Gene-Environment Interactions with Ozone and Non-atopic Asthma

项目摘要 流行病学研究提供的证据表明，臭氧（O3）暴露与以下疾病的发生有关： 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 表明环境O3暴露与非特应性儿童的嗜酸性气道炎症有关。 实验室研究表明，O3暴露会导致非人类的粘液细胞化生/增生， 灵长类、大鼠和小鼠。这包括我们小组最近的结果，表明经典近交系 暴露于0.8 ppm O3（4小时/天）9天的小鼠品系发生嗜酸性气道炎症， 此外还有粘液细胞增生。因此，三个物种和人类的数据为以下方面提供了强有力的依据： 研究旨在了解O3®非特应性哮喘相关的潜在机制。 我们对机制问题采取遗传学的方法。我们调查了来自协作十字架的菌株， (CC)小鼠群体，一组新的重组近交系，并确定了一个品系，CC002， 在反复暴露于O3后，其嗜酸性粒细胞炎症水平比其他菌株高得多 测试至今。暴露于O3的CC002小鼠也出现了粘液细胞化生和气道过度增生。 反应性（AHR），哮喘的另外两种标志性表型。因此，CC002提供了一种新的遗传模型 我们可以利用它来确定O3诱导的非特应性哮喘相关性的潜在机制。在 第一个目标，我们将测试的假设，CC002的夸大反应，O3暴露是由于 通过alarmins的上皮信号传导失调，其反过来刺激2型先天淋巴样细胞（ILC2）。我们 将确定在O3暴露中上皮来源的先天性细胞因子（IL-1a/B、IL-25、IL-33、TSLP）是否增加。 CC002小鼠与非应答株相比，如果这些细胞因子的中和减弱O3诱导的 气道炎症和AHR。接下来，我们将确定ILC2数量和激活是否在O3中增加- 暴露的CC002小鼠，并且如果ILC2的消耗或ILC2衍生的细胞因子（IL-5、IL-13）的抑制改善， O3诱导的CC002小鼠气道炎症和AHR。为了全面表征分子 为了确定上皮细胞、ILC2和其他白细胞对O3的反应，我们将进行单细胞RNA测序。使用 这种创新和强大的方法，我们将量化基因表达和细胞组成的差异， +/-O3，在菌株内和菌株之间。我们还将通过测试来检查CC002反应的特异性。 该菌株是否对其他暴露，包括内毒素，香烟烟雾， 和屋尘螨过敏原在第二个目标中，我们将鉴定使CC002对以下敏感的遗传基因座： 氧气我们将进行数量性状基因座作图，以确定携带CC002等位基因的染色体区域 与O3敏感性相关，随后进行生物信息学分析，以确定高优先级候选基因， 每个轨迹总的来说，我们的工作将揭示O3暴露与糖尿病之间联系的潜在机制。 非特应性哮喘表型的发展，并确定潜在的治疗靶向途径。

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.