Introduction

For several decades, bone marrow-derived macrophages were considered as the only large phagocytes involved in homeostasis, tissue healing, and defence against pathogens. Emerging evidence has overturned this dogma and has shown that resident macrophages (rMacs) are also fundamental players in a plethora of functions and cellular interactions both in homeostasis and in the modulation of the inflammatory response following injury and in tissue remodelling. Originating from the yolk sac or fetal liver progenitors [45], tissue rMacs inhabit various organs such as the bone marrow [58], lungs [76], liver [12], pancreas [17], brain [96], and heart [34]. Differently from circulating immune cells, rMac retain tissue-specific features. This population is made up of macrophages ontogenetically older than bone marrow-derived macrophages [95], they are evolutionarily conserved [30] and present throughout the lifetime. They can proliferate in situ and this process is exacerbated during inflammation [41]. In murine cardiac tissue, resident cardiac macrophages (rcMac) are reported to constitute up to 5–10% of the non-myocyte population, a percentage that increases dramatically following cardiac damage [50, 89]. With their peculiar spindle-like morphology, these resident immune cells take part in a large variety of physiological mechanisms which indeed include efferocytosis [26] but also immune surveillance, cardiac conduction [78]. These technologies have mostly been applied to murine models and they have identified different waves of rcMac formation [78]. Distinct lineages of rcMacs exist within the ventricular myocardium of the develo** heart and playing as essential regulators during cardiac development [64]. According to their cardiac localization and origin, it is possible to identify at least two distinct subsets of macrophages, CCR2− and CCR2+ (C–C chemokine receptor type 2) [64]. CCR2− cells originate from yolk sac progenitors, whereas CCR2+ derive from fetal monocyte progenitors, which is also reflected in their divergent gene expression profiles [64]. CCR2− cells are the first macrophage population appearing in the cardiac tissue at embryonic day 12.5 (E12.5), whereas CCR2+ inhabits the heart at E14.5. These cells are also confined in different regions of the heart [64]. More specifically, CCR2− are mostly found within the myocardial wall and in proximity to the coronary vasculature, whereas CCR2+ are in the trabecular projection of the endocardium [64]. These macrophages remain in the cardiac tissue for their entire life-span. For their embryonic origin and intrinsic self-renewal capacity, CCR2− rMacs are also defined as “resident population”. On the contrary, CCR2+ subset originates from haematopoiesis and their number is ensured by recruitment of circulating monocytes. For this reason, this subset is also defined as “non-resident population” [64]. Clinically, the association of CCR2+ macrophages abundance on LV remodeling and cardiac function has been shown in patient with heart failure [11].

During their development and in response to different environmental stimuli and functional responses, macrophages can be activated and functionally categorized into certain subgroups including M1, or M2 phenotypes. It is important to reiterate that this classification does not appropriately depict the in vivo spectrum of macrophage sub-populations present in both the healthy and diseased myocardium. In vitro this heterogeneity is reduced and the stimulation is applied in a more controlled environment, as such this simplified definition of M1/M2 is more acceptable. M1 or “classical” activated macrophages are pro-inflammatory phagocytic cells involved in the initial stages of inflammation and this phenotype is generally attributed by infiltrating monocytes [107]. Differently, the M2 or “alternative” activated macrophages are anti-inflammatory cells implicated in the resolution of the inflammatory process [88] and normally rcMacs in steady-state heart reflect this phenotype [107]. In vitro, M1 cells are known to secrete pro-inflammatory cytokines such as nitric oxide (NO), tumor necrosis factor (TNF-α), and interleukin 12p70 (IL-12p70) thus eliciting a robust inflammatory response [110]. On the contrary, the M2 in vitro activation leads to anti-inflammatory cytokines secretion which includes transforming growth factor (TGF-β), interleukin 10 (IL-10), and arginase-1 (Arg1). These cytokines support the repression of the inflammatory response, favour tissue healing and collagen deposition [74, 110]. The M1 or M2 phenotype is not permanent and can change. It was recently reported that rcMacs (mostly M2) can transition to M1-like phenotype in aged mice [69]. The M1/M2 paradigm was not only proposed based on the activation status, but it was also confirmed by distinct metabolic profiles, alterations in cell morphology [16], gene transcription [66] and functional efferocytosis [33, 37, 47, 54].

