Malassezia spp. are commensal fungi, normally colonizing the human skin, which sometimes become involved in skin disorders, like pityriasis versicolor (PV). Dermatophytes are strictly pathogenic fungi responsible for dermatophytosis. During PV or dermatophytosis, filamentous fungal structures named hyphae invade the cornified layer of the epidermis while inflammatory responses are induced in host cells. Models of infection by Malassezia furfur or by Trichophyton benhamiae on Reconstructed Human Epidermis (RHE) have been described as representative of in vivo fungal invasion and skin inflammation. During infection, keratinocytes are the first cell type encountered by developing fungal hyphae and reacts through the secretion of pro-inflammatory factors and antimicrobial peptides as players of innate immunity. However, how keratinocytes perceive the presence of fungi and respond is still unknown. The Toll-like receptor 2 (TLR2) basally expressed in keratinocytes has been reported being able to recognize fungal motives like phospholipomannan in Candida albicans. This work aims to better characterize the precise role of TLR2 expressed by keratinocytes when recognizing Malassezia and dermatophyte fungal motives, and how TLR2 induces inflammatory responses during infection.
For this purpose, TLR2-/--N/TERT-keratinocytes have been previously generated in our lab using the CRISPR/Cas9 method. As a first step, the procedures of infection by M. furfur and T. benhamiae were adapted for RHE made of N/TERT-keratinocytes (N/TERT-RHE). Indeed, weaker barrier function is observed in N/TERT-RHE in comparison with RHE reconstructed with primary keratinocytes (primary RHE), on which the infection procedure was originally established. Unexpectedly, we observed that M. furfur does not easily infect N/TERT-RHE in comparison to primary RHE, requiring a longer incubation period and an increased lipid supply. Regarding T. benhamiae, the development of infection appeared similar in N/TERT-RHE and in primary RHE. In a second step, RHE reconstructed with unedited TLR2+/+-N/TERT-keratinocytes (TLR2+/+-RHE) or from TLR2-/--N/TERT-keratinocytes (TLR2-/--RHE) were infected by T. benhamiae. No difference was observed in the infection process between TLR2+/+- and TLR2-/--RHE. Some decrease in the expression or release of tested markers were sometimes observed but no clear difference was observed in the signaling pathways activated following T. benhamiae infection between TLR2+/+-RHE and TLR2-/--RHE. Overall, our results suggest that, in addition to TLR2, other receptors also appear involved in the recognition of T. benhamiae and in the induction of inflammatory responses.
Role of TLR2 in the induction of inflammation in the context of cutaneous fungal infection
VAN der GUCHT, M. (Auteur). 15 janv. 2024
Student thesis: Master types › Master en sciences biologiques