Until recently, it was thought that macrophages exclusively originate from hematopoietic stem cell (HSC)-derived monocytes in the bone marrow and are released into the peripheral blood circulation (12). The egression of monocytes from bone marrow into blood requires the expression of the C-C chemokine receptor type 2 (CCR2) (13, 14). Blood monocytes can be subdivided into two subtypes: classical and non-classical monocytes. Classical monocytes are circulating for several days in the blood before leaving the circulation by diapedesis and entering tissues in steady state to replenish the tissue macrophage populations under inflammatory conditions. Non-classical monocytes predominantly remain in the circulation (13) and engage in long-term migration along the endothelium with or against the flow, a process termed patrolling (14). These monocyte subpopulations are reviewed in detail in section 2.2, including their various expression of surface markers.

However, besides macrophages originating and renewing from HSCs, some macrophages develop in the early embryo before the development of HSCs. These cells are termed erythromyeloid progenitors (EMP) (15). It was shown in mice, that macrophages develop in the yolk sac beginning from embryonic day 8.5 (E8.5) (16, 17). Further, the transcription factor myeloblastosis (Myb) is required for the development of HSCs in the bone marrow as well as for the development of all CD11b^high monocytes and CD11b^high macrophages. For the development of yolk sac-derived F4/80⁺ macrophages in several tissues, including liver Kupffer cells, epidermal Langerhans cells as well as microglia cell populations, Myb was dispensable. In adult mice, these populations can persist independently of HSCs, suggesting that a lineage of tissue macrophages is derived from the yolk sac and is genetically distinct from HSC progeny (18). Additionally, it was shown in mice, that the majority of adult tissue-resident macrophages in various organs like liver, brain, lung and skin originates from an Angiopoietin-1 receptor⁺ (Tie2) cellular pathway generating Colony stimulating factor 1 receptor⁺ (Csf1r) EMPs, which are distinct from HSCs (19, 20). It has been shown that during inflammation there is an expansion of both HSC- and EMP-derived macrophages, presumably performing different functions at different stages of the inflammatory process.

In the cornea, the CCR2^- population is already is present in the cornea at E12.5, which are similar to yolk sac-derived macrophages, whereas the CCR2⁺ population does not appear in the cornea until E17.5. Besides the different phenotype and gene expression profile, the role of these populations in corneal wound healing is different. Whereas CCR2⁺ macrophages seem to act pro-inflammatory in an early stage of corneal wound healing, CCR2^- macrophages seem to act anti-inflammatory during the later stage of wound healing (21).

In mice, the antigenic differentiation of two monocyte subsets was first achieved after the observation that monocytes could be subdivided according to their expression of CCR2, L-selectin (CD62L) and CX3C chemokine receptor 1 (CX3CR1) (13, 22–25). CCR2⁺CD62L⁺CX3CR1^low expressing monocytes have pro-inflammatory characteristics and are recruited into the tissue during inflammation, e.g., for host defense, whereas CCR2^-CD62L^-CX3CR1^high monocytes/macrophages have anti-inflammatory properties and replenish the tissue resident macrophage population, mediate wound healing and patrol the vasculature (13, 26–28). CCR2⁺CD62L⁺CX3CR1^low expressing monocytes are known to be the “inflammatory” subset, whereas CCR2^-CX3CR1^high expressing monocytes are considered as the “resident” subset. Besides CCR2 as a marker for monocytes/macrophages, Geissmann et al. identified an additional marker, lymphocyte antigen 6C (Ly6C), for CCR2⁺ monocytes/macrophages (13). In particular, CCR2⁺CD62L⁺CX3CR1^lowLy6C⁺ monocytes seem to have an outstanding importance for the infiltration into inflamed tissues (29). These cells produce pro-inflammatory cytokines and chemokines, including tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, IL-12, IL-23, and C-C motif chemokine 11 (CCL11) (30, 31). Additionally, cells express high levels of Triggering receptor expressed on myeloid cells 1 (TREM1), which can potently amplify pro-inflammatory responses (32).

