Abstract
BACKGROUNDMethotrexate (MTX) is the cornerstone of treatment in the management of autoimmune arthritis, including juvenile idiopathic arthritis (JIA). Despite being in use for over 35 years the mechanism through which MTX inhibits inflammation remains unclear. In this study we investigate activation of JAK/STAT signaling in fibroblast‐like synoviocytes from patients with JIA and the effect of MTX.METHODSSynoviocytes were isolated from the synovial fluid of juvenile idiopathic arthritis patients following routine arthrocentesis and maintained in culture. Synoviocytes were treated with MTX and assessed for cellular levels of MTX and its polyglutamated metabolites, as well as intermediates of nucleotide biosynthesis, including dUMP and ZMP. Analytes were detected by UPLC‐MS/MS. JAK/STAT signaling was assessed in synoviocytes and A549 human lung carcinoma cells by Western blotting and immunofluorescence. Experiments were performed in triplicate and statistical testing was done by unpaired t test analysis.RESULTSMTX was actively taken up and metabolized in synoviocytes to form polyglutamated metabolites of MTX, however, MTX failed to cause measurable inhibition of de novo nucleotide synthesis by dUMP and ZMP analysis. Synoviocytes displayed a high level of STAT phosphorylation with 58.8±4.8% of total measureable STAT3 signal and 69.9±7.3% of total measureable STAT5 signal. In contrast, phosphorylation of STAT3 and STAT5 was undetectable in A549 cells and treatment with 100 ng/mL of recombinant IL‐6 resulted in a marked increase in STAT phosphorylation with 64.4±6.0% of measureable STAT3 signal and 76.6±8.5% of measureable STAT5 signal. Treatment with MTX resulted in inhibition of STAT3 and STAT5 phosphorylation by 38.1±3.4% (p<0.05) and 61.2±7.8% (p<0.05) in primary synoviocytes and 65.3±2.8 (p<0.05) and 92.8±8.1% (p<0.01) in IL‐6 stimulated A549 cells, respectively. In comparison, phosphorylation of STAT3 and STAT5 in response to IL‐6 stimulation in A549 cells was decreased by 91.6±11.3% (p<0.001) and 94.5±12.3% (p<0.001) by 50 μM of JAK Inhibitor I (CAS 457081‐03‐7). In A549 cells inhibition of STAT phosphorylation by MTX was completely reversed by co‐treatment with 100 μM folinic acid.CONCLUSIONThese findings support constitutive activation of JAK/STAT signaling in fibroblast‐like synoviocytes isolated from patients with JIA that is inhibited by treatment with MTX. Although the underlying biochemical mechanism through which MTX inhibits JAK/STAT signaling is not known, it appears to be dependent on the anti‐folate activity of MTX. Together, this data supports inhibition of JAK/STAT signaling as a possible mechanism of anti‐inflammatory activity of MTX.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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