The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF. SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant. The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways. These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.

中文翻译：

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IL6/sIL6R 通过调节转录和转录后机制调节滑膜成纤维细胞中的 TNFα 炎症反应

特异性白细胞介素 6 抑制剂的临床疗效证实了 IL6 在类风湿性关节炎 (RA) 中的核心作用。然而，IL6 的局部作用，特别是在滑膜成纤维细胞 (SF) 中作为 IL6/sIL6R 信号的直接细胞靶标，尚未得到很好的表征。该研究的目的是表征 TNFα 和 IL6/sIL6R 信号之间的串扰对 SF 的效应促炎症反应。SF 系用 TNFα、IL6/sIL6R 或两者一起刺激，时间和剂量为每个实验指定，并且在指定时，用抑制剂放线菌素 D、阿达木单抗、鲁索替尼和放线菌酮处理细胞。通过定量RT-PCR分析细胞因子、趋化因子和基质金属蛋白酶(MMP)的mRNA表达。通过ELISA测量培养物上清液中IL8/CXCL8和CCL8的水平。使用来自 SF 培养物的条件培养基通过 transwell 评估单核和多形核细胞迁移测定。如相应的图例所示进行统计分析，并且 p 值 < 0.05 被认为具有统计学意义。用 IL6/sIL6R 和 TNFα 刺激 SF，协同促进单细胞和淋巴细胞趋化因子（如 IL6、CCL8 和 CCL2）以及基质降解酶（如 MMP1）的表达，同时抑制中枢中性粒细胞趋化因子（如 IL8）的诱导/CXCL8。趋化因子表达模式的这些变化导致 SF 对多形核 (PMN) 的减少和单核细胞 (MNC) 的化学吸引力增加。基因时间表达的机制分析表明，这两种因子介导的协同调节主要是通过 IL6/sIL6R 激活的从头转录机制诱导的。此外，我们还证明了 TNFα 和 IL6/sIL6R 的合作部分是由通过 JAK/STAT 通路传递信号的次级因子的表达所介导的。这些结果表明 TNFα 诱导的 SF 中对 IL6 的高度协调反应，并提供了对 IL6/sIL6R 在 RA 背景下的作用的额外见解，突出了 IL6/sIL6R 对 SF 与其他炎症细胞相互作用的贡献。我们还证明了 TNFα 和 IL6/sIL6R 的合作部分是由通过 JAK/STAT 通路的二级因子信号传导的表达介导的。这些结果表明 TNFα 诱导的 SF 中对 IL6 的高度协调反应，并提供了对 IL6/sIL6R 在 RA 背景下的作用的额外见解，突出了 IL6/sIL6R 对 SF 与其他炎症细胞相互作用的贡献。我们还证明了 TNFα 和 IL6/sIL6R 的合作部分是由通过 JAK/STAT 通路的二级因子信号传导的表达介导的。这些结果表明 TNFα 诱导的 SF 中对 IL6 的高度协调反应，并提供了对 IL6/sIL6R 在 RA 背景下的作用的额外见解，突出了 IL6/sIL6R 对 SF 与其他炎症细胞相互作用的贡献。