Hypothesis

Chronic low‑level IFN‑I signaling in aged tissues drives a self‑reinforcing loop whereby IFN‑induced suppression of Foxo1 in γδ T cells compromises mitochondrial antioxidant defenses, leading to mtDNA efflux, cGAS‑STING activation, and further IFN‑I production.

Mechanistic Rationale

• IFN‑I signaling via IFNAR activates PI3K‑AKT, which phosphorylates and excludes Foxo1 from the nucleus in γδ T cells [1]
• Foxo1 directly transcribes Sod2 (mitochondrial superoxide dismutase) and Ppargc1a (a regulator of mitochondrial biogenesis) [2]; its loss reduces SOD2 activity and increases mitochondrial ROS
• Elevated ROS oxidizes mitochondrial DNA, promoting its release into the cytosol through VDAC or Bax/Bak pores
• Cytosolic mtDNA engages cGAS, generating cGAMP that activates STING, IRF3, and STAT1, amplifying IFN‑β transcription—a positive feedback loop
• IFN‑I also induces caspase‑11 and GBP2, priming non‑canonical inflammasome activation [3]; mtDNA release further fuels NLRP3 inflammasome assembly via oxidized mtDNA, increasing IL‑1β/IL‑18
• Dietary restriction restores chromatin accessibility and reduces IFN signatures [2]; we propose that DR enhances mitophagy (via AMPK‑ULK1) and NAD⁺‑dependent SIRT3 activity, preserving Foxo1‑Sod2 axis and limiting mtDNA efflux

Testable Predictions

1. In aged mice, γδ T cells will show decreased Foxo1 nuclear localization, lower SOD2 protein, higher mitochondrial ROS, and increased cytosolic mtDNA compared with young counterparts
2. Pharmacological scavenging of mtDNA (e.g., using anti‑mtDNA antibodies or Oligo‑dT‑based scavengers) will reduce IFN‑β mRNA and ISG expression in aged tissues without altering basal IFN‑I production in young mice
3. Overexpression of Foxo1 specifically in γδ T cells (via Foxo1‑ transgenic or CRISPRa) will rescue SOD2 levels, diminish mtDNA release, lower cGAS‑STING signaling, and attenuate IL‑1β/IL‑18 elevation
4. Mitochondrial-targeted antioxidant MitoTEMPO will mimic the effects of DR on chromatin accessibility at IFN‑stimulated loci, as measured by ATAC‑seq

Experimental Approach

• Isolate γδ T cells from young (3 mo) and aged (24 mo) C57BL/6 mice; assess Foxo1 localization (imaging flow cytometry), SOD2 activity (enzyme assay), MitoSOX fluorescence, and cytosolic mtDNA (qPCR after subcellular fractionation)
• Treat aged mice with Oligo‑dT‑based mtDNA scavenger or MitoTEMPO for 2 weeks; measure serum IFN‑β, ISG signatures (RNA‑seq), and inflammasome cytokines (ELISA)
• Generate Foxo1‑fl/‑ mice crossed with Tcrd‑Cre‑ERT2 for inducible γδ‑T‑cell‑specific Foxo1 overexpression; repeat aging and readouts
• Perform ATAC‑seq and RNA‑seq on sorted γδ T cells to evaluate chromatin changes at IFN‑responsive promoters and correlate with Foxo1 binding motifs

If mtDNA efflux is required for the IFN‑I amplification loop, disrupting it should break the cycle and improve immune homeostasis in aging, providing a mechanistic link between the IFN‑Foxo1 axis, mitochondrial quality control, and inflammaging