Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism. Issue 5 (3rd February 2018)

• Record Type: Journal Article
• Authors: Municio, Cristina; Dominguez-Soto, Ángeles; Fuentelsaz-Romero, Sara; Lamana, Amalia; Montes, Nuria; Cuevas, Víctor D; Campos, Raquel García; Pablos, José L; González-Álvaro, Isidoro; Puig-Kröger, Amaya
• Is Part Of: Annals of the rheumatic diseases. Volume 77:Issue 5(2018)
• Page Start: 752
• Page End: 759
• Publication Date: 2018-02-03
• Subjects: methotrexate -- rheumatoid arthritis -- dmards (synthetic); Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2017-212537
• Languages: English
• ISSNs: 0003-4967

Abstract

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.