infarction (MI), the inflammatory responses are induced and assist to repair ischaemic injury and restore tissue integrity, but excessive inflammatory processes promote abnormal cardiac remodelling and progress towards heart failure. Thus, a timely resolution of inflammation and a firmly regulated balance between regulatory and inflammatory mechanisms can be helpful. Molecular‐ and cellular‐based approaches modulating immune response post‐MI have emerged as a promising therapeutic strategy. Exosomes are essential mediators of cell‐to‐cell communications, which are effective in modulating immune responses and immune cells following MI, improving the repair process of infarcted myocardium and maintaining ventricular function via the crosstalk among immune cells or between immune cells and myocardial cells. The present review aimed to seek the role of immune cell‐secreted exosomes in infarcted myocardium post‐MI, together with mechanisms behind their repairing impact on the damaged myocardium. The exosomes we focus on are secreted by classic immune cells including macrophages, dendritic cells, regulatory T cells and CD4+ T cells; however, further research is demanded to determine the role of exosomes secreted by other immune cells, such as B cells, neutrophils and mast cells, in infarcted myocardium after MI. This knowledge can assist in the development of future therapeutic strategies, which may benefit MI patients.

the vasculature and overexpression of adhesion molecules like integrins to recruit immune cells. Indeed, injured cardiomyocytes post-MI undertake the necrotic process and release the dangerassociated molecular patterns (DAMPs) into the extracellular environment, which promote an acute inflammatory response to digest and clear necrotic debris and extracellular matrix (ECM) tissue. 3 DAMPs, such as heat shock proteins like high mobility group box 1, induce responses of the immune system via binding to cognate pattern recognition receptors, including nucleotide-binding oligomerization domain-like receptors and toll-like receptor/interleukin 1 receptors (TLR/IL1R), on living cardiomyocytes. 4 The transient and intense MI-promoted inflammatory responses play critical roles in cardiac repair and clearing the infarcted area of injured and dead cells and ECM debris. 9 After MI, the inflammatory process occurs during two different temporal phases, including an early inflammatory phase followed by a reparative phase. 9 Neutrophils are the first immune cells recruited to the myocardium.
These immune cells induce macrophage infiltration and have critical role in the cardiac repair and survival. 9 In the early phase of inflammatory process immediately after MI, the pro-inflammatory chemokines and cytokines are secreted from M1 macrophages to provoke inflammatory responses to degrade and remove necrotic or injured cells. Through the following days, the inflammatory phase gently shifts to the reparative phase involving inflammation resolution, neovascularization and scar generation. The switching to the reparative stage needs the in time inhibition of the inflammatory process through the activity of immune-suppressive lymphocytes and antiinflammatory M2 macrophages. 9 During the reparative phase, cardiac macrophages polarize to M2 phenotype secreting pro-fibrotic and anti-inflammatory cytokines like IL-10 and transforming growth factor-beta (TGFβ), which inhibit inflammation and induce tissue repair. M2 macrophages also trigger ECM generation and angiogenesis via secreting the vascular endothelial growth factor (VEGF) and TGFβ. 10 In addition, bone marrow-derived dendritic cells infiltrate the necrotic regions of the myocardium, mainly through the reparative phase. 11,12 Infiltrated dendritic cells (DCs) seem to regulate macrophage homeostasis after MI, therefore, modulating the postinfarction healing process. 12 Besides, T cells are also found to involve in the inflammatory process to varying degrees in the infracted myocardium after MI. 13 Although the inflammatory process leads to clearing damaged myocardial cells and facilitating scar formation, an inefficient early immune response can cause the fatal cardiac rupture while excessive or prolonged inflammation response promotes the ECM degradation and the adverse cardiac remodelling causing heart failure (HF). 14 Of note, a regulated balance between the promotion and suppression of inflammatory responses post-MI provides a subsequent proliferative phase of healing in the infarcted area. Thus, a timely resolution of inflammation and a firmly regulated balance between regulatory and inflammatory mechanisms is critical. Immune cells, rather than damaged cardiomyocytes, are the principal modulators of such balance. Therefore, modulating inflammation response after MI may provide a potential approach to diminish myocardial dysfunction. Notably, there is growing evidence showing the essential role of immune cellderived exosomes in these functions, [15][16][17][18] and thus, in the present review, we aimed to summarize the immunomodulatory impacts of immune cell-secreted exosomes on cardiac repair after MI.

