Although numerous wound healing models have been established, a model to study re-epithelialization in vitro, without disrupting the underlying dermal compartment, is lacking. Therefore, we established a new standardized human ex vivo model based on application of negative pressure, inducing blistering and consequently dermal epidermal separation. Effects of this treatment for epidermal regeneration and immune cells were investigated. Immunostaining revealed type IV collagen expression on the remaining dermal compartment, indicating that the basement membrane and thus the underlying dermis remained intact after removal of the blister roof epidermis. Keratinocytes were absent in the wounded area as evidenced by a lack of K14 staining. Four to six days after removal of the epidermis and culture, the wound was completely covered again with one to three-cell thick K14+ keratinocyte layers. Keratinocytes (∼20%) covering the initial wound bed expressed the endogenous cell proliferation marker Ki67, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing several epidermal differentiation markers (e.g., K10, filaggrin, DSG-1, CDSN). While application of negative pressure did not affect the morphology, phenotype and density of Langerhans cells, we observed more T cells in the blister roof epidermis as compared to normal epidermis. Additionally, we identified several populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating an influx upon pressure. Whether and how this contributes to the wound healing process is currently under investigation. This model recapitulates the main features of epithelial wound regeneration, and thus can be applied for testing wound healing therapies and investigating the underlying mechanisms in the future.

TH9 cells are considered to be a novel T cell subset, identified by the production of IL-9. These cells are mainly detected within the skin-homing TH population in peripheral blood and play a role in the pathogenesis of inflammatory skin diseases. As a better understanding of their biology may contribute to the development of novel therapeutic approaches, we adapted a skin T cell culture system to investigate the effects of IL-9 skewing cytokines on skin resident T cells. Healthy human skin biopsies were cultured on cell foam matrices (grids) either in the presence of IL-2 and IL-15 (standard condition) or IL-2, IL-4 and TGF-beta (TH9-promoting condition). Both culture conditions favored the proliferation of CD45RO+ T cells. TH9-promoting conditions yielded significantly higher numbers of CD4+ T cells as compared to standard conditions, while the frequency of Ki-67+ cells was similar under both conditions after 4 weeks. A differential expression of the skin-resident T cell markers CD69, CD103, and the skin homing receptors CLA and CCR4 was found on T cells using either culture condition. Paired analysis of T cells freshly isolated from skin and after 4 weeks under TH9-promoting culture conditions indicated drastic and significant downregulation of CD69 under both culture conditions and a trend towards upregulation of CD103 under TH9-promoting conditions. Significantly more T cells produced IL-9 under TH9-promoting conditions as compared to standard conditions upon CD3/CD28 stimulation and PMA/ionomycin restimulation. CD3+CD56+ cells were identified under standard conditions but not under TH9-promoting conditions. We show for the first time that IL-2, IL-4 and TGF-beta promote the expansion of IL-9-producing T cells from healthy human skin thus enabling further studies about their functional role in the skin.

Prevention of infections by using antiseptics is a key element in professional wound management. Ideal agents for the topical treatment of skin wounds should have anti-microbial efficacy without negative influence on wound healing. Octenidine dihydrochloride (OCT) has become a widely used antiseptic in modern wound care, yet its effects on skin physiology are mostly unknown. We have tested its impact on skin cells upon topical application on ex vivo non-tape-stripped and tape-stripped (simulation of wound situation) human skin explants. H&E and immunofluorescence staining revealed that standard OCT concentrations alter neither human skin architecture nor the viability of skin cells upon a culture period of 72 hours. Immunohistochemistry of epidermal sheets revealed that not only the epidermis of explants but also CD207+ Langerhans cells (LCs) in OCT-treated skin remained morphologically intact and comparable to controls throughout the culture duration. OCT also inhibited the upregulation of the maturation marker CD83 on LCs and prevented their emigration in tape-stripped skin accompanied by low MIP-3α levels. Additionally, OCT revealed strong anti-inflammatory capacity as shown by the inhibition of IL-8 and IL-33 upregulation in skin explant cultures. OCT largely blocked the upregulation of Gro-α, involved in the processes of wound healing and angiogenesis without significantly influencing the production of VEGF involved in vasculogenesis and angiogenesis. In conclusion, our data provide novel insights into the host response to OCT within the biologically relevant environment of viable human skin, suggesting, in addition to its known antimicrobial activity, an anti-inflammatory action that might contribute to the wound healing influence of OCT.

Adult human skin is home to huge numbers of T cells that as pivotal part of the immune system fulfill important roles in immune surveillance and play a key role in cutaneous immunity. T cells are already present in fetal skin, yet their function remains elusive. The aim of this study is to delineate the nature and significance of fetal skin T cells and to compare it with T cells in adult skin. As T cells are extremely rare in fetal skin we compared established and novel isolation techniques and succeeded to enrich them in sufficient numbers to better characterize them. The highest T cell yield was achieved with an automatic tissue dissociator in combination with an appropriate dissociation kit. This methodology preserved surface markers such as CD4, CD8, CD45RA and CD45RO upon single cell isolation. Flow cytometry analysis showed that prenatal CD3+ T cells were predominantly positive for CD4 and that higher CD8+ T cell percentages were found in fetal compared to adult skin. Further examination showed that most of the fetal skin T cells had a naive phenotype, while adult T cells largely expressed a memory phenotype. Moreover, we were able to expand sizable numbers of fetal T cells in vitro, using different culture conditions, which principally preserved the phenotype before culture. These studies will expand our understanding of the formation of important prerequisites for a functional skin immune system and consequently help to better understand many human skin diseases characterized by T cell activation and proliferation.