Search

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.

In this study, the human immune response mechanisms againstSporothrix brasiliensisandSporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction ofS. brasiliensisandS. schenckiiwith human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis ofSporothrixyeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these twoSporothrixyeasts 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 theSporothrixyeast 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)-1β secretion by hMDM in response to bothS. brasiliensisandS. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against theseSporothrixspecies. Nevertheless, the structural differences in the PRMs of the twoSporothrixspecies, 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 andviaCR3 receptor mediating an inflammatory response toSporothrixspecies.

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., The Dectin‐1 cluster of C‐type lectin receptors (CLRs) are structurally related receptors that recognise a diverse array of ligands and are capable of intracellular signalling, inducing a wide variety of cellular responses. These CLRs are involved in numerous processes that are important for the regulation of health and disease .

Environmental factors, particularly fungi, influence the pathogenesis of allergic airway inflammation, but the mechanisms underlying these effects are still unclear. Melanin is one fungal component which is thought to modulate pulmonary inflammation. We recently identified a novel C-type lectin receptor, MelLec (Clec1a), which recognizes fungal 1,8-dihydroxynaphthalene (DHN)-melanin and is able to regulate inflammatory responses. Here we show that MelLec promotes pulmonary allergic inflammation and drives the development of Th17 T-cells in response to spores of Aspergillus fumigatus. Unexpectedly, we found that MelLec deficiency was protective, with MelLec-/- animals showing normal weight gain and significantly reduced pulmonary inflammation in our allergic model. The lungs of treated MelLec-/- mice displayed significantly reduced inflammatory foci and reduced bronchial wall thickening, which correlated with a reduced cellular influx (particularly neutrophils and inflammatory monocytes) and levels of inflammatory cytokines and chemokines. Notably, fungal burdens were increased in MelLec-/- animals, without apparent adverse effects, and there were no alterations in the survival of these mice. Characterization of the pulmonary T-cell populations, revealed a significant reduction in Th17 cells, and no alterations in Th2, Th1 or Treg cells. Thus, our data reveal that while MelLec is required to control pulmonary fungal burden, the inflammatory responses mediated by this receptor negatively impact the animal welfare in this allergic model.

Funding Information: We thank P. Asamaphan, A. Clark, and B. Kerscher for providing NIH overexpression cell lines, S. Yamasaki for the anti‐Mincle antibody, the staff of the University of Aberdeen animal facility for the care for our animals, and the Iain Fraser Cytometry Centre at the University of Aberdeen for their assistance. This work was supported by funding from the Wellcome Trust (102705, 217163), the Medical Research Council Centre for Medical Mycology, and the University of Exeter (MR/N006364/2).

Environmental factors, particularly fungi, influence the pathogenesis of allergic airway inflammation, but the mechanisms underlying these effects are still unclear. Melanin is one fungal component which is thought to modulate pulmonary inflammation. We recently identified a novel C-type lectin receptor, MelLec (Clec1a), which recognizes fungal 1,8-dihydroxynaphthalene (DHN)-melanin and is able to regulate inflammatory responses. Here we show that MelLec promotes pulmonary allergic inflammation and drives the development of Th17 T-cells in response to spores of Aspergillus fumigatus. Unexpectedly, we found that MelLec deficiency was protective, with MelLec-/- animals showing normal weight gain and significantly reduced pulmonary inflammation in our allergic model. The lungs of treated MelLec-/- mice displayed significantly reduced inflammatory foci and reduced bronchial wall thickening, which correlated with a reduced cellular influx (particularly neutrophils and inflammatory monocytes) and levels of inflammatory cytokines and chemokines. Notably, fungal burdens were increased in MelLec-/- animals, without apparent adverse effects, and there were no alterations in the survival of these mice. Characterization of the pulmonary T-cell populations, revealed a significant reduction in Th17 cells, and no alterations in Th2, Th1 or Treg cells. Thus, our data reveal that while MelLec is required to control pulmonary fungal burden, the inflammatory responses mediated by this receptor negatively impact the animal welfare in this allergic model.

