Transgenerational Susceptibility to Asthma from Air Pollution Exposure

描述（由申请人提供）：问题：我们知道父母哮喘是儿童哮喘的主要危险因素（母亲或父亲），并且在怀孕期间接触化学物质和污染物会增加未来疾病的风险。相比之下，尚不清楚妊娠期暴露于发育中的胎儿是否会造成真正的跨代影响。F3后代（F1胎儿及其F2卵子的后代）哮喘易感性增加。解决方案：我们的实验室已经建立了哮喘易感性母婴传播的可靠模型。正常怀孕小鼠单次暴露于柴油废气颗粒（DEP）会导致F1后代的过敏反应增强和哮喘样表型，并与后代树突状细胞（dc）的表观遗传变化有关。这项基础研究为测试哮喘易感性的跨代遗传是否会在妊娠暴露于典型环境毒素后发生提供了一个强大的平台。目的1将研究妊娠暴露于柴油废气颗粒（DEP）的F0母鼠对F2和F3后代哮喘易感性的跨代传递。在所有3代幼犬中，我们将测量过敏性气道炎症（BAL嗜酸性粒细胞、细胞因子、组织学）和气道高反应性（AHR），这是在一个故意的次优过敏原（卵清蛋白）致敏和气溶胶刺激方案之后，对正常幼犬的影响最小。剂量和时间研究将在使用已经显示具有F1效应的参数进行初步试验之后进行。Aim 2将绘制F1、F2和F3代“易感”dc的DNA甲基组变化。这一目标建立在先前的研究基础上，表明暴露于depa的母亲所生的哮喘易感幼崽的F1 dc中存在明显的甲基组。我们将追踪暴露于dep母鼠F1至F2和F3后代哮喘易感性持续（或消失）的甲基组变化。为此，我们将使用减少代表性亚硫酸盐测序（RRBS）来量化和绘制脾脏dc中的DNA甲基化变化，并通过焦磷酸测序对选定的靶点进行额外验证。生物信息学分析将确定每一代甲基化变化的模式和程度，并允许与在窝群中观察到的表型（过敏性炎症的易感性和程度，AHR）直接相关。意义：本项目将确定哮喘易感性是否存在跨代遗传，为更好地分析妊娠暴露效应的持续或解决机制奠定基础。

DESCRIPTION (provided by applicant): The Problem: We know that parental asthma is a major risk factor for asthma in children (maternal > paternal), and that exposures to chemicals and pollutants during pregnancy can increase risk of future disease. In contrast, it remains unknown whether pregnancy exposures of the developing fetus can cause true transgenerational effects---i.e. increased asthma susceptibility in the F3 offspring (progeny of the F1 fetus and its F2 ova). The Solution: Our laboratory has already established a robust model of maternal transmission of asthma susceptibility. A single exposure of normal pregnant mice to diesel exhaust particles (DEP) causes enhanced allergic responses and an asthma-like phenotype in F1 offspring, linked to epigenetic changes in offspring dendritic cells (DCs.) this groundwork provides a robust platform to test whether transgenerational inheritance of asthma susceptibility will follow pregnancy-exposure to a prototypical environmental toxin. Aim 1 will investigate transgenerational transmission of asthma susceptibility to F2 and F3 offspring after a single pregnancy exposure to diesel exhaust particles (DEP) of F0 mother mice. For all 3 generations, we will measure allergic airway inflammation (BAL eosinophils, cytokines, histology) and airway hyperresponsiveness (AHR) after an intentionally sub-optimal allergen (ovalbumin) sensitization and aerosol challenge protocol that has minimal effects in normal pups. Dose and timing studies will follow initial trials using parameters already shown to have F1 effects. Aim 2 will map DNA methylome changes in 'susceptible' DCs from F1, F2 and F3 generations. This aim builds on prior work showing a distinct methylome in F1 DCs of asthma-susceptible pups born to DEP-exposed mothers. We will track the methylome changes that accompany persistence (or disappearance) of asthma susceptibility from F1 to F2 and F3 progeny of DEP-exposed mother mice. To do this, we will use reduced-representation bisulfite sequencing (RRBS) to quantify and map DNA methylation changes in splenic DCs, with additional validation of selected targets by pyrosequencing. Bioinformatic analysis will identify patterns and magnitude of methylation changes seen in every generation, and allow direct correlation to the phenotype observed in littermates (susceptibility and magnitude of allergic inflammation, AHR). Significance: The project will determine if transgenerational inheritance of asthma susceptibility occurs, and lays the foundation for better mechanistic analysis of persistence or resolution of pregnancy exposure effects.