Division of Pulmonary and Critical Care Medicine, Department of Medicine.
Department of Biochemistry and Molecular Genetics, and.
JCI Insight. 2018 Sep 6;3(17). doi: 10.1172/jci.insight.123287.
Pneumonia represents the leading infectious cause of death in the United States. Foxp3+ regulatory T cells promote recovery from severe pneumonia in mice, but T cell responses in patients with pneumonia remain incompletely characterized because of the limited ability to serially sample the distal airspaces and perform multidimensional molecular assessments on the small numbers of recovered cells. As T cell function is governed by their transcriptional and epigenetic landscape, we developed a method to safely perform high-resolution transcriptional and DNA methylation profiling of T cell subsets from the alveoli of critically ill patients. Our method involves nonbronchoscopic bronchoalveolar lavage combined with multiparameter fluorescence-activated cell sorting, unsupervised low-input RNA-sequencing, and a modified reduced-representation bisulfite sequencing protocol. Here, we demonstrate the safety and feasibility of our method and use it to validate functional genomic elements that were predicted by mouse models. Because of its potential for widespread application, our techniques allow unprecedented insights into the biology of human pneumonia.
肺炎是美国主要的传染性致死病因。Foxp3+调节性 T 细胞可促进小鼠严重肺炎的恢复，但由于难以连续采集远端气腔样本，以及对有限数量的恢复细胞进行多维分子评估，肺炎患者的 T 细胞反应仍未完全明确。由于 T 细胞功能受其转录和表观遗传特征的控制，我们开发了一种安全的方法，可从重症患者的肺泡中对 T 细胞亚群进行高分辨率转录组和 DNA 甲基化分析。我们的方法包括非支气管镜支气管肺泡灌洗，联合多参数荧光激活细胞分选、无监督低输入 RNA 测序和改良的低覆盖度亚硫酸氢盐测序方案。在这里，我们证明了我们的方法的安全性和可行性，并使用它来验证通过小鼠模型预测的功能基因组元件。由于其具有广泛应用的潜力，我们的技术使人们能够以前所未有的方式深入了解人类肺炎的生物学特性。
