Dermatophytosis is a superficial fungal infection of keratinized structures. Its incidence is estimated around 20% in the global human population and has increased during the last decade. Despite such threatening incidence, precise information is still lacking about keratinocyte responses devoted to alert immune components and fight against infection. In order to identify and characterize such responses, fungal infection of reconstructed human epidermis (RHE) was undertaken. Arthrospores of the anthropophilic Trichophyton rubrum dermatophyte were topically seeded on RHE. Infected tissue was then maintained for 4 days in culture. Levels of expression and release of pro-inflammatory cytokines and antimicrobial peptides by keratinocytes were respectively assessed by RT-qPCR on RNA from tissue extracts and by ELISA on culture media. Integrity of the epidermal barrier was monitored using measurement of trans-epithelial electrical resistance or of dye-permeation through the RHE using Lucifer Yellow. During the first 3 days of infection, no response can be detected using these assays. On the fourth day after inoculating RHE with pathogens, cytokines (TNFa, IL-8, TSLP) are revealed in culture media and levels of mRNA encoding cytokines (TSLP, IL-1a, IL-1b, IL-8) or antimicrobial peptides (b-defensin-2, b-defensin-3, S100A7) are increased in keratinocytes. Simultaneously, the epidermal barrier of the RHE weakens. Altogether, these results suggest that dermatophytes inoculated on RHE initially remain confined inside the stratum corneum for 3 days after starting the infection, likely blocked in the superficial layer by an efficient barrier. On the fourth day, dermatophyte infection results into a barrier disruption in the RHE by a still unknown mechanism. Alteration of the epidermal barrier, allowing intimate contact between fungal processes and living keratinocytes, appears responsible for activation of epidermal, as well as immune defenses.

Analysis of keratinocyte response to Trichophyton rubrum dermatophyte infection in a model of reconstructed human epidermis