基于CD4+CD25+Treg受体Axl及其诱生下调脓毒症二重感染的实验研究

Sepsis is a life-threatening systemic inflammatory response resulted from microbial infection. It is well known that sepsis usually cause only a transient pro-inflammatroy period followed by a more prolonged period of immune suppression. Sepsis induced immunosuppression is a major factor in the host’s susceptibility to secondary infections. In sepsis, CD4+CD25+Treg cell activity increased and was associated with decreased Th1 pro-inflammatory response, contributing to sepsis-induced immunoparalysis, but the mechanism remains largely elusive. Axl is a receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. Axl was not expression in normal T lymphocytes, but our previous study observed the expression of Axl in CD4+CD25+Tregs. Moreover, Axl receptor activation can enhance the suppressive activity of CD4+CD25+Tregs by upregulating the expression of CTLA-4 and Foxp3, and may play a great role in sepsis-induced immunoparalysis and secondary infections. Therefore, the mechanism of Axl expression in Treg differentiation, and the effect of inhibiting Axl and its generation on sepsis-induced immunoparalysis as well as secondary infections will be investigate in this project. This project would help to further study the mechanism of immunoparalysis in sepsis, and provide a new strategy for controlling the suppression acitivity of Treg and secondary infection after sepsis.

脓毒症致病过程中，机体的免疫功能受抑，导致二重感染的发生率显著增加，已经成为脓毒症治疗的新难题。研究证实，脓毒症中CD4+CD25+调节性T细胞（Treg）免疫抑制活性显著增强，是脓毒症免疫麻痹形成的关键环节，但其机理尚不清楚，且缺乏有效的干预手段。项目组前期发现，酪氨酸激酶受体Axl表达于CD4+CD25+Treg，但在初始T淋巴细胞中不表达。此外，激活Axl能够增强Treg的免疫抑制活性，提示初始T淋巴细胞向Treg分化诱导了Axl受体产生，且在Treg免疫抑制活性强化中发挥重要作用。本项目拟从整体动物水平、细胞水平和分子水平三个层次探讨CD4+CD25+Treg分化过程中受体Axl的诱生机制，分析阻断Axl受体及其诱生对脓毒症CD4+CD25+Treg免疫抑制活性和二重感染的影响。本项目将从全新角度认识免疫麻痹形成的机理，并为脓毒症二重感染防治提供新的途径和线索。

研究证实，脓毒症患者的过度炎症反应往往很短暂，继而伴随着持续的、难以纠正的免疫功能紊乱和免疫缺陷状态。脓毒症的“免疫麻痹”状态使得机体难以有效清除病原菌，患者难以从初始感染中恢复，同时可能导致二次感染的发生。CD4+CD25+调节性T细胞（Tregs）是一类具有免疫调节功能的T细胞亚群，其免疫抑制功能强化已经被证实是脓毒症后免疫麻痹的重要环节，然而其机理尚不清楚，也缺乏有效的干预措施。TAM受体是新近发现的一个受体酪氨酸激酶（RTKs）亚家族，包括三个成员：Tyro3、Axl和Mer，其作为机体炎症“刹车蛋白”已经得以证实。本项目首先分析了TAM受体在Tregs中的表达规律。在此基础上，观察了脓毒症中CD4+CD25+Treg 受体Axl 的表达情况，分析Axl与Treg免疫抑制功能的关系；通过体外实验研究Treg 受体Axl 诱生与Sp1/Sp3的关系；进一步阐述阻断Axl信号通路对脓毒症中Treg细胞免疫抑制功能和二次感染的影响；此外，探讨了丙酮酸乙酯作为多靶点药物在脓毒症Treg免疫功能紊乱和二次感染中的应用价值。项目研究发现：1）CD4+CD25+Treg高表达Axl，低表达Mer，不表达Tyro3；TAM配体Gas6能够有效增强Tregs的免疫抑制功能，阻断Axl能够抑制该效应。2）脓毒症中CD4+CD25+Treg细胞Axl受体的表达显著上调，其下游信号分子Stat5也显著活化，并与Tregs细胞免疫功能强化密切相关。3）在CD4+CD25-T细胞向Tregs细胞分化过程中，Axl表达增加，Sp1/Sp3水平显著升高；抑制Sp1/Sp3能够有效减低CD4+CD25-T细胞诱导分化后Axl和Foxp3的水平。4）采用anti-CD25抗体干预以及阻断Axl信号通路均能够减低Treg免疫功能强化所致的脓毒症“免疫麻痹”，下调脓毒症动物对二重感染的易感性。5）免疫调节药物丙酮酸乙酯能够改善脓毒症免疫功能紊乱和二重感染的发生，其效应与其对Tregs的免疫调节作用有关。项目从全新角度认识免疫麻痹形成的机理，并为脓毒症二重感染防治提供新的途径和线索。