γδ T cells respond directly and selectively to the skin commensal yeast Malassezia for IL-17-dependent fungal control.

Stable microbial colonization of the skin depends on tight control by the host immune system. The lipid-dependent yeast Malassezia typically colonizes skin as a harmless commensal and is subject to host type 17 immunosurveillance, but this fungus has also been associated with diverse skin pathologies in both humans and animals. Using a murine model of Malassezia exposure, we show that Vγ4+ dermal γδ T cells expand rapidly and are the major source of IL-17A mediating fungal control in colonized skin. A pool of memory-like Malassezia-responsive Vγ4+ T cells persisted in the skin, were enriched in draining lymph nodes even after fungal clearance, and were protective upon fungal re-exposure up to several weeks later. Induction of γδT17 immunity depended on IL-23 and IL-1 family cytokine signalling, whereas Toll-like and C-type lectin receptors were dispensable. Furthermore, Vγ4+ T cells from Malassezia-exposed hosts were able to respond directly and selectively to Malassezia-derived ligands, independently of antigen-presenting host cells. The fungal moieties detected were shared across diverse species of the Malassezia genus, but not conserved in other Basidiomycota or Ascomycota. These data provide novel mechanistic insight into the induction and maintenance of type 17 immunosurveillance of skin commensal colonization that has significant implications for cutaneous health.

The human pathobiont Malassezia furfur secreted protease Mfsap1 regulates cell dispersal and exacerbates skin inflammation.

Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, is not well characterized. In this study, we compared the expression level of secreted proteases, lipases, phospholipases, and sphingomyelinases of Malassezia globosa in healthy subjects and seborrheic dermatitis or atopic dermatitis patients. We observed upregulated gene expression of the previously characterized secretory aspartyl protease MGSAP1 in both diseased groups, in lesional and non-lesional skin sites, as compared to healthy subjects. To explore the functional roles of MGSAP1 in skin disease, we generated a knockout mutant of the homologous protease MFSAP1 in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, further highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.

Epidermal barrier impairment predisposes for excessive growth of the allergy-associated yeast Malassezia on murine skin.

BACKGROUND: The skin barrier is vital for protection against environmental threats including insults caused by skin-resident microbes. Dysregulation of this barrier is a hallmark of atopic dermatitis (AD) and ichthyosis, with variable consequences for host immune control of colonizing commensals and opportunistic pathogens. While Malassezia is the most abundant commensal fungus of the skin, little is known about the host control of this fungus in inflammatory skin diseases. METHODS: In this experimental study, MC903-treated mice were colonized with Malassezia spp. to assess the host-fungal interactions in atopic dermatitis. Additional murine models of AD and ichthyosis, including tape stripping, K5-Nrf2 overexpression and flaky tail mice, were employed to confirm and expand the findings. Skin fungal counts were enumerated. High parameter flow cytometry was used to characterize the antifungal response in the AD-like skin. Structural and functional alterations in the skin barrier were determined by histology and transcriptomics of bulk skin. Finally, differential expression of metabolic genes in Malassezia in atopic and control skin was quantified. RESULTS: Malassezia grows excessively in AD-like skin. Fungal overgrowth could, however, not be explained by the altered immune status of the atopic skin. Instead, we found that by upregulating key metabolic genes in the altered cutaneous niche, Malassezia acquired enhanced fitness to efficiently colonise the impaired skin barrier. CONCLUSIONS: This study provides evidence that structural and metabolic changes in the dysfunctional epidermal barrier environment provide increased accessibility and an altered lipid profile, to which the lipid-dependent yeast adapts for enhanced nutrient assimilation. Our findings reveal fundamental insights into the implication of the mycobiota in the pathogenesis of common skin barrier disorders.

New insights into immunity to skin fungi shape our understanding of health and disease.

Fungi represent an integral part of the skin microbiota. Their complex interaction network with the host shapes protective immunity during homeostasis. If host defences are breached, skin-resident fungi including Malassezia and Candida, and environmental fungi such as dermatophytes can cause cutaneous infections. In addition, fungi are associated with diverse non-infectious skin disorders. Despite their multiple roles in health and disease, fungi remain elusive and understudied, and the mechanisms underlying the emergence of pathological conditions linked to fungi are largely unclear. The identification of IL-17 as an important antifungal effector mechanism represents a milestone for understanding homeostatic antifungal immunity. At the same time, host-adverse, disease-promoting roles of IL-17 have been delineated, as in psoriasis. Fungal dysbiosis represents another feature of many pathological skin conditions with an unknown causal link of intra- and interkingdom interactions to disease pathogenesis. The emergence of new fungal pathogens such as Candida auris highlights the need for more research into fungal immunology to understand how antifungal responses shape health and diseases. Recent technological advances for genetically manipulating fungi to target immunomodulatory fungal determinants, multi-omics approaches for studying immune cells in the human skin, and novel experimental models open up a promising future for skin fungal immunity.

HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction.

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Trained immunity and tolerance in innate lymphoid cells, monocytes, and dendritic cells during allergen-specific immunotherapy.

BACKGROUND: Despite the efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated. OBJECTIVE: Here, we have performed a comprehensive longitudinal analysis of the systemic innate immune cell repertoire during the course of AIT. METHODS: Patients with allergy received standard preseasonal subcutaneous AIT with allergoids to birch and/or grass. Healthy controls were monitored without any intervention. Flow cytometry of innate lymphoid cell (ILC), natural killer cell, monocyte cell, and dendritic cell (DC) subsets was performed at baseline, 3 months (birch season), 6 months (grass seasons), and 12 months after the therapy in patients or at similar seasonal time points in controls. Additional analyses were performed in the third-year birch and grass season. RESULTS: We observed a durable decrease in group 2 ILCs and an increase of group 1 ILCs after AIT, with dynamic changes in their composition. We found that an expansion of CD127+CD25++ clusters caused observed shifts in the heterogeneity of group 1 ILCs. In addition, we observed development of CD127+CD25++c-Kit+ group 3 ILC clusters. Moreover, we found an increase in the number of intermediate monocytes in parallel with a reduction in nonclassical monocytes during the first year after AIT. Classical and intermediate monocytes presented significant heterogeneity in patients with allergy, but AIT reduced the HLA-DR++ clusters. Finally, an increase in plasmacytoid DCs and CD141+ myeloid DCs was observed in individuals with allergy, whereas the number of CD1c+ myeloid DCs was reduced during the first year of AIT. CONCLUSION: AIT induces changes in the composition and heterogeneity of circulating innate immune cells and brings them to the level observed in healthy individuals. Monitoring of ILCs, monocytes, and DCs during AIT might serve as a novel biomarker strategy.

Tight junction, mucin, and inflammasome-related molecules are differentially expressed in eosinophilic, mixed, and neutrophilic experimental asthma in mice.

BACKGROUND: Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct in vivo comparison has not been performed. METHODS: We developed mouse models representing three different phenotypes of allergic airway inflammation-eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT-PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air-liquid interface were used to study the mechanisms revealed in the in vivo models. RESULTS: By whole-genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome-related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo-1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation. Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1, and IL-1ß was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine-IL-1ß-may transiently impair epithelial barrier function, while IL-1ß and IL-17 increase mucin expressions in primary human bronchial epithelial cells. CONCLUSION: Our findings suggest that differential expression of TJ, mucin, and inflammasome-related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic.

Card9 and MyD88 differentially regulate Th17 immunity to the commensal yeast Malassezia in the murine skin.

The fungal community of the skin microbiome is dominated by a single genus, Malassezia. Besides its symbiotic lifestyle at the host interface, this commensal yeast has also been associated with diverse inflammatory skin diseases in humans and pet animals. Stable colonization is maintained by antifungal type 17 immunity. The mechanisms driving Th17 responses to Malassezia remain, however, unclear. Here, we show that the C-type lectin receptors Mincle, Dectin-1, and Dectin-2 recognize conserved patterns in the cell wall of Malassezia and induce dendritic cell activation in vitro, while only Dectin-2 is required for Th17 activation during experimental skin colonization in vivo. In contrast, Toll-like receptor recognition was redundant in this context. Instead, inflammatory IL-1 family cytokines signaling via MyD88 were also implicated in Th17 activation in a T cell-intrinsic manner. Taken together, we characterized the pathways contributing to protective immunity against the most abundant member of the skin mycobiome. This knowledge contributes to the understanding of barrier immunity and its regulation by commensals and is relevant considering how aberrant immune responses are associated with severe skin pathologies.

IL-12 regulates type 3 immunity through interfollicular keratinocytes in psoriasiform inflammation.

Psoriasis is a chronic inflammatory skin disorder underpinned by dysregulated cytokine signaling. Drugs neutralizing the common p40 subunit of interleukin-12 (IL-12) and IL-23 represented a therapeutic breakthrough; however, new drugs that block the IL-23p19 subunit and spare IL-12 are more effective, suggesting a regulatory function of IL-12. To pinpoint the cell type and underlying mechanism of IL-12­mediated immune regulation in psoriasis, we generated a conditional Il12rb2-knockout (KO)/reporter mouse strain. We detected Il12rb2 expression in T cells and a specific subset of interfollicular (IF) keratinocytes. Analysis of single-cell RNA-sequencing (scRNAseq) data from patients with psoriasis confirmed a similar expression pattern in the human skin. Deletion of Il12rb2 across the hematopoietic compartment did not alter the development of Aldara-induced psoriasiform inflammation. However, depletion of Il12rb2 in keratinocytes exacerbated disease development, phenocopying the Il12rb2 germline knockout. Protective IL-12 signaling blocked the hyperproliferation of keratinocytes, maintained skin barrier integrity, and diminished disease-driving IL-23/type 3 immune circuits. In line, specific IL-23p19 blockade led to a more profound reduction of psoriatic keratinocyte expression signatures in the skin of patients with psoriasis than combined IL-12/IL-23 inhibition. Collectively, we provide a potential explanation for the superior efficacy of IL-23p19 inhibitors in psoriasis and describe an unperceived role of IL-12 in maintaining skin epithelial cell homeostasis.

