Background/Purpose: Methotrexate (MTX) is an anti-metabolite inhibitor of folate-dependent biochemical pathways and is effective in reducing disease activity in autoimmune arthritis. This work seeks to investigate the relationship between dysregulation of folates and response to MTX in the collagen-induced arthritis (CIA) mouse model, with respect to biomarker identification and drug development.

Methods: Arthritis was induced in male DBA/1 mice at 7-9 weeks of age by intradermal injection of chicken-collagen in complete Freund’s adjuvant (Day 0) with a booster injection at Day 19 (n=25) and compared to healthy controls (n=5). Subcutaneous MTX injections of 0, 2, 10, 20, and 50 mg/kg were given weekly beginning at Day 14 for a total of 6 weeks. Arthritis disease activity was assessed by paw volume measurement and a 16-point clinical disease score. Mice were sacrificed at Day 54 and tissue samples were collected. Erythrocyte and liver tissue samples were evaluated for folate content by ultra-performance liquid chromatography tandem mass spectrometry. Liver tissue samples were analyzed for folic acid (FA), tetrahydrofolate (THF), 5-methyl-THF (5mTHF) and formyl-THF (fTHF), and RBCs were analyzed for 5mTHF content.

Results: Induction of arthritis in mice had no significant effect on RBC 5mTHF levels, but caused a marked dysregulation of liver folate. Compared to healthy controls, untreated disease mice were found to have a 37% reduction in liver 5mTHF (8.89±1.84 vs 5.58±1.10 pmol/mg, p=0.01) and a 57% increase in liver fTHF (3.54±0.63 vs 5.57±0.68 pmol/mg, p=0.003), with no significant impact on liver THF or FA content. Treatment with MTX resulted in a dose-dependent reduction in RBC 5mTHF, with a 32% reduction in the 50 mg/mL treatment group (p<0.05) that is similar to those observed in patients treated with low-dose MTX. Interestingly, MTX at 50 mg/mL also caused a 42% increase in liver 5mTHF (p=0.03) and a 47% reduction in liver fTHF (p=0.003), resulting in levels similar to those seen in the healthy control mice. Decreased paw volumes across the animals studied were associated with reduced RBC 5mTHF (r=0.63, p=0.004), increased liver 5mTHF (r=-0.62, p=0.002), and reduced liver fTHF (r=0.48, p=0.03). Similarly, reduced disease activity scores were associated with reduced RBC 5mTHF (r=0.59, p=0.008) and increased liver 5mTHF (r=-0.67, p=0.0006).

Conclusion: Together, this data suggests that induction of autoimmune arthritis is associated with the dysregulation of tissue folates in the CIA mouse model, and is at least partially reversed by treatment with MTX. The increased ratio of fTHF to 5mTHF is hypothesized to reflect increased oxidation of folates under pro-inflammatory conditions. The finding that measurements of disease activity are associated with the effect of MTX on folates supports the role of folates as a therapeutic target and biomarker of drug response in autoimmune arthritis.