PT  - JOURNAL ARTICLE
AU  - Cristina Municio
AU  - Blanca Soler Palacios
AU  - Lizbeth Estrada-Capetillo
AU  - Alberto Benguria
AU  - Ana Dopazo
AU  - Elena García-Lorenzo
AU  - Salvador Fernández-Arroyo
AU  - Jorge Joven
AU  - María Eugenia Miranda-Carús
AU  - Isidoro González-Álvaro
AU  - Amaya Puig-Kröger
TI  - Methotrexate selectively targets human proinflammatory macrophages through a thymidylate synthase/p53 axis
AID  - 10.1136/annrheumdis-2015-208736
DP  - 2016 Dec 01
TA  - Annals of the Rheumatic Diseases
PG  - 2157--2165
VI  - 75
IP  - 12
4099  - http://ard.bmj.com/content/75/12/2157.short
4100  - http://ard.bmj.com/content/75/12/2157.full
SO  - Ann Rheum Dis2016 Dec 01; 75
AB  - 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.