Defective macrophage plasticity exacerbates progression of inflammation in patients with rheumatoid arthritis

Background Rheumatoid arthritis (RA) affects over 1% of the global population. Current therapies cannot cure the disease, and only 20% of patients achieve long-term, drug-free remission. Recent studies highlight a dual role for synovial macrophages, which comprise both proinflammatory and remission-associated subsets. However, how macrophage plasticity and metabolic programming drive RA pathogenesis remains poorly understood. This study investigated how phenotypic and functional shifts in macrophage subsets contribute to chronic joint inflammation and bone erosion. Methods Peripheral blood CD14+ monocytes from RA patients and healthy controls (HC) were differentiated into M1, M2a, M2b, M2c, and M2d macrophage subsets. To simulate the inflamed joint, cells were cultured under both atmospheric oxygen and hypoxic conditions. Phenotypic and chemokine receptor profiles were evaluated using RT-qPCR and multiparametric flow cytometry for a panel of markers, including CD14, CD11c, CD38, CD86, CD163, CD206, CD209, MerTK, IRF4, CCR1, CCR2, CCR4, CCR5, and CCR8. Metabolic characterization was performed via flow cytometric assessment of protein synthesis. To validate these phenotypes in vivo , scRNA-seq analysis (GSE200815) was conducted on synovial tissue macrophages from RA and HC individuals. Finally, the functional capacity of each macrophage subtype to differentiate into bone-resorbing osteoclasts was assessed. Results scRNA-seq data identified M1, M2a, M2b, M2c, and M2d clusters at comparable frequencies between HC and RA synovial tissues. However, RA macrophage subsets exhibited a distinct proinflammatory shift, characterized by increased CD86 and VEGF expression, and reduced MerTK expression. In vitro , RA-derived macrophages displayed altered metabolic activity and a similar pathogenic phenotype, which was particularly pronounced in the M2c subset compared to HCs. Hypoxia significantly exacerbated these phenotypic discrepancies in RA M2c macrophages. Functional assays revealed that all macrophage subsets, except M2c, successfully differentiated into osteoclasts, with RA-derived cells yielding significantly higher numbers of pathogenic osteoclasts. Conclusion Dysregulated phenotypic, metabolic, and functional plasticity causes RA macrophages to adopt intermediate, pathogenic phenotypes. These alterations may impair tissue resolution, enhance osteoclastogenesis, and sustain chronic inflammation in the arthritic joint.