ID2 Regulates Macrophage Polarization and Mediates Methotrexate Responsiveness in Rheumatoid Arthritis

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA), but patient responses are highly variable. Macrophages are central drivers of RA inflammation, yet how monocyte/macrophage heterogeneity and transcriptional programs influence MTX responsiveness remains unclear. Single-cell RNA sequencing was performed on peripheral blood mononuclear cells from MTX-responsive and MTX-unresponsive RA patients to define immune composition and macrophage-like monocyte subsets. Polarization states were assessed using gene-signature scoring and pseudotime analysis. The role of inhibitor of differentiation 2 (ID2) was examined in LPS-stimulated RA patient-derived macrophages using gain- and loss-of-function approaches, cytokine assays and chromatin immunoprecipitation assays. Although overall immune cell distributions were similar between groups, macrophage-like monocytes from MTX-unresponsive patients exhibited higher M1 polarization scores. PLBD1⁺ and CDKN1A⁺ macrophage-like monocytes were enriched in MTX-unresponsive patients, whereas RLIM⁺ macrophage-like monocytes were reduced. Integrative analyses identified ID2 as a regulator associated with MTX resistance. ID2 expression was elevated in macrophage-like monocytes from MTX-unresponsive patients and positively correlated with M1 polarization. In vitro, MTX suppressed ID2 expression and inflammatory activation. ID2 knockdown reduced pro-inflammatory responses, whereas overexpression attenuated the inhibitory effects of MTX. ID2 was found to regulate EEF1A1 transcription. ID2 promotes pro-inflammatory macrophage polarization and may contribute to MTX resistance in RA.