Macrophages were first discovered in 1884 by the Russian-French biologist, Ilya Mechnikov, who observed a specific population of large white blood cells engulfing and digesting bacteria in a process that he referred to as phagocytosis, and thus named them macrophages [1]. For decades, these cells were believed to populate the body tissues (tissue-resident macrophages) through continuous blood-circulating monocytes that were generated from progenitor cells in the adult bone marrow (BM). This cellular lineage of macrophages, the "mononuclear phagocyte system" represents the promonocytes and their precursors in the BM, monocytes in the peripheral blood, and macrophages and dendritic cells in tissues [2]. In recent years however, our knowledge of the ontogeny of the cell types within the mononuclear phagocyte system has changed dramatically with studies indicating that many tissue-resident macrophages populations actually arise from the embryo through embryonic precursors that take residence in body tissues prior to birth, and maintain themselves locally throughout adulthood, independent of the contribution from the BM-derived precursors [3]. These findings are significant in our ability to understand the origin, the developmental pathways, and the homeostatic processes that regulate tissue-resident macrophages which in turn enables the design of future intervention strategies to modulate macrophage functions at specific sites.

One source of macrophages is from the mononuclear phagocyte system (MPS), a term introduced by van Furth and Cohn in 1968. The MPS is defined as a cell lineage which originates from bone marrow progenitor cells and gives rise to blood monocytes, tissue macrophages and dendritic cells [4]. Thus, the process of generating a macrophage from the MPS begins with a promonocyte in the BM which undergoes a differentiation process into a monocyte that is ready to enter the systemic circulation. After a short period (<48h) in the circulation [5], these newly formed monocytes rapidly infiltrate into peripheral tissues where a majority of them differentiate into macrophages or dendritic cells (DC) [6, 7]. Although circulating blood monocytes were believed to be the sole source of precursor tissue macrophages for a long time [8], studies in the past few years have challenged this notion. These studies indicate that the majority of macrophages in tissues actually originate from embryonic precursors that are derived from the embryo yolk sac, and the embryo itself before birth, not from BM-derived monocytes [9-11].

Moreover, these macrophage populations are maintained in the adult tissues through the ability of these embryonic macrophages to retain a long-term capacity for self-renewal. These studies support the idea that embryonic origin tissue macrophages constitute a pool of macrophages that is independent of the monocytes that are produced in the BM in postnatal life [8]. Consequently, this discovery questioned the view that the macrophages cell of origin evolved from a common progenitor, and the emerging evidence suggest that two primary distinct sources of macrophages exist in almost all tissues; tissue-resident macrophages of embryonic origin, which self-renew in situ , and the monocyte-derived macrophages, which indeed are replenished by the BM [11].

Several studies showed that macrophage populations exhibit distinct transcriptional signatures [22, 23] and epigenetic marks [23, 24] that are specific to their tissue of residence. In addition, macrophages that derive from embryonic progenitors or from BM monocytes exhibit heterogeneity both in vitro [25] and in vivo [22, 23], thus, understanding how factors that are unique to their origin and tissue environment should be integrated to define their functional capacities. Furthermore, knowledge of the macrophage cell of origin increases our ability to distinguish macrophage populations from other cell populations in a given tissue site that may exhibit similar phenotype and or function [6]. Table 1 summarizes the commonly used macrophage markers and antibodies against them.

