UBC James Hogg Research Centre, Providence Heart + Lung Institute, St, Paul's Hospital, Vancouver, BC, Canada.
BMC Genomics. 2010 Jun 4;11:358. doi: 10.1186/1471-2164-11-358.
Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o- cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o- cells was evaluated using Agilent Whole Human Genome microarrays.
Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e.g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated).
We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility.
烟曲霉（A. fumigatus）是一种无处不在的真菌，通过释放大量空气传播的分生孢子（孢子）进行无性繁殖，这些孢子很容易被呼吸。在过敏和免疫功能低下的个体中，A. fumigatus 可引起广泛的疾病，包括变应性支气管肺曲霉病、曲霉肿和侵袭性曲霉病。先前的研究表明，A. fumigatus 分生孢子被巨噬细胞和肺上皮细胞内化；然而，气道上皮细胞对分生孢子的确切转录反应目前尚不清楚。因此，本研究旨在确定人类支气管上皮细胞系（16HBE14o-）与 A. fumigatus 分生孢子相互作用后对转录组的反应。我们使用荧光激活细胞分选（FACS）从表达绿色荧光蛋白的结合和/或内化 A. fumigatus 分生孢子的 16HBE14o-细胞中分离出没有孢子的细胞。然后分离总 RNA，并使用安捷伦全人类基因组微阵列评估 16HBE14o-细胞的转录组。
免疫荧光染色和制霉菌素保护试验表明，16HBE14o-细胞在共孵育 6 小时内内化了 30-50%结合的分生孢子。在对同一细胞培养物进行 FAC 分选以分离与分生孢子相关的细胞和没有分生孢子的细胞后，全基因组分析显示了一组 889 个基因在具有分生孢子的细胞中表现出差异表达。具体而言，这些 16HBE14o-细胞的基因转录本水平升高，这些基因与修复和炎症过程相关（例如，基质金属蛋白酶、趋化因子和谷胱甘肽 S-转移酶）。此外，差异表达的基因显著富集了基因本体论术语，包括：染色质组装、G 蛋白偶联受体结合、趋化因子活性和谷胱甘肽代谢过程（上调）；细胞周期阶段、有丝分裂和细胞内细胞器（下调）。
我们展示了一种使用 FACs 分析来自同一细胞群体中感染和未感染细胞转录组的方法，该方法将为未来描述 A. fumigatus 等病原体与人源细胞之间的特定相互作用提供框架，这些人源细胞来自有或没有潜在疾病易感性的个体。
