title: Methotrexate selectively targets human proinflammatory macrophages through a thymidylate synthase/p53 axis.
abstract: OBJECTIVE
  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.
authors.affiliation: Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Municio
authors.affiliation: Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
authors.forename: Blanca
authors.initials: B
authors.lastname: Soler Palacios
authors.affiliation: Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
authors.forename: Lizbeth
authors.initials: L
authors.lastname: Estrada-Capetillo
authors.affiliation: Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Benguria
authors.affiliation: Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Dopazo
authors.affiliation: Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: García-Lorenzo
authors.affiliation: Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Reus, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Fernández-Arroyo
authors.affiliation: Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Reus, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Joven
authors.affiliation: Departamento de Reumatología, Hospital Universitario La Paz-IdiPAZ, Madrid, Spain.
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: Miranda-Carús
authors.affiliation: Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain.
authors.forename: Isidoro
authors.initials: I
authors.lastname: González-Álvaro
authors.affiliation: Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
authors.forename: Amaya
authors.initials: A
authors.lastname: Puig-Kröger
mesh_terms: D018501:Antirheumatic Agents / Q000494:pharmacology*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy*; D006801:Humans; D008264:Macrophages / Q000187:drug effects*; D008727:Methotrexate / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D013940:Thymidylate Synthase / Q000378:metabolism; D059467:Transcriptome; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D018501:Antirheumatic Agents; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; D008727:Methotrexate
keywords: DMARDs (synthetic); Methotrexate; Rheumatoid Arthritis