Figure 4.

Differential ex vivo depletion of MTX by gut microbiomes from patients with new-onset RA, and correlation between remaining drug concentrations and future clinical response. Fecal samples from responders and nonresponders to MTX were incubated with MTX (100 μg/ml) at a temperature of 37°C under anaerobic conditions for 72 hours (a–c) or 48 hours (d–f). Abundance of MTX was measured in the supernatant using nuclear magnetic resonance (NMR) spectroscopy (a–c) or liquid chromatography mass spectroscopy (LC-MS) (d–f). a, Clustering of samples from nonresponders to MTX (red; n = 13) separately from samples from responders to MTX (blue; n = 9), determined by orthogonal partial least squares (OPLS) analysis based on the integration of 28 signals identified on the NMR spectra. b, OPLS model of the correlation of change in the Disease Activity Score in 28 joints (DAS28) with change in NMR signals. c, Significant correlation of the mean abundance (spectral intensity in AU) of the 2 peaks corresponding to MTX (B8_6200 and B1_9825) with future clinical response (DAS28) in patients with new-onset RA (rho = 0.619, P < 0.05 by Spearman’s 2-s ided rank correlation test). The blue line shows the mean linear regression; shading indicates 95% confidence intervals. d, Levels of MTX at the indicated time points after incubation with fecal samples from RA patients (n = 30). Most patients whose fecal microbiota rapidly depleted MTX did not have an adequate response to treatment. e, Proportion of nonresponders and responders to MTX who were fast metabolizers. f, Significant correlation of MTX elimination rate (slope of a linear fit to a semi-log plot of MTX concentration versus time) with future clinical response (DAS28 score) in patients with new-onset RA (rho = −0.37, P = 0.04 by Spearman’s 1-sided rank correlation test). See Figure 1 for other definitions.