Capitalizing on the fact that during embryonic development, transcription factor usage and surface-marker expression differ between yolk-sac-derived and definitive- hematopoietic stem cells (HSC)-derived macrophages, specific tools were developed to define macrophages ontogeny. For example, definitive-HSC are entirely dependent on the transcription factor MYB, whereas yolk-sac-derived progenitors develop independently of MYB [26]. Genetic fate-mapping techniques give us the ability to identify and track different embryonic macrophage populations into adulthood precisely, and when combined with parabiotic (two organisms sharing the blood circulation) and adoptive-transplant studies, we can discern the relationship between macrophages and circulating blood monocytes. Fate-mapping studies utilize the principle of genetic recombination to permanently label cells (and all subsequent progeny) based on the recombination-induced expression of a reporter gene that is under the control of a constitutive promoter (typically Rosa26). Genetic techniques evolved to include inducible systems, where temporally controlling recombination facilitates the accurate labeling of embryonic populations and tracking them into adulthood [8]. In the assessment of many adult resident tissue macrophages, challenges remain because while these may originate during embryonic development, they exhibit differential expression of cell-surface markers as the animal matures, thus hindering the ability to precisely track the macrophage populations. For example, embryonically established tissue macrophages express the C-X3-C motif chemokine receptor, CX3CR1, during development in lung and peritoneal macrophages and liver Kupffer cells, but lose this expression after birth [27].

Several transient waves of hematopoietic cells are produced before the establishment of hematopoietic stem cells in the BM during late gestation, which correspond to days E6.5-E10.5 in the mouse [28, 29]. These embryonic waves are differentially regulated in both time and space and exhibit distinct lineage potentials that determine the hematopoietic populations in adulthood. They can also overlap in time and space and are therefore difficult to separate clearly, even with the most recent fate-mapping tools available. Table 2 below delineates the different embryonic waves in the mouse as they relate to the markers that are used to distinguish each of these progenitor populations. These waves include primitive hematopoiesis in the Yolk-sac (YS), and definitive hematopoiesis, which comprises a transient definitive stage, leading to the production of multi-lineage erythro-myeloid progenitors (EMPs) and lymphoid-myeloid progenitors (LMPs). The definitive stage is characterized by the creation of HSCs in the aorta-gonad-mesonephros (AGM). These transient progenitors establish themselves transiently in the fetal liver (FL) during the mid to late stages of hematopoiesis and remain difficult to separate clearly, even with the most recent fate-mapping tools available [3]. The primitive, transient definitive, and definitive waves of fetal hematopoiesis sequentially generate progenitors that can seed the FL, where they differentiate and expand to create the local macrophage population.

In mice, the first hematopoietic progenitors appear in the extra-embryonic YS blood islands at around embryonic age 7.25 (E7.25). This wave gives rise to mainly nucleated erythrocytes and megakaryocyte progenitors [30]. They express CD41 and colony-stimulating factor-1receptor (CSF-1R) and are independent of the transcription factor C-Myb, a signature of myeloid/macrophage commitment [31]. The expression of RUNX1, the Runt-related transcription factor 1, is noted explicitly at the E7.25 derived progenitors which give rise to microglia, brain-macrophages [32]. Upon the establishment of blood circulation at around E8.5, the EMPs differentiate into primitive macrophages as well as primitive erythrocytes and granulocytes. Primitive macrophages seed all fetal tissues, in particular, the head where they will give rise to future brain microglia that can continuously self-renew throughout adulthood [33, 34].

This wave constitutes the earlier lineage-restricted HSC-independent progenitors that seed the FL at E10.5. Because these progenitors arise concurrently with the transition of primitive to definitive erythropoiesis, they are considered a form of a transient stage of the definitive hematopoiesis [9, 35, 36]. This wave includes progenitors that are sequentially acquiring myeloid, and then lymphoid potential, without exhibiting the long-term reconstitution potential of the HSCs. E9.5 YS progenitors in the adult spleen express CD93 and CD19 [37, 38].

