Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Fc-conjugated C-type lectin receptors: Tools for understanding host-pathogen interactions.

The use of soluble fusion proteins of pattern recognition receptors (PRRs) used in the detection of exogenous and endogenous ligands has helped resolve the roles of PRRs in the innate immune response to pathogens, how they shape the adaptive immune response, and function in maintaining homeostasis. Using the immunoglobulin (Ig) crystallizable fragment (Fc) domain as a fusion partner, the PRR fusion proteins are soluble, stable, easily purified, have increased affinity due to the Fc homodimerization properties, and consequently have been used in a wide range of applications such as flow cytometry, screening of protein and glycan arrays, and immunofluorescent microscopy. This review will predominantly focus on the recognition of pathogens by the cell membrane-expressed glycan-binding proteins of the C-type lectin receptor (CLR) subgroup of PRRs. PRRs bind to conserved pathogen-associated molecular patterns (PAMPs), such as glycans, usually located within or on the outer surface of the pathogen. Significantly, many glycans structures are identical on both host and pathogen (e.g. the Lewis (Le) X glycan), allowing the use of Fc CLR fusion proteins with known endogenous and/or exogenous ligands as tools to identify pathogen structures that are able to interact with the immune system. Screens of highly purified pathogen-derived cell wall components have enabled identification of many unique PAMP structures recognized by CLRs. This review highlights studies using Fc CLR fusion proteins, with emphasis on the PAMPs found in fungi, bacteria, viruses, and parasites. The structure and unique features of the different CLR families is presented using examples from a broad range of microbes whenever possible.

C-type lectin receptors in antifungal immunity: Current knowledge and future developments.

C-type lectin receptors (CLRs) constitute a category of innate immune receptors that play an essential role in the antifungal immune response. For over two decades, scientists have uncovered what are the fungal ligands recognized by CLRs and how these receptors initiate the immune response. Such studies have allowed the identification of genetic polymorphisms in genes encoding for CLRs or for proteins involved in the signalisation cascade they trigger. Nevertheless, our understanding of how these receptors functions and the full extent of their function during the antifungal immune response is still at its infancy. In this review, we summarize some of the main findings about CLRs in antifungal immunity and discuss what the future might hold for the field.

Characterization of antifungal C-type lectin receptor expression on murine epithelial and endothelial cells in mucosal tissues.

Our data reveal that selection of enzymes for generating single cell suspensions from murine tissues influences detection of surface expression of antifungal CLRs. Using a method that most preserves receptor expression, we show that non-myeloid expression of antifungal CLRs is limited to MelLec on endothelial cells in murine mucosal tissues.

Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the ß-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.

PAMPs of the Fungal Cell Wall and Mammalian PRRs.

Fungi are opportunistic pathogens that infect immunocompromised patients and are responsible for an estimated 1.5 million deaths every year. The antifungal innate immune response is mediated through the recognition of pathogen-associated molecular patterns (PAMPs) by the host's pattern recognition receptors (PRRs). PRRs are immune receptors that ensure the internalisation and the killing of fungal pathogens. They also mount the inflammatory response, which contributes to initiate and polarise the adaptive response, controlled by lymphocytes. Both the innate and adaptive immune responses are required to control fungal infections. The immune recognition of fungal pathogen primarily occurs at the interface between the membrane of innate immune cells and the fungal cell wall, which contains a number of PAMPs. This chapter will focus on describing the main mammalian PRRs that have been shown to bind to PAMPs from the fungal cell wall of the four main fungal pathogens: Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Pneumocystis jirovecii. We will describe these receptors, their functions and ligands to provide the reader with an overview of how the immune system recognises fungal pathogens and responds to them.

Synthesis of the Fungal Metabolite YWA1 and Related Constructs as Tools to Study MelLec-Mediated Immune Response to Aspergillus Infections†.

We describe the chemical synthesis of the fungal naphthopyrones YWA1 and fonsecin B, as well as their functionalization with an amine-spacer arm and the conjugation of the resulting molecules to three different functional tags (i.e., biotin, Oregon green, 1-[3-(succinimidyloxycarbonyl)benzyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]pyridinium bromide (PyMPO)). The naphthopyrone-biotin and -PyMPO constructs maintained the ability to bind the C-type lectin receptor MelLec, whose interaction with immunologically active fungal metabolites (i.e., 1,8-dihydroxynaphthalene-(DHN)-melanin and YWA1) is a key step in host recognition and induction of protective immune responses against Aspergillus fumigatus. The fluorescent Fonsecin B-PyMPO construct 21 was used to selectively visualize MelLec-expressing cells, thus validating the potential of this strategy for studying the role and functions of MelLec in immunity.

