Hypothesis

Chronic mTOR inhibition extends lifespan by mimicking nutrient scarcity, but it simultaneously blocks the mTORC1‑dependent metabolic‑epigenetic rewiring required for trained immunity. We hypothesize that pulsatile (intermittent) mTOR inhibition—alternating short periods of drug exposure with drug‑free windows—will retain the longevity‑promoting stress response (autophagy, AMPK/FOXO activation) while permitting periodic mTORC1 activity sufficient to drive the glycolytic shift and H3K4me3/H3K27ac deposition needed for monocyte/macrophage trained immunity. Consequently, animals receiving intermittent rapamycin will show lifespan extension comparable to continuous treatment and retain or enhance β‑glucan‑induced innate immune memory, whereas continuous rapamycin will impair this memory.

Mechanistic Rationale

• mTORC1 activity is necessary for HIF‑1α stabilization and the glycolytic burst that fuels acetyl‑CoA production for histone acetylation at immune gene promoters (e.g., TNFα, IL6) during trained immunity [3, 4].
• Autophagy and FOXO-mediated stress resistance are activated rapidly upon mTORC1 inhibition and can be sustained with brief inhibitory pulses [1, 2], suggesting that the longevity program does not require continuous suppression.
• Epigenetic marks underlying trained immunity (H3K4me3, H3K27ac) are dynamic and can be reestablished when mTORC1 signaling rebounds, provided the initial stimulus (e.g., β‑glucan) is present during the active window.

Testable Predictions

1. Lifespan: Mice receiving intermittent rapamycin (e.g., 5 days on/2 days off) will have median survival not significantly different from those on continuous rapamycin (p>0.05, log‑rank test).
2. Autophagy/FOXO: Both regimens will show comparable increases in LC3‑II/I ratio and nuclear FOXO3 in liver and muscle tissue [1, 2].
3. Trained Immunity: After a primary β‑glucan challenge, monocytes from intermittently treated mice will exhibit a secondary cytokine response (TNFα, IL‑6) equal to or greater than untreated controls, whereas continuously treated mice will show a blunted response [3, 4].
4. Epigenetic Markers: H3K4me3 and H3K27ac enrichment at the TNFα and IL6 promoters will be restored in intermittently treated mice during the drug‑free window but remain suppressed in continuously treated mice.

Experimental Design (falsifiable)

• Subjects: 20‑month-old C57BL/6 mice, n=15 per group (continuous rapamycin, intermittent rapamycin, vehicle).
• Drug regimen: Rapamycin encapsulated in chow to achieve plasma ~5 ng/mL; continuous group receives it ad libitum; intermittent group receives it for 5 consecutive days followed by 2 drug‑free days, repeated for 6 months.
• Readouts: Survival monitoring; tissue collection for LC3‑II/I Western blot and FOXO3 immunofluorescence; bone‑marrow‑derived monocytes isolated after primary β‑glucan (1 mg/kg, i.p.) exposure; secondary challenge 7 days later with LPS (100 ng/mL) and cytokine ELISA; ChIP‑qPCR for H3K4me3/H3K27ac at TNFα/IL6 promoters.
• Statistical analysis: Kaplan‑Meier with log‑rank test for survival; ANOVA with Tukey post‑hoc for cellular and molecular assays.

Potential Outcomes and Interpretation

• If intermittent rapamycin matches continuous rapamycin in lifespan extension and restores trained immunity metrics, the hypothesis is supported, indicating that longevity and immune readiness can be uncoupled via dosing schedule.
• If intermittent dosing fails to extend lifespan despite preserving autophagy, the hypothesis is refuted, suggesting that continuous mTOR suppression is required for the longevity program.
• If intermittent dosing preserves lifespan but does not rescue trained immunity, the hypothesis is partially falsified, implying that the epigenetic reprogramming for immune memory demands more sustained or differently timed mTORC1 activity than can be achieved with simple on/off cycles.

This framework directly tests whether the trade‑off between lifespan extension and innate immune memory is a pharmacological artifact of continuous mTOR inhibition or an inevitable consequence of mimicking scarcity.