Gene-Environment Interactions with Ozone and Non-atopic Asthma

项目总结 流行病学研究提供的证据表明，臭氧(O_3)暴露与心脏病的发生有关 哮喘，特别是非特应性(非过敏性)亚型。这些发现得到了临床研究的支持。 显示环境中的臭氧暴露与非特应性儿童的嗜酸性呼吸道炎症有关。 实验室研究表明，接触臭氧会导致非人类粘液细胞的化生/增殖 灵长类动物，老鼠和老鼠。这包括我们的团队最近的研究结果表明，经典近亲繁殖 暴露于0.8ppm臭氧(每天4小时)9天的小鼠品系出现嗜酸性气道炎 除粘液细胞增生外。因此，三个物种和人类的数据提供了强有力的理由 旨在了解臭氧®非特应性哮喘关联的潜在机制的研究。 我们对机制问题采取了遗传学的方法。我们调查了协力十字组织的菌株 (CC)小鼠群体，一组新的重组近交系菌株，并鉴定了一种菌株CC002，该菌株 在反复接触臭氧后表现出比任何其他菌株更高的嗜酸性炎症水平 目前为止已经测试过了。接触臭氧的CC002小鼠也出现粘液细胞化生和呼吸道高反应性。 反应性(AHR)，哮喘的另外两个标志性表型。因此，CC002提供了一种新的遗传模型 我们可以利用这一点来确定臭氧诱导的非特应性哮喘关联的潜在机制。在 首先，我们将检验CC002的S对臭氧暴露的夸大反应是由于 调节失调的上皮细胞信号通过警报转而刺激2型固有淋巴样细胞(ILC2s)。我们 将确定暴露在臭氧中的上皮来源的固有细胞因子(IL-1a/b、IL-25、IL-33、TSLP)是否增加 CC002小鼠与无应答菌株的比较，以及这些细胞因子的中和是否减弱了臭氧诱导的 呼吸道炎症和AHR。接下来，我们将确定在臭氧中ILC2的数量和激活是否增加- 暴露于CC002小鼠，如果ILC2耗尽或抑制ILC2衍生的细胞因子(IL-5、IL-13)可改善 臭氧诱导的CC002小鼠气道炎症和AHR。对分子进行综合表征 对于上皮细胞、ILC2s和其他白细胞对臭氧的反应，我们将进行单细胞RNA测序。vbl.使用 这一创新和强大的方法，我们将量化基因表达和细胞组成的差异 /-O_3，菌株内和菌株间都是如此。我们还将通过测试来检验CC002的S响应的特异性 这种菌株对其他暴露是否表现出夸大的反应，包括内毒素、香烟烟雾、 和屋尘螨过敏原。在第二个目标中，我们将确定使CC002对 臭氧。我们将进行数量性状基因座定位，以确定含有CC002等位基因的染色体区域 与臭氧敏感性相关，然后进行生物信息学分析，以确定高优先级候选基因 每一个轨迹。总而言之，我们的工作将揭示臭氧暴露和空气污染之间的联系机制。 发展非特应性哮喘的表型，并确定潜在的靶向治疗途径。

PROJECT SUMMARY Epidemiologic studies have provided evidence that ozone (O3) exposure is associated with the development of asthma, in particular the non-atopic (non-allergic) sub-type. These findings are supported by clinical studies showing that ambient O3 exposure is associated with eosinophilic airway inflammation in non-atopic children. Laboratory studies have shown that O3 exposure causes mucous cell metaplasia/hyperplasia in non-human primates, rats and mice. This includes recent results from our groups demonstrating that classical inbred strains of mice exposed to 0.8 ppm O3 (4 hours/day) for 9 days develop eosinophilic airway inflammation in addition to mucous cell hyperplasia. Thus, data in three species and humans provide strong rationale for studies geared towards understanding the underlying mechanisms of the O3 ® non-atopic asthma association. We take a genetic approach to the question of mechanism. We surveyed strains from the Collaborative Cross (CC) mouse population, a new panel of recombinant inbred strains and identified one strain, CC002, that exhibited much higher levels of eosinophilic inflammation after repeated O3 exposure than any other strain tested to date. O3 exposed CC002 mice also developed mucous cell metaplasia and airway hyper- responsiveness (AHR), two other hallmark phenotypes of asthma. Thus, CC002 provides a new genetic model that we can exploit to identify mechanisms underlying the of O3-induced non-atopic asthma association. In the first Aim, we will test the hypothesis that CC002’s exaggerated response to O3 exposure are due to dysregulated epithelial signaling via alarmins that in turn stimulates type 2 innate lymphoid cells (ILC2s). We will determine if epithelial-derived innate cytokines (IL-1a/b, IL-25, IL-33, TSLP) are increased in O3-exposed CC002 mice compared to non-responder strains, and if neutralization of these cytokines attenuates O3-induced airway inflammation and AHR. Next, we will determine if ILC2 numbers and activation are increased in O3- exposed CC002 mice, and if depletion of ILC2s or inhibition of ILC2-derived cytokines (IL-5, IL-13) ameliorates O3-induced airway inflammation and AHR in CC002 mice. To comprehensively characterize molecular responses to O3 in epithelia, ILC2s, and other leukocytes, we will perform single cell RNA-sequencing. Using this innovative and powerful approach, we will quantify differences in gene expression and cellular composition +/- O3, both within and between strains. We will also examine the specificity of CC002’s response by testing whether this strain exhibits exaggerated responses to other exposures, including endotoxin, cigarette smoke, and house dust mite allergen. In the second aim, we will identify the genetic loci that render CC002 sensitive to O3. We will perform quantitative trait locus mapping to identify chromosomal regions harboring CC002 alleles associated with O3 sensitivity, followed by bioinformatic analyses to identify high priority candidate genes at each locus. In total, our work will reveal mechanisms underlying the association between O3 exposure and the development of non-atopic asthma phenotypes and identify potential pathways to target therapeutically.