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 β-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., Author summary Invasive fungal infections remain an important health problem in immunocompromised patients. Immune recognition of fungal pathogens involves binding of specific cell wall components by pathogen recognition receptors (PRRs) and subsequent activation of immune defences. Some cell wall components are conserved among fungal species while other components are species-specific and phenotypically diverse. The fungal cell wall is dynamic and capable of changing its composition and organization when adapting to different growth niches and environmental stresses. Differences in the composition of the cell wall lead to differential immune recognition by the host. Understanding how changes in the cell wall composition affect recognition by PRRs is likely to be of major diagnostic and clinical relevance. Here we address this fundamental question using four soluble immune receptor-probes which recognize mannans and β-glucan in the cell wall. We use this novel methodology to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces was not correlated with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes on fungal cell surfaces are differentially distributed within and between the cell walls of fungal pathogens.

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.

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, Excessive superoxide production by CF phagocytes against A. fumigatus is associated with clinical disease severity http://ow.ly/Elwy30i8mLe

Innate immunity is constructed around genetically encoded receptors that survey the intracellular and extracellular environments for signs of invading microorganisms. These receptors recognise the invader and through complex intracellular networks of molecular signaling, they destroy the threat whilst instructing effective adaptive immune responses. Many of these receptors, like the Toll-like receptors in particular, are well-known for their ability to mediate downstream responses upon recognition of exogenous or endogenous ligands; however, the emerging family known as the C-type lectin-like receptors contains many members that have a huge impact on immune and homeostatic regulation. Of particular interest here are the C-type lectin-like receptors that make up the Dectin-1 cluster and their intracellular signaling motifs that mediate their functions. In this review, we aim to draw together current knowledge of ligands, motifs and signaling pathways, present downstream of Dectin-1 cluster receptors, and discuss how these dictate their role within biological systems.

Zymosan is a beta-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a beta-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the beta-glucan receptor mediating this activity. To address the role of the recently described beta-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte beta-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of beta-glucans for therapeutic drug design.

Beta-glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for beta-glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for beta-glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non-Toll-like pattern-recognition receptor required for the induction of protective immune responses.

11 pags, 7 figs, 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was funded by the Wellcome Trust, the National Research Foundation, the Deutscher Akademischer Austauschdienst, the University of Cape Town, the UK Research Council Basic Technology Initiative “Glycoar-rays” (GRS/79268), and the UK Medical Research Council. A. S. P is a fellowof the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal) and M. A. C. of the Consejo Superior de Investigaciones Cientificas, Programe “Junta para la Ampliación de Estudios” (JaeDoc/098/2011) cofinanced by the Fondo Social Europeo.

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-α, 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.

Recognition of microbial products by germ-line-encoded PRR initiates immune responses, but how PRR mediate specific host responses to infectious agents is poorly understood. We and others have proposed that specificity is achieved by collaborative responses mediated between different PRR. One such example comprises the fungal beta-glucan receptor Dectin-1, which collaborates with TLR to induce TNF production. We show here that collaborative responses mediated by Dectin-1 and TLR2 are more extensive than first appreciated, and result in enhanced IL-23, IL-6 and IL-10 production in DC, while down-regulating IL-12 relative to the levels produced by TLR ligation alone. Such down-regulation occurred with multiple MyD88-coupled TLR, was dependent on signaling through Dectin-1 and also occurred in macrophages. These findings explain how fungi can induce IL-23 and IL-6, while suppressing IL-12, a combination which has previously been shown to contribute to the development of Th17 responses found during fungal infections. Furthermore, these data reveal how the collaboration of different PRR can tailor specific responses to infectious agents.

Fungal infections represent a significant health burden, especially in immunocompromised individuals, yet many of the underlying immunological mechanisms involved in the recognition and control of these pathogens are unclear. The identification of the Toll-like receptors (TLRs) has shed new insights on innate microbial recognition and the initiation of immune responses; however, recent evidence indicates that the 'non-TLR' receptors also have a significant role in these processes, particularly in antifungal immunity. Of interest are members of the C-type lectin-receptor family, including the mannose receptor, dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), Dectin-1, Dectin-2 and the collectins. Here, we review the roles of each of these receptors, describing how they contribute to fungal recognition, uptake and killing and also participate in the induction and/or modulation of the host immune response.

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, p0.001), 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, p0.01). 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.

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.

