Methotrexate Impacts Conserved Pathways in Diverse Human Gut Bacteria Leading to Decreased Host Immune Activation

Immunomodulatory drugs inhibit bacterial growth, yet their mechanism of action, spectrum, and clinical relevance remain unknown. Here, we show that the first-line rheumatoid arthritis (RA) drug methotrexate (MTX) broadly alters the human gut microbiota. Drug sensitivity varied between strains. Growth experiments, transcriptomics, and metabolomics suggest that the mechanism of action is conserved between mammalian and bacterial cells. RA patient microbiotas were sensitive to MTX and changes in gut bacterial taxa and gene family abundance were distinct between responders and non-responders. Transplantation of post-treatment samples into germ-free mice given an inflammatory trigger led to reduced immune activation relative to pre-treatment controls, enabling the identification of MTX-modulated bacterial taxa associated with intestinal and splenic immune cells. These results show how conservation in cellular pathways across domains of life can result in broad off-target effects of drugs on the human gut microbiota with downstream consequences for immune function.