Chapter 3 of 3

Molecular regulators: NLRP3, MIF, TGF-β

Three major molecular axes orchestrate the macrophage-fibroblast dialogue and determine the outcome of corneal healing ^{[Yaghmour 2026]}.

Key molecular regulators of corneal healing: NLRP3 inflammasome, MIF, TGF-β, with their effects, consequences for healing and therapeutic targets — Figure 6. Three key molecular regulators — NLRP3, MIF, TGF-β — and their respective therapeutic targets (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

NLRP3: amplifier of inflammation

Activation of the NLRP3 inflammasome by DAMPs and PAMPs triggers assembly of NLRP3-ASC-caspase-1, which cleaves pro-IL-1β and pro-IL-18 into their active forms. In mice, Xu et al. showed that NLRP3 activation in infiltrating macrophages amplifies TGF-β1 expression by the corneal epithelium, creating a feed-forward loop that sustains myofibroblast survival and collagen deposition; genetic or pharmacological inhibition of this pathway (e.g., MCC950) reduces stromal haze in murine models ^{[Xu 2022]}.

MIF: the pro-fibrotic lock

Macrophage migration inhibitory factor (MIF) acts as an upstream regulator of sustained macrophage activation in ocular tissues. It promotes macrophage retention at the injury site and amplifies pro-inflammatory signalling via the NF-κB and STAT3 pathways, reinforcing IL-1β and TNF-α production while actively opposing deactivation and resolution programmes. MIF thus acts as a true molecular checkpoint that locks macrophages into a pathogenic state — a promising therapeutic target for restoring proper immune timing ^{[Yaghmour 2026]}. Its receptors — CD74, CXCR2, CXCR4 — are detectable in ocular fluids in inflammatory contexts ^{[Taguchi 2001]}.

TGF-β: master cytokine

TGF-β acts through two complementary pathways once bound to its TβRI/TβRII receptors: the canonical Smad2/3 pathway, which activates transcription of pro-fibrotic genes (COL1A1, FN1, α-SMA); and non-canonical pathways (MAPK, Rho/ROCK, PI3K/Akt) that directly drive myofibroblast activation, contraction and matrix deposition. As detailed in the physiology chapter of this course, TGF-β has a double face: essential for resolution of inflammation and repair at low dose, it becomes one of the main drivers of fibrosis when its activity is excessive or prolonged.

Three targets, one logic

NLRP3, MIF and TGF-β converge on the same pathological lock: macrophage inflammation that fails to switch off. Inhibiting them only makes sense within that window of persistence — not during the early inflammatory phase, which is essential for host defence.

Fibrosis & macrophage-fibroblast dialogue

Excessive matrix deposition driven by persistent pro-fibrotic macrophages does more than fill the stroma: it alters its mechanical properties, closing the loop back onto the macrophages themselves.

Key mechanisms of corneal fibrosis: NLRP3 inflammasome, MIF, TGF-β, matrix stiffening and feedback loop, macrophage-fibroblast dialogue — Figure 4. From macrophage activation to the fibrotic scar: a feedback loop between matrix stiffening and mechanical macrophage activation (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

Excess deposition of collagen I, III and V, fibronectin and proteoglycans (decorin, biglycan) stiffens the stroma and disrupts its remodelling. This increased stiffness is sensed by macrophages via integrins, the YAP/TAZ pathway, NF-κB and mechanoreceptors — a form of mechanotransduction that further activates macrophages and amplifies the fibrotic response, independently of any new classical inflammatory signal.

Cellular interactions and communication: bidirectional dialogue between macrophages and fibroblasts, feedback loops — Figure 7. The macrophage-fibroblast dialogue is bidirectional: macrophages direct fibroblast behaviour, which in turn influences the polarization of recruited macrophages (original illustration, Pr É. Gabison's team, after Yaghmour *et al.* 2026). Click to enlarge ⤢

This dialogue is bidirectional. On one side, pro-resolving M2 macrophages secrete IL-10, TGF-β, PDGF, VEGF, PGE₂, resolvins and lipoxins, which direct fibroblasts toward proliferation, migration, controlled differentiation and balanced matrix synthesis — favouring restoration of transparency. On the other, fibroblasts themselves secrete IL-6, IL-33, GM-CSF, TGF-β, hyaluronic acid and fractalkine (CX3CL1), which direct the polarization of newly recruited macrophages — toward a reparative M2 profile in a favourable context, or toward a deleterious M1 profile if the balance tips, with resulting persistent inflammation, matrix degradation and, eventually, fibrosis and opacification.

