Background: Methotrexate (MTX) is the main rheumatoid arthritis (RA) therapy. However, the molecular pathways directly modulated by MTX and transcriptomic features that distinguish clinical responders from non-responders remain unclear. This study aimed to define MTX‑associated transcriptional programs and baseline synovial gene expression pathways associated with a clinical response to MTX-treated RA through cross‑tissue transcriptomic integration.

Methods: Peripheral blood gene expression profiles collected before and after MTX treatment were obtained from the Gene Expression Omnibus (GSE35455) database. Differential gene expression analysis was used to identify upregulated and downregulated genes, and functional enrichment analyses were conducted for each gene set. Clinical response–associated transcriptional signatures were independently evaluated on a dataset of baseline synovial tissue from MTX‑treated patients with RA (GSE45867). Gene set enrichment analysis was used to identify pathways associated with the MTX response.

Results: In the GSE35455 dataset, MTX treatment was associated with significant differential expression of 730 genes, which were predominantly downregulated. MTX-downregulated genes showed limited enrichment in classical Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways but demonstrated significant suppression of inflammatory signaling modules, including TNF-α signaling via NF-κB. In contrast, MTX-upregulated genes were enriched for metabolic stress responses, including Hypoxia and Glycolysis pathways, and innate immune response modules. Disease and phenotype enrichment analyses of the upregulated genes highlighted immune-related disease annotations and lymphoid tissue abnormalities. In synovial tissue (GSE45867), MTX responders exhibited strong baseline enrichment of interferon-stimulated gene programs, cytokine-regulated JAK-STAT signaling pathways, and immune activation signatures.

Conclusions: MTX induces broad immunomodulatory transcriptional effects characterized by the suppression of inflammatory signaling networks, and baseline activation of interferon- and cytokine-responsive programs in the synovial tissue. This cross-tissue integration provides mechanistic insights into MTX response heterogeneity in RA.