152 Chapter 5 RESULTS Burn injury is followed by a strong local increase in granulocytes and moderate increase in monocytes and lymphocytes Local immune effects of burn trauma were investigated in burn tissue that was debrided during routine surgical procedures (subject and sample characteristics shown in Supplementary Table 1). We selected viable sections of tissue biopsies and neglected necrotized or blackened segments to ensure the isolation of viable cells. CD45 immunohistochemical (IHC) staining showed an extreme infiltration of leukocytes (CD45+ cells) in burn tissue (Figure 1A). The majority of leukocytes were viable after isolation from healthy skin (90.3% ± 6.6) and burn tissue (89.1% ± 10.5) (Supplementary Figure 2A). Flow cytometry (FCM)-based quantification revealed that the increase in leukocyte numbers was most abundant at post burn week (PBW) 2-3 (Figure 1B). As a result, the percentage of CD45- cells, which include fibroblasts, keratinocytes and endothelial cells, was lower in burn tissue from PBW 2-3 than in healthy skin (Supplementary Figure 2B). The leukocytes isolated from healthy skin consisted of approximately 25% granulocytes, 55% monocytic cells (monocytes and macrophages) and 20% lymphocytes (Figure 1C). In burn tissue from PBW 1, there were 52% granulocytes, while for the proportion of monocytic cells was 29%. The lymphocytes fraction in burn tissue was similar to healthy skin (19%). In burn tissue from PBW 2-4, the portion of granulocytes was still enlarged (55-62%), while the fraction of monocytic cells decreased further to 13-16% and the lymphocyte fraction increased (24-31%). During PBW 1-3, absolute number of granulocytes, monocytic cells and lymphocytes rose and declined only at PBW 4 (Figure 1D). Multiplex spatial phenotyping of healthy skin and burn tissue sections using CD3 and CD15 revealed dense areas populated with granulocytes and T cells in burn tissue (Figures 1E,F).
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