655 Epidermal response to bacterial and yeast adhesion: RHE model in co-colture with THP-1 cells

Abstract

The skin is colonized by bacteria and yeasts most of which are harmless or even beneficial. In vitro Reconstructed Human Epidermis (RHE) infected with commensal and pathogens has been developed and the keratinocytes mediated bacterial adhesion and defence mechanisms have been investigated focusing barrier properties impairment, innate immunity response and biofilm formation. RHE tissues were infected with Staphylococcus aureus -MRSA, S.epidermidis, Malassezia Globosa, At 6h and 24h time points after colonization the following parameters were measured: barrier function and fence properties (Biotin assay, claudin-1, integrinb1), bacterial counts, ultrastructural morphology of bacterial phenotype (planktonic or biofilm) by SEM analisys. SEM analysis indicates that cocci were adherent to the epidermal surface after 6h, proliferated and produced a dense biofilm structure after 24h; cocci were not able on intact RHE to penetrate deeper epidermal layers; they were able to modify the mineral elements (Iron) content on RHE surface. M.globosa deeply penetrated the SC and viable epidermis. Barrier function was impaired by S.aureus but not by S.epidermids. The co-culture model of RHE colonized with S. aureus and monocytes (THP-1) was setted up to elicit a Th2 response and to amplify S. aureus invasion modifications observed in an atopic phenotype at transcriptional level. The model takes into account the Keratinocyte innate and inflammatory response, the adaptive immunomediated response (co-colture with monocytes) in presence of a S. aureus stable colonization mimicking the feature of AD lesion. THP-1-RHE co-culture model in presence of S. Aureus colonies allows TSLP over expression suggesting Th-2 response activation, down regulation of Filaggrin and Defensin beta 2 (DEFB4), down regulation of immune-response genes TLR2. ITGA6 e ITGB1 were also down regulated compared to the control.