This wave is first established during embryonic development starting with the emergence of a small number of HSCs from the AGM at E10.5 in murine embryos or 5 weeks in human embryos [39]. After E9.5 in the mouse, new waves of hematopoietic progenitors emerge in the hemogenic endothelium (HE) of the embryo proper, and then in the AGM region and the placenta [40-42]. These progenitors seed the FL at approximately E10.5 [43, 44] to establish definitive hematopoiesis [45, 46]. Thus, the FL becomes the major hematopoietic organ after E11.5, where all hematopoietic lineages are generated; however, the FL itself does not produce progenitors de novo, but only recruits progenitors to initiate definitive hematopoiesis [47]. There is no specific expression marker to characterize embryonic HSC progeny except for Flt3 which could be used to follow the progeny of LMPs [48]. Schulz et al highlighted that a significant difference between primitive and definitive hematopoiesis is increased expression of the transcription factor Myb [26], however, Myb expression is not unique to this wave of hematopoiesis. The combination of the marker Sca-1, the hallmark of HSCs, and new markers such as those from the SLAM family (CD150, CD48, CD229, and CD244) have much helped clarify the characterization of HSCs as Lin− kit+ Sca-1+ CD150+ CD48− CD244− [49, 50].

Embryonic and neonatal macrophages play essential roles in tissue remodeling during development and compared to adult-monocyte-derived macrophages, they have a reduced capacity to generate inflammatory responses. Studies indicate that embryonic macrophages play a crucial role in regulating embryonic vascular growth and patterning, which is demonstrated by reduced CNS vascular complexity and branching in as early as E11.5 in macrophage-deficient embryos, and also a role in the clearance of senescent cells during embryonic development [51-54].

The origin of bone marrow-derived macrophages is from the definitive wave of embryogenesis described above. Accordingly, hematopoietic progenitors, which arise from the intraembryonic hemogenic endothelium give rise to fetal HSCs in the aorta, gonads, and mesonephros regions. These cells then migrate through the newly formed blood circulation and colonize the FL where they establish definitive hematopoiesis and seed the fetal BM, the site of adult BM HSCs production [48]. Fetal monocytes continue to participate in the tissue-resident macrophage network until hematopoiesis switches completely from the fetal liver to the bone marrow. Once adult hematopoiesis begins to take place in the bone marrow-generating monocytes, specific tissues, such as the dermis, heart peritoneum, and the gut, continue to recruit adult monocytes from the bone marrow to create resident macrophages and replace the embryonic-derived macrophages with time.

Studies of the MPS ontogeny discussed above revealed that monocytes and macrophages arise from a macrophage and dendritic cell progenitors (MDPs) which are present in the BM. As shown in Table 3, these cells are defined as c-kit+ CX3CR1+ Flt3+ CD115+ lineage [55]. These MDPs further differentiate through a newly described common monocyte precursor (cMoP), which is defined as c-kit+ CX3CR1+ Flt3− CD115+ lineage [56] that gives rise to the two main subsets of circulating monocytes that are distinguished by the expression of Ly6C; (1) the classical Ly6C^hi monocytes, and (2) Ly6C^lo nonclassical monocytes, which differentiate from Ly6C^hi monocytes in a Nr4a1-dependent transcriptional program [27, 56, 57]. These monocyte subsets are highly conserved and transcriptionally closely resemble human monocyte subsets, and the human counterparts of these monocyte subsets are termed classical (CD14++ CD16−), intermediate (CD14+ CD16+), and non-classical (CD14dim CD16++) monocytes. In addition to CD14 and CD16, the most informative markers that discriminate among these three monocyte populations are CCR2, CD36, HLA-DR, and CD11c [58]. The nonclassical subset, Ly6C^lo is unusual in that they appear to primarily function within the vasculature as “patrolling” cells that clear damaged endothelial cells and maintain the integrity of the vasculature [59] in a manner dependent upon LFA-1 integrin [60]. These cells do not express the core, mature macrophage signature of mRNA transcripts, including Mer tyrosine kinase [22, 61]. The classical Ly6C^hi monocytes are referred as "inflammatory monocytes", because they can enter resting tissues, patrol the extravascular tissues, and pick up antigens for transport to draining lymph nodes and remain relatively undifferentiated. Once they enter the tissue, Ly6C^hi monocytes can take on many characteristics of Ly6C^lo monocytes [62, 63]. Thus, monocytes of both Ly6C subsets carry out a patrolling function; the classical monocyte subset patrols the extravascular tissues, and the nonclassical subset patrols the intravascular space. This patrolling feature distinguishes monocytes from macrophages, which have a limited ability to emigrate in comparison to monocytes.

