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.
- 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.
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