Fate-mapping studies as well as single cell analyses were recently able to provide major insights into the heterogeneity of macrophages. In this context, a study performed by Yona et al. demonstrated, that tissue resident macrophage populations, including peritoneal, splenic and lung macrophages, as well as liver Kupffer cells, are established prior to birth and are disconnected from monocyte input in adult steady state (27). Furthermore, this study demonstrated that in steady state monocytes with a CX3CR1^intLy6C⁺ expression form a short-lived obligatory precursor intermediate for the generation of Ly6C^- monocytes, which dynamically control the lifespan of their progenitors (27). A very recent study from Wieghofer et al., provided further insight into the heterogeneity of macrophages in various tissues of the eye, including the cornea (33). In this study, a combination of three techniques, single-cell RNA sequencing, embryonic and adult cell fate mapping and parabiosis with the use of reporter mouse lines was deployed to compare the transcriptional profiles, origin and turnover characteristics of retinal microglia, and resident macrophages in the ciliary body as well as in the cornea (33). Out of 17 different clusters containing CD45⁺CD3^-CD19^-Ly6G^- cells, five clusters were significantly enriched in the cornea. Furthermore, this study showed, that all investigated compartments of the adult murine eye contained macrophages of prenatal origin that derive either from the yolk sac and/or the fetal liver to various degrees. However, in the cornea of adult mice, macrophages are continuously replaced with cells derived from the definitive hematopoiesis with a short turnover (33).

A number of markers that are expressed by macrophages can be used for characterization and localization during experimental set ups, including F4/80, CD11b and CD68 (34–36). Besides macrophages, F4/80 is also a marker for microglial cells (37). Additionally, myeloid dendritic cells, blood monocytes and eosinophilic granulocytes also express low levels of F4/80 (38), whereas CD11b is also expressed on neutrophils, peritoneal B1 cells, CD8⁺ dendritic cells (DCs), natural killer cells (NK) and a subset of CD8⁺ T cells (39–41). CD11b is also highly expressed in CD4⁺ conventional DCs and in conventional DCs type 2 regardless of their CD4 expression (42). CD68 is also present on basophils, dendritic cells, fibroblasts, Langerhans cells, mast cells, CD34⁺ progenitor cells, neutrophils, osteoclasts, activated platelets and B and T cells (43, 44). Additional markers widely used for the characterization of tissue resident macrophages are CD64 and Mer tyrosine kinase (MerTK). A combination of different markers including CD11b, F4/80, CD64, MerTK and CD68 for flow cytometry is commonly used to characterize tissue-resident macrophages (45). Furthermore, the characterization of macrophages is also possible using immunofluorescence. For example, Saylor et al. developed an automated, multiplexed staining approach including anti-CD68, -CD163, -CD206, -CD11b, and -CD11c antibodies to identify macrophages in tumor tissue (46).

Interestingly, macrophages share the marker Lymphatic Vascular Endothelial Hyaluronan Receptor 1 (LYVE-1) with lymphatic endothelial cells (LECs) (47). Therefore, these cell types also have to be discriminated, e.g. by using further specific markers for LECs such as the transcription factor Prospero homeobox protein (Prox-1) (48) and the membrane glycoprotein Podoplanin (6, 49, 50). Besides lymphatic vascular endothelium, LYVE-1 serves for both, macrophages and LECs as receptor during hyaluronate metabolism and angiogenesis (2, 3, 6, 51–53). Intriguingly, a recent study of Chakarov et al. has shown that two independent monocyte-derived tissue resident macrophage populations exist across various tissues with specific niche-dependent phenotypes and functional programming, distinguished by their LYVE-1, MHC II and CX3CR1 expression pattern (54). LYVE-1^lowMHC II^highCX3CR1^high macrophages are preferentially located, but conserved, in sub tissular niches located adjacent to nerve fibers, whereas LYVE-1^highMHC II^lowCX3CR1^low macrophages are preferentially located adjacent to blood vessels (54). LYVE-1^lowMHC II^highCX3CR1^high macrophages exhibit potent immune-regulatory potential, while LYVE-1^highMHC II^lowCX3CR1^low macrophages are able to express higher levels of genes which are involved in wound healing, repair, and fibrosis, as well as blood vessel morphology and leukocyte migration (54).