Each organ has its particular composition of embryonically derived and adult-derived macrophage subsets, and a unique pattern of circulating monocyte replacement of resident macrophages after birth. In a review on tissue-resident macrophages in steady state, Ginhoux and Guilliams described a classification of tissue-resident macrophage ontogeny based on the dynamics of monocyte recruitment into tissues. Accordingly, they categorized the primary tissues as closed - with no steady-state monocyte recruitment as is the case of the brain, epidermis, lung and liver; or open tissues - that recruit and differentiate bone marrow-derived monocytes into macrophages with kinetics that are specific to each tissue. The open-slow kinetics represent the dynamics in the heart and pancreas, and the open-fast dynamics in the gut and the dermis [11]. One unique category of the macrophage cell population is designated for Kupffer cells of the liver which are only temporarily “open” at birth but remain “closed” during adulthood, thus receiving only a minor contribution of neonatal monocytes [3, 27, 64]. Table 4 below organizes the different macrophage cells in the major adult body tissues based on this classification - see the BM monocyte recruitment dynamics column. Each of the main tissues is also described in regard to specific macrophage composition, markers, and unique functions. Chakarov S et al uncovered two distince types of interstitial macrophages (Lyve1^lo MHCII^hi and Lyve1^hi MHCII^lo) in murine lung, fat, heart and dermis [65]. In addition to the body tissues discussed below, macrophages from other body tissues / organs have been studied extensively. For example, Rowe SE et al detected spleen macrophages in mice with an F4/80 antibody [66] and Dai H et al isolated mouse spleen macrophages with anti-F4/80 beads from Miltenyi Biotec [67]. ElTanbouly MA et al identified macrophages from mouse spleens as CD11b+ F4/80+ cells [68]. MOMA-2 antibody, a generic marker of monocytes or macrophages, is very commonly used [69]. EX Han et al used F4/80 and CD68 as rat macrophage markers [70].

Brain microglia are derived entirely from yolk sac macrophages; therefore, in the steady state, their functions can be directly attributed to this lineage. After birth, a massive expansion of microglia cell numbers is driven exclusively through in situ proliferation via M-CSF and the CX3CR1 ligand IL-34 without monocyte input [71-74]. Resident microglia have critical homeostatic functions in regulating synaptic development. As in the embryo, microglia retain their ability to alter neuronal circuitry during the postnatal period, where they are critical for synaptic pruning through engulfment of neuronal synapses, in addition to phagocytosis of apoptotic neurons [75] - activities that are necessary for healthy brain development. Importantly, microglia dynamically interact with neurons at both pre- and postsynaptic sites through the extension of microglial processes, where prolonged contact leads to neuronal elimination [76]. The expression of CX3CR1 is critical in controlling microglia numbers, synaptic pruning, and functional brain connectivity as determined by Cx3cr1 deficient mice studies [77]. Brain microglia is discussed in detail in another article.

