Transcriptional and functional architecture of the whole neutrophil compartment [RNA-seq Hoxb8]

In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils. Our study provides a model that delineates the global architecture of the neutrophil compartment in mammals, and stablishes a robust framework for further exploration of neutrophil biology. Overall design: HoxB8-immortalized myeloid progenitors were routinely tested for mycoplasma contamination and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 30 µM ß-mercaptoethanol (Thermo Fisher Scientific), 1% supernatant from SCF-producing CHO cells, and 1 µM ß-estradiol (Sigma-Aldrich) to maintain the progenitor state. Neutrophil differentiation was initiated by ß-estradiol withdrawal and continued culture in medium supplemented with 1% SCF-containing supernatant. Differentiation into neutrophils was achieved by culturing cells in RPMI 1640 medium containing 10% FCS, 30 µM ß-mercaptoethanol, 4% SCF supernatant, and 20 ng ml?¹ granulocyte colony-stimulating factor (G-CSF) under standard tissue culture conditions (37 °C, 5% CO2) To generate .JunB Overexpressing HoxB8 cells we used Lentiviral vectors for Junb overexpression. Cells were co-transfected with (i) a transfer plasmid containing the Junb cDNA under the control of the human PGK promoter, (ii) packaging plasmid psPax2, and (iii) envelope plasmid pMD2.G encoding VSV-G.