Effects of cytokines involved in a microenvironment mimicking atopic dermatitis in a well standardized three-dimensional model of normal human skin

Atopic dermatitis (AD) is one of the most common chronic skin inflammatory disorders, driven by several pro-inflammatory cytokines among which interleukin (IL)-4, IL-13, and IL-22 are the most involved mediators. Tight junctions (TJs) are targeted in AD from both a morphological and a molecular level, leading an epidermal barrier disruption, which is one of the clinical manifestations of AD. TJs seal adjacent keratinocytes in the granular layer and are composed by transmembrane proteins such as occludin and claudins, in addition to cytosolic scaffold proteins. The present study was aimed at evaluating the early effects of IL-4 and IL-13 using a well-standardized three-dimensional model of normal human skin. Skin explants were obtained from plastic surgery of healthy women (n=5) 20-40 years old after informed consent, and cultured overnight in Dulbecco’s modified Eagle’s medium and treated with 50 ng/mL IL-4, 50 ng/mL IL-13. Secondly, the samples were harvested 24 hours after the cytokine incubation and in parallel processed for morphological and molecular biology analysis through both Western Blot. Compared to control samples, a decrease of the expression of occludin was found in samples incubated with either IL-4 or IL-13. TEM analysis revealed an increase of the intercellular spaces and chromatine condensation only in samples treated with IL-4, with well preserved desmosomes. Conversely, samples incubated with IL-13 showed similar features as control ones. All these results suggest the usefulness of this experimental model, which could be used in order to study and evaluate the early and direct effects of several pro-inflammatory cytokines involved in AD, providing a well and standardize method for pre-clinic dermatology research.