Background: Dermatophytosis, or tinea, is a poorly studied cutaneous fungal infection encountered by 70% of the population during their lifetime. The anthropophilic species of dermatophyte, Trichophyton rubrum is the main cause of tinea and its prevalence is continuously rising. This superficial fungal infection affects the keratinized structures of the host, namely hair, nails and epidermis. The epidermis is the body’s primary defense structure against pathogens and keratinocytes express Toll-like receptors (TLRs) able to initiate innate and adaptive immune responses when facing infection. Particularly, TLR-2 is incriminated as the main receptor recognizing fungal components and leading to inflammatory responses through the induction of its downstream signaling pathways.
Aim: The goal of this study is to assess the specific involvement of the TLR-2 receptor during dermatophytosis caused by T. rubrum and to determine the signaling pathways induced.
Methods: Firstly, read-outs of TLR-2 activation in keratinocytes are determined by the used of zymosan, a specific agonist of TLR-2. In practice, inflammatory responses and signaling pathways activated are monitored after keratinocytes stimulation by zymosan through different techniques including IL-8 ELISA assay, (RT)-qPCR, Western blot analysis and NF-κB translocation. In parallel, keratinocyte monolayers are infected with T. rubrum conidia and responses and signaling pathways are analyzed. Secondly, an edited N/TERT keratinocytes cell line deleted for the TLR2-coding gene is generated via CRISPR/Cas9 method. The clones obtained are cultured as monolayers and as reconstructed human epidermis (RHE), and treated with zymosan or infected by T. rubrum conidia. Responses and signaling pathways are then assessed and compared to those measured in non-edited N/TERT keratinocytes.
Results: In keratinocyte monolayers, the TLR-2 activation by zymosan resulted in IL-8 release, increase of the protein abundance of TLR-2, pIκB and pERK, and overexpression of inflammatory factors such as IL-8, IL-1α, IL-1β and β-defensin 2. Similar results were obtained with keratinocyte monolayers infected with T. rubrum. Regarding to N/TERT keratinocytes edition, four N/TERT TLR2-/- keratinocyte clones have been generated and characterized by sequencing. Two clones TLR2-/- have been used to reconstruct human epidermis. During infection of TLR2-/- RHE, signaling pathways and inflammatory responses are detected but in a lower extent compared to TLR2+/+ RHE.
Conclusion: Through zymosan treatment, read-outs of TLR-2 activation could be identified. Some of these results are induced during infection by T. rubrum on keratinocyte monolayers or RHE, and appeared reduced when using TLR2-/- keratinocytes. Altogether, these results seem to demonstrate an implication of TLR-2 during dermatophytosis, although TLR-2 appeared not essential for the induction of inflammation. The generated date encourage further research to precise the role of TLR-2 in the recognition of fungal material and in the induction of signaling pathways leading to an immune response.
Cutaneous fungal infections: analysis of TLR receptors involved in the epidermal response
DENIL, E. (Auteur). 18 janv. 2023
Student thesis: Master types › Master en sciences biomédicales à finalité spécialisée en recherche préclinique