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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.

Author information

1
Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
2
Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
3
Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain.
4
Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Reus, Spain.
5
Departamento de Reumatología, Hospital Universitario La Paz-IdiPAZ, Madrid, Spain.

Abstract

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

PMID:
26920997
DOI:
10.1136/annrheumdis-2015-208736
[Indexed for MEDLINE]

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