Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
SciLifeLab, University of Gothenburg, Gothenburg, Sweden.
Arthritis Res Ther. 2024 Oct 11;26(1):178. doi: 10.1186/s13075-024-03406-6.
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.
活化的成纤维样滑膜细胞（FLS）是类风湿关节炎（RA）滑膜炎和结构关节损伤的驱动因素。尽管使用了改善病情的药物，但在实际环境中，只有约 50%的 RA 患者达到缓解。我们采用了一种无偏倚的方法来研究标准治疗药物甲氨蝶呤（MTX）和 Janus 激酶抑制剂托法替尼（TOFA）对 RA-FLS 基因表达的影响，以确定未靶向的疾病介质。
通过白细胞介素-1β（IL-1β）或血小板衍生生长因子+IL-1β刺激，在存在或不存在 MTX 或 TOFA 的情况下，以及是否存在其他抑制剂，将原代 RA-FLS 激活。在直接和间接系统中进行滑膜细胞共培养。收集细胞进行 RNA 测序或 qPCR，并分析上清液中的蛋白浓度。
在 MTX 处理的活化 RA-FLS 中，有 6350 个基因差异表达，大多数上调，在 TOFA 处理的样本中，有 970 个基因差异表达，大多数下调。通路分析表明，MTX 对“癌症的分子机制”影响最大，TOFA 对“干扰素信号”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，如白细胞介素-1α（IL1A）和粒细胞-巨噬细胞集落刺激因子（CSF2）。MTX 在活化的 RA-FLS 中促进 CSF2 的表达在 48 小时达到峰值，可以通过 NF-κB 或 AP-1 转录因子介导，并且可以被 IL-1 抑制剂（IRAK4 抑制剂和 anakinra）阻断。在共培养环境中，MTX 处理活化的 RA-FLS 诱导巨噬细胞中 IL1B 的表达。
MTX 治疗诱导活化的 RA-FLS 分泌 IL-1，通过自分泌信号增强其 GM-CSF 的释放。MTX 的这种意外作用可能导致滑膜炎的持续存在。
