Background/Purpose: The trillions of microorganisms (microbiota) found within the human gut play a critical role in shaping the immune system, yet these complex microbial communities are also highly sensitive to numerous environmental factors. While much of the focus to date has been on dietary intake, emerging data has begun to suggest that the use of pharmaceutical drugs, even those that are not considered to be antibiotics, can alter the human gut microbiota with unknown consequences for treatment outcomes. Here, we examine the effect of methotrexate (MTX), which is first-line therapy for rheumatoid arthritis (RA), on microbiota composition and physiology as well as downstream consequences on the host’s immune system.

Methods: To examine the effect of MTX on human gut microbiota composition in vivo, gnotobiotic mice were colonized with microbiota from either healthy or RA patient donors and treated with oral MTX at varying doses. We performed high-throughput sequencing of the 16S rRNA gene to examine changes in taxonomic composition. We looked at the effects of varying the route of administration or co-administration with folic acid. To determine the direct effects of MTX on bacterial growth, we treated monocultures of 42 different human gut isolates with MTX and monitored growth using optical density. To identify MTX-induced transcriptional changes, we performed RNA-Seq on 4 isolates treated with MTX.  To determine the effects of MTX on bacterial taxonomy in vivo, fecal samples from RA patients (100% fulfilling ACR criteria) starting MTX were profiled longitudinally using 16S sequencing. To determine if MTX-induced shifts reduce the inflammatory potential of the RA microbiome, microbiota from RA patient donors either before or after MTX initiation were transplanted into germ-free mice. These mice were challenged with an inflammatory trigger with dextran-sodium sulfate (DSS), and immune profiling was done to assess inflammation.

Results: MTX altered community composition in vivo in humanized mice, leading to reduced Bacteroidetes and increased Firmicutes. Varying the route of drug administration or co-administration with folic acid produced similar, reproducible shifts. MTX directly altered growth of many human gut isolates. At the phylum level, Bacteroidetes tended to be more sensitive than Firmicutes, recapitulating the trends observed in gnotobiotic mice in vivo. RNA-Seq revealed that MTX alters transcriptional pathways involved in purine and pyrimidine metabolism. Furthermore, longitudinal analyses of the in vivo gut microbiotas of RA patients showed that MTX-induced shifts in bacterial relative abundance are associated with improved drug response. Transplant experiments revealed that mice harboring MTX-altered microbiota exhibited a reduced Th17 and Th1 response to DSS compared to mice harboring pre-treatment microbiota, suggesting that MTX alters the inflammatory potential of the RA patient microbiome.

Conclusion:

Together, these results suggest that the mechanism-of-action of non-antibiotic drugs may be due in part to off-target effects on the gut microbiota, while providing a critical first step towards explaining long-standing differences in drug response between patients.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/perturbation-of-the-human-gut-microbiome-by-methotrexate-contributes-to-the-resolution-of-inflammation-and-autoimmune-disease/