Macrophage lineages in heart development and regeneration

Introduction

The contribution of macrophage lineages to the development of the heart is a relatively recent area of focus, with emerging data on critical roles in coronary and lymphatic vessel development and postnatal heart regeneration. In the past few years, there is increasing appreciation of multiple macrophage lineages with origins and gene expression profiles that change during embryonic and fetal development, as well as after birth, in the myocardium and heart valves with roles in tissue homeostasis and injury response. Here, we focus on macrophage lineages which have been identified to be critical in the developing cardiovascular system. While single cell RNA sequencing (scRNAseq) of embryonic hearts provides evidence for other immune cell types, including B-cells and T-cells, even less is known of the functions and contributions of these cell types to the developing heart (Feng et al., 2022). The most well-defined functions of macrophages in heart development are in coronary vessel remodeling and in the nodes of the specialized conduction system (Hulsmans et al., 2017; Leid et al., 2016). A caveat is that most information is based on studies in mice, although immune cells have been noted in single cell gene profiling of embryonic chicken hearts and fetal human hearts (Mantri et al., 2021; Miao et al., 2020). While the functions of macrophages in the developing cardiovascular system are beginning to be discovered, much remains unknown as to how they contribute to specific cell functions with dynamic regulation over time.

Macrophage lineage transitions in the developing heart

During mammalian embryonic development, hematopoietic cells, including macrophages, are generated first in yolk sac (YS) endothelial cells beginning at ~embryonic day (E)7.5 in mice, followed by definitive hematopoiesis in aorta-gonad-mesonephros (AGM) beginning at ~E10.5, and then in the fetal liver after ~E12.5 (Ginhoux and Guilliams, 2016; Lee and Ginhoux, 2022; Liu et al., 2022). At the same time, the heart begins to form with the initial differentiation of the cardiac crescent at approximately E7.5, followed by formation of the beating looped heart tube at E10.5, and the beginning of atrioventricular septation at E12.5 (Buckingham et al., 2005; Evans et al., 2010). By E10.5, there is a functional cardiovascular system evident in unidirectional blood flow through the heart and vessels that is essential for embryonic viability. Postnatally, the main source of new myeloid lineage immune cells shifts to the bone marrow by approximately one month after birth (Epelman et al., 2014; Ginhoux and Guilliams, 2016; Gula and Ratajska, 2022). Thus, there are three waves of hematopoiesis with origins in the embryonic YS/AGM, fetal liver, and postnatal bone marrow (Lee and Ginhoux, 2022). Improved lineage analysis with sophisticated mouse genetic tools has provided more details and higher resolution information on where and when macrophages arise and are present in the developing heart (Liu et al., 2022).

With the initiation of cardiovascular function, hematopoietic cells, primarily macrophages from the YS and AGM, circulate throughout the embryo and take up residence in multiple organs, including the heart (Figure 1). The initial cardiac macrophages were identified as expressing the surface antigen F4/80 at E10.5 and increase up to E14.5, predominantly on the surface of the heart in the subepicardial space (Balmer et al., 2014; Gula et al., 2021; Liu et al., 2022). F4/80 positive macrophages also are present within the myocardium and developing valves in proximity to endocardial endothelial cells at E12.5-E15.5 (Liu et al., 2022). Valvular macrophages were initially reported to arise from hemogenic endocardial endothelial cells based on Nuclear Factor of Activated T-cells 1 (Nfatc1)-Cre lineage positivity and Runx1 immunostaining (Shigeta et al., 2019). Subsequent lineage tracing with Nfatc1-Cre inducible lines demonstrated recombination in endothelial cells of the YS and AGM, as well as circulating white blood cells, supporting extracardiac origin of resident cardiac macrophages (Liu et al., 2022). Likewise, cardiac macrophages are not labeled with natriuretic peptide receptor 3 (Npr3)+ endocardial and Wilm’s tumor 1 (WT1)+ epicardial-derived Cre+ lineages or an intercellular genetic tracing system for endocardial-derived lineage cells, further supporting their extracardiac origin (Liu et al., 2022) (Liu et al., 2023). However, these studies are limited by the specificity and efficiency of the Cre-driver lines and could miss a rare population of valvular macrophages that arise from adjacent hemogenic endothelial progenitors (Nakano and Liu, 2023). Altogether, the lineage tracing studies demonstrate that the vast majority of embryonic cardiac macrophages arise from primitive and definitive hematopoiesis from the YS and AGM-derived progenitors. These tissue resident macrophages persist after birth and self-renew by proliferation alongside infiltrating bone marrow-derived myeloid lineage cells present in mature organs (Molawi et al., 2014; Sattler and Rosenthal, 2016).

