New biomedical approaches for studying (patho)physiological conditions of healthy and inflamed skin in vitro

Abstract

Atopic dermatitis (AD) is the most common skin disease worldwide and the prevalence continues to increase in industrialized countries. However, due to the complex interplay between barrier disruption and the skin immune system, the pathogenesis of AD is still not fully understood. The use of human-based in vitro skin equivalents grown from patient-derived cells is a promising alternative approach to investigate pathogenic parameters in AD. However, skin biopsies, that are necessary for the collection of patient cells, are often associated with wound healing complications and scar formations. In this thesis, it was shown for the first time that both keratinocytes and fibroblasts can also be isolated from plucked scalp hair follicles and successfully used for generation of skin equivalents. Moreover, the established method laid the foundation for the generation of skin disease models with the help of patient cells. Over the past years, the impact of fibroblasts for disease induction and/or maintenance has been increasingly recognized. However, little is known about their actual contribution in AD. Hence, the impact of AD patient-derived fibroblasts, isolated from plucked hair follicles, on the tissue homeostasis of human-based skin equivalents was investigated. Interestingly, a subset of AD patient-derived fibroblasts induced characteristic features of AD in the skin equivalents such as hyperproliferation, altered expression of tight junction and skin barrier proteins. Notably, the expression of AD-related proteins such as thymic stromal lymphopoietin (TSLP) and protease-activated receptor 2 (PAR2) were significantly increased. A reduced expression of differentiation-associated cytokine leukemia inhibitor factor (LIF) seems to be linked to these effects. Exposure of hyperproliferative skin equivalents to CD4+ T cells resulted in T cell migration into the dermal equivalent, which was not observed in the equivalents grown from normal fibroblasts. Surprisingly, the addition of T cells to the disease equivalents improved the stratum corneum lipid profiles and distinctly attenuated PAR2 expression, probably as a result of increased LIF signaling due to the T cells. In addition to their effects in the skin equivalents, their angiogenic impact was investigated by co-cultivation with dermal microvascular endothelial cells. Interestingly, AD fibroblasts facilitated to the formation of an increased number of endothelial tubes and thus appear to be involved in angiogenesis. Overall, the results of this doctoral thesis demonstrate the pathogenic effects of AD fibroblasts. The major donor differences among the patients support the opinion that AD is a highly heterogeneous disease, which increases the difficulty of unraveling the pathomechanism of AD. In order to a better understanding of this multifactorial disease, the development of a mast cell-competent skin equivalent was introduced. This model might be a promising biomedical approach for the investigation of complex cell-cell interactions in AD lesions, which is currently poorly understood.