Hypothesis

Aging shifts Type I IFN signaling from STAT1‑ to STAT3‑dominant transcription, which we propose increases mitochondrial reactive oxygen species (mtROS) production in innate immune cells, leading to oxidation‑driven release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA activates the cGAS‑STING pathway, thereby amplifying chronic IFN‑I generation and reinforcing the STAT3 bias. This positive feedback loop locks tissues into a low‑grade interferonopathy that exhausts adaptive immunity and diminishes vaccine‑induced antibody titers.

Mechanistic Model

1. STAT3‑driven transcriptional program – In aged monocytes and dendritic cells, STAT3 upregulates genes involved in glycolysis and NADPH oxidase activity (e.g., NOX2) while repressing PGC‑1α–mediated mitochondrial biogenesis (2,3). This skews cellular metabolism toward a pro‑oxidant state.
2. mtROS‑mediated mtDNA release – Elevated mtROS oxidize cardiolipin and impair the inner mitochondrial membrane, promoting formation of mitochondrial‑derived vesicles that escape mitophagy and dump oxidized mtDNA into the cytosol (7).
3. cGAS‑STING amplification – Cytosolic mtDNA engages cGAS, producing cyclic GMP‑AMP that activates STING, which in turn drives IRF3‑dependent IFN‑β transcription. In aged cells, STAT3 preferentially binds the IFNAR‑associated promoter regions, biasing downstream signaling toward STAT3 phosphorylation rather than STAT1 (1).
4. Feedback to STAT3 – Persistent IFN‑I signaling sustains STAT3 activation via JAK‑STAT cross‑talk, further elevating mtROS production and completing the loop.

Testable Predictions

• Prediction 1: In aged human PBMCs, pharmacological inhibition of STAT3 (e.g., with stattic) will reduce mtROS levels and cytosolic mtDNA detection by >40 % compared with DMSO control.
• Prediction 2: STING knockout or inhibition in aged mice will normalize the STAT1/STAT3 ratio of IFN‑stimulated gene expression after low‑dose IFN‑α treatment, restoring antiviral ISG profiles.
• Prediction 3: Elderly vaccine recipients who exhibit high baseline mtDNA in serum will show lower seroconversion rates; attenuating mtDNA release with mitochondria‑targeted antioxidants (e.g., MitoQ) will improve antibody titers.

Experimental Approach

• Human cohort: Collect blood from donors aged 20‑35 (young) and 65‑80 (old). Measure phospho‑STAT1, phospho‑STAT3, mtROS (MitoSOX), cytosolic mtDNA (qPCR for mtDNA‑nDNA ratio), and serum IFN‑α. Treat ex vivo monocytes with STAT3 inhibitor, STING inhibitor (H‑151), or MitoQ and assess changes.
• Mouse model: Use aged (>18 mo) WT and STING‑deficient mice. Administer low‑dose poly(I:C) to mimic viral challenge, then track ISG expression (IFIT1, MX1) and STAT1/STAT3 phosphorylation. Include a group receiving MitoQ in drinking water.
• Vaccination arm: Immunize aged mice with inactivated influenza vaccine; correlate baseline serum mtDNA with hemagglutination inhibition titers. Intervene with MitoQ or STAT3 inhibitor prior to vaccination and evaluate efficacy.

If predictions hold, the data would support a model where age‑dependent STAT3 skewing fuels a mitochondrial‑DNA‑STING interferon loop that drives inflammaging and compromises vaccine responsiveness, offering precise intervention points (STAT3, STING, mtROS) distinct from global IFN blockade.