Engineered Vesicles Reprogram Macrophage Metabolism to Fight Chronic Inflammation

Chronic inflammation drives tissue destruction in diseases like periodontitis, partly because macrophages become locked in a hyperactive, pro-inflammatory (M1) state characterized by excessive glycolysis and impaired mitochondrial oxidative phosphorylation (OXPHOS). A critical metabolic switch is pyruvate kinase M2 (PKM2): in its tetrameric form (Tet-PKM2), it channels glucose-derived carbons into the TCA cycle and OXPHOS, supporting anti-inflammatory (M2) macrophage function. The researchers first confirmed that Tet-PKM2 is sharply downregulated in gingival tissue from human periodontitis patients compared with healthy donors, with immunoelectron microscopy and metabolomic analysis revealing concurrent disruption of pyruvate metabolism and accumulation of pro-inflammatory metabolites.

To therapeutically restore Tet-PKM2, the team developed bioengineered large extracellular vesicles (LEVs). PKM2-overexpressing HEK293T cells were treated with TEPP-46, a small-molecule allosteric activator that stabilizes the tetrameric conformation, enabling Tet-PKM2 to be packaged preferentially into the cells' secreted LEVs. The resulting Tet-PKM2-enriched LEVs were then surface-modified with tannic acid (TA), a plant-derived polyphenol that confers pH-responsive charge reversal: neutral at physiological pH but switching to disrupt lysosomal membranes under the acidic conditions of the lysosome, enabling cargo escape into the cytoplasm.

In vitro experiments using LPS-activated macrophages demonstrated that LEVs^Tet-PKM2@TA significantly outperformed unmodified LEVs in lysosomal escape efficiency. Treatment rescued pyruvate kinase activity, reduced lactate accumulation, restored TCA cycle metabolite flux, elevated mitochondrial membrane potential, increased ATP production, and suppressed pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) while enhancing anti-inflammatory markers (IL-10, Arg-1). Targeted metabolomic profiling confirmed a broad shift away from aerobic glycolysis toward OXPHOS-dependent metabolism.

In a mouse ligature-induced periodontitis model, local injection of LEVs^Tet-PKM2@TA reduced alveolar bone loss as measured by micro-CT, promoted periodontal ligament and cementum regeneration on histology, and shifted the tissue macrophage population toward M2 phenotypes. Biosafety assessments of major organs showed no adverse effects, supporting the platform's translational potential.

This work establishes a proof-of-concept for protein-level immunometabolic reprogramming using bioengineered EVs, addressing the longstanding challenge of lysosomal degradation that limits EV-based therapeutic delivery. The approach is notable for its use of a naturally derived surface modifier (tannic acid) and a clinically relevant disease model, though translation to humans will require further pharmacokinetic, dosing, and safety studies.