RNAi Screen in Air Pollutant-Enhanced Influenza Infection

描述(由申请人提供)：问题：空气污染增加了对流感感染的易感性，但机制尚不明确。潜在的公共卫生影响很大，因为未来的流感流行和大流行将涉及污染水平较高的城市地区，从而加剧感染的传播和严重程度。机会：全基因组RNAi筛查可以发现流感病毒感染所必需的宿主基因和细胞功能。我们的理由是，RNAi功能性基因筛查方法也可以为空气污染暴露如何改变流感易感性提供新的见解。在试点研究中，我们证实了可溶形式的残油飞灰(ROFA)作为城市空气污染的替代品，增强了流感病毒诱导的肺上皮细胞死亡。这种基于细胞的分析最初用于功能性基因筛选，揭示了新基因的参与，并支持了可行性。该项目的目标是进行RNAi筛选，以确定介导污染增加对流感感染易感性的关键细胞基因。解决方案：目标1：使用一种新的慢病毒RNAi文库，我们将进行全基因组筛选，以确定在单独感染流感、单独暴露柴油尾气和感染前暴露于污染物后，其‘敲除’抑制会导致更高存活率的基因。目标2：使用与目标1相同的全基因组筛选策略，我们将识别在流感感染后暴露于污染物时介导增强细胞死亡的基因集。Aim 3将通过以下测试验证由10个最佳候选基因组成的小组(根据功能效应的大小和生物信息学分析优先考虑生物相关性和新颖性)：1)肺上皮细胞目标基因的表达，2)单个shRNA的有效敲除，3)单个基因敲除的表型(尽管污染暴露，但对流感的抵抗力增强)；4)在呼吸道上皮(包括原代人肺上皮细胞)中类似的表达和功能分析。意义：这一探索性项目将建立在有希望的试点数据的基础上，以确定调节流感空气污染物增强的功能定义基因。这一结果将为环境暴露如何影响这种重要感染开辟新的具体机制研究路线。 与公共卫生相关：这一探索性项目将建立在有希望的试点数据的基础上，以确定调节流感空气污染物增强的功能定义基因。这一结果将为环境暴露如何影响这种重要感染开辟新的具体机制研究路线。

DESCRIPTION (provided by applicant): The Problem: Air pollution enhances susceptibility to influenza infection, but mechanisms are poorly defined. The potential public health impact is high, since future influenza epidemics and pandemics will involve urban areas with high levels of pollution exacerbating the spread and severity of infection. The Opportunity: Genome-wide RNAi screens allow discovery of host genes and cellular functions essential for infection by influenza viruses. We reason that the RNAi functional genetic screening approach could also provide new insights into how air pollution exposures modify susceptibility to influenza. In pilot studies, we established that the soluble form of residual oil fly ash (ROFA) a surrogate for urban air pollution, enhances influenza virus-induced cell death of lung epithelium. Initial use of this cell-based assay for functional genetic screening reveals involvement of novel genes and supports feasibility. The goal of this project is to perform RNAi screens to identify critical cellular genes which mediate pollution-enhanced susceptibility to influenza infection. The Solution: Aim 1: Using a novel lentiviral RNAi library, we will perform genome-wide screening to identify genes whose 'knockdown' inhibition results in greater survival after influenza infection alone, diesel exhaust exposure alone and pre-infection exposure to pollutant. Aim 2: Using the same genome-wide screen strategy as in aim 1, we will identify gene sets that mediate enhanced cell death upon exposure to pollutant after influenza infection. Aim 3 will validate a panel of 10 top candidates (selected for magnitude of functional effect, and bioinformatics analysis to prioritize biologic relevance and novelty) by testing for: 1) expression of the target gene by lung epithelial cells, 2) effective knockdown by individual shRNAs, and 3) replication of the phenotype (increased resistance to influenza despite pollutant exposure) by individual gene knockdown; 4) similar assays of expression and function in airway epithelium (including primary human lung epithelial cells). Significance: This exploratory project will build on promising pilot data to identify functionally defined genes that mediate air pollutant enhancement of influenza. The results will open new lines of specific mechanistic research in how environmental exposures impact this important infection. PUBLIC HEALTH RELEVANCE: This exploratory project will build on promising pilot data to identify functionally defined genes that mediate air pollutant enhancement of influenza. The results will open new lines of specific mechanistic research in how environmental exposures impact this important infection.