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.

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.

IL-23 supports host defense against systemic Candida albicans infection by ensuring myeloid cell survival.

The opportunistic fungal pathogen Candida albicans can cause invasive infections in susceptible hosts and the innate immune system, in particular myeloid cell-mediated immunity, is critical for rapid immune protection and host survival during systemic candidiasis. Using a mouse model of the human disease, we identified a novel role of IL-23 in antifungal defense. IL-23-deficient mice are highly susceptible to systemic infection with C. albicans. We found that this results from a drastic reduction in all subsets of myeloid cells in the infected kidney, which in turn leads to rapid fungal overgrowth and renal tissue injury. The loss in myeloid cells is not due to a defect in emergency myelopoiesis or the recruitment of newly generated cells to the site of infection but, rather, is a consequence of impaired survival of myeloid cells at the site of infection. In fact, the absence of a functional IL-23 pathway causes massive myeloid cell apoptosis upon C. albicans infection. Importantly, IL-23 protects myeloid cells from apoptosis independently of the IL-23-IL-17 immune axis and independently of lymphocytes and innate lymphoid cells. Instead, our results suggest that IL-23 acts in a partially autocrine but not cell-intrinsic manner within the myeloid compartment to promote host protection from systemic candidiasis. Collectively, our data highlight an unprecedented and non-canonical role of IL-23 in securing survival of myeloid cells, which is key for maintaining sufficient numbers of cells at the site of infection to ensure efficient host protection.

Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction.

Animal models are crucial for infectious disease research. They provide an important basis for analyzing the full spectrum of interactions that occur between microbes and their host in vivo in a tissue-specific manner. Pathogenic fungi are increasingly recognized as a serious threat for humans and exploiting such infection models have greatly improved our understanding of fungal pathogenicity. Species of the genus Malassezia are the most abundant fungi of the human skin microbiota and they are also associated with the development of severe inflammatory skin disorders such as seborrheic dermatitis and atopic dermatitis. However, a causative link between Malassezia and disease pathogenesis remains unknown, a fact that can be attributed to the poor knowledge of the complex crosstalk of Malassezia with the skin immune system. This protocol describes the establishment of an experimental mouse model that allows studying the interaction of Malassezia with the mammalian skin in vivo. It outlines the method for cultivating Malassezia spp. under laboratory conditions, how to infect the murine skin with Malassezia spp. and how to assess the outcome of infection by means of the skin inflammation and fungal burden analyses. The model described here works in fully immunocompetent animals and does not rely on immune suppressive or antibiotic pretreatment of the animals. It is furthermore adaptable to virtually all genetically modified mouse strains and can be combined with other skin disease models. These features make this infection model a very powerful tool for studying in detail the innate and adaptive immune response of the host against Malassezia in the skin in vivo.

Role of NS1 and TLR3 in Pathogenesis and Immunity of WNV.

West Nile Virus (WNV) is a mosquito-transmitted flavivirus which causes encephalitis especially in elderly and immunocompromised individuals. Previous studies have suggested the protective role of the Toll-like receptor 3 (TLR3) pathway against WNV entry into the brain, while the WNV non-structural protein 1 (NS1) interferes with the TLR3 signaling pathway, besides being a component of viral genome replication machinery. In this study, we investigated whether immunization with NS1 could protect against WNV neuroinvasion in the context of TLR3 deficiency. We immunized mice with either an intact or deleted TLR3 system (TLR3KO) with WNV envelope glycoprotein (gE) protein, NS1, or a combination of gE and NS1. Immunization with gE or gE/NS1, but not with NS1 alone, induced WNV neutralizing antibodies and protected against WNV brain invasion and inflammation. The presence of intact TLR3 signaling had no apparent effect on WNV brain invasion. However, mock-immunized TLR3KO mice had higher inflammatory cell invasion upon WNV brain infection than NS1-immunized TLR3KO mice and wild type mice. Thus, immunization against NS1 may reduce brain inflammation in a context of TLR3 signaling deficiency.

Interleukin-17 in Antifungal Immunity.

The field of IL-17 biology has received much attention over the last decade owing to the pathogenic role of this cytokine in psoriasis and other autoinflammatory disorders and the successful implementation of IL-17-targeting therapies in patients suffering from these diseases. IL-17-mediated pathologies are contrasted by the important host beneficial effects of this cytokine. IL-17 is essential for regulating microbial colonization in barrier tissues. Rare congenital defects in the IL-17 pathway exemplify the relevance of IL-17 in protective immunity against the opportunistic fungal pathogen C. albicans. However, more recently, evidence is accumulating that IL-17 can also provide protection against fungi other than C. albicans. Importantly, protective IL-17 responses directed against commensal fungi can, under certain conditions, promote inflammation with detrimental consequences for the host, thereby assigning fungi a new role as disease-promoting factors apart from their role as potential infectious agents.

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.

IL-17 Takes Center Stage in Dermatophytosis.

The cytokine IL-17 plays a critical role in host defense against fungal infections. So far, clinical relevance for IL-17 antifungal activity focused on mucocutaneous candidiasis. Burstein and colleagues now provide evidence that type 17 immunity is also essential for defense against dermatophytosis.

