基于AhR通路调控DNA甲基化研究苯并[a]芘暴露对哮喘小鼠DCs的影响及分子机制

Epidemiologic studies suggest that exposure to environmental pollutants is associated with an increase in prevalence of asthma. Benzo[a]pyrene (B[a]P) is used as a representative of polycyclic aromatic hydrocarbons (PAHs) and it can induce asthma. However, the effects of B[a]P on the function of DCs in a mouse model of allergic asthma are unknown. Epigenetics provide a promising approach for improving understanding the relationship between environmental pollutants and genetic susceptibility. Therefore, the present study is carried out to explored the effects and mechanisms of B[a]P on DNA methylation in DCs. The study contains three parts: ① Effect on bronchial response to B[a]P exposure and dust mite allergen (Der f1、2) in sensitized asthma mouse; ② The effects of B[a]P expsure on DNA methylation in the DCs of allergic mouse are systematically analyzed in the level of global DNA methylation, in the level of specific genes and in the level of new DNA methylation sites; ③ The role of aryl hydrocarbon receptor (AhR) and other possible mechanisms in the B[a]P induced abnormal DNA methylation is investigated with the use of transgenic mice model. Thus, we propose “B[a]P priming hypothesis", providing a new view and theoretical basis to understand a modest increase in risk for air pollution in relation to asthma.

流行病学研究表明哮喘发病率的升高与室内环境的污染密切相关。B[a]P作为环境中常见的多环芳烃污染物，能够引起哮喘的发生，但其对免疫细胞（包括树突状细胞）的功能作用尚未清楚。表观遗传学作为联系环境因素与遗传易感因素之间的纽带，其在免疫系统发挥功能的过程中担当重要角色。本课题以尘螨诱导的哮喘小鼠为模型，基于AhR通路调控DNA甲基化研究B[a]P暴露对哮喘小鼠DCs功能的影响，从而诱导并增加过敏性疾病的发生：①明确B[a]P暴露可增加尘螨过敏原的致敏性；②从基因组整体水平甲基化、基因特异位点甲基化和新甲基化位点上系统分析B[a]P对哮喘小鼠DCs基因甲基化的改变；③利用AhRd转基因小鼠模型探讨AhR通路和其他可能机制在B[a]P暴露对DCs DNA甲基化过程中的作用。本课题提出哮喘发病的“B[a]P诱发及叠加效应假说”，拟加深对B[a]P诱导过敏性反应的认识，为预防过敏性哮喘提供理论依据。

与过敏原共存的环境污染物与哮喘的加重密切相关。然而，潜在的分子机制仍然难以捉摸。我们试图确定苯并[a]芘(BaP)与屋尘螨提取液( house dust mite extract，HDM )对BALB/c小鼠气道炎症表型联合暴露是否能增强HDM诱导的哮喘及其潜在机制。采用HDM诱导的小鼠哮喘模型，观察BaP对哮喘小鼠气道高反应性、过敏性炎症和上皮来源细胞因子的影响。进一步分析BaP对HDM诱导的哮喘小鼠肺部差异基因的变化和甲基化水平的影响。我们还研究了芳香烃受体(AhR)信号在BaP促进的HDM诱导的DNA甲基化、细胞因子的产生和过敏性炎症中的作用。研究显示，与HDM相比，BaP与HDM共暴露可导致哮喘小鼠的气道高反应性和肺部炎症反应加重。IL-4、IL-5和IL-17α在这些小鼠的呼吸道中的表达也增加。此外，BaP与Derf1联合暴露可激活AhR信号转导通路，增加AhR和CYP1A1的表达。此外，BaP联合暴露诱导的哮喘小鼠肺部基因的表达。采用转录组测序检测并比较肺部的基因表达谱差异。通过Gene Ontology（GO）寻找DCs功能异常和DNA甲基化调控的基因。通过KEGG pathway分析差异基因影响的主要调控机制和重点调控的蛋白：细胞因子-细胞因子受体相互作用和趋化因子信号通路的DEG数最高。用实时定量PCR验证筛选的差异基因mRNA的表达水平。BaP可通过AhR轴促进Der f1诱导的上皮细胞因子释放。①明确B[a]P暴露可增加尘螨过敏原的致敏性；②从基因组整体水平甲基化、基因特异位点甲基化和新甲基化位点上系统分析B[a]P对哮喘小鼠DCs基因甲基化的改变。