C-type lectin receptors of the Dectin-1 cluster: Physiological roles and involvement in disease.

C-type lectin receptors (CLRs) are essential for multicellular existence, having diverse functions ranging from embryonic development to immune function. One subgroup of CLRs is the Dectin-1 cluster, comprising of seven receptors including MICL, CLEC-2, CLEC-12B, CLEC-9A, MelLec, Dectin-1, and LOX-1. Reflecting the larger CLR family, the Dectin-1 cluster of receptors has a broad range of ligands and functions, but importantly, is involved in numerous pathophysiological processes that regulate health and disease. Indeed, these receptors have been implicated in development, infection, regulation of inflammation, allergy, transplantation tolerance, cancer, cardiovascular disease, arthritis, and other autoimmune diseases. In this mini-review, we discuss the latest advancements in elucidating the function(s) of each of the Dectin-1 cluster CLRs, focussing on their physiological roles and involvement in disease.

C-Type Lectin Receptors in Antifungal Immunity.

Most fungal species are harmless to humans and some exist as commensals on mucocutaneous surfaces. Yet many fungi are opportunistic pathogens, causing life-threatening invasive infections when the immune system becomes compromised. The fungal cell wall contains conserved pathogen-associated molecular patterns (PAMPs), which allow the immune system to distinguish between self (endogenous molecular patterns) and foreign material. Sensing of invasive microbial pathogens is achieved through recognition of PAMPs by pattern recognition receptors (PRRs). One of the predominant fungal-sensing PRRs is the C-type lectin receptor (CLR) family. These receptors bind to structures present on the fungal cell wall, eliciting various innate immune responses as well as shaping adaptive immunity. In this chapter, we specifically focus on the four major human fungal pathogens, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Pneumocystis jirovecii, reviewing our current understanding of the CLRs that are involved in their recognition and protection of the host.

Mycobacterial receptor, Clec4d (CLECSF8, MCL), is coregulated with Mincle and upregulated on mouse myeloid cells following microbial challenge.

The C-type lectin receptor (CTLR), Clec4d (MCL, CLECSF8), is a member of the Dectin-2 cluster of CTLRs, which also includes the related receptors Mincle and Dectin-2. Like Mincle, Clec4d recognizes mycobacterial cord factor, trehalose dimycolate, and we recently demonstrated its key role in anti-mycobacterial immunity in mouse and man. Here, we characterized receptor expression in naïve mice, under inflammatory conditions, and during Mycobacterium bovis BCG infection using newly generated monoclonal antibodies. In naïve mice, Clec4d was predominantly expressed on myeloid cells within the peritoneal cavity, blood, and bone marrow. Unexpectedly, basal expression of Clec4d was very low on leukocytes in the lung. However, receptor expression was significantly upregulated on pulmonary myeloid cells during M. bovis BCG infection. Moreover, Clec4d expression could be strongly induced in vitro and in vivo by various microbial stimuli, including TLR agonists, but not exogenous cytokines. Notably, we show that Clec4d requires association with the signaling adaptor FcRγ and Mincle, but not Dectin-2, for surface expression. In addition, we provide evidence that Clec4d and Mincle, but not Dectin-2, are interdependently coregulated during inflammation and infection. These data show that Clec4d is an inducible myeloid-expressed CTLR in mice, whose expression is tightly linked to that of Mincle.

MICL controls inflammation in rheumatoid arthritis.

