Activated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs, only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor, tofacitinib (TOFA), on gene expression in RA-FLS, in order to identify untargeted disease mediators. Primary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA, with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR, and supernatants were analyzed for protein concentrations. Six thousand three hundred fifty genes were differentially expressed, the majority being upregulated, in MTX-treated activated RA-FLS and 970 genes, the majority being downregulated, in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor, GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h, could be mediated via either NF-κB or AP-1 transcription factors, and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting, MTX-treatment of activated RA-FLS induced IL1B expression in macrophages. MTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis.

中文翻译：

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甲氨蝶呤通过自分泌白细胞介素 1 信号传导促进类风湿关节炎成纤维细胞样滑膜细胞释放粒细胞-巨噬细胞集落刺激因子


活化的成纤维细胞样滑膜细胞 （FLS） 是类风湿性关节炎 （RA） 滑膜炎和结构性关节损伤的驱动因素。尽管使用了改善疾病的药物，但在现实世界中，只有大约 50% 的 RA 患者达到缓解。我们使用无偏倚的方法研究了标准护理甲氨蝶呤 （MTX） 和 Janus 激酶抑制剂托法替布 （TOFA） 对 RA-FLS 基因表达的影响，以识别非靶向疾病介质。在存在或不存在 MTX 或 TOFA 的情况下，通过白细胞介素-1β （IL-1β） 或血小板衍生生长因子 + IL-1β 刺激原发性 RA-FLS 激活，有或没有额外的抑制剂。滑膜细胞的共培养在直接和间接系统中进行。收集细胞用于 RNA 测序或 qPCR，并分析上清液的蛋白质浓度。在 MTX 处理的活化 RA-FLS 中，有 6350 个基因差异表达，其中大多数上调，在 TOFA 处理的样品中，有 970 个基因下调。通路分析显示，MTX 对“癌症的分子机制”的影响最大，TOFA 对“干扰素信号传导”的影响最大。对疾病相关基因的靶向分析表明，MTX 增加了细胞周期调节基因的表达，但也增加了促炎介质如 IL-1α （IL1A） 和粒细胞-巨噬细胞集落刺激因子 GM-CSF （CSF2） 的表达。MTX 促进的活化 RA-FLS 中 CSF2 的表达在 48 小时达到峰值，可通过 NF-κB 或 AP-1 转录因子介导，并被 IL-1 抑制剂 （IRAK4 抑制剂和阿那白滞素） 消除。在共培养环境中，活化的 RA-FLS 的 MTX 处理诱导了巨噬细胞中的 IL1B 表达。 MTX 治疗诱导激活的 RA-FLS 分泌 IL-1，通过自分泌信号传导增加它们对 GM-CSF 的释放。MTX 的这种意外影响可能导致滑膜炎的持续存在。