# Monocytes from inflammatory arthritis patients accumulate methotrexate and their transcriptome predicts methotrexate clinical response

## TL;DR

This study shows that methotrexate accumulates in monocytes from arthritis patients and that gene patterns in these cells can predict treatment response.

## Contribution

The study identifies monocyte transcriptome changes and gene markers that predict methotrexate effectiveness in arthritis patients.

## Key findings

- Methotrexate suppresses inflammation in whole blood 5 days after treatment.
- Methotrexate polyglutamates accumulate in monocytes and alter their gene expression.
- Non-classical monocyte genes predict better methotrexate clinical response.

## Abstract

Monocytes and monocyte-derived macrophages play a key pathogenic role in inflammatory arthritis. Methotrexate (MTX) is the first-line disease-modifying antirheumatic drug (DMARD) for arthritis, yet the mechanisms and kinetics of its effects on monocyte/macrophages remain poorly understood. We have now investigated the temporal dynamics of the MTX's anti-inflammatory action by initially performing a phase I clinical trial (METOMAC) on healthy individuals following a single MTX dose, which revealed that MTX plasma levels peak at 1 h after MTX treatment, with maximal suppression of LPS-induced IL-1β and IL-6 in whole blood 5 days after MTX exposure. Building on these findings, we performed an observational clinical study (METOMAC-PAC) on 29 DMARD-naïve early arthritis patients receiving a weekly dose of MTX over a 3 month period. The METOMAC-PAC study revealed that MTX polyglutamates (MTX-PG) accumulates in peripheral blood monocytes, where MTX-PG is detected as early as 5 days post-treatment, and modulates the monocyte gene profile after 4 MTX doses, with specific enrichment of the aryl-hydrocarbon receptor (AhR)-molecular signature and genes coding for anti-inflammatory factors. Importantly, stratification of METOMAC-PAC patients according to their clinical response revealed that Good Responders exhibit increased expression of “non-classical monocyte”-specific genes (MAF, FCGR3B, ICAM4) both before and after MTX treatment, while Partial Responders patients showed a higher baseline expression of genes preferentially expressed by “classical monocytes”. Our results elucidate the kinetics of the anti-inflammatory action of MTX, demonstrate that MTX modulates the monocyte transcriptional signature in vivo, and identify “non-classical monocyte”-associated genes as predictors for an effective MTX clinical response.

The online version contains supplementary material available at 10.1007/s00018-026-06162-9.