Various markers have been used to identify macrophage lineage subtypes and origins in the developing heart (Figure 1). The earliest multipotential erythro-myeloid progenitors (EMPs) in the YS and AGM express the receptor tyrosine kinase cKIT and macrophage colony stimulating factor 1 receptor (Csf1r). Subsequent immature macrophage progenitors express the fractalkine receptor C-X3-C motif chemokine receptor 1 (Cx3Cr1), which has been used to track and manipulate early macrophage lineage cells. Cx3Cr1 lineage cells are present in the YS as early as E8.5, are detected in circulating blood by E9, and infiltrate target organs, including the heart, soon thereafter (Stremmel et al., 2018). Differentiated tissue resident macrophages express the marker F4/80 and these cells are present in epicardial, endocardial and valvular regions of the heart by E10.5 (Gula et al., 2021; Liu et al., 2022). After E12.5, fetal liver-derived monocytes present in the heart express low levels of F4/80, but highly express the more mature surface integrin CD11b, as detected by fluorescent cell sorting (Epelman et al., 2014). Additional subtypes of macrophages express Ly6c hi/lo and major histocompatibility complex (MHC) II (Epelman et al., 2014). The YS-derived resident tissue macrophages persist throughout development, self-renew and are maintained in the heart, more than most other organs, as part of the innate immune system in adults (Epelman et al., 2014; Lee and Ginhoux, 2022).

In the weeks after birth, bone marrow hematopoietic stem cells mature to produce multiple myeloid lineages, including circulating monocyte-derived macrophages, that migrate to target organs, including the heart. Myeloid lineage investment is dependent on expression of the CC-motif chemokine receptor 2 (CCR2) that recognizes chemokines including CC-motif chemokine ligand 2 (CCL2), also called monocyte chemoattractant protein-1 (MCP-1) (Epelman et al., 2014). Within the heart, these bone marrow-derived cells express higher levels of MHCII and CD206 than embryonic YS-derived macrophages and are detectable few weeks after birth in mice. (Epelman et al., 2014; Hulin et al., 2019; Molawi et al., 2014).

Single cell transcriptomic analysis of developing and neonatal hearts confirms presence of multiple immune cell lineages in mice, humans, and chicken embryos (Cui et al., 2019; Mantri et al., 2021; Mehdiabadi et al., 2022; Quaife-Ryan et al., 2017). In mouse embryos, clusters of cells with immune signatures are present by E10.5 and express myeloid markers Csf1r and Cx3Cr1 (Cahill et al., 2021). By spatial transcriptomics of chicken embryo hearts, immune cells consist of predominantly macrophages and dendritic cells within the epicardium and valve regions, distinct from circulating blood within the heart chambers as indicated by erythrocyte gene expression profiles (Mantri et al., 2021). At later fetal and postnatal stages in mice, B, T, and NK cells, as well as mast cells, are present in myocardial tissue, in addition to prevalent myeloid lineage macrophages and dendritic cells (Skelly et al., 2018; Suryawanshi et al., 2020). In postnatal mice, immune cells make up ~10% cells of remodeling heart valves at P7 and P30, consisting predominantly of macrophages, with additional B, T, dendritic and mast cells (Hulin et al., 2019). Likewise, in the developing myocardium, single cell RNAseq analysis has identified multiple immune cell types, including macrophages, as being present, but relative numbers and macrophage subtypes are yet to be reported using this approach (Feng et al., 2022). In the whole adult mouse heart, immune cells defined as expressing the hematopoietic cell marker CD45 make up ~5-10% of noncardiomyocytes (Pinto et al., 2016), and most of the CD45+ cells are macrophages (Cahill et al., 2021). Thus, diverse populations of immune cells, predominantly macrophages, are present in the developing and mature heart as detected by single cell transcriptomic studies.

