Regulatory Genomics of Ozone Air Pollution Response in Vitro and In Vivo

项目摘要 暴露在环境空气污染物臭氧(臭氧)中与心肺疾病和死亡有关， 使其成为一个重要的公共卫生问题。受控暴露研究表明，急性臭氧暴露会导致 呼吸道炎症、上皮损伤和一过性肺功能下降。这些研究还 表明受试者在臭氧反应中表现出高度可重复性的差异，这表明 环境相互作用(GxE)。候选基因研究已经为少数基因提供了GxE的证据， 然而，基因变异在基因组其余部分中的作用在很大程度上是未知的。这种数据差距限制了我们的能力 识别易感人群，并深入了解臭氧引起不良影响的机制。这里, 我们提出了一项建议，利用体外培养的人支气管上皮细胞(HBECs)来解决这一数据差距。HBEC 是呼吸道中第一个与臭氧相互作用的细胞，我们已经证明，在体外，hBECs暴露在臭氧中 上调关键致炎基因(如CXCL8)的表达，反映体内的反应。我们 假设臭氧诱导的炎症变化与hBEC基因表达的差异有关， 而基线和臭氧后基因表达的个体间差异具有遗传基础，即 表达数量性状基因座(EQTL)。此外，我们假设一些eQTL是由单个 影响染色质可及性(CaQTL)的核苷酸多态性(SNP)。在目标1中，我们将建立良好的- 从300名不同性别和性别的银行肺组织供者身上分离出在气液界面上生长的hBEC。 不同的祖先，然后将他们暴露在臭氧和过滤空气(FA)中，并测量关键的hBEC O3响应表型 (例如，IL-8的产生、氧化应激、脂质过氧化、屏障功能和细胞毒性)。我们将对基因进行分析 混合RNA-seq和单细胞RNA-seq在FA和臭氧暴露的hBECs中的表达 诱导/抑制基因及其细胞类型特异性。基因分型后，我们将在基线上定位eQTL (MRNAFA)、响应eQTL(mRNA03-mRNAFA)和所有hBEC O3响应表型的QTL，然后使用 用于识别符合假设因果模型的SNP和基因的中介分析：O_3+SNP→[m RNA]→hBEC O_3 反应表型。在目标2中，我们将执行atac-seq来表征臭氧如何改变染色质的可及性 在hBEC中，然后定位基线和响应caQTL。我们将进行多组数据集成(eQTL，caQTL， →染色质可及性→[→]hBEC O_3反应表型。最后，在目标3中，我们将验证新的基因和基因调控机制 体内和体外臭氧响应的潜在变化。我们将确定关键的SNPs如何影响基因调控 以及在体外，敲除相应基因是否会改变臭氧的反应。对于体内验证，我们将 191人数据集中感兴趣的SNPs与臭氧诱导的中性粒细胞募集的相关性检验 接触臭氧的志愿者。总之，我们的工作将确定基因变异和基因调控机制， 影响对臭氧诱导的呼吸道炎症的敏感性。

Project Summary Exposure to the ambient air pollutant ozone (O3) is associated with cardiopulmonary morbidity and mortality, rendering it an important public health issue. Controlled exposure studies show that acute O3 exposure causes airway inflammation, epithelial injury, and a transient decrease in lung function. These studies have also demonstrated that subjects exhibit highly reproducible differences in O3 response, suggestive of gene-by- environment interactions (GxE). Candidate gene studies have provided evidence of GxE for a handful of genes, however, the role of genetic variants in the rest of the genome is largely unknown. This data gap limits our ability to identify susceptible individuals and gain insight into mechanisms by which O3 causes adverse effects. Here, we put forth a proposal to address this data gap using human bronchial epithelial cells (hBECs) in vitro. hBECs are the first cells of the respiratory tract to interact with O3, and we have shown that hBECs exposed to O3 in vitro upregulate the expression of key pro-inflammatory genes (e.g., CXCL8), mirroring the in vivo response. We hypothesize that variation in O3-induced inflammation is associated with differences in hBEC gene expression, and that inter-individual differences in gene expression at baseline and after O3 have a genetic basis, i.e., are expression quantitative trait loci (eQTL). Further, we hypothesize that some eQTL are caused by single nucleotide polymorphisms (SNPs) that affect chromatin accessibility (caQTL). In Aim 1, we will establish well- differentiated hBEC cultures, grown at air-liquid interface, from 300 banked lung tissue donors of both sexes and diverse ancestries, then expose them to O3 vs. filtered air (FA) and measure key hBEC O3 response phenotypes (e.g. IL-8 production, oxidative stress, lipid peroxidation, barrier function, and cytotoxicity). We will profile gene expression in FA and O3-exposed hBECs using both bulk RNA-seq and single cell RNA-seq to identify O3- induced/repressed genes and their cell-type specificity. After genotyping, we will map eQTL at baseline (mRNAFA), response eQTL (mRNAO3-mRNAFA), and QTL for all hBEC O3 response phenotypes, then use mediation analyses to identify SNPs and genes fitting a putative causal model: O3+SNP → [mRNA] → hBEC O3 response phenotype. In Aim 2, we will perform ATAC-seq to characterize how O3 alters chromatin accessibility in hBECs, then map baseline and response caQTL. We will perform multi-omic data integration (eQTL, caQTL, QTL) to identify gene regulatory models of O3 response, i.e., O3+SNP→chromatin accessibility→[mRNA]→hBEC O3 response phenotype. Finally, in Aim 3, we will validate novel genes and gene regulatory mechanisms underlying variation in O3 response in vitro and in vivo. We will determine how key SNPs affect gene regulation and whether knocking down the corresponding genes alters O3 response in vitro. For in vivo validation, we will test for association between SNPs of interest and O3-induced neutrophil recruitment in a dataset of 191 human volunteers exposed to O3. In total, our work will identify genetic variants and gene regulatory mechanisms that influence susceptibility to O3-induced airway inflammation.