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 activity is a key regulator of neutrophil ROS and NET production, cytotoxic molecules which are both implicated in the pathogenesis of immune-mediated inflammatory diseases such as rheumatoid arthritis (RA). Targeted inhibition of PFKFB3 expression blocked the production of ROS and NETs in a dose-dependent manner in both RA and HC neutrophils (p<0.01). RA neutrophils were more sensitive to lower concentrations of PFKFB3 inhibition. We also demonstrated that RA neutrophils retain ROS and NET production in culture conditions which mimic the low glucose environments encountered in the RA synovial joint. By dissecting the intricate interplay between PFKFB3, glycolysis, and neutrophil effector functions, this study advances the understanding of the molecular mechanisms governing innate immune responses and identifies PFKF3B as a potential therapeutic target for conditions characterized by dysregulated neutrophil activity.

The authors have declared no competing interest.

MFA was funded by a Versus Arthritis and Masonic Charitable Fund PhD scholarship (Grant No. 22193). GA was funded by a University of Liverpool CIMA MRes scholarship and a Dunhill Medical Trust and University of Liverpool PhD Scholarship. AN was funded by a Connect Immune Research and The Lorna & Yuti Chernajovsky Biomedical Research Foundation Grant (Grant No. 22925). HLW was funded by a Versus Arthritis Career Development Fellowship (Grant No. 21430). The equipment and software licences used in the Shared Research Facility for NMR metabolomics were funded by the MRC (Grant No. MR/M009114/1).

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The study was approved by the University of Liverpool Central University Research Ethics Committee C and NRES Committee North West (Greater Manchester West, Manchester, UK) for respectively the isolation of neutrophils from HC and RA blood. All participants gave written, informed consent in accordance with the declaration of Helsinki.

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