123 Systemic Immune Response in Burn Patients Figure 2. Unsupervised FlowSOM analysis of granulocyte and monocyte subtypes after severe burn injury. FlowSOM plots present proportions of populations and the expression of markers that were used in the innate flow cytometry panel (CD10, CD11b, CD14, CD15 and CD16). (A) Cluster structure based on flow cytometry data of 10 healthy controls and 7 burn wound patients that were observed for 4 weeks. The most pronounced subtypes are encircled by dashed lines: CD16+ monocytes (node 1), CD10bright neutrophils (nodes 2-7), CD10dim neutrophils (nodes 8-13), CD16¯ granulocytes (node 14), CD14brightCD16¯ monocytes (node 16). FlowSOM plots of: (B) Week 1; (C) Week 2; (D) Week 3; (E) Week 4 after burn; (F) Healthy controls. We verified the unsupervised findings by supervised flow cytometry analysis of data from all patients. The leukocyte increase after burn injury was indeed due to a rise in neutrophil numbers and was associated with shifts in maturation stage (Figure 3A). Eosinophil numbers (CD9+CD15+CD16¯ granulocytes) increased over time but only to a small extent (Figure 3B). The high number of immature neutrophils at 0–3 days after injury decreased after PBD 6, but remained higher than in healthy controls until PBD 34–36. Mature neutrophil counts increased at PBD 4 and remained elevated from PBD 7 onward (Figure 3C,D). Supervised analysis confirmed the persistent increase in classical monocytes, but also revealed an increase in intermediate CD14brightCD16+ and nonclassical CD14dimCD16+ monocytes. These data demonstrate that burn trauma induced a continuous release of (immature) neutrophils and monocyte subtypes. 4
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