52 TESTING CONTROL A PAS M FURFUR M. FURFUR + KTZ
B LY
Figure 2: Representative images of reconstructed human epidermis uncolonized (Control) or colonized byMalassezia furfur in the presence of Ketoconazole (M. furfur + KTZ) or not (M. furfur). A: Periodic acid-Schiff staining of paraffin-embedded epidermis. B: Lucifer yellow fluorescence after out/in epidermal barrier diffusion assay
the epidermal barrier (Figure 2A). A slight thickening of the epidermis was also observed in the presence of the yeast. Additionally, as expected, addition of KTZ weakened the multiplication of M. furfur, therefore reducing the invasion of the epidermal barrier. In order to evaluate this barrier disruption,
we performed a transepidermal Lucifer yellow (LY) diffusion assay on colonized RHE. LY is essentially a fluorescent dye able to diffuse from the outside to the inside layers of a tissue if the intercellular spaces are enlarged, typically like in disrupted RHE. Both the quantification of fluorescence in
the culture media after diffusion of the dye throughout the RHE and the quantification of remaining fluorescence in paraffin-embedded RHE, reflect the permeability of the RHE. In the case of M. furfur-colonized RHE, LY applied on the stratum corneum was able to diffuse to the basal layer, as observed by fluorescent images (Figure 2B) and by quantification in the culture media (Figure 3). In contrast, treatment with KTZ reduced M.
furfur intrusion to allow an accumulation of green dye in the top layer of the RHE (Figure 2B)
450 400 350 300 250 200 150 100 50 0
and a resulting lower quantity of fluorescence in the culture media (Figure 3). Altogether, those data confirmed that the invasive yeast clearly declined the outside/inside epidermal barrier of the RHE, in a KTZ-limiting way. Impact on gene expression The observation of the disrupted barrier led us to investigate deeper the impact of M. furfur on the response of the tissue to this invasive stress. To this end, we conducted a RT-qPCR gene expression analysis (Figure 4) and observed that the expression of genes playing role in the structure of the barrier such as loricrin, filaggrin, desmoglein, or keratin-1, -10 and -14 was statistically downregulated. Additionally, the expression of genes coding
for the matrix metalloproteinases MMP-1 and -9 involved in the turnover of extracellular matrix components were upregulated. Regarding the expression of inflammatory genes, we observed that the expression of the interleukins IL-1- alpha, IL-1-beta, IL-8, and IL-23A genes were upregulated, up to 140-fold time for the IL-8 gene. Similarly, the expression fold change of
defensin genes like the Human Defensin DEFB4A gene or the antimicrobial psoriasin
200 150 100
35 -5
15 0
S100A7 gene were around 150 time overexpressed. This extended transcriptomic analysis confirmed the effective activation of the immune response following M. furfur colonization, as well as an impairment of the epidermal structure, at the gene level.
Secretion of protein biomarkers In order to support those transcriptomic data, we aimed to quantify the release of some proinflammatory mediators. As described earlier, the RT-qPCR analysis revealed a particularly significant overexpression of IL-8 and DEFB4A genes. Given the major involvement of these two
biomarkers in the response to infection, we selected two specific ELISA assays measuring the Human Beta Defensin 2 (HBD-2, encoded by the gene DEFB4A) and the IL-8 cytokines to define the respective levels of those proteins in the culture media of RHE colonized with M. furfur, in the presence or absence of KTZ. In harmony with the previously obtained
RT-qPCR results, ELISA assays demonstrated that M. furfur significantly induces the secretion of HBD-2 and IL-8 proteins in the culture media, by a respective fivefold and tenfold change
M. furfur
Figure 3: Quantification of out/in Lucifer Yellow diffusion through the stratum corneum of reconstructed human epidermis uncolonized (Control) or colonized byMalassezia furfur in the presence of Ketoconazole (M. furfur + KTZ) or not (M. furfur).
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M. furfur
M. furfur + KTZ
Figure 4: Analysis of inflammatory, innate immunity and epidermal genes differentially expressed in reconstructed human epidermis colonized by Malassezia furfur during three days. Changes in gene expression are expressed as fold to control (uncolonized reconstructed human epidermis). DEFB4A: Human Beta Defensin 4A - DSG1: Desmoglein 1 - FLG: Filaggrin - IL’s: Interleukins - KRT’s: Keratins - LOR: Loricrin - MMP’s: Matrix Metalloproteinases - S100A7: Psoriasin
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Lucifer Yellow diffusion (% relative to Control)
mRNA expression (fold change to control)
DEFB4A
S100A7 IL-1 alpha IL-1 beta
IL-8 IL-23A
MMP-1 MMP-9 KRT-1 KRT-10 KRT-14 LOR FLG DSG1
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