尘螨免疫治疗对呼吸道上皮细胞β防御素和树突状细胞的调节作用

Allergen specific immunotherapy (ASIT) is the only treatment that can modify the natural course of allergic asthma. Clinical investigation has shown a significant reduction in occurrence of asthma exacerbation induced by pulmonary infection in patients with ASIT with dust mite vaccince . This might be associated with the enhancement of innate immunity function. We have found Fim H protein of enterobacter in dust mite vaccine, which can protect from lower airway infection by up-regulating the expression of mouse beta-definsin-3 (homolog for human beta-defensin [HBD]-2) of airway epithelium in mouse model. However, the detail mechanism has not been clarified. We propose the hypothesis of two-way regulations of house dust mite vaccination. This project is designed to analyze the levels of HBD-2 in induced sputum, nasal lavage, and secretory mediators by the dendrictic cells (DC), DC subtype, ratio of T helper cell (Th) 1 and Th2 in alveolar lavage, and level of HBD-2 mRNA in airway epithelium in allergic patients before and after ASIT. Associations between clinical improvements including symptom and medication scores and pulmonary functions and the above pulmonary immunological biomarkers are evaluated. The project also applies for in vitro cellular experiments, asthmatic mouse model and Toll like receptor (TLR) 4 knockout mice to investigate signal pathway of HBD-2 expression, regulation of pattern of DC subtype exchange in airway mucosa, and pivotal factors in inducing DC maturation and modulating Th polarization. We would be able to reveal the regulatory mechanisms of respiratory innate immunity with ASIT in this project.

特异性免疫治疗（ASIT）是过敏性哮喘的唯一对因疗法，临床显示尘螨ASIT使感染诱发哮喘加重的发生率下降，与固有免疫增强有关。我们前期研究发现尘螨疫苗含肠杆菌菌毛（Fim）H蛋白（JACI，2014），可上调小鼠肺上皮细胞β防御素-3（人β防御素[HBD] -2同系物）表达，增加肺部感染的抵抗力，其机制未明，故提出“尘螨疫苗双功能假说”。本研究对尘螨过敏哮喘患者进行ASIT，分析治疗前后诱导痰、鼻腔灌洗液HBD-2蛋白、树突状细胞（DC）分泌因子水平、DC亚型和Th1/Th2细胞比例及肺上皮细胞HBD-2 mRNA水平，观察上述指标与症状、用药及肺功能变化的关系；通过体外细胞实验、小鼠哮喘动物模型及Toll样受体（TLR）4基因敲除鼠，研究尘螨疫苗诱导HBD-2表达信号通路、调节气道黏膜DC亚型转换方式以及诱导DC成熟并调控Th分化的方向，揭示ASIT对呼吸道固有免疫的调节机制。

尘螨是我国过敏性鼻炎和哮喘患者最主要的致敏原，为了解尘螨本身及其致敏机制，本项目首先经过高通量测序技术检测屋尘螨的全基因组，发掘了19种屋尘螨新过敏原，并发现屋尘螨和粉尘螨在主要过敏原表达量上存在显著差异。接着原核表达纯化了Der f 20、21、31、33等新过敏原，并进行了相关致敏机制研究，发现Der f 20过敏原可刺激树突状细胞（DCs）的TIM4的表达；Der f 31可通过TLR2调节肺部上皮细胞TSLP、IL-33的表达，从而使ILC-2细胞表达增加促使过敏的发生；Der f 33 具有上调DCs分泌CD80和TNF-α而致敏的功能。项目前期阐明尘螨过敏原基因及其致敏机制，对项目后期尘螨疫苗ASIT提高机体固有免疫和免疫治疗过敏性疾病相关机制的研究，具有重要的指导作用。.在尘螨基因组测序及其致敏机制研究的基础上，本项目通过动物和细胞实验探讨了尘螨过敏原疫苗ASIT增强机体免疫的机制。研究发现：尘螨疫苗ASIT能有效减轻尘螨诱导的过敏性哮喘及气道炎症，并通过上调小鼠抗菌肽CRAMP和小鼠beta防御素分子MBD-3表达，而增强哮喘小鼠肺部抗菌的免疫应答；尘螨疫苗中FimH蛋白能够启动DCs表达IL-10，诱导CD4+CD25+FoxP3+ T细胞（Treg）细胞分化增殖，增强尘螨疫苗ASIT后过敏原免疫耐受的效果。此外临床研究调查，我们发现尘螨疫苗免疫治疗后能明显增强患者抵抗下呼吸道感染能力，而对呼吸道感染的预防不明显；进一步临床实验研究发现接受屋尘螨特异性免疫治疗的过敏性鼻炎和/或支气管哮喘患者的血中特异性IgG4、sIgE和IgE-FAB浓度与患者临床症状指标密切相关。本项目研究结果将为尘螨过敏性疾病精准诊疗提供理论依据。