| E XOSOME S
A vital route of intercellular communication is provided by exosomes that are released by cells to extracellular space and participate in various physiological processes such as immune regulation. 19 Exosomes are nano-sized (30-120 nm) lipid bilayer extracellular vesicles that are secreted by almost all cell types and carry a wide range of biologically active cargos, including cell-specific proteins, lipids and genetic materials like mRNAs and microRNAs (miRs), to the target cells. 20 Exosomes can target specific cell types, depending on their origin cell, molecular contents and surface antigens. By transporting biologically active cellular constituents from the donor to the recipient cells, exosomes modulate the function and behaviour of recipient cells under both normal and pathological conditions. 21,22 Exosomes participate in cell-to-cell communication through various routes, including receptor/ligand interaction in which exosomal transmembrane proteins bind to the signalling receptors of recipient cells 23 ; fusion with the plasma membrane of recipient cells and deliver their contents into cytosol; or cellular internalization via caveolin-or clathrin-dependent endocytosis 24,25 as well as cellular uptake through phagocytosis or micropinocytosis. 26,27 With exosomes being growingly studied and used to cardiovascular disorders like MI, 28 it is expectable that, considering wellestablished cell-based immunotherapies, immune cell-derived exosomes will provide new effectual substitutes for patients.
Exosome-based cell-free therapeutic approaches that recapitulate and even intensify the impacts of cell therapies show numerous advantages. The main concerns regarding cell therapy, which can be bypassed via exosomes, are the possibility for incidence of cellular embolism following intravenous injection, hardness of passing through the blood-brain barrier, ectopic tissue formation, infusionrelated toxicity resulted from transplanted cells embedded in the pulmonary microvasculature, the low cell viability, the immune rejection and tumorigenicity, as well as safety and ethical issues. [29][30][31][32] Regarding the handling and manufacturing processes, exosomebased therapeutic approaches demand simple preparation and sterilization steps and require easier storage conditions, 28 considerably decreasing the total cost in comparison with cell-based therapies.
Additionally, the general stability and safety of exosomes secreted by various cell types have been approved in a number of clinical trials (50). However, up to now, there have been no clinical trials evaluating exosomes for treating cardiovascular disease, and thus, the safety and practicability of exosome-based therapeutic approaches in this field remain to be approved.
The present paper would review the emerging role of immune cell-derived exosomes in infarcted myocardium after MI, together with mechanisms underlying their repairing effects on the injured myocardium ( Table 1). The exosomes we focus on are secreted by classic immune cells including macrophages, dendritic cells, regulatory T cells (Tregs) and CD4 + T cells; however, further research is demanded to determine the role of exosomes secreted by other immune cells, such as B cells, neutrophils and mast cells, in infarcted myocardium after MI. This knowledge can assist in the development of future therapeutic strategies, which may benefit MI patients.

| The role of dendritic cells in postmyocardial infarction
DCs are the most efficient member of the professional antigenpresenting cells (APCs) that play as the key and impressive immunoregulators modulating various lines of immune cells in innate and adaptive immunity. 33 Emerging data indicate that DCs have critical roles in the pathophysiological mechanisms of various cardiovascular disorders, especially MI. 34 In the infarcted myocardium, DCs are essential for the recruitment and activation of immune cells, especially T cells and macrophages, accompanied by a marked elevation of inflammatory cytokines. 35 Notably, it has been recently found that the infiltration of DCs is remarkably increased in the infarcted myocardium and, in turn, the frequency of circulating DCs is decreased in post-MI. 11,12,36 After MI, DCs migrate to the watershed infarcts and participate in the activation of lymphocytes and the promotion of immune responses. 35,37 In vivo studies have shown that DC ablation in mice leads to the increased and sustained production of inflammatory cytokines like TNFα, IL1β   in CD4 + cells, which led to greater and more rapid migration and recruitment of additional DC-EXs into the spleen. 51 Activated CD4 + T cells have been found to play a determinant role in improving myocardial wound healing post-MI. 13 Notably, injected splenic CD4 + T cells activated by MI-DC-EXs or activated CD4 + T cells that had uptaken DC-EXs could deliver into the infarcted myocardium and eventually improve the cardiac function of mice post-MI. 51 As the number of anti-inflammatory CD4 + Foxp3 + Tregs between CD4 + T cell population in heart-draining lymph nodes is known to be elevated in the wake of MI, 51 it can be postulated that DC-EXs likely activate cardioprotective Tregs after infarction and, thereby, beneficially impact wound healing and survival.

