The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans

The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-alpha by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.     

Role of Mincle in Alveolar Macrophage-Dependent Innate Immunity against Mycobacterial Infections in Mice

The role of macrophage-inducible C-type lectin Mincle in lung innate immunity against mycobacterial infection is incompletely defined. In this study, we show that wild-type (WT) mice responded with a delayed Mincle induction on resident alveolar macrophages and newly immigrating exudate macrophages to infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), peaking by days 14-21 posttreatment. As compared with WT mice, Mincle knockout (KO) mice exhibited decreased proinflammatory mediator responses and leukocyte recruitment upon M. bovis BCG challenge, and they demonstrated increased mycobacterial loads in pulmonary and extrapulmonary organ systems. Secondary mycobacterial infection on day 14 after primary BCG challenge led to increased cytokine gene expression in sorted alveolar macrophages of WT mice, but not Mincle KO mice, resulting in substantially reduced alveolar neutrophil recruitment and increased mycobacterial loads in the lungs of Mincle KO mice. Collectively, these data show that WT mice respond with a relatively late Mincle expression on lung sentinel cells to M. bovis BCG infection. Moreover, M. bovis BCG-induced upregulation of C-type lectin Mincle on professional phagocytes critically shapes antimycobacterial responses in both pulmonary and extrapulmonary organ systems of mice, which may be important for elucidating the role of Mincle in the control of mycobacterial dissemination in mice.     

Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses

C-type lectin receptors (CLRs) are an emerging family of pattern recognition receptors that recognizes pathogens or damaged tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. MS analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells expressed a set of inflammatory genes in response to cholesterol crystals, and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, whereas other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and that they activate innate immune responses.     

Dectin-1 interaction with tetraspanin CD37 inhibits IL-6 production

C-type lectins are pattern-recognition receptors important for pathogen binding and uptake by APCs. Evidence is accumulating that integration of incoming cellular signals in APCs is regulated by grouping of receptors and signaling molecules into organized membrane complexes, such as lipid rafts and tetraspanin microdomains. In this study, we demonstrate that C-type lectin dectin-1 functionally interacts with leukocyte-specific tetraspanin CD37. Dectin-1 and CD37 colocalize on the surface of human APCs. Importantly, macrophages of CD37-deficient (CD37(-/-)) mice express decreased dectin-1 membrane levels, due to increased dectin-1 internalization. Furthermore, transfection of CD37 into a macrophage cell line elevated endogenous dectin-1 surface expression. Although CD37 deficiency does not affect dectin-1-mediated phagocytosis, we observed a striking 10-fold increase of dectin-1-induced IL-6 production in CD37(-/-) macrophages compared with wild-type cells, despite reduced dectin-1 cell surface expression. Importantly, the observed increase in IL-6 production was specific for dectin-1, because signaling via other pattern-recognition receptors was unaffected in CD37(-/-) macrophages and because the dectin-1 ligand curdlan was used. Taken together, these findings show that tetraspanin CD37 is important for dectin-1 stabilization in APC membranes and controls dectin-1-mediated IL-6 production.     

Distinct functions of DC-SIGN and its homologues L-SIGN (DC-SIGNR) and mSIGNR1 in pathogen recognition and immune regulation

Antigen presenting cells express C-type lectins that are involved in pathogen capture, processing and antigen presentation to induce immune responses against these pathogens. However, it is becoming clear that pathogens have evolved to subvert the function of some C-type lectins to escape immune surveillance. An important C-type lectin family is represented by DC-SIGN and its homologues in human and mouse. Here we discuss the structure in relation to the pathogen binding specificity of the SIGN receptors and the function of these receptors in mouse and humans.     

C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6′-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.     

Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas

As an initial step in the functional analysis of lectins in the Pacific oyster, Crassostrea gigas, we attempted to obtain the full coding sequences of C. gigas lectins and conduct tissue expression analyses. To obtain lectin genes quickly, we identified C. gigas expressed sequence tags that coded for lectins in GenBank, and selected three encoding partial sequences of C-type lectin 1 (CgCLec-1), galectin (CgGal) and fucolectin. We obtained full open reading frames of CgCLec-1 and CgGal cDNAs by RACE-PCR. CgCLec-1 is a typical C-type lectin with a signal peptide and C-type lectin domain. CgCLec-1 mRNA was expressed only in specialized basophilic cells involved with digestive enzyme secretion in the digestive gland, suggesting that CgCLec-1 is secreted into the lumen of the digestive diverticula. CgGal is a prototype galectin with a single galactose-binding domain that was expressed in all of the tissues examined. As suggested for vertebrate galectin-1 (prototype galectin), CgGal may function in general cell activities such as cell adhesion. Fucolectin in C. gigas was expressed specifically in the gonads, indicating a possible function in gonadal development. CgCLec-1 and CgGal expression in hemocytes was not upregulated after injecting Vibrio tubiashii into adductor muscle, suggesting that bacterial infection does not induce synthesis of these lectins. Of the three lectins examined, CgCLec-1 is an interesting target for future investigations of innate immunity in the digestive system of C. gigas.     

