Modelling epidermal infection by Malassezia spp. yeasts using reconstructed human tissue

Abstract

The human skin microbiome consists in the whole commensal microorganisms actually living on the skin and is composed of numerous microorganisms such as bacteria, archaea, viruses, and fungi. Malassezia yeasts are part of the skin commensal microbiome and, due to their lipophilic nature, colonize sebum rich regions of the skin (e.g. neck, face). The upper most layer of the human skin, namely the epidermis, both living in symbiosis.
Even though being harmless in most individuals, Malassezia spp. can be involved in several skin disorders. Notably, in pityriasis versicolor, Malassezia spp. proliferate and invade the cornified layer which is the upper most layer of the epidermis leading to pigmentation alterations and mild itching However, mechanisms underlying the shift between commensal and pathogen for Malassezia yeasts are still poorly understood. Furthermore, because of the long-lasting antifungal treatment required to treat these diseases, Malassezia spp. are prone to develop drug resistance phenotype. Thus, it is important to understand Malassezia infection processes and their interactions with the human epidermal cells (i.e. keratinocytes) to develop new therapeutic strategies.

Here, we developed an in vitro model of Malassezia infection by topical application of Malassezia furfur yeasts onto a reconstructed human epidermis (RHE). In presence of a source of lipids, M. furfur was able to invade and proliferate over the tissue, as assessed by morphological analyses and quantitative PCR. This infection model was representative of in vivo human pityriasis versicolor lesions, with invasion limited to the cornified layer, allowing the study of infection mechanisms deployed by Malassezia yeasts, as well as the subsequent responses of keratinocytes.

Notably, morphological analyses revealed the presence of extracellular matrix between Malassezia yeasts, maybe biofilm, as well as the formation of hyphae. During the invasion of the tissue by Malassezia, the epidermal barrier integrity of RHE was altered, as assessed by trans-epithelial electrical resistance and Lucifer Yellow permeability measurements. Simultaneously, keratinocytes of infected RHE responded to the presence of yeasts by increased mRNA expression, assessed by RT-qPCR after total RNA extraction, and release, evaluated by ELISA assay on culture medium, of several pro-inflammatory cytokines (i.e. IL-8, TSLP, TNF⍺, IL-1⍺, IL-1β, IL-17C) and antimicrobial peptides (AMPS; i.e. hBD2, hBD3, Ribo7, S100A7).

Overexpression of Toll-like receptor 2 by keratinocytes of infected RHE, assessed by RT-qPCR after total RNA extraction, suggested the involvement of this receptor in the detection of Malassezia by keratinocytes and possibly in the subsequent induction of cytokines and AMPs expression and release. However, previous studies also revealed the existence of Malassezia indirect interactions with keratinocytes, notably through the release of extracellular vesicles or indoles by Malassezia yeasts. Even though not tested in our study, our results suggest that these interactions could take place early after inoculation, but further studies are required to confirm this hypothesis.

Date of Award	19 Jan 2021
Original language	English
Awarding Institution	University of Namur
Supervisor	Yves Poumay (Supervisor)