Center for Pharmacogenomics, Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America.
Texas Biomedical Research Institute, San Antonio, Texas, United States of America.
PLoS One. 2018 May 30;13(5):e0198221. doi: 10.1371/journal.pone.0198221. eCollection 2018.
Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq's reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.
人类肺泡巨噬细胞（HAM）是结核分枝杆菌（M.tb）感染时细菌栖息地和免疫反应的主要细胞，而人外周血单核细胞衍生的巨噬细胞（MDM）则是研究 M.tb 与巨噬细胞相互作用的模型。在这里，我们使用靶向 RNA-Seq 方法来测量新鲜获得的 HAM（在 6 小时内使用）和在感染 M.tb 后 6 天培养的 MDM 的转录组范围内的 RNA 表达模式随时间的变化（2、24 和 72 小时），来自三个供体的未感染和感染细胞中各有三个。Ion AmpliSeq™ Transcriptome Human Gene Expression Kit（AmpliSeq）使用针对 18574 个 mRNAs 和 2228 个非编码 RNA（ncRNA）的引物，总共针对 20802 个转录物。AmpliSeqTM 产生高度精确和可重复的基因表达谱（R2>0.99）。利用 AmpliSeq 的可重复性，我们建立了 HAM 与 MDM 之间明确的定量 RNA 表达模式，包括部分在 MDM 和 HAM 之间不同的网络和途径中的显著 M.tb 诱导基因。在所有时间点，未感染的 MDM 和 HAM 之间检测到的表达基因数量相似，在包括炎症和免疫功能在内的常见途径中，但也存在经典途径的差异。特别是在 2 小时时，与免疫反应相关的多个基因在未感染的 HAM 或 MDM 中优先表达，而 HAM 的 RNA 谱在培养过程中随着时间的推移接近 MDM 的谱，突出了新鲜获得的 HAM 的独特 RNA 表达谱。在感染 M.tb 后，MDM 比 HAM 表现出更大的转录反应，上调或下调的基因数量增加了 2 到 10 倍以上。结果确定了两种不同的人类巨噬细胞类型中参与细胞对 M.tb 反应的关键基因。后续的生物信息学分析表明，大约 30%的反应基因在 GTEx 中有表达数量性状基因座（eQTLs），这些常见的 DNA 变体可以影响宿主基因表达对 M.tb 的易感性或抗性，用 TREM1 基因簇和 IL-10 来说明。
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