Transcriptional Diversity Among Alveolar Neutrophils During Pneumonia

Abstract

Neutrophils are transcriptionally dynamic during acute pneumonia. Our prior work demonstrated that 1733 genes were differentially expressed between peripheral blood and alveolar neutrophils during pneumococcal pneumonia. In multiple settings of chronic inflammation or malignancy, functionally distinct neutrophil subsets have been defined. This current study was designed to test the hypothesis that acute pulmonary infection leads to the development of multiple functionally distinct neutrophil subsets in the alveolar space. METHODS: Neutrophils were isolated from blood and BAL fluid from two C57BL/6 mice infected with S. pneumoniae for 24 hours. Single cell RNA sequencing was performed, utilizing the 10x Genomic Chromium and Illumina NextSeq 500 systems. Clusters were generated using Celda and visualized using tSNE dimensionally reduction. Gene expression patterns were analyzed within and between Celda-defined cell clusters. RESULTS AND CONCLUSIONS: Sequences were obtained from an average of 3200 cells per sample and 30k reads per cell, resulting in 28,692 features, aligned to 3,481 genes among 12,790 cells. Non-neutrophils were identified by known sequence markers and excluded from further analysis. Peripheral blood neutrophils had a distinct transcriptome from BAL neutrophils, consistent with prior bulk transcriptomic data. The peripheral blood neutrophil transcriptome was largely uniform, whereas subsets of alveolar neutrophils demonstrated significant transcriptomic diversity. Variable genes included central regulators of inflammation and apoptosis, and secreted chemokines and cytokines.