Figure 1. Phenotypic analysis of whole blood neutrophils reveals distinct neutrophil populations in mild and severe COVID-19.

Fresh whole blood from healthy controls and patients with COVID-19 was subjected to phenotypic analysis using a combination of markers identifying the activation, maturation, and immunomodulatory status of neutrophils. (A) Total neutrophil count from patients with mild (n = 41) and severe (n = 29) COVID-19. (B) Flow cytometric gating strategy for identification of neutrophils in whole blood. (C) A total of 63,000 cells concatenated from 6 healthy controls (HC) and 10 patients with mild COVID-19 and 5 patients with severe COVID-19 were visualized using uniform manifold approximation and projection (UMAP) for dimensionality reduction. The UMAPs of all samples and separate study groups are shown. (D) Median fluorescence intensity (MFI) heatmap of surface markers projected on UMAP. (E) Distribution of identified FlowSOM populations overlaid on the UMAPs. (F) Frequency of the FlowSOM populations within HC (n = 6) and patients with mild (n = 10) and severe (n = 5) COVID-19. (G) Heatmap of selected surface markers in the 8 FlowSOM populations (Pop). (H) The HyperFinder plugin in FlowJo was used to determine the shortest gating strategy for the identified FlowSOM populations. Fraction of mature-homeostatic (HC, n = 6; mild C-19, n = 10; sev C-19, n = 5), aged (HC, n = 6; mild C-19, n = 17; sev C-19, n = 6), immunosuppressive (HC, n = 6; mild C-19, n = 10; sev C-19, n = 8), immature non-degranulated (HC, n = 6; mild C-19, n = 10; sev C-19, n = 9), immature (HC, n = 6; mild C-19, n = 18; sev C-19, n = 8), immature-activated (HC, n = 6; mild C-19, n = 15; sev C-19, n = 7). Statistical analysis performed using 2-tailed Mann-Whitney test (A). Differences between HC and mild and severe groups were analyzed using ordinary 1-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01. Median with IQR is shown.