Corneal macrophages and limbal stem cells: emerging roles in ocular surface regeneration

Abstract

Background

Corneal integrity and visual acuity rely on limbal stem cells (LSCs) and immune homeostasis. Macrophages, as critical immune regulators, are increasingly recognized for their roles in stem cell niche maintenance and tissue regeneration. Recent advances in human single-cell and spatial transcriptomic profiling have further revealed previously underappreciated macrophage heterogeneity, context-dependent activation states, and neuroimmune interactions within the limbal niche.

Objective

To review current evidence on the localization, plasticity, and immuno­modulatory functions of corneal macrophages—with emphasis on human-derived insights, emerging neuroimmune mechanisms, and their interactions with LSCs during homeostasis and injury.

Methods

This narrative review integrates findings from the immunology, stem cell biology, and ophthalmology literature. It focuses on macrophage-derived cytokines, metabolic mediators, and microenvironmental cues that influence LSC behavior, and incorporates recent human single-cell, spatial transcriptomic, and regenerative immunology studies as well as therapeutic strategies modulating macrophage phenotype.

Results

Macrophages exhibit dynamic and spectrum-like polarization during corneal inflammation and repair, extending beyond the traditional M1/M2 dichotomy. Their secreted factors—including TNF-α, IL-6, TGF-β, IL-10, and NO, and Arg1-derived metabolites—affect LSC proliferation, migration, and differentiation in a dose-, time-, and context-dependent manner. Therapeutic reprogramming of macrophages via pharmacological agents, stem cell-derived exosomes, α-MSH-based neuroimmune modulation, and bioactive compounds enhances corneal regeneration.

Conclusion

Macrophages serve as pivotal modulators of the limbal niche. Targeting macrophage–stem cell crosstalk represents a promising avenue for restoring niche stability and preventing limbal stem cell deficiency. Future progress will rely on human single-cell atlases, immune microenvironment profiling, and macrophage-targeted immunoregenerative strategies.

Graphical Abstract

1. Introduction

The cornea relies on limbal stem cells (LSCs) to maintain epithelial integrity and regenerate after injury. These stem cells reside within the limbal niche—a specialized epithelial–stromal interface essential for sustaining tissue homeostasis. Disruption of this niche by inflammatory insults, mechanical trauma, or degenerative conditions can lead to limbal stem cell deficiency (LSCD), manifested by persistent epithelial defects, conjunctivalization, neovascularization, and progressive vision loss.

Increasing evidence indicates that the limbal niche is an immune-responsive microenvironment in which macrophages act as essential regulators. Under homeostasis, resident macrophages support epithelial quiescence and preserve stromal architecture. Upon injury, they undergo rapid polarization and release cytokines, growth factors, and metabolic mediators that shape LSC proliferation, differentiation, and survival. This immune–stem cell interplay has become a determining factor controlling corneal inflammation and repair outcomes. Recent human single-cell sequencing, spatial transcriptomics, and immune regenerative profiling now provide unprecedented resolution for defining macrophage diversity and immune regenerative interactions within the human ocular surface [1–3].

Given the clinical relevance of inflammatory imbalance in LSCD, a deeper understanding of macrophage phenotypes and their mechanistic influence on LSC fate is critical for developing novel immunomodulatory therapies. However, current literature remains fragmented, particularly regarding human macrophage heterogeneity, neuroimmune regulation, and niche-specific microenvironmental cues. This review summarizes current knowledge of corneal macrophage biology, emphasizing their roles within the limbal stem cell niche and their translational potential for regenerative ophthalmology.

2. Macrophages

Macrophages are pivotal effectors of the mononuclear phagocyte system (MPS), originating from hematopoietic progenitors and serving essential roles in both innate immunity and the initiation of adaptive immune responses. Broadly, they can be categorized into two distinct populations: tissue-resident macrophages, which derive from yolk sac progenitors, fetal liver precursors, or through local self-renewal mechanisms; and infiltrating macrophages, which arise from bone marrow–derived monocytes and are recruited to sites of injury or infection [4].

Beyond their role in immune surveillance, macrophages are integral mediators of tissue repair and regeneration [5,6]. Upon tissue damage or pathogen invasion, host cells release damage-associated molecular patterns (DAMPs), while pathogens contribute pathogen-associated molecular patterns (PAMPs), both of which activate inflammatory pathways. These signals coordinate the recruitment and activation of a variety of cell types—including neutrophils, lymphocytes, epithelial and endothelial cells, fibroblasts, stem cells, and macrophages themselves—thus initiating complex cascades of tissue remodeling. Under tightly regulated conditions, this response facilitates resolution of inflammation and restoration of tissue homeostasis [7]. Macrophages function as central hubs in this process, balancing pro- and anti-inflammatory signals. Dysregulated macrophage activity—such as aberrant polarization, excessive cytokine expression, or impaired resolution mechanisms—can lead to fibrosis, scarring, or even organ dysfunction [8].

Traditionally recognized for their phagocytic capacity in clearing apoptotic cells and pathogens, macrophages are now acknowledged as key participants in all stages of tissue repair—from the inflammatory phase to proliferation, remodeling, and resolution [5,7,8]. Experimental evidence supports this paradigm. In neonatal mice, macrophage depletion via clodronate liposomes significantly impaired cardiac regeneration after myocardial infarction, resulting in fibrosis and reduced cardiac function [9]. In salamander limb regeneration models, early macrophage depletion prior to amputation disrupted wound healing and led to regenerative failure, whereas late-stage depletion after blastema formation did not abolish regeneration but delayed tissue restoration [10]. In skeletal muscle, macrophage-derived signals are crucial for activating satellite cell proliferation after injury [11]. Similarly, in the aging central nervous system, recruitment of young monocytes has been shown to enhance remyelination capacity [12]. Collectively, these findings underscore the indispensable role of macrophages in establishing a regenerative microenvironment and guiding effective tissue restoration.

One of the hallmarks of macrophages is their phenotypic plasticity. In response to local cues, they can polarize into distinct functional states. M1 macrophages, induced by interferon-γ (IFN-γ) and microbial ligands such as lipopolysaccharide (LPS), exhibit pro-inflammatory, cytotoxic, and antimicrobial properties. In contrast, M2 macrophages, stimulated by interleukin-4 (IL-4) and interleukin-13 (IL-13), are involved in anti-inflammatory signaling, angiogenesis, extracellular matrix remodeling, and tissue repair. M1 macrophages secrete high levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12), with limited interleukin-10 (IL-10) production. Conversely, M2 macrophages express elevated levels of IL-10 and transforming growth factor-β (TGF-β), while exhibiting low IL-12 expression. These polarization states are not binary but exist along a spectrum, with functional heterogeneity shaped by origin, microenvironmental context, and dynamic signaling interactions. Although these paradigms were largely established in animal models, recent human single-cell datasets suggest a far more refined and heterogeneous macrophage landscape within the ocular surface [3]. In vivo confocal microscopy has further revealed distinct macrophage morphologies and activation states in human corneas under physiological conditions and in response to environmental variation [13].

3. The cornea

The cornea forms the anterior transparent segment of the eye and, together with the sclera, constitutes the outer fibrous tunic. Anatomically, it consists of five distinct layers from anterior to posterior: the epithelium, anterior limiting lamina, stroma, posterior limiting lamina, and endothelium [14]. The cornea and its overlying tear film together account for the majority of the eye’s total dioptric power and function as the principal optical interface guiding light toward the retina [15]. The epithelium and basement membrane provide the primary barrier against dehydration, microbial invasion, ultraviolet radiation, and mechanical insults, and help attenuate ultraviolet radiation [16,17]. Beneath the epithelium, the anterior limiting lamina is an acellular, collagen-rich sheet that contributes to mechanical resilience but is readily disrupted during chemical corneal burns, with limited capacity for regeneration [18,19]. The stroma constitutes approximately 90% of the corneal thickness and confers transparency and biomechanical strength through its highly ordered collagen lamellae and keratocyte-regulated extracellular matrix dynamics [20]. The endothelium [21,22] maintains stromal hydration through ionic pumping activity but possesses minimal regenerative potential in adulthood, rendering it susceptible to inflammation, trauma, or surgical intervention.

Corneal transparency depends on continuous epithelial renewal and the strict regulation of ocular surface inflammation [23–25]. Disruption of this balance by trauma, infection, or chronic inflammatory signaling leads to epithelial instability, stromal disorganization, and progressive loss of clarity, underscoring the essential role of immune–epithelial homeostasis in preserving corneal function.

