Hypothesis

Chronic thiol oxidation in aging monocytes drives irreversible liquid‑liquid phase separation (LLPS) of glycolytic enzymes, converting protective stress‑granule‑like condensates into stable aggregates that lock the cell into a pro‑inflammatory trained‑immunity state.

Mechanistic rationale

In monocytes, metabolic reprogramming during trained immunity increases flux through glycolysis and the pentose‑phosphate pathway, elevating NADH/NAD+ ratio and generating reactive oxygen species (ROS) [4]. Oxidation of cysteine residues in intrinsically disordered regions (IDRs) of enzymes such as PKM2, GAPDH, and LDHA promotes multivalent interactions that nucleate LLPS, analogous to stress granule formation driven by G3BP1, FUS, and TDP‑43 [1][2][3]. Under acute stress, these condensates are reversible, sequestering excess enzymes and mRNAs to buffer metabolic spikes while allowing rapid disassembly via chaperones and autophagy [5]. However, age‑associated accumulation of sulfinic and sulfonic modifications shifts the equilibrium toward solid, amyloid‑like aggregates [6]. Once solidified, the granules can no longer release their cargo, leading to persistent sequestration of glycolytic enzymes, a bottleneck in ATP production, and compensatory HIF‑1α stabilization that sustains trained‑immunity epigenetic programs (e.g., H3K27ac at promoters of IL6, TNF) [7][8]. Thus, what begins as a proteostatic containment strategy becomes a maladaptive scaffold that sustains inflammatory signaling.

Testable predictions

1. Oxidation dependence – Treating human monocytes with a mild oxidant (diamide, 50 µM, 30 min) will increase cytosolic puncta of PKM2‑GFP that co‑localize with ubiquitin and thioflavin‑S, whereas pretreatment with N‑acetylcysteine will abolish puncta formation [4].
2. Reversibility threshold – Acute LPS exposure (4 h) yields liquid‑like droplets that recover after washout; chronic LPS (24 h) or aged donor monocytes show resistance to 1 M urea solubilization, indicating a solid state [5][6].
3. Functional impact – Monocytes in which LLPS is genetically blocked (PKM2 C‑mutant lacking cysteines) will display reduced HIF‑1α accumulation and lower H3K27ac at inflammatory promoters after β‑glucan training, accompanied by decreased IL‑1β secretion upon secondary challenge [7][8].
4. In vivo relevance – Aged mice treated with a brain‑penetrant thiol‑reducing agent (e.g., ER‑730) will exhibit fewer PKM2 aggregates in bone‑marrow‑derived monocytes and attenuated trained‑immunity‑mediated protection against secondary infection, confirming the causal link between oxidation‑driven LLPS and maladaptive memory [8].

Falsifiability

If oxidant‑induced PKM2 puncta remain fully reversible after 24 h in aged monocytes, or if preventing LLPS fails to alter trained‑immunity epigenetic marks, the hypothesis would be refuted, indicating that monocyte aggregates are epiphenomena rather than drivers of trained immunity.