Background/Purpose: Despite advances in targeted therapeutics, methotrexate (MTX) remains the first-line therapy in the treatment of rheumatoid arthritis (RA) and other inflammatory arthritides. However, more than 50% of patients do not achieve clinically adequate improvement on MTX alone. For decades, investigators have sought to identify determinants of MTX response, and while multiple factors have been implicated (e.g., genetics, environment), the molecular mechanisms underpinning MTX response are incompletely understood. We sought to investigate the extent to which the human gut microbiome contributes to variation in MTX response. We previously showed that among RA patients, the gut microbiome is associated with MTX efficacy. Further, we found microbial communities from RA patients variably depleted MTX ex vivo, and that depletion was negatively associated with future MTX efficacy. We also identified multiple representative human gut bacterial strains that directly deplete MTX in vitro. Given these clinical associations and supportive ex vivo and in vitro findings, here we quantify the extent to which the human gut microbiome contributes to MTX pharmacokinetics (PK) in vivo and whether this is linked to treatment outcomes in autoimmunity.

Methods: Germ-free mice were variably colonized with human or murine microbiota. We quantified levels of MTX and its metabolites in circulation and stool using LC-MS over an 8- to 24-hour time course. We used stable isotope labeling to identify novel MTX metabolites. We harvested strains from RA patients to test their MTX metabolism abilities in vitro. We used specific-pathogen-free (SPF) SKG mice reared in different facilities to test the impact of the gut microbiota on MTX pharmacology and clinical arthritis scores.

Results: MTX PK profiles significantly differed between germ-free and colonized mice in vivo. Further, germ-free mice colonized with different patient microbiota significantly differed in MTX plasma profiles. We harvested over 300 bacterial strains from RA patients representing 46 unique species; of these, 12 (26%) metabolized MTX in vitro. Stable isotope labeling studies revealed 3 novel MTX metabolites, and one of these novel metabolites was found to be the major product of microbial metabolism. Finally, studying SPF SKG mice reared at two different facilities with differing microbiota, we found that clinical response to MTX differed by facility, that co-housing reduced this difference, and that germ-free mice colonized with these different mouse microbiotas showed significantly different MTX PK profiles, suggesting that microbial MTX metabolism is linked to treatment outcomes in vivo.

Conclusion: We provide evidence that the human gut microbiome causally contributes to MTX pharmacology in vivo through known and novel mechanisms and raise the possibility that microbial metabolism can be modulated to impact MTX pharmacology and treatment outcomes in patients. These results are a first step towards understanding and manipulating the human gut microbiome in the treatment of autoimmunity and the advancement of precision medicine for millions of patients globally taking MTX for autoimmunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-gut-microbiome-shapes-mtx-pharmacology-and-is-linked-to-treatment-outcomes/