Host Responses to Malassezia spp. in the Mammalian Skin

The skin of mammalian organisms is home for a myriad of microbes. Many of these commensals are thought to have beneficial effects on the host by critically contributing to immune homeostasis. Consequently, dysbiosis can have detrimental effects for the host that may manifest with inflammatory diseases at the barrier tissue. Besides bacteria, fungi make an important contribution to the microbiota and among these, the yeast Malassezia widely dominates in most areas of the skin in healthy individuals. There is accumulating evidence that Malassezia spp. are involved in a variety of skin disorders in humans ranging from non- or mildly inflammatory conditions such as dandruff and pityriasis versicolor to more severe inflammatory skin diseases like seborrheic eczema and atopic dermatitis. In addition, Malassezia is strongly linked to the development of dermatitis and otitis externa in dogs. However, the association of Malassezia spp. with such diseases remains poorly characterized. Until now, studies on the fungus–host interaction remain sparse and they are mostly limited to experiments with isolated host cells in vitro. They suggest a multifaceted crosstalk of Malassezia spp. with the skin by direct activation of the host via conserved pattern recognition receptors and indirectly via the release of fungus-derived metabolites that can modulate the function of hematopoietic and/or non-hematopoietic cells in the barrier tissue. In this review, we discuss our current understanding of the host response to Malassezia spp. in the mammalian skin.

FiGURe 1 | Interaction of Malassezia spp. with the mammalian skin. Direct interactions involve various PRRs, which recognize fungal cell wall constituents and are distinctly expressed on the surface of non-hematopoietic (i.e., keratinocytes) and hematopoietic cells (i.e., Langerhans cells, mast cells) of the skin. Spatial and temporal signal integration of different PRR signals results in the induction of inflammation and immunity or, alternatively, in the regulation and tolerance of the host toward Malassezia spp. Indirect interactions of Malassezia spp. with the skin include fungus-derived metabolites such as irritant fatty acids on the one hand and indoles that are potent agonists for the AhR, which is expressed by various skin cells, on the other hand. KCs, keratinocytes; LCs, Langerhans cells; DCs, dendritic cells; Mϕ, macrophages; MCs, mast cells; SC, stratum corneum; AhR, aryl hydrocarbon receptor. maintained over time, despite the skin's exposure to the external environment (11). It is currently unknown whether Malassezia spp. play a mutualistic role and may thus contribute to immune homeostasis of the host. Apart from their commensal nature, Malassezia spp. are also associated with common skin disorders such as pityriasis versicolor and seborrheic dermatitis as well as more severe inflammatory skin pathologies including atopic eczema and atopic dermatitis in humans (10) and dermatitis and otitis externa in animals, most frequently in dogs (12). The composition of the skin mycobiome can vary under pathological conditions and some species of Malassezia such as M. sympodialis and Malassezia furfur are found to be enriched in certain skin disorders (10). To date, a causative link between Malassezia and disease development has only been made for Pityriasis versicolor, while the role of the yeast in other pathologies remains correlative (10,13,14). Changes in the degree of colonization in diseased compared to healthy skin have been documented in dogs (15) but remain uncertain in humans (16).
The pathophysiology of Malassezia-associated skin conditions is largely unknown. The lack of knowledge on the cellular and molecular interactions between Malassezia spp. and the host preclude a better understanding of the factors determining commensalism versus disease. Herein, we review the current knowledge with regard to how the host recognizes Malassezia spp. and responds to it (Figure 1).

SenSinG OF Malassezia spp. BY THe HOST
Through their localization in the skin, Malassezia spp. interact primarily with keratinocytes, tissue-resident dendritic cells (DCs), and macrophages, as well as with myeloid cells that are recruited to the skin under inflammatory conditions. Activation of DCs is key for induction of adaptive immunity and memory formation. The fungus is recognized by the host either directly through interaction of fungal cell wall components with membrane bound pattern recognition receptors (PRRs) or indirectly through soluble metabolites that are released by Malassezia spp. The set of receptors expressed by the hematopoietic and the nonhematopoietic compartment are largely distinct.