Beta-glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian beta-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble beta-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

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

beta-Glucans are structural components of fungal cell walls, which have a stimulatory effect on the immune system. Although a number of receptors for these carbohydrates have been proposed, the recently identified C-type lectin-like receptor, Dectin-1, appears to play a central role. Dectin-1 is expressed on phagocytic cells, including macrophages and neutrophils, and mediates both the internalization and cellular responses to beta-glucan, through unique mechanisms. Dectin-1 can recognize and respond to live fungal pathogens and is being increasingly appreciated as having a key role in the innate responses to these pathogens. In addition to its exogenous ligands, Dectin-1 can recognize an unidentified endogenous ligand on T cells and may act as a co-stimulatory molecule, although its function in these responses is less clear. This review will highlight the current knowledge of Dectin-1 and its potential role in antifungal immunity, as well as deficiencies in our understanding.

Podoplanin, a small transmembrane glycoprotein, is expressed at high levels on lymphatic endothelial cells, kidney podocytes and lung type I alveolar cells, but absent on blood endothelial cells. It has also been described on splenic and peritoneal macrophages, but the functional significance of this is unknown [1]. Podoplanin is an endogenous ligand for CLEC-2, a C-type lectin expressed on platelets and at a lower level on subsets of other hematopoietic cells [2–4]. The interaction of CLEC-2 on platelets with podoplanin on lymphatic endothelial cells is essential for the separation of lymphatic and blood vessels [5,6]. In the present study, we further investigated the expression of podoplanin on macrophages and explored the ability of macrophage-expressed podoplanin to function as a CLEC-2 ligand. We probed various macrophage populations with a podoplanin-specific antibody and FcCLEC-2 (a probe for CLEC-2 ligands [7]). We were unable to detect specific binding of either the podoplanin antibody or FcCLEC-2 on RAW264.7 macrophages, bone marrow-derived macrophages (BMDMs) and tissue-resident macrophages, including alveolar and resident peritoneal macrophages, indicating the absence of podoplanin (Fig. 1A). Conversely, podoplanin was detected on thioglycollate-elicited ‘inflammatory’ peritoneal macrophages and lipopolysaccharide (LPS)-treated RAW264.7 cells using the same two ligands (Fig. 1A). Podoplanin expression was also markedly up-regulated on BMDMs following stimulation with LPS and weakly up-regulated in response to the TLR2/1 and TLR2/6 agonists, Pam3CSK4 and Pam2CSK4, respectively (Fig. 1B). There was also a slight, but significant, up-regulation induced by the inflammatory cytokine tumor necrosis factor (TNF), but not by several other cytokines tested (Fig. 1B). These results demonstrate that podoplanin is expressed on inflammatory but not tissue-resident macrophages, and is up-regulated in response to TLR stimulation and some inflammatory cytokines. The present results extend a previous report of podoplanin expression on macrophages [1], by revealing that expression is limited to inflammatory subsets. Fig. 1 Podoplanin expression on macrophages. (A) Anti-podoplanin and FcCLEC-2 staining of various macrophage populations as indicated. Resident and thioglycollate-elicited peritoneal macrophages and resident alveolar macrophages were isolated from Balb/c mice ... To investigate whether macrophage-expressed podoplanin could induce CLEC-2 activation we used a reporter system based on BWZ.36 cells expressing a chimeric CLEC-2/CD3ζ receptor linked to β-galactosidase expression and IL-2 secretion [8]. Incubation of these cells with podoplanin-expressing macrophages induced IL-2 and β-galactosidase, whereas podoplanin-negative macrophages had no effect (Figs 1C and D). We also investigated whether podoplanin-expressing macrophages could directly induce platelet activation (determined by analysing aggregation and ATP secretion). Untreated podoplanin-negative RAW264.7 macrophages had no effect on platelet aggregation or ATP secretion, whereas the subsequent addition of thrombin induced powerful activation. In contrast, LPS-activated RAW264.7 macrophages stimulated platelet aggregation and ATP secretion after a delay of several minutes (Fig. 1E). A delay in activation is reminiscent of platelet activation by the CLEC-2 ligand rhodocytin [2]. The subsequent lack of an effect of addition of thrombin confirmed that these platelets had undergone full activation. Aggregation and secretion induced by LPS-stimulated RAW264.7 cells was blocked by the αIIbβ3 inhibitor, lotrafiban, confirming integrin-dependent platelet activation, and by the Src kinase inhibitor, PP1, consistent with CLEC-2-mediated platelet activation (Fig. 1F). In line with this, activation was blocked in CLEC-2-deficient mouse platelets [9], whereas thrombin was still able to induce full activation of these cells (Fig. 1G). Another mechanism by which platelets and macrophages interact is via binding of P-selectin on activated platelets to PSGL-1 on macrophages. We investigated whether blocking this interaction would modulate platelet activation triggered by podoplanin-expressing macrophages; however, the inclusion of a blocking antibody against PSGL-1 did not have any effect on platelet aggregation or secretion (data not shown). These results demonstrate that inflammatory macrophages expressing podoplanin can directly activate platelets via CLEC-2. This could represent a novel mechanism for extravascular platelet activation during clotting, wound healing and vascular inflammatory processes such as atherosclerosis. Expression of podoplanin promotes migration of a variety of cell types, including tumor, MDCK and lymphatic endothelial cells [10–12]. The presence of podoplanin on macrophages could therefore underlie a mechanism through which platelets regulate migration of inflammatory macrophages as well as other functions.

