160 Chapter 5 Figure 5. Local NK and B cell response to burn injury. Flow cytometry-based quantification of: (A) Absolute number of NK cells (CD56+ lymphocytes) per mg tissue; (B) Percentage of NK cells that are CD16¯ and CD16+; (C) Absolute number of B cells (CD19+ lymphocytes) per mg tissue. (D) Unsupervised clustering of NK and B cells in healthy skin and burn tissue, 4 clusters are highlighted. Node size represents relative size of population and node diagram shows expression level of markers. (E) Percentage of NK or B cells within each cluster. Error bars in E show boxplot, p values were calculated using Mann-Whitney U statistical test, significant differences are indicated by black asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. Immune cell infiltration coincides with high levels of cytokines, chemokines and growth factors The concentrations of 37 soluble immune factors were determined in homogenates of burn tissue using Luminex immunoassay (raw data is presented in Supplementary Figure 3). Figure 6 shows an overview of these results using volcano plots and heatmaps at 4 time intervals after burn injury. In burn tissue there was an extremely high expression of IL-6, IL-1β, IFN-γ and TNF-α compared to healthy skin. The levels of these factors were persistently high, but for IL-6 and IFN-γ the levels declined at the later time intervals. Interestingly, increased levels of IL-12p40 and IL-5 were found only late after burn injury
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