DCs表面PIR-A/PIR-B受体平衡失调在Th17/Treg分化和创伤免疫紊乱中的作用

Trauma contributes to the imbalance of immune function, and its formation mechanism has not been fully elucidated. Complementary PIR-A/PIR-B receptor is a newly discovered DCs surface receptor involved in regulating immune response. Our previous experiment showed that PIR-A/PIR-B receptor expression on the DCs surface was abnormal after trauma, recently found that PIR-A/PIR-B receptor was involved in the balance of the DCs mediated Th17/Treg, and the balance is the key to maintaining immune steady state, this balance disorders can cause immune imbalance, so we speculated that PIR-A/PIR-B receptor can participate in the immune dysfunction after trauma by regulating the balance of Th17/Treg. Aims of this study were to investigate: (1) the expression of PIR-A/PIR-B activity on the DCs surface and Th17/Treg cells in the peripheral blood of traumatic patients, and (2) in vitro interferent the balance of PIR-A/PIR-B receptor activity can regulate the imbalance of Th17/Treg cells and improve the immune function, and (3) to explore the molecular mechanism of Th17/Treg imbalance, and (4) in the animal experimental to study “overexpression or silence of PIR-A/PIR-B” DCs can reverse the imbalance of Th17/Treg cells and improve immune function after trauma, so as to provide a new strategy for treating the immune dysfunction after trauma.

创伤可致机体免疫失衡，其发病机制未完全阐明。PIR-A/PIR-B作为新发现DCs表面受体参与调节免疫反应。我们前期研究发现，创伤后DCs表面刺激性PIR-A和抑制性PIR-B受体表达异常，最近又发现PIR-A/PIR-B参与DCs介导Th17/Treg平衡，而该平衡是维持免疫稳态的关键，此平衡失调可致免疫失衡，因此我们推测PIR-A/PIR-B受体通过调控DCs介导Th17/Treg平衡参与创伤免疫紊乱发生。为此本课题拟进行以下研究：（1）明确创伤后存在PIR-A/PIR-B表达异常和Th17/Treg失衡；（2）体外观察靶向干扰PIR-A/PIR-B表达对Th17/Treg平衡和免疫功能影响；（3）探讨PIR-A/PIR-B调控Th17/Treg平衡的分子机制；（4）在体观察过表达/沉默PIR-A/PIR-B能否纠正创伤后Th17/Treg失衡和免疫紊乱，为创伤免疫失衡防治提供新策略。

创伤可致机体免疫失衡，其发病机制未完全阐明。本实验从T细胞亚群平衡角度，观察创伤对小鼠脾脏Th17/Treg细胞、细胞因子和转录因子表达的影响；从DCs表面受体表达入手，观察创伤对小鼠脾脏DCs免疫功能影响；并探讨DCs表面Pir-B分子调控创伤后Th17/Treg失衡的分子机制。制作创伤出血小鼠模型，采用免疫磁珠分选CD4+T细胞和CD11c+DCs；MTT法检测T细胞增殖，流式检测Th17/Treg细胞；ELISA检测IL-17和TGF-β含量；PCR检测子RoRγt和Foxp3 mRNA表达；并分析DCs细胞数量、表面分子MHC-II、CD80、CD86和抑制性分子Pir-B表达，ELISA检测DCs分泌IL-6、IL-10和IL-12含量；通过体外靶向过表达或沉默Pir-B，观察上述指标变化。结果发现：小鼠脾脏T淋巴细胞增殖明显降低，且最低水平出现在创伤后3天；创伤小鼠脾脏Th17细胞比例、IL-17及RoRγt mRNA表达明显降低，而Treg细胞比例、TGF-β和Foxp3 mRNA表达明显增加；创伤小鼠脾脏DCs细胞数量无明显变化；创伤小鼠脾脏DCs表面MHC-II、CD86表达明显下降，IL-6和IL-12含量明显减少；创伤小鼠脾脏DCs表面Pir-B表达明显增加；创伤DCs转染siRNA-Pir-B后1、2、3和5天，siRNA-DC组Pir-BmRNA和蛋白表达明显降低；转染后3天，siRNA-DC组MHC-II表达和IL-6含量明显增加；与初始T细胞共培养后，siRNA-DC组Th17细胞比例和IL-17含量明显增加，Treg细胞比例明显降低，TGF-β含量无明显变化；共培养体系中加入1µg、5µg和10µg anti-IL-6抗体后，与Saline组比较，5µg和10µg anti-IL-6组Th17细胞比例明显降低，IL-17含量、Treg细胞和TGF-β含量无明显变化。结论：创伤出血致小鼠脾脏Th17/Treg细胞失衡，参与创伤后免疫抑制形成；创伤出血抑制DCs功能，促进抑制性Pir-B分子表达增加；靶向沉默Pir-B表达，可恢复DCs介导的Th17/Treg失衡和改善免疫功能，且该调控作用是通过IL-6通路来实现的。通过本项目研究进一步阐明创伤免疫抑制发生机制，并为临床创伤后免疫功能紊乱防治提供理论依据。