Architecture of the neutrophil compartment in mice [scRNA-seq rewilded]

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: 8 weeks old Male BALB/C and 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, lung and tumors were enzymatically digested to generate cell suspensions that 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). 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 neutrophil suspensions were Taged (BD Multiplexing Kit) and pooled in equal proportion. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to the manufacturer instructions. Briefly, cell suspensions from each condition were incubated with Sample Tags (BD) for 20 minutes at RT. Cells were then washed three times and pooled in a single tube. Viability and cell concentration using the Countess III cell counter (Thermofisher). 60,000 cells were loaded into a Rhapsody Single Cell Analysis System cartridge. Cell capture and cDNA synthesis were performed according to manufacturer's instructions; cells were isolated into nanowells by gravity, then cells lysed and mRNAs together with sample tags oligos were released and captured by beads present in the nanowells. Each bead contained a unique oligo named “cell label” to identify each individual bead. All beads present in the cartridge were collected and cDNA synthesis took place in a single reaction. At this point each cDNA and Sample Tag oligo were attached to its corresponding “cell label” oligo. Two separated indexed libraries were prepared for whole transcriptome analysis and Sample Tag demultiplexing following manufacturer's instructions. The average size of the libraries was calculated using the 2100 Bioanalyzer (Agilent) and the concentration was determined using the Qubit fluorometer (Thermofisher). Finally, libraries were combined and sequenced together in a paired-end run (60x42) using a NextSeq 2000 system (Illumina) and a P2 flow cell. Output files were processed with NextSeq 1000/2000 Control Software Suite v1.4.1. FastQ files for each sample were obtained using BCL Convert v3.6.3 software (Illumina).