Tofacitinib repairs inflammation and mitochondrial dysregulation in GM-CSF-reprogrammed RA macrophages

Rheumatoid arthritis (RA) exhibits heterogeneous endotypes, complicating treatment strategies. GM-CSF and GM-CSFRα are enriched in RA synovial CD68⁺macrophages (MΦs), and are implicated in acute and chronic disease stages. Since anti-TNFi and anti-IL6R therapies did not effectively suppress GM-CSF/GM-CSFRα expression or the GM-CSF-associated landscape, we explored alternative therapeutic strategies to target GM-CSF function using RA blood, synovial tissues, and preclinical models. We demonstrate that GM-CSF-MΦs reprogrammed in RA blood and synovial tissue share a distinct IL1β⁺S100A⁺HIF1⁺IL10ˡᵒNFIL3/6ˡᵒ expression profile, manifested by mitochondrial oxidative stress and fragmentation. To correct the metabolic imbalance of GM-CSF-MΦs, cells were treated with a complex I inhibitor (i) or a glucose uptake blocker. Complex Ii did not broadly alter the inflammatory or metabolic networks or affect the mitochondrial dynamics remodeled by GM-CSF-MΦs. While the glucose uptake inhibitor (HK2i) reduced glycolysis-derived ATP, it had limited efficacy in restricting the inflammatory signature or restoring TCA enzymes in GM-CSF-MΦs. In contrast, tofacitinib achieved broad-spectrum effects by downregulating GM-CSFRα expression and inhibiting STAT5 signaling. Moreover, tofacitinib redirected RA blood and synovial IL1β⁺S100A⁺HIF1⁺IL10ˡᵒNFIL3/6ˡᵒMΦs into a regulatory phenotype, reversing oxidative stress and mitochondrial fragmentation. In preclinical models, local GM-CSF overexpression induced MΦ-directed joint inflammation and metabolic dysregulation. Consistently, Tofacitinib reversed GM-CSF-differentiated murine IL1β⁺HBEGF⁺HIF1⁺MΦs by impeding STAT5 signaling, correcting metabolic dysregulation, and repairing mitochondrial fragmentation. In conclusion, anti-TNFi, anti-IL6R, and metabolic-targeted therapies were largely ineffective in modifying GM-CSF-MΦ pathology. Conversely, tofacitinib deactivation of STAT5 attenuates GM-CSF-MΦ-triggered inflammation and mitochondrial malfunction by restoring regulatory markers and rebalancing oxidative phosphorylation in RA specimens and/or preclinical models.