Neutrophil development at single cell resolution

Abstract

Neutrophils are involved in bactericidal functions and play essential roles in host defense response. It has been identified that many genes (e.g. GFI1, ELANE, HAX1, and CSF3R) are critical for the development of neutrophils. Individuals who carry any dysfunctional mutations of these genes cause severe congenital neutropenia, which is characterized as impaired maturation of neutrophils. A supply shortage of neutrophils can lead to serious consequence, and it may cause death due to severe infection. What’s more, elderly people are more susceptible to pathogen invading that is partially resulted from dysfunctional development of neutrophils. However, current studies uncovered limited information for potential molecular regulations, it hence is absolute necessary to elucidate further underlying mechanisms of neutrophil development in young and aging. In this study, recently developed state-of-art single cell technique was employed for profiling transcriptome of cells in bone marrow and peripheral blood. To comprehensively identify developmental trajectory of neutrophils, a novel quality control strategy was proposed in consideration of natural attributes of low RNA content for mature neutrophils. Applying this strategy for a published dataset of single RNA sequencing (scRNA-seq) resulted more than 21, 000 newly identified cells, which confirmed its tremendous power for recognizing low RNA content cells. Adopting this strategy, we profiled single cell transcriptome from 106, 077 cells in bone marrow and peripheral blood of young and aging mice. Initially, developmental trajectory of neutrophils in bone marrow under homeostasis of young mice was characterized in detail. Our results revealed a series of extraordinary heterogeneous neutrophil subsets of four previously reported stages (proNeu, preNeu, immNeu, and matNeu), which indicated their complex dynamic developmental progress. And these neutrophil subsets in peripheral blood compartment exhibited enormous phenotypic differences. Along their differentiation lineage, we identified some increasingly expressed potential driver genes (e.g. Gfi1, Cebpe, Jdp2, and Tfec) with probable important roles during fate determination. And expression landscape of neutrophil development was found gradually altered, which represented their strictly regulated maturation processes. Furthermore, our cellular communication analysis investigated their complex characters in cell-cell interactions of secreted signaling, cell-cell contact, and ECM-receptor. Of our great interests are IL1, CCL, and CXCL signaling pathways, which showed distinct outgoing and incoming communication patterns for these neutrophil subsets. Notably, our results interpreted developmental dependent molecular basis of their signaling transduction. Our comparative exploration of single cell transcriptome in aging mice confirmed dramatical alterations of neutrophil development, including their trajectory and transcriptional phenotypes. We found aging introduced oxidative stress to neutrophils and reduced their ability in bacterial recognition, migration, and activation. Besides, aging related alterations of signaling pathways, including IL1, CCL, and CXCL, were identified from our comparative cellular communication analysis. Interestingly, our results revealed aging related decreased signaling effects of IL1 and CCL for neutrophil subsets in consist of their decreasingly expressed receptors but despite of their increasingly expressed ligands. In summary, this study uncovered neutrophil heterogeneity in bone marrow and peripheral blood of mice, and identified aging related alterations that reflected molecular bases of their dysfunctional states.