Asthma susceptibility due to environmental programing of innate immunity in utero

描述(由申请人提供)：产前环境暴露日益被认为是哮喘的重要危险因素。严重暴露包括城市空气污染及其成分-柴油废气。这些机制的特征并不明确。该项目旨在解决这一知识差距。为了建立一个力学研究的框架，我们开发了一个小鼠模型。在这个模型中，怀孕小鼠反复暴露在柴油废气颗粒物(DEP)中，使其后代对出生后的过敏原-卵清蛋白(OVA)过敏。这些后代在受到次优OVA致敏和挑战时会患上哮喘。相比之下，在未暴露的水坝中出生的幼崽在低于最佳OVA暴露时不会患上哮喘。我们的初步数据表明，在这个模型中，哮喘是由NK细胞介导的，NK细胞产生IL-5、IL-13和IL-17。使用特定抗体耗尽这些细胞可以预防哮喘。我们还发现IL-5/IL-13/IL-17 NK细胞在哮喘患者中升高。除了产生IL-5、IL-13和IL-17外，致病小鼠NK细胞的特征是芳香烃受体(AhR)特征转录本(AHRR、Cyp1a1和CyP1b1)的表达增加。AHR是一种已知对DEP的有机化合物，即多环芳烃(PAH)做出反应的转录因子。AHR还可以调节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提供了母体接触DEP和子代NK细胞产生细胞因子之间的分子联系。基于这些数据，我们的主要假设是，在子宫内暴露于柴油废气通过依赖于AhR启动NK细胞来促进哮喘易感性。我们提出以下具体目标：1)研究NK细胞在产前暴露于DEP诱导的哮喘易感性中的重要性；2)研究芳香烃受体在NK细胞激活和哮喘风险母婴传播中的作用；3)检测致病DEP效应在第一代和后代中的持久性；4)检测IL-5/IL-13/IL-17 NK细胞和AhR/AHRR/CYP1B1 NK细胞在人类哮喘中的相关性。为了实现这些目标，我们将使用NK细胞缺陷小鼠、没有适应性免疫的小鼠和具有NK细胞特异性AhR缺失的小鼠。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身上。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的方法。为了研究接触DEP的跨代影响，我们将检测F2和F3后代的哮喘易感性。为了确定人类相关性，我们将检测哮喘儿童和成人的IL-5/IL-13/IL-17 NK细胞和AhR/AHRR/CYP1B1 NK细胞。我们将把NK细胞的结果与PAH-DNA加合物的数量(环境暴露的一种衡量标准)联系起来。利用体外培养系统，我们将确定DEP是否能将非过敏性非哮喘供者的非极化NK细胞转化为产生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.