A plethora of functional macrophage phenotypes exist. For a long time, macrophage phenotypes were classified into two polarized macrophage subtypes, depending on their activation state. The “classical” activation of macrophages occurs via stimulation by pro-inflammatory mediators, e.g. Interferon (IFN)-γ, TNF-α or lipopolysaccharides (LPS). These macrophages show an increased expression of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12, increased antigen presentation and production of nitrogen and oxygen radicals as well as increased microbicidal activity (55). This macrophage phenotype occurs primarily in early phases of inflammatory responses. In contrast, “alternative” activation of macrophages is mediated by the Type 2 cytokines IL-4 and IL-13 which induce the expression of hallmark genes such as Retnla (resistin-like molecule alpha), Chil3 (chitinase-like 3), and Arg1 (arginase 1) (55, 56). IL-4/IL-13-activated macrophages show an increased activity in signaling pathways that are important for the termination of an immune response, leading to an increase of the expression of prophagocytic, antioxidant and motility-enhancing factors, while the expression of pro-inflammatory factors is decreased (55). Furthermore, IL-4Rα-activated macrophages are crucially involved in tissue repair, evidenced by defective skin wound healing in Il4ra ^fl/− Lyz2-cre mice (57). In this study, IL-4Rα-activated macrophages were shown to have a major impact on the collagen-modifying function of fibroblasts and thereby on scar formation (57). Macrophages in Il4ra ^fl/− Lyz2-cre mice fail to initiate an essential repair program rather than an unrestrained pro-inflammatory response which was reported in prior studies by Chen et al. (58). It is clear, however, that this subdivision is an oversimplification and only reflects two extremes of polarization and that in tissues a wide range of activation states exist in parallel (56).

Corneal avascularity is highly important for the maintenance of corneal transparency, ensuring the basis of good visual acuity. However, a variety of diseases and surgical manipulations can lead to the breakdown of the hem- and lymphangiogenic privilege of the cornea resulting in pathological corneal hem- and lymphangiogenesis. Diseases that can be associated with corneal neovascularization include inflammatory disorders, corneal graft rejection after transplantation, infectious keratitis, contact lens-related hypoxia, alkali burns, stromal ulceration, or limbal stem cell deficiency. In these conditions, the balance between pro-angiogenic and anti-angiogenic factors is disturbed and leads to an upregulation of pro-angiogenic factors, and a downregulation of anti-angiogenic factors followed by neovascularization (120–122). Blood vessels directly reduce corneal transparency if growing into the optical zone or due to secondary effects such as hemorrhage and lipid exudation through immature and leaky capillaries. Unlike blood vessels, clinically invisible lymphatic vessels do not reduce the transparency of the cornea. However, they contribute to various inflammatory diseases of the ocular surface, including corneal transplant rejection, dry eye disease (DED) and ocular allergy (7, 123). In those diseases, the corneal lymphatic vessels facilitate the migration of APC from the ocular surface to the regional lymph nodes, which induces undesired immune responses (124–126). On the other hand, lymphatics may also be involved in draining excess tissue fluid thus contributing to corneal transparency and vision (9).

Macrophages play a pivotal role in corneal neovascularization. Macrophages are able to secrete paracrine factors, such as VEGF-A, which promotes hem- and lymphangiogenesis by binding on VEGFR-2 (2), and VEGF-C and VEGF-D, which promote lymphangiogenesis by binding to VEGFR-3 (127, 128). In addition, macrophages also express VEGFR-1 and VEGFR-3 which both may mediate chemotactic effects in myeloid cells and thereby perpetuate an inflammatory hem- and lymphangiogenic response (“immune amplification”) (2). Notably, so far it is not reported in the literature whether macrophage-derived VEGF-A is critical for corneal vascularization, and it is unclear by which other mediator macrophages precisely might mediate corneal vascularization. Besides secreting lymphangiogenic and angiogenic growth factors (2), macrophages also directly contribute to corneal lymph vessel formation by integrating into newly formed corneal lymphatic vessels (3). That means macrophages have a dual important role in mediating corneal lymphangiogenesis (129). Furthermore, macrophages seem to be essential also for maintenance of (corneal) lymphatics (130). It was shown that depletion of macrophages significantly reduces corneal hem- and lymphangiogenesis (2, 131, 132). Recent studies have also identified distinct functions of early- versus late-phase corneal wound macrophages in hem- and lymphangiogenesis: whereas early-phase wound macrophages are essential for the initiation and progression of injury-mediated corneal hem- and lymphangiogenesis, late-phase wound macrophages control the maintenance of established corneal lymphatic vessels, but not blood vessels (10, 11). Furthermore, studies indicate that the type of corneal damage controls the hem- and lymphangiogenic potential of corneal macrophages: whereas an acute perforating incision injury induced wound macrophages with lymphangiogenic, but not hemangiogenic potential with an increased expression of VEGF-C and D, suture placement into the corneal stroma provoked wound macrophages with hem- and lymphangiogenic potential ( Figure 1B ). Interestingly, in a model of Pseudomonas aeruginosa-induced bacterial keratitis, corneal hemangiogenesis was induced in early as well as late stages, whereas lymphangiogenesis was induced solely in late stages, which was strongly dependent on corneal macrophages ( Figure 1C ) (10). Taken together, the hem- and lymphangiogenic potential of corneal wound macrophages is determined by the type of the corneal damage and the phase of corneal injury (11). However, based on currently available data, it seems not possible to speculate or conclude whether different macrophage populations or different activation phenotypes are separately regulating corneal hem- and/or lymphangiogenesis.

