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风险因素(母亲和父亲)，怀孕期间暴露于化学品和污染物会增加未来疾病的风险。相比之下，发育中胎儿的妊娠暴露是否会造成真正的跨代影响-即增加F3后代(F1胎儿及其F2卵子的后代)的哮喘易感性。解决方案：我们的实验室已经建立了一个强大的哮喘易感性母体传播模型。正常妊娠小鼠一次暴露于柴油废气颗粒物(DEP)会导致F1后代过敏反应增强和哮喘样表型，这与后代树突状细胞(DC)的表观遗传学变化有关。这一基础工作提供了一个强大的平台，以测试怀孕后暴露于典型环境毒素是否会导致哮喘易感性的跨代遗传。目的1研究单次孕鼠暴露于F0母鼠柴油废气颗粒物(DEP)后，F2和F3子代对哮喘易感性的跨代传播。对于所有三代，我们将测量过敏性呼吸道炎症(BAL嗜酸性粒细胞，细胞因子，组织学)和呼吸道高反应性(AHR)，在故意次优变应原(卵清蛋白)致敏和气雾剂激发方案后，对正常幼犬的影响最小。剂量和时间研究将在使用已被证明具有F1效应的参数的初步试验之后进行。AIM 2将定位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工作基础上，该工作显示了暴露于DEP的母亲所生的哮喘易感幼崽的F1 DC中存在明显的甲基组。我们将跟踪伴随哮喘易感性持续(或消失)的甲基组变化，从接触DEP的母鼠的F1到F2和F3后代。为此，我们将使用简化代表性亚硫酸氢盐测序(RRBS)来量化和绘制脾DC中DNA甲基化的变化，并通过焦磷酸测序进一步验证选定的靶点。生物信息学分析将确定在每一代中看到的甲基化变化的模式和程度，并允许与在窝产仔中观察到的表型(过敏性炎症的易感性和程度，AHR)直接相关。意义：该项目将确定是否发生哮喘易感性的跨代遗传，并为更好地对持续或解决怀孕暴露影响的机制分析奠定基础。

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.