242 Chapter 8 proportion of CD14highCD16+ (intermediate) monocytic cells in burn tissue at post burn week 3 could indicate a relevant shift towards more M2-like macrophages. This shift that is needed to support wound healing [75] might be delayed after burn injury, slowing down the healing process. Inflammatory mediators that typify M1 macrophage activity (e.g. IL-1β, IL-6, IFN-γ, TNF-α, MCP-1 and IL-8) were increased in burn tissue, while the increase of M2 mediators (e.g. IL-4, IL-10, IL-13, TGF-β1 and VEGF-A) was limited [74–77]. On the other hand, investigation of the expression of M1 (CD40 and CD80) and M2 markers (CD163 and CD206) on macrophages revealed that most macrophages in burn tissue express markers of both subtypes. It was therefore impossible to classify macrophages based on these cell markers. Williams et al. found that increased number of M1 monocytes and decreased M2 monocytes was associated with slow wound healing and hypertrophic scar formation [78]. Because the classification of macrophages based on their marker expression is difficult, it is suggested to include analysis of gene expression and functionality as well [79,80]. Altogether, the composition of monocytes/macrophages and cytokine profile after burns might support M1 activity, while limiting or delaying M2 activity. This in turn will negatively affect wound repair functions. To study how burn injury exactly affects macrophage differentiation and how the different macrophage subtypes influence wound healing, macrophage functionality should be investigated. This research might include more determinative markers for macrophage polarization and analysis of protein and gene expression signatures. Other researchers described the use of markers such as CD11c, CD40, CD80, CD86, HLA-DR, iNOS, to study M1-like macrophages and CD163, CD204 (macrophage scavenger receptor 1), CD206 (mannose receptor), CD192 (CCR2), CD181 (CXCR1), CD182 (CXCR2), CD209 (DC-SIGN) for M2-like macrophages [77,81–84]. Nevertheless, some researchers have suggested that the binary M1/M2 classification might be too simplistic to catch the complexity of wound healing. Therefore, functional assays are required to better understand the behavior of monocytes/macrophages in blood and wound of burn patients during the different stages of wound healing. For example, the effect of burn-induced DAMPs on macrophages polarization and secretion profile could be investigated. In Chapter 7, we utilized our FSE models to study the effect of burn injury on the phenotype and cytokine production of PBMNC-derived monocytes. We showed monocytes differentiated into macrophages and that burn injury increased the amount of HLA-DR+ macrophages and inflammatory cytokines such as IL-1β, IL-6, IL-8 and IL-12p70. In future experiments, these macrophages could be polarized before they are added to the models to study the effect of different subtypes or compositions on wound healing. Ultimately, manipulation of the monocyte/
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