%0 Journal Article
%A Municio, Cristina 
%A Dominguez-Soto, Ángeles 
%A Fuentelsaz-Romero, Sara 
%A Lamana Domínguez, Amalia
%A Montes, Nuria 
%A Cuevas, Víctor 
%A García Campos. Raquel 
%A Pablos Álvarez, José Luis
%A González-Álvaro, Isidoro 
%A Puig-Kröger, Amaya 
%T Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism
%D 2018
%@ 0003-4967
%U https://hdl.handle.net/20.500.14352/94316
%X 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.
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