Vimentin及其磷酸化修饰促进克罗恩病发生发展的机制研究

Innate immunity is closely involved in the development of Crohn’s disease. Using a proteomic and mass spectrometry approach, the expression level and phosphorylation of Vimentin was significantly increased in pathologic tissue from patients with Crohn’s disease, as compared with control tissues. In our experiments, transduction of Vimentin increased inflammatory reaction and expression of pattern recognition receptor (Dectin-1, TLR2 and NOD2) in macrophages. Introduction of adenovirus-mediated Vimentin and phosphorylation mutant Vimentin aggravated dextran sulfate sodium -induced colitis and pathologic features versus Vector in mice, with enhanced inflammation, inflammasomes and augmented ROS in peritoneal macrophages and impaired epithelial protection. The role of phosphorylation mutant Vimentin was more prominent in these pathological manifestations. However, such pathologies were significantly attenuated upon administration of ROS inhibitor APO and inflammasome inhibitor arglabin. Previous studies and our results suggest that Vimentin and phosphorylated Vimentin could promote the development of inflammatory bowel disease through mediation of ROS and inflammasome. In our next step, we will assess whether Vimentin and phosphorylated Vimentin promotes inflammatory bowel disease development, using Vimentin deficient mouse, Vimentin and phosphorylation Vimentin mutant transgenic mouse, with mechanisms clarification. Personalized mechanisms will be probed with in vitro and in vivo experiments using samples from bank of inflammatory bowel disease, Rui Jin Hospital.

固有免疫在克罗恩病（CD）发病中起重要作用。我们用TLR2和Dectin-1等免疫沉淀-质谱和二维电泳-质谱发现CD组织中Vimentin水平及磷酸化修饰比正常对照增高。实验中，Vimentin诱导巨噬细胞炎症，上调其交互作用固有免疫受体表达。小鼠体内过表达Vimentin和磷酸化突变体Vimentin(E)腺病毒可促进葡聚糖硫酸钠诱导的肠炎病变；小鼠巨噬细胞炎性反应、炎性小体和ROS均增强。这些表型中Vimentin(E)作用更明显。而抑制ROS和炎性小体能降低上述病理表现。结合文献提示Vimentin通过固有免疫受体-氧化应激和炎性小体机制介导，促进CD发病，磷酸化 Vimentin可能效应更显著。后续将以体内外实验，用Vimentin敲除/敲入/磷酸化突变体敲入等小鼠和通路抑制方案，明确Vimentin及其磷酸化对CD发生的影响，阐明机制，揭示与CD发病和严重度有关的个体机制。

本项目在前期工作基础上，通过蛋白质质谱分析发现Vimentin作为模式识别受体TLR-2和Dectin-1的潜在配体，在肠道炎症区域高表达，并在CD患者病变黏膜得到验证。细胞水平研究发现，敲低Vimentin引起小鼠巨噬细胞RAW264.7细胞分泌活性caspase-1和IL-1β水平明显下降，而Vimentin重组蛋白能诱导人外周血单核细胞系THP-1细胞表达固有免疫受体（Dectin-1、NOD2及TLR2）以及炎症因子（TNF-、IL-1、IL-6、IFN-γ、MCP-1）。进一步研究发现CD发病与Vimentin的磷酸化修饰关系密切。动物模型证实Vimentin及其磷酸化修饰能促进DSS诱导的肠炎病变。临床与基础相结合，发现CD活动期和缓解期及正常人群唾液菌群差异，为今后建立以唾液菌为基础的无创CD活动性标志物奠定基础，实现从免疫学前沿理论到临床转化型研究的衔接。