Background/Purpose: Methotrexate (MTX) is the most frequently used DMARD in the treatment of rheumatoid arthritis (RA). There are known links between the use of MTX and accelerated development of rheumatoid nodules in RA, although the mechanisms are unclear. Little is known of the potential for an anti-folate mode of action by MTX within nodule tissue. Previous studies indicate that adenosine release and genetic factors may be important to nodule development. However, the potential for these mechanisms to operate within nodules is unknown. The objective of this study was to determine whether MTX affects the expression of genes encoding adenosine receptors, MTX transporters and metabolising enzymes within subcutaneous rheumatoid nodules.

Methods: Subcutaneous nodule tissues (n=23) were obtained following elective surgery from 21 patients with RA, including 16 nodules from 15 patients receiving MTX and 7 nodules from 6 patients without MTX therapy.  Plasma levels for 15 cytokines were measured in paired bloods from 12 patients using Bio-Plex Pro human Th17 assays (BioRad). Quantitative real-time PCR assays (Applied Biosystems) were used to determine the expression of SLC19A1, ABCB1, ABCC1, ABCG2, GGH, FPGS, MTR, MTRR and TYMS, ADORs A1, A2A, A2B, and A3 genes within each nodule. Differences in normalised gene expression between groups was determined by Mann Whitney U tests and Spearman correlations between gene expression used to establish gene connectivity.

Results: All genes examined were expressed in all 23 nodule tissues. Amongst all genes, only the expression of MTR, encoding 5-methyltetrahydrofolate-homocysteine methyl transferase, was affected by MTX therapy, with reduced MTR expression in nodules from patients on MTX therapy, compared to those not receiving MTX (1.682ng vs 3.625ng respectively; p=0.023). The potential for gene co-regulation and connectivity was determined based on gene expression correlations. Greatest connectivity was established for TYMS, where expression significantly correlated, positively with GGH and FPGS and negatively with ADORA1, ABCG2 and MTR expression. MTX therapy impacted on this connectivity maintaining especially the TYMS with MTR and ADDORA1 negative correlations. Finally we found no evidence for an effect from interleukin (IL)-23, IL-6, TNFalpha or IFNgamma in plasma on gene expression within nodules.

Conclusion: The genes responsible for MTX transport, metabolism and mode of action are expressed in subcutaneous rheumatoid nodules. Amongst the genes investigated, MTX therapy impacts only on MTR gene expression, which is significantly reduced in nodules by MTX therapy. Plasma levels of key cytokines driving RA have no impact on nodule gene expression. Our data demonstrate the potential for MTX to exert its anti-inflammatory effects witin nodules; that MTX influences connectivity between gene expresion in nodules; and suggest that, through MTR expression, DNA methylation might be an underlying mechanism important to the formation of this extraarticular inflammatory lesion.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-of-the-genes-facilitating-methotrexate-action-within-rheumatoid-subcutaneous-nodules/