Chapter 2 of 3

Recruitment & polarization signals

The recruitment of circulating monocytes and their polarization into functional macrophages are orchestrated by chemical and mechanical signals specific to the injured corneal microenvironment ^{[Yaghmour 2026]}.

Epithelial injury releases DAMPs (HMGB1, ATP) and pro-inflammatory cytokines, generating a chemokine gradient — CCL2/MCP-1, CCL5/RANTES, CX3CL1 (fractalkine) — relayed by M-CSF and IL-1β/TNF-α. Circulating monocytes adhere to the limbal vascular endothelium and then transmigrate to the injury site; local hypoxia further amplifies this recruitment.

Recruitment and polarization of macrophages in the cornea: recruitment chemokines, M1-M2 polarization spectrum, functional activities according to the healing phase — Figure 8. Recruitment of monocytes/macrophages from the blood, polarization along a dynamic M1 → M2 spectrum, and functional activities across the four phases of corneal healing (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

Once recruited, macrophages polarize along a functional spectrum, historically simplified into two poles:

M1 (pro-inflammatory pole)

induced by LPS, IFN-γ, TNF-α, DAMPs and hypoxia; markers iNOS, CD86, IL-1β, IL-6, TNF-α, IL-12, CXCL9/10. Antimicrobial defence, amplification of inflammation, cell recruitment.

M2 (pro-resolving pole)

induced by IL-4, IL-13, IL-10, TGF-β, glucocorticoids and resolvins; markers Arg-1, CD206, IL-10, TGF-β, CCL17, CCL18, MerTK. Resolution of inflammation, repair, homeostasis.

Between these two poles, intermediate states reflect metabolic and functional plasticity: the response adapts continuously to the corneal microenvironment rather than flipping between fixed states. It is precisely this plasticity that the M1/M2 classification, useful as a conceptual starting point, does not fully capture ^{[Martinez & Gordon 2014]}.

A classification worth nuancing

The M1/M2 framework remains pedagogically useful, but the review by Yaghmour et al. stresses that it is a simplification of a much richer continuum of macrophage programmes, shaped by tissue context, signal kinetics and local cellular interactions.

Transcriptomic states: a continuum

Single-cell transcriptomic profiling has revealed substantial heterogeneity among corneal macrophage populations, organized not into two sharp categories but into a five-state continuum: pro-inflammatory M1 → intermediate state 1 → intermediate state 2 → pro-resolving M2 → pro-fibrotic M2 ^{[Yaghmour 2026]}.

Transcriptomic states of corneal macrophages: five-state continuum, transcriptional markers, main cytokines, functions and localization over time — Figure 5. Transcriptomic continuum of corneal macrophages: markers, cytokines, dominant functions and localization/migration across the four phases of healing (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

Key molecular features along the transcriptomic continuum
State	Transcriptional markers	Main cytokines	Main functions
Pro-inflammatory M1	NF-κB, IRF5, STAT1 ↑	IL-1β, TNF-α, IL-6, IL-12, IL-23	Antimicrobial defence, inflammation, cell recruitment
Intermediate 1	STAT3, KLF4 ↑	IL-10, TGF-β (low), IL-1ra	Transition, fine regulation of inflammation
Intermediate 2	PPARγ, c-MAF ↑	IL-10, TGF-β, lipid mediators (resolvins, PGE₂)	Resolution, efferocytosis, remodeling
Pro-resolving M2	PPARγ, IRF4, STAT6 ↑	IL-10, TGF-β, VEGF, PDGF	Resolution, repair, homeostasis
Pro-fibrotic M2	TGFBR2, SMAD3, YAP/TAZ ↑	TGF-β, CTGF, PDGF, LOX	Myofibroblast activation, matrix deposition, fibrosis

These states are also distributed in space and time: pro-inflammatory macrophages predominate in the epithelium and superficial stroma during the early phase (0-3 days); intermediate states progressively migrate deeper during early repair (3-7 days); pro-resolving macrophages settle in at resolution (> 7 days); if this transition fails, pro-fibrotic macrophages persist adjacent to Descemet's membrane and the endothelium, weeks to months later.

Key message

The coordinated transition between these transcriptional states is essential for effective, non-fibrotic healing. A blockade in either the pro-inflammatory or the pro-fibrotic state leads, in both cases, to pathological healing.

M2 pro-resolving subtypes

M2 macrophages are not a single entity: they subdivide into specialized subpopulations with complementary functions, defined by their inducing signals ^{[Yaghmour 2026]}.

M2 pro-resolving macrophage subtypes: M2a, M2b, M2c, M2d, M2eff, with their activating signals, functions and role in the cornea — Figure 3. Five M2 subtypes, their activating signals and their proposed role in the cornea — context-dependent plasticity driving healing (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

M2a to M2eff: five profiles

Functional heterogeneity of M2 subtypes (after Yaghmour *et al.* 2026, Table 3)
Subtype	Activating stimulus	Primary function
M2a (classical)	IL-4, IL-13	Wound healing, collagen synthesis; prolonged activity may promote fibrosis
M2b (immune regulator)	Immune complexes + TLR	Mixed pro- and anti-inflammatory response; regulates the magnitude of the immune response rather than suppressing it
M2c (deactivation)	IL-10, TGF-β, glucocorticoids	Matrix remodeling, immunosuppression, efficient efferocytosis — terminates inflammation
M2d (angiogenic)	TLR ligands, A2a agonists	Promotes angiogenesis; double-edged role, pathological if regulatory constraints fail
M2eff (efferocytic)	Phagocytosis of apoptotic cells	Clearing cellular debris, secretion of IL-10/TGF-β post-inflammation

An important nuance — the M2d case

The M2d subtype illustrates the ambivalence of M2 macrophages well: its pro-angiogenic capacity can favour tissue repair in some contexts, but it is also the mechanism by which macrophages breach the cornea's avascular privilege when regulatory signals fail — opening the door to pathological neovascularization.

