Transcriptional and functional architecture of the whole neutrophil compartment [bulkRNAseq_human_donors]

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. Bone marrow samples were obtained from healthy donors under informed consent approved by the ethics committee of the University Hospital Tübingen. CD34⁺ HSPCs were isolated through Ficoll gradient centrifugation followed by magnetic bead-based separation using the EasySep™ Human CD34⁺ Cell Selection Kit II (Stem Cell Technologies, #17856). CD34⁺ cells (n=4; purity 95.4 ± 1.9%) were cultured at a density of 5 × 10⁵ cells/ml in StemSpan™ SFEM II hematopoietic stem cell medium (Stem Cell Technologies, #09655), supplemented with 1% penicillin/streptomycin (P/S), 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml TPO, 50 ng/ml SCF, and 50 ng/ml FLT-3L (all cytokines purchased from R&D Systems). Cells were cultured under standard conditions (37°C, 5% CO₂) and frozen for future use. For granulocytic differentiation in vitro, cells were seeded at a density of 2 × 10⁵ cells/ml. During the first 8 days of differentiation (days 0–7), cells were maintained in a myeloid cell expansion medium - RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin (P/S), 5 ng/ml SCF, 5 ng/ml IL-3, and 1 ng/ml G-CSF. The medium was changed every two days. On day 8 of culture, the medium was replaced with a granulocytic cell differentiation medium (RPMI 1640 supplemented with 10% FCS, 1% P/S, and 1 ng/ml G-CSF). The medium was changed every two days until day 14. On day 13 of differentiation, cells were collected and counted. 800,000 cells were lysed for RNA isolation, 50,000 cells for FACS, and 40,000 cells for cytospins. The remaining cells were resuspended in fresh granulocytic differentiation medium at a seed density of 2 × 10⁵ cells/ml and divided into four groups. Group one was maintained in granulocytic differentiation medium, group two was treated with 10 ng/ml TGF-β, group three with 10 ng/ml IFNβ (refreshed after 24h), and group 4 with 10 ng/ml GM-CSF. RNA-seq analyses were performed 48 hours after stimulation.