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

项目摘要 暴露于环境空气污染物臭氧（O3）与心肺发病率和死亡率相关， 使其成为重要的公共卫生问题。受控暴露研究表明，急性O3暴露导致 气道炎症、上皮损伤和肺功能的短暂下降。这些研究还 研究表明，受试者在O3反应中表现出高度可重复的差异，这表明基因- 环境相互作用（GxE）。候选基因研究为少数基因提供了GxE的证据， 然而，遗传变异在基因组其余部分中的作用在很大程度上是未知的。这个数据缺口限制了我们的能力 识别易感个体并深入了解O3引起不良影响的机制。在这里， 我们提出了一个在体外使用人支气管上皮细胞（hBECs）来解决这一数据缺口的建议。hBECs 是呼吸道中第一个与O3相互作用的细胞，我们已经证明，体外暴露于O3的hBEC 上调关键促炎基因的表达（例如，CXCL 8），反映了体内反应。我们 假设O3诱导炎症变化与hBEC基因表达的差异有关， 并且在基线和O3后基因表达的个体间差异具有遗传基础，即，是 表达数量性状基因座（eQTL）。此外，我们假设一些eQTL是由单一的 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- 来自300个储存的肺组织供体的分化的hBEC培养物，在空气-液体界面生长， 不同的祖先，然后将他们暴露于O3与过滤空气（FA），并测量关键的hBEC O3反应表型 (e.g. IL-8产生、氧化应激、脂质过氧化、屏障功能和细胞毒性）。我们会侧写吉恩 使用批量RNA-seq和单细胞RNA-seq在FA和O3暴露的hBEC中表达以鉴定O3- 诱导/抑制基因及其细胞类型特异性。基因分型后，我们将在基线定位eQTL （mRNAFA）、应答eQTL（mRNAO 3-mRNAFA）和所有hBEC O3应答表型的QTL，然后使用 中介分析，以确定SNP和基因拟合假定的因果模型：O3+SNP → [mRNA] → hBEC O3 反应表型在目标2中，我们将执行ATAC-seq来表征O3如何改变染色质可及性 在hBECs中，然后绘制基线和响应caQTL。我们将进行多组学数据整合（eQTL，caQTL， QTL）来鉴定O3响应的基因调控模型，即，O3+SNP→染色质可及性→[mRNA]→hBEC O3反应表型。最后，在目标3中，我们将验证新基因和基因调控机制 在体外和体内O3响应的潜在变化。我们将确定关键SNPs如何影响基因调控 以及敲除相应的基因是否会改变体外的O3反应。对于体内验证，我们将 在191人数据集中测试感兴趣的SNP与O3诱导的中性粒细胞募集之间的关联 暴露在O3中的志愿者总之，我们的工作将确定遗传变异和基因调控机制， 影响对O3诱导气道炎症的易感性。

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.