Abstract
Malassezia is the most abundant commensal fungus colonising our skin and the skin of many other mammals. The lipid rich environment of the skin provides an ideal niche for this lipiddependent yeast to grow. At the same time, cutaneous lipids are an integral part of the hydrophobic skin barrier protecting us from physical, chemical, and microbial threats and preventing excessive dehydration. Besides commensal colonisation of the skin, Malassezia has been linked to several skin pathologies including dandruff, seborrheic dermatitis, pityriasis versicolor and atopic dermatitis. The aim of this thesis was to elucidate the immunological response to Malassezia in healthy skin and in atopic dermatitis. First, we elucidated the host immune response to Malassezia under homeostatic conditions in adult experimental mice. Mice exposed to Malassezia on the ear skin for the first time mounted an inflammatory immune response. This response was characterised by early infiltration of neutrophils followed by monocytes and eosinophils. By day 7 post fungal association, lymphocytes mediated protective immunity against Malassezia by producing the cytokine IL-17. We further found an essential and dominating role of γδ T cells in mediating this antifungal immune response. In the absence of γδ T cells, IL-17 levels were strongly reduced, and fungal clearance was delayed. We found the γδ T cells to belong to the Vγ4+ subset. Malassezia-responsive Vγ4+ γδ T cells were not only found in the skin but were also present at high numbers in the ear-draining lymph nodes (dLN), where they were sustained long after fungal clearance. These memory-like γδ T cells were effective upon re-encounter of the fungus at least one month after fungal clearance. Blocking lymphocyte trafficking from the dLN to the colonised ear and, thereby, preventing these memory-like cells from entering the Malassezia challenged skin, led to an impairment in fungal control. The emergence of Malassezia-responsive Vγ4+ γδ T cells did not depend on CD11c+ antigen presenting cells, nor did we detect significant activation of the T cell receptor (TCR) upon Malassezia antigen presentation by dendritic cells. The response also did not depend on Toll-like receptor or on Card9-dependent C-type lectin receptor signalling either. The γδ T cell response did dependent on IL-23 and partially on IL-1 family cytokines. γδ T cells after prior Malassezia exposure responded directly to Malassezia-derived structures, which appeared to be secreted, heat-stable, and highly conserved among Malassezia species but no other fungi tested. After assessing the homeostatic response to Malassezia in the murine model of skin colonisation, we investigated the role of Malassezia in atopic dermatitis (AD). With a life-time prevalence of around 20% AD is a common chronic inflammatory skin disease that is marked by a disturbed skin barrier and a type 2 immune bias. AD poses a great physical and 4 psychological burden to patients due to its nature of evoking itch and eczema as well as an increased risk of developing secondary bacterial infections. Since sensitisation to Malassezia is common in AD, we sought to determine the role of Malassezia in a model of AD by epicutaneous application of calcipotriol (MC903). We found that AD-like skin showed excessive growth of Malassezia and an apparent dysfunction of antifungal immune control. Type 2 immunity did not interfere with effective fungal clearance as TSLP- and MyD88- deficient animals with AD-like skin displayed the same fungal overgrowth despite the lack of TSLP and IL-33-induced type 2 cytokine signalling. Additionally, when increasing IL-4 levels systemically by injecting IL-4 anti-IL-4 antibody complexes, fungal clearance was not negatively affected. We did find that MC903-treatment repressed the IL-17 response. Still, while baseline fungal burdens were increased in IL-17A/F-deficient compared to wildtype mice, the AD-like skin retained higher fungal loads even in the absence of IL-17. While neither excessive type 2 immunity nor deficient type 17 immunity were able to fully explain the fungal overgrowth, we investigated changes in the skin barrier function. RNA-Sequencing of bulk ear skin revealed a signature of cellular stress, wound healing, hyperkeratosis and altered lipid metabolism. Mice with an increased keratinocyte stress response facilitated by constitutive expression of Nrf2 in K5 + keratinocytes also showed hyperkeratosis and fungal overgrowth. Skin barrier disruption by tape stripping favoured Malassezia growth as well. Flaky tail mice, which display an AD phenotype due to mutations in Tmem79 and filaggrin genes, carried more Malassezia after association compared to mice with an intact skin barrier. Characteristic changes in flaky tail mice affect the lipid metabolism and encompass a switch to shorter-chain fatty acids, which, according to the literature, can be more efficiently used by the fungus as nutrient source. We found that Malassezia-genes related to lipid uptake and processing were upregulated under AD-like conditions. Altogether, this indicates that Malassezia can exploit the AD skin, which provides it with a niche for efficient colonisation due to the disrupted skin barrier and increased nutrient availability. Lastly, we could detect increased expression of different Malassezia secretory hydrolases in human atopic and seborrheic dermatitis patients compared to healthy controls. Targeted deletion of one major secretory hydrolase gene, i.e. Malassezia furfur secretory aspartyl protease 1 (MFSAP1), resulted in less skin inflammation in fungus associated mice, while the presence of MFSAP1 caused epidermal hyperplasia and neutrophil infiltration. In conclusion, this thesis provides insights into the γδ T cell-mediated protective type 17 immune response to the abundant fungus Malassezia. It further presents mechanisms of fungal adaptation to the atopic skin environment exploiting the more easily accessible impaired skin barrier as a niche for enhanced nutrient acquisition, and it describes potential enzymatic virulence factors overexpressed by Malassezia in atopic skin.
Ruchti, Fiorella