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Atopic dermatitis (AD) skin is characterized by over-expression of interleukin (IL)-4, IL-13 and IL-25. When methyl-β-cyclodextrin (MβCD) treatment preceded exposure to these interleukins, combination of both treatments was found to mimic hallmarks of AD in vitro, such as barrier weakening, histological alterations and typical signaling responses in a reconstructed human epidermis (RHE). However, the respective role of each IL and whether any of them is critical when combined with MβCD treatment was unknown. Therefore, this work aimed to distinguish RHE responses after exposure to MβCD and each one of the three IL reported to mimic typical features of AD. IL-4 incubation preceded by MβCD was found responsible for altered histology, as well as for barrier alterations, evidenced by electrical resistance and dye permeation measurements. This combination further decreased loricrin (LOR) immunoreactivity, whereas mainly IL-25, combined to MβCD treatment, was able to downregulate filaggrin (FLG) mRNA level. Carbonic anhydrase II (CA2) and hyaluronan synthase 3 (HAS3), two other markers up-regulated in AD, were also induced when MβCD treatment was followed by IL-4, whilst the expression of neural epidermal growth factor-like 2 (NELL2) was up-regulated by paired IL-4 and IL-13. In conclusion, multiple features of AD were found in this in vitro model mainly when treatment of RHE by IL-4 was conducted after preliminary MβCD incubation.

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Keratinocyte monolayers, cultured in immersed conditions, constitute a frequently used in vitro model system to study keratinocytes behaviour in response to environmental assaults. However, monolayers lack the keratinocyte terminal differentiation and the organization of the epidermal tissue, which are observed in vivo. Advancements of in vitro techniques were used to reconstruct three-dimensional equivalents that mimic human epidermis in terms of layering, differentiation and barrier function. Here, we update a published method and illustrate the progressive morphogenesis responsible for in vitro reconstruction. The analysis of cell proliferation, expression of differentiation markers and barrier efficacy demonstrate the excellent similarity of the reconstructed tissue with normal human epidermis. Availability of epidermal tissue during its reconstruction phase in culture appears crucial for studies intending to challenge the barrier function., (© 2012 John Wiley & Sons A/S.)