Microbial metabolism of methotrexate produces a STAT3 signaling molecule that alleviates gut inflammation

Methotrexate (MTX) is a gold standard therapy for inflammatory diseases but has mixed therapeutic success in inflammatory bowel disease (IBD). The gut microbiota impact MTX efficacy and toxicity by metabolizing MTX and altering its bioavailability, yet how inflammation alters this metabolism, and how the resulting metabolites act on the host, remain poorly understood. Here, we find that gut microbes, notably Clostridium asparagiforme, metabolize MTX via carboxypeptidase G2 (CPDG2) to produce deoxyaminopteroic acid (DAMPA). In intestinal epithelial cells, DAMPA preserves mitochondrial health through mitochondrial signal transducer and activator of transcription 3 (STAT3) signaling and enhances mitophagy. DAMPA administration attenuates intestinal inflammation in vivo, and remodels colonic mitochondrial pathways. IBD mouse models show reduced microbial CPDG2 and DAMPA, while patients with IBD show reduced CPDG2 gene abundance and suppressed DAMPA-enriched mitochondrial pathways, potentially explaining MTX outcomes in IBD. Together, these findings reframe microbial MTX metabolism from passive drug detoxification to active regulation of host mitochondrial and inflammatory homeostasis.