4. The limbal stem cell niche

The limbus, located at the junction of the cornea, conjunctiva, and sclera, contains the palisades of Vogt—specialized epithelial–stromal ridges that support LSC maintenance, protection, and activation [26,27]. LSCs situated at the base of these structures give rise to transient amplifying cells that migrate centripetally to replenish the corneal epithelium [28]. Their behavior is orchestrated by stromal fibroblasts, melanocytes, Langerhans cells, resident and infiltrating macrophages, vascular endothelial cells, extracellular matrix (ECM) components, basement membrane structures, and soluble signaling mediators [1,26].

External insults such as chemical or thermal burns, ocular cicatricial pemphigoid, pterygium, Stevens–Johnson syndrome, or iatrogenic trauma induce persistent inflammation, which destabilizes limbal homeostasis and leads to progressive LSC depletion. Elevated nitric oxide (NO) and reactive nitrogen species generated under high inflammatory burden further compromise LSC function by impairing mitochondrial metabolism and promoting nitrosative stress [29,30]. As discussed in later sections, NO exhibits dose- and context-dependent effects, which help reconcile conflicting findings regarding its role in epithelial repair.

Macrophages have emerged as central regulators within this microenvironment. Through phenotype-dependent secretion of cytokines, growth factors, and matrix-remodeling enzymes, polarized macrophage subsets modulate LSC proliferation, migration, differentiation, and survival. A detailed understanding of these macrophage–LSC interactions will be essential for developing targeted immunoregulatory therapies capable of restoring limbal niche integrity and supporting corneal regeneration.

Beyond stromal and immune components, the limbal stem cell niche is also regulated by the trophic and sensory influence of corneal nerves. Nerve fibers originating from the ophthalmic branch of the trigeminal nerve form a dense subepithelial plexus at the limbus and anterior stroma, releasing neuropeptides and growth factors—including substance P (SP), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF)—that sustain LSC proliferation, migration, and survival [31,32].

Animal studies have provided direct evidence of the neurotrophic dependence of limbal stem cells. In murine models, trigeminal nerve transection or chemical denervation markedly reduced the expression of stemness markers (ABCG2, p63α) and delayed corneal epithelial healing, while exogenous supplementation with SP or NGF restored stem cell activity and epithelial integrity [31,33]. Denervation disrupts the spatial association between stromal macrophages and corneal nerves, highlighting that neuroimmune interactions are integral to maintaining limbal niche stability [34].

These findings demonstrate that corneal nerves not only provide neurotrophic support for LSCs but also integrate with immune and stromal elements to maintain limbal homeostasis. This neuro-stem cell-immune axis represents a critical regulatory pathway for ocular surface integrity and offers potential therapeutic targets for neurotrophic keratopathy and limbal stem cell deficiency.

5. Corneal macrophages under physiological conditions

In the healthy cornea, macrophages are spatially segregated and distributed within the stromal layer, primarily localizing to the anterior and posterior regions between the anterior limiting lamina and the posterior limiting lamina, where they form close associations with resident keratocytes [3,35]. This spatial distribution implies a degree of compartmentalization, with distinct subsets contributing to immune surveillance, stromal maintenance, and tissue-resident immune homeostasis across stromal subdomains. Corneal macrophages are commonly identified by surface markers such as F4/80, CD68, CD11b, and CX3CR1 [35–37]. In murine corneas, particularly following epithelial debridement, immunofluorescent co-labeling with CD45, CD11b, and MHC class II reveals that the majority of CD45⁺ stromal cells co-express CD11b, with approximately 40–50% also positive for MHC II, suggesting context-dependent antigen-presenting capability [38,39].

In the human cornea, CD45⁺CD11b⁺CD11c^- macrophages have been distinguished from dendritic cells based on their lack of CD11c expression [40,41]. Further spatial resolution has been achieved using CX3CR1-GFP reporter mice, which allow in vivo visualization of stromal macrophage networks via intravital confocal microscopy [36]. Interestingly, a subset of CD45⁺ stromal cells also express CD34—a marker typically associated with hematopoietic progenitors and endothelial precursors [38,42]. Flow cytometry data indicate that CD34⁺ cells represent approximately 3% of stromal leukocytes, paralleling the frequency of CD45⁺ cells and suggesting the existence of a rare progenitor-like population with potential roles in immune regulation or stromal remodeling.

Phenotypic diversity among corneal macrophages has been further elucidated through flow cytometric profiling using markers such as CD14, CD11b, CD206, CD163, CD209, and CD86. Expression of CD206 and CD163 is indicative of an M2-like phenotype, often associated with anti-inflammatory activity and extracellular matrix remodeling, whereas CD86 denotes M1-like macrophages with pro-inflammatory characteristics [43]. This heterogeneity reflects the high plasticity and niche-dependent specialization of corneal macrophages, shaped by the cornea’s avascularity and lack of conventional lymphatic drainage—factors central to its immune-privileged status [44].

Unlike the skin, which harbors a complex array of mononuclear phagocytes—including tissue-resident macrophages, monocytes, and dendritic cells—the corneal stroma comprises a streamlined yet functionally adaptable population derived primarily from the monocyte–macrophage lineage [35]. This relative cellular simplicity underscores the central role of macrophages in maintaining local immune equilibrium. Importantly, macrophages located adjacent to limbal vasculature and lymphatic vessels are strategically positioned to interact with the LSC niche. Along with other niche components—such as stromal fibroblasts, melanocytes, Langerhans cells, vascular endothelial cells, and the basement membrane—these macrophages contribute to a finely regulated microenvironment that governs LSC quiescence, activation, and differentiation. Through their secretion of cytokines, growth factors, and matrix-remodeling enzymes, stromal macrophages not only support epithelial renewal but also preserve stromal clarity and reinforce long-term corneal transparency [26].

6. Advances in in vivo imaging of corneal stromal macrophages

Traditional histological and flow cytometric analyses have long defined the corneal macrophage landscape in terms of static phenotypic markers. However, recent breakthroughs in in vivo optical imaging have provided a dynamic, real-time perspective on these cells within their native microenvironment. Functional in vivo confocal microscopy and related high-resolution modalities now permit single-cell-level, noninvasive visualization of immune populations in the transparent human cornea under physiological conditions. These technologies have revealed that stromal macrophages—once regarded as sessile, dendritiform residents—undergo continuous morphodynamic remodeling and display measurable motility within the stromal lattice.

Time-lapse imaging demonstrates cyclical extension and retraction of cellular processes, a phenomenon quantified as the “dancing index,” indicative of active immune surveillance, dynamic stromal sampling, and sustained interaction within the keratocyte network [13,45].

Quantitative analyses derived from functional in vivo confocal microscopy (Fun-IVCM) have introduced morphodynamic descriptors such as dendrite-probing speed, circularity, and macrophage motility indices, which serve as sensitive biomarkers of innate immune activity. Seasonal imaging studies have further demonstrated that these parameters fluctuate with environmental variables—including ambient temperature, humidity, and airborne particulate load—underscoring high environmental sensitivity and adaptive responsiveness of corneal stromal macrophages [13]. Notably, innate immune populations such as macrophages and dendritic cells exhibit pronounced season-dependent modulation, whereas adaptive epithelial T cells remain comparatively static, supporting the concept that resident myeloid cells function as dynamic biosensors that calibrate corneal immune homeostasis in response to external fluctuations.

Importantly, in vivo imaging has established a translational platform linking morphodynamic data with molecular and clinical correlates. Integration of Fun-IVCM-derived parameters with tear cytokine profiling and ocular surface imaging has begun to delineate the differential behavior of stromal macrophages under homeostatic versus perturbed conditions [3]. These imaging advances not only redefine the spatial and functional architecture of the corneal immune microenvironment but also enable mechanistic exploration of macrophage–stromal cell crosstalk, neuroimmune interactions, and environmental drivers of tissue-resident immunity in ocular surface health and disease.

7. Corneal macrophages under pathological conditions

7.1. Overview of inflammatory and reparative dynamics

Macrophages have been shown to exhibit temporally regulated, biphasic activity in response to tissue injury. During the initial inflammatory phase, they act as professional phagocytes, clearing necrotic debris and invading pathogens. As the response transitions toward resolution, macrophages adopt a reparative phenotype and secrete a diverse array of bioactive mediators—including interleukins, TNF-α, TGF-β family, platelet-derived growth factor (PDGF), and NO—as well as ECM-modifying enzymes such as collagenases, elastases, and plasminogen activators. These factors collectively orchestrate immune cell recruitment, stromal activation, lymphangiogenesis, ECM remodeling, and epithelial regeneration [8,46].

