156 Chapter 5 (CD14highCD16+) monocytic cells increased. Next, we analyzed the macrophages (CD68+ cells) within the monocytic cell population and found a steady increase in macrophages over time after burn injury (Figure 3C-E). By both IHC and FCM we could detect a significant increase in macrophages at PBW 3. Macrophage phenotype was further investigated by analyzing CD40 and CD80 expression (indicative for pro-inflammatory phenotype) and CD163 and CD206 expression (hallmarks for pro-healing) (Supplementary Figure 2D). The only significant difference we observed was a reduction of CD40+ macrophages at PBW 3 (Supplementary Figure 2D). Using FlowSOM analysis of the FCM data, we identified macrophage subtypes with different expression patterns: CD163¯ macrophages (cluster 1), CD163+ macrophages with a low or moderate expression of CD40, CD80 and CD206 (cluster 2) and CD163+ macrophages with a moderate to high expression of CD40, CD80 and CD206 (cluster 3) (Figure 3F). A significant increase in macrophages in cluster 2 was observed in burn tissue at PBW1 and 3 (Figure 3G). Overall, this analysis demonstrated that burn injury increased the number of macrophages and changed their composition.
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