Single-cell analysis reveals the range of transcriptional states of circulating human neutrophils

Abstract

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. To date, neutrophils have been characterized based on discrete parameters including cell-surface markers, buoyancy, maturation status, or tissue localization. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two endpoints defined by either relative transcriptional inactivity (Nh2) or high expression of type I interferon-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.

Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases

“…This approach has been used to characterize neutrophil development from granulocyte–monocyte progenitors in the bone marrow, which mature into myelocytes and eventually into mature neutrophils that are released into the circulation. Recent advances in single-cell technologies have enabled researchers to analyse neutrophil heterogeneity and neutrophil dynamics with unprecedented granularity, revealing the presence of distinct transcriptional subsets in health and disease in both humans and mice 8 – 13 .…”

“…Recently, neutrophil subpopulations in healthy humans and mice have been described, including a subset of neutrophils that express higher levels of type I interferon-stimulated genes (ISGs) than other neutrophil subsets 10 , 13 , 23 . Although the physiological and/or pathogenic roles of this interferon-responsive neutrophil subset remain to be determined, it is possible that they represent primed cells that may more readily combat infections and/or patrol the blood.…”

“…For example, LDGs produce higher levels of some cytokines, including type I interferons, and are prone to increased NET formation with vasculopathic and immunostimulatory features 30 . LDGs, as defined by density gradient, contain a mix of cells that are at various transcriptional states, likely representing different origin, state of maturation and/or effector functions 13 , 29 , 31 . These cells are discussed in more detail below in relation to disease states such as systemic lupus erythematosus (SLE).…”

“…New technologies, such as multi-omic sequencing, have enabled researchers to study immune cells with unprecedented granularity and detail, providing insights into the regulation and function of neutrophils in health and disease at the single-cell level. However, it is clear that neutrophils require special protocols, including practical handling and analysis of data, even when using modern ‘-omics’ assays 13 .…”

Cross-species analysis identifies conserved transcriptional mechanisms of neutrophil maturation

“…However, to date the transcriptional states of zebrafish neutrophils during maturation and associated functions in vivo have not been mapped to their human equivalents, thus limiting cross-species comparisons. Comparative transcriptomic studies have been hampered by under-representation of neutrophils in many human datasets due to technical difficulties and by a lack of suitable zebrafish lines allowing effective sorting of neutrophils comprising multiple maturation stages 26 .…”