Importantly, this tightly timed, phase-specific sequence of macrophage activation—rather than a simple inflammatory-reparative switch—constitutes the mechanistic foundation for corneal repair, reflecting the cornea’s unique requirement for temporal precision, given its avascularity, optical vulnerability, and low tolerance for persistent inflammation. This overview establishes the temporal and mechanistic context for the disease-specific polarization patterns discussed in the following subsections.

7.2. Pathology-specific polarization patterns

7.2.1. Diabetic keratopathy

In diabetic keratopathy, chronic hyperglycemia and oxidative stress reprogram corneal stromal macrophages toward a sustained M1-polarized phenotype, characterized by upregulation of IL-1β, TNF-α, and reactive oxygen species (ROS)-mediated matrix degradation. This persistent inflammatory state disrupts epithelial–stromal communication, alters extracellular matrix homeostasis, and significantly delays wound healing [47,48].

Beyond direct epithelial effects, accumulating evidence indicates that immune dysregulation in diabetic corneas critically contributes to sensory nerve degeneration, thereby amplifying tissue dysfunction. Activated macrophages and pro-inflammatory cytokines impair corneal nerve integrity and regeneration, leading to reduced neurotrophic support for the limbal niche. Given the established dependence of LSCs on corneal innervation for maintenance of stemness and regenerative capacity, nerve damage represents a key intermediary through which immune activation exacerbates LSC dysfunction. Together, these processes establish a pathogenic immune-nerve–LSC axis in diabetic keratopathy, in which immune-driven nerve injury and impaired LSC function mutually reinforce disease progression [47].

Therapeutic interventions targeting this inflammatory imbalance—such as restoration of insulin signaling, antioxidant strategies, or immunoregulatory cytokines such as the neuroimmune peptide α-MSH—have been shown to attenuate pro-inflammatory immune activation, reduce neural and stromal injury, and promote reparative tissue responses [49]. Early human studies using in vivo confocal microscopy and tear immunoprofiling have identified immune activation patterns and nerve alterations that parallel those seen in diabetic mouse models, reinforcing the translational relevance and cross-species conservation of this immune-nerve–LSC axis.

7.2.2. Infectious keratitis

Infectious keratitis, a leading cause of global visual impairment, elicits strong innate immune responses upon microbial invasion. Viral, bacterial, protozoal, and fungal pathogens such as adenoviruses, Pseudomonas aeruginosa, fungi, and Acanthamoeba are common etiologic agents [50,51]. Lipopolysaccharide from Gram-negative bacteria activates resident corneal macrophages via Toll-like receptor 4 (TLR4), upregulating colony-stimulating factor 1 receptor (CSF1R) and, under certain conditions, inducing apoptotic cell death [52]. In murine models, the stromal proteoglycan lumican modulates macrophage activation, and lumican-deficient mice exhibit impaired bacterial clearance and exaggerated inflammation due to aberrant macrophage polarization [53]. Although most mechanistic insights in infectious keratitis derive from animal models, early human imaging studies using in vivo confocal microscopy have documented macrophage and dendritic cell activation patterns that are consistent with these experimental findings, supporting their translational relevance [13].

7.2.3. Autoimmune and chronic inflammatory ocular surface diseases

Macrophages play pivotal roles in autoimmune-mediated ocular surface diseases [54]. In a model of dry eye disease (DED) associated with autoimmune regulator deficiency, local macrophage depletion via subconjunctival clodronate liposome injection reduced corneal epithelial damage and stromal fibrosis, emphasizing the pathogenic contribution of pro-inflammatory macrophages [55,56]. Dysregulation of the IL-6–Treg axis perpetuates this chronic inflammation [57], while IL-10-expressing tolerogenic dendritic cells or macrophages have shown potential to restore immune balance and preserve limbal stem cell integrity. In immune-mediated graft-versus-host disease (GVHD), corneal dendritic cell density increases concurrently with nerve fiber loss, indicating early neuroimmune disruption [58]. Human in vivo confocal microscopy (IVCM) enables real-time visualization of these immune changes, serving as a valuable clinical tool for early detection [13].

7.2.4. Corneal transplantation immunity

During corneal transplantation, macrophages critically determine graft fate by modulating inflammatory and reparative processes. Macrophage depletion impairs epithelial wound closure and attenuates neovascularization, whereas adoptive macrophage transfer enhances epithelial healing and graft integration [59]. M1 macrophages promote inflammation and angiogenesis via IL-1β and TNF-α [60], whereas M2 macrophages secrete IL-10 and TGF-β to support immune tolerance and epithelial regeneration [61]. Macrophage composition differs between high-risk and low-risk graft beds, suggesting microenvironment-dependent programming. Therapeutic modulation through CSF1R inhibition or IL-10 administration has potential to enhance graft survival and preserve the limbal stem cell microenvironment, but requires rigorous preclinical and clinical validation.

7.2.5. Neuroimmune crosstalk

Given the cornea’s dense sensory innervation, neuroimmune interactions critically shape local immune responses. Following central corneal injury, limbal macrophages rapidly disengage from nerve bundles within two hours, revealing a bidirectional communication axis between sensory nerves and stromal immune cells [34,62]. This axis influences cytokine release, macrophage activation states, and ultimately LSC niche stability. Recent human single-cell studies suggest subtype-specific neuroimmune interactions that may differ from those inferred from animal models [41]. Corneal nerves rapidly respond to epithelial injury and modulate immune activation, coordinating macrophage mobilization and resolution of inflammation. These findings support an integrated neuroimmune circuitry in which sensory nerves contribute to maintaining LSC stemness and limbal niche homeostasis.

7.3. Critical appraisal of current evidence

Although substantial advances have been made in characterizing macrophage behavior across corneal pathologies, significant methodological and translational limitations remain. Much of the current understanding is derived from animal models, which differ fundamentally from human corneas in stromal organization, immune composition, and neuroregenerative potential, thereby constraining the direct applicability of polarization kinetics to human disease [3]. For example, murine corneas exhibit markedly accelerated nerve regeneration compared with humans, complicating interpretation of temporal M1–M2 transitions in diabetic keratopathy.

Moreover, the widely used M1/M2 classification lacks sufficient granularity to capture the transcriptional and functional diversity revealed by emerging single-cell technologies [41]. Recent human ocular single-cell and spatial transcriptomic studies have further illustrated the unexpectedly complex immune architecture of the ocular surface and revealed previously unrecognized myeloid populations and injury-associated transcriptional states [37,44].

Heterogeneity in experimental design, disease induction methods, and macrophage markers continues to contribute to inconsistent and occasionally conflicting findings across studies. Collectively, these limitations emphasize the need for rigorous, human-based investigations to delineate macrophage lineage diversity, temporal dynamics, and context-dependent roles in corneal immunity.

7.4. Knowledge gaps in corneal macrophage biology

While interest in macrophage-mediated corneal pathology has grown substantially, several key knowledge gaps continue to hinder mechanistic insight and clinical translation.

First, high-resolution profiling of human corneal macrophages remains insufficient. Existing data are largely extrapolated from animal studies or bulk analyses, both of which obscure the molecular heterogeneity and spatial compartmentalization of human macrophage subpopulations. Although single-cell studies [63] in ocular surface disorders have revealed unexpectedly complex immune microenvironments, human cornea–focused datasets remain limited. More recent spatial and single-cell resources across ocular tissues provide initial evidence of tissue-specific macrophage states but still lack corneal-level resolution [37,44].

Second, the clinical relevance of macrophage polarization states remains poorly defined. Preclinical models implicate M1/M2 dynamics in disease progression; however, phenotype-specific biomarkers, imaging correlates, or associations with disease severity and therapeutic responsiveness in humans remain unclear. Early in vivo imaging studies documenting neuroimmune alterations in human corneal disease [13] represent progress but remain insufficient for clinical stratification.

Third, microenvironmental regulation of macrophage plasticity remains underexplored. Mechanical cues, extracellular matrix stiffness, metabolic gradients, and hypoxia modulate macrophage phenotypes in other tissues [13], yet their specific contributions to corneal inflammation, fibrosis, or nerve regeneration—particularly in the context of the steep oxygen and nutrient gradients across the cornea—remain undefined.

