Spermidine‑eIF5A‑TFEB Axis Controls a Sterile‑Inflammation Feedback Loop that Drives Immune‑Mediated Aging

Background

Aging reduces spermidine levels, which diminishes eIF5A hypusination and lowers TFEB transcription in T and B cells, impairing autophagy and promoting senescence‑associated secretory phenotype (SASP) 2. Defective mitophagy in aged phagocytes leads to mitochondrial DNA release, activating cGAS‑STING and NLRP3 inflammasomes, thereby increasing IL‑1β, IL‑6 and TNF‑α 4. Chronic interferon‑stimulated gene (ISG) signaling can suppress TFEB expression via STAT1‑mediated transcriptional repression, creating a potential double‑negative loop that amplifies inflammaging.

Hypothesis

We hypothesize that age‑related spermidine depletion triggers a two‑step feedback loop: (1) reduced eIF5A hypusination lowers TFEB translation, causing autophagic failure and mitochondrial damage; (2) mitochondrial DNA‑driven cGAS‑STING/NLRP3 activation induces type I interferon production, which STAT1‑dependently represses TFEB transcription, further worsening autophagy. Restoring spermidine‑eIF5A activity will break this loop by simultaneously boosting TFEB translation and suppressing STAT1‑mediated TFEB repression, thereby reducing inflammasome activation and SASP.

Mechanistic Model

1. Low spermidine → decreased eIF5A hypusination → selective translation deficit of TFEB mRNA (and other autophagy‑related transcripts).
2. Autophagy loss → accumulation of damaged mitochondria → mtDNA efflux → cGAS‑STING activation → IRF3‑dependent IFN‑β production.
3. IFN‑β → JAK‑STAT1 signaling → STAT1 binds TFEB promoter → transcriptional repression.
4. Reduced TFEB → further autophagy decline → sustained inflammasome activation (NLRP3) → IL‑1β/IL‑18 release → SASP propagation. Thus, spermidine controls both the translational and transcriptional arms of TFEB regulation, positioning it as a node that links metabolic sensing to sterile inflammation.

Predictions

• In aged murine immune cells, spermidine supplementation will increase eIF5A hypusination, TFEB protein, and autophagic flux, while decreasing phospho‑STAT1 levels and IFN‑β secretion.
• Inhibition of eIF5A hypusination (using GC7) in young immune cells will phenocopy the aged state: reduced TFEB, elevated mtDNA‑cGAS‑STING signaling, and heightened NLRP3 activity, even in the presence of normal spermidine.
• STAT1 knockdown in aged immune cells will rescue TFEB expression and autophagy despite low spermidine, and will attenuate inflammasome‑dependent cytokine release.
• Conversely, overexpressing a transcription‑defective TFEB mutant will not suppress inflammasome activation, indicating that TFEB’s transcriptional activity—not just its presence—is required to break the loop.

Experimental Approach

1. Isolate CD4+ T cells and bone‑marrow‑derived macrophages from young (3 mo) and aged (24 mo) mice.
2. Treat cells with spermidine (10 µM), GC7 (1 µM), or vehicle; assess eIF5A hypusination (Western blot for hypusinated eIF5A), TFEB mRNA and protein (qPCR, immunoblot), autophagic flux (LC3‑II turnover with bafilomycin A1), mtDNA in cytosol (qPCR), phospho‑STAT1, IFN‑β ELISA, and NLRP3 inflammasome activation (ASC speck formation, caspase‑1 activity, IL‑1β release).
3. Perform parallel experiments with STAT1 siRNA or CRISPR‑Cas9 knockout to test epistatic relationships.
4. In vivo, administer spermidine supplementation in aged mice for 8 weeks, then measure circulating IFN‑β, IL‑1β, and tissue‑specific senescence markers (p16^INK4a^, SA‑β‑gal).

Potential Outcomes

• If spermidine restores TFEB and reduces inflammasome activity without lowering phospho‑STAT1, the hypothesis is falsified, indicating that TFEB acts downstream of IFN signaling.
• If STAT1 loss rescues autophagy despite persistent spermidine deficiency, the transcriptional repression arm is confirmed as essential.
• Failure of TFEB overexpression to inhibit inflammasome activation would suggest that TFEB’s lysosomal functions, not merely its transcriptional role, are required to mitigate mitochondrial damage.

This framework provides a testable, mechanistic link between metabolic sensing (spermidine/eIF5A), transcriptional control (TFEB), and sterile inflammasome signaling, offering a precise node for interventions aimed at interrupting immune‑driven aging.