Tissue-specific IL-17-mediated immunity in experimental dermatophytosis

BURSTEIN VL; BECCACECE I; CHIAPELLO LS

Salvador

Congreso; International Congress of International Society of Human and Animal Mycology (ISHAM) and Infecciones Fúngicas en la Práctica Clínica de Latino América (INFOCUS); 2019

ISHAM

ObjectiveTo investigate the tissue-restricted antifungal immunity and the regulatory mechanisms of skin inflammation during an experimental model of epicutaneous infection with Microsporum dermatophytes.MethodsEight to ten-week C57BL/6, IL-17RA-/-, IL-17A-GFP reporter, Lang-EGFPDTR male mice were infected on depilated and slightly abraded back with a Microsporum canis or M. gypseum (N. gypsea) hyphae suspension. After diferent days post-infection (dpi), mice were euthanized and the following parameters were evaluated: histopathological analysis, skin fungal burden (colony forming units/skin), extracutaneous fungal dissemination (organ culture), skin cell populations (flow cytometry), cytokine production by skin-draining lymph node (sdLN) cells and by epidermal cell populations (flow cytometry and ELISA). For conditional depletion of langerin-expressing cells (Lang+ cells), mice were intraperitoneally injected with diphtheria toxin (500 ng). For IFN-γ in vivo neutralization, mice were injected with anti-IFN-γ on 3 and 6 dpi (R4-6A2, 100 µg). To inhibit lymphocyte recruitment to skin, mice were injected with fingolimod (FTY720, 40 µg). T-student test or ANOVA were used for statistical analysis.ResultsExperimental epicutaneous dermatophytosis mimics mild inflammatory human disease and is characterized by epidermal-limited invasion and neutrophil recruitment (p<0.0001). Furthermore, an specific Th17 immunity was induced in sdLNs (p<0.001) mainly produced by CD4+T lymphocytes (p<0.005) with langerin-expressing cells partially contributing to this response (p<0.002). IL-17-mediated immunity limited dermatophyte superficial overgrowth (p<0.01) however this type-17 response was not relevant for controlling pathogen dissemination to deeper tissuesnor affected neutrophil recruitment to the infection site (p<0.0001), demonstrating that IL-17 immunity and neutrophil infiltration are uncoupled and would contribute independently in skin. Interestingly, we found that dermatophyte infection of IL-17RA-deficient mice promoted an exaggerated skin inflammation (TNF p<0.0001, CXCL1 p<0.05, neutrophil influx p<0.0001) and a shift to a Th1-mediated response (p<0.01). Strikingly, in vivo IFN-γ neutralization in IL-17RA KO mice supressed fungal overgrowth (p<0.05), neutrophil recruitment to skin (p<0.0001) and TNF epidermal levels (p<0.0001). Moreover, anti-IFN-γ-treated KO mice restored the normal skin architecture along with up-regulation of IL-17 pathway-related cytokines like IL-22 (p<0.05) and IL-23 (p<0.001) suggesting that IFN-γ actively suppresses the control of dermatophyte proliferation in the absence of a functional IL-17 pathway and revealing an alternative mechanism by which IL-17 regulates cutaneous antifungal immunityEven though langerin-expressing cell depletion decreased Th17 cells frequency in lymph nodes (p<0.002) it did not modified skin fungal burden suggesting that other skin cell population locally produce IL-17A. To further insight into this phenomenon, we infected IL-17A-GFP reporter mice and observed that hematopoietic cells, specifically TCRβ+ (p<0.05) and TCRγδ+ lymphocytes (p<0.05), were the only IL-17-producing subsets and, after inhibiting lymphocyte egress from LN (FTY720 treatment), we demonstrated that IL-17A production in dermatophytosis was mainly due to tissue-resident TCRβ+CD69+ (p<0.05) and TCRγδ+ CD69+ (p<0.05) T cell populations.ConclusionOur investigations are the first that directly demonstrate the role of IL-17-mediated immunity in dermatophytosis and paves the way to further dissect tissue-specific immunity that deals with fungal pathogens.