Hypothesis In aged human monocytes, β‑glucan–induced trained immunity cannot activate autophagy because persistent mTORC1 signaling actively blocks the initiation complex; simultaneous low‑dose rapamycin restores autophagy flux and enables β‑glucan to drive epigenetic reprogramming.

Rationale

• Autophagy decline in aging is due to active suppression via mTORC1 hyperactivation, RUBCN upregulation, and transcriptional repression, while the core machinery remains intact {1}{2}{3}.
• Trained immunity in young monocytes requires autophagy for β‑glucan‑mediated chromatin remodeling {5}.
• No data test whether β‑glucan can overcome age‑specific autophagy checkpoints.

Novel Mechanistic Insight β‑glucan engages Dectin‑1 → Syk → CARD9 → NF‑κB and also stimulates glycolysis, which normally activates AMPK and inhibits mTORC1 via TSC2. In aged monocytes, constitutive mTORC1 activity (driven by lysosomal amino acid sensing and growth factor signaling) phosphorylates ULK1 at Ser757, rendering it unresponsive to AMPK. Additionally, age‑associated increase in RUBCN binds VPS34, blocking PI3P production downstream of ULK1. Thus, even if β‑glucan raises AMPK, the downstream block persists. Pharmacologic mTORC1 inhibition removes the ULK1 inhibition, allowing AMPK‑ULK1 axis to function and permitting VPS34 activity when RUBCN is concurrently downregulated by β‑glucan‑induced miR‑30a (hypothesized). This creates a permissive window for autophagy initiation and subsequent lysosomal biogenesis via TFEB nuclear translocation.

Predictions

1. β‑glucan alone will not increase LC3‑II turnover or decrease p62 in monocytes from donors >65 y, whereas it will in <30 y donors.
2. Phospho‑S6K (mTORC1 readout) will remain high after β‑glucan in aged cells; AMPK phosphorylation (Thr172) will be transient but insufficient to reduce p‑S6K.
3. Adding low‑dose rapamycin (10 nM) to β‑glucan will reduce p‑S6K, increase LC3‑II flux, promote TFEB nuclear localization, and enhance H3K27ac at glycolytic gene promoters (e.g., HK2, PFKFB3).
4. The combined treatment will amplify trained immunity phenotypes: heightened IL‑1β and TNF‑α production upon secondary LPS challenge and increased oxidative burst.

Experimental Design

• Isolate CD14⁺ monocytes from young (20‑30 y) and old (65‑80 y) donors (n = 6 per group).
• Treat cells for 24 h with: (i) vehicle, (ii) β‑glucan (10 µg/mL), (iii) rapamycin (10 nM), (iv) β‑glucan + rapamycin.
• Assess: autophagy flux (LC3‑II/I by Western blot with bafilomycin A1 block, mCherry‑GFP‑LC3 microscopy), mTORC1 activity (p‑S6K Thr389), AMPK activity (p‑AMPK Thr172), RUBCN protein levels, TFEB localization (immunofluorescence), and trained immunity markers (H3K27ac ChIP‑qPCR at promoters of IL6, TNF; cytokine secretion after LPS restimulation).
• Include controls: chloroquine (lysosomal inhibition) to distinguish flux vs. accumulation, and siRNA against RUBCN to test its contribution.

Potential Outcomes

• If β‑glucan fails to induce autophagy in aged monocytes but succeeds when combined with rapamycin, the hypothesis is supported, indicating that mTORC1‑mediated suppression is dominant over irreversible damage.
• If β‑glucan alone restores autophagy in aged cells, the hypothesis is refuted, suggesting that age‑related autophagy loss is more passive or that other pathways compensate.
• If rapamycin does not enhance β‑glucan‑induced autophagy despite reducing p‑S6K, then downstream blocks (e.g., RUBCN, transcriptional repression) are likely predominant, redirecting focus to those mechanisms.

This framework directly tests whether age‑related autophagy suppression is a reversible signaling barrier that can be overcome by trained immunity stimuli when mTORC1 is restrained, linking immunometabolic training to lysosomal homeostasis in aging.