Epidermal Langerhans cells are unique macrophages that share many features with dendritic cells (DCs). After many years of debate about their classification, they are categorized as macrophages with DC functions. This is because they phenotypically share many features with DCs such as dendritic cell morphology, the ability to take up and process foreign antigens in steady-state conditions and inflammation and to present to cells of the adaptive immune system in the lymph nodes. This later function suggests that they are motile, like DCs, a feature that macrophages usually lack. They remain classified as macrophages because their ontology relates them to tissue-resident macrophages [78]. Recently, a highly elegant lineage tracing study validated the dual identity of Langerhans cells. The authors showed that Langerhans cells express the DC marker Zbtb46, while they originate from a Mafb-expressing progenitor, which indicate a macrophage origin [79]. Thus, Langerhans cells represent a highly unique cell type that share features with DCs but arise from a different origin. Langerhans cells are essential immune surveillance and homeostasis players, and they also induce tolerance or mediate inflammation and thus impact the pathology. Their unique capacity to self-renew within the epidermis, while also being able to migrate to lymph nodes to present antigen, place Langerhans cells in a key position to sample the local environment and decide on the appropriate cutaneous immune response. Langerhans cells are critically dependent upon the local production of IL-34 acting through the M-CSF receptor [72, 80]. In addition, distinct TGF-β signaling pathways are involved in the development of Langerhans cells and the maintenance of their homeostasis [81]. Unlike mouse Langerhans cells, the human cells express low levels of CD11c and no F4/80, whereas they express CD1a and CD1c, two MHC-I-related molecules that are involved in the presentation of lipid antigens [82].

Lung alveolar macrophages (AMF) reside on the epithelial surface of the lung, and in contrast to other resident macrophage populations, they are in direct contact with the environment, which includes commensal bacteria, inhaled particulates, and host-epithelial- derived factors, such as surfactants. Before birth, F4/80(hi) CD11b(lo) primitive macrophages and Ly6C(hi) CD11b(hi) fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. Embryonically derived fetal monocytes appear to colonize the lung shortly after birth and differentiate into alveolar macrophages in a granulocyte macrophages colony-stimulating factor (GM-CSF)-dependent process [85]. GM-CSF also induces PPAR-γ gene expression, a crucial transcription factor for AMF development which contributes to the unique gene-expression profile of lung macrophages [86]. Alveolar macrophages usually live independently of blood monocyte input; however, if alveolar macrophages are depleted, repopulation by in situ proliferation independent of blood monocyte input occurs [87]. Pulmonary surfactant is produced by alveolar epithelial cells, and together with other pathways, it acts to suppress alveolar macrophage activation during resting conditions [88]. An essential role of alveolar macrophages is the ability to phagocytose excessive surfactant proteins and prevent alveolar proteinosis [85].

Liver Kupffer cells originate from yolk sac-derived precursors during embryogenesis and monocyte-derived macrophages. Kupffer cells are essential for maintaining tissue homeostasis and ensuring rapid responses to hepatic injury. Because the liver is continuously exposed to antigens from the intestine and to low levels of bacterial endotoxins, Kupffer cells help suppress an ‘accidental’ activation of the immune system and play a significant role in maintaining immunological tolerance in the liver and in providing an anti-inflammatory micromilieu during homeostasis [89]. Kupffer cells are activated by exposure to lipopolysaccharide, interferon-gamma, or IL-4/IL-13 [90, 91]. Following their activation, they modulate inflammation and recruit immune cells [92]. The Interferon regulatory factor 5 (IRF5) has recently emerged as an critical proinflammatory transcription factor that is involved in their activation under acute and chronic inflammation [93]. In human liver, CD68 has been proposed as a marker; however, it is not an exclusive marker for Kupffer cells [94], but more recently, CD163L was proposed as a marker for tissue-resident macrophages [95].

Cardiac Macrophages play critical roles in homeostatic maintenance of the myocardium under normal conditions and tissue repair after injury. In the steady-state heart, resident cardiac macrophages remove senescent and dying cells and facilitate electrical conduction [96]. Cardiac macrophages originate from the embryo yolk-sac and fetal monocytes progenitors and give rise to adult cardiac macrophages; however, the embryonic resident macrophages can then be replaced by BM and spleen-derived monocytes, especially after heart injury such as cardiac ischemia [97]. This type of heart injury may stimulate the recruitment of a large number of macrophages, resulting in about one-third increase in cardiac macrophages [96]. As such, cardiac macrophages are ontologically and functionally distinct, as there are several subsets with mixed ontological origins [97]. Dick et al used fate mapping, parabiosis, and single-cell transcriptomics to demonstrate that at steady state, changes in the levels of four markers may reveal the hierarchy of monocyte contribution to functionally distinct cardiac macrophage subsets; cardiac macrophages that self-renew with negligible blood monocyte input are represented by TIMD4+ LYVE1+ MHC-IIlo CCR2−, monocytes partially replaced resident macrophages are TIMD4– LYVE1– MHC-IIhi CCR2− , and entirely replaced macrophages are TIMD4− LYVE1− MHC-IIhi CCR2+ [98]. FC Simões et al sorted Ly6G F4/80+ cells from single cell cardiac tissue suspensions as macrophages [99].

