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Published on: August 4, 2023
Nfe2l2-knockout mouse model exhibits oxidative stress-associated stromal degeneration and inflammatory activation
Xiaoxue Li1,2, Jianing Gu2,3, Xihao Sun1,2,3
1Aier Academy of Ophthalmology, Central South University, Changsha, China.
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.
Insights
Loss of NRF2 in mice causes keratoconus-like corneal changes, including thinning and nerve damage. NRF2 activation may offer therapeutic benefits for this eye condition.
Area of Science:
- Ophthalmology
- Genetics
- Molecular Biology
Background:
- Oxidative stress is implicated in keratoconus (KC) pathogenesis.
- NRF2 is a critical regulator of antioxidant responses.
- An in vivo model linking NRF2 deficiency to KC was previously unavailable.
Purpose of the Study:
- To establish and characterize an in vivo model of KC using NRF2-deficient mice.
- To investigate the role of NRF2 in corneal integrity and pathology.
- To explore NRF2 activation as a potential therapeutic strategy for KC.
Main Methods:
- Generated CRISPR/Cas9 Nfe2l2-knockout (KO) mice.
- Corneal assessments included OCT, histology, TEM, ROS assays, and single-cell RNA-seq.
- In vitro experiments utilized NRF2 activator RTA-408.
Main Results:
- Nfe2l2 loss induced central corneal thinning, epithelial disruption, stromal changes, and nerve damage.
- Accumulated reactive oxygen species (ROS) and increased inflammatory markers were observed.
- Single-cell profiling revealed ECM degradation, EMT-like changes, and immune dysregulation.
- NRF2 activation partially reversed oxidative stress and inflammation in vitro.
Conclusions:
- NRF2 deficiency drives KC-like pathology through oxidative stress, inflammation, and ECM degradation.
- The Nfe2l2-KO mouse model mimics key features of human KC.
- NRF2 supplementation shows potential for mitigating KC-related pathological changes.