隐球菌病患者单核细胞中LINC00997-HuR-CCR2轴调控免疫应答的机制及免疫干预研究

Cryptococcosis is a serious disease with high mortality and limited therapy effects. The patients are often accompanied by Impaired immune function. Monocytes/macrophages play important roles in activation of immune effector cells and clearance of cryptococcus neoformans. Our previous studies have showed that the immune response functions of monocytes from patients with Cryptococcosis descended significantly; LINC00997 and chemokine receptor CCR2 were down-expressed and related to disease course; LINC00997 could bind with HuR and promote inflammatory cytokines; LINC00997 over-expression in mice infected models could enhance the defensive immune responses. This project aims to explore these Scientific problems: 1.the regulation functions of lncRNA LINC00997 in monocytes during host anti-cryptococcus-infection process; 2.the mechanism of LINC00997 regulating on HuR-CCR2 pathway; 3.the mechanism of DNA methylation and transcription factor regulating on LINC00997 expression; 4.using mice models to explore the regulation effect and mechanism of LINC00997 on immune response against cryptococcus in vivo. These investigations would provide scientific basises of new pathway to further immunotherapy.

新生隐球菌病死亡率高，治疗较为棘手，患者常伴有免疫功能受损。单核/巨噬细胞在激活免疫效应细胞、清除新生隐球菌中起重要作用。前期研究发现，隐球菌病患者单核细胞抗隐球菌免疫应答显著下降； LINC00997与趋化因子受体CCR2表达显著下调，与病情相关； LINC00997可与HuR蛋白结合，促进炎性因子分泌；慢病毒LINC00997过表达载体可促进隐球菌病小鼠模型抗新生隐球菌感染的免疫应答。本项目拟进一步解决如下关键问题：1、新生隐球菌病患者单核细胞中LINC00997发挥的调控功能；2、LINC00997调控HuR-CCR2通路的分子机制；3、DNA甲基化和重要转录因子对LINC00997在单核细胞表达的调控机制；4、利用小鼠模型，研究LINC00997调控新生隐球菌感染体内免疫应答的作用及机制。本项目将为探索免疫治疗新途径提供科学依据。

结题按计划顺利完成。已发表专业论文17篇，其中SCI论文11篇，研究主要结果如下：.（1）新生隐球菌感染宿主单核细胞过程中，lncRNA LINC00997所发挥的功能特征。.LINC00997 siRNA腺病毒载体滴度为1.5×109TU/ml；过表达LINC00997之后，Elavl1/HuR蛋白表达明显受抑制，IL-1β、CCL7和CCL2表达显著升高。在灭活隐球菌刺激下，LINC00997可促进幼稚CD4+T增殖及IFN-γ分泌，抑制IL-4分泌。..（2）LINC00997如何调控HuR-CCR2 信号途径。.过表达LINC00997可促进隐球菌感染患者单核细胞中CCL7和CCL2的mRNA水平，并下调转录相关蛋白Elav11/HuR蛋白水平。构建HuR-/-单核细胞，过表达LINC00997组中CCL2、CCL7、IFN-γ表达显著上调，IL-4表达下降。..（3）DNA甲基化和重要转录因子对LINC00997在单核细胞表达的调控机制。.隐球菌病患者LINC00997启动子区甲基化频率与正常对照组相比，差异无统计学意义。miR-17-5p可正向调控隐球菌感染后单核细胞中LINC00997、CCL2、CCL7、IFN-γ表达，负向调控HuR和IL-4的表达，对IL-10无明显调控作用。..（4）利用小鼠模型，研究LINC00997调控新生隐球菌感染体内免疫应答的作用及其机制。.LINC00997过表达载体可显著减轻隐球菌感染7天、14天、21天后小鼠肺部和脑部的菌落负荷。隐球菌感染21天后，LINC00997过表达组的外周血中IFN-γ水平明显升高，IL-4和IL-10水平明显下降。