Pancreas macrophages are found in two intrapancreatic regions; in islets of Langerhans, and the inter-acinar stroma, and these different populations exhibit distinct origin and phenotypic properties. These two regions represent the pancreas endocrine component (islets of Langerhans) that secretes hormones into the bloodstream, and the exocrine component (acinar glands) that secretes digestive enzymes into the intestine. The islets macrophages are derived from definitive hematopoiesis, depend on CSF-1, and they can be distinguished based on high expression of CD11c, MHC-II, F4/80, CD11b, CD64, CX3CR1, and CD68. The inter-acinar stroma population is heterogeneous and comprised of three major sets that can be profiled based on the expression of the surface markers F4/80 and CD11b (F4/80+ CD11b+, F4/80- CD11b+, F4/80- CD11b-) [100]. The function of pancreatic macrophages is speculated to be involved in the regulation of angiogenesis or lymphogenesis. One specific scenario is that in the high proteolytic and bicarbonate-rich environment of the stroma or the hormone-rich environment of the islets of Langerhans for example, these macrophage-rich populations serve a distinct environment that leads to functional specialization and support for the different cells that are present in these extreme environments [23, 24].

Gut Macrophages represent the most abundant source of resident tissue macrophages [101, 102]. Gut-resident Macrophages are an essential immune population that is capable of integrating and interpreting different food-derived, commensal-derived, pathogen-derived, and host-derived signals in their environment. Moreover, under inflammatory response, BM-derived gut macrophages and resident macrophages play crucial roles in the control of infection. Thus, the dynamic regulation of the intestinal macrophage pool is at the center of long-term health [103]. BM-derived macrophages that replenish the resident pool in a healthy gut are identified as CX3CR1^hiMHCII^hiLy6C^low [104], and they have multiple crucial functions in gut homeostasis including Treg expansion, epithelial maintenance, luminal sampling, and bacterial killing [105, 106]. As noted in Table 4, the gut and dermis are considered open tissues with fast kinetics of recruitment and differentiation of bone marrow-derived monocytes into macrophages [11]. And although at birth, there are embryonically derived macrophages present in the gut, these are replaced by cells derived from an influx of CCR2-dependent Ly6C^hi monocytes [107]. IL-10 receptor signaling on macrophages is one pathway that is crucial for instructing macrophage function in the gastrointestinal mucosa [108, 109].

The immune system within the skin is located in the epidermis and the dermis, and while immunity in the epidermis is mediated via Langerhans cells (described above), the effectiveness of the dermis immunity depends on the close communication between several immune cell types residents of the dermis including dendritic cell subpopulations, T cells, and macrophages [110]. Dermal macrophages are occupied with the clearance of senescent cells, extracellular debris, and maintenance of tissue homeostasis [102, 111]. In addition, involvement in graft versus host disease (GVHD) has been described in terms of their innate immune function by providing critical inflammatory signals such as TNF-α in response to IFN-γ and LPS [112, 113], Recently, however, dermal macrophages were also implicated in the induction of GVHD that was promoted by antigen-specific stimulation of allogeneic T cell responses, a function reserved to DCs. Using tandem transplant experiments in mice model, which require the induction of GVHD [114], the authors show that dermal macrophages outlive all other cutaneous antigen-presenting cells and can enhance GVHD through antigen-specific means by presenting host tissue antigens [115]. Macrophages show consistent CD163 and CD14 expression, lower CD45 and HLA-DR expression, and no CD1a or CD1c expression. They produce IL-1 and IL-6 and synthesize TNF-α and IL-23 upon stimulation [116].