TA B L E 1 Molecular targets and mechanisms underlying impacts of exosomal miRs secreting by various immune cells after MI
Besides, there is evidence showing the key role of DC-EXs in angiogenesis following MI. 52 During the early phase after MI, angiogenesis plays an important role in the recovery of the injured myocardium and myocardial remodelling as well as maintenance of cardiac function 53,54 through healing the microvascular bed and preventing further necrosis and apoptosis of ischaemic myocardial tissue, which may be damaged, hibernating or stunned following MI. 53 Notably, DC ablation has been found to deteriorate angiogenesis and cardiac function in mice post-MI, 12

| The role of macrophages in postmyocardial infarction
Macrophages are a group of heterogeneous monocyte/macrophage lineage cells due to the plasticity of the cells in the various milieu.
Macrophages display diverse phenotypes in response to different stimuli, and they are thus classified into two subsets, including classically activated macrophages (M1) produce pro-inflammatory cytokines 55 and alternatively polarized M2 macrophages suppressing immune responses, supporting Th2 immunity, and promoting tissue regeneration and remodelling as well as wound healing. 56 Macrophage modulation has been known to provide a vital reg- Macrophages can also abundantly secrete EXs that show both pro-and anti-inflammatory activity in diverse contexts, partly because of previously activated macrophages that have already been primed with stimuli. There have been investigations comparing EXs secreted from M1 (M1-EXs) and M2 (M2-EXs) macrophages. 74,75 There is evidence that shows, similar to their parent cells, M2-EXs exhibit contrary functions to M1-Exos in the tumour biology 76,77 and MI, as discussed in the following sections.

| The role of M1-EXs in postmyocardial infarction
Macrophages through the crosstalks with cardiac fibroblasts induce cardiac remodelling after MI. 78,79 Of note, EXs secreted from infiltrated macrophages (Mac-EXs) were shown to impact the function of fibroblasts. In the mouse heart post-MI, it was found that cardiac Mac-EXs containing miR-155 could move into cardiac fibroblasts and mediate macrophage-fibroblast crosstalk. 80 It can be further supported by a study that showed MI significantly elevates the levels of mir-155 in cardiac fibroblasts. 81 Notably, it was shown that mir-155 expression is highly up-regulated in cardiac cells in response to angiotensin II, which can result in the active packaging of mir-155 into cardiac Mac-EXs that are taken up by fibroblasts. 80 In mechanism, angiotensin II-stimulated macrophages were found to secrete mir-  80 Mac-EXs containing miR-155 could also reduce the collagen generation and induce cardiac rupture, which is mediated by SOCS1 and SoS1 targeting. 81 Interestingly, such effects were reversed in miR-155-deficient mice, which showed a marked decrease in the incidence of cardiac rupture and an enhanced cardiac function, elucidating the critical role of cardiac Mac-EX-mir-155. 80 Further study indicated that miR-155-enriched EXs are also highly secreted from pro-inflammatory M1 macrophages after MI and exert an antiangiogenic impact and accelerate MI damage. 82

| The role of M2-EXs in postmyocardial infarction
The MI, due to occlusion of coronary arteries, results in transmural myocardial ischaemia that, in turn, leads to irreversible death of cardiomyocytes and myocardial injury or necrosis.
Though in time and absolute myocardial reperfusion is the most effectual approach to protect the myocardium after MI, restoration of the coronary blood urges further irreversible damage to the myocardium and it leads to final infarct size (INF), termed as 'myocardial ischemia/reperfusion' (MI/R) injury. 85 The pathophysiology of MI/R injury is not precisely known. A reduced potential of inner mitochondrial membrane and elevated levels of reactive oxygen species (ROS), inorganic phosphate or calcium ions is found in MI/R injury, which may result in mitochondrial permeability transition pore (MPTP) and further increase cell apoptosis. 86 Although using the antithrombotic and antiplatelet drugs are the standard strategy to preserve the patency of the infarct-related coronary artery, there is no therapeutic approach to sufficiently protect against MI/R injury, while the life-threatening reperfusion attributes to more than 50% of the final INF. 87 Therefore, developing novel therapeutics better able to decrease infarct size represents a major challenge. 86 Switching harmful and inflammatory M1 macrophages towards anti-inflammatory M2 macrophages with ameliorating effects is found to protect against various kinds of I/R injuries. [88][89][90] Emerging studies suggest that exosomes may serve as key mediators in MI/R injury. It has been recently shown that M2-EXs can protect MI/R injury. 75 M2-EXs were found to transport high levels of miR-148a into cardiomyocytes and, consequently, increase the viability of injured cardiomyocytes and alleviate Ca 2+ overload and dysregulation of cardiac enzymes known as MI biomarkers like creatine kinase, creatine kinase myocardial band (CK-MB) and lactate dehydrogenase, leading to a reduction in infarct size and MI/R injury after experimental MI. 75 Such effects are mediated by down-regulating the thioredoxin-interacting protein (TXNIP) and inactivating the TLR4/NF-κB/NLRP3 inflammasome signalling pathway. 75 Mechanistically, miR-148 can reduce the TXNIP via directly binding to its 3' untranslated region (3'UTR). 91 TXNIP plays the important role in redox homeostasis and has increased levels in MI/R, whose overexpression can cause cellular apoptosis by elevating levels of ROS and oxidative stress. 92 TXNIP has been documented to closely link to the phosphorylation and activation of TLR4/NF-κB/NLRP3 inflammasome pathway 93-95 that is correlated with various cardiovascular diseases. 96 Furthermore, there is evidence that shows M2-EXs could modulate the signalling of various biomolecules from M1-EXs, thereby inhibiting the inflammatory function of M1 macrophages in post-MI. For instance, the expression level of pro-inflammatory miR-155, which is highly expressed in M1 macrophages (as discussed in the last section), is lower in M2 phenotype than in M1, 97,98 and it has been found that M2-EXs cytokines, such as VEGF, reduce the expression levels of cellular and exosomal miR-155 in macrophages. 82

