Hypothesis

Aged tissues exhibit a shift from STAT1‑dominant to STAT3/5‑dominant JAK‑STAT signaling after chronic interferon exposure. Basal interferon‑stimived gene (ISG) expression remains high because STAT3/5 continue to drive a subset of ISGs, whereas acute interferon responsiveness appears blunted because STAT1 phosphorylation is rapidly removed by elevated SHP2 phosphatase activity and JAK2 is sequestered away from plasma‑membrane receptors. It's this isoform switch that creates the paradox of high basal ISGs with poor inducible responses.

Mechanistic basis

• Chronic IFN in aging sustains low‑level JAK2 activity that preferentially phosphorylates STAT3 and STAT5 due to altered receptor complex composition (e.g., increased gp130‑type cytokine receptor subunits) [https://doi.org/10.1101/2025.04.21.649782].
• Persistent STAT3/5 signaling maintains transcription of ISGs such as Irf7, Oas1a and Gsis that contribute to the observed basal ISG elevation [https://doi.org/10.1101/gr.240093.118].
• Upon acute IFN rechallenge, aged cells show a rapid rise in SHP2 (PTPN6) phosphatase activity that dephosphorylates pSTAT1 more efficiently than pSTAT3/5, truncating the canonical STAT1‑driven inflammatory transcriptional burst.
• Simultaneously, JAK2 is redirected to intracellular pools (e.g., endosomes) where it sustains STAT3/5 signaling but cannot efficiently couple to IFNAR1/2 complexes, limiting new STAT1 activation.

Predictions and experimental design

1. Phospho‑STAT kinetics – In young vs. aged mouse spleen, liver, and muscle satellite cells, measure pSTAT1, pSTAT3, pSTAT5 at 0, 5, 15, 30, 60 min after a single IFN‑β pulse. We'll expect aged cells to display higher basal pSTAT3/5, comparable or lower pSTAT1 at baseline, and a faster decay of pSTAT1 after stimulation.
2. Phosphatase dependence – Treat aged ex‑vivo tissues with a selective SHP2 inhibitor (e.g., SHP099) before IFN rechallenge. We'll predict pSTAT1 decay will be slowed to youthful levels and acute ISG induction (e.g., Ccl5, Irf1) will be restored without affecting basal STAT3/5‑driven ISGs.
3. JAK2 localization – Use subcellular fractionation or proximity ligation assay to quantify membrane‑associated JAK2 in young and aged cells after IFN. We'll predict aged cells will show reduced membrane JAK2 despite unchanged total JAK2, correlating with impaired STAT1 phosphorylation.
4. STAT isoform requirement – Knock‑down STAT3 or STAT5 in aged muscle satellite cells using siRNA. We'll predict basal ISG expression will drop, confirming their role in maintaining the elevated ISG signature, while acute IFN responsiveness (pSTAT1) remains unchanged.

Potential outcomes and interpretation

• If pSTAT1 decay is normalized by SHP2 inhibition and basal ISGs remain high, the data support a model where phosphatase‑mediated STAT1 silencing, not global JAK‑STAT exhaustion, creates the interferon paradox.
• If JAK2 membrane relocation correlates with rescued STAT1 phosphorylation, it indicates that receptor‑proximal re‑wiring, rather than simple down‑regulation, governs age‑related signaling bias.
• If STAT3/5 knock‑down reduces basal ISGs but does not affect pSTAT1 kinetics, it confirms that the persistent ISG transcriptome is driven by alternative STAT isoforms, separating basal elevation from acute responsiveness defects.

This hypothesis is falsifiable: failure of SHP2 inhibition to restore pSTAT1 dynamics, or lack of altered JAK2 localization, would refute the proposed mechanism and point toward alternative explanations such as global JAK degradation or SOCS‑mediated broad suppression.