Key points

The macrophage-fibroblast dialogue is essential for functional, non-fibrotic healing. It is the balance of signals — not the existence of this dialogue itself — that determines the inflammation-resolution-fibrosis continuum. Therapies targeting these interactions pave the way for novel anti-fibrotic strategies.

Therapeutic implications, summary & references

Recognizing macrophages as temporal regulators of corneal healing opens the way to a timing-based immunotherapy, rather than global immune suppression ^{[Yaghmour 2026]}. Two broad strategies are being explored:

Macrophage cell therapy (introduction of exogenous or reprogrammed macrophages): an interesting proof of mechanistic concept, but its translational applicability remains uncertain.
Manipulation of host macrophages — the favoured approach: reprogramming resident and recruited populations in situ, without introducing exogenous cells, through modulation of IL-1/TGF-β/MIF signalling, delivery of immunoregulatory factors, metabolic reprogramming, or mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs). The goal is not to suppress macrophage activity, but to promote a timely transition toward resolution-phase programmes.

Therapeutic targets associated with the three molecular regulators
Pathway	Targets / agents	Intended effect
NLRP3 inflammasome	NLRP3, ASC, caspase-1 (e.g., MCC950)	↓ Inflammation, ↓ TGF-β, ↓ fibrosis
MIF	MIF or its receptors (CD74, CXCR2, CXCR4)	Limiting inflammation and fibrosis
TGF-β / Smad	TGF-β/Smad pathway or its effectors	Prevention of fibrosis and opacification

Summary & key points

The cornea harbours resident macrophages (surveillance, homeostasis) and recruits monocyte-derived macrophages (acute defence) — two non-redundant roles.
The response follows a four-phase timeline: inflammation (0-3 days), repair (3-7 days), resolution (> 7 days), or chronic drift (weeks-months) if the transition fails.
The M1/M2 framework is a simplification of a five-state transcriptomic continuum and of five M2 subtypes (M2a-M2d, M2eff) with distinct functions.
NLRP3, MIF and TGF-β form a molecular triangle that locks macrophages into a pro-fibrotic state when their activity persists.
The bidirectional macrophage-fibroblast dialogue, amplified by matrix stiffening (YAP/TAZ mechanotransduction), self-perpetuates fibrosis once triggered.
The therapeutic target is not macrophage suppression, but restoring their proper timing — favouring a rapid transition toward resolution states.

Key takeaway

A macrophage is neither "good" nor "bad" in itself: it is its timing that decides the outcome. A necessary defender on day 1, it becomes the chief architect of fibrosis if it fails — or is unable — to stand down in time.

Original teaching course. Primary source: systematic review by Yaghmour, Arabpour, Al-Khudari & Djalilian (Life, 2026), Department of Ophthalmology and Visual Science, University of Illinois Chicago — open-access article.

Primary source

Yaghmour A, Arabpour Z, Al-Khudari H, Djalilian A. When Macrophages Heal and When They Scar: Timing in Corneal Fibrosis. Life 2026;16(7):1090. doi:10.3390/life16071090 (open access).

Supporting references cited in this course

Streilein JW. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nat Rev Immunol 2003;3(11):879-889.
Wynn TA, Vannella KM. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity 2016;44(3):450-462.
Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 2014;6:13.
Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol 2013;229(2):176-185.
Xu J, Chen P, Luan X, et al. The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium. Invest Ophthalmol Vis Sci 2022;63(9):15.
Taguchi C, Sugita S, Tagawa Y, Nishihira J, Mochizuki M. Macrophage migration inhibitory factor in ocular fluids of patients with uveitis. Br J Ophthalmol 2001;85(11):1367-1371.
Ambati BK, Nozaki M, Singh N, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature 2006;443(7114):993-997.

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