Involvement of the anti-inflammatory protein TSG-6 in the context of a fungal dermatophyte infection in a reconstructed human epidermis model

    Student thesis: Master typesMaster en sciences biomédicales à finalité spécialisée en recherche préclinique

    Résumé

    Background: Dermatophytosis is a widespread superficial fungal infection of keratinized structures of the host. Despite its increasing prevalence, mechanisms underlying the infection are still unestablished. Moreover, TSG-6 protein, a hyaladherin with anti-inflammatory properties, appears to be overexpressed in several and diverse inflammatory conditions, as in the dermatophytosis.
    Aim: The main objective of this master thesis is thus studying the involvement of TSG-6 protein in the pathophysiology of the dermatophytosis, especially in a fungal dermatophytosis produced by Trichophyton rubrum.
    Methods: The model of dermatophytosis induced by T. rubrum previously developed on reconstructed human epidermis (RHE) produced from primary keratinocytes has been adapted for immortalized N/TERT keratinocytes, edited for TSG-6 (TSG-6-/-) and non-edited (TSG-6+/+). The development of infection by T. rubrum on TSG-6+/+ and TSG-6-/- RHE was characterized through morphological observation, assessment of fungal load by absolute quantification of infecting agents via qPCR, and measurement of the epidermal barrier integrity by transepithelial electrical resistance and Lucifer Yellow permeability. Simultaneously, keratinocyte responses were analysed by measuring the expression and release of cytokines and antimicrobial peptides, respectively via RT-qPCR and ELISA. Because Toll-like receptors (TLR) have been demonstrated to be involved in the recognition of fungal elements and in the implementation of the responses of keratinocytes, some signaling pathways downstream of TLR were investigated by Western blotting.
    Results: The infection by T. rubrum appeared to develop quicker in TSG-6-/- RHE than in TSG-6+/+ RHE. As demonstrated by morphological studies and assessment of the fungal load, fungal elements reach the living layers of TSG-6-/- RHE within three days following infection while they remain confined in the stratum corneum of TSG-6+/+ RHE. The impact of the infection onto the epidermal barrier integrity, as well as responses of keratinocytes, seems similar between TSG-6+/+ and TSG-6-/- RHE. On another hand, HA release in the underlying culture medium of TSG-6-/- RHE is increased, without any variation in expression level of enzymes involved in HA synthesis or degradation. Finally, a delay in the phosphorylation of ERK, which is notably a component of the TLR signaling pathway, is observed during the infection of TSG-6-/- RHE.
    Conclusion: Altogether, our results suggest a delay in the development and implementation of the immune response in TSG-6-/- RHE, resulting into a more rapid invasion of fungal elements inside the tissue. As TSG-6 protein is involved in the retention of HA, the ECM is probably disorganized in TSG-6-/- RHE. This disorganization seems to induce a delay, or an incorrect presentation of molecules produced by fungal elements to TLRs, leading to a delay in the establishment of immune responses.
    la date de réponse17 janv. 2022
    langue originaleAnglais
    L'institution diplômante
    • Universite de Namur
    SuperviseurYves Poumay (Promoteur)

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