Single-cell RNA sequencing and binary hierarchical clustering define lung interstitial macrophage heterogeneity in response to hypoxia.
Single-cell RNA sequencing and binary hierarchical clustering define lung interstitial macrophage heterogeneity in response to hypoxia.
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单细胞RNA测序和二元分层聚类定义了肺间质巨噬细胞对缺氧反应的异质性。
DOI：
10.1152/ajplung.00104.2022
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发表时间：
2022-07-01
影响因子：
4.9
通讯作者：
Stenmark, Kurt R.
中科院分区：
文献类型：
--
作者：
Campbell, Nzali, V;Mickael, Claudia;Kumar, Sushil;Zhang, Hui;Campbell, Ian L.;Gillen, Austin E.;Trentin, Caio O.;Diener, Katrina;Gao, Bifeng;Kheyfets, Vitaly O.;Gu, Sue;Kumar, Rahul;Phang, Tzu;Brown, R. Dale;Graham, Brian B.;Stenmark, Kurt R.
尽管巨噬细胞会导致缺氧肺动脉高压（PH），但很少有研究在单细胞水平上检测体内缺氧后肺间质巨噬细胞（IM）的分子表型。我们的目的是确定IM多样性及其与缺氧诱导ph的关系。我们假设整合单细胞RNA测序（scRNAseq）和二元层次聚类（BHC）可以解决正常稳态条件下和缺氧暴露引起的变化下IM异质性。Cx3cr1GFP/+报告小鼠暴露于常氧条件（~ 21%）或暴露于缺氧1天（D1）或7天（D7）（~ 10%）。我们使用流式细胞术分离Cx3cr1+ IMs，并使用10X Genomics平台进行scRNAseq、Cell Ranger、Seurat、ClusterMap、monocle、独创性通路分析和Fisher精确检验（q值< 0.05）进行功能研究。n = 374例（正常缺氧），n = 2526例（D1）和n = 1211例（D7） IMs被纳入分析。我们确定了三种正常缺氧相关的细胞类型，五种缺氧相关的细胞类型出现在D1，三种出现在D7。我们描述了一种假定的住院医师训练的先天IM的存在，它存在于常氧环境中，在D1时短暂耗尽，并在持续缺氧7天后恢复。我们还定义了一个罕见的假定致病群体，该群体与在D7出现的PH发育相关的转录本有关。最后，我们描述了BHC与scRNAseq的成功整合，以确定IM的异质性及其与ph的关系。这些结果揭示了住院医师训练的先天IM如何变得更加异质性，但最终习惯于缺氧。
Few studies have examined lung interstitial macrophage (IM) molecular phenotypes after being exposed to hypoxia in vivo at the single-cell level, even though macrophages contribute to hypoxic pulmonary hypertension (PH). We aimed to determine IM diversity and its association with hypoxia-induced PH. We hypothesized that integrating single-cell RNA sequencing (scRNAseq) and binary hierarchal clustering (BHC) could resolve IM heterogeneity under normal homeostatic conditions and changes induced by hypoxia exposure. Cx3cr1GFP/+ reporter mice were exposed to normoxic conditions (∼21% ) or exposed to 1 day (D1) or 7 days (D7) of hypoxia (∼10% ). We used flow cytometry to isolate Cx3cr1+ IMs and the 10X Genomics platform for scRNAseq, Cell Ranger, Seurat, ClusterMap, monocle, ingenuity pathway analysis, and Fisher’s exact test (q value < 0.05) for functional investigations. n = 374 (normoxia), n = 2,526 (D1), and n = 1,211 (D7) IMs were included in the analyses. We identified three normoxia-related cell types, five hypoxia-associated cell types that emerged at D1, and three that appeared at D7. We describe the existence of a putative resident trained innate IM, which is present in normoxia, transiently depleted at D1, and recovered after 7 days of sustained hypoxia. We also define a rare putative pathogenic population associated with transcripts implicated in PH development that emerges at D7. In closing, we describe the successful integration of BHC with scRNAseq to determine IM heterogeneity and its association with PH. These results shed light on how resident-trained innate IMs become more heterogeneous but ultimately accustomed to hypoxia.
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影响因子：
16
作者：
Martinon, F;Burns, K;Tschopp, J
通讯作者：
Tschopp, J
ClusterMap：比较不同实验条件下的多个单细胞 RNA-Seq 数据集
影响因子：
5.8
作者：
Gao, Xin;Hu, Deqing;Li, Hua
通讯作者：
Li, Hua
影响因子：
14.2
作者：
Gordon, Elizabeth M.;Yao, Xianglan;Xu, Haitao;Karkowsky, William;Kaler, Maryann;Kalchiem-Dekel, Or;Barochia, Amisha V.;Gao, Meixia;Keeran, Karen J.;Jeffries, Kenneth R.;Levine, Stewart J.
通讯作者：
Levine, Stewart J.
影响因子：
5.8
作者：
Osorio, Daniel;Cai, James J.
通讯作者：
Cai, James J.
影响因子：
56.9
作者：
Altman, JD;Moss, PAH;Davis, MM
通讯作者：
Davis, MM