Hypothesis

Transient type I interferon (IFN‑I) secreted by senescent fibroblasts creates a paracrine niche that primes adjacent stem cells for proliferative regeneration; sustained IFN‑I signaling, however, drives STAT1‑dependent epigenetic silencing of Wnt antagonists, leading to stem‑cell exhaustion and fibrosis. The therapeutic goal is not to eliminate senescent cells but to modulate the duration/intensity of their IFN‑I output.

Rationale

Young senescent fibroblasts recruited during wound healing secrete protective SASP factors such as PDGF‑AA and TGF‑β that promote contraction and remodeling [1]. These same cells also produce low‑level IFN‑I that enforces acute immune surveillance [3]. When IFN‑I signaling is brief, it induces a transient ISG profile that upregulates STAT1‑mediated expression of CXCR4 on stem cells, enhancing their recruitment to the injury site without triggering exhaustion. Chronic IFN‑I, by contrast, maintains STAT1 phosphorylation, leading to heterochromatin formation at promoters of Wnt inhibitors (e.g., DKK1, SFRP1) and consequent β‑catenin hyperactivity that pushes stem cells toward senescence or apoptosis [4]). This dichotomy explains why intermittent senolytic dosing preserves regenerative capacity while continuous depletion delays healing [2]).

Predictions

1. In a murine excisional wound model, genetic ablation of IFN‑β specifically in senescent fibroblasts (p16‑CreERT2;Ifnb1^fl/fl) will reduce early stem‑cell recruitment and impair wound closure, despite normal numbers of senescent cells.
2. Pharmacologic blockade of IFNAR1 with an antibody administered only during the first 48 h post‑injury will phenocopy the genetic deficit, whereas delayed blockade (starting day 4) will improve collagen deposition and reduce α‑SMA‑positive myofibroblasts.
3. Single‑cell RNA‑seq of wound‑derived stem cells will show a transient IFN‑stimulated gene signature (Isg15, Mx1) correlated with heightened CXCR4 expression in the transient‑IFN condition, and a persistent STAT1‑driven repression of Wnt antagonists in the chronic‑IFN condition.
4. Senolytic treatment (navitoclax) given after the transient IFN window (day 3) will not exacerbate healing defects, whereas the same treatment initiated at day 0 will abolish the early IFN‑I stem‑cell priming signal and mimic the IFN‑β fibroblast knockout phenotype.

Experimental Approach

• Generate p16‑CreERT2;Ifnb1^fl/fl mice; induce senescence locally with 4‑OHT and laser ablation; track senescence via p16‑LacZ reporter.
• Administer anti‑IFNAR1 (MAR1‑5A3) or isotype control at defined intervals; measure wound area, histology, and stem‑cell flow cytometry (Lin⁻Sca1⁺cKit⁺).
• Perform scRNA‑seq on FACS‑sorted stem cells at 24 h, 72 h, and 7 d; compute IFN‑response and Wnt‑antagonist scores.
• Use senolytic (navitoclax 50 mg/kg i.p.) on separate cohorts; compare healing kinetics with and without IFN‑β fibroblast deletion.

Falsification: If transient IFN‑I from senescent fibroblasts is dispensable for stem‑cell recruitment (no difference in early CXCR4⁺ stem‑cell numbers or wound closure between Ifnb1‑fibroblast KO and controls), the hypothesis is refuted. Conversely, if chronic IFN‑I blockade fails to restore Wnt antagonist expression or reduce fibrosis, the proposed epigenetic mechanism is invalid.