Gene-environment Interaction And Pulmonary Disease: Translational Studies

在人类肺部疾病的发病机制中，遗传背景和氧化环境刺激之间的相互作用在很大程度上尚未被探索。对氧化应激的生物反应是许多炎症性疾病的关键机制。在这个项目中，我们已经开始了一些研究计划，这些计划是从我们的小鼠炎症和急性肺损伤模型演变而来的。在第一个项目中，我们与Francine Kauffmann博士和Rachel Nadif博士(巴黎INSERM)建立了强有力的合作，以调查不同暴露于环境氧化剂煤尘和香烟烟雾中的煤炭工人尘肺病(CWP)的遗传基础。考夫曼和纳迪夫博士在253名煤矿工人中进行了一项前瞻性流行病学研究，其中包括对环境刺激的反应的量化表型，这些表型可能与煤工尘肺(一种炎症性肺部疾病)有关。研究了六种氧化应激标志物作为暴露反应的中间表型，包括红细胞谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶活性。研究的氧化剂暴露是吸烟习惯和累积粉尘暴露，通过工作历史和环境暴露测量来评估。疾病表型包括第一次调查时的亚临床计算机断层扫描评分，以及每隔5年两次的X射线灌注评分，以评估已确诊的煤工尘肺。我的实验室从招募到这项研究的每个人身上提取了血液，并分离出了DNA。我们开始研究煤工尘肺易感性的遗传基础，通过评估选定的表型与下列类别或类别的基因的多态之间的关联：先天免疫、炎症和抗氧化剂。对矿工进行了一些候选基因中常见功能性单核苷酸多态(SNPs)的基因分型，发现SNPs与疾病结局显著相关。结果提示，遗传背景与环境暴露和中间反应表型的交互作用是煤矿工人疾病发病机制的重要组成部分。 已经建立了第二个项目，以调查急性呼吸窘迫综合征(ARDS)不良后果易感性的遗传基础。ARDS是成人和新生儿的一种主要急性肺部疾病，以非心源性水肿和炎症为特征。ARDS的死亡率从50%到80%不等。ARDS的发病率没有很好的确定，但估计每10万人中约有2-8例。易感机制尚不清楚，也没有特效的治疗方法。我们在近交系小鼠中的研究发现，转录因子NRF2是ARDS模型高氧性肺损伤易感性的候选基因。因此，我们假设，NRF2功能丧失的多态易导致与ALI/ARDS相关的氧化性肺损伤。到目前为止，NRF2的基因组序列还没有得到很好的描述，只有少数SNPs定位于该基因的编码区。在本研究中，对NRF2基因的部分编码区和1kb的启动子进行了扩增、测序和比较，以定位NRF2的多态。随后，对潜在重要的已鉴定的SNPs进行了功能相关性NRF2表达或结合活性的体外评估。我们正在使用类似的方法来鉴定和评估Keap1基因中的SNPs，Keap1是一种细胞内的NRF2抑制蛋白。在一项平行的研究中，我们已经开始评估NRF2的一个目标基因，即第二阶段抗氧化剂NQO1(NAD(P)H：苯醌氧化还原酶1)中的SNPs。我们已经在NQO1启动子中发现了新的SNPs，它们与细胞对氧化刺激的反应具有功能相关性。通过与宾夕法尼亚大学的Jason Christie博士的合作，NRF2和NQO1中功能相关的SNPs已经通过协会评估了它们在ALI患者疾病发病机制中的重要性。 我们的第三个项目旨在评估宿主对呼吸道合胞病毒反应的遗传机制，并对小鼠和人类进行先天免疫研究。本项目旨在研究Toll样受体和抗氧化防御在呼吸道合胞病毒(RSV)感染和疾病进展中的作用。由于TLR4和NRF2似乎对抵御RSV免疫挑战至关重要，因此与约翰·霍普金斯大学的Fernando Polack博士合作，在阿根廷布宜诺斯艾利斯招募的一组感染RSV的婴儿中，测试了基因功能丧失将促进在原发RSV感染期间发生下呼吸道感染的假设。 我们的第四个项目旨在研究抗氧化基因在极低出生体重(VLBW)婴儿支气管肺发育不良(BPD)发病机制中的作用。BPD是婴儿慢性肺部疾病最常见的原因。与费尔南多·波拉克博士合作，我们正在前瞻性地研究候选抗氧化剂基因在BPD易感性中的作用。我们相信，了解抗氧化剂基因在BPD易感性中的作用，可以立即识别需要个性化医学方法的易感人群，评估现有的具有潜在预防和/或治疗价值的基因靶向抗氧化剂，并在不久的将来将这些观察扩展到其他重要的氧化应激相关疾病(例如COPD、肺癌、哮喘)。

Interaction between genetic background and oxidative environmental stimuli in the pathogenesis of human lung disease has been largely unexplored. Biological response to oxidative stress is a key mechanism in numerous inflammatory diseases. In this program, we have begun a number of research initiatives that evolved from our mouse modeling of inflammation and acute lung injury. In the first project, we have extablished strong collaboration with Drs. Francine Kauffmann and Rachel Nadif (INSERM, Paris) to investigate the genetic basis of susceptibility to coal workers pneumoconiosis (CWP) in individuals differentially exposed to environmental oxidants coal dust and cigarette smoke. Drs. Kauffmann and Nadif have undertaken a prospective epidemiologic study in 253 coal miners, which included quantitative phenotypes of response to environmental stimuli that may be involved in CWP, an inflammatory lung disease. Six oxidative stress markers were studied as intermediate phenotypes of response to exposure, including erythrocyte glutathione peroxidase (GSH-Px) and catalase activities. Oxidant exposures studied were smoking habits and cumulative dust exposure assessed by job history and ambient measures of exposure. Disease phenotypes included subclinical computed tomography score at the first survey and X-ray profusion grades twice 5 years apart to assess established CWP. My laboratory obtained blood from each of the individuals recruited to the study and DNA was isolated. We began to investigate the genetic basis for CWP susceptibility by evaluating association of selected phenotypes with polymorphisms in the following classes or categories of genes: innate immunity, inflammation, and antioxidant. Miners were genotyped for common functional single nucleotide polymorphisms (SNPs) in a number of candidate genes and have found