Single-cell RNA sequencing and binary hierarchical clustering define lung interstitial macrophage heterogeneity in response to hypoxia.

ABSTRACT

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% [Formula see text]) or exposed to 1 day (D1) or 7 days (D7) of hypoxia (∼10% [Formula see text]). 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.