程序性坏死信号通路调控免疫老化T细胞功能参与衰老相关炎症的研究

Age-related inflammation (or so-called inflamm-ageing state) plays a key role in a large number of diseases in the elderly. However, the cellular and molecular mechanisms of origining and maintaining of this inflamm-ageing state is elusive yet. Immunosenescent T cells are prone to produce higher pro-inflammatory cytokiens, which could promote inflammation and contibute to inflamm-ageing state. Our and others' previous study revealed that RIP1 and RIP3 mediated programmed necrosis, i.e. necroptosis, of which the pathway was checked by FADD, was involved in the regulation of T cells' functions, such as acitvation induced cell death (AICD) in the process of proliferation and contraction. Interestingly, our preliminary data showed that the expression level of RIP3, the key molecule of necroptosis, were decreased in the aged CD4+ T and CD8+ T cells, when compared with their young couterparts. Those observations indicated that the alteration of necroptosis pathway might affect the functions of senescent T cells. Thus, this proposal is aimed to explore the possible role of necroptosis in regulating the functions of the immunosenecent T cells, and the related immunological significances on the inflamm-ageing state. By in vitro T cell stimulation assay, ex vivo T cells' adoptive transfer assay and in vivo influenza virus infection in mouse,we will apply the knockout mice of FADD, RIP1, RIP3 or double knockout of FADD and RIP1, and wildtype mice as contorl, to investigate the possibility of necroptosis pathway regulating the aged CD4+ T cells' function, such as activation, differentiation, proliferation and contraction, especially production of the proinflammatory cytokines. Those data might help to understood the relationship between the cell death pathway of necroptosis and the immunosenescent CD4+ T cells' functions, and shed light on a new mechanism which could contribute to the age-related inflammation. Ultimately, new clues could be expected to figure out new targets for effective therapeutic approahces of inflamm-ageing related diseases.

衰老相关炎症状态在诸多老年性疾病致病中起关键作用，但其本身发生、维持的分子细胞机制尚不明了,免疫老化T细胞在其中可能发挥重要作用。数据显示，免疫老化T细胞易受刺激而产生多种促炎细胞因子，故可参与衰老相关炎症过程；课题组前期工作发现,程序性坏死（necroptosis）信号通路是调控年轻T细胞功能的重要机制之一；老年CD4+ 及CD8+ T细胞中程序性坏死关键分子RIP3的表达水平显著减少。据此推测，程序性坏死通路在老年T细胞中的变化可能影响其功能。本课题拟运用T细胞体外刺激、过继性转移、流感病毒感染小鼠等方法，利用基因敲除小鼠模型，剖析程序性坏死信号通路是否参与调控老年T细胞的活化、分化、增殖、缩减等功能，特别是促炎细胞因子的分泌。研究旨在阐明程序性坏死信号通路调控免疫老化T细胞功能的可能机制，探讨该调控作用对衰老相关炎症形成的免疫学意义，为寻找相关老年炎性疾病临床治疗的新靶点提供线索。

炎症在诸多老年感染性疾病的致病中起关键作用，但相关分子细胞机制尚需进一步阐明。本研究以流感及衣原体感染小鼠为模型，探索了炎症小体及程序性坏死通路在感染炎症发生中的作用。1) 分析了NLRP3炎症小体及其下游IL-1信号通路在流感病毒感染中的作用及机制。敲除炎症小体关键成分NLRP3, caspase-1或ASC的小鼠比野生型的小鼠对H7N9流感病毒感染的抵抗力更好，表现为更少的致死率、更少的肺部炎症病理（Scientific Report 2017;7:7625）； IL-1受体敲除小鼠也有相似的现象（中华微生物与免疫学杂志2016;36:887-893）。因此 NLRP3炎症小体在H7N9流感病毒感染过程中起病理性作用，可能是由于 caspase-1 活化及 IL-1 信号通路介导的过度炎症有关。2) 分析了在LPS/ATP刺激或衣原体感染的小鼠骨髓来源的巨噬细胞BMDM中，RIP3在协助Caspase-1 介导的IL-1β生成中的作用（BioMed Research International 2017;2017:1592365）。结果显示，RIP3在LPS/ATP刺激BMDM产生IL-1β过程中发挥作用，但不在衣原体感染的BMDM产生IL-1β过程中发挥作用；两种方法刺激BMDM后均可发生细胞焦亡（pyroptosis）, 但衣原体感染情形中无需RIP3参与。3）分析了程序性坏死关键分子RIP3在甲型流感病毒H1N1 PR8感染中的作用。 结果显示流感病毒感染后RIP3-/-组小鼠感染后死亡率较野生型组小鼠显著降低；RIP3-/- 组小鼠肺组织损伤较轻，炎症浸润较少，肺部炎症细胞因子也较野生型组相对减少，提示 RIP3分子可能在 H1N1 PR8 流感病毒感染中发挥促炎症病理作用（中国实验动物学报 2018;26:727-733）。总之，本研究探讨了感染炎症发生过程中炎症小体和细胞程序性坏死通路的作用；发表了4篇论著，其中2篇SCI论文，2篇中文核心论文，培养研究生4人。后续研究仍在继续中，用细胞程序性坏死通路关键分子RIP3-/- 小鼠、MLKL-/-小鼠以及RIP3-/-MLKL -/-双敲除小鼠比较流感病毒感染后老年T细胞应答能力与机体炎症病理的差异，数据正在整理中。以上研究对揭示病原体感染机体的T细胞反应相关的信号通路与分子机制有一定意义。