Abstract Rationale Eosinophils are key effector cells in allergic asthma, yet their metabolic reprogramming during activation remains poorly defined. Lactate, once considered a waste product of glycolysis, has emerged as a signaling molecule and substrate for protein lysine lactylation, linking metabolism to epigenetic and transcriptional regulation. However, the role of lactate metabolism in eosinophil function and asthma pathogenesis is largely unexplored. Methods Mouse eosinophils were isolated and activated with IL-33/IL-4/GM-CSF. Transcriptomic and metabolomic profiling were performed to assess metabolic changes. ¹³C-labeled glucose and glutamine tracing was used to determine lactate sources. Lactate levels in supernatants were measured, and MCT4 involvement was evaluated using inhibitors. Functional effects of lactate modulation on IL-5 and IL-13 secretion were assessed. Global lysine lactylation and CREB1 modifications were analyzed by Western blot and mass spectrometry. CREB1 K122 lactylation was functionally validated in 293T cells using acetylation-mimetic and lactylation-mimetic mutants. Pharmacological inhibitors of CREB and p300 were used to assess downstream effects. Clinical relevance was examined via public transcriptomic data from asthma patients. Results Eosinophil activation enhanced glycolysis and increased lactate production predominantly from glucose. Lactate was actively exported via MCT4. Exogenous lactate or inhibition of its export promoted IL-5 and IL-13 secretion, while blocking lactate generation suppressed inflammation. Activation increased global protein lactylation. Lactylome analysis identified over 400 differentially modified proteins, including CREB1 at K122—adjacent to its activating S133 phosphorylation site. Modulating lactate levels altered CREB1 phosphorylation. Mimicking K122 lactylation enhanced p-CREB levels. Inhibiting CREB or p300 reversed lactate-induced cytokine upregulation. Asthma patient data showed elevated expression of LDHA and SLC16A3 (MCT4) in sputum. Conclusion Activated eosinophils undergo glycolytic reprogramming to produce and secrete lactate, which in turn acts as a signaling metabolite that enhances inflammatory function. Lactate promotes CREB1 K122 lactylation, facilitating its phosphorylation and transcriptional activity, forming a “lactate-lactylation-CREB1” axis that drives eosinophil activation. This study reveals a novel metabolic-epigenetic regulatory loop in eosinophil biology with potential therapeutic implications for asthma. This abstract is funded by: U 2 2 A 2 0 2 6 5
Wang et al. (Fri,) studied this question.