Nfe2l2-knockout mouse model exhibits oxidative stress-associated stromal degeneration and inflammatory activation resembling keratoconus pathology
TL;DR
Data support a feed-forward axis in which NRF2 deficiency drives oxidative stress, inflammation, and extracellular-matrix degradation that extends to corneal innervation, while NRF2 supplementation partially mitigates these pathological changes.
Abstract
Oxidative stress is central to keratoconus (KC) pathogenesis, and NRF2 is a key antioxidant regulator. However, an in vivo model linking NRF2 loss to KC has been lacking. We generated CRISPR/Cas9 Nfe2l2-knockout (KO) mice and assessed 4-month-old corneas by slit-lamp fluorescein staining, optical coherence tomography (OCT), histology/immunofluorescence and transmission electron microscopy (TEM), reactive oxygen species (ROS) assays, βIII-tubulin whole-mounts, and whole-cornea single-cell RNA-seq with Seurat/CellChat. Nfe2l2 loss caused central corneal thinning, increased fluorescein uptake, and disrupted epithelial tight junctions; the stroma exhibited reduced keratocyte density, disorganized collagen, and depleted proteoglycans. ROS accumulated while antioxidant effectors (Hmox1, Aldh3a1) declined; inflammatory/fibrotic markers (ICAM1, iNOS, α-SMA) and immune infiltration increased, mirroring clinical KC. The subbasal nerve plexus was markedly reduced and disorganized. Single-cell profiling revealed loss of extracellular matrix (ECM) -maintenance programs in keratocytes, epithelial-to-mesenchymal transition (EMT)-like epithelial changes, reduced limbal stemness, endothelial dysfunction, immune polarization, and remodeled intercellular signaling (attenuated FN1/OCLN; augmented APP/CALCR). Importantly, pharmacological activation of NRF2 with the activator RTA-408 in complementary in vitro rescue experiments partially reversed oxidative stress and inflammation. These data support a feed-forward axis in which NRF2 deficiency drives oxidative stress, inflammation, and extracellular-matrix degradation that extends to corneal innervation, while NRF2 supplementation partially mitigates these pathological changes. The Nfe2l2-ko mouse exhibits several pathological features similar to those observed in human keratoconus and provides a platform to investigate disease mechanisms and evaluate NRF2-targeted interventions.