219 Immune Cell Model for Burn Immune Reactions an innate and an adaptive immune reaction to burn injury. Cells of the innate immune system such as neutrophils and monocytes/macrophages are actively involved in the acute inflammatory phase after burn injury, while cells of the adaptive immune system such as T cells are crucial for regulation of ongoing inflammation [5,39]. In this study, we analyzed our immune cell model using microscopy and flow cytometry and focused on changes in cell phenotype and cytokine production. Unlike other models, we used human primary monocytes and were able to analyze them by flow cytometry 1 week after culturing them in FSEs and investigate the effect of burn injury. This model brings us another step closer to a more realistic skin model that is useful for the study of burninduced inflammation and therapeutic interventions to improve burn wound healing. Here, we showed that monocytes differentiated into macrophages within 7 days of culture in FSEs. Based on immunohistochemistry, we observed that monocytes in FSEs upregulated their CD68 expression over time while CD14 expression decreased, which is in line with findings of Smith et al. [40]. The percentage of HLA-DR+ macrophages was higher in burn-injured FSEs than in uninjured FSEs, which could be a reaction of the macrophages to the burn injury. Notably, in monocytes/macrophages cultured in suspension there was an even higher percentage of HLA-DR+ cells, which might have been induced by culturing on cell repellent surface. HLA-DR is an MHC class II receptor that is upregulated upon inflammatory stimuli and typifies M1 activity [41]. Furthermore, IL-1β production was only increased in burn-injured FSEs with macrophages, which is a characteristic M1 cytokine [41,42]. Also, CD163 expression, indicative of M2 activation, appeared to be slightly decreased in burn-injured FSEs, but this was not statistically significant. We observed much donor variation for markers CD14 and CD11b, which could be related to distinct activation or differentiation rates of different PBMC donors. Overall, monocytes differentiate into macrophages upon culture in FSEs and there was an indication that burn injury enhanced this polarization further, but more research is needed to clarify this finding. Other researchers have developed comparable skin models with macrophages to study skin diseases such as inflammatory skin disorders or carcinoma. Chung et al. co-cultured FSEs with RAW264.7 cells to simulate skin inflammatory responses [43]. In this model, the FSE was placed on a transwell membrane while RAW264.7 cells were cultured underneath the transwell. Using this co-culture system, the researchers demonstrated interactions between the skin cells and macrophages that affect cytokine production and the degree of inflammation. Griffoni et al. [44] made co-cultures of polarized PBMC-derived macrophages and FSEs and checked cell viability and expression of M1 and M2 specific genes. These researchers stressed the need for appropriate culture media to create more standardized and accurate immunocompetent skin models. Linde et al. created a 7
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