Langerin+ DCs regulate innate IL-17 production in the oral mucosa during Candida albicans-mediated infection.

The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αß and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1ß, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa.

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.

Assessment of Immune Responses to Fungal Infections: Identification and Characterization of Immune Cells in the Infected Tissue.

The immune system is important to protect the host from fungal infections. Diverse cell types belonging to the innate or adaptive branch of the immune system act in a tightly coordinated and tissue-specific manner. Experimental mouse models of fungal infections have proved essential for assessing the protective principles against different fungal pathogens. Besides pathological, histological, biochemical and molecular parameters, the analysis of phenotypic and functional aspects of immune cells in infected tissues is key for understanding the mechanisms of antifungal defense. In this chapter, we describe a method based on flow cytometry to assess innate and adaptive immune cells isolated from an in vivo context in a qualitative and quantitative manner.

IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease.

Interleukin 17-Mediated Host Defense against Candida albicans.

Candida albicans is part of the normal microbiota in most healthy individuals. However, it can cause opportunistic infections if host defenses are breached, with symptoms ranging from superficial lesions to severe systemic disease. The study of rare congenital defects in patients with chronic mucocutaneous candidiasis led to the identification of interleukin-17 (IL-17) as a key factor in host defense against mucosal fungal infection. Experimental infections in mice confirmed the critical role of IL-17 in mucocutaneous immunity against C. albicans. Research on mouse models has also contributed importantly to our current understanding of the regulation of IL-17 production by different cellular sources and its effector functions in distinct tissues. In this review, we highlight recent findings on IL-17-mediated immunity against C. albicans in mouse and man.

The late endosomal adaptor molecule p14 (LAMTOR2) regulates TGFß1-mediated homeostasis of Langerhans cells.

Langerhans cells (LCs), a sub-population of dendritic cells (DCs) in the skin, participate in the regulation of immunity and peripheral tolerance. The adaptor molecule p14 is part of the late endosomal/lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin (mTOR) activator/regulator (LAMTOR) complex, which mediates the activation of lysosome-associated extracellular signaling-regulated kinase (ERK) and the mTOR cascade. In previous work, we demonstrated that CD11c-specific deficiency of p14 disrupts LC homeostasis by affecting the LAMTOR-mediated ERK and mTOR signaling. In this study, we extended our analysis on p14 deficiency specifically in LCs. Langerin-specific ablation of p14 caused a complete loss of LCs, accompanied by an increased maturational phenotype of LCs. The absence of LCs in p14-deficient mice reduced contact hypersensitivity (CHS) responses to the contact sensitizer trinitrochlorobenzene. Analysis using bone marrow-derived DCs (BMDCs) revealed that p14 deficiency in DCs/LCs interfered with the LC-relevant transforming growth factor ß1 (TGFß1) pathway, by lowering TGFß receptor II expression on BMDCs and LCs, as well as surface binding of TGFß1 on BMDCs. We conclude that p14 deficiency affects TGFß1 sensitivity of LCs, which is mandatory for their homeostasis and subsequently for their immunological function during CHS.

Langerhans cells: an update.

Langerhans cells belong to the family of dendritic cells, professional antigen-presenting cells, and populate the skin and epithelia of mammals. It was the extensive investigation of this particular dendritic cell subpopulation in earlier days, which contributed crucially to the current understanding of the regulation of antigen processing and presentation, a concept, which was termed "the Langerhans cell paradigm". Extensive research during the last decades has revealed that Langerhans cells might not only be involved in the induction of adaptive immune responses but also in the maintenance of peripheral tolerance in order to prevent auto-immunity. In addition it appeared that Langerhans cells represent a rather extravagant dendritic cell population with a unique origin and homeostatic regulation. This review highlights the most important findings about Langerhans cell ontogeny and homeostasis as well as their function in the immune system.

LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling.

The receptor tyrosine kinase Flt3 and its ligand are crucial for dendritic cell (DC) homeostasis by activating downstream effectors including mammalian target of Rapamycin (mTOR) signalling. LAMTOR2 is a member of the Ragulator/LAMTOR complex known to regulate mTOR and extracellular signal-regulated kinase activation on the late endosome as well as endosomal biogenesis. Here we show in mice that conditional ablation of LAMTOR2 in DCs results in a severe disturbance of the DC compartment caused by accumulation of Flt3 on the cell surface. This results in an increased downstream activation of the AKT/mTOR signalling pathway and subsequently to a massive expansion of conventional DCs and plasmacytoid DCs in ageing mice. Finally, we can revert the symptoms in vivo by inhibiting the activation of Flt3 and its downstream target mTOR.

The late endosomal adaptor molecule p14 (LAMTOR2) represents a novel regulator of Langerhans cell homeostasis.

