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

Ann Rheum Dis. 2016 Dec;75(12):2157-2165. doi: 10.1136/annrheumdis-2015-208736. Epub 2016 Feb 26.


Objectives: Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MØ) or M-CSF (M-MØ), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively.

Methods: The transcriptomic profile of both human macrophage subtypes exposed to 50 nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints.

Results: MTX exclusively modulated gene expression in proinflammatory GM-MØ, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS+ GM-MØ are susceptible to MTX, whereas anti-inflammatory TSlow/- M-MØ and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS+ GM-MØ. Importantly, TS and p53 were found to be expressed by CD163+/TNFα+ GM-CSF-polarised macrophages from RA joints but not from normal synovium.

Conclusions: Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro.

Keywords: DMARDs (synthetic); Methotrexate; Rheumatoid Arthritis.

MeSH terms

  • Antirheumatic Agents / pharmacology*
  • Arthritis, Rheumatoid / drug therapy*
  • Humans
  • Macrophages / drug effects*
  • Methotrexate / pharmacology*
  • Signal Transduction / drug effects*
  • Thymidylate Synthase / metabolism
  • Transcriptome
  • Tumor Suppressor Protein p53 / metabolism


  • Antirheumatic Agents
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Thymidylate Synthase
  • Methotrexate