Origins and Hallmarks of Macrophages: Development, Homeostasis, and Disease

Introduction and Perspectives

Macrophages, originally identified by Metchnikoff by their phagocytic nature, are ancient cells in Metazoan phylogeny. In adult mammals, they are found in all tissues where they display great anatomical and functional diversity. In tissues, they are organized in defined patterns with each cell occupying its own territory, a type of tissue within a tissue. Although several attempts have been made to classify macrophages, the most successful definition is the mononuclear phagocytic system (MPS), which encompasses these highly phagocytic cells and their bone marrow (BM) progenitors. In the MPS schema, adult macrophages are defined as end cells of the mononuclear phagocytic lineage with tissue macrophages deriving from circulating monocytes that originate in the BM. However, this definition is inadequate as macrophages have several origins during ontogeny and each of these different lineages persist into adulthood where they display great diversity ¹. Other classifications have included binary classifications that refer to inflammatory states. These include the activated (AM) and alternatively activated (AAM) or M1 or M2 ^2,3 defined by responses to the cytokines IFNγ and activation of TLRs and IL4/IL13 respectively. While this is a useful heuristic that may reflect extreme states such as in AMs in THI mediated immune responses or AAM in parasitic infections ² it cannot represent the complex in vivo milieu for most macrophage types where numerous cytokines and growth factors interact to define the final differentiated state of macrophages. Indeed transcriptional profiling of resident macrophages by the “Immunological Genome Project” find that these populations show great transcriptional diversity with minimal overlap suggesting many unique classes¹.

Macrophages have roles in almost every aspect of an organism’s biology ranging from development, homeostasis, to repair through to immune responses to pathogens. Resident macrophages regulate tissue homeostasis by acting as sentinels and responding to changes in physiology as well as challenges from outside. During these homeostatic adaptations, macrophages of different phenotypes can also be recruited from the monocyte reservoirs of blood, spleen and bone marrow ⁴, and perhaps, from resident tissue progenitors or through local proliferation ^5,6. Unfortunately, in many cases, these homeostatic and reparative functions can be subverted by continuous insult, resulting in causal association of macrophages with disease states, such as fibrosis, obesity and cancer (Figure 1). Macrophages, therefore, are an incredibly diverse set of cells constantly shifting their functional state to new set points in response to changes in tissue physiology or environmental challenges. They should not even be considered as one cell type but be subdivided into different functional subsets with acknowledgement to their different origins.

This review will not dwell on macrophage responses to pathogens that have been discussed extensively by others^2,7,8, but will emphasize the homeostatic mechanisms by which macrophages contribute to physiological and pathophysiologic adaptations in mammals. It will define the hallmarks of “macrophage” performing particular functions, taking into account new insights into the diversity of their lineages, identity and regulation. This phenotypic diversity is essential to understand because macrophages are central to many disease states, and have emerged as important therapeutic targets in many diseases.

