Hypothesis

Aged fibroblasts remain interferon‑hyporesponsive because chronic low‑grade inflammation keeps USP18 constitutively high, which in turn recruits PRC2 to deposit H3K27me3 at ISG promoters, creating a self‑reinforcing epigenetic block that's distinct from transient USP18‑mediated desensitization.

Mechanistic Model

1. Persistent USP18 expression – Inflammaging elevates basal IFN‑β levels, maintaining JAK‑STAT activity just above the threshold that triggers USP18 transcription without inducing full ISG activation, and it's this low‑level signaling that sustains USP18.

2. USP18‑PRC2 interaction – USP18 doesn't just block STAT1 binding; it also directly binds the EZH2 subunit of PRC2 via its C‑terminal ubiquitin‑like domain, targeting the complex to STAT‑occupied ISG promoters.

3. Epigenetic locking – PRC2 catalyzes H3K27me3, which compacts chromatin and prevents ISGF3 access even after USP18 levels fall, and this doesn't require ongoing IFN stimulation.

4. Feedback isolation – The cell‑cycle gating that normally separates early ISGF3 priming from late USP18 feedback can't be overridden by cyclin‑dependent kinase activity because the USP18‑PRC2 loop operates independently, locking cells in a prolonged state.

Predictions & Tests

• Prediction 1: USP18 knock‑down in aged fibroblasts will reduce H3K27me3 at ISG promoters and restore poly(I:C)‑induced ISG transcription.
  • Test: siRNA against USP18 followed by ChIP‑qPCR for H3K27me3 at ISG15 and MX1 promoters after 6 h poly(I:C).
• Prediction 2: Pharmacologic inhibition of EZH2 (e.g., GSK126) will rescue ISG induction without altering USP18 levels, and we're expecting a rapid rebound in antiviral genes.
  • Test: Treat aged fibroblasts with GSK126, then measure ISG mRNA after IFN‑β stimulation.
• Prediction 3: Cells forced into a transient IFN pulse (short‑duration, high‑dose) won't acquire the H3K27me3 mark, whereas chronic low‑dose exposure will.
  • Test: Compare histone marks after 4 h high‑dose IFN‑β vs 48 h low‑dose IFN‑β.
• Prediction 4: USP18 mutants unable to bind EZH2 can't induce H3K27me3 despite being expressed.
  • Test: Reconstitute USP18‑KO fibroblasts with wild‑type or EZH2‑binding‑deficient USP18 and assess ISG responsiveness.

Potential Interventions

• USP18‑EZH2 interface blockers – Small molecules or peptides that disrupt the USP18‑EZH2 interaction could prevent epigenetic silencing while preserving USP18’s regulatory role in acute IFN signaling, and they don't affect basal USP18 expression.
• Targeted epigenetics – CRISPR‑dCas9‑KRAB or dCas9‑TET1 delivered to ISG promoters to erase H3K27me3 may transiently restore antiviral states in aged tissues, but it's unclear how long the effect lasts.

This hypothesis shifts the focus from receptor exhaustion to a USP18‑driven epigenetic lock, offering clear, falsifiable experiments that can be performed in primary aged fibroblasts or tissue explants, and it's ready for immediate testing.