Another possibility of macrophages promoting hemangiogenesis is VEGF-independent, as macrophages might act as bridge cells on the tips of sprouting lymphatics guiding the cells into finding and anastomosing with tip cells from other sprouting lymphatics (133, 134). However, this pathway has not been shown for corneal neovascularization so far.

It should be noted that corneal neovascularization might also occur independent of macrophages. An example is corneal lymphangiogenesis induced by Herpes simplex virus 1 (HSV-1) (135). It was shown in this context that lymphangiogenesis depends on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Importantly, macrophages were not the source of VEGF-A and did not play a role in the induction of the lymphangiogenic response. Infected epithelial cells were discovered as the primary source of VEGF-A in this model, suggesting that HSV-1 directly induces vascularization of the cornea through up-regulation of epithelial VEGF-A expression and not via macrophages ( Figure 1C ).

We have recently demonstrated an important role of Interleukin-10 (IL-10)-activated macrophages in inflammatory corneal neovascularization. In particular, we could show that the multifunctional cytokine IL-10, which acts anti-inflammatory as well as immune-regulatory, controls the corneal lymphangiogenesis and the resolution of corneal inflammation via macrophages (8). In healthy corneas, the expression of IL-10 was unverifiable, however macrophages which infiltrated inflamed corneas after corneal injury showed a strong increase in IL-10 expression. In vitro stimulation of macrophages with IL-10 led to an anti-inflammatory, but surprisingly pro-lymphangiogenic phenotype, characterized by an upregulation of VEGF-C. In IL-10 deficient mice, corneal injury resulted in both reduced expression of VEGF-C and reduced corneal lymphangiogenesis. However, the loss of IL-10 had no effect on corneal hemangiogenesis (8). The deletion of the central mediator of IL-10 signaling, Signal transducer and activator of transcription 3 (Stat3), specifically in myeloid cells resulted in reduced corneal lymphangiogenesis and persistent corneal inflammation in injured corneas, reinforcing the critical role of IL-10⁺ macrophages in the regulation of corneal lymphangiogenesis and inflammation (8). These findings indicate that IL-10 leads to an anti-inflammatory but pro-lymphangiogenic VEGF-C secreting macrophage phenotype during an inflammatory corneal response. These macrophages can induce the activation and growth of lymphatic vessels, leading to an egress of inflammatory cells and the termination of the local inflammatory response (8).