Fourth, neuroimmune communication mechanisms are incompletely characterized. Although recent evidence demonstrates rapid bidirectional crosstalk between nerves and macrophages after corneal injury [3], the molecular mediators involved—neuropeptides, axonal cytokines, mechanosensitive ion channels—remain to be fully elucidated.

Finally, macrophage-targeted therapies lack translational development. While metabolic modulation, cytokine-based strategies, and myeloid reprogramming show promise in systemic inflammatory diseases [64–66], their adaptation to ocular delivery systems and safety frameworks is still at an early stage.

7.5. Future directions

Looking ahead, advancing the field will require coordinated efforts that bridge high-resolution immune profiling, mechanistic investigation, and translational innovation. A central priority is the development of comprehensive, human-specific atlases of corneal macrophages. Approaches such as single-cell RNA sequencing, spatial transcriptomics, and multiplex imaging will be essential for defining macrophage subsets, lineage trajectories, and niche-specific interactions within the human cornea [2,37]. Notably, emerging human single-cell and spatial transcriptomic datasets demonstrate metabolic and biomechanical features that differ substantially from those in mouse tissues, underscoring the need for cross-species integrative analyses [63].

A parallel line of progress involves establishing dynamic imaging platforms capable of monitoring macrophage behavior in real time. Advances in adaptive optics, high-resolution in vivo confocal microscopy, and label-free imaging may enable longitudinal visualization of macrophage activation, migration, and neuroimmune communication in human subjects [13], thus linking cellular dynamics with clinical phenotypes.

Another critical direction is the mechanistic dissection of microenvironmental cues—including extracellular matrix mechanics, metabolic rewiring, and neurosensory pathways—that shape macrophage plasticity. Evidence from non-ocular tissues highlights the influence of biomechanical and metabolic signals on macrophage fate decisions, offering a conceptual framework for investigating similar regulatory systems in corneal inflammation, nerve regeneration, and fibrotic remodeling.

Translational advancement will depend on the design, optimization, and safety evaluation of macrophage-focused therapeutic strategies. Candidate approaches include cytokine modulation, metabolic targeting, nanoparticle-mediated immune reprogramming, and engineered macrophage transplantation [8]. The development of targeted ocular delivery systems and rigorous preclinical testing will be essential steps toward clinical implementation.

In parallel, increasing attention has focused on cell-based immunomodulatory therapies as a translational bridge between immune regulation and tissue regeneration. Mesenchymal stem cell (MSC)–based therapies, amniotic membrane transplantation (AMT), and emerging myeloid-derived cell approaches exert their primary therapeutic effects by reprogramming the inflammatory microenvironment—suppressing M1-dominant responses, promoting pro-resolving macrophage phenotypes, and restoring immune homeostasis within the limbal niche—thereby creating permissive conditions for subsequent epithelial and stromal regeneration. Neuroimmune modulators such as α-MSH, discussed above in disease-relevant chemical injury models, exemplify this immune-first regenerative paradigm. Notably, some of these strategies, particularly AMT, are already integrated into routine clinical practice for ocular surface reconstruction, underscoring the feasibility and translational relevance of immune-first regenerative paradigms.

Finally, integrating macrophage modulation into regenerative and surgical interventions—such as limbal stem cell therapies, bioengineered stromal scaffolds, and corneal transplantation—may enhance epithelial repair, attenuate chronic inflammation, and improve long-term functional outcomes. Recent insights into macrophage–epithelium and macrophage–stem cell interactions underscore the therapeutic potential of harnessing immune-regenerative crosstalk.

Collectively, these strategic directions outline a coherent research pathway that links high-resolution human immunology with mechanistic and translational discovery, providing the conceptual bridge to subsequent sections addressing macrophage regulation of limbal stem cell function and ocular surface regeneration.

8. Macrophages and stem cells

Beyond their well-established functions in tissue repair, remodeling, and fibrosis regulation, macrophages have emerged as pivotal modulators of stem cell fate across multiple organ systems—including the skin, liver, and gastrointestinal tract—positioning the macrophage–stem cell axis as a fundamental element of regenerative biology. In murine models of cutaneous injury, macrophage depletion significantly impairs wound closure, primarily due to the loss of macrophage-derived signaling molecules such as TNF-α and Wnt ligands, notably Wnt3a and Wnt7b. These factors activate Lgr5⁺ hair follicle stem cells via a TNF–AKT–β-catenin cascade, promoting cell cycle re-entry and facilitating follicular regeneration [67].

Similarly, in models of chronic hepatic injury, macrophages critically regulate the self-renewal, migration, and differentiation of hepatic progenitor cells. Phagocytic macrophages expressing Wnt3a can reprogram mature hepatocytes into progenitor-like cells through canonical Wnt signaling, thereby contributing to liver regeneration [8,68]. In the intestinal epithelium, macrophage-derived extracellular vesicles (EVs), enriched in Wnt ligands, support the survival and proliferation of intestinal stem cells (ISCs) following radiation-induced damage, thereby sustaining niche integrity and epithelial renewal [69].

These examples underscore the indispensable role of macrophages in constructing regenerative microenvironments that sustain stem cell viability and function. However, while these mechanisms are increasingly well-characterized in systemic organs, their application to ocular surface regeneration—particularly within the LSC niche—remains relatively underexplored.

Specifically, how macrophages regulate LSC fate—encompassing proliferation, quiescence, differentiation, and directed migration—has yet to be fully elucidated. Key mechanistic questions remain: How do tissue-resident versus infiltrating macrophage populations influence LSC behavior during homeostasis and inflammation? Which molecular pathways mediate this communication? Is the interaction primarily contact-dependent, paracrine-driven, or a combination of both?

One prevailing hypothesis suggests that macrophages may traverse the porous epithelial basement membrane in the limbus to establish direct cellular contact with LSCs, thereby influencing fate decisions during corneal repair. Alternatively, macrophages may exert paracrine control via the release of soluble mediators—including anti-inflammatory cytokines (e.g. IL-10, TGF-β), growth factors (e.g. VEGF), and chemokines—that modulate LSC dynamics from a distance. It is plausible that both direct cell–cell interactions and paracrine signaling act synergistically to preserve niche homeostasis and guide regenerative outcomes.

To decipher the precise role of macrophages in the LSC niche, future studies should employ advanced tools such as high-resolution in vivo imaging, lineage-tracing models, spatial transcriptomics, and ex vivo organotypic cultures. A deeper understanding of macrophage–LSC crosstalk could uncover novel immunomodulatory targets to enhance corneal regeneration and inform the development of cell-based therapies for ocular surface reconstruction.

9. Macrophage-mediated regulation of limbal stem cell function

Although previous sections have outlined the cellular composition of the limbal niche, recent experimental studies reveal that macrophages exert a far more direct influence on LSC maintenance than previously appreciated. Findings from depletion, polarization, adoptive-transfer, and co-culture models now provide important mechanistic insights into this interaction.

Accumulating evidence positions macrophages as active and phenotype-dependent regulators of LSC dynamics, rather than passive inflammatory bystanders. Among the most compelling data are those from macrophage-depletion models, where selective elimination of tissue-resident macrophages using CSF1R inhibitors or clodronate liposomes consistently delays corneal epithelial resurfacing, reduces proliferation of p63⁺/ABCG2⁺ progenitors, and diminishes colony-forming efficiency. These observations indicate that macrophage presence is essential for sustaining LSC competence under both homeostatic and reparative conditions [59].

Macrophage phenotype further shapes the behavior of LSCs in distinct and often opposing ways. Experimentally induced M1-dominant inflammatory activation—typically via LPS or IFN-γ—suppresses LSC proliferation, enhances apoptotic signaling, and triggers oxidative stress- and DNA damage-associated pathways within the limbal epithelium [70,71]. In contrast, M2-like reparative macrophages induced by IL-4, IL-13, corticosteroids, or metabolic conditioning preserve stemness-related transcriptional programs, promote epithelial stratification, and accelerate wound closure. These phenotype-dependent patterns underscore the functional plasticity of macrophages and illustrate how inflammatory skewing of the limbal microenvironment can destabilize LSC homeostasis.