Contributions of macrophage lineages to cardiac organogenesis

Cardiac macrophages were first identified morphologically in electron microscopy studies and associated with regions of cell death in the outflow tract and epicardium (Manasek, 1969; Pexieder, 1975). In embryonic rat heart organ cultures beginning at E12, macrophages identified by expression of Griffonia simplicifolia isolectin B4 (GAS) were detected in the subepicardial region of the outflow tract and have phagocytic activity prior to apoptotic activity (Sorokin et al., 1994). More recently, myocardial macrophages were identified by fluorescence-activated cell sorting (FACS) and categorized into subpopulations by surface markers related to developmental origins (Epelman et al., 2014).

While extensive data are available on the presence and diversity of macrophage populations in the developing heart, less is known of their specific functions in cardiac organogenesis. It has been hypothesized that cardiac resident macrophages have phagocytic functions related to clearing apoptotic cells and tissue remodeling, but this has not been demonstrated definitively (Gula and Ratajska, 2022). Dynamic regulation of tissue resident macrophage subpopulations and localization has been demonstrated in endocardial cushions and remodeling heart valves, but their contributions to heart valve development are not fully known (Liu et al., 2022; Shigeta et al., 2019). On the surface of the developing myocardium, CD45+ immune cells, also positive for the monocyte marker CD68 and lymphatic vessel endothelial HA-receptor (Lyve1), are present in the subepicardial space and have been implicated in coronary vessel remodeling and lymphatic endothelial cell plexus formation (Balmer et al., 2014; Cahill et al., 2021; Gula et al., 2021; Leid et al., 2016). Surface macrophages are also located in the outflow tract near regions of apoptosis, but specific functions in tissue remodeling have not been demonstrated (Gula et al., 2021). Cell lineage analysis and ablation studies also demonstrated a requirement for macrophages in formation and function of the atrioventricular node of the cardiac conduction system (Hulsmans et al., 2017). Together, these studies provide initial evidence for diverse regulatory functions of macrophages in tissue remodeling and maturation of cardiac valves, vasculature, and specialized conduction system necessary for organogenesis and function of the heart (Figure 2).

Macrophages in cardiomyocyte maturation and heart regeneration after birth

The period after birth in mice is characterized by maturation and cell cycle arrest of cardiomyocytes, increased fibroblast proliferation, and ECM production, along with infiltration of bone marrow-derived monocytes that occurs with the maturation of the immune system (Hortells et al., 2019; Lavine et al., 2018; Velayutham et al., 2020). Embryonic macrophage lineages in heart muscle are expanded in the neonatal period and retain proangiogenic and reparative functions as resident macrophages in the myocardium throughout life (Lavine et al., 2014). Transcriptional analysis of sorted leucocytes from neonatal and adult hearts demonstrates developmental maturation in macrophage gene expression associated with neonatal regenerative versus adult fibrotic response after injury (Quaife-Ryan et al., 2017). Moreover, by single cell gene expression analysis there is evidence for cell-cell communication between macrophages and other cardiac cell types that increases with postnatal age and cardiac injury (Whitehead and Engler, 2021). In general, the neonatal heart has an immature adaptive immune system and reduced pro-inflammatory function in comparison to the mature heart (Sattler and Rosenthal, 2016). However, there is limited understanding of the specific contributions of embryonic macrophages and maturing myeloid lineages to postnatal heart maturation or neonatal versus adult injury response (Figure 3).

