Evasion or subversion of host immune responses have been shown for a variety of microorganisms, and this might be the case for Trichophyton rubrum, the most common pathogenic fungus causing chronic dermatophytosis in humans. Keratinocytes, the main epidermal cells, have important roles as a first defense against microbial challenges in local immune reactions. Epidermal keratinocytes express several Toll-like receptors and produce host defense peptides, cytokines and chemokines in response to various stimuli. We analyzed the expression of Toll-Like receptor TLR2, TLR4, TLR6, and Human Beta Defensin (HBD)-1, HBD-2, Interleukin IL-1b and IL-8 production, when exposing primary keratinocyte cultures to T. rubrum. We observed changes in size and granularity of keratinocytes stimulated with either whole conidia or conidial homogenates compared to other treatments. Intact conidia decreased keratinocytes’ TLR2 and TLR6 expression without affecting that of TLR4, while conidial homogenates increased the expression of these three receptors. Interestingly, whole conidia decreased HBD-1 and HBD-2 production, whereas conidial homogenate increased it. No changes were observed in IL-1b and IL-8 production after stimulation with conidia or conidial homogenate. CONCLUSIONS. Our results suggest that: 1) Keratinocytes can recognize and respond to cell wall components of T. rubrum; 2) Viable intact conidia inhibit TLR-2 and TLR6 expression and decrease HBD-1 and HBD-2 production; 3) Conidial homogenate from T. rubrum increases the expression of TLR2, TLR4 and TLR6 and induces HBD-1 and HBD-2 production; 4) Therefore, innate immune functions of keratinocytes as the first level of local skin immunity are apparently manipulated by T. rubrum, likely to ensure its establishment, persistence and survival.

Keratinocytes from human skin respond as typical immune cells after stimulation with Trichophyton rubrum. Trichophyton rubrum is the main agent causing dermatophytosis (1). Keratinocytes are considered to be the first physical barrier of defense against pathogens (2). But not only a physical barrier. They recognize antigens through Toll like receptors (TLR) (3). The activation of this TLR, present on the surface of the keratinocytes, induce the expression of different pro-inflammatory cytokines, costimulatory molecules and antimicrobial peptides such as beta β-defensins (4). The main objective of this work is to determine if lipopolysaccharides of G-bacteria (LPS), lipotheichoic acid from G+ bacteria (LTA), and conidias, isolated from T. rubrum were able to activate the expression of TLR2 and TLR6 on the cell surface of a primary culture of human keratinocytes through Flow cytometry. Furthermore we are looking for the presence of β-defensins 1 and 2, IL1b and IL-8 in the supernatant, of the above mentioned culture of cells, by Western blot. From the flow cytometry data, the preliminary results showed an important dispersion in terms of proliferation, increase in size and granularity of keratinocytes, from primary cultures of skin from healthy donors, stimulated 6 hours with conidias of T. rubrum, and LTA, but not when non stimulated, or stimulated with LPS (Fig 1). When keratinocytes from primary cultures of skin from healthy donors were cultivated 48 hours, it was found dispersion in terms of proliferation, increase in size and granularity when stimulated with conidias of T. rubrum, and LPS but not when non stimulated, or stimulated with LTA (Fig 2). The keratinocytes expressed increased levels of TLR2 and TLR6 when were stimulated with LTA and less to T. rubrum, in the 6 hours cultures, but this last cells still showed increased size (Fig 3). The Keratinocytes expressed increased levels of TLR2 in the 48 hours cultures when were stimulated with LPS and T. rubrum.(Fig 4) Besides, β-defensin-2 was detected in the supernatant of cultures of keratinocytes stimulated with LPS (Fig 5). It can preliminary be concluded that keratinocytes from primary cultures of human skin from healthy donors, are cells that respond as typical immune cells, after stimulation with T. rubrum, LTA and LPS in different conditions, and that this mechanism may be very important, for the protection of local environment. .