烟曲霉硫氧还蛋白还原酶GliT抗侵袭性曲霉病的免疫保护作用及机制研究

Vaccination is a novel approach against invasive aspergillosis (IA), and a vaccine that effectively protects immunocompromised patients from IA is the key of successful therapy. However, a few of immunoprotective antigens from Aspergillus have been identified up to now, and their protective mechanism against aspergillosis is still unknown. In previous study, we found that thioredoxin reductase GliT (TR), a novel immunodominant antigen of Aspergillus fumigatus, strongly reacted with the IA patient's sera. We had assessed this protein for ability to activate Th cytokine production by human peripheral CD4+ T lymphocytes, and found that TR could elicited most robust strong IFN-γ responses in healthy donors, which indicated that TR might be a potential protective antigen. Thus we plan to establish TR vaccination mice model, evaluate the protective efficacy against Aspergillus fumigatus pulmonary infection in corticosteroid-immunosuppressed mice by survival rate, analyze the differences in gene expression for Tbet, Gata3, Rorc, Foxp3, ifn-γ and il4 in spleen CD4+T cells by real-time RT-PCR, and cytokine levels for IFN-γ, IL-4, IL-17A/F and IL-10 in lung homogenates by ELISA from vaccinated mice and nonimmunized mice, assess antifungal protection by adoptive transfer of anti-TR antibodies or purified splenic CD4+ T cells from TR-vaccinated animals into nonimmunized recipients. We try to investigate TR's protective mechanism and evaluate in particular the roles of antibodies and T cells, then provide new methods and theoretical basis for the therapy of IA.

预防接种正成为侵袭性曲霉病（IA）防治的新手段，寻找高效的保护性抗原是成功的关键。但迄今鉴定的曲霉保护性抗原极少,且其作用机制不明。课题组前期研究新发现一个烟曲霉强免疫反应性抗原，即硫氧还蛋白还原酶GliT（TR），该蛋白可体外诱导人PMBC产生具有保护作用的Th1型细胞因子IFN-γ,而不产生Th2细胞因子，提示该蛋白可能是保护性抗原。本项目拟用重组TR蛋白预先免疫小鼠，用皮质类固醇造成免疫抑制,用存活率评估抗烟曲霉呼吸道感染的保护效能。通过比较分析曲霉感染的免疫鼠与未免疫鼠间肺组织匀浆IFN-γ、IL-4、IL-17A/F、IL-10水平和脾CD4+T细胞中其上游基因Tbet、Gata3、Rorc、Foxp3、ifnr、il4的表达差异；过继转移抗TR抗体或TR免疫鼠脾CD4+T细胞至未免疫鼠来阐明TR免疫保护作用的机制，为IA防治提供新的手段和理论依据。

本课题组在前期研究中用免疫蛋白质组学技术首次筛选鉴定了一个新的强免疫反应性烟曲霉抗原，即硫氧还蛋白还原酶GliT（TR）。ELISPOT结果显示重组烟曲霉TR可以诱导健康人外周血单个核细胞（PBMC）产生强IFN-γ应答而几乎不产生IL-4应答。提示烟曲霉硫氧还蛋白还原酶GliT是一个保护性抗原。之后，C57BL/6小鼠随机分为免疫组和对照组，免疫组小鼠用重组TR免疫2次，对照组用PBS代替抗原注射。感染烟曲霉前用环磷酰胺和地塞米松对全部小鼠进行免疫抑制处理，通过气管壁穿刺法向气管内注入烟曲霉孢子悬液，建立IA疾病模型。用烟曲霉孢子攻击后，TR免疫组小鼠存活率为为64.7％，而对照组全部死亡（p＜0.001）。TR免疫组肺组织匀浆烟曲霉菌落计数结果示，免疫组小鼠组织真菌载量明显低于对照组（p＜0.01）。组织病理学检观察：死亡小鼠肺肿胀出血、肺泡间隔增宽、大量炎症细胞聚集、组织灶状坏死，并有大量烟曲霉菌丝存在，而存活小鼠肺组织损伤程度轻，且组织间无菌丝。肺泡灌洗液细胞分类结果示，免疫组小鼠单个核细胞显著高于对照组小鼠（p＜0.01）。免疫组小鼠肺组织匀浆中IFN-γ含量显著高于对照组小鼠(p<0.05)；而IL-4、IL-10和IL-17A/F的含量显著降低(p<0.05)。免疫组小鼠脾脏CD4+ T细胞中Tbet、Foxp3和ifnr表达量显著高于对照组小鼠(p<0.01)；而Gata3、Rorc和 il4表达量显著降低(p<0.01)。过继转移抗TR抗体不能提供免疫保护作用，但过继TR免疫鼠脾CD4+T细胞可有一定的保护性作用。上述结果说明，烟曲霉重组TR对侵袭性曲霉病小鼠有中度的免疫保护作用，其作用机制为激活Th1型应答，活化Treg细胞，且通过抑制Th17细胞分泌IL-17A/F，以减轻机体因炎症反应造成的病理损伤。从肺组织炎细胞募集情况和过继转移实验来看，单个核细胞可能在抵抗烟曲霉感染中发挥了重要作用；而且过继转移TR免疫CD4+T细胞有免疫保护作用，而抗TR抗体没有免疫保护作用。因此，烟曲霉重组硫氧还蛋白还原酶可作为一个有潜力的疫苗候选抗原，为IA的免疫治疗提供新的方法。