Background

MiR-146a has been shown to negatively regulate innate immune, inflammatory response and antiviral pathway, however, its role in the tolerogenic responses remains largely unknown. This study aimed to investigate the role of miR-146a in the OVA-induced allergic inflammation of dendritic cells (DCs).

Methods

Bone marrow-derived DCs (BMDCs) were treated with OVA (100 µg/ml) for 24 h. MiR-146a expressions were assessed by quantitative RT-PCR. BMDCs were transfected with miR-146a mimics or inhibitor. Cell surface markers were analyzed by flow cytometry. Cytokine levels were determined by ELISA assay. Mixed lymphocyte culture assay was adopted to assess CD4 + T-cell differentiation. The 3ʹ UTR luciferase reporter assay was utilized to determine the miRNA target sequence.

Results

OVA treatment significantly up-regulated miR-146a in BMDCs in a dose- and time-dependent manner. In the OVA-treated DCs, overexpression of miR-146a (mimics transfection) down-regulated the surface markers (CD80, CD86) and increased production of anti-inflammatory cytokines TGF-β1 and IL-10 but decreased pro-inflammatory cytokine IL-12. MiR-146a overexpression promoted immature DC to induce regulatory T cells (Treg) differentiation. By contrast, transfection of miR-146a inhibitor into DC exhibited the opposite trends. Notch1 was a direct target of miR-146a, and Notch1 knock-down induced similar effects as miR-146a mimics transfection in BMDCs. Moreover, the effect of miR-146a inhibitor on OVA-induced DC was attenuated by Notch1 knock-down.

Conclusion

miRNA-146a promoted tolerogenic properties of DCs, at least partially, through targeting Notch1 signaling.

中文翻译：

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MiR-146a通过靶向Notch1信号来促进树突状细胞的耐受性。

背景

已显示MiR-146a负调节先天免疫，炎症反应和抗病毒途径，但是，其在致耐受反应中的作用仍然未知。这项研究旨在调查miR-146a在OVA诱导的树突状细胞（DC）过敏性炎症中的作用。

方法

骨髓衍生DC（BMDC）用OVA（100 µg / ml）处理24小时。通过定量RT-PCR评估MiR-146a表达。用miR-146a模拟物或抑制剂转染BMDC。通过流式细胞仪分析细胞表面标志物。通过ELISA测定来确定细胞因子水平。采用混合淋巴细胞培养法评估CD4 + T细胞分化。利用3′UTR荧光素酶报告基因测定来确定miRNA靶序列。

结果

OVA治疗以剂量和时间依赖性方式显着上调BMDC中的miR-146a。在OVA处理的DC中，miR-146a的过度表达（模拟转染）下调了表面标志物（CD80，CD86）并增加了抗炎性细胞因子TGF-β1和IL-10的产生，但降低了炎性前细胞因子IL- 12 MiR-146a过表达促进未成熟DC诱导调节性T细胞（Treg）分化。相比之下，miR-146a抑制剂转染DC表现出相反的趋势。Notch1是miR-146a的直接靶标，Notch1敲除诱导的作用与miR-146a模仿BMDC中的转染类似。此外，NotR1敲低减弱了miR-146a抑制剂对OVA诱导的DC的作用。

结论

miRNA-146a通过靶向Notch1信号传导至少部分地促进了DC的致耐受性。