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.

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.

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.

The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization.

Immune inertness of Aspergillus fumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to ß-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host-fungal interaction.

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.

Aspergillus-induced superoxide production by cystic fibrosis phagocytes is associated with disease severity.

Aspergillus fumigatus infects up to 50% of cystic fibrosis (CF) patients and may play a role in progressive lung disease. As cystic fibrosis transmembrane conductance regulator is expressed in cells of the innate immune system, we hypothesised that impaired antifungal immune responses play a role in CF-related Aspergillus lung disease. Peripheral blood mononuclear cells, polymorphonuclear cells (PMN) and monocytes were isolated from blood samples taken from CF patients and healthy volunteers. Live-cell imaging and colorimetric assays were used to assess antifungal activity in vitro. Production of reactive oxygen species (ROS) was measured using luminol-induced chemiluminescence and was related to clinical metrics as collected by case report forms. CF phagocytes are as effective as those from healthy controls with regards to phagocytosis, killing and restricting germination of A. fumigatus conidia. ROS production by CF phagocytes was up to four-fold greater than healthy controls (p<0.05). This effect could not be replicated in healthy phagocytes by priming with lipopolysaccharide or serum from CF donors. Increased production of ROS against A. fumigatus by CF PMN was associated with an increased number of clinical exacerbations in the previous year (p=0.007) and reduced lung function (by forced expiratory volume in 1 s) (p=0.014). CF phagocytes mount an intrinsic exaggerated release of ROS upon A. fumigatus stimulation which is associated with clinical disease severity.

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.

C-type lectins in immunity and homeostasis.

The C-type lectins are a superfamily of proteins that recognize a broad repertoire of ligands and that regulate a diverse range of physiological functions. Most research attention has focused on the ability of C-type lectins to function in innate and adaptive antimicrobial immune responses, but these proteins are increasingly being recognized to have a major role in autoimmune diseases and to contribute to many other aspects of multicellular existence. Defects in these molecules lead to developmental and physiological abnormalities, as well as altered susceptibility to infectious and non-infectious diseases. In this Review, we present an overview of the roles of C-type lectins in immunity and homeostasis, with an emphasis on the most exciting recent discoveries.

Signalling through MyD88 drives surface expression of the mycobacterial receptors MCL (Clecsf8, Clec4d) and Mincle (Clec4e) following microbial stimulation.

The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this "two-step" model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity.

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.

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.

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.

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

Dectin-1 promotes fungicidal activity of human neutrophils.

Human polymorphonuclear leukocytes (PMN) are a first line of defense against fungal infections. PMN express numerous pattern recognition receptors (PRR) that facilitate identification of invading microorganisms and ultimately promote resolution of disease. Dectin-1 (beta-glucan receptor) is a PRR expressed on several cell types and has been studied on monocytes and macrophages. However, the role played by dectin-1 in the recognition and killing of fungi by PMN is unknown. We investigated the ability of dectin-1 to mediate human PMN phagocytosis and fungicidal activity. Dectin-1 was expressed on the surface of PMN from all subjects tested (n=29) and in an intracellular compartment that co-sedimented with azurophilic granules in Percoll density gradients. Soluble beta-glucan and mAb GE2 (anti-dectin-1) inhibited binding and phagocytosis of zymosan by human PMN (e.g., ingestion was inhibited 40.1% by 30 min, p<0.001), and blocked reactive oxygen species production. Notably, soluble beta-glucan and GE2 inhibited phagocytosis and killing of Candida albicans by PMN (inhibition of killing was 54.8% for beta-glucan and 36.2% for GE2, p<0.01). Our results reveal a mechanism whereby PMN dectin-1 plays a key role in the recognition and killing of fungal pathogens by the innate immune system.

Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.

Expression of functionally different dectin-1 isoforms by murine macrophages.

Dectin-1 is a specific receptor for beta-glucans and a major receptor for fungal particles on macrophages (Mphi). It is a type II membrane receptor that has a C-terminal, NK-like, C-type lectin-like domain separated from the cell membrane by a short stalk region and a cytoplasmic immunoreceptor tyrosine-based activation-like motif. We observed functional differences in dectin-1-dependent recognition of fungal particles by Mphi from different mouse strains. RT-PCR analysis revealed that mice have at least two splice forms of dectin-1, generated by differential usage of exon 3, encoding the full-length dectin-1A and a stalkless Mphi dectin-1B. Mphi from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mphi from C57BL/6 and related mice mainly expressed the smaller isoform. NIH-3T3 fibroblast and RAW264.7 macrophage cell lines stably expressing either isoform were able to bind and phagocytose zymosan at 37 degrees C. However, binding by the smaller dectin-1B isoform was significantly affected at lower temperatures. These properties were shared by the equivalent human isoforms. The relative ability of each of the isoforms to induce TNF-alpha production in RAW264.7 Mphi was also found to be different. These results are the first evidence that dectin-1 isoforms are functionally distinct and indicate that differential isoform usage may represent a mechanism of regulating cellular responses to beta-glucans.

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages.

We recently demonstrated that the beta-glucan receptor Dectin-1 (betaGR) was the major nonopsonic beta-glucan receptor on macrophages (Mphi) for the yeast-derived particle zymosan. However, on resident peritoneal Mphi, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mphi mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mphi and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mphi. Both SIGNR1 and betaGR cooperated in the nonopsonic recognition of zymosan by these Mphi. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mphi, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as betaGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mphi expressing SIGNR1 resulted in TNF-alpha production. Treatment of RAW 264.7 Mphi expressing SIGNR1, which express low levels of betaGR, with beta-glucans had little effect on binding or TNF-alpha production, indicating that there was no absolute requirement for betaGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mphi.

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes.

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide.

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.