芳香烃受体在严重创伤后树突状细胞介导调节性T细胞分化紊乱中的作用机制研究

Trauma threatens human health severely and sepsis is a major pattern of traumatic complication. In the cases where the patients are rescued successfully in the early stage of severe trauma, the prevention and cure of sepsis appears to be the key factor in determining whether they can survive and be rehabilitated finally. Therefore, it is fundamentally significant to explore systematically the characters of immunocyte, intervention factor and regulatory mechanismity during the early stage of severe trauma, and to examine whether and how they help postponing or even preventing the development of traumatic sepsis. Although Dentritic cells (DC) and regulatory T cells (Treg) play important roles with respect to the occurrence and development of sepsis after severe trauma, the strategy of new intervention target in regulating DC-Treg differentiation and its mechanism are yet to be further examined. The current literature on this topic and our preliminary research suggest that, expression of aryl hydrocarbon receptor (AHR) changes significantly in each type of DC, and moreover, it can directly affect the function of Treg and in turn cause the ratio of Treg to change. As such, we submit the following hypotheses. Firstly, AHR may regulate the DC role of antigen presentation and decide the result of Treg differentiation after the occurrence of severe trauma, and it may even influence the development of sepsis and multiple organ dysfunction syndrome (MODS). Secondly, AHR probably acts as a novel intervention target to improve the state of DC and Treg abnormal differentiation, and relieve or prevent partly the harmful prognosis in sepsis. Thirdly, the mechanism is likely to be related with IL-2 as well as JAK/STAT signal pathway. In this investigation, we will use AHR KO , Foxp3 GFPKI, and DBA/2 mice imported to our laboratory to perform a series of in vivo experiments. We will treat the animals with fracture and hemorrhage to mimic severe trauma, then put them under different infection conditions by lipopolysaccharide (LPS) stimulation or cecal ligation and puncture (CLP). Upon the occurrence and during the development of traumatic sepsis, we will explore the influence of AHR on the biological features of DC and Treg (including its abnormal differentiation), such as number, ratio ,distribution and function. In addition, we will also identify the role of AHR regulator, leflunomide, in relieving the prognosis of traumatic sepsis as an intervention target. This investigation aims to provide a theoretical basis as well as a new direction as to how to effectively rebuild homeostasis and maintain immunological balance after the occurrence of severe trauma. The research methodologies to be used in this investigation include without limitation, fluorescence activated cell sorter (FACS) analysis, enzyme linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), co-immunoprecipitation (CO-IP) and electrophoresis mobility shift assay (EMSA).

严重创伤导致的机体免疫功能紊乱将直接影响后续感染介导的脓毒症的发生发展进程，其中免疫抑制是引发创伤性脓毒症患者病情恶化的重要原因。AHR、DC和Treg分别在维持机体免疫平衡和促进免疫抑制中发挥了重要作用，但它们之间的关系和机制还不清楚。申请人根据文献报道和前期工作结果认为，AhR可以通过对DC抗原递呈作用的调控决定Treg细胞分化结局，使机体适应性免疫功能发生改变，进而影响患者的病情发展，该过程可能与IL-2和JAK/STAT信号途径有关。提示AHR可能作为改善DC-Treg分化紊乱状态的干预靶点，以缓解或部分逆转创伤性脓毒症的不良预后。本项目拟以AHRKO小鼠为主要实验动物，系统探讨严重创伤后AhR对DC和Treg的数量、比例、分布和功能上的影响规律，揭示AHR介导DC参与Treg分化调控的分子机制，为严重创伤后及时纠正机体免疫失衡、重建内环境稳态和早期防治脓毒症提供理论基础和新思路。

系统探索严重创伤发生后至脓毒症发生之前时间段机体免疫细胞特点、影响/干预因素和调控机制，将有效延缓甚至阻止创伤性脓毒症的发生和不良预后，是创伤和重症医学长期面临的重要基础课题。本项目主要探讨了严重创伤后芳香烃受体（Aryl hydrocarbon receptor, AHR, AhR）通过树突状细胞（dendritic cell, DC）调控调节性T细胞（regulatory T cell, Treg）的作用机制，同时结合实验实际对相关内容进行了调整和拓展，引进、研发和培育了一批转基因小鼠，优化完善了部分实验技术，揭示了严重创伤后AhR能够影响严重创伤后骨髓来源DC（bone marrow derived DC, BMDC）的生物学特征，其机制可能与Musculin（MSC）有关；阐明了胍丁胺（agmatine, AGM）可以调控巨噬细胞抗炎、抗氧化功能发挥治疗效应，其中磷脂酰肌醇-3-激酶/蛋白激酶（phosphoinositide 3-kinase protein kinase B, PI3K/AKt）、核因子NF-E2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）/血红素氧合酶-1（hemo oxygenease-1, HO-1）途径以及HO-1催化终产物皆参与了该作用；确证了MSC可能作为严重创伤后恢复机体内环境免疫稳态[包括辅助性T细胞（helper T cell, Th）17和分泌白细胞介素（interleukin, IL）-22的3型固有淋巴细胞（group 3 innate lymphoid cells, ILC3s, ILC3）]的潜在调节靶点，通过免疫保护改善脓毒症和炎症性肠病（inflammatory bowel disease, IBD）的病理损害。该研究能够促进有效调控机体免疫功能关键因子的深度挖掘，有利于纠正严重创伤后机体免疫失衡状态，延缓或阻止创伤性脓毒症的发生和发展，进一步改善伤员预后，从而为临床救治和免疫调理严重创伤并发症提供理论基础和新策略。截止2018年12月31日，本项目发表论文7篇，其中SCI论文1篇、专家共识1篇、已接受待发表1篇；申请国家发明专利1项；以本项目为基础申请并获得重庆市科委基金2项；培养技术员4名，指导硕士研究生1名。