Macrophage Origins Rewritten

Ontologically, the MPS has been proposed to arise from a rigid temporal succession of macrophage progenitors ⁹. In mice, these first develop at embryonic (e) day 8 from the primitive ectoderm of the yolk sac and give rise to macrophages that do not have a monocytic progenitor. This primitive system is followed by definitive hematopoiesis in the fetal liver, which is initially seeded by hematopoietic progenitors from the yolk sac and subsequently from the hematogenic endothelium of the aorto-gonadal-mesonephros region of the embryo. Thereafter, during embryogenesis the fetal liver is the source of definitive hematopoiesis generating circulating monocytes. Coincident with the post-natal formation of bone, fetal liver hematopoiesis declines and is replaced by BM hematopoiesis. This definitive hematopoiesis is the source of circulating monocytes (resident Ly6c- and inflammatory Ly6c+ in mice) and from which it has been considered that all resident macrophages in tissues are derived ⁴. However, this model for the formation of the MPS has been challenged (Figure 2). First, lineage-tracing experiments have shown that Langerhans cells (LC) and microglia are primarily derived from the yolk sac progenitors while LCs have a mixed origin from YS and fetal liver ^10,11. Second, even in the absence of hematopoietic stem cells, yolk sac progenitors were capable of giving rise to the major tissue resident population of macrophages (defined as F4/80 bright) in skin, spleen, pancreas, liver, brain and lung. In a few tissues, such as kidney and lung, macrophages were shown to have a chimeric origin (HSC- and yolk sac-derived), whereas other cells, such as classical dendritic cells (DC) and lesser populations of F4/80^low cells, were continuously replaced by BM-derived progenitors ⁶. These data indicate that there are at least three lineages of macrophages in the mouse arising at different stages of development and persisting in the adult. It also calls into question the function of circulating monocytes because, at least in mice, these cells do not seed the majority of the adult tissues with macrophages. In fact, complete loss of CD16⁺ monocytes in humans appears to be of little consequence ¹². Thus the function of monocytes needs to be defined with the possibility that patrolling monocytes (Ly6c-) act to maintain vessel integrity and to detect pathogens while inflammatory monocytes (Ly6c+) are recruited only to sites of infection or injury or to tissues that have continuous cyclical recruitment of macrophages such as the uterus.

Regardless of their origin, genetic and cell culture studies indicate the major lineage regulator of almost all macrophages is the colony stimulating factor 1 receptor, CSF1R. This class III transmembrane tyrosine kinase receptor is expressed on most, if not all, mononuclear phagocytic cells, and a reporter mouse expressing GFP from the Csf1r locus illustrates their relative abundance (5–20% of cells) and tissue distribution ¹³. Csf1r expression and its requirement for differentiation distinguish macrophages from many but not all DCs sub-types ¹⁴. Targeted ablation of the Csf1r causes severe depletion of macrophages in many tissues, such as brain, skin, bone, testis, ovary etc. Moreover, an initial comparison of the Csf1r null mice with a spontaneous mutant that lacks its cognate ligand CSF1 (Csf1^op/op mice) demonstrated that all phenotypes in the receptor null were also found in the ligand null, indicating that CSF1 only has a single receptor ¹⁵. However, the phenotype of the Csf1r null mice is more severe than that of the Csf1 null mice, including the complete loss of microglia and LCs ^10,16 in the Csf1r null mice, which suggested the presence of another ligand. Indeed, IL34, with a distinct but overlapping pattern of expression with Csf1, was recently identified as an additional ligand for the CSF1R ¹⁷. Targeted ablation of Il34 resulted in loss of microglia and LCs, but had little impact on BM, liver, or splenic macrophages ¹⁸.

Despite the importance of the CSF1R in macrophage specification, Csf1r null mutant mice still have some tissue macrophages, such as in the spleen, indicating the existence of other macrophage growth factors. Potential candidates include GM-CSF and IL3 that act as macrophage growth factors in tissue culture. However, mice lacking GM-CSF or IL3 do not exhibit significant defects in their tissue macrophages except alveolar ones that are regulated by GM-CSF ¹⁹. VEGF is another candidate regulator of macrophages because it can compensate for the loss of Csf1 in osteoclast development in vivo ²⁰. In contrast to CSF-1 that is found in all tissues and serum and is a basal regulator of macrophage number through a negative feedback loop ¹⁵, GM-CSF is not a steady-state ligand and appears to be synthesized in response to challenge ²¹. GM-CSF and FLT3L regulate the maturation of DC populations with the notable exception of LCs, whose development is dependent on Csf1r ²². Recent genomic profiling of LCs place them closer to macrophages than DCs and this data together with their lineage dependence on Csf1r, may require changes in classification ¹⁴. DCs will not be discussed further in this review but their biology and lineages have been extensively covered recently ¹⁴.

