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

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. 8 weeks old Male C57Bl6 mice were sacrificed via CO2, and blood, spleen, bone marrow, and lung were collected. 1ml of blood was collected in BD Microtainer tubes containing EDTA (365974, BD), and red blood cell were lysed in 10ml of 1X ACK buffer for 10min on ice. The spleen and lung cell suspensions were washed, resuspended in 1ml of 1X ACK buffer and incubated for 5min on ice to get rid of the red blood cells. The cell suspension was washed, resuspended in cold staining buffer, and kept on ice. The bone marrow was harvested by flushing the femur’s shaft with cold staining buffer and filtered to obtain a single cell suspension. The cells were washed and red blood cell were lysed using 1ml of 1X ACK buffer (5min on ice). After lysis, the cells were further washed, resuspended in cold staining buffer, and kept on ice. Cell suspensions were incubated with Fc block (TruStain FcX PLUS – 156603 – Biolegend) for 10min on ice, and for another 30min with an antibody cocktail containing: anti-Ly6G_PE (127608 - Biolegend), anti-CD11b_APC (101211 - Biolegend), TotalSeq_A Hashtag antibodies cocktail (Biolegend). The hashtag antibodies used were: TotalSeq_A0301 (155801, Biolegend – spleen), TotalSeq_A0302 (155803, Biolegend – bone marrow), TotalSeq_A0303 (155805, Biolegend – blood), TotalSeq_A0310 (155819, Biolegend – lung). Before acquisition and sorting, single cell suspensions were incubated for 15min with Dapi (NBP2311561, Novus Biologicals). Live-CD11b+-Ly6G+ neutrophils were sorted out. After sorting, the 4 neutrophil suspensions were enumerated and pooled in equal proportion. After that, we proceded with the library preparation.