Background. Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6- MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis(MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. Methodology. The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. aviumusing a radiometric (14CO2 BACTEC®) detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (% -ΔcGI). Principal Findings. The positive control antibiotic (clarithromycin) has ≥85% 2DcGI at a concentration of 0.5 μg/ml. The negative control (ampicillin) has minimal inhibition at 64 μg/ml. MAP ATCC 19698 shows ≥80% ΔcGI for both agents by 4 μg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. Conclusions. We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % -ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti- MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.
Greenstein, R. J., Su, L., Haroutunian, V., Shahidi, A., & Brown, S. T. (2007). On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis. PLoS ONE, 2(1). https://doi.org/10.1371/journal.pone.0000161