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Basic and translational research
Extended report
Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism
  1. Cristina Municio1,
  2. Ángeles Dominguez-Soto2,
  3. Sara Fuentelsaz-Romero1,
  4. Amalia Lamana3,
  5. Nuria Montes3,
  6. Víctor D Cuevas2,
  7. Raquel García Campos1,
  8. José L Pablos4,
  9. Isidoro González-Álvaro3,
  10. Amaya Puig-Kröger1
  1. 1 Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
  2. 2 Centro de Investigaciones Biologicas, Madrid, Spain
  3. 3 Servicio de Reumatología, Hospital Universitario La Princesa, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Madrid, Spain
  4. 4 Servicio de Reumatología, Instituto de Investigación Hospital Universitario 12 de Octubre, Universidad Complutense de Madrid, Madrid, Spain
  1. Correspondence to Dr Amaya Puig-Kröger, Laboratorio de Inmuno-Metabolismo, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain; amaya.puig{at}salud.madrid.org

Abstract

Objectives Methotrexate (MTX) is the anchor drug for treatment of rheumatoid arthritis (RA), but the mechanism of its anti-inflammatory action is not fully understood. In RA, macrophages display a proinflammatory polarisation profile that resembles granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages and the response to MTX is only observed in thymidylate synthase+ GM-CSF-dependent macrophages. To determine the molecular basis for the MTX anti-inflammatory action, we explored toll-like receptor (TLR), RA synovial fluid (RASF) and tumour necrosis factor receptor (TNFR)-initiated signalling in MTX-exposed GM-CSF-primed macrophages.

Methods Intracellular responses to TLR ligands, TNFα or RASF stimulation in long-term low-dose MTX-exposed human macrophages were determined through quantitative real-time PCR, western blot, ELISA and siRNA-mediated knockdown approaches. The role of MTX in vivo was assessed in patients with arthritis under MTX monotherapy and in a murine sepsis model.

Results MTX conditioned macrophages towards a tolerant state, diminishing interleukin (IL)-6 and IL-1β production in LPS, LTA, TNFα or RASF-challenged macrophages. MTX attenuated LPS-induced MAPK and NF-κB activation, and toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF1)-dependent signalling. Conversely, MTX increased the expression of the NF-κB suppressor A20 (TNFAIP3), itself a RA-susceptibility gene. Mechanistically, MTX-induced macrophage tolerance was dependent on A20, as siRNA-mediated knockdown of A20 reversed the MTX-induced reduction of IL-6 expression. In vivo, TNFAIP3 expression was significantly higher in peripheral blood cells of MTX-responsive individuals from a cohort of patients with arthritis under MTX monotherapy, whereas MTX-treated mice exhibited reduced inflammatory responses to LPS.

Conclusions MTX impairs macrophage proinflammatory responses through upregulation of A20 expression. The A20-mediated MTX-induced innate tolerance might limit inflammation in the RA synovial context, and positions A20 as a potential MTX-response biomarker.

  • methotrexate
  • rheumatoid arthritis
  • dmards (synthetic)

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

https://doi.org/10.1136/annrheumdis-2017-212537

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    Footnotes

    • CM and ÁD-S contributed equally.

    • Handling editor Josef S Smolen

    • Contributors CM and AD-S designed research, performed research and analysed data; SF-R, AL, NM, VDC and RGC performed research and analysed data; JLP participated in the research; IG-A designed research and analysed data; AP-K conceived the study, designed research, performed some research, analysed data and wrote the paper. All authors had final approval of the version.

    • Funding This work was supported by grants from Instituto de Salud Carlos III/FEDER (PI14/00075 and PI17/00037) to AP-K, PI14/00422 to IG-A, RIER RD16 to JLP, IG-A and AP-K, Ministerio de Economía y Competitividad SAF2014-54423-R to ALC. FEDER, Fondo Europeo de Desarrollo Regional: una manera de hacer Europa. SF-R and AP-K are supported by FIBHGM.

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval This study was approved by Research Ethics Committee of Hospital Universitario La Princesa (PI-518).

    • Provenance and peer review Not commissioned; externally peer reviewed.