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

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

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Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain.
Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Reus, Spain.
Departamento de Reumatología, Hospital Universitario La Paz-IdiPAZ, Madrid, Spain.



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.


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.


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.


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.


DMARDs (synthetic); Methotrexate; Rheumatoid Arthritis

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