Notch信号在呼吸道病毒疫苗增强性肺部免疫病理中的作用及机制研究

Respiratory syncytial virus（RSV）is the most important pathogen of lower respiratory tract disease in infants and young children. RSV infection of infants immunized with RSV vaccine led to more severe pulmonary immunopathology, which is a major obstacle for the development of safe and effective vaccines. Notch signaling is an important mechanism, by which immune cells, such as T cells and dendritic cells, activate and develop. In previous studies, it was found that abnormal innate immune and imbalanced T celullar memory responses induced by vaccines, especial Th2-biased and Th17-biased memory responses, were associated with the vaccine-enhanced pulmonary immunopathology, and new adjuvants could impair the vaccine-enhanced immunopathology by regulating immune memory responses. In addition, we found that the Notch signaling in these immune cells might be involved in the pathgenosis of the vaccine-enhanced disease. Mice infected with Pneumonia virus of mice(PVM) can exhibit a few clinical features and immunopathology in RSV infected infants, while mice infected with RSV cann't do it. The current study will in vivo or in vitro investigate the effect of Notch signaling on innate immune response and development of Th1/Th2/Th17 memory cells induced by the formalin-inactivated PVM vaccine with TLR or IL-27 ligand as an adjuvant, and on pulmonary immunopathology mediated by innate immune and adaptive immune memory responses activated by RSV challenge, which may help to clarify the role and mechanism of Notch signaling in the vaccine-enhanced pulmonary immunopathology. It may be an important theoretic basis for studying safe and effective RSV vaccines.

呼吸道合胞病毒（RSV）是婴幼儿下呼吸道感染的最重要病原,接种疫苗者感染病毒后导致加重的肺部免疫病理，是疫苗成功的主要障碍。Notch信号是调节T、DC等免疫细胞活化和分化的重要机制。前期研究发现疫苗诱导的固有免疫异常、T细胞记忆应答不平衡、尤其是Th2和Th17优势应答是疫苗增强性免疫病理的重要因素；佐剂通过调节记忆应答而减轻免疫病理；机制可能与免疫细胞的Notch信号有关。小鼠肺炎病毒（PVM）感染小鼠能呈现RSV感染幼儿的症状及病理，RSV感染小鼠则不能。本项目拟用PVM/小鼠模型，以福尔马林灭活的PVM为模型疫苗，以TLR配体和IL-27为佐剂，体内外研究Notch信号对疫苗诱导的固有免疫和Th1/Th2/Th17记忆细胞的影响、及对病毒感染时固有免疫和免疫记忆被激活而导致免疫病理的影响，阐明Notch信号在疫苗增强性肺部免疫病理中的作用及机制，为研制安全有效的RSV疫苗奠定基础

呼吸道合胞病毒疫苗增强性免疫病理与Th细胞免疫应答不平衡有关，Notch信号是决定Th细胞分化的关键机制，本课题以福尔马林灭活的小鼠肺炎病毒（FI-PVM）为疫苗模型，研究了Notch信号在疫苗诱导的Th细胞免疫记忆过程中及病毒攻击过程中的作用，结果表明：Notch信号在疫苗诱导的记忆细胞Th1、Th2、Th17过程中及病毒攻击时激活Th1、Th2、Th17记忆细胞过程中发挥重要作用；提示Notch信号可能通过影响疫苗诱导的Th细胞免疫记忆应答而影响病毒感染时疫苗增强性免疫病理。.疫苗诱导细胞免疫应答必须首先激活抗原提呈细胞，FI-PVM刺激小鼠巨噬细胞系RAW264.7高表达抗原提呈相关分子MHCⅡ、CD80和CD86，以及Notch3、Notch4 、配体Jagged1、Jagged2、和靶基因Deltex和HES1，表明FI-PVM在激活抗原提呈细胞时激活了Notch信号。.用抑制剂抑制RAW264.7细胞的Notch信号后，细胞表面MHC-II分子和CD80分子双阳性和单阳性细胞百分率均降低，同时趋化因子和细胞因子如MCP-1、IL-6等均受到明显抑制，表明抑制Notch信号使得巨噬细胞的抗原提呈活性降低。.将Notch信号野生型或抑制型的RAW264.7细胞与Notch信号野生型或抑制型的FI-PVM特异性CD4+T记忆细胞共培养，与这两种野生型细胞共培养相比，Th1、Th2、Th17型的转录因子和细胞因子的表达和分泌均受到显著抑制。且单独抑制RAW264.7或CD4+T记忆细胞的Notch信号，对Th1、Th2、Th17型转录因子和细胞因子的表达和分泌有显著不同的影响。表明Notch信号在记忆细胞的激活和分化过程中发挥重要作用，且对Th1、Th2、Th17细胞的分化有不同的影响。.以TLR9配体CpG为佐剂时得到与上述一致的结果。.用PVM刺激野生型或Notch信号抑制型RAW264.7细胞，然后与CD4+T记忆细胞共培养，结果提示Notch信号在病毒攻击时对Th1、Th2、Th17记忆细胞的激活过程中同样发挥重要作用。.本课题研究结果初步表明，Notch信号在呼吸道病毒疫苗诱导免疫记忆应答过程中和病毒攻击后激活记忆细胞应答过程中发挥重要作用，提示Notch信号可能通过影响疫苗诱导的Th细胞免疫记忆应答而影响病毒感染时疫苗增强性免疫病理