As outlined from the descriptions of tissue macrophages above, most resident tissue macrophages can maintain themselves indefinitely without monocyte input, but circulating monocytes can readily repopulate the macrophage niche. Also, examples of tissue-specific factors and signaling that drive highly specialized macrophage functions irrespective of their ontological origin, suggest that there are tremendous plasticity and redundancy in the mononuclear phagocyte system. As Table 4 outlines, macrophages in the different tissues display various immune-related functions based on the resident organ function, and they express unique markers, produce specific cytokines, and are sensitive to individual signals. In some tissues, the wide range of immunological functions even challenged the identification of macrophages from other immune cells such as DCs (in epidermal Langerhans cells, and macrophages of the dermis). This knowledge as a whole is significant in our ability to design future intervention strategies to modulate macrophage functions at specific tissue sites.

Although macrophages are terminally differentiated cells, both macrophages and their monocytic precursors can change their functional state in response to microenvironmental cues, and exhibit a different activation state. Thus, in addition to their unique profiles in the tissues, macrophages can be functionally polarized into different activation/functional states. Unlike lymphocytes where phenotypic changes are “fixed” by changes in transcription programs after exposure to polarizing cytokines, macrophages have a plastic gene expression profile that is influenced by the type, concentration, and duration of exposure to the stimulating agents [117-119]. Macrophages are also renowned for their apparent phenotypic heterogeneity and for the diverse activities in which they engage [120]. These two factors are important to consider in studies, and present a challenge since many of these activities appear to be opposing in nature: pro- versus anti-inflammatory effects, immunogenic versus tolerogenic activities, and tissue destruction versus tissue-repair [121]. Moreover, the functional pattern expressed by macrophages changes with time as reported for example after LPS stimulation, which result in significant differences between genes that are expressed early (up to 6 h) versus late (12–24 h or later) [122]. To address the heterogeneous features of macrophages upon activation, a sub-classification of macrophages was devised.

In the 1990s, Nathan CF et al discovered that the cytokine interleukin IL-4 induced different effects on macrophage gene expression compared to that of interferon IFN-gamma and lipopolysaccharide (LPS) [123]. A few years later, observing similar heterogeneous effects on macrophages, Mills et al proposed to classify macrophages as either M1, to designate the proinflammatory macrophages or M2, as the alternatively activated macrophages [124]. Accordingly, M1 was thought to mainly produce the antimicrobial and pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-1, IL-6, and Nitric Oxide Synthase 2 (NOS2), which are important for early immune defense. In contrast, M2 was characterized by high levels of IL-10, Arginase 1 (ARG1) and the Manose receptor C-type1 (MRC1), possibly contributing to tissue repair and for the resolution of inflammation [125]. The M1 versus M2 designation is a widely utilized paradigm in mice, and it appears to be conserved to some degree in humans [126]. For instance, AA Kapralov et al reported that M1 and M2 cells have different susceptibility to ferroptosis [127]. However, studies now demonstrate that treatment of human blood monocytes with a variety of stimuli results in a spectrum of macrophage phenotypes rather than two distinct subsets [25, 128]. Thus, the M1/M2 classification of macrophages is now considered an oversimplified approach that does not adequately describe the spectrum of macrophage populations, including the identification of tumor-associated macrophages (TAMs) which do not fit into the criteria for M1 or M2 macrophages [129], or macrophages that express T cell receptors (TCR) and CD169. Moreover, it has been demonstrated that the process of polarization from the M1 state to the M2 is reversible. Davis MJ et al demonstrated that macrophages are capable of complete and rapid repolarization from M2 to M1 depending on the chemokine environment, in a mechanism that involves rewiring of the signaling networks at both the transcriptional and translational levels [130, 131]. It is now appreciated that the M2 group encompasses a functionally diverse group of macrophages that can be further subdivided into four subsets; M2a, M2b, M2c, and M2d based on their distinct gene expression profiles. As shown in table 5 below, the M2a subtype is elicited by IL-4, IL-13 or fungal and helminth infections. M2b by IL-1 receptor ligands, immune complexes and LPS, M2c by IL-10, TGF-b and glucocorticoids, and the M2d is elicited by IL-6 and adenosine [132].