significant association of SNPs and disease outcome. Results suggest that interactions of genetic background with environmental exposure and intermediate response phenotypes are important components in the pathogenesis of disease (CWP) in coal miners. A second project has been established to investigate the genetic basis of susceptibility to adverse outcomes of acute respiratory distress syndrome (ARDS). ARDS is a major acute lung disease in adults and neonates, and is characterized by noncardiogenic edema and inflammation. The mortality rate for ARDS ranges from 50-80%. The incidence of ARDS is not well established, but approximately 2-8 cases per 100,000 are estimated. The mechanisms of susceptibility are unclear, and there are no specific therapies. Our studies in inbred mice have identified the transcription factor NRF2 as a candidate gene for susceptibility to hyperoxic lung injury, a model of ARDS. We have hypothesized, therefore, that loss-of-function polymorphisms in NRF2 predispose to oxidative lung damage associated with ALI/ARDS. To date, the genomic sequence of NRF2 is not well characterized and only a few SNPs have been localized to coding regions within the gene. In the current study, portions of the coding region and 1-kb of the promoter were amplified, sequenced and compared in order to locate NRF2polymorphisms. Subsequently, potentially important identified SNPs were evaluated in vitro for functional relevance NRF2 expression or binding activity. We are using a similar approach to identify and evaluate SNPs in the gene for KEAP1, an intracellular NRF2 inibitory protein. In a parallel investigation we have begun to evaluate SNPs in a target gene of NRF2, the phase II antioxidant NQO1 (NAD(P)H:quinone oxidoreductase1). We have identified novel SNPs in the promoter of NQO1 that have functional relevance to the cellular response to oxidant stimule. Functionally relevant SNPs in NRF2 and NQO1 have been evaluated by association for their importance in disease pathogenesis in a population of ALI patients through a collaboration with Dr. Jason Christie (University of Pennsylvania). Our third project is designed to evaluate genetic mechanisms of host response to respiratory syncytial virus and innate immunity studies in mice and humans. This project is designed to investigate the role of toll-like receptors and antioxidant defense in respiratory syncytial virus (RSV) infection and disease progression. Because TLR4 and NRF2 appear to be critical to protection against RSV immune challenge, a collaboration has been established with Dr. Fernando Polack (Johns Hopkins University) to test the hypothesis that loss of gene function will enhance development of lower respiratory tract infection during primary RSV infection in a cohort of infected infants recruited in Buenos Aires, Argentina. Our fourth project has been designed to investigate the role of antioxidant genes in the pathogenesis of bronchopulmonary dysplasia (BPD) in very low birth weight (VLBW) infants. BPD is the most frequent cause of chronic pulmonary illness in infants. In collaboration with Dr. Fernando Polack, we are prospectively examining the role of candidate antioxidant genes in susceptibility to BPD. We believe that understanding the role of antioxidant genes in susceptiblity to BPD can lead to immmediate identification of susceptible populations requiring a personalized medical approach, evaluation of available gene-targeting antioxidant agents of potential preventive and/or therapeutic value, and extension of these observations to other important oxidant stress-related diseases in the near future (e.g. COPD, lung cancer, asthma).