Additional mechanistic insight comes from depletion and reconstitution studies in severe ocular surface injury. In murine models of alkali-induced limbal damage, pharmacologic depletion of tissue-resident macrophages using CSF1R inhibitors exacerbates limbal stem cell loss, promotes stromal inflammation, and worsens corneal opacity, whereas repopulation of the limbal niche by resident macrophages restores epithelial regeneration and partially preserves LSC markers [72]. These findings indicate that macrophage phenotype and ontogeny—not simply cell number—are critical determinants of limbal stem cell survival and recovery after injury.

Complementary in vitro studies have delineated several paracrine pathways underlying macrophage–LSC crosstalk. Co-culture experiments show that reparative macrophages enhance LSC proliferation, Ki-67 expression, and clonal expansion while promoting epithelial-sheet formation. Inflammatory macrophages, by contrast, induce LSC senescence, metabolic dysfunction, and loss of stemness markers. Conditioned medium assays implicate IL-6, IL-10, TGF-β, and Wnt-associated signals as key mediators of these effects [19,27]. Moreover, recent single-cell profiling of the human limbal niche highlights macrophages as a major immunoregulatory population that likely interacts directly with epithelial stem and progenitor cells [2].

Taken together, these findings establish macrophages as direct and phenotype-dependent regulators of LSC maintenance and epithelial regeneration. Perturbations in macrophage homeostasis—whether through depletion, chronic inflammation, or impaired polarization—compromise LSC function, whereas restoration of reparative macrophage populations can partially rescue stem cell dysfunction. This body of evidence provides a strong mechanistic rationale for targeting macrophage activity in future therapeutic strategies for limbal stem cell deficiency and other ocular surface disorders.

10. Emerging immunomodulatory strategies in macrophage microenvironment and ocular surface regeneration

Building upon the mechanistic insights presented in Section 8 regarding macrophage–stem cell interactions, several therapeutic approaches aim to reprogram macrophage-centered immune microenvironments to promote ocular surface regeneration. This section highlights three complementary, translation-oriented strategies—neuropeptide-based immunomodulation, MSC–derived paracrine therapy, and bioactive amniotic membrane (AM) scaffolds—that converge on restoring immune homeostasis, limiting oxidative injury, and enabling functional epithelial and stromal repair.

10.1. Neuroimmune modulation via α‑melanocyte‑stimulating hormone (α‑MSH)

The neuropeptide α-MSH is an endogenous melanocortin with potent anti-inflammatory and cytoprotective functions across ocular tissues. α-MSH suppresses NF-κB–driven transcription, promotes a pro-resolving macrophage phenotype through MC1R/MC3R signaling, and activates the Nrf2–HO-1 antioxidant pathway. These coordinated mechanisms attenuate leukocyte infiltration, oxidative stress, and fibrosis, while supporting epithelial regeneration and preservation of stromal transparency. Recent evidence in ophthalmic models positions α-MSH as a prototypical neuroimmune modulator linking macrophage reprogramming, redox homeostasis, and tissue repair [73].

Importantly, its therapeutic benefit has been demonstrated in a nitrogen mustard–induced chemical injury model that recapitulates features of mustard keratopathy and limbal epithelial compromise: systemic α-MSH reduced corneal epitheliopathy, preserved limbal epithelial cell density, mitigated corneal edema, and decreased apoptosis at both early and late time points, supporting a disease-relevant role for α-MSH in preventing or partially reversing LSCD-like pathology [74].

In preclinical settings, α-MSH improved epithelial recovery and limited endothelial and stromal damage after injury, with protective effects that are complementary to standard anti-inflammatory therapies. Notably, these effects directly counteract the immune-driven nerve injury and limbal stem cell dysfunction described in diabetic and chemically induced corneal pathologies, positioning α-MSH as a mechanism-based intervention within the neuroimmune–LSC axis. Sustained-release platforms—such as hydrogels, therapeutic lenses, or periocular depots—may be required to maintain therapeutically relevant exposure.

10.2. Mesenchymal stem cell-derived paracrine immunoregulation

MSCs exert most of their therapeutic activity through paracrine immunomodulation rather than long-term engraftment. Their secretome—comprising IL-10, TGF-β, prostaglandin E2, and extracellular vesicles enriched with regulatory microRNAs—reprograms corneal macrophages toward a pro-resolving M2-like phenotype, attenuates neutrophil recruitment, and mitigates pro-angiogenic signaling. Through these macrophage-directed mechanisms, MSC therapy enhances corneal re-epithelialization, limits neovascularization and stromal haze, and supports restoration of tissue homeostasis across injury and transplantation models.

Importantly, MSC-derived exosomes recapitulate many of the immunoregulatory and reparative benefits of MSCs while minimizing the risks associated with live-cell transplantation, positioning them as a promising next-generation cell-free therapeutic approach. Key outstanding challenges include standardizing MSC sources and manufacturing protocols, optimizing dose and delivery route (topical, subconjunctival, or systemic), and defining the durability and reversibility of macrophage reprogramming. Controlled clinical trials will be essential to benchmark MSC-based immunotherapies against current anti-inflammatory standards of care.

10.3. Amniotic membrane as a bioactive anti‑inflammatory and regenerative scaffold

The amniotic membrane (AM) provides more than a physical substrate for epithelial migration; it functions as a bioactive matrix enriched with growth factors (e.g., EGF, HGF, TGF-β) and immunoregulatory mediators that suppress M1 macrophage activation, promote M2-driven resolution, and reduce oxidative injury to epithelial progenitors. Clinical and translational studies demonstrate that AM—whether cryopreserved, dehydrated, or device-mounted—shortens epithelial closure time, reduces pain and stromal scarring, and improves visual outcomes in persistent epithelial defects, infectious keratitis, and other ocular surface disorders.

Beyond monotherapy, AM has been explored as a potential delivery platform for localized, sustained release of immunomodulatory agents such as α-MSH analogues or MSC-derived exosomes, thereby enhancing therapeutic precision within the limbal–corneal niche [75–78]. These insights expand the anti-inflammatory and immunoregulatory dimensions of ocular surface regenerative therapy and strengthen the translational relevance of this review.

11. Macrophage polarization during inflammatory responses

Inflammation triggered by tissue injury unfolds in three temporally overlapping yet functionally distinct phases: initiation, amplification, and resolution. During the early pro-inflammatory phase, innate immune cells—primarily neutrophils and monocyte-derived macrophages—are rapidly recruited to the injury site, where they initiate pathogen clearance and secrete signals that orchestrate the early stages of tissue repair. This is followed by a reparative phase, marked by attenuation of inflammation and a shift in macrophage phenotype toward tissue remodeling and regeneration. Ultimately, the resolution phase is characterized by clearance of immune cells and restoration of tissue homeostasis [7].

The temporally distinct functions of macrophages across these phases have been demonstrated in various organ-specific injury models. In murine hepatic fibrosis, for instance, early-phase depletion of macrophages reduces fibrotic scar formation, whereas macrophage ablation after ECM deposition impairs scar remodeling and delays matrix resolution [79]. Similarly, in models of cutaneous wound healing, the timing of macrophage depletion yields markedly different outcomes: early removal impairs granulation tissue formation and re-epithelialization; intermediate-phase depletion reduces neovascularization and increases hemorrhagic risk; and late-stage ablation disrupts matrix organization and scar architecture [80].

These context- and time-dependent effects are underpinned by the phenotypic plasticity of macrophages in response to local environmental cues. During the early inflammatory phase, macrophages predominantly adopt a classically activated M1 phenotype in response to IFN-γ, LPS, and DAMPs. M1 macrophages enhance microbial clearance, present antigens, promote Th1 polarization, and degrade injured tissue. As inflammation resolves, macrophages shift toward an alternatively activated M2 phenotype—typically induced by IL-4 and IL-13—characterized by phagocytosis of apoptotic cells, promotion of Th2 responses, fibrosis control, matrix remodeling, angiogenesis, and support of tissue regeneration. Although this M1–M2 framework remains useful for conceptualizing phase-specific functions, recent single-cell studies demonstrate that human macrophage states form a transcriptional continuum rather than discrete binary categories.

This M1–M2 paradigm has important implications for the regulation of stem cells within regenerative niches. As the cytokine milieu and ECM landscape evolve during inflammation, macrophage activation states and secretory profiles also shift, thereby altering the behavior of local stem and progenitor cells. Macrophages release a complex array of bioactive molecules—including cytokines (e.g., IL-1β, TNF-α, IL-10, TGF-β), chemokines, growth factors (e.g., VEGF, FGF, PDGF), and proteases—that together define the immunologic and structural features of the tissue microenvironment.