Another important aspect of macrophage biology is the heterogeneity in origin and phenotype following cardiac injury or during tissue remodelling. In this context, several markers are efficiently used to distinguish infiltrating and rcMacs, unfortunately, they are often not consistently expressed across animal species thus complicating the translation of research findings. Transgenic animals with fluorophore-labelled macrophages [33] or Cre-loxP macrophage reporter mice [99] can be helpful to provide informative data of specific cell types and overcome technical issues associated with antibody combinations. To date, one of the most common markers to discriminate resident and non-rMacs is CCR2−/+ which is conserved in human, rat, and mouse [10, 34]. Other options are C-X-3-C Motif Chemokine Receptor 1 (CX3CR1) and the major histocompatibility complex class II (MHCII). Using these markers, CX3CR1+MHCII− embryonic macrophages were identified in hearts from new-born mice and it was demonstrated how they tent to progressively diversify by increasing MHCII expression and decreasing CX3CR1 expression during aging [79]. In human, HLA-DR represents human homologue of MHC-II and human cardiac macrophages could be subdivided into three distinct subsets (CCR2+HLA-DRlow; CCR2+HLA-DRhigh; CCR−HLA-DRhigh) based on CCR2 and HLA-DR [11]. Alternatively, lymphocyte antigen 6 complex locus C (Ly6C) and MHCII were also used to efficiently distinguish four distinct subgroups of murine macrophages [39, 111]. Ly6C−/CCR2−/MHCIIhigh and MHCIIlow were shown to label macrophages deriving from the yolk sac, while Ly6C+CCR2− and Ly6C+CCR2+ are macrophages deriving from haematopoiesis [39, 111]. In rats, Ly6C marker is replaced by CD43high/low [1], whereas for human samples the equivalent marker is CD14 [11]. Recently, TIMD4 (T-cell immunoglobulin and mucin domain containing 4) and LYVE1 (Lymphatic vessel endothelial receptor 1) were identified as new markers for murine rcMacs [28].

Other common macrophage markers in human, mouse, and rat are CD68 [20, 29, 46, 112], MerTK (myeloid-epithelial-reproductive tyrosine kinase) [38], Mac-3 [70], galactose-specific lectin 3 (Galectin 3) [85] and CD163 [1, 29], these markers, however, do not discriminate between resident and non-rMacs. Other options are F4/80 [8, 105] which is mouse-specific and CD169 [29, 112] or CD64 [38] used for rat and mouse tissue [6, 98]. In human specimens, EMR1 (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1) is the homolog of F4/80, and it labels both macrophages and granulocytes [4, 48]. CD11b (ITGAM) is also not sufficiently specific as it targets monocytes, neutrophils, and natural killer cells (NK cells) [70, 113]. A completely different set of markers is used to discriminate in vitro M1 and M2 macrophages. Inducible nitric oxidase (iNOS/NOS2) has been considered for several years a standard M1 marker [94]. Recently, the classic perception of CMs being the sole cellular units able to propagate the cardiac electrical impulse has been revisited. Indeed, it has been reported that the action potential propagation and CM contraction can be altered also by cell–cell interactions and communication between stromal cells and CMs [59]. Fibroblasts (FBs) play a key role in CM contraction in both physiological and pathological conditions [93]. Several in vitro studies have demonstrated the crucial role of direct contact between cardiac FBs and CMs to regulate electronic coupling [59, 86]. More recently, Hulsmans et al. were the first to demonstrate that rcMacs are also important mediators in this process and can alter electrical conduction [18]. These EV-YF1-enriched exosomes target macrophages leading to an increase in the production and secretion of IL-10 [18]. In a co-culture of CMs and macrophages, it was observed that the overexpression of EV-YF1 in macrophages, results in a cardioprotective outcome through IL-10 secretion [18]. In vivo, rats subjected to MI and treated with EV-YF1 displayed a reduction of the infarct area, highlighting the cardioprotective effect of this Y RNA fragment [18]. Similarly, exosomes secreted by CDCs can be enriched with different miRNA including miRNA-181b which were shown to induce macrophage polarization towards a cardioprotective phenotype with associated beneficial effect at a tissue level [24]. miR-155 is overexpressed in cardiac macrophages. In a mouse model of myocarditis, this specific microRNA was highly expressed by infiltrating macrophages [22]. The systemic knockdown of miR-155 leads to reduced infiltration of monocyte-derived macrophages and reduced cardiac damage [22]. In line with this, the pro-inflammatory role miR-155 has been confirmed also in a pressure-overload mouse model [84]. Mice with a deletion of miR-155 in macrophages show reduced hypertrophy and inflammation [84], suggesting its potential for therapeutic applications. Differently, lncRNA-Macrophages M2 polarization (MMP2P) is upregulated in M2, but not in M1 macrophages [19]. Moreover, the knockdown of lncRNA-MMP2P inhibits the polarization of macrophages towards the M2 phenotype by decreasing the phosphorylation of signal transducer and activator of transcription 6 (STAT6) [19].

Conclusions

Recent technological developments and contemporary immunological techniques are offering new opportunities to identify and study the roles and contribution of rcMac in respect to recruited monocytes and other cardiac cells. These novel approaches have already allowed scientist to better understand rcMac origin, phenotypic profile and their functional contribution in myocardial function. Basic and pre-clinical studies which involve the use of drugs or non-coding RNAs also demonstrated the potential of rcMac to regulate cellular interactions thus suggesting their use to modulate and potentially prevent tissue remodelling. The emerging evidence is also highlighting the detrimental effects induced by uncontrolled responses of this cell type. The future of macrophage-modulated therapy will have to take advantage of the mechanistic pathways that coordinate tissue repair and exploit them to develop more precise and effective therapeutic strategies.