高致病性流感病毒在老龄化宿主中诱导失衡性免疫应答的机制研究

The aging results in the immunosenescence and becomes more vulnerable to infection, which is recently exemplified by H7N9 infection that causes more severe outcomes in clinic in elder patients. Our previous report showed H7N9 infection induced hypercytokinemia (such as IL-6, IL-8, and macrophage inflammatory protein-1β) especially in the aged patients with a minimal type I interferon response, the functional deficiency of IFITM3 further exacerbates the dysregulated immune responses. Based on our previous established animal influenza infection model in mice（H3N2/H1N1/H7N9）, we will further develop influenza infection model in aged mice. By employing the transcriptomic and Epigenomic tools, we will screen and identify the differentially expressed genes and miRNA between adult and aged mice after H7N9 infection. In parallel, we will also identify the differentially expressed genes and miRNA between H7N9-infected adults and elder subjects during natural H7N9 infection. The similarly regulated genes in both aged mice and aged human will be analyzed for their signaling pathways and the signaling pathway clustered with differentially expressed genes will be targeted for siRNA to knock down and to re-direct the dysregulated immune responses. The most influential genes and pathways will be identified as our targets. Meanwhile, the similarly up-regulated miRNA in elder mice and human will be selected for their functional analysis to determine their role in the dysregulated resposes. This study will unfold the basis for the development of new drugs and immune intervention strategies which could improve the health of aged individuals.

老年化进程中机体免疫功能逐渐衰退且抵御感染能力显著降低。典型案例为人感染新型高致病性禽流感病毒（HPAIs）H7N9，老年人易感且预后不佳。本团队前期发现H7N9感染可诱导老年体内高水平炎症因子（IL-6,IL-8及MIP-1b）并伴有IFITM3介导的I型IFN抗病毒通路损伤。.本课题将在前期构建的流感病毒（H3N2/H1N1/H7N9）成年小鼠感染模型基础上，建立老年小鼠H7N9感染模型。以H7N9感染的成年鼠为参照，分析H7N9感染老年小鼠与成年小鼠时，参与免疫应答如抗原提呈细胞与炎症活化的重要细胞群DCs, NK, T细胞与抗体应答的差异性；通过基因转录组学与表观遗传技术筛选NF-kB/I型IFN通路中差异化表达的分子（DE-genes），并在老年与成年人H7N9感染者的组织标本中加以印证。在小鼠肺组织动态定位炎症应答始动细胞与关键分子，随后在体外细胞水平验证关键分子对始动细胞的信号传导通路的调控作用，从而明确老年宿主H7N9感染导致偏向性诱导炎症应答的分子机制，并为老年病毒感染的免疫干预策略提供生物学靶点。

老年人群因衰老而导致免疫功能减退，流感病毒感染后发病率高、病程进展快速，年龄与恢复时间有显著的正相关。本团队前期研究表明，老年H7N9感染者体内存在高水平炎症反应造成呼吸系统严重损伤导致临床转归不佳。为进一步解析老年宿主诱导偏向性免疫应答及其调控机制，本研究利用不同致病性流感病毒（H7N9、H9N2），探索建立老年小鼠的流感病毒感染差异化临床转归模型。由于老年小鼠感染H7N9后，2-5天内体重即下降30%，生存时间不超过6天；而感染H9N2后第9天体重降低30%，14天内死亡率为50%；同期对照组的成年小鼠感染H9N2体重下降10%和死亡率为0。提示老年小鼠/H9N2流感病毒模型更能全程再现老年宿主流感病毒感染的疾病进程。. 本研究发现老年动物感染H9N2后转归不良者，第1天病毒达到峰值，3天内体内病毒复制失控，肺组织毒载量是成年小鼠的6倍多；肺内、外周血中炎症因子风暴以及肺脏病理损伤均显著重于成年小鼠；老年小鼠体内所产生的针对H9流感蛋白特异性中和抗体分泌水平显著弱于成年对照组（p=0.0085）。后者与临床转归不良的老年H7N9感染者，在感染后第10天只有少数（3/12）中和抗体滴度达到1:40（显著低于成年对照的3/6）这一现象相互印证。. 通过基因转录组学技术筛选到了188个影响小鼠流感病毒感染后的临床转归的差异化表达基因，可能影响宿主诱导偏向性免疫应答，以及临床转归。发现老年小鼠感染H9N2后转归不佳者， Tff2蛋白（成年小鼠中对流感病毒感染损伤具有修复作用的关键分子），肺内表达上调显著低于成年对照，可能参与调控老年小鼠诱导偏向性免疫应答，影响肺脏损伤修复。. 本研究建立了老年小鼠流感病毒感染的动物模型，有效再现了老年宿主在流感病毒感染免疫应答中偏向性诱导炎症和免疫应答，初步发现影响临床转归的关键分子，为老年宿主高致病流感病毒感染损伤的免疫干预策略提供了生物学靶点。