AbstractThe most crucial role of the human skin is to maintain an effective barrier between the organism and the environment. This barrier function is however perturbed in many inflammatory skin conditions like atopic dermatitis (AD), a disease we are focusing on in this PhD thesis. AD is one of the most common chronic inflammatory skin disease and the most common form of eczema in childhood, whose prevalence has considerably increased during the last decades, while currently no fully effective treatments have been found. Better understanding of the disease as well as evaluation of potential therapeutic actives is thus required. Therefore, the main goal of this research has been to reproduce epidermal features of AD in an in vitro model.
In a first part, we transferred the use of methyl-β-cyclodextrin (MβCD) from monolayer cultures of keratinocytes to reconstructed epidermis (RHE), because MβCD is a molecule able to deplete cholesterol from plasma membrane and which was reported by Conny Mathay and colleagues to reproduce some characteristics of AD at the transcriptomic level in monolayer cultures. Most of the major changes reported at the transcriptomic level in monolayer cultures of keratinocyte were also observed in RHE. Because AD pathology is characterized by epidermal barrier alterations, RHE were first incubated with the MβCD molecule, on day 5 of reconstruction when the barrier begins to form in the tissue. Such conditions however did not reveal any impairment to reconstruct a functional epidermal barrier. Furthermore, at day 11, when RHE closely mimic the in vivo epidermal situation, the tissues that had been incubated in the presence of MβCD at day 5 disappointedly appeared similar to the non-treated RHE.
Therefore, given the importance of the Th2 dysregulated immune response in AD, it was chosen to produce cholesterol-depleted RHE at day 11 and incubate them with three Th2-related cytokines (IL-4, IL-13 and IL-25) previously reported as playing important roles in the development of AD, as well as altering function of the epidermal barrier. When combining both treatments (MβCD and the three interleukins), several essential epidermal features were observed. Indeed, RHE treated that way exhibited spongiosis, disappearing granular layer, alteration of the barrier function, as well as dysregulated expression levels for genes involved in AD pathogenesis. While trying to identify individual roles for each component used to create AD-like alterations, incubation with IL-4 following cholesterol depletion from plasma membrane was found inducing most of the observed alterations, whereas the overall combination of all compounds studied was inducing prominent alterations in RHE.
In summary, the “AD RHE model” that has been progressively developed and analysed during this work, using a combination of plasma membrane cholesterol depletion in order to challenge keratinocytes before subsequent incubation with IL-4, IL-13 and IL-25, reveals potential for a better understanding of epidermal features that characterize AD. Finally, although some refinements should certainly be made to the proposed model, it may nevertheless be considered for eventual in vitro screening of cosmetic or therapeutic compounds in the context of AD and the linked altered epidermal barrier.
|Date of Award||20 Dec 2016|
|Sponsors||STRATICELL SCREENING TECHNOLOGIES & Programme Partenariat Public-Privé Région Wallonne|
|Supervisor||Yves POUMAY (Supervisor), Catherine Lambert De Rouvroit (Co-Supervisor), Carine MICHIELS (President), JEAN-PIERRE GILLET (Jury), Johanna Brandner (Jury), Michel Salmon (Jury) & Arjen F. Nikkels (Jury)|
Attachment to an Research Institute in UNAMUR
- reconstructed human epidermis
- atopic dermatitis