BACKGROUND: Myeloid inhibitory C-type lectin-like receptor (MICL, Clec12A) is a C-type lectin receptor (CLR) expressed predominantly by myeloid cells. Previous studies have suggested that MICL is involved in controlling inflammation. OBJECTIVE: To determine the role of this CLR in inflammatory pathology using Clec12A(-/-) mice. METHODS: Clec12A(-/-) mice were generated commercially and primarily characterised using the collagen antibody-induced arthritis (CAIA) model. Mechanisms and progress of disease were characterised by clinical scoring, histology, flow cytometry, irradiation bone-marrow chimera generation, administration of blocking antibodies and in vivo imaging. Characterisation of MICL in patients with rheumatoid arthritis (RA) was determined by immunohistochemistry and single nucleotide polymorphism analysis. Anti-MICL antibodies were detected in patient serum by ELISA and dot-blot analysis. RESULTS: MICL-deficient animals did not present with pan-immune dysfunction, but exhibited markedly exacerbated inflammation during CAIA, owing to the inappropriate activation of myeloid cells. Polymorphisms of MICL were not associated with disease in patients with RA, but this CLR was the target of autoantibodies in a subset of patients with RA. In wild-type mice the administration of such antibodies recapitulated the Clec12A(-/-) phenotype. CONCLUSIONS: MICL plays an essential role in regulating inflammation during arthritis and is an autoantigen in a subset of patients with RA. These data suggest an entirely new mechanism underlying RA pathogenesis, whereby the threshold of myeloid cell activation can be modulated by autoantibodies that bind to cell membrane-expressed inhibitory receptors.

The Dectin-2 family of C-type lectin-like receptors: an update.

Myeloid and non-myeloid cells express members of the C-type lectin-like receptor (CTLR) family, which mediate crucial cellular functions during immunity and homeostasis. Of relevance here is the dendritic cell-associated C-type lectin-2 (Dectin-2) family of CTLRs, which includes blood dendritic cell antigen 2 (BDCA-2), dendritic cell immunoactivating receptor (DCAR), dendritic cell immunoreceptor (DCIR), Dectin-2, C-type lectin superfamily 8 (CLECSF8) and macrophage-inducible C-type lectin (Mincle). These CTLRs possess a single extracellular conserved C-type lectin-like domain and are capable of mediating intracellular signalling either directly, through integral signalling domains, or indirectly, by associating with signalling adaptor molecules. These receptors recognize a diverse range of endogenous and exogenous ligands, and can function as pattern recognition receptors for several classes of pathogens including fungi, bacteria and parasites, driving both innate and adaptive immunity. In this review, we summarize our knowledge of each of these receptors, highlighting the exciting discoveries that have been made in recent years.

The C-type lectin receptor CLECSF8 (CLEC4D) is expressed by myeloid cells and triggers cellular activation through Syk kinase.

CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.

Development of Negative Controls for Fc-C-Type Lectin Receptor Probes.

Fc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of ß-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind ß-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding to Candida albicans yeasts, Aspergillus fumigatus resting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate that A. fumigatus spores expose a low amount of ß-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes. IMPORTANCE While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a ß-glucan containing particle, and 2 human pathogenic fungi, Candida albicans yeasts and Aspergillus fumigatus conidia. We show that A. fumigatus conidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.

Human dectin-1 deficiency and mucocutaneous fungal infections.

Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2.

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing ß-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

CLEC-2 is a phagocytic activation receptor expressed on murine peripheral blood neutrophils.

CLEC-2 is a member of the "dectin-1 cluster" of C-type lectin-like receptors and was originally thought to be restricted to platelets. In this study, we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of Ab-coated beads and the production of proinflammatory cytokines, including TNF-alpha, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like dectin-1, CLEC-2 can recruit the signaling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.

Quantifying Receptor-Mediated Phagocytosis and Inflammatory Responses to Fungi in Immune Cells.

Phagocytosis and cytokine production are important processes by which innate immune cells, especially professional phagocytes such as neutrophils and macrophages, control and regulate immunity to fungi. These cellular responses are initiated when conserved pathogen components, known as pathogen-associated molecular patterns (PAMPs), are recognized by pattern-recognition receptors (PRRs), which include members of the C-type lectin receptor (CLR) family that are able to bind to fungal cell wall-derived carbohydrates. Phagocytosis and cytokine production can be quantitatively examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, using in vitro based assays with primary-derived murine cells and cell lines. Here, we describe a flow cytometry-based method using transduced cell lines to assess the ability of CLRs to mediate internalization, using A. fumigatus conidia and the ß-1,3 glucan receptor, Dectin-1 (CLEC7A), as an example. The use of ELISA-based assays to measure cytokine production by immune cells that are induced in response to fungi and methods for isolating and culturing primary macrophages from various murine tissues are described.

Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.