The novel endocytic and phagocytic C-Type lectin receptor DCL-1/CD302 on macrophages is colocalized with F-actin, suggesting a role in cell adhesion and migration

C-type lectin receptors play important roles in mononuclear phagocytes, which link innate and adaptive immunity. In this study we describe characterization of the novel type I transmembrane C-type lectin DCL-1/CD302 at the molecular and cellular levels. DCL-1 protein was highly conserved among the human, mouse, and rat orthologs. The human DCL-1 (hDCl-1) gene, composed of six exons, was located in a cluster of type I transmembrane C-type lectin genes on chromosomal band 2q24. Multiple tissue expression array, RT-PCR, and FACS analysis using new anti-hDCL-1 mAbs established that DCL-1 expression in leukocytes was restricted to monocytes, macrophages, granulocytes, and dendritic cells, although DCL-1 mRNA was present in many tissues. Stable hDCL-1 Chinese hamster ovary cell transfectants endocytosed FITC-conjugated anti-hDCL-1 mAb rapidly (t(1/2) = 20 min) and phagocytosed anti-hDCL-1 mAb-coated microbeads, indicating that DCL-1 may act as an Ag uptake receptor. However, anti-DCL-1 mAb-coated microbead binding and subsequent phagocytic uptake by macrophages was approximately 8-fold less efficient than that of anti-macrophage mannose receptor (MMR/CD206) or anti-DEC-205/CD205 mAb-coated microbeads. Confocal studies showed that DCL-1 colocalized with F-actin in filopodia, lamellipodia, and podosomes in macrophages and that this was unaffected by cytochalasin D, whereas the MMR/CD206 and DEC-205/CD205 did not colocalize with F-actin. Furthermore, when transiently expressed in COS-1 cells, DCL-1-EGFP colocalized with F-actin at the cellular cortex and microvilli. These data suggest that hDCL-1 is an unconventional lectin receptor that plays roles not only in endocytosis/phagocytosis but also in cell adhesion and migration and thus may become a target for therapeutic manipulation.     

Identification of lectin-like receptors expressed by antigen presenting cells and neutrophils and their mapping to a novel gene complex

In an experimental rat model, we recently mapped an arthritis susceptibility locus to the distal part of Chromosome 4 containing genes predicted to encode C-type lectin superfamily (CLSF) receptors. Here we report the cDNA cloning and positional arrangement of these receptor genes, which represent rat orthologues to human Mincle and DCIR and to mouse MCL and Dectin-2, as well as four novel receptors DCIR2, DCIR3, DCIR4 and DCAR1, not previously reported in other species. We furthermore report the cDNA cloning of human Dectin-2 and MCL, and of the mouse orthologues to the novel rat receptors. Similar to the killer-cell lectin-like receptors (KLR) some of these receptors exhibit structural features suggesting that they regulate leukocyte reactivity; e.g., human DCIR and rodent DCIR1 and DCIR2 carry an immunoreceptor tyrosine-based inhibitory motif (ITIM), predicting inhibitory function, and conversely, in all three species Mincle has a positively charged amino acid in the transmembrane region, suggesting activating function. Sequence comparisons show that the receptors form a discrete family, more closely related to group II CLSF receptors than to the group V KLR. Their distance to the KLR is underscored by their preservation of evolutionary conserved calcium/saccharide binding residues, present in group II and lacking in group V CLSF and their cellular expression patterns, with most of the genes preferentially expressed by professional antigen-presenting cells (dendritic cells, macrophages and B cells) and neutrophils. In all three species, the genes map together, forming an evolutionary conserved gene complex, which we call the antigen presenting lectin-like receptor complex (APLEC).     

The C-Type Lectin Receptor Mincle Binds to Streptococcus pneumoniae but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in Streptococcus pneumonia infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca²⁺-dependent, and serotype-specific binding of Mincle to S. pneumonia. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize S. pneumonia but is not required for the anti-pneumococcal innate immune response.     

Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell- or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose- and N-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.     

Simple affinity chromatographic procedure to purify beta-galactoside binding lectins

Affinity chromatography based on the commercial resin Sepharose CL-6B was used to isolate new C1-beta-type lectins from crude preparations of snake venoms (Bothrops jararaca, Bothrops jararacussu, Bothrops newiedi, Bothrops moojeni, Lachesis muta rhombeata). Most of the C-type lectins could be eluted with almost 100% recovery using the competitor isopropyl-beta-D-thiogalactoside (IPTG) or through Ca2+ sequestration with EDTA. The lectin yield varied considerably among the different snake species, but B. newiedi venom was a particularly rich source of lectin, retaining 2.7 mg of lectin by milliliter of resin in saturating conditions. C1-alpha-lectins from Crotalus durisus terrificus venom, from the jack fruit (jacalin) and from bread fruit seeds extract (frutalin) had no affinity, either with or without Ca2+ added, for Sepharose CL-6B, showing that the resin is specific for C1-beta-type lectins. Sepharose CL-6B used as galactose-affinity chromatography provides a simple and fast method for isolating C-type beta-galactoside binding lectins from crude sample preparations.     

Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins

Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.     

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall

Galactofuranosyl residues are present in various microorganisms but not in mammals. In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides were bridged by disulfide bonds. The hIntL gene was split into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus. hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin. These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca(2+)-dependent lectins. Recombinant hIntL revealed affinities to d-pentoses and a d-galactofuranosyl residue in the presence of Ca(2+), and recognized the bacterial arabinogalactan of Nocardia containing d-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host.     

Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle^-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.