Asthma susceptibility due to environmental programing of innate immunity in utero

描述（由申请人提供）：产前环境暴露越来越被认为是哮喘的重要危险因素。关键的暴露包括城市空气污染及其组成部分——柴油废气。这些机制还没有很好地描述。本项目旨在解决这一知识缺口。为了建立一个机制研究的框架，我们开发了一个小鼠模型。在这个模型中，怀孕小鼠反复暴露于柴油废气颗粒（DEP）会使它们的后代对产后过敏原卵清蛋白（OVA）过敏。这些后代在受到不理想的卵细胞致敏和刺激时发生哮喘。相比之下，出生在未暴露的水坝的幼崽在次优的OVA暴露下不会患哮喘。我们的初步数据表明，该模型中的哮喘是由产生IL-5、IL-13和IL-17的NK细胞介导的。使用特异性抗体清除这些细胞可以预防哮喘。我们还发现IL-5/IL-13/IL-17+ NK细胞在人类哮喘中增加。除了IL-5、IL-13和IL-17的产生外，致病小鼠NK细胞的特点是芳烃受体（AhR）特征转录本（AhRR、Cyp1a1和Cyp1b1）的表达增加。AhR是一种转录因子，已知对DEP的有机化合物，即多环芳烃（PAH）有反应。AhR也被认为可以调节IL-17基因的转录。一些报道认为AhR在IL-5和IL-13的产生中起积极作用。因此，AhR提供了母体暴露于DEP和后代NK细胞中细胞因子产生之间的分子联系。基于这些数据，我们的首要假设是，在子宫内暴露于柴油废气通过ahr依赖的NK细胞启动促进哮喘易感性。我们提出以下具体目标：1)研究NK细胞在产前暴露DEP诱导哮喘易感性中的重要性；2)研究芳烃受体在NK细胞活化及哮喘母婴传播中的作用；3)检验致病性DEP效应在第一代和后代生命中的持久性；4)检测IL- 5/IL-13/IL-17+ NK细胞和AhR/AhRR/Cyp1b1+ NK细胞在人哮喘中的相关性。为了达到这些目标，我们将使用NK细胞缺陷小鼠、没有适应性免疫的小鼠和NK细胞特异性缺失AhR的小鼠。我们还将进行NK细胞从产前暴露的后代转移到未暴露的小鼠。为了研究在后代一生中哮喘易感性的保存，我们将使用交叉培养和延迟过敏原暴露方法。为了研究DEP暴露的跨代影响，我们将检查F2和F3后代的哮喘易感性。为了确定人类相关性，我们将在幼儿和成人哮喘患者中测量IL-5/IL-13/IL-17+ NK细胞和AhR/AhRR/Cyp1b1+ NK细胞。我们将NK细胞结果与多环芳烃- dna加合物（一种环境暴露的测量）的数量相关联。使用体外培养系统，我们将确定DEP是否可以将来自非过敏性非哮喘供体的非极化NK细胞扭曲为产生IL-5/IL- 13/IL-17的细胞。然后，我们将使用击倒方法研究AhR在这一过程中的作用。

DESCRIPTION (provided by applicant): Prenatal environmental exposures are increasingly recognized as important risk factors for asthma. Critical exposures include urban air pollution and its component - diesel exhaust. The mechanisms are poorly characterized. This project seeks to address this gap in knowledge. To build a framework for mechanistic studies we have developed a mouse model. In this model, repeated exposure of pregnant mice to diesel exhaust particles (DEP) renders their offspring hypersensitive to the postnatal allergen - ovalbumin (OVA). These offspring develop asthma when subjected to suboptimal OVA sensitization and challenge. In contrast, pups born to unexposed dams do not develop asthma upon suboptimal OVA exposure. Our preliminary data indicate that asthma in this model is mediated by NK cells that produce IL-5, IL-13 and IL-17. Depletion of these cells using specific antibodies prevents asthma. We also show that IL-5/IL-13/IL-17+ NK cells are increased in human asthma. In addition to IL-5, IL-13 and IL-17 production, the pathogenic mouse NK cells are characterized by increased expression of the aryl hydrocarbon receptor (AhR) signature transcripts (AhRR, Cyp1a1 and Cyp1b1). AhR is a transcriptional factor that is known to respond to organic compounds of DEP, namely, polycyclic aromatic hydrocarbons (PAH). AhR is also known to regulate the IL-17 gene transcription. Some reports suggest the positive role of AhR in IL-5 and IL-13 production. Thus, AhR provides a molecular link between maternal exposure to DEP and cytokine production in offspring NK cells. Based on these data, our overarching hypothesis is that in utero exposure to diesel exhaust promotes asthma susceptibility through AhR-dependent priming of NK cells. We propose following specific aims: 1) Examine the importance of NK cells in asthma susceptibility induced via prenatal exposure to DEP; 2) Study the role of the aryl hydrocarbon receptor in NK cell activation and maternal-fetal transmission of asthma risk; 3) Examine the persistence of the pathogenic DEP effect through the life of first and subsequent generations; 4) Examine the relevance of IL- 5/IL-13/IL-17+ NK cells and AhR/AhRR/Cyp1b1+ NK cells in human asthma. To meet these goals we will use NK cell-deficient mice, mice without adaptive immunity and mice with NK cell-specific deletion of AhR. We will also perform transfers of NK cells from prenatally exposed offspring to unexposed mice. To study preservation of asthma susceptibility throughout the offspring life we will use cross-fostering and delayed allergen exposure approaches. To study transgenerational effects of DEP exposure, we will examine asthma susceptibility in F2 and F3 offspring. To determine human relevance we will measure IL-5/IL-13/IL-17+ NK cells and AhR/AhRR/Cyp1b1+ NK cells in young children and adults with asthma. We will correlate NK cell results with amounts of PAH-DNA adducts (a measure of environmental exposure). Using an in vitro culture system we will determine whether DEP can skew non-polarized NK cells from non-allergic non-asthmatic donors into IL-5/IL- 13/IL-17-producing cells. We will then study the role of AhR in this process using a knockdown approach.