213 Immune Cell Model for Burn Immune Reactions Monocytes differentiated into macrophages in full skin equivalents and upregulated their HLA-DR expression upon burn injury Unstimulated monocytes were added to (burn-injured) FSEs to simulate an innate immune response to burn injury. About 2.5 × 105 monocytes were administered to the dermal side of the FSEs to prevent the cells from adhering to the culture transwell (see Figure 6B for procedure). Unstimulated monocytes cultured in suspension or in a matrix without skin cells served as controls. Using microscopy, we confirmed that the monocytes were present in the FSE, irrespective of burn injury. Monocytes seemed to lose or downregulate the monocyte marker CD14 (data not shown) and upregulate the expression of macrophage marker CD68 in the FSEs upon culture regardless of burn injury (Figure 2A), suggesting that these cells differentiated into macrophages. To study these monocyte-derived macrophages in more detail, FSEs that were cultured for 7 days were dissociated and the cells were isolated. Using flow cytometry, we studied the number of macrophages and the expression of several markers: CD68 (macrophage marker), CD14 (monocyte marker), CD11b (activation), HLA-DR (M1 differentiation) and CD163 (M2 differentiation). Uninjured FSEs contained on average 8.0 ×104 CD68+ cells (macrophages) (Figure 2B). There was high variation in the percentages of CD68+ macrophages that were CD14+ and CD11b+ (Figure 2C,D). There were samples with a low percentage of CD14+ and CD11b+ macrophages and samples with a high percentage. This level or rate of macrophage differentiation and activation in the FSE (with or without burn injury) is likely donor-dependent. The percentages of HLA-DR+ and CD163+ macrophages were lower in the FSEs than in cells cultured in the absence of skin cells (Figure 2E,F). Burn injury seemed to increase the average number of macrophages (1.6 × 105), although this did not reach significance. The percentage of CD14+ macrophages was significantly decreased after burn injury. Interestingly, burn injury significantly increased the percentage of HLA-DR+ macrophages and appeared to decrease the percentage of CD163+ macrophages in the FSEs. Thus, we generated a human FSE model with keratinocytes and fibroblasts incorporating unstimulated monocytes that actively differentiated into macrophages upon culture. Burn injury appeared to stimulate differentiation into M1 macrophages. 7
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