Host Immunity to Malassezia in Health and Disease.

The microbiota plays an integral role in shaping physical and functional aspects of the skin. While a healthy microbiota contributes to the maintenance of immune homeostasis, dysbiosis can result in the development of diverse skin pathologies. This dichotomous feature of the skin microbiota holds true not only for bacteria, but also for fungi that colonize the skin. As such, the yeast Malassezia, which is by far the most abundant component of the skin mycobiota, is associated with a variety of skin disorders, of which some can be chronic and severe and have a significant impact on the quality of life of those affected. Understanding the causative relationship between Malassezia and the development of such skin disorders requires in-depth knowledge of the mechanism by which the immune system interacts with and responds to the fungus. In this review, we will discuss recent advances in our understanding of the immune response to Malassezia and how the implicated cells and cytokine pathways prevent uncontrolled fungal growth to maintain commensalism in the mammalian skin. We also review how the antifungal response is currently thought to affect the development and severity of inflammatory disorders of the skin and at distant sites.

A Novel Mycovirus Evokes Transcriptional Rewiring in the Fungus Malassezia and Stimulates Beta Interferon Production in Macrophages.

Mycoviruses infect fungi, and while most persist asymptomatically, there are examples of mycoviruses having both beneficial and detrimental effects on their host. Virus-infected Saccharomyces and Ustilago strains exhibit a killer phenotype conferring a growth advantage over uninfected strains and other competing yeast species, whereas hypovirus-infected Cryphonectria parasitica displays defects in growth, sporulation, and virulence. In this study, we identify a double-stranded RNA (dsRNA) mycovirus in five Malassezia species. Sequence analysis reveals it to be a totivirus with two dsRNA segments: a larger 4.5-kb segment with genes encoding components for viral replication and maintenance, and a smaller 1.4-kb segment encoding a novel protein. Furthermore, transcriptome sequencing (RNA-seq) of virus-infected versus virus-cured Malassezia sympodialis revealed an upregulation of dozens of ribosomal components in the cell, suggesting the virus modifies the transcriptional and translational landscapes of the cell. Given that Malassezia is the most abundant fungus on human skin, we assessed the impact of the mycovirus in a murine epicutaneous infection model. Although infection with virus-infected strains was not associated with an increased inflammatory response, we did observe enhanced skin colonization in one of two virus-infected M. sympodialis strains. Noteworthy, beta interferon expression was significantly upregulated in bone marrow-derived macrophages when challenged with virus-infected, compared to virus-cured, M. sympodialis, suggesting that the presence of the virus can induce an immunological response. Although many recent studies have illuminated how widespread mycoviruses are, there are relatively few in-depth studies about their impact on disease caused by the host fungus. We describe here a novel mycovirus in Malassezia and its possible implications in pathogenicity.IMPORTANCEMalassezia species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of clinical skin disorders. Recent studies have reported associations of Malassezia with Crohn's disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because M. sympodialis has lost genes involved in RNA interference (RNAi), we hypothesized Malassezia could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several Malassezia species and characterized the MsMV1 mycovirus of M. sympodialis We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of Malassezia, focusing on a previously unidentified novel mycovirus.

The Skin Commensal Yeast Malassezia Triggers a Type 17 Response that Coordinates Anti-fungal Immunity and Exacerbates Skin Inflammation.

Commensal fungi of the mammalian skin, such as those of the genus Malassezia, are associated with atopic dermatitis and other common inflammatory skin disorders. Understanding of the causative relationship between fungal commensalism and disease manifestation remains incomplete. By developing a murine epicutaneous infection model, we found Malassezia spp. selectively induce IL-17 and related cytokines. This response is key in preventing fungal overgrowth on the skin, as disruption of the IL-23-IL-17 axis compromises Malassezia-specific cutaneous immunity. Under conditions of impaired skin integrity, mimicking a hallmark of atopic dermatitis, the presence of Malassezia dramatically aggravates cutaneous inflammation, which again was IL-23 and IL-17 dependent. Consistently, we found a CCR6+ Th17 subset of memory T cells to be Malassezia specific in both healthy individuals and atopic dermatitis patients, whereby the latter showed enhanced frequency of these cells. Thus, the Malassezia-induced type 17 response is pivotal in orchestrating antifungal immunity and in actively promoting skin inflammation.