OBJECTIVE: The folate antagonist methotrexate (MTX) is an anchor drug in the 
treatment of rheumatoid arthritis (RA), but its mechanism of action with regard 
to the impact on folate metabolism remains elusive. The aim of the present study 
was to investigate the cellular pharmacologic impact of MTX on peripheral blood 
cells, by comparing MTX-treated RA patients to MTX-naive RA patients and healthy 
controls.
METHODS: Gene expression microarray data were used to investigate the expression 
of 17 folate pathway genes by peripheral blood cells from a cohort of 25 RA 
patients treated with MTX, 10 MTX-naive RA patients starting treatment with MTX, 
and 15 healthy controls (test cohort). Multiplex real-time polymerase chain 
reaction was used to validate the results in an independent cohort, consisting 
of 151 RA patients treated with MTX, 28 MTX-naive RA patients starting treatment 
with MTX, and 24 healthy controls (validation cohort).
RESULTS: Multiple folate metabolism-related genes were consistently and 
significantly altered between the 3 groups in both cohorts. Concurrent with 
evidence of an immune-activation gene signature in MTX-naive RA patients, 
significant up-regulation of the folate-metabolizing enzymes γ-glutamyl 
hydrolase and dihydrofolate reductase, as well as the MTX/folate efflux 
transporters ABCC2 and ABCC5, was observed in the MTX-naive RA group compared to 
healthy controls. Strikingly, MTX treatment of RA patients normalized these 
differential gene expression levels to the levels observed in healthy controls.
CONCLUSION: These results suggest that under inflammatory conditions, basal 
folate metabolism in the peripheral blood cells of RA patients is markedly 
up-regulated, and treatment with MTX restores folate metabolism to normal 
levels. Identification of this novel gene signature provides insight into the 
mechanism of action of MTX, thus paving the way for development of novel folate 
metabolism-targeted therapies.