Orellana, D. A. | bioRxiv Science

bioRxiv2026

Multi-cohort Analysis Reveals Microbiome Signatures Associated with Drug Response in New-Onset Rheumatoid Arthritis

The human gut microbiome influences treatment outcomes, but whether microbiome signatures of drug response generalize across cohorts remains unclear. Here, we perform a multi-cohort analysis (3 cohorts, N=100 patients) to determine whether cross-cohort microbial signatures are associated with methotrexate (MTX) response in new-onset rheumatoid arthritis (RA) patients. Pre-treatment gut microbiome community structure and function differed by future MTX response status, with MTX-nonresponders (MTX-NR) showing Bacteroides caccae depletion and Ruminococcus bromii enrichment. Multiple microbial functions were depleted in MTX-NR, including nucleotide metabolism, one-carbon pool by folate, and histidine metabolism. Notably, candidate MTX-degrading genes were enriched in MTX-NR. Microbiome functional profiles outperformed clinical metrics in predicting future MTX response. These results show that consistent microbiome signatures are associated with MTX response across different RA cohorts and pave the way for microbiome-based precision medicine in newly diagnosed RA patients.

microbiology↗

bioRxiv2025

The gut microbiome shapes pharmacology and treatment outcomes for a key anti-inflammatory therapy

The human gut microbiome encodes a formidable metabolic repertoire that harvests nutrients from the diet, but these same pathways may also metabolize medications. Indeed, large screens have revealed extensive microbial metabolism of drugs in vitro, but the pharmacologic and clinical repercussions of microbiota-mediated metabolism in vivo remain to be discerned. As a proof-of-concept, we investigate how human gut microbes contribute to in vivo pharmacology and efficacy of a key anti-inflammatory drug, methotrexate (MTX). Specifically, we demonstrate that the gut microbiome shapes drug pharmacology in vivo in mice, both by directly metabolizing the drug and by inducing host pathways that promote drug metabolism. Moreover, interindividual variation in the human gut microbiome contributes to variation in pharmacokinetic (PK) profiles. When we quantified metabolites produced by microbes, we unexpectedly identified novel MTX metabolites, one of which, p-methylaminobenzoyl-L-glutamic acid (pMABG), was a major byproduct of microbial metabolism both in vitro and in vivo. Further, we find that a large proportion of patient-associated microbes are capable of metabolizing MTX. Finally, we show that microbial metabolism of MTX is linked to PK profiles and disease outcomes in a mouse model of inflammatory arthritis. Taken together, these findings provide evidence that the human gut microbiome causally contributes to drug pharmacology in vivo for a key anti-inflammatory drug through known and novel mechanisms. Our studies provide a framework for elucidating the clinical relevance of drug microbial metabolism in the context of treatment response. 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 taking MTX for immune or inflammatory conditions. HighlightsO_LIThe gut microbiome impacts methotrexate (MTX) pharmacology in mice C_LIO_LIThe human gut microbiome contributes to interindividual variation in MTX pharmacology C_LIO_LIHuman gut microbes produce novel MTX metabolites, pMABG and 6-MPDA C_LIO_LIMicrobial metabolism of MTX is linked to treatment outcomes C_LI

pharmacology and toxicology↗