Understanding how macrophage polarization influences stem cell behavior is crucial for advancing immunomodulatory regenerative therapies. Unraveling these molecular interactions may enable precise reprogramming of macrophage phenotypes to promote regeneration while minimizing fibrotic remodeling and long-term scarring. Recent advances in single-cell and spatial omics approaches are beginning to map macrophage polarization states directly in human tissues, offering opportunities to validate these concepts in the limbal niche and to identify therapeutic macrophage states.

12. Inflammatory cytokines and stem cells

During tissue repair, M1 macrophages initiate the early immune response by releasing a diverse array of pro-inflammatory mediators. These include cytokines such as TNF-α, interleukins (IL-1β, IL-6, IL-12), as well as chemokines including CXCL1, CXCL3, CXCL5, and CXCL8. M1 macrophages also produce IL-12/IL-23 heterodimers, ROS, and NO, which collectively promote pathogen clearance, activate immune cascades, and amplify inflammatory signaling. While these responses are essential for host defense, prolonged or excessive M1 activation can disrupt tissue homeostasis and impede regeneration.

In contrast, M2 macrophages contribute to the resolution phase of inflammation and promote tissue remodeling. They secrete anti-inflammatory and reparative cytokines such as IL-10, IL-4, CCL12, and CCL18, and express a suite of surface molecules implicated in immune regulation and extracellular matrix interactions, including dectin-1 (CLEC7A), CD206 (mannose receptor), scavenger receptors A and B1, CD163, CCR2, CXCR1/2, and DC-SIGN (CD209). These receptors and secreted factors support matrix turnover, angiogenesis, and epithelial regeneration, thereby aligning the M2 phenotype with reparative tissue outcomes.

The limbal basement membrane constitutes a vital structural and signaling interface for LSCs, regulating their adhesion, spatial orientation, and maintenance of quiescence. In addition to serving as a mechanical scaffold, it facilitates the localized presentation and gradient distribution of cytokines and growth factors secreted by neighboring niche constituents such as macrophages, fibroblasts, and vascular endothelial cells. Through these spatially restricted cues, the basement membrane maintains LSC identity and supports corneal epithelial renewal.

Despite increasing recognition of macrophage–LSC interactions, the precise molecular contributions of macrophage-derived cytokines, chemokines, proteases, and growth factors within the limbal niche remain incompletely understood. The complexity of the cellular constituents and the dynamic interplay of immune and epithelial signals underscore the need for a deeper mechanistic understanding of this regulatory axis. Clarifying the immunological crosstalk within the limbal niche—particularly the influence of inflammatory cytokines on stem cell fate—could inform the development of targeted therapies for corneal repair and regenerative modulation.

In the subsequent section, we summarize selected inflammatory mediators with confirmed or proposed roles in regulating LSC biology. As these cytokines and signaling pathways are discussed below, readers are encouraged to refer to Table 1 for a consolidated overview. Table 1 outlines their primary sources, associated signaling pathways, and specific effects on LSC proliferation, migration, differentiation, or maintenance.

Table 1.

Macrophage-associated mediators regulating limbal stem cells.

Mediator	Primary source	Principal signaling pathways	Effects on LSCs	Supporting evidence
IL-1β	M1 macrophages	IL-1RI/NF-κB	Suppresses CFE and stemness, promotes differentiation	71
TNF-α	M1 macrophages	TNF/AKT/β-catenin, NF-κB	Context-dependent regulation of LSC survival and regenerative capacity	70, 71
NO	M1 macrophages (iNOS)	NO/cGMP, ERK, p38 MAPK	Dose-dependent support or impairment of epithelial regeneration	82–86
IL-6	M2 Macrophages, stromal fibroblasts	IL-6R/STAT3	Increases CFE, maintains ABCG2/p63α expression, promotes regeneration	91
TGF-β	M2 macrophages, niche cells	TGF-β/Smad2/3, BMP/Smad1/5/8	Regulates LSC fate, excessive signaling induces EMT	102–104
IL-10	M2 macrophages, regulatory T cells	IL-10R-STAT3, OXPHOS	Supports stem-cell maintenance via anti-inflammatory and metabolic effects (indirect)	105, 106
Arginase-1	M2 macrophages	Arg1/l-ornithine, ERK	Facilitates epithelial/neural progenitor cell migration and differentiation, potential relevance to LSC niche (indirect)	107, 108

12.1. IL-1β and stem cells

Pro-inflammatory cytokines—including interleukin-1α (IL-1α), interleukin-1β (IL-1β), TNF-α, IFN-γ, monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-6, and interleukin-8 (IL-8)—serve as central mediators of corneal inflammation, immune activation, and wound healing [44,81]. Among these, IL-1β, a canonical product of M1 macrophages, is generated through caspase-1-mediated cleavage of its inactive precursor and signals via interleukin-1 receptor type I (IL-1RI) to regulate immune cell recruitment, epithelial apoptosis, and turnover of the ocular surface barrier.

In progenitor-enriched corneal epithelial cells, IL-1β imposes a pronounced anti-regenerative influence. Exposure of TKE2 cells to IL-1β reduces colony-forming efficiency and clone size, suppresses ΔNp63 and Importin-13 expression, and induces aberrant differentiation toward K3/K12-positive lineages. Consistently, topical IL-1β administration delays epithelial resurfacing in debridement models, highlighting its capacity to impair LSC-driven regeneration [71].

Although classically regarded as a pro-inflammatory cytokine, IL-1β demonstrates context-dependent signaling properties: transient, low-intensity activation may contribute to early epithelial activation, whereas sustained or excessive signaling perturbs limbal homeostasis and promotes stem cell exhaustion. These findings collectively establish IL-1β as a key mediator linking chronic inflammatory stress to disruption of LSC function within the limbal niche.

12.2. TNF-α and stem cells

TNF-α is a pleiotropic cytokine predominantly produced by activated macrophages and monocytes, playing essential roles in innate defense, leukocyte recruitment, and stromal remodeling [65]. In contrast to IL-1β, whose chronic effects are largely suppressive, TNF-α exerts a characteristic biphasic influence on epithelial stem cell populations that is strongly dependent on dose, exposure duration, and local microenvironmental context.

In progenitor-enriched corneal epithelial cultures, sustained TNF-α exposure impairs regenerative capacity, reducing colony-forming efficiency, suppressing ΔNp63 and Importin-13 expression, and promoting premature differentiation. In vivo, topical TNF-α delays epithelial resurfacing following debridement injury [71], underscoring its capacity to disrupt LSC-mediated repair under persistent inflammatory stress.

Beyond these inhibitory effects, TNF-α also functions as a transient regenerative cue in specific injury settings. After cutaneous epithelial damage, TNF-α expression rises rapidly and co-localizes with infiltrating macrophages in regions where quiescent Lgr5⁺ hair follicle stem cells become activated. Transient TNF-α signaling facilitates this activation through AKT–β-catenin pathways, whereas prolonged signaling induces progenitor dysfunction and impaired wound healing [67]. Recent ocular studies extend this concept to the limbal niche: pharmacologic suppression of TNF-α signaling protects LSCs from inflammation-induced exhaustion and reduces conjunctivalization after chemical burns [70], highlighting its context-dependent influence on limbal homeostasis.

Collectively, these findings underscore that TNF-α functions as both a regenerative cue and a pathological driver, depending on concentration, timing, and microenvironmental context. Therapeutic strategies aimed at temporally and spatially modulating TNF-α signaling—rather than global inhibition—may therefore optimize epithelial repair while minimizing adverse effects on LSC homeostasis.

12.3. NO and stem cells

Following the discussion of classical pro-inflammatory mediators such as IL-1β and TNF-α, increasing attention has shifted toward immunometabolic regulators that exert potent yet context-dependent effects on limbal stem cell biology. Among these regulators, NO—primarily produced by inducible nitric oxide synthase (iNOS) in M1 macrophages—has emerged as a key bidirectional modulator within the inflamed limbal niche.

NO is a pleiotropic, highly diffusible signaling molecule generated by three nitric oxide synthase isoforms—endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms. Its diverse physiological and pathological functions in immunity, metabolism, and stem cell regulation have been well documented [82]. In stem cell biology, the NO–cyclic guanosine monophosphate (cGMP) pathway modulates stem/progenitor differentiation programs, particularly in neural repair contexts, including promoting neuronal and glial differentiation of human embryonic stem cells [83]. In cutaneous epithelia, NO enhances epidermal stem cell proliferation via the cGMP–FOXG1–c-Myc signaling pathway and regulates cytoskeletal remodeling and motility through Rho GTPase activation [84,85].

