Laboratorio de Inmuno-Metabolismo, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
Ann Rheum Dis. 2016 Dec;75(12):2157-2165. doi: 10.1136/annrheumdis-2015-208736. Epub 2016 Feb 26.
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.
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.
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 TS 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.
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.
甲氨蝶呤（MTX）在癌症中作为抗增殖剂，在类风湿关节炎（RA）中作为抗炎药物。尽管巨噬细胞对 RA 病理有重要贡献，但它们对 MTX 的反应尚不清楚。为了确定 MTX 反应标志物，我们探索了其对 GM-CSF（GM-MØ）或 M-CSF（M-MØ）极化的巨噬细胞的转录效应，GM-MØ 和 M-MØ 分别类似于 RA 和正常关节中发现的促炎性和抗炎性巨噬细胞。
使用基因表达微阵列确定长期和短期方案下两种人巨噬细胞亚型暴露于 50 nM MTX 时的转录组谱，并通过定量实时 PCR 和 ELISA 进行验证。通过药理学、siRNA 介导的敲低方法、多谷氨酸化-MTX 检测的代谢组学、western blot 和 RA 和正常关节的免疫荧光确定参与巨噬细胞 MTX 反应性的分子途径。
MTX 仅在促炎性 GM-MØ 中调节基因表达，其影响 757 个基因的表达，并在 mRNA 和蛋白水平诱导 CCL20 和 LIF。药理学和 siRNA 介导的方法表明，巨噬细胞亚群特异性 MTX 反应性与胸苷酸合成酶（TS）表达相关，因为促炎性 TS GM-MØ 对 MTX 敏感，而抗炎性 TS M-MØ 和单核细胞对 MTX 耐药。此外，发现 p53 活性介导促炎性 TS GM-MØ 中依赖 TS 的 MTX 反应性。重要的是，在 RA 关节而非正常滑膜中发现 CD163/TNFα GM-CSF 极化的巨噬细胞中表达 TS 和 p53。
巨噬细胞对 MTX 的反应是极化依赖性的，并由 TS-p53 轴决定。CCL20 和 LIF 构成体外 MTX 反应性的新型巨噬细胞标志物。