In their basal state, resident tissue macrophages show great diversity in their morphologies, transcriptional profiles, anatomical locations, and functional capabilities ²³. This functional heterogeneity likely results from the dynamic crosstalk between resident tissue macrophages and the client cells that they support. To understand this macrophage diversity there must be an understanding of transcriptional regulation. The most important of these transcription factors is PU1, Spl1, a member of the ETS family whose loss following targeted mutation results in complete depletion of Cd11b+ F4/80+ macrophages including those derived from the YS ⁶. However, Pu.1 action is not limited to macrophages as B cells are also severely depleted in these null mutant mice. Similarly other members of the ETS family are also involved in macrophage differentiation including Ets2 that positively regulates the Csf1r promoter. In adults, Mafb (c-Maf) is required for the local proliferation that maintains resident macrophages ⁴. In the differentiation of osteoclasts, Fos and Mitf are required ²⁴, whereas Gata-2 is also required for monocyte development but not resident macrophage populations ²⁵. However, little is known about the transcriptional control of the differentiation of the diverse tissue macrophages, such as those in the liver and brain ¹³. Rather most research has focused on their functional activation in response to environmental challenges ²³, topics that will be discussed in following sections. Nevertheless, the recent transcriptional profiling of resident macrophages has identified many candidate transcription factors including those that may regulate core macrophage-associated genes such as MiT (Micropthalmia) family members, Tcef3, C/EBPa, Bach1, Creg-1 and those unique to sub-populations including Gata6 and Spi-c whose targeted gene ablation will undoubtedly define sub-sets of macrophages and their unique activities ¹.

Macrophages in Development

Metchnikoff proposed that macrophages participate in the maintenance of tissue integrity and homoeostasis. This necessitated that macrophages be able to discriminate self from non-self, sense tissue damage, and recognize invading pathogens, an insight that led to the concept of innate immunity for which he was awarded the Nobel Prize. The properties that included sensing inside from out together with motility throughout the organism, phagocytosis and degradation were later sequestered to instruct the acquired immune system as it evolved to more efficiently deal with ever changing pathogenic challenges. This enhanced sophistication of the immune system probably resulted in the evolution of DCs as specialized mononuclear phagocytes to interface with the acquired immune system. Indeed, in mammals, DCs appear to be focused upon initiating tissue immune responses, while tissue macrophages on homeostasis and tissue integrity ⁹.

Emphasis on the immunological and repair aspects of macrophage function has rather overshadowed their importance in the development of many tissues. For instance, studies of Csf1 null mutant mice, which lack many macrophage populations, have revealed a cluster of developmental abnormalities ¹⁹. Most notable amongst them is the development of osteopetrosis, which is caused by the loss of bone reabsorbing macrophages known as osteoclasts. This phenotype, which is also observed in Spl1 null mice, is axiomatic for the roles of macrophages in development, in that cell fate decisions are unchanged but the tissue remodeling and expression of growth factors is lost. Specifically, while bone formation is intact in Csf1 or Spl1 null mice, the bones are not sculpted to form the cavities where hematopoiesis can ensue ¹⁹. Consequently, the functional integrity of the bones, in terms of load bearing and hematopoiesis, is compromised. Csf1 null mice survive to adulthood because of extra-medullary hematopoiesis in the spleen and liver ¹⁹, and as mice age, osteoclastogenesis is rescued by compensatory expression of VEGF ²⁰.

Remodeling deficiencies in the absence of macrophages have also been noted in several other tissues, including the mammary gland, kidney and pancreas suggesting a general requirement for macrophages in tissue patterning and branching morphogenesis ^19,26. In the mammary gland, the best studied of these tissues, macrophages are recruited to the growing ductal structure and their loss results in a slower rate of outgrowth and limited branching, phenotypes that are re-iterated during the mammary growth caused by pregnancy ¹⁹. This stems in part from the failure to remodel the extracellular matrix during the outgrowth of the ductal structures. However, recent studies have also implicated macrophages in maintaining the viability and function of mammary stem cells, which reside at the tip of the duct known as the terminal end bud and are responsible for the outgrowth of this structure ²⁷. Similar roles for macrophages in stem cell biology has been suggested in the maintenance of intestinal integrity and its regeneration after damage ²⁸, whereas a sub-population of macrophages in the hematopoietic niche regulates the dynamics of hematopoietic stem cell release and differentiation ²⁹. Furthermore in regenerating livers, macrophages specify hepatic progenitor fate through the expression of WNT ligands and antagonism of Notch signaling ³⁰. Macrophage control of stem cell function is clearly an emerging and important research area.