Within the ocular surface, iNOS expression has been detected in both limbal fibroblasts and tissue-resident macrophages, correlating with the LSC viability and niche function. In alkali-injury models, treatments that reduce infiltration of iNOS⁺ macrophages also diminish NO overproduction, thereby attenuating acute inflammation and enhancing corneal repair [86]. Importantly, recent findings highlight the dualistic nature of NO signaling in corneal regeneration. At low, physiological ranges, exogenous NO donors have been shown to enhance human corneal epithelial cell viability and migration and to accelerate wound closure in alkali-burn models [29,87]. By contrast, higher NO levels are associated with increased oxidative stress and reduced epithelial cell survival, implying that excessive or sustained NO production may compromise stem cell function and tissue repair [29].

Interventions targeting NO signaling further illustrate this dose-dependent complexity. Inhibition of nitric oxide synthase activity can abrogate NO-mediated improvements in epithelial healing, whereas controlled low-dose NO delivery facilitates epithelial sheet expansion and reduces corneal opacity in experimental injury models [29,87].

Taken together, these findings position NO as a key immunometabolic regulator linking macrophage activation to LSC behavior. The bidirectional consequences of NO—supporting epithelial homeostasis at physiologic concentrations while impairing regeneration when overproduced—underscore the need for refined therapeutic strategies aimed at modulating NO pathways to optimize corneal repair and protect the limbal niche under inflammatory stress.

12.4. IL-6 and stem cells

IL-6 is a pleiotropic cytokine with well-established roles in immune modulation and chronic inflammation. Beyond its classical pro-inflammatory functions, IL-6 has emerged as a key regulator of stem cell behavior, influencing self-renewal, MSC recruitment, and the maintenance of cancer stem-like properties.

In the central nervous system (CNS), IL-6 signaling demonstrates context-dependent effects on neural stem and progenitor cells, particularly in directing astrocytic lineage commitment. In murine models of spinal cord injury, blockade of IL-6 signaling with the anti–mouse IL-6 receptor monoclonal antibody (MR16-1) altered the post-traumatic inflammatory milieu, reduced secondary tissue damage, promoted axonal regeneration and/or sprouting, and improved functional outcomes, indicating that modulation of IL-6 signaling can have beneficial effects on neural tissue regeneration [88–90].

In the context of the ocular surface, IL-6 similarly plays an essential role in modulating LSC dynamics. In a serum-free co-culture system that mimics epithelial–stromal interactions using human limbal epithelial cells (HLEs) and proliferative limbal fibroblasts, co-culture led to markedly increased IL-6 secretion, which was associated with enhanced expression of stem cell markers (ABCG2, p63α) and elevated colony-forming efficiency [91]. Mechanistically, IL-6 induced time-dependent phosphorylation of signal transducer and activator of transcription 3 (STAT3), and inhibition of either IL-6 or STAT3 signaling pathways significantly reduced clonogenic potential [91].

Collectively, these findings highlight IL-6 as a critical paracrine mediator within the limbal stem cell niche. Acting predominantly through the IL-6/STAT3 axis, it supports progenitor maintenance and promotes epithelial regeneration. Targeted modulation of this pathway may offer therapeutic leverage for enhancing corneal repair and stem cell–based regenerative strategies.

12.5. TGF-β and stem cells

TGF-β—comprising the isoforms TGF-β1, TGF-β2, and TGF-β3—is a central member of the TGF-β superfamily, which also includes bone morphogenetic proteins (BMPs) and related ligands. This signaling network plays a pivotal role in regulating stem cell behavior in both embryonic and adult tissues. In MSCs, TGF-β signaling influences lineage specification and differentiation, while under defined conditions, it also supports the maintenance of an undifferentiated phenotype [92–94]. In embryonic stem cells, TGF-β is critical for sustaining pluripotency [95,96].

Within the corneal limbal niche, TGF-β signaling components display region-specific expression. Both human and rodent studies have shown that TGF-β receptors—particularly TGF-βRI and TGF-βRII—are preferentially localized to basal epithelial cells in the limbus compared to the central cornea [97,98]. Immunohistochemical studies have confirmed the expression of TGF-β1 [99], TGF-β2 [100], and their corresponding receptors in the limbal epithelium. Additionally, BMP4 is prominently expressed in the limbal region, suggesting an active role for BMP-mediated signaling in LSC maintenance [101].

Canonical TGF-β signaling is initiated when TGF-β ligands bind to TGF-βRII, which recruits and phosphorylates TGF-βRI. This activates downstream Smad proteins: TGF-β ligands preferentially induce Smad2/3 phosphorylation, whereas BMPs activate Smad1/5/8. These complexes translocate to the nucleus and regulate gene transcription programs governing stem cell fate [102].

In ex vivo LSC expansion models, particularly those utilizing human amniotic membrane (HAM) scaffolds, tight regulation of TGF-β signaling is critical for preserving epithelial progenitor identity. Excessive activation of TGF-β/Smad signaling has been shown to drive epithelial–mesenchymal transition (EMT) in limbal progenitor cells, leading to loss of epithelial characteristics and compromised clonogenic capacity. Activation of Smad2/3-dependent TGF-β signaling triggers EMT-associated transcriptional programs, whereas pathway inhibition or counter-regulatory mechanisms—such as Notch-mediated induction of Smad7—effectively restrain TGF-β-assisted EMT and preserve limbal epithelial progenitor cell phenotype and regenerative potential under ex vivo culture conditions [102,103]. These findings highlight the strong dose- and context-dependent effects of TGF-β signaling within the limbal niche. While tightly controlled basal signaling may contribute to epithelial homeostasis, excessive or sustained TGF-β activation destabilizes limbal stem cell identity by promoting EMT and epithelial lineage erosion, underscoring the necessity of balanced TGF-β/BMP signaling for successful LSC maintenance and expansion.

Three-dimensional co-culture systems incorporating limbal epithelial and stromal cells have further elucidated the crosstalk between TGF-β/BMP and Wnt pathways. Under proliferative conditions, BMP4 expression and Smad1/5/8 activation are observed in LSCs. Inhibition of BMP signaling using Noggin results in nuclear β-catenin accumulation, reduced clonogenic potential, and upregulation of differentiation markers such as cytokeratin 12 (K12), indicating that BMP activity antagonizes Wnt signaling to maintain stemness [104].

In summary, the precise regulation of TGF-β and BMP signaling—particularly in relation to Wnt pathway interactions—is crucial for preserving LSC quiescence and preventing pathological EMT. Dysregulation of this signaling axis disrupts epithelial integrity and contributes to limbal niche failure and ocular surface disease.

12.6. IL-10 and stem cells

IL-10 is a potent anti-inflammatory cytokine primarily secreted by alternatively activated M2 macrophages. It plays a pivotal role in the resolution of inflammation and the promotion of tissue repair. In addition to its immunoregulatory functions, IL-10 has recently emerged as a key modulator of stem cell metabolism and function.

Mechanistically, IL-10 induces a metabolic reprogramming from aerobic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS), a shift that facilitates osteogenic differentiation in human dental pulp stem cells (DPSCs). Pharmacologic inhibition of OXPHOS abrogates this IL-10-driven differentiation, underscoring the critical role of metabolic context in stem cell fate decisions. Notably, this effect is concentration-dependent, with both subthreshold and supraphysiologic levels of IL-10 failing to elicit a robust osteogenic response [105].

In the intestinal epithelium, IL-10 similarly exerts pro-regenerative effects. Single-cell transcriptomic profiling of intestinal organoid models has revealed heterogeneous IL-10 expression across various epithelial subtypes, including Lgr5⁺ intestinal stem cells (ISCs), transit-amplifying cells, and terminally differentiated lineages. Administration of exogenous IL-10 enhances stem-like gene expression and clonogenic capacity of ISCs. Although this study was conducted in an inflammatory context, it highlighted a broader paradigm of immune-derived IL-10 acting as a paracrine regulator of epithelial stem cell maintenance and niche stability. Importantly, regulatory T cell–derived IL-10 was identified as a key paracrine signal for maintaining ISC niche stability and epithelial homeostasis [106].

