中枢神经系统内Th17细胞的浸润在铅神经毒性损伤中的作用及机制研究

Environmental lead pollution induced lead poisoning in children has become a social problems which seriously threaten our children's health. Our previous studies found that lead causes microglial activation，resulting in regionally neuron injury, but the role of peripheral T lymphocytes in lead induced neurotoxicity has not been elucidated. Recently infiltration of T-helper 17 cells in CNS were demonstrated to be essential in the pathogenesis of numerous neurological diseases, such as AD and PD. We found that RORγt, the specific transcriptional factor of Th17 cells, was upregulated at mRNA expression level by lead-exposure; and the cytokines, IL-17 and IL-22, were also upregulated by lead. These results indicated that Th17 cells may contribute to the pathogenesis of neurotoxicity caused by lead, but the underlying mechanisms need to be studied. With advanced molecular biological and immunological technologies, we will investigate the effect of lead exposure on infiltration of Th17 cells in CNS，and to explore the relationship between Th17 and lead induced neurotoxicity in vivo and in vitro. We propose that lead may affect the Th17 differentiation and normal function by regulating RORγt expression, resulting in the efficient transmigration of Th17 across BBB, which ultimately leads to neuroinflammation by proinflammatory factors and neuronal injury by ICAM-1/LFA-1 mediated intercellular communication between Th17 and neurons. This study will be helpful to clarify the immunological mechanisms of lead-induced neuron injury and provide new effective measures to prevent the neurotoxicity induced by lead exposure.

环境铅污染导致儿童铅中毒已成为严重威胁我国儿童健康的重要社会问题。前期发现，小胶质细胞活化引起的炎性反应是铅神经毒性的重要机制之一，却一直忽视了外周免疫细胞在铅神经损伤中的作用。新近研究表明，Th17细胞可以原发和继发的炎性成分浸润中枢神经系统，在多种神经退行性疾病启动及调控中发挥重要作用。我们发现铅暴露可引起海马区Th17关键转录因子RORγt、细胞因子IL-17/IL-22表达增加，提示Th17的浸润可能与铅神经毒性有关，但其具体机制尚待阐明。本项目拟以神经-免疫相互作用为突破口，利用铅暴露动物细胞模型，观察铅对Th17浸润中枢神经系统的影响，阐明RORγt调控的Th17功能改变在铅神经损伤中的核心作用，揭示Th17浸润后通过促进神经炎症和ICAM-1/LFA-1介导的细胞间相互作用引起神经损伤的关键机制，为铅毒性神经免疫学发病机制的阐明，寻找新的防护靶点提供关键的理论依据和科学参考。

环境铅污染严重危害人类健康。前期发现，小胶质细胞活化引起的炎性反应是铅神经毒性的重要机制之一，却一直忽视了外周免疫细胞在铅神经损伤中的作用。深入研究铅暴露诱导神经炎性的发生及具体机制对阐明铅神经毒性具有重要意义。本项目以神经-免疫相互作用为突破口，利用铅暴露模型，观察铅对Th17浸润中枢神经系统的影响，阐明Th17功能改变在铅神经损伤中的核心作用。我们首先建立了体内铅暴露动物模型，发现铅暴露可引起血脑屏障通透性改变，海马神经元损伤及胶质细胞活化；同时发现铅暴露后，脑内Th17主要细胞因子IL17表达升高，脑内Th17细胞亚群的比例显著高于对照组。铅暴露还可引起海马/皮质bdnf表达降低，部分炎性因子、黏附分子、趋化因子等表达水平升高。进一步采用CRISPR/Cas9 技术，构建了IL17a基因剔除小鼠。分别使用WT和IL17a基因剔除小鼠建立铅暴露的动物模型。结果发现，与铅暴露WT小鼠相比，IL17a基因剔除小鼠血脑屏障通透性损伤、海马神经元损伤均有所改善。水迷宫结果发现，与对照组相比，铅暴露可引起WT小鼠空间学习记忆能力损伤；而铅暴露后IL17a基因剔除小鼠目标象限停留时间与对照组之间无显著差异。流式细胞术检测未发现IL17a基因剔除小鼠脑内有明显淋巴细胞浸润。相关细胞因子表达检测发现，与铅暴露后WT小鼠相比，铅暴露后IL17a基因剔除小鼠皮质bdnf表达水平升高，对teck和mip1 mRNA 表达水平无显著影响。免疫荧光染色进一步发现，与WT小鼠相比，IL17a基因剔除小鼠铅暴露后海马ICAM-1/LFA1共定位与表达有所改善，提示ICAM-1/LFA1直接作用可能参与了IL17a介导的铅神经毒性损伤。综上，本项目阐明了Th17/IL-17轴在铅神经毒性损伤中的核心作用，揭示铅暴露后浸润中枢神经系统的Th17细胞不仅可通过释放致炎相关因子促进神经炎症发生，还会通过LFA-1/ICAM-1介导的细胞间相互作用造成神经元的直接损伤，为铅毒性神经免疫学发病机制的阐明，寻找新的防护靶点提供关键的理论依据和科学参考。