Direct Recognition of Malassezia spp. by Surface-Bound Receptors
The fungal cell wall is rich in carbohydrates and glycoproteins that are recognized by PRRs of the family of Syk-coupled C-type lectin receptor (CLR), which are expressed primarily by myeloid cells (17,18). Binding to these receptors results in ligand internalization and activation of multiple signaling pathways, including the MAPK, NF-κB, and NFAT pathways as well as the inflammasome.
The polysaccharides of the Malassezia cell wall are organized differently than in other fungal species analyzed to date (19,20). Moreover, the cell wall is surrounded by a lipid-rich outer layer (21). Several CLRs have been shown to respond to Malassezia spp. in vitro. The two FcRγ-associated receptors Dectin-2 and Mincle sense Malassezia spp., albeit through recognition of distinct ligands (22). While Mincle binds to two distinct glycolipids in Malassezia, Dectin-2 recognizes the fungus through α-1,2-linked mannose. High-mannose binding is a general feature of Dectin-2, which is reported to recognize a variety of fungi, including Candida albicans, Saccharomyces cerevisiae, Blastomyces dermatitidis, Aspergillus fumigatus, Cryptococcus neoformans, and Fonsecaea pedrosoi (23). In contrast, Malassezia spp. were initially found to be unique agonists of Mincle when a large panel of 50 different fungi was tested in a glycoconjugate microarray (24). More recently, other fungi such as Pneumocystis carinii, F. pedrosoi, and Fonsecaea monomorpha were also reported to engage Mincle (25)(26)(27), in addition to bacterial ligands (28)(29)(30)(31)(32), mammalian alarmins released from damaged cells (33,34) and even cholesterol crystals (35,36). Mincle is thus a highly pleiotropic receptor, which can bind chemically and structurally distinct ligands through at least two complementary binding sites (37)(38)(39)(40). The β-glucan receptor Dectin-1, which was the first member of the family of Syk-coupled CLRs to be identified (41), was also found to sense Malassezia and was linked to the activation of the NLRP3 inflammasome (42). Finally, Langerin was suggested to act as a receptor for Malassezia in the skin due to its prominent expression by epidermal Langerhans cells and by a subset of dermal DCs. Direct binding of the fungus to recombinant Langerin was indeed observed (43,44). Activation of myeloid cells by Malassezia spp. via these different CLRs was shown to induce the secretion of proinflammatory cytokines. However, the relative contribution of individual receptors to fungal control in vivo during commensalism and in infectious settings remains to be determined. At least partial redundancy of receptors that signal via the same pathway may occur, similarly to what was found for other fungi (45,46). Dissecting the role of Mincle in the context of Malassezia spp. in more detail will also be interesting in light of its reported antagonizing activity, e.g., in response to Fonsacaea spp. (27), and thus this receptor may also mediate regulatory or inhibitory responses to Malassezia spp.
In addition to CLRs, Toll-like receptors (TLRs), and in particular TLR2, also contribute to fungal recognition by the host. TLR2 was implicated in sensing of Malassezia spp. and inducing a proinflammatory response characterized by the release of cytokines, chemokines and antimicrobial peptides by keratinocytes (47)(48)(49)(50).
The proinflammatory response is generally enhanced by lipid removal from the yeast to enhance exposure of fungal cell wall carbohydrates (51,52). In contrast, thymic stromal lymphopoietin secretion from keratinocytes was found to be induced specifically by the lipid layer components of M. restricta and M. globosa but not by yeasts that were depleted of lipids (53).