These findings position IL-10 as a dual-function mediator that attenuates inflammatory injury while supporting stem cell proliferation, survival, and niche integrity. While its roles in intestinal and mesenchymal systems are increasingly recognized, the function of IL-10 in regulating LSCs remains poorly defined. Given its potent immunosuppressive and pro-reparative properties, further investigation is warranted to elucidate the contribution of IL-10 to LSC maintenance and corneal epithelial regeneration in the context of ocular surface inflammation and injury.

12.7. Arginase-1 and stem cells

L-Arginine metabolism in macrophages bifurcates into two principal enzymatic pathways, each closely aligned with distinct polarization states and corresponding immunological functions. Classically activated M1 macrophages primarily express iNOS, converting L-arginine into NO and reactive oxygen species (ROS), thereby sustaining pro-inflammatory responses essential for microbial clearance and early-phase wound defense.

In contrast, alternatively activated M2 macrophages are characterized by robust expression of arginase-1 (Arg1), an enzyme that competes with iNOS for the common substrate L-arginine. Arg1 hydrolyzes L-arginine to yield urea and L-ornithine—the latter serving as a critical precursor for the synthesis of proline and polyamines. These metabolites contribute to collagen biosynthesis, cellular proliferation, and extracellular matrix remodeling, thus supporting the anti-inflammatory and pro-reparative functions of M2 macrophages.

Recent studies have revealed that polyamines derived from L-ornithine enhance epithelial differentiation in human amniotic epithelial cells via activation of the ERK pathway [107]. Moreover, L-ornithine has been shown to facilitate directed migration of neural stem/progenitor cells through α-actinin-4–mediated cytoskeletal reorganization [108]. These findings suggest that the Arg1– L-ornithine pathway has the capacity to influence epithelial and neural progentior behavior, raising the possibility that Arg1-expressing macrophages may contribute to the establishment of a regenerative microenvironment.

The Arg1–L-ornithine axis thus represents a compelling mechanism of macrophage–stem cell communication. Whether Arg1-expressing macrophages within the corneal limbus directly modulate LSC proliferation, migration, or lineage specification remains to be elucidated. Further mechanistic studies are warranted to explore this metabolic interplay within the limbal niche and its impact on LSC maintenance and corneal regeneration.

13. Reversing macrophage polarization to promote tissue repair

Macrophage polarization is orchestrated by distinct signaling cascades and transcriptional programs that enable dynamic transitions between pro-inflammatory and reparative phenotypes. M1 macrophages are regulated by transcription factors such as STAT1, NF-κB, and interferon regulatory factors (IRF3/4/5), along with microRNAs including miR-155 and miR-223, which drive antimicrobial activity and inflammatory cytokine production. In contrast, M2 macrophages are governed by STAT6, the CREB–C/EBPβ axis, peroxisome proliferator-activated receptor gamma (PPAR-γ), and hypoxia-inducible factor 2α (HIF-2α), promoting matrix remodeling, angiogenesis, and resolution of inflammation Targeted modulation of these regulatory networks can reprogram macrophage phenotype and represents a promising therapeutic avenue for restoring tissue homeostasis and regeneration within the limbal niche.

Growing evidence indicates that dysregulated macrophage polarization contributes directly to limbal niche disruption across major ocular surface diseases. In chemical or alkali burns, a rapid and persistent M1-dominant response—driven by DAMPs, reactive oxygen species, and necrotic debris—results in excessive TNF-α/IL-1β production, degradation of limbal stromal architecture, and apoptosis of epithelial progenitors. Experimental strategies that shift this response toward an M2-like reparative state, including IL-4/IL-13 conditioning, MSC-derived factors, and metabolic reprogramming, have been shown to reduce inflammation and partially restore LSC-supportive signals in alkali-burn models.

A similar imbalance characterizes autoimmune and severe DED, where chronic exposure to IFN-γ and IL-17 maintains macrophages in a sustained M1-skewed state while reducing reparative subsets. This shift alters cytokine composition, oxidative status, and metabolic signaling within the limbal microenvironment, ultimately compromising epithelial stability and LSC maintenance. Restoring M2 polarization through melatonin, MSC-derived extracellular vesicles, and targeted cytokine modulation has proven effective in reducing ocular surface inflammation and supporting epithelial barrier repair in preclinical DED models.

Post-infectious corneal scarring provides another example in which macrophage dysfunction drives pathological remodeling. After bacterial or viral keratitis, macrophages can remain locked in a profibrotic, TGF-β–rich state that promotes stromal fibrosis, myofibroblast persistence, and angiogenesis, ultimately distorting the limbal niche. In experimental systems, reversing this profibrotic polarization attenuates stromal scarring and enhances epithelial regeneration.

In addition to disease-specific alterations, numerous pharmacological and naturally derived compounds have demonstrated efficacy in modulating macrophage polarization. PPAR agonists such as fenofibrate and gemfibrozil (PPARα agonists) suppress M1 polarization by attenuating Toll-like receptor 4 (TLR4) signaling and modulating PPARα-dependent pathways, including β-defensin 1 expression [109]. WY14643, a selective PPARα agonist, has been shown to promote M2-like phenotypes by upregulating Arg1, Ym1, CD206, and TGF-β, while suppressing iNOS and other pro-inflammatory mediators [110]. Natural bioactive molecules—including azithromycin [111,112], cocoa-derived polyphenols [113], resveratrol, polydatin, geraniin, compound A, CP-25, and aloe-emodin [114]—exert immunoregulatory effects that support M1-to-M2 transition through immunoregulatory and antioxidative mechanisms.

Mesenchymal stem cells are well-recognized for their immunomodulatory capacity and contribute to macrophage reprogramming via paracrine signaling. Transplantation of gingiva-derived MSCs into wound sites enhanced local M2 macrophage recruitment, elevated IL-10 and IL-6 secretion, and reduced TNF-α levels, resulting in accelerated cutaneous wound healing [115]. MSC-derived exosomes enriched with specific microRNAs, such as miR-223, promote M2 polarization and contribute to the establishment of a regenerative microenvironment [116].

Together, these findings underscore macrophage plasticity as a central determinant of limbal niche stability and pathological remodeling. Therapeutic strategies that restore macrophage balance—particularly in diseases characterized by chronic inflammation, fibrosis, or immune dysregulation—represent a compelling direction for improving endogenous repair and augmenting LSC-based regenerative therapies. Future integration of macrophage-reprogramming strategies with human single-cell and spatial transcriptomic data may further refine patient-specific immunoregenerative approaches and inform precision stratification in ocular surface disease.

14. Conclusions

Macrophages exhibit extraordinary plasticity and functional heterogeneity, enabling them to regulate immune responses, extracellular matrix remodeling, and epithelial regeneration throughout all phases of corneal inflammation and repair. Their capacity to polarize into pro-inflammatory or reparative phenotypes critically influences LSC behavior and corneal homeostasis. Deciphering the signaling pathways and molecular mediators that govern macrophage activation and macrophage–stem cell crosstalk is pivotal for the design of targeted immunoregenerative strategies.

In the context of ocular surface diseases, therapeutic modulation of macrophage phenotype offers substantial promise: by enhancing antimicrobial defense, reducing chronic inflammation and stromal fibrosis, and supporting LSC-driven epithelial renewal, macrophage-centered approaches may improve corneal healing and long-term visual outcomes. Emerging tools—including small-molecule modulators, stem cell–derived exosomes, and bioengineered scaffolds—offer translational potential to precisely reprogram macrophage function within the limbal niche. At the same time, it is important to recognize that much of the current evidence derives from animal models and ex vivo systems, highlighting the need for validation in human tissues and carefully designed clinical studies.

Altogether, advancing our understanding of the macrophage–LSC axis may accelerate the development of next-generation therapies to restore ocular surface integrity and preserve vision in inflammatory or degenerative eye diseases, and provide a mechanistic foundation for rational clinical trial design in macrophage-targeted ocular immunotherapy.

Funding Statement

This study was supported by the China Postdoctoral Science Foundation (Grant No. 2023M733238), Henan Provincial Science and Technology Research Project (Grant No. 242102310015), Henan Provincial Medical Science and Technology Joint Construction Project (Grant No. LHGJ2023170), Natural Science Foundation of Henan Province (Grant No. 252300421593). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosure statement

The authors declare no competing interests arising from commercial or financial associations.

Data availability statement

This manuscript was prepared using previously published literature and does not report any new data. As no datasets were generated or analyzed during the current study, data sharing is not applicable.