scRNA-seq–based neutrophil subset description and comparison to murine subsets

Neutrophil substate 0, the most abundant substate, is characterized by the highest expression of CXCR2 and CXCL8, along with Fc gamma receptors like FCGR2A, FCGR3A, and FCGR3B. In line with a more advanced maturity, substate 0 neutrophils express high levels of CXCR4 (85). Substate 1 expressed proinflammatory markers (GCA, IL1B, SOD2, C5AR1, and TNFRSF1B) and interferon-stimulated genes (IFITM2 and MXD) (Fig. 1M and fig. S2, A to C). Substate 2 neutrophils are defined by up-regulation of the translational machinery, including eukaryotic translation EEF1A1 and ribosomal proteins, suggesting high translational activity in line with a younger substate (fig. S2B). Neutrophil maturation markers such as CXCR2, MNDA, or FCGR3B/CD16, CD10/MME, as well as l-selectin/SELL are less abundantly expressed in substate 2 (Fig. 1N). Neutrophil substate 3 exhibits a pro-inflammatory transcriptomic phenotype with up-regulation of MMP9, several S100 proteins, and genes promoting NET formation (PADI4, HIST1H2AC, and H2AC6; Fig. 1, L and M, and fig. S2A). Last, substate 9 neutrophils transcribe high levels of genes implicated in interferon signaling and was characterized by expression of activation markers like l-selectin (SELL) and CD177, respectively, and S100 proteins (Fig. 1, M and O, and fig. S2A).

Comparing both patient data and the reanalyzed data from Xie et al. (21), we find highly conserved populations, such as the ISG-expressing populations G5b (mouse), hG5b (human), and substate 9 (human) (Fig. 3, J to L). Similarly, substate 0 neutrophils show signatures comparable with populations G5c and hG5c, while cell state 3 neutrophils resembled murine and human populations G5a and hG5a (Fig. 3, K and L) (21). Shown violin plots are downsampled and include a similar number of cells per condition.