Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.

中文翻译：

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miR-221-3p通过抑制JAK3 / STAT3激活，将M2-巨噬细胞转变为促炎功能。

目的：巨噬细胞通常分为促炎（M1）和消炎（M2）功能类型。有证据表明，在类风湿关节炎（RA）滑膜中，巨噬细胞具有炎性表型（M1）。MicroRNA（miRs）在调节先天免疫细胞的炎症反应中起关键作用，在RA患者中发现其水平失调。在这里，我们探讨了调节M2巨噬细胞炎症功能的miR。方法：通过qPCR分析RA，其他炎性关节炎（OIA），骨关节炎（OA）和健康供体（HD）的临床样本中miR-221-3p和miR-155-5p的表达谱。用miR-模拟物和抑制剂转染体外产生的巨噬细胞。在有或没有LPS处理的情况下，对过表达miR-221-3p模拟物的M2巨噬细胞进行了RNA测序的转录组分析。使用ELISA在TLR2 / TLR3 / TLR4-刺激下在M1-和M2-巨噬细胞中测量IL-6，IL-10，IL-12，IL-8和CXCL13的分泌。通过免疫印迹评估包括NF-κB，IRF3，MAPK和JAK3 / STAT3在内的炎症途径。通过萤光素酶报告基因检测miR-221-3p与JAK3 3'UTR的预期靶位点的直接靶相互作用。结果：RA与OA或OIA相比，滑膜组织和体液中的miR-221-3p升高。在M1中，miR-221-3p和miR-155-5p的内源性表达水平高于源自RA患者或HD的M2巨噬细胞。M1和M2巨噬细胞的TLR4刺激导致miR-221-3p的下调，但miR-155-5p的上调。转染了miR-221-3p模拟物的M2巨噬细胞分泌的IL-10和CXCL13较少，但IL-6和IL-8较多，表现出JAK3蛋白下调和pSTAT3活化降低。JAK3被鉴定为巨噬细胞中miR-221-3p的新直接靶标。M2巨噬细胞中miR-221-3p / miR-155-5p模拟物的共转染可增加M1特异性IL-12的分泌。结论：miR-221-3p通过直接靶向JAK3充当TLR4诱导的炎症性M2巨噬细胞功能的调节剂。如在RA患者滑膜中所见，miR-221-3p表达失调导致抗炎反应减弱，并驱动M2巨噬细胞表现出M1细胞因子谱。JAK3被鉴定为巨噬细胞中miR-221-3p的新直接靶标。M2巨噬细胞中miR-221-3p / miR-155-5p模拟物的共转染可增加M1特异性IL-12的分泌。结论：miR-221-3p通过直接靶向JAK3充当TLR4诱导的炎症性M2巨噬细胞功能的调节剂。如在RA患者滑膜中所见，miR-221-3p表达失调导致抗炎反应减弱，并驱动M2巨噬细胞表现出M1细胞因子谱。JAK3被鉴定为巨噬细胞中miR-221-3p的新直接靶标。M2巨噬细胞中miR-221-3p / miR-155-5p模拟物的共转染可增加M1特异性IL-12的分泌。结论：miR-221-3p通过直接靶向JAK3充当TLR4诱导的炎症性M2巨噬细胞功能的调节剂。如在RA患者滑膜中所见，miR-221-3p表达失调导致抗炎反应减弱，并驱动M2巨噬细胞表现出M1细胞因子谱。