A major transcription factor, which is stabilized during physiological skin wound healing and which is required for the induction of Vegfa, is Hypoxia-Inducible Factor 1 α (HIF-1α) (170, 171). Stabilization of HIF-1α is impaired in a diabetic environment and in aged mice, conditions with a typically impaired wound healing response. Interestingly, angiogenesis and wound closure are improved in diabetic mice when HIF-1α is stabilized (170–174). Up to date, in classic repair models in the cornea (e.g. corneal incision injury, suture-induced corneal neovascularization) a functional impact of HIF-1α stabilization on neovascularization and repair has not been described. Of note, in a mouse model of corneal HSV−1 infection, hypoxia in the cornea and subsequent stabilization of HIF-1α in immune cells has been shown (175). However, whether HIF-1α is activated specifically in macrophages and whether this has a functional impact in this infection model, remains open. Chen et al. could show an inhibition of VEGF expression and corneal neovascularization by shRNA targeting HIF-1α in a mouse model of closed eye contact lens wear (176). In skin, wound healing studies using mouse models with cell type-specific Hif1a gene deletion revealed that endothelial cell-, fibroblast-, and epidermis-specific HIF-1α are critical for Vegfa expression, angiogenesis, and timely wound closure (177–179). However, direct evidence that myeloid cell-derived Vegfa expression depends on HIF-1α activation in early phase wound macrophages is lacking. Yet, the critical role of HIF-1α in regulating Vegfa expression in macrophages and the inflammatory phenotype of skin macrophages is well documented (180), proposing that HIF-1α might regulate Vegfa expression in early phase wound macrophages. Both, hypoxia and inflammatory stimuli such as TNF-α, IL-1β, and bacterial products have been shown to stabilize HIF-1α in a NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)-dependent manner (181, 182). Interestingly, by day 1 after injury the wound tissue was shown to be normoxic, while macrophages already expressed Vegfa (183), indicating that in the very early phase of healing other signals than hypoxia might induce an angiogenic phenotype in macrophages. Recently, mitochondrial metabolism has been identified as critical regulator of HIF-1α activation in macrophages in vitro. Under inflammatory conditions, macrophages repurpose their mitochondria from ATP production towards production of mitochondrial reactive oxygen species (mtROS), which stabilize HIF-1α independently of hypoxia (184). In future studies it will be interesting to understand whether mtROS operate in wound macrophages to mediate the wound angiogenic response.

As revealed by imaging of lymphatic vessels in Vegfr3 reporter mice (Vegfr3 ^EGFPLuc), lymphangiogenesis is a transient process during skin wound healing, peaking in the mid-phase of healing and returning back to basal levels after re-epithelialization is completed (166). In experimental mouse models of skin wound healing and contact hypersensitivity, treatment with the synthetic glucocorticoid dexamethasone blocks lymphangiogenesis, showing that lymphangiogenesis is tightly connected with the inflammatory response in the skin (166). A similar finding was reported in corneal repair; after suture placement in the cornea corticosteroids were identified as strong inhibitors of corneal lymphangiogenesis (152). Furthermore, by phase-specific macrophage depletion our group has shown that in the injured cornea early-phase macrophages are essential for initiation of lymphangiogenesis (11). Yet, the assessment of lymphangiogenesis in skin wounds upon diphtheria toxin-mediated macrophage ablation is lacking (11, 167–169). However, similar to the inflamed cornea, independent groups identified F4/80⁺/LYVE-1⁺ lymphatic structures in early granulation tissue after excisional punch injury, indicating that macrophages contribute to lymphatic vessels during physiological skin repair (185, 186). The crucial function of macrophages in regulating lymphangiogenesis in skin wounds was further demonstrated by treating wounds of diabetic mice with IL-1β-activated macrophages, which resulted in the formation of granulation tissue and of new F4/80⁺/LYVE-1⁺ lymphatic vessel structures (186). Further, macrophages are well-known sources of lymphangiogenic paracrine factors. The critical impact of macrophage-derived VEGF-A, VEGF-C, and VEGF-D on lymphangiogenesis was shown by Kataru et al. in an ear skin inflammation model (187). Following the intradermal injection of Toll-like receptor (TLR) ligands, depletion of macrophages by clodronate or blockade of VEGF−A or VEGF−C/D resulted in significantly attenuated lymphangiogenesis in the inflamed skin and impaired inflammation resolution (187). Expression of Vegfc in skin macrophages has been shown to be controlled by the transcription factor tonicity-responsive enhancer-binding protein (TonEBP, also known as NFAT5) (188). In this study, deletion of Nfat5 specifically in myeloid cells prevented high salt diet-induced Vegfc expression and subsequently lymphangiogenesis in the skin (188). Interestingly, in a mouse model of bacterial skin infection it was found that salt accumulated at the site of the skin lesion drives inflammatory macrophage activation via TonEBP to facilitate pathogen removal (189). Whether myeloid cell-specific TonEBP has a function during skin and corneal repair is unknown. It will be interesting to study in the future the interrelationship between salt concentrations, TonEBP activation, lymphangiogenesis, and macrophage activation in both tissues.