indirect interaction
Specific products of Malassezia metabolic pathways are thought to act as virulence factors promoting inflammation and pathology, while others downregulate the production of inflammatory mediators and thereby contribute to immune regulation. Fungal strains with altered production of such factors have been linked to Malassezia-associated skin disorders (54)(55)(56).
Malassezia-derived lipases and phospholipases, which are required to assimilate host-derived lipids, can initiate an inflammatory response in the skin by releasing unsaturated free fatty acids from the sebum lipids (57)(58)(59)(60). Oleic acid has irritant and desquamative effects on keratinocytes (61)(62)(63), whereas arachidonic acid produces proinflammatory eicosanoids and leads to inflammation and damage to the stratum corneum, thereby contributing to the disruption of the epithelial barrier function and induction of abnormal keratinization (64).
Malassezia furfur is able to convert tryptophan into a variety of indole alkaloids. This pathway is mainly active if tryptophan is the sole source of nitrogen (65). M. furfur-derived indoles including malassezin, indirubin, and indolo [3,2-b] carbazole (ICZ) serve as potent ligands for the host aryl hydrocarbon receptor (AhR) and thereby potentially modify the function of all cells in the epidermis expressing this receptor (54,55,66,67). For example, some tryptophan metabolites can promote apoptosis of melanocytes (68) or inhibit the respiratory burst in neutrophils (69). Given the broad spectrum of biological responses that are influenced by AhR activity, M. furfur may engage this pathway to modulate inflammation and/or promote skin immune homeostasis (70) but may also promote skin pathology (71) or even contribute to carcinogenesis (72). The significance of yeast-derived indoles in each of these contexts remains to be demonstrated in vivo.
innATe iMMUniTY TO Malassezia spp.
The majority of what is currently known about the host response to Malassezia spp. is based on in vitro studies with isolated myeloid cells or keratinocyte cell lines. Stimulation of these cells with Malassezia yeast leads to the induction of mainly proinflammatory cytokines, chemokines, and antimicrobial peptides (22,24,(47)(48)(49)(50)(51)(52)(73)(74)(75)(76). In line with an inflammatory character of the innate response to the fungus, the intraperitoneal injection of Malassezia into mice results in the recruitment of neutrophils to the peritoneum (24). Only few studies have examined regulatory cytokines such as IL-10 and TGF-β by the yeast (24,49,51,74,77), but these may be relevant with regard to the role of Malassezia spp. as a skin commensal.
Given the association of Malassezia spp. with inflammatory skin disorders and allergic responses, the fungus may also interact with mast cells. Progenitor cell-derived mast cells from atopic patients show increased release of proinflammatory cytokines upon stimulation with Malassezia (76) and are enriched in the skin of atopic eczema patients where they are positioned in the superficial dermis and can interact with the fungus (78). Mast cell activation in response to Malassezia spp. has also been reported in studies with bone-marrow-derived mast cells. These cells are directly activated by the fungus in a TLR2-dependent manner and release inflammatory mediators and cytokines (79). Moreover, the crosslinking of the high-affinity IgE receptor (FcεRI) by antigen-bound IgE can induce mast cell degranulation (79). Therefore, mast cells may contribute to further barrier disruption and thereby amplify the inflammatory response. The access of Malassezia to immune cells in the skin may be facilitated by disruption of the epithelial barrier as it frequently occurs during chronic inflammation. Moreover, Malassezia spp. were reported to release nanovesicles/exosomes that contain immunogenic proteins and trigger increased release of cytokines by DCs (80).
ADAPTive iMMUniTY TO Malassezia spp.
As a commensal, Malassezia interacts continuously with the immune system. Therefore, cellular and humoral immune memory to the fungus can be evidenced in healthy individuals (81). Although there are fewer studies related to dogs when compared with humans, dogs also develop cellular and humoral immune responses to their commensal yeast, M. pachydermatis (82)(83)(84). Generally, the adaptive immune responses are heightened and qualitatively distinct in patients with Malassezia-associated diseases.