As professional phagocytes, macrophages perform critical functions in the remodeling of tissues, both during development and in the adult animal. For instance, during erythropoiesis, maturing erythroblasts are surrounded by macrophages, which ingest the extruded erythrocyte nuclei. Remarkably, this function of macrophages is critically important because in its absence, erythropoiesis is blocked and lethality ensues ³¹. Macrophages also make decisions about hematopoietic egress from the bone marrow through engulfing cells that do not express the CD47 ligand ³². They also maintain hematopoietic steady state via engulfment of neutrophils and erythrocytes in the spleen and liver and the failure of this activity results in neutropenia, splenomegaly and reduced body weight ³³. Phagocytosis particularly of apoptotic cells is obviously central to macrophage function and this is emphasized by the build up in macrophage-depleted mice of such cells during development for example during the resolution of the inter-digit areas during limb formation ³⁴. However, there is no apparent consequence to this phenomenon as non-professional phagocytes clear excess apoptotic cells. Despite this, macrophages have evolved to “eat” cells, and to suppress inflammation and auto-immunity in response to self-antigens that might arise during homeostasis ³⁵.

Macrophages also regulate angiogenesis through a variety of mechanisms. This has been most extensively studied in the eye during its development. Early in the postnatal period during regression of the hyaloid vasculature, macrophages identify and instruct vascular endothelial cells (VEC) to undergo apoptosis if these cells do not receive a counter-balancing signal from pericytes to survive. Wnt7b synthesized by macrophages delivers this cell death signal to the VECs and either its absence or the absence of macrophages, there is vascular over-growth ³⁶. WNT secretion is also required later in retinal vasculature development but in this case macrophage synthesized Wnt5a, a non-canonical WNT, induces expression of soluble VEGFR1 through an autocrine mechanism that titrates VEGF and thereby reduces vascular complexity so that the vascular system is appropriately patterned ³⁷. Furthermore, at other times of ocular development macrophages regulate vascular complexity. In this circumstance macrophage synthesized VEGFC reinforces Notch signaling ³⁸. In addition, during angiogenesis in the hindbrain macrophages enhance the anastomosis of tip and stalk cells to give functional vessels ³⁹. These macrophage functions are not restricted to the vascular arm of the circulatory system since they also play roles in lymphangiogenesis during development ⁴⁰ and in adults they play a significant role in maintaining fluid balance through their synthesis of VEGFC ⁴¹.

Brain development is also influenced by macrophages. These macrophages called microglia depend on CSF1R signaling for their presence ^10,16. In the absence of this signaling there are no microglia and the brains of these mice have significant structural defects as they mature ¹⁶. Both CSF1 and IL34 are expressed by neurons in a mutually restricted pattern of expression with IL34 being the major factor for microglial differentiation and viability ^10,42. The disruption of architecture in the Csf1r null mouse brain together with well-documented deficiencies in neuronal processing regulating olfaction and the reproductive axis in the hypothalamus in Csf1 null mice strongly suggest that microglia are involved in the development of neuronal circuitry and maintenance of brain structure ^16,19. Indeed microglia have been shown to promote neuron viability ¹⁹, modulate neuronal activity ⁴³, to prune synapse during development ⁴⁴ as well as express a range of neuronal growth and survival factors including NGF ¹⁹. This conjecture is reinforced because a hypomorphic mutation in the CSF1R in humans is responsible for hereditary diffuse leukoencephalopathy with spheroids that results from loss of myelin sheaves and axonal destruction ⁴⁵. These trophic activities of microglia are also consistent with macrophage roles in neuroprotection following injury in a variety of contexts. These effects include the promotion of survival and proliferation of retinal progenitor cells and the regeneration of adult sensory neurons ^46–48. However, caution needs to be exercised in attributing all the phenotypes observed in the brains of Csf1r mutant mice or humans to the loss of microglia as Csf1r expression has been reported on neuronal stem cells and their development in vivo is regulated by CSF1R ⁴². Nevertheless, it seems likely that microglia play important roles in the development of neuronal circuitry though their effects on the proliferation, survival and connectivity of neurons ⁴³ or through effects on myelination or by modulating angiogenesis and fluid balance in the brain ¹⁶.

