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程度高的城市地区，从而加剧感染的传播和严重程度。机遇：全基因组 RNAi 筛选可以发现流感病毒感染所必需的宿主基因和细胞功能。我们认为 RNAi 功能基因筛查方法还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，我们确定可溶形式的残留油飞灰（ROFA）是城市空气污染的替代品，可增强流感病毒诱导的肺上皮细胞死亡。这种基于细胞的检测方法最初用于功能遗传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，以确定介导污染增强的流感感染易感性的关键细胞基因。解决方案：目标 1：使用新型慢病毒 RNAi 文库，我们将进行全基因组筛选，以鉴定那些“敲低”抑制可在单独流感感染、单独柴油机尾气暴露和感染前暴露于污染物后提高存活率的基因。目标 2：使用与目标 1 相同的全基因组筛选策略，我们将鉴定在流感感染后暴露于污染物时介导细胞死亡增强的基因组。目标 3 将验证由 10 个顶级候选者组成的小组（根据功能效果的大小进行选择，并通过生物信息学分析来优先考虑生物学相关性和新颖性），方法是测试：1）肺上皮细胞的靶基因表达，2）单个 shRNA 的有效敲低，以及 3）单个基因敲低的表型复制（尽管暴露于污染物，但对流感的抵抗力增强）； 4)气道上皮（包括原代人肺上皮细胞）中表达和功能的类似测定。意义：该探索性项目将建立在有希望的试点数据的基础上，以确定介导流感空气污染物增强的功能定义基因。这些结果将为环境暴露如何影响这种重要感染的具体机制研究开辟新的思路。

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.