Single-cell gene expression profiling of interstitial macrophages exposed to hypoxia

Employing single-cell transcriptomic analyses, we investigated the repertoire and functional profiles of pulmonary interstitial macrophages in response to exposure at 0, 1, 3, 7, and 21 days of hypobaric hypoxia (at an altitude of 5,486 meters). The repertoire and functional profiles of interstitial macrophages (IMs) undergo dynamic changes based on the duration of hypoxia exposure. Acute hypoxia leads to type 1 acute inflammatory responses in most IM populations, which largely subside by Day 7. The resolution of the inflammatory phase is succeeded by the accumulation of dysregulated IMs, contributing to abnormal pulmonary vascular repair and remodeling. The classical and alternative complement pathways may play distinct roles in regulating early inflammation and later vascular remodeling phases in pulmonary hypertension (PH) pathogenesis. These findings highlight the dynamic nature of IMs, emphasizing that understanding the timing and mechanisms through which they influence PH pathogenesis will contribute to the development of IM-specific therapeutic targets. Overall design: Cx3cr1+/GFP reporter mice were crossbred with C57BL/6J and raised. To eliminate potential confounding factors associated with adaptation to reduced oxygen levels at Denver altitude, Cx3cr1+/GFP animals were placed in a sea-level chamber (~21% fraction of inspired oxygen (FiO2)) at 5 weeks of age for 4 weeks to acclimate to sea-level conditions. Subsequently, animals were kept at sea level (Day 0) or exposed to hypoxic conditions (simulated altitude of 5,486 meters; ~10% FiO2) for 1, 3, 7, or 21 days. At each time point, the lungs of a pair of age- and sex-matched mice (1 male and 1 female) were harvested for subsequent analyses. Pulmonary interstitial macrophages were sorted by flow cytometry as previously described [PMID:]. Single cells were captured using a Chromium Box (10X Genomics, Pleasanton, CA). Libraries were sequenced using the Illumina NovaSEQ 6000 sequencer (Illumina, Inc., San Diego, CA). Subsequently, RNA sequence reads were aligned to the Mus musculus GRCm39 reference genome using Cell Ranger v6 (10x Genomics). Downstream analysis was performed using the Seurat package in R.