The examples cited above indicate a few of the roles for macrophages in normal development and these are likely to expand with further study. Phenotypically in mice, macrophages are all Cd11b+, CD68+ CSF1R+ F4/80+ and phagocytic and their activities are through the temporal and spatial delivery of developmentally important molecules such VEGFs and WNTs as well as proteases. These developmental roles of macrophages are re-capitulated in repair as described below but are also intimately involved in chronic conditions that lead to pathologies as well as the development and progression of malignancies.

Macrophages in Metabolic Homeostasis

Mammalian metabolic organs like liver, pancreas, and adipose tissue are composed of parenchymal and stromal cells, including macrophages, which function together to maintain metabolic homeostasis ⁴⁹. By regulating this interaction, mammals are able make dramatic adaptations to changes in their environment and nutrient intake. For instance, during bacterial infection, innate activation of macrophages results in secretion of pro-inflammatory cytokines, such as TNFα, IL6 and IL1β, which collectively promote peripheral insulin resistance to decrease nutrient storage^50,51. This metabolic adaptation is absolutely necessary for mounting an effective defense against bacterial and viral pathogens because nearly all activated immune cells preferentially utilize glycolysis to fuel their functions in host defense. However, this adaptive strategy of nutrient reallocation becomes maladaptive in the setting of diet-induced obesity, a state characterized by chronic low-grade macrophage-mediated inflammation^51,52. In the sections below, we provide a general framework for understanding the pleiotropic functions performed by macrophages to maintain metabolic homeostasis (Figure 3). Although our current knowledge in this area is primarily derived from studies in obese insulin resistant mice, it is likely that tissue resident macrophages also participate in facilitating metabolic adaptations in healthy animals.

Macrophages in disease

When tissues are damaged following infection or injury, inflammatory monocytes (Ly6c+ in mice) are recruited from the circulation and differentiate into macrophages as they migrate into the affected tissues ⁴. These recruited macrophages often exhibit a pro-inflammatory phenotype in the early stages of a wound healing response. They secrete a variety of inflammatory mediators including, TNFα, IL1, and nitric oxide (NO), which activate anti-microbial defense mechanisms, including oxidative processes that contribute to the killing of invading organisms ⁷. They also produce IL12 and IL23, which direct the differentiation and expansion of anti-microbial T_H1 and T_H17 cells that help drive inflammatory responses forward ³. Although these inflammatory macrophages are initially beneficial because they facilitate the clearance of invading organisms, they also trigger substantial collateral tissue damage because of the toxic activity of reactive oxygen and nitrogen species and of T_H1 and T_H17 cells ⁷¹. Indeed, if the inflammatory macrophage response is not quickly controlled, it can become pathogenic and contribute to disease progression, as is seen in many chronic inflammatory and autoimmune diseases ^72,73. To counteract the tissue damaging potential of the inflammatory macrophage response, macrophages undergo apoptosis or switch into an anti-inflammatory or suppressive phenotype that dampens the pro-inflammatory response, while facilitating wound healing ⁷. These regulatory macrophages often produce ligands associated with development such as WNT ligands, that are essential for tissue repair ⁷⁴. It is becoming increasingly clear that the mechanisms that regulate the transformation of inflammatory macrophages into an anti-inflammatory cell or suppressive macrophages back into a proinflammatory phenotype has a major impact on the progression and resolution of many chronic diseases, as discussed in the following sections (Figure 4).

