996 Atopic dermatitis model on RHE in presence of S.aureus and infiltrating immuno-competent cells

Abstract

Atopic dermatitis (AD) is a common skin disease linked to a dysregulation of the immune system and an impaired epidermal barrier. AD lesions show an increased susceptibility to infection by S. aureus, which triggers the production of thymic stromal lymphopoietin (TSLP) by keratinocytes. Based on Reconstructed Human Epidermis (RHE) with a double porosity polycarbonate filter allowing THP-1 monocyte infiltration, a unique in vitro model has been developed to better investigate the cutaneous immune response activated by S. aureus in AD. RHE with impaired barrier were colonized with S. aureus at 2x106 CFU/insert. THP-1 infiltrated the RHE for 4 hours. Tissue response was assessed after 4 hours (atopic rash), 16 and 48 hours (delayed inflammation) by qRT-PCR of inflammatory (TNF-α, TSLP, TLR-2), antimicrobial (human β-defensin 2) response and barrier (filaggrin, CLDN1, ZO-1) markers. Markers of THP-1 differentiation in APC were evaluated (CD86, CD14 and CD11b) as well as Th1/Th2-associated cytokines (TNF-α, IFN-γ, IL-4, IL-5, IL-8, IL-10, IL-13). For selected biomarkers protein expression was confirmed by immunolabelling. THP-1 successfully infiltrated the colonized RHE and differentiated towards a dendritic phenotype. A significant increase of the expression of IL-8, TLR-2 and TNF-α indicated an inflammation in the THP-1-RHE model in presence of S. aureus. TSLP expression presented a 20-fold increase while filaggrin expression decreased when THP-1 were in direct contact with the keratinocytes. S. aureus colonization of the RHE co-cultured with THP-1 also decreased filaggrin expression. Human β-defensin 2 increased by more than 100-fold in the AD model, reflecting keratinocyte reaction to S. aureus. The THP-1-RHE cell migration model seems to fully recapitulate the features of AD in vitro by taking into account the keratinocyte innate and inflammatory response and the immune-mediated response in presence of a S. aureus stable colonization.