The small molecule inhibitor 3PO is a modulator of neutrophil metabolism, ROS production, and NET release.

Neutrophils are key effector leukocytes of the innate immune system and play a pivotal role in defending the host against microbial infections. Recent studies have identified a crucial link between glycolysis and neutrophil cellular functions. Using human neutrophils, we have investigated the intricate relationship between glycolysis, extracellular glucose availability, and the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), in the regulation of reactive oxygen species (ROS) and neutrophil extracellular trap (NET) production. We have identified that PFKFB3 is elevated in rheumatoid arthritis (RA) neutrophils and that the small molecule PFKFB3 inhibitor 3PO is a key regulator of neutrophil ROS and NET production. 3PO blocked the production of ROS and NETs in a dose-dependent manner in both RA and healthy neutrophils (P < 0.01), and RA neutrophils were more sensitive to lower concentrations of 3PO. Bacterial killing was only partially inhibited by 3PO, and the proportion of live neutrophils after 24 h incubation was unchanged. Using NMR metabolomics, we identified that 3PO increases the concentration of lactate, phenylalanine, and L-glutamine in neutrophils, as well as significantly decreasing intracellular glutathione (adj. P-value < 0.05). We also demonstrated that RA neutrophils produce ROS and NETs in culture conditions which mimic the low glucose environments encountered in RA synovial joints. Our results also suggest that 3PO may have molecular targets beyond PFKFB3. By dissecting the intricate interplay between metabolism and neutrophil effector functions, this study advances the understanding of the molecular mechanisms governing pro-inflammatory neutrophil responses and identifies 3PO as a potential therapeutic for conditions characterized by dysregulated neutrophil activation.