Humoral Responses
During steady state, Malassezia-specific antibodies are predominantly of the IgG and IgM isotypes (81). In contrast, although Malassezia-specific IgE is not usually detected in healthy individuals, it is common in atopic patients (85). A positive correlation was found between the sensitization to Malassezia-specific IgE and the severity of atopic dermatitis (86,87). Similar observations were made in atopic dogs (83,84). However, whether the IgE response plays a pathogenic role in atopic and other Malassezia-associated inflammatory disorders or rather serves as a marker for the severity of disease remains unclear.

T Cell Responses
Patients with atopic dermatitis often show positive skin prick test and atopic patch test reactions to Malassezia (85). T cellresponsiveness to Malassezia in such patients was associated with a Th2 response (88), in line with the classical paradigm of Th2-polarized allergic T cells. GATA3 + T cells were identified in pityriasis versicolor lesions (89) and likewise Malassezia-specific T cell in allergic dogs were found to be strongly polarized toward a type 2 response (82). More recently, other T helper cell subsets such as Th17 and Th22 cells have been found enriched in allergic individuals (90,91) as well as in non-allergic immune-mediated skin diseases such as psoriasis (92). Consistent with this notion, Malassezia-reactive skin homing T cells from Malasseziasensitized atopic dermatitis patients comprise not only Th1 and Th2 subsets but also IL-17-and IL-22-secreting cells (93). Of note, IL-4/IL-17 coproducers have also been described in the context of atopic eczema especially in children (94). Importantly, Th17 differentiation is a hallmark of T cell responses induced by CLR signaling (46) and T cells directed against other fungi, in particular Candida spp., belong predominantly to the Th17 subset (95). Whether and how IL-17 and/or IL-22 may contribute to pathogenicity in atopic dermatitis remains to be determined. It is also unknown to which subset Malassezia-specific T cells belong in healthy individuals and to what extent T cell plasticity contributes to sensitization.

Malassezia Allergens
To date, 13 Malassezia-derived allergens have been identified from M. furfur and M. sympodialis (3,96). Interestingly, more allergens are released from M. sympodialis when cultured at the increased pH conditions of atopic skin compared with culture at the pH of healthy skin (97). Several of the known allergens belong to a class of phylogenetically highly conserved proteins and display a high degree of homology with the corresponding mammalian proteins. Cross-reactivity between Malasseziaderived allergens and endogenous human proteins (e.g., thioredoxin, manganese-dependent superoxide dismutase) has been indeed demonstrated (93,98,99). Therefore, the induction of autoreactive T cells by Malassezia allergens may play a role in sustained inflammation.

COnCLUSiOn
Malassezia spp. have been implicated in various pathologies. Yet, direct evidence for a causal relationship between Malassezia spp. and the mammalian host remains elusive. For instance, it is unclear whether Malassezia actively promotes atopic dermatitis or whether the inflammatory environment in the atopic skin triggers a dysregulated immune response toward the fungus.
At the basis of this is the key question of what determines the balance between commensalism and pathogenicity of Malassezia spp. The answer likely relates to changes occurring in both the fungus (55) (e.g., variable secretion of AhR agonists) and in the host (e.g., barrier defects, changes in immune polarization) which are responsible for promoting the development of pathology. Changes in the environment such as seasonal variations in sebum production have also been linked to altered disease prevalence (100).
Inter-species variations in the skin mycobiome may further contribute as different species of Malassezia can induce variable inflammatory responses (51,75,101). Moreover, Malassezia spp. have been shown to display a large intra-species diversity (73) similarly to what is known for other opportunistic fungal pathogens (102), and thus the exact composition of Malassezia strains and species present in an individual at a given time may contribute to different outcomes in the interaction between the fungus and the host. The recently completed assembly and detailed annotation of the genome of M. sympodialis makes an important contribution to approach this complexity (103). Future research will help fill the important gaps in our knowledge on the pathophysiology of and the host response to Malassezia in vivo. Enhanced understanding of host-Malassezia interactions may contribute to improved diagnostic and therapeutic options for patients affected by Malassezia-associated pathologies.

AUTHOR COnTRiBUTiOnS
Both authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

FUnDinG
This work was supported by the University of Zürich.