Hypothesis

Rapamycin extends lifespan by mimicking nutrient scarcity, but chronic activation of the integrated stress response (ISR) downstream of mTORC1 inhibition impairs acute immune defenses. We hypothesize that intermittent pharmacological relief of ISR activation (e.g., with ISRIB) will preserve rapamycin’s autophagy‑driven longevity benefits while restoring resistance to bacterial and viral challenges.

Mechanistic Rationale

Rapamycin inhibits mTORC1, shifting cells toward catabolic programs and inducing autophagy through ULK1 activation [1][2]. This inhibition also activates AMPK, which can phosphorylate and activate eIF2α kinases such as PERK and GCN2, leading to sustained eIF2α‑P [3]. Persistent eIF2α‑P reduces global protein synthesis, favoring ATF4‑driven transcription of stress‑adaptation genes but simultaneously limiting rapid synthesis of cytokines, antimicrobial peptides, and neutrophil effector proteins. Recent work shows rapamycin‑treated mice exhibit delayed clearance of Listeria monocytogenes and heightened mortality after influenza infection, correlating with lowered serum IL‑6 and neutrophil extracellular trap formation [4]. Transient ISR inhibition with ISRIB restores eIF2α‑B activity, re‑establishing translation of immune‑effector mRNAs without blocking autophagy flux ISRIB study. Thus, the longevity program triggered by famine signaling can be uncoupled from its immunosuppressive side‑effect.

Experimental Design

Animals: 20‑month‑old C57BL/6 mice, n=15 per group. Groups: (1) Control chow; (2) Rapamycin (14 ppm diet); (3) Rapamycin + ISRIB (0.5 mg/kg i.p. twice weekly); (4) ISRIB alone. Duration: 6 months treatment. Readouts:

• Lifespan and frailty index (clinical scoring).
• Autophagy flux in liver and muscle (LC3‑II/I ratio, p62 degradation after chloroquine chase).
• eIF2α‑P levels (Western blot) and ATF4 target gene expression (qPCR).
• Immune competence: sublethal intraperitoneal LPS (5 µg/g) survival; bacterial CFU burden in spleen and liver 24 h post‑Listeria (1×10⁴ CFU) challenge; viral titer after low‑dose influenza A (PR8) intranasal inoculation.
• Serum cytokines (IL‑6, TNF‑α, IFN‑γ) at baseline and 6 h post‑challenge.

Predictions and Falsifiability

If the hypothesis is correct, the rapamycin + ISRIB group will:

• Show lifespan extension comparable to rapamycin alone (≈10‑15 % increase).
• Maintain elevated autophagy markers similar to rapamycin monotherapy.
• Exhibit significantly lower eIF2α‑P and higher ATF4‑independent translation of immune effectors relative to rapamycin alone.
• Demonstrate improved survival after LPS, faster bacterial clearance, and reduced influenza mortality, matching or exceeding control levels.

Falsification occurs if:

• Rapamycin + ISRIB fails to extend lifespan beyond control, indicating ISRIB interferes with longevity pathways.
• Autophagy flux is suppressed in the combination group, showing ISRIB blocks the core benefit of rapamycin.
• Infection outcomes remain unchanged or worsen despite ISRIB, indicating chronic ISR activation is not the limiting factor for immune deficiency in this context.

This design directly tests whether uncoupling famine‑signaling from chronic ISR activation can yield a healthier, longer life without the hidden cost of compromised defense.