| The role of Treg-secreted exosomes in postmyocardial infarction
The maintenance of cardiac function after the MI needs structural repair. Myocardial repair is a complicated process relying on the activation of inflammatory response that acts as a double-edged sword. On the one hand, uncontrolled inflammatory response causes an enlargement in the size of myocardial infarct size and deteriorates reverse cardiac remodelling. On the other hand, insufficient inflammatory response influences macrophage-mediated phagocytosis of necrotic debris, which in turn impacts myocardial repair. 10 Indeed, the cardiac repair response can be promoted only after subsiding the inflammatory response.
Tregs, as a particular subtype of CD4 + lymphocytes with immunosup-

| The role of CD4 + T cell-secreted exosomes in post-myocardial infarction
Although the sterile inflammation promoted by necrotic cardiomyocytes activates post-MI cardiac repair by the macrophage recruitment and the production of pro-inflammatory cytokines and chemokines, 111,112 the sustained inflammation that is mainly controlled by cardiac-infiltrated CD4 + T cells participates in the development of cardiac fibrosis and dysfunction. 113 Notably, immune therapeutics inhibiting CD4 + T cells have been found to prevent fibrotic pathology and dysfunction in the ischemic heart. 114 The detrimental impacts of cardiac-infiltrated CD4 + T cells have also been detected in pressure overload-promoted cardiac fibrosis and hypertrophy, which was found to be reversed by genetic inactivation of CD4 + T cells. 115 Altogether, such results emphasize the importance of cardiac activated CD4 + T cells in the abnormal cardiac remodelling.

Exosomes secreted from activated CD4 + T cells (T-activated EXs)
have been identified as the important mediators in the myofibroblast activation, which is the core element for cardiac fibrotic remodelling post-MI. 116 It was shown that T-activated EXs containing miR-142-3p are highly uptaken by cardiac fibroblasts and thereby deteriorate cardiac fibrosis post-MI. 116 The exosomal miR-142-3p is found to directly target and down-regulate the adenomatous polyposis coli (APC), which leads to induction of β-catenin degradation in the cytoplasm, resulting in the inhibition of the canonical WNT signalling cascade and, thereby, triggering myofibroblast activation and fibrogenesis. 116 The APC protein is a positive regulator for the glycogen synthase kinaseβ (GSKβ), a key protein included in cardiac remodelling. 117 Thus, the T-activated EXs can confer the pro-fibrotic effects through modulating APC-GSKβ-β-catenin signal cascade mediated by miR-142-3p. Besides, the myofibroblasts activated by T-activated EXs-miR-142-3p might release bioactive molecules, such as TGF, 118,119 to induce the hypertrophic response of cardiomyocytes, leading to pathological cardiac remodelling. 116 Supporting is the in vivo study on the MI mice that revealed the intravenously in- EXs are the critical signal carriers in cardiac fibrosis following MI, and exosomal miR-142-3p serves as the signal conductor, providing a potential therapeutic target for treating MI-related cardiac fibrosis. 116

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no data sets were generated or analysed during the current study.