Background/Purpose: Early treatment initiation in rheumatoid arthritis (RA) is fundamental to avoid chronic joint destruction and disability. Despite remarkable advances in RA therapeutics, oral methotrexate (MTX) remains the anchor drug and mainstay of treatment worldwide. However, MTX bioavailability has a wide inter-individual variability and >50% of patients with moderate or severe RA show no or suboptimal improvement in their symptoms in response to MTX. The reasons for these disparities in treatment response remain unclear. Prior studies have shown that the biotransformation of MTX is altered in germ-free and microbiome-depleted mice, prompting us to hypothesize that inter-individual differences in the human gut microbiome could impact drug bioavailability and thus clinical efficacy. We sought to determine differences in the microbiome of drug-naïve, new onset RA (NORA) patients that could predict response to MTX therapy.

Methods: We enrolled 27 drug-naïve, NORA patients priori to MTX initiation (test cohort),  and classified them as either MTX-responders (MTX-R; 39% of the cohort) or non-responders (MTX-NR; 61%) based on a stringent definition of clinical response (delta improvement of DAS28 >1.8 by month 4). We performed 16S rRNA gene and Shotgun Metagenomic sequencing on the baseline gut microbiomes of these NORA patients and confirmed the results in an independent validation cohort (n=31). NMR and LC-MS were performed in ex vivo incubations to measure the capacity of each NORA microbiome to metabolize MTX.   

Results: Our analysis revealed significant associations between the abundance of gut bacterial taxa and future MTX response. Patients that responded to therapy had significantly lower microbial diversity (p< 0.05). A significant difference in overall gut microbial community structure was also observed between groups (Bray-Curtis distance; PERMANOVA < 0.05). At the class level, we observed statistically higher abundance of Clostridia and lower abundance of Bacteroidia in MTX-NR (p< 0.05; q< 0.2). Furthermore, the baseline metagenome separated most MTX-R from MTX-NR (PCoA; PERMANOVA p< 0.05). We identified 8 microbial modules and 23 pathways, whose abundance significantly differed between groups (p< 0.05, q< 0.2), including genes related with purine and MTX metabolism, indicating a major difference in metabolic and biosynthetic potential between the microbiome of MTX-R and MTX-NR patients. Machine learning techniques were applied to this metagenomic data, resulting in a robust model based on bacterial gene abundance that accurately predicted response to MTX in an independent cohort. Finally, MTX available levels remaining after ex vivo incubation with distal gut samples from pre-treatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a direct effect of the gut microbiome on MTX bioavailability and response to therapy.

Conclusion: Together, these results provide the first step towards predicting response to oral MTX in NORA patients and support the utility of the gut microbiome as a prognostic tool and perhaps even as a target for manipulation in the treatment of rheumatic and autoimmune disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-pre-treatment-gut-microbiome-predicts-early-response-to-methotrexate-in-rheumatoid-arthritis/