TESTING
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3D modelling of Malassezia furfur skin interaction
Valérie Borrel, Antoine Florin, Maëlle Garion, Christel Boudry, Michel Salmon - Straticell
Malassezia furfur is a lipid-dependent yeast naturally living on the skin. Despite its tolerance by the immune system under healthy condition, M. furfur overgrowth is associated with skin disorders such as dandruff. Dandruff and its more severe form seborrheic dermatitis (SD), are common chronic inflammatory conditions characterized by an abnormal shedding of the skin in seborrheic areas of the body. While dandruff is restricted to the scalp, SD also affects oily rich areas of the face, causing itching, pruritic lesions and erythema. Environmental and internal stress factors,
dysregulation of the immune system and fungal colonization are amongst the main contributors to the development of SD and dandruff.1
It has
been described that in dandruff, the quantity of Malassezia can increase up to 1.5 to 2 times its normal level.2 This huge spread of yeast highly invades the
epidermis, with detrimental consequences on the skin barrier function. Because the epidermis is the principal barrier against the penetration of chemicals and pathogens, any disruption increases microbial invasion and triggers both innate and acquired immune response.3 Keratinocytes are playing a central role
in this response by secreting key signaling molecules like antimicrobial peptides to overcome pathogenic infections, as well as various cytokines and interleukins to recruit inflammatory cells. In the case ofMalassezia overgrowth, previous studies have reported the expression of some defensins and pro-inflammatory cytokines in response to infection.4,5,6
SD and dandruff are highly disconformable
scalp disorders. Above the itchy feeling, flaking skin is socially embarrassing, forcing patients to expanse large amount of money on treatments. Given the association of M. furfur with SD and dandruff, one treatment option is to prevent its expansion using broad spectrum antimycotic agents like Ketoconazole (KTZ). KTZ increases the fungal membrane fluidity,
therefore limiting the yeast multiplication. Recently, KTZ has demonstrated its efficacy to reduce the quantity ofMalassezia in SD while restoring the skin microbial communities.7,8 Nowadays, new antifungal solutions
are entering the market, with a need to demonstrate their efficacy to reduce the growth of M. furfur and restore the skin barrier. Organotypic 3D skin models have become
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essential tools to understand the biological activity of compounds on the skin. In order to accelerate the knowledge about
Malassezia-host interactions, we decided to develop a 3D skin model that replicates Malassezia infection. Here, we present the successful colonization of reconstructed human
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epidermis (RHE) with a living strain of M. furfur. After confirming the effective growth of the
yeast on the stratum corneum of the RHE, the response of the tissue to this colonization has been studied by monitoring key biomarkers.
M. furfur 0 hour
M. furfur M. furfur + KTZ 72 hour
Figure 1: Malassezia furfur growth on the stratum corneum of reconstructed human epidermis in the presence of Ketoconazole (M. furfur + KTZ) or not (M. furfur). After 72 hours, yeasts were harvested and plated on an adapted solid microbiological media for the counting of colony-forming units (CFU)
Colonization of RHE with M. furfur and barrier disruption For the development of this new 3D model, human epidermis were reconstructed from primary normal human epidermal keratinocytes (NHEK). At the end of the reconstruction, M. furfur was laid on the stratum corneum in a lipid mixture representative of the ecological niche of this lipophilic yeast. As growth control conditions, the yeast was applied in the absence or presence of KTZ. After three days, yeasts were harvested from the top layer of RHE, and plated on an adapted solid microbiological media for the counting of colony-forming units (CFU). A sevenfold growth was counted in the absence of KTZ (Figure 1). However, in the presence of this antimycotic
agent, the growth was reduced fourfold compared to the initial yeast inoculum. These results confirmed the survival and growth of M. furfur on the RHE, and the efficacy of KTZ to effectively reduce this multiplication. From a histological point of view, the yeast- specific periodic acid-Schiff (PAS) staining of paraffin-embedded RHE demonstrated that after three days of growth, M. furfur passed the stratum corneum of the RHE to clearly invade
November 2022 PERSONAL CARE
CFU counting (log CFU/cm2
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