基于TLR7通路AIV抗原触发鸭和鸡DCs分化成熟差异的分子机制

The influenza virus can activate dendritic cells through the TLR7/MyD88 signaling pathway to enhance the body's immune response to influenza antigen between human and mouse. Ducks receiving an inactivated influenza vaccine showed lower antibody titers than chickens, however, the reason is still unclear now. In view of this, H9N2 AIV will be used as the model antigen to induce the dendritic cells of duck and chicken, activate or inhibit the TLR7 receptor on the dendritic cells, Phenotypic changes, efficiency of antigen phagocytosis and expression of TLR7 receptor-related effective proteins of dendritic cells in duck and chicken were been detected through the methods of FCM, ELISA, qPCR and so on.To clarify the effects of influenza antigen on the differentiation and maturation in duck and chicken. Analysis of cell phenotype changes and expression of IL-1β, IL-6 and IFN-α by overexpression or silencing of TLR7-IκB and TLR7-IRF-7 nodal proteins in dendritic cell. To elucidate the effects of proinflammatory cytokines and type I interferon-mediated TLR7 signaling pathway on differentiation and maturation of duck and chicken dendritic cells, and to explore their molecular regulatory mechanisms. In order to explore the mechanisms of duck showed low immune response to inactivated influenza vaccine and provide a reference for prevention and control for the epidemic of avian influenza.

流感病毒可以通过TLR7/MyD88信号通路释放干扰素和炎性细胞因子使人和小鼠的树突状细胞活化，增强机体对流感抗原的免疫应答能力。禽流感灭活疫苗对鸭的免疫抗体效价往往低于鸡，但原因目前仍不清楚。鉴于此，本研究将以H9N2 AIV作为模式抗原诱导鸭和鸡的树突状细胞，激活或抑制树突状细胞上TLR7受体。通过FCM、ELISA和qPCR等方法检测鸭和鸡树突状细胞表型变化、抗原吞噬的效率和TLR7受体相关效应蛋白的表达情况，分析流感抗原对鸭和鸡树突状细胞分化成熟的影响。并通过过表达或沉默TLR7-IκB和TLR7-IRF-7节点蛋白，分析树突状细胞表型的变化和IL-1β、IL-6和IFN-α的表达情况，阐明促炎性细胞因子和I型干扰素所介导的TLR7信号通路对鸭和鸡树突状细胞分化成熟的影响，并探讨其分子调控机制。为禽流感疫情的防控和研究鸭对流感疫苗免疫应答低下的机制提供参考。

鉴于禽流感灭活疫苗免疫鸭后，其抗体效价往往低于鸡，而树突状细胞TLRs信号通路的激活可以提高小鼠对流感疫苗的免疫应答效果。本研究建立了鸭和鸡外周血单核源树突状细胞（MoDCs）体外培养体系和相关基因的荧光定量检测方法。比较分析了H9N2流感病毒灭活抗原（H9N2 IAIV）对鸭和鸡MoDCs中MHC-II、TLR7和部分细胞因子mRNA表达及吞噬H9N2 IAIV能力的影响。结果表明：与鸭MoDCs相比，H9N2 IAIV诱导/上调鸡MoDCs成熟、TLR7和IL-1β mRNA的表达以及吞噬H9N2 IAIV的能力更强。将TLR7信号通路激活剂与H9N2 IAIV联合刺激鸭MoDCs，显示TLR7激活剂联合刺激组可以明显上调鸭MoDCs中IRF7、NF-κB和MHC-II mRNA水平，并提高鸭MoDCs对H9N2 IAIV的吞噬效率，表明TLR7信号通路激活可以有效上调鸭MoDCs的成熟和吞噬H9N2抗原的能力。. 将TLR2激活剂（Pam2CSK4）、TLR3 激活剂（Poly(I:C)）和TLR7 激活剂（Imiquimod）不同组合分别与H9N2 IAIV联合刺激鸭MoDCs。发现Poly(I:C)-H9N2 IAIV和Imiquimod-H9N2 IAIV组中MHC-II mRNA水平明显高于Pam2CSK4-H9N2 IAIV组，但这三组鸭MoDCs吞噬H9N2 IAIV的百分比没有明显差异，提示鸭MoDCs的成熟程度与其吞噬H9N2抗原的能力并非完全线性关系。相对于Imiquimod-H9N2 IAIV组，IRF7、NF-κB mRNA水平和鸭MoDCs吞噬H9N2 IAIV的百分比在Pam2CSK4-Imiquimod-H9N2 IAIV和Poly(I:C)-imiquimod-H9N2 IAIV组没有明显上调，但在Pam2CSK4-poly(I:C)-imiquimod-H9N2 IAIV组却明显上调。提示并不是所有TLRs激活剂组合都有协同增效效应。结合本项目的研究基础发现IFN-γ、IL-6、TNF-α、MHC-II mRNA水平和抗体滴度在Pam2CSK4-poly(I:C)-imiquimod-H9N2 IAIV组中最高，提示上述细胞因子对鸭（MoDCs）TLRs信号通路激活和免疫应答有积极作用，但具体机制还需进一步研究。