The right balance between these subtypes, more than the isolated presence of any one of them, determines effective repair: resolution of inflammation, orderly matrix remodeling and restoration of corneal transparency.

Glossary of abbreviations used in this course

Scientific abbreviations and acronyms used throughout the 10 pages of this course, listed alphabetically.

95% CI: 95% confidence interval
AAV: adeno-associated virus (gene-therapy viral vector)
ABCG2: ABCG2 transporter, a limbal stem-cell marker
AMT: amniotic membrane transplantation
anti-VEGF: anti-angiogenic treatment targeting VEGF
ASC: inflammasome adaptor protein (Apoptosis-associated Speck-like protein containing a CARD)
BrdU: bromodeoxyuridine, a cell-proliferation marker
CCL2: CCL2 chemokine, synonym of MCP-1; recruits circulating monocytes
CCR2: receptor for CCL2; marker of newly recruited monocytes/macrophages
CD147: cluster of differentiation 147; synonym of EMMPRIN and basigin
CD163: M2 macrophage marker; scavenger receptor for haemoglobin-haptoglobin
CD206: mannose receptor; marker of M2 polarization
CD74: cell-surface receptor for MIF
CDVA: corrected distance visual acuity
CGRP: calcitonin gene-related peptide
CTGF: connective tissue growth factor
CX3CL1: fractalkine, a chemokine involved in monocyte/macrophage recruitment
CXCR2/CXCR4: chemokine receptors, also receptors for MIF
DAMP: damage-associated molecular pattern
DESI: direct epithelial–stromal interaction
EGF: epidermal growth factor
EGFR: epidermal growth factor receptor
EMMPRIN: extracellular matrix metalloproteinase inducer; synonym of CD147
ETDRS: reference visual-acuity scale used in clinical research (Early Treatment Diabetic Retinopathy Study)
FasL: Fas ligand, an apoptosis-inducing protein
FISH: fluorescence in situ hybridisation
GAG: glycosaminoglycan(s)
GM-CSF: granulocyte-macrophage colony-stimulating factor
GVHD: graft-versus-host disease
HGF: hepatocyte growth factor
HIF-3α: hypoxia-inducible factor, 3-alpha subunit
HMGB1: high mobility group box 1, an alarmin released by injured cells (DAMP)
IFN-γ: interferon gamma
IGF-1: insulin-like growth factor 1
IL-1: interleukin-1
iNOS: inducible nitric oxide synthase
IPAS: inhibitory PAS domain protein
KGF: keratinocyte growth factor
LASIK: laser-assisted in situ keratomileusis
LogMAR: logarithmic visual-acuity scale
LOX: lysyl oxidase, a collagen cross-linking enzyme
LPS: bacterial lipopolysaccharide
M1: classically activated macrophage, pro-inflammatory profile
M2: alternatively activated macrophage, pro-resolving/reparative profile (subtypes M2a-M2d, M2eff)
MEN2B: multiple endocrine neoplasia type 2B
MerTK: receptor tyrosine kinase involved in efferocytosis
MIF: macrophage migration inhibitory factor
MRI: magnetic resonance imaging
NGF: nerve growth factor
NK: neurotrophic keratitis
NLRP3: NLRP3 inflammasome (NOD-, LRP- and pyrin domain-containing protein 3)
NO: nitric oxide
NSAID: nonsteroidal anti-inflammatory drug
PACAP: pituitary adenylate cyclase-activating polypeptide
PAF: platelet-activating factor
PAMP: pathogen-associated molecular pattern
PDGF: platelet-derived growth factor
PED: persistent epithelial defect
PEDF: pigment epithelium-derived factor
PGE₂: prostaglandin E2
PRK: photorefractive keratectomy (laser surface ablation)
pro-NGF: precursor form of NGF
RGTA: matrix-therapy ReGeneraTing Agent, e.g. Cacicol
rhNGF: recombinant human NGF (cenegermin)
ROS: reactive oxygen species
Smad: signalling proteins of the TGF-β pathway
SPK: superficial punctate keratitis
sVEGFR-1: soluble form of VEGF receptor-1 (= sFlt-1)
sVEGFR-3: soluble form of VEGF receptor-3
TGF-β: transforming growth factor beta
TIMP: tissue inhibitor of metalloproteinases
TLR: Toll-like receptor
TNF-α: tumour necrosis factor alpha
TRPV4: transient receptor potential vanilloid 4, a mechanosensitive ion channel
uPA: urokinase-type plasminogen activator
V1: ophthalmic branch of the trigeminal nerve
VEGF: vascular endothelial growth factor
VEGF-C: lymphangiogenic isoform of VEGF
VIP: vasoactive intestinal peptide
YAP/TAZ: Hippo pathway effectors, mechanosensors of matrix stiffness
α-SMA: alpha-smooth muscle actin, a myofibroblast marker