Perspectives

The understanding of macrophage biology is rapidly expanding with an appreciation of their diverse origins, transcriptional complexity, lability and phenotypic switching according to homeostatic demands and in response to insult. Macrophages are involved in almost every disease and thus represent extremely attractive therapeutic targets that either augment or inhibit their responses. However for these therapies to be effective it is necessary to understand macrophage diversity and define the cells according to anatomical location and function as well as the regulation of the particular set-points that defines the particular recognizable macrophages such as microglia, osteoclasts and Kuppffer cells. In fact the recognition of multiple origins (YS, FL, BM) may result in the conclusion that there is no such thing as a “macrophage” but rather clades of cells that have similar characteristics but with different origins. In fact, their different origins may allow unique opportunities to selectively target the recruited ones in the context of the chronic diseases discussed above thereby inhibiting the pathology without disturbance of resident macrophages and thereby normal homeostasis. To define these similarities and differences it will be necessary to determine proteomes and transcriptomes of particular subtypes as was recently performed for resident macrophages ¹. Clearly the rapidly advancing field of genomic analysis will provide unique insights and novel ways of defining macrophage types. Furthermore macrophage biology in humans is poorly developed because of the obvious technical limitations of getting fresh material for FACS and the over-reliance in functional and genomic studies on cell lines such as the myelomonocytic leukemic cell line THP1 ¹²³ or the in vitro differentiation of circulating monocytes by CSF-1. Significant differences also exist between human and mouse macrophages for example the inability of human macrophages to increase arginase-1 expression that is an important marker of IL4-regulated macrophages in mice ³. These differences render even binary classifications such as M1 and M2 wanting let alone more complex ones. Definition of human macrophage diversity is just beginning ¹²⁴ with several ongoing sequencing efforts and these efforts will enhance the essential need to translate mouse biology into the human context.

Considerable advances in defining macrophage biology have recently been made using mouse genetic approaches. For example the use of fluorescently labeled macrophages by expressing GFP from the Csf1r promoter to define and in some case live image them using intravital microscopy ¹²⁵. Furthermore, the development of macrophage restricted cre recombinases for example expressed from the LysM or Csf1r promoters and the ability to ablate macrophages through the expression of the Diptheria toxin receptor that sensitized mouse cells to the toxic effect of Diptheria toxin ¹¹⁹ or using miRNAs to direct expression of HSV TK in macrophages has been key to defining functions. While these systems have given major insights into macrophage function caution needs to be exercised as none of the promoters are uniquely expressed in macrophages and they are also expressed in most macrophage types thereby making it difficult to discriminate the functions of sub-classes of macrophages. As we move forward specific promoters will be developed to ablate genes in particular sub-sets and more sophisticated lineage tracing to follow fates and sub-type switching will be possible through photoactivatable flours such as Dendra2 that allow single or a few cells to be tracked ¹²⁵.

Therapeutic targeting of macrophages is now well underway ^21,23. Most of the therapies are targeted at pan-macrophage markers such as the CSF1R. Reagents either inhibiting the ligand, ligand binding or the tyrosine kinase activity are various stages of clinical trials in cancer, including small molecules and mAbs directed against CSF1R ²¹. Other strategies in fibrosis and cancer have been to target recruitment of macrophages particularly through inhibition of inflammatory monocyte trafficking with anti-CCL2 or CCR2 antibodies. In other circumstances, the protective effects of recombinant human serum amyloid P (SAP; also known as pentraxin 2) in IPF and post-surgical scarring in patients treated for glaucoma is thought to be through the reduction of inflammation and fibrosis by inducing the production of IL-10 in regulatory macrophages ¹⁰⁷. Neutralization of GM-CSF using antibodies is being tested in phase II trials for multiple sclerosis and Rheumatoid arthritis ²¹. In the future it seems it will be possible to exploit the inherent plasticity of macrophages to adjust their set points to control obesity by down-modulating inflammatory cytokines, to resolve fibrosis by inducing the differentiation of resolving macrophages and to treat cancer by converting macrophages from their trophic to an immunologically activated anti-tumoral state.