BackgroundRheumatoid arthritis (RA) is an aggressive, systemic autoimmune disease in which overactivated macrophages play a critical role in its pathogenesis. This study aimed to explore the potential role of glycolytic reprogramming in the production of proinflammatory cytokines by macrophages in RA.MethodsThe Seahorse assay was conducted on RA or healthy control (HC) serum-treated human monocyte-derived macrophages (HMDMs) to evaluate glycolysis levels. RNA sequencing was performed to identify activated signaling pathways and key molecules in HMDMs stimulated by RA serum. The proinflammatory cytokines and hypoxia-inducible factor 1 alpha (HIF-1 alpha) were verified by Western blotting and quantitative polymerase chain reaction (qPCR).ResultsWe found that HMDMs stimulated with RA serum showed higher aerobic glycolysis levels than those treated with HC serum, along with higher expression of glycolysis-related genes, including hexokinase2 (HK2), pyruvate kinase L/R (PKLR), and phosphoglycerate kinase 1 (PGK1). Furthermore, RA serum-treated macrophages exhibited a higher level of interleukin-1 beta (IL-1 beta), and the expression of IL-1 beta positively correlated with HK2. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) or HK2 knockdown significantly suppressed IL-1 beta production in macrophages. The HIF-1 alpha-associated signaling pathways and HIF-1 alpha protein levels were also elevated in RA serum-treated macrophages. Inhibition of glycolysis by 3BrPA or knockdown of HK2 reduced HIF-1 alpha. Inhibiting HIF-1 alpha can suppress IL-1 beta production of RA serum-treated macrophages, and vice versa. TNF-alpha and IL-1 beta enhanced HIF-1 alpha and IL-1 beta expression in macrophages, an effect attenuated by glycolysis inhibition. Blocking TNF-alpha and IL-1 beta in RA serum diminished both glycolysis and IL-1 beta production.ConclusionOur findings demonstrate that RA serum triggers aerobic glycolysis in macrophages, which promotes HIF-1 alpha to drive IL-1 beta production. Notably, IL-1 beta within RA serum amplifies its own expression via this glycolysis-HIF-1 alpha axis, establishing a pathogenic positive feedback loop in RA.