Gut-Driven Systemic Signaling Accelerates Corneal Stromal Aging via Keratocyte Senescence

Hypothesis

Age‑related corneal stromal thinning and keratocyte loss are driven not by autonomous ocular wear but by circulating gut‑derived factors that re‑program keratocyte phenotype toward a senescent, pro‑fibrotic state.

Mechanistic Rationale

1. Gut‑originating agonists – LPS, secondary bile acids (e.g., deoxycholic acid), and specific short‑chain fatty acids (SCFAs) increase with age‑related dysbiosis and cross the intestinal barrier into systemic circulation.2
2. Ocular exposure – These molecules reach the anterior segment via limbal vasculature and tear film, where they can bind Toll‑like receptors (TLR4) on keratocytes and corneal epithelial cells.3
3. Signal transduction – TLR4 activation triggers NF‑κB and MAPK pathways, up‑regulating TGF‑β1 and IL‑6 secretion from keratocytes.4
4. Cellular outcome – Chronic TGF‑β/Smad signaling induces keratocyte senescence (p16^INK4a^+, SA‑β‑gal+) and a shift to a myofibroblast‑like phenotype, promoting excessive collagen cross‑linking and fibril disorganization.5
5. Feedback loop – Senescent keratocytes secrete SASP factors (IL‑1β, MMP‑9) that further impair stromal transparency and accelerate collagen glycation (e.g., glucosepane formation).6

This cascade positions the gut as an upstream regulator of corneal aging, analogous to the gut‑retina axis demonstrated in AMD.2

Testable Predictions

• Prediction 1: Age‑matched mice receiving fecal microbiota transplants (FMT) from young donors will exhibit higher keratocyte density and thinner collagen fibrils than mice receiving FMT from aged donors, independent of local ocular inflammation.
• Prediction 2: Pharmacological neutralization of circulating LPS (using polymyxin B) or blockade of TLR4 in aged mice will reduce keratocyte senescence markers and preserve stromal transparency.
• Prediction 3: Elevation of specific beneficial SCFAs (e.g., butyrate) via dietary supplementation will attenuate TGF‑β1 upregulation in keratocytes and slow age‑related collagen cross‑link accumulation.

Experimental Design (mouse model)

1. Groups (n=10 per group): young FMT, aged FMT, aged FMT + anti‑LPS, aged FMT + TLR4 inhibitor, aged FMT + butyrate supplementation, and sham‑treated aged controls.
2. Interventions administered orally for 8 weeks.
3. Outcomes measured at baseline and endpoint:
   • Keratocyte density via in vivo confocal microscopy (cells/mm²).
   • Corneal collagen fibril diameter and organization by second‑harmonic generation imaging.
   • Levels of LPS, IL‑6, TGF‑β1, and SASP cytokines in tears and aqueous humor (ELISA).
   • Senescence markers (p16^INK4a^, SA‑β‑gal) in keratocyte isolates (immunohistochemistry/qPCR).
   • Functional read‑out: corneal hysteresis and central thickness (Ocular Response Analyzer).

Potential Outcomes and Falsifiability

• If aged FMT recipients show no significant difference in keratocyte density or collagen structure compared to young FMT recipients, and TLR4/LPS blockade fails to alter senescence markers, the hypothesis is falsified.
• Conversely, a consistent rescue of stromal metrics by gut‑targeted interventions would support a gut‑to‑cornea signaling axis and suggest that longevity strategies targeting the microbiome may directly mitigate corneal aging.

Implications

Confirming this axis would shift the paradigm from treating the cornea as an isolated, passively aging tissue to recognizing it as a downstream endpoint of systemic inflammatory and metabolic cues originating in the gut. It would also provide a mechanistic rationale for incorporating microbiome modulation into ocular health regimens aimed at preserving vision throughout the lifespan.