Chronic interferon signaling in aged lymphatic endothelial cells drives persistent ISG15 expression via cGAS-STING activation from telomere dysfunction【1】. While JAK-STAT hyperactivation is documented in senescent cells and aged tendon stem/progenitor cells【3】【4】, defective responsiveness also appears in chronic inflammation and aging【5】, creating ambiguity between active feedback regulation and pathway exhaustion. We hypothesize that the apparent exhaustion observed in aged lymphatic endothelium stems from IFNAR receptor depletion rather than upregulated phosphatases or USP18-mediated deISGylation, and that intermittent JAK blockade allows receptor recycling, thereby restoring STAT phosphorylation capacity.

Rationale

• Aged tissues show sustained ISG15 through cGAS-STING, not acute viral responses, indicating a distinct upstream trigger【1】.
• Negative feedback mechanisms (SHP1/2, TC-PTP, USP18) exist, yet JAK inhibitors like AG490 reduce senescence markers without fully blocking the pathway, suggesting receptor loss may masquerade as feedback【4】【6】.
• Lymphatic endothelial cells in the nasopharyngeal plexus exhibit heightened type I interferon signaling in aging (synthetic observation), providing a accessible model to test receptor dynamics.

Testable Prediction If receptor depletion underlies the exhausted phenotype, intermittent exposure to a JAK inhibitor will preserve IFNAR surface expression and improve STAT1/2 phosphorylation kinetics after IFN washout, whereas continuous inhibition or no inhibition will not rescue signaling.

Experimental Design

1. Isolate lymphatic endothelial cells from young (3 mo) and aged (24 mo) mice nasopharyngeal plexus.
2. Pre‑treat cells with either: (a) vehicle, (b) continuous AG490 (10 µM), or (c) intermittent AG490 (10 µM for 1 h every 6 h) for 24 h.
3. Stimulate with IFN‑α (100 U/mL) for 30 min, wash, and allow recovery for 0, 15, 30, 60, 120 min.
4. Measure at each time point:
   • Surface IFNAR1 and IFNAR2 levels by flow cytometry.
   • Phospho‑STAT1 (Y701) and phospho‑STAT2 (Y690) by western blot or phospho‑flow.
   • ISG15 and MX1 mRNA by qPCR.
5. Include controls for lysosomal degradation (chloroquine) and recycling inhibition (monensin) to dissect trafficking routes.

Expected Outcomes

• If receptor depletion drives exhaustion: Intermittent AG490 will maintain higher IFNAR surface levels during chronic IFN exposure, leading to faster and greater pSTAT recovery compared to continuous AG490 or vehicle. ISG induction will be correspondingly restored in aged cells to levels resembling young cells.
• If active feedback predominates: Intermittent JAK blockade will not improve IFNAR surface expression or pSTAT kinetics; recovery will resemble continuous blockade or vehicle, indicating that phosphatases/USP18 dominate.

Falsifiability A null result—no significant difference in IFNAR surface density or pSTAT recovery between intermittent and continuous JAK inhibition across aged and young lymphatic endothelial cells—would falsify the hypothesis, supporting the view that observed exhaustion is primarily due to upregulated negative feedback rather than receptor loss.

Broader Impact Confirming receptor recycling as a key mechanism would reframe interventions for age‑related interferonopathies, suggesting that pulsatile JAK inhibition could rejuvenate tissue‑specific immune responsiveness without broadly suppressing host defense.