220 Chapter 7 human skin squamous cell carcinoma model with PBMC-derived macrophages that was cultured up to 3 weeks. The researchers analyzed macrophage polarization and found M2 activation of macrophages in the tumor model [45]. In another proof-of-principle study, Bechetoille et al. produced an FSE with anti-inflammatory dermal-type macrophages and studied the effect of co-culture on cytokine production and phagocytic potential of the macrophages [46]. As opposed to these models, we studied the effect of burn injury on PBMC-derived monocytes by means of flow cytometry and cytokine production. When pre-activated T cells were added to the FSE, a portion of T cells actively migrated into the FSEs. In this population of migrated T cells, the percentage of Th1 receptor CXCR3 expressing cells [47] and Th17 receptors CCR4/CCR6 [48] expressing cells was increased, regardless of burn injury. This coincided with increased levels of pro-inflammatory cytokines IFN-γ, IL-6, IL-8, IL-12p70, IL-17A and IP-10. It has been shown before that the production of chemokines such as IP-10 can be induced by IFN-γ, especially in inflamed tissue [49,50]. IP-10 is a chemoattractant for T cells and binds to only one receptor, namely CXCR3 [51]. The decrease in IP-10 production in the burn-injured FSEs could be related to a loss of keratinocytes caused by the burn injury. The percentage of CD25+CD127¯ T cells, possibly Tregs, was also increased. Simultaneously, levels of IL-4, IL-10 and TGF-β1 were elevated. The production of IL-10 was, however, reduced in the burn-injured FSEs. This could also be related to the destruction of keratinocytes as about 18% of the model was burn injured or be the result of impaired regulatory activity caused by burn injury. Nevertheless, more research is needed to elucidate this. Our FSE with T cells was based on previous work, where T cells were cultured in a skin model to study cross-talk between keratinocytes and T cells [35]. Other researchers used similar skin models to focus on skin diseases such as psoriasis or atopic dermatitis [35,52,53]. In studies by Shin et al. and Lorthois et al. T cells were stimulated towards Th1/Th17 to study their role in psoriatic skin models [52,53]. Our model was unique for the study of burn injury. Of the pre-activated T cell pool, only a small portion of T cells migrated into the FSEs. This could be related to incomplete activation of T cells, T cell death, insufficient migratory activity, or suboptimal isolation from the FSEs. The isolation of T cells from FSEs was performed in the absence of collagenase, because collagenase treatment is known to affect the presence of chemokine receptors. This might have limited the yield of T cells compared to monocytes/macrophages from FSEs. Migratory activity of T cells can be enhanced by adding an additional chemotactic stimulus such as T cell chemokines MIP-1α (CCL3), MIP-1β (CCL4) and RANTES (CCL5) [54]. Finally, our T cell preparation technique could be improved by magnetic or fluorescence cell sorting to establish an enriched population of T cells before placing the cells in the model.
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