Methotrexate selectively targets human proinflammatory macrophages through a thymidylate synthase/p53 axis

Area of Science:

• Immunology
• Pharmacology
• Molecular Biology

Background:

• Macrophages play a key role in rheumatoid arthritis (RA) pathogenesis.
• The response of macrophages to methotrexate (MTX), a common RA drug, is not well understood.
• Understanding macrophage response to MTX is crucial for optimizing RA treatment.

Purpose of the Study:

• To investigate the transcriptional effects of MTX on different macrophage subtypes.
• To identify potential markers for MTX responsiveness in macrophages.
• To elucidate the molecular mechanisms underlying macrophage MTX sensitivity or resistance.

Main Methods:

• Gene expression microarrays were used to profile human macrophage subtypes (GM-MØ and M-MØ) treated with MTX.
• Quantitative real-time PCR, ELISA, pharmacological inhibition, siRNA knockdown, metabolomics, western blot, and immunofluorescence were employed for validation and pathway analysis.
• Macrophage subtypes were polarized using GM-CSF (proinflammatory) or M-CSF (anti-inflammatory).

Main Results:

• MTX exclusively modulated gene expression in proinflammatory GM-MØ, affecting 757 genes and inducing CCL20 and LIF.
• Macrophage subset-specific MTX responsiveness correlated with thymidylate synthase (TS) expression; TS+ GM-MØ were sensitive, while TSlow/- M-MØ and monocytes were refractory.
• TS-dependent MTX responsiveness in GM-MØ was mediated by p53 activity. TS and p53 were found in RA synovial macrophages but not normal ones.

Conclusions:

• Macrophage response to MTX is polarization-dependent, governed by the TS-p53 axis.
• CCL20 and LIF are identified as novel in vitro markers for MTX responsiveness in macrophages.
• These findings provide insights into MTX efficacy and potential biomarkers in RA treatment.