We recently identified dectin-1 (βGR) as a major β-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate β-glucans. Using a novel mAb (2A11) raised against βGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mφ) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the βGR, but at lower levels. Alveolar Mφ, like inflammatory Mφ, exhibited the highest surface expression of βGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mφ expressed much lower levels of βGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mφ suggested the existence of an additional β-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mφ. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mφ mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mφ.

New discoveries reveal crucial roles for the Dectin-2 family in many aspects of the immune response., 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 innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus , and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung.

Contains fulltext : 88534.pdf (Publisher’s version ) (Closed access) BACKGROUND: The occurrence of oropharyngeal candidiasis (OPC) in combination with HIV disease progression is a very common phenomenon. However, not all HIV-infected patients develop OPC, even when they progress to low CD4 T-cell counts. Because T-cell immunity is defective in AIDS, the innate defence mechanisms are likely to have a central role in antifungal immunity in these patients. We investigated whether genetic variations in the innate immune genes DECTIN-1, TLR2, TLR4, TIRAP, and CASPASE-12 are associated with the presence of OPC in HIV-infected subjects from East Africa. METHODS: A total of 225 HIV patients were genotyped for several single nucleotide polymorphisms (SNPs), and this was correlated with the occurrence of OPC in these patients. In addition, primary immune cells obtained from individuals with different genotypes were stimulated with Candida albicans, and cytokine production was measured. RESULTS: The analysis revealed that no significant differences in the polymorphism frequencies could be observed, although a tendency toward a protective effect on OPC of the DECTIN-1 I223S SNP was apparent. Furthermore, interferon gamma production capacity was markedly lower in cells bearing the DECTIN-1 SNP I223S. It could also be demonstrated that the 223S mutated form of the DECTIN-1 gene exhibits a lower capacity to bind zymosan. CONCLUSIONS: These data demonstrate that common polymorphisms of TLR2, TLR4, TIRAP, and CASPASE-12 do not influence susceptibility to OPC in HIV-infected patients in East Africa but suggest an immunomodulatory effect of the I223S SNP on dectin-1 function and possibly the susceptibility to OPC in HIV patients.

Contains fulltext : 80438.pdf (Publisher’s version ) (Open Access) 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.

Contains fulltext : 70889.pdf (Publisher’s version ) (Closed access) 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.

Background C-type lectin-like receptors (CTLRs) play critical roles in immunity and homeostasis by recognising a great variety of microbial or endogenous ligands [1]. CLECSF8 is a member of the Dectin–2 family of CTLRs. Previous research indicates that CLECSF8 associates with the FcRg adaptor, which is essential for surface expression and signalling through the SYK/CARD9 pathway. Recently, the mycobacterial cord factor (TDM, trehalose-6,6’-dimycolate) was identified as the ligand of CLECSF8, as shown previously for the closely related CTLR Mincle [2]. Indeed, we recently showed that CLECSF8 plays a critical role in human and murine anti-mycobacterial immunity [3]. In this study, we characterised CLECSF8 expression in the mouse under naive and inflammatory conditions.

Dectin-1 is a specific receptor for β-glucans and a major receptor for fungal particles on macrophages (Mφ). 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 Mφ 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 Mφ dectin-1B. Mφ from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mφ 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°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-α production in RAW264.7 Mφ 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 β-glucans.

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 ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.