193 Epidermal invasion by Malassezia spp. yeasts in 3D-reconstructed tissue

Abstract

Being usually harmless commensal components of the skin microbiome, Malassezia spp. sometimes become involved in human disorders like pityriasis versicolor where yeasts proliferate and invade the cornified layer, leading to alterations in pigmentation and mild itching. Mechanisms underlying the shift from commensal to pathogenic yeasts are still unclear. Experimental tools that could enable studies of the infection process used by Malassezia are required in order to understand their interactions with keratinocytes and better manage these kind of diseases and their treatments. Here, an in vitro model using reconstructed human epidermis (RHE) is described, based on topical application of Malassezia furfur individuals grown in suspension. Seeded on RHE preincubated with olive oil, M. furfur is able to proliferate and invade the tissue, as assessed respectively by quantitative PCR of yeast-specific DNA and histology of RHE using PAS-staining. This model exhibits lesions mimicking human pityriasis versicolor, with invasion limited to the cornified layer. Most notably, the formation of hyphae is observed. Simultaneous with the developing infection, the integrity of the epidermal barrier of RHE is altered, as revealed by lower trans-epithelial electrical resistance and increased permeability to Lucifer Yellow. Keratinocytes in infected RHE react to the presence of yeasts by enhancing mRNA expression and release of several pro-inflammatory cytokines (i.e. IL-8, TSLP, TNFα, IL-1α, IL-1β, IL-17C) and antimicrobial peptides (AMPS; i.e. hBD2, hBD3, Ribo7, S100A7) into the culture medium underneath the tissue. Overexpression of Toll-Like Receptor (TLR) 2 by keratinocytes in infected RHE suggests involvement of this TLR in the detection of Malassezia by epidermal cells, as well as in triggering expression and release of cytokines and AMPs. This model provides experimental conditions to further investigate changes in Malassezia’s phenotype, when individuals behave as pathogens, and to identify how keratinocytes perceive and react when intimately exposed to them.