Macrophages are polarized towards the M1 phenotype by LPS, IFN-g, and GM-CSF stimulation, which induces secretion of large amounts of cytokines such as IL-1b, TNF, IL-12, IL-18, IL-23, and drive antigen-specific cell inflammatory responses. They also secrete the Th1 cell-attracting chemokines CXCL9 and CXCL10, and express high levels of major histocompatibility complex class II (MHC II), CD68 marker, and co-stimulatory molecules CD80 and CD86 (see Table 5). M1 macrophages up-regulate the expression of the intracellular protein suppressor of cytokine signaling 3 (SOCS3) and activate inducible NOS2 to produce nitric oxide from L-arginine [133, 134]. In a disease state, they are implicated in initiating and sustaining the inflammatory response and are thus crucial for health.

The M2 macrophages are induced by fungal cells, immune complexes, helminth infections, complement components, apoptotic cells, macrophage colony stimulating factor (MCSF), and by IL-4, IL-13, IL-10 and TGF-b. M2 macrophages secret high levels of IL-10 and low levels of IL-12 (IL-12^lowIL-10^high), and the chemokines CCL17, CCL22, and CCL24. In addition, they express high levels of scavenger mannose and galactose E-type and C-type receptors and have the capacity to repurpose arginine metabolism to express ornithine and polyamine, which promotes growth. As outlined in table 5, the M2 macrophages can be further subdivided into M2a, M2b, M2c, and M2d, and each subgroup is uniquely activated by different inducers, which also stimulate specific cytokines and chemokines secretion.

Some of the markers that are used to define the M1 and M2 polarization vary between mouse and human macrophages such as arginase-1 [126] ; however, some molecules are conveniently conserved such as the multifunctional enzyme transglutaminase 2 (TGM2) [126]. One of the most debated issues in the context of human macrophage polarization is the identification of unique or restricted markers that may be used for research and clinical purposes to reflect the distinct biological role/s of each subset. The most frequently utilized markers for the M1 and M2 subsets in human samples are shown in table 6 below- adapted from references [126, 135].

Macrophages are intriguing immune cells with complex ontogeny, developmental stages, and functions. The ongoing aim to label these cells and designate specific markers to track their cell of origin, tissue location, a particular function or activation states has been met with several layers of complexity which are termed in the literature “plastic” and “heterogeneous.” It is now clear that several factors must be considered before deciding on the best markers that can be used in each macrophage study. As outlined in the different tables in this review, macrophage markers are different during development, in the adult tissue, when monocytes replenish them, or when they become immune activated and polarized. And although there are now five categories of macrophages activation states (M1, M2a, M2b, M2c, M2d) each with different designated markers, there is still lack of agreement that these categories sufficiently describe activated macrophages. Additional challenges are introduced in the study of microglia - brain macrophages - and their activation from an inaccessible organ when trying to study progressive neurodegenerative disorders such as AD for example. The study of microglia activity and polarization is met with the limitation of working mostly with ex vivo brain samples and relying on antibodies that may specifically work well with fixed human tissue sections. Despite all these challenges, researchers now have an extensive repertoire of markers to work with, and as advances in imaging techniques, novel preservation techniques, and possibly fluid biomarker advancements, we can expect that breakthroughs in our understanding of macrophages and macrophages markers will increase and lead to the development of targeted therapies.