Effect of miR-494-3p on LPS-treated intestinal epithelial cells isolated from C57BL/6 mice

The microenvironment of injured mucosa has important effects on intestinal stem cell self-renewal and reconstruction of epithelial barrier function in inflammatory bowel disease (IBD). However, the precise status of the interactions between intestinal epithelial cell (IEC) injury, particularly intestinal crypt absence, and microenvironment in IBD is not completely understood. We identified miR-494-3p as important for protection of colonic stemness in intestinal inflammation colonic organoid culture. A novel cytokine-cytokine receptor, EDA-A2/EDA2R, could suppress colonic stemness and epithelial repair during IBD. During intestinal inflammation, high level of LP macrophage-derived EDA-A2 inhibited the nuclear β-catenin/c-Myc axis and organoid growth by targeting EDA2R in colonic crypt stem cells. We further demonstrated that the pro-inflammatory cytokines IL-1β and IL-6 are capable of stimulating macrophages to release EDA-A2 during colitis. Secondly, we identified the cross-talk among IECs, colonic crypts, and lamina propria (LP) macrophages in miR-494-3p-mediated colitis. Furthermore, our study showed that miR-494-3p deficiency in IECs promoted LP macrophage recruitment and M1 activation in DSS-induced colitis mice. In addition, we identified miR-494-3p as critical to dampening IEC injury; specifically, miR-494-3p inhibited inflammation-induced IKKβ/NF-κB activation by targeting the IKKβ 3’UTR in IECs. As such, administration of adequate amounts of a miR-494-3p agomire attenuate colitis in vivo. Consistent with this inference, we showed that miR-494-3p levels were decreased in colonic crypts and serum in colitis mice, and loss of miR-494 potentiated the severity of colonic colitis. Our clinical data on the interactions between miR-494-3p levels in serum exosomes & colonic tissues and associated outcomes support the clinical relevance of miR-494-3p in IBD. The miR-494-3p agomir system, which we designed permits local delivery in vivo in this study, significantly ameliorated the severity of colonic colitis. Our findings no only uncover a miR-494-3p-mediated cross-talk mechanism by which inflamed colonic LP macrophages integrate signals from IECs to regulate colonic stemness and colonic epithelial repair/homeostasis. The miR-494-3p agomir may serve as a potential therapeutic approach in IBD.

损伤黏膜微环境对炎症性肠病（inflammatory bowel disease, IBD）中肠干细胞（intestinal stem cell）的自我更新及上皮屏障功能重建具有重要调控作用。然而，目前对于炎症性肠病中肠上皮细胞（intestinal epithelial cell, IEC）损伤（尤其是肠隐窝（intestinal crypt）缺失）与微环境之间的相互作用机制仍未完全阐明。我们在结肠类器官培养（colonic organoid culture）中鉴定出miR-494-3p对肠道炎症环境下结肠干细胞干性（colonic stemness）具有保护作用。一种新型细胞因子-细胞因子受体EDA-A2/EDA2R可在炎症性肠病进程中抑制结肠干细胞干性与上皮修复。在肠道炎症状态下，固有层（lamina propria, LP）巨噬细胞来源的高水平EDA-A2可通过靶向结肠隐窝干细胞表面的EDA2R，抑制核β-连环蛋白/c-Myc通路（nuclear β-catenin/c-Myc axis）及类器官生长。我们进一步证实，在结肠炎发生过程中，促炎细胞因子IL-1β与IL-6可诱导巨噬细胞释放EDA-A2。其次，我们阐明了miR-494-3p介导的肠上皮细胞、结肠隐窝与固有层巨噬细胞之间的交叉对话机制。此外，我们的研究显示，在葡聚糖硫酸钠诱导的结肠炎小鼠模型中，肠上皮细胞内miR-494-3p的缺失可促进固有层巨噬细胞招募及M1型巨噬细胞激活。我们还鉴定出miR-494-3p可显著减轻肠上皮细胞损伤：具体而言，miR-494-3p可通过靶向肠上皮细胞内的IKKβ 3’非翻译区（3’UTR），抑制炎症诱导的IκB激酶β/核因子κB（IKKβ/NF-κB）通路激活。因此，体内给予足量的miR-494-3p激动剂（agomir）可减轻结肠炎症状。与该推论一致的是，我们发现结肠炎小鼠的结肠隐窝与血清中miR-494-3p水平均显著降低，而miR-494的缺失会加重结肠炎的严重程度。我们针对血清外泌体（serum exosomes）与结肠组织中miR-494-3p水平及其与临床结局的相关性开展的临床数据研究，证实了miR-494-3p在炎症性肠病中的临床相关性。本研究中我们设计的miR-494-3p激动剂递送系统可实现体内局部递送，显著缓解结肠炎的严重程度。我们的研究结果不仅揭示了一种由miR-494-3p介导的交叉对话机制：即炎症状态下的结肠固有层巨噬细胞如何整合肠上皮细胞的信号，以调控结肠干细胞干性与结肠上皮修复及稳态（homeostasis）。miR-494-3p激动剂有望成为炎症性肠病的潜在治疗手段。