Single-Cell Transcriptome Analysis of Peripheral Neutrophils From Patients With Idiopathic Pulmonary Arterial Hypertension.

Abstract

Idiopathic pulmonary hypertension (IPAH) is a rare and devastating disease often accompanied by persistent inflammation and immune responses. We aim to provide a reference atlas of neutrophils to facilitate a better understanding of cellular phenotypes and discovery of candidate genes. Peripheral neutrophils from naive patients with IPAH and matched controls were profiled. Whole-exon sequencing was performed to exclude known genetic mutations before establishing single-cell RNA sequencing. Marker genes were validated by flow cytometry and histology in a separate validation cohort. Seurat clustering analysis revealed that the landscape of neutrophils encompassed 5 clusters, including 1 progenitor, 1 transition, and 3 functional clusters. The intercorrelated genes in patients with IPAH were mainly enriched in antigen processing presentation and natural killer cell mediated cytotoxicity. We identified and validated differentially upregulated genes, including matrix metallopeptidase 9 (MMP9), ISG15 ubiquitin-like modifier (ISG15), and C-X-C motif ligand 8 (CXCL8). The positive proportions and fluorescence quantification of these genes were significantly increased in CD16+ neutrophils in patients with IPAH. The higher proportion of positive MMP9 (matrix metallopeptidase 9) neutrophils increased mortality risk after adjustment for age and sex. Patients with higher proportions of positive MMP9 neutrophils had worse survival, while the fraction of ISG15- or C-X-C motif chemokine ligand 8-positive expression neutrophils failed to predict outcome. Our study yields a comprehensive dataset of the landscape of neutrophils in patients with IPAH. The predictive values of a neutrophil cluster characterized by higher MMP9 expression indicate a functional role for neutrophil-specific matrix metalloproteinases in the pathogenesis of pulmonary arterial hypertension.