Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
Centro de Investigaciones Biologicas, Madrid, Spain.
Ann Rheum Dis. 2018 May;77(5):752-759. doi: 10.1136/annrheumdis-2017-212537. Epub 2018 Feb 3.
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.
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.
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 (), 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, 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.
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.
甲氨蝶呤（MTX）是治疗类风湿关节炎（RA）的基础药物，但它的抗炎作用机制尚未完全阐明。在 RA 中，巨噬细胞表现出促炎极化表型，类似于粒细胞-巨噬细胞集落刺激因子（GM-CSF）分化的巨噬细胞，并且只有在胸苷酸合酶 GM-CSF 依赖性巨噬细胞中才观察到 MTX 的反应。为了确定 MTX 抗炎作用的分子基础，我们研究了 Toll 样受体（TLR）、RA 滑液（RASF）和肿瘤坏死因子受体（TNFR）在 MTX 暴露的 GM-CSF 预刺激巨噬细胞中的信号转导。
通过定量实时 PCR、western blot、ELISA 和 siRNA 介导的敲低方法，确定 TLR 配体、TNFα 或 RASF 刺激在长期低剂量 MTX 暴露的人巨噬细胞中的细胞内反应。在接受 MTX 单药治疗的关节炎患者和小鼠脓毒症模型中评估 MTX 的体内作用。
MTX 使巨噬细胞向耐受状态转变，减少 LPS、LTA、TNFα 或 RASF 刺激的巨噬细胞中白细胞介素（IL）-6 和 IL-1β 的产生。MTX 减弱了 LPS 诱导的 MAPK 和 NF-κB 激活，以及 Toll/IL-1R 域包含衔接蛋白诱导 IFN-β（TRIF1）依赖性信号转导。相反，MTX 增加了 NF-κB 抑制剂 A20 的表达（），A20 本身是 RA 易感性基因。从接受 MTX 单药治疗的关节炎患者队列中，机制上，MTX 诱导的巨噬细胞耐受依赖于 A20，因为 A20 的 siRNA 介导敲低逆转了 MTX 诱导的 IL-6 表达减少。在体内，在接受 MTX 治疗的关节炎患者的外周血细胞中，表达明显高于接受 MTX 治疗的关节炎患者，而 MTX 处理的小鼠对 LPS 的炎症反应降低。
MTX 通过上调 A20 的表达来抑制巨噬细胞的促炎反应。A20 介导的 MTX 诱导的先天耐受可能限制 RA 滑膜中的炎症，并且将 A20 定位为潜在的 MTX 反应生物标志物。