The neonatal mammalian heart regenerates after injury resulting in the formation of new muscle in the absence of fibrosis or scarring (Porrello et al., 2011). Approximately one week after birth in mice and pigs, the heart loses the capacity to regenerate and cardiac injury leads to fibrosis, scar formation, and dysfunction (Porrello et al., 2011; Ye et al., 2018; Zhu et al., 2018). The loss of regenerative capacity occurs at the same time as maturation of the immune system, which has been hypothesized to contribute to scarring and pathologic fibrotic response in the nonregenerative heart (Quaife-Ryan et al., 2017; Sattler and Rosenthal, 2016). In neonatal mice, inhibition of macrophage function with chlodronate liposome treatment inhibits new muscle formation after injury in the postnatal period, which was linked to macrophage-dependent angiogenesis during the regeneration process (Aurora et al., 2014). Similarly, in zebrafish that maintain a cardiac regenerative response in adults, depletion of macrophages prior to injury inhibits cardiac regeneration (Lai et al., 2019). In neonatal mice, macrophages promote cardiomyocyte proliferation and regeneration through secretion of oncostatin M that promotes cardiomyocyte Yap activation (Li et al., 2020). However, in adults proinflammatory myeloid monocyte-derived macrophages are recruited to the heart after injury and contribute to scarring and pathologic remodeling (Lavine et al., 2014). Interestingly transplantation of neonatal macrophages into adult injured myocardium promotes regenerative repair, supporting a beneficial paracrine signaling between neonatal, but not adult, cardiac macrophages and cardiomyocytes (Li et al., 2021). The apparent proregenerative capacity of neonatal resident cardiac macrophages has made them an attractive target for stimulation of cardiac repair after injury in adults (Nahrendorf, 2018).

Summary and conclusions

Changes in macrophage lineage origins, composition, and gene expression profiles have been demonstrated in the developing heart, including in the postnatal period, but there is limited knowledge on their specific functions in cardiac organogenesis.

While the requirements for macrophages have been defined, the specific cellular functions of macrophages in heart development are less well known. Initially macrophages were implicated in tissue remodeling in apoptotic regions of the heart, but phagocytic functions have not been definitively demonstrated (Manasek, 1969; Pexieder, 1975). More recently, macrophage-secreted factors, such as Igf2 or oncostatin M, have been associated with angiogenic remodeling and cardiac regeneration (Leid et al., 2016; Li et al., 2020). Single cell gene expression profiling has provided additional evidence for candidate secreted factors that mediate cell-cell interactions between macrophages and other cell types (Whitehead and Engler, 2021), which is an important area of research for future understanding of immune cell contributions to cardiac tissue remodeling and repair mechanisms. In general, specific functions and cellular contributions of macrophages to development of the heart are not well defined and need to be studied further.

Cell ablation studies have demonstrated specific requirements for embryonic macrophages in coronary and lymphatic vessel development, specialized conduction system maturation, and regeneration after neonatal injury. The developmental origins of macrophage lineages changes over time, with embryonic lineages having more reparative and remodeling functions in comparison to the more mature bone marrow derived myeloid lineages. However, embryonic macrophage lineages persist in the myocardium and heart valves as resident cardiac macrophages in adults. Thus, enhancing reparative abilities of embryonic and neonatal macrophage lineages has been invoked as a therapeutic opportunity for adult cardiac injury response (Sattler and Rosenthal, 2016). In addition, harnessing the developmental potential of macrophages also could promote cardiac angiogenesis or contribute to prevention of conduction anomalies. Conversely, genetic or pharmacologic inhibition of the chemokine receptor CCR2, preventing organ targeting of monocyte-derived macrophages, ameliorates myxomatous valve disease of Marfan syndrome, cardiac fibrosis, and atherosclerosis (Kim et al., 2020; Nahrendorf, 2018; Xu and Yutzey, 2022). Thus, inhibition of pathologic functions of mature myeloid-derived macrophages is an attractive strategy for limiting the progression of congenital valve disease or pathologic fibrotic remodeling after cardiac injury (Figure 3).

Despite extensive macrophage lineage tracing and ablation studies, there is limited information on their specific functions and mechanistic contributions to cardiac organogenesis and heart maturation after birth. This is likely due to the necessity of systemic macrophages for postnatal viability, together with the relative inability to conditionally target macrophage subpopulations in specific organs. In addition, increased knowledge of neonatal heart immune cell subpopulations and gene expression profiles at the single cell level also are needed. There is great interest in recapitulating neonatal macrophage contributions to promote more effective adult cardiac repair after injury (Nahrendorf, 2018; Simões and Riley, 2022). Thus, lessons from embryonic macrophage contributions to developmental remodeling and myocardial repair may lead to new therapeutic strategies in treatment of cardiovascular disease.