Basic research on ex vivo modification of dendritic cell functions for development of treatment for inflammatory bowel diseases

本课题旨在阐明树突状细胞（DC）在正常小鼠胃肠道和人胃肠道中对正常肠道植物群的低反应性的功能，以开发治疗炎症性肠病的新药物。1.在小鼠溃疡性结肠炎（UC）模型中，DC的募集有助于疾病的持续，该作用可被光敏素β受体可溶性嵌合蛋白有效阻断。用脂多糖（LPS）刺激，这是由于缺乏MD-2，MD-2是通过Toll样受体4进行LPS信号传导的必需分子。进一步分析UC患者结肠细胞MD-2和细胞因子的异常表达。3.过继转移的DC定位于肠系膜淋巴结。4.由于逆转录病毒载体在研究过程中暴露出明显的毒副作用，因此我们寻找其他方法来体外修饰DC的功能。我们分析了霍乱毒素（CT）对DC的作用，发现CT能够通过与细胞表面神经节苷脂GM 1结合激活NF-κB而完全激活DC。我们创建了新的双mCT（dmCT），其在ADP-核糖基转移酶活性中心（E112 K）和COOH-末端KDEL中具有两个氨基酸取代（E112K/KDEV或E112K/KDGL）。上述一系列实验结果为通过体外修饰树突状细胞的功能来开发治疗炎症性肠病的新药物提供了重要的基础知识。

The purpose of this project was to clarify the function of dendritic cells (DC) in normal murine gastrointestinal (GI) tract as well as human GI tract in terms of the characteristic hyporesponsiveness to normal commensal flora, to develop a new remedy for inflammatory bowel disease.1.In a mouse model for ulcerative colitis (UC), recruitment of dendritic cells contributed to the perpetuation of disease, which was efficiently blocked by a soluble form chimeric protein of lymphotoxin β receptor.2.Human colonic macrophages and dendritic cells showed low cytokine responses after. stimulation with lipopolysaccharides (LPS), which was due to lack of MD-2, an essential molecule for LPS-signaling via toll like receptor 4. Further analysis of colonic cell obtained from UC patients showed aberrant expression of MD-2 and cytokines.3.We found that adoptively transferred DCs were localized in mesenteric lymph nodes. It indicated the possibility of modulation of mucosal immunity by exogenous DC.4.Because the significant side effect of retroviral vector was revealed during the research period, we searched other methods to modify function of DC ex vivo. We analyzed the effect of cholera toxin (CT) and CT was able to fully activate DC by activation NF-κB which was dependent on the binding to cell surface GM1 ganglioside.5.To develop a methods to deliver molecules into DC utilizing intracelluer transport, we created new double mCTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). KDEL mutant was transported to Golgi but not to ER.Results of these serial experiment listed above provided important and basic knowledge to develop new remedies for the treatment of inflammatory bowel disease by modifying the function of dendritic cells ex vivo.