Supplementary Material for: The Effects of Human Beta-Defensins on Skin Cells in vitro

<p><b><i>Background:</i></b> Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. <b><i>Objective:</i></b> We aimed to investigate the effects of human β-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. <b><i>Methods:</i></b> Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. <b><i>Results:</i></b> We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. <b><i>Conclusion:</i></b> Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining.