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Extended report
Methotrexate selectively targets human proinflammatory macrophages through a thymidylate synthase/p53 axis
  1. Cristina Municio1,
  2. Blanca Soler Palacios1,
  3. Lizbeth Estrada-Capetillo1,
  4. Alberto Benguria2,
  5. Ana Dopazo2,
  6. Elena García-Lorenzo3,
  7. Salvador Fernández-Arroyo4,
  8. Jorge Joven4,
  9. María Eugenia Miranda-Carús5,
  10. Isidoro González-Álvaro3,
  11. Amaya Puig-Kröger1
  1. 1Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
  2. 2Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
  3. 3Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain
  4. 4Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Reus, Spain
  5. 5Departamento de Reumatología, Hospital Universitario La Paz-IdiPAZ, Madrid, Spain
  1. Correspondence to Dr Amaya Puig-Kröger, Laboratorio de Inmuno-Metabolismo, Instituto de Investigación Sanitaria Gregorio Marañón, c/Doctor Esquerdo 46, Madrid 28007, Spain; amaya.puig{at}


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.

  • Methotrexate
  • Rheumatoid Arthritis
  • DMARDs (synthetic)

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  • Handling editor Tore K Kvien

  • CM and BSP contributed equally.

  • Contributors CM, BSP and LE-C: designed research, performed research and analysed data. AB and AD: analysed microarray data. SF-A: performed research and analysed data. JJ, MEM-C, EG-L and IG-A contributed vital reagents and materials and analysed data. AP-K conceived the study, designed research, analysed data and wrote the paper. All authors approved the final version.

  • Funding This work was supported by grant PI14/00075 from Instituto de Salud Carlos III/FEDER to AP-K, and grants S2010/BMD2350 from Comunidad de Madrid/FEDER (RAPHYME Program) (MEM-C and AP-K) and RIER RD12/0009/FEDER (MEM-C, IG-A and AP-K) (FEDER, Fondo Europeo de Desarrollo Regional: una manera de hacer Europa). AP-K is supported by FIBHGM and LEC is supported by CONACYT.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.