Langerhans cells (LCs) are dendritic cells (DCs) residing in epithelia, where they critically regulate immunity and tolerance. The p14 adaptor molecule is part of the late endosomal/LAMTOR (lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin [mTOR] activator/regulator) complex, thereby contributing to the signal transduction of the extracellular signaling-regulated kinase (ERK) and the mTOR cascade. Furthermore, p14 represents an important regulator for endosomal sorting processes within the cell. Mutated, dysfunctional p14 leads to a human immunodeficiency disorder with endosomal/lysosomal defects in immune cells. Because p14 participates in the regulation of endosomal trafficking, growth factor signaling, and cell proliferation, we investigated the role of p14 in mouse DCs/LCs using a conditional knockout mouse model. p14-deficient animals displayed a virtually complete loss of LCs in the epidermis early after birth due to impaired proliferation and increased apoptosis of LCs. Repopulation analysis after application of contact sensitizer leads to the recruitment of a transient LC population, predominantly consisting of short-term LCs. The underlying molecular mechanism involves the p14-mediated disruption of the LAMTOR complex which results in the malfunction of both ERK and mTOR signal pathways. Hence, we conclude that p14 acts as a novel and essential regulator of LC homeostasis in vivo.

Cytip regulates dendritic-cell function in contact hypersensitivity.

Cytohesin-interacting protein (Cytip) is induced during dendritic cell (DC) maturation and in T cells upon activation. It has also been shown to be involved in the regulation of immune responses. Here, we evaluated the functional consequences of Cytip deficiency in DCs using Cytip knockout (KO) mice. No difference in DC subpopulations in the skin draining lymph nodes (LNs) was found between Cytip KO mice and their wild-type counterparts, excluding a role in DC development. To investigate the function of Cytip in DCs in vivo, we used 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) as a model system. In the sensitization as well as in the elicitation phase, DCs derived from Cytip KO mice induced an increased inflammatory reaction indicated by more pronounced ear swelling. Furthermore, IL-12 production was increased in Cytip KO bone marrow-derived DCs (BMDCs) after CpG stimulation. Additionally, Cytip-deficient DCs loaded with ovalbumin induced stronger proliferation of antigen-specific CD4(+) and CD8(+) T cells in vitro. Finally, migration of skin DCs was not altered after TNCB application due to Cytip deficiency. Taken together, these data suggest a suppressive function for Cytip in mouse DCs in limiting immune responses.

Langerhans cells and dermal dendritic cells capture protein antigens in the skin: possible targets for vaccination through the skin.

Dendritic cells capture and process antigen and present it to T lymphocytes in the lymphoid organs. Dendritic cells of the skin, including epidermal Langerhans cells, langerin(+) and langerin(negative) dermal dendritic cells are ideally positioned to take up pathogens that enter the body through the skin or vaccines that are administered into (intradermal) or onto (epicutaneous) the skin. The antigen uptake properties of skin dendritic cells have not thoroughly been studied yet. We therefore investigated the uptake of the fluorochrome-conjugated model antigen ovalbumin (OVA) by skin dendritic cells in the mouse. OVA was readily taken up by immature Langerhans cells both in situ and in cell suspensions. When offered to Langerhans cells in situ either by "bathing" skin explants in OVA-containing culture medium or by intradermal injection they retained the captured OVA for at least 2-3 days when migrating into the culture medium and, importantly, into the draining lymph nodes. Also langerin(+) and - to a larger extent - langerin(negative) skin dendritic cells took up and transported OVA to the lymph nodes. Interestingly, mature Langerhans cells were still capable of ingesting substantial amounts of OVA, indicating that predominantly receptor-mediated endocytosis is operative in these cells. Unlike macropinocytosis, this pathway of endocytosis is not shut down upon dendritic cell maturation. These observations indicate that in intradermal vaccination schemes, Langerhans cells from the epidermis are prominently involved. They were recently shown to possess the capacity to induce functional cytotoxic T lymphocytes. Furthermore, the potential to markedly enhance antigen uptake and processing by targeting antigen to c-type lectin receptors on Langerhans cells was also recently demonstrated. Our data provide a rationale and an incentive to explore in more detail antigen targeting to Langerhans cells with the aim of harnessing it for immunotherapy.

Langerhans cells as targets for immunotherapy against skin cancer.

Cancer is the second most common cause of death in the world. Treatment of cancer is very challenging and immunotherapy has been developed as a potential way to fight cancer. The main obstacle with immunotherapy is that cancer cells evolve from healthy body cells in response to an accumulation of genetic mutations. As a consequence, the immune system struggles to detect the abnormal cells as they are mainly recognized as self. This implies that equipping the immune system to eliminate cancer cells is tricky, yet represents a very efficient way to constrain the growth of tumors. We became interested in developing immunotherapeutical strategies against skin cancer in the context of our observations that Langerhans cells (LC) are very potent antigen presenting cells and are able to incorporate protein antigens and present them to CD4(+) and CD8(+) T cells in the skin-draining lymph nodes. As a consequence, we developed an immunization strategy through the skin, termed epicutaneous immunization. Protein antigen applied onto barrier-disrupted skin induces long-lasting cytotoxic T-cell responses, potent enough to control and inhibit tumor growth. In this review, we suggest that immunization strategies through the skin could be a promising new approach for the treatment of skin cancer.