Mincle: 20 years of a versatile sensor of insults

Mincle: 20 years of a versatile sensor of insults Abstract Macrophage-inducible C-type lectin, better known as Mincle, is a member of the C-type lectin receptor family and is encoded by Clec4e. Mincle was an orphan receptor for a long time after having been discovered as a lipopolysaccharide-induced protein, yet later an adjuvant glycolipid in mycobacteria—trehalose dimycolate—was identified as a ligand. Ligands for Mincle were also found existing in bacteria, fungi and even mammals. When confronted with foreign elements, Mincle can recognize characteristic pathogen-associated molecular patterns, mostly glycolipids, from Mycobacterium tuberculosis and other pathogens, and thus induce immune responses against infection. To maintain self-homeostasis, Mincle can recognize lipid-based damage-associated molecular patterns, thereby monitoring the internal environment. The mechanism by which Mincle functions in the immune system is also becoming more clear along with the identification of its ligands. Being expressed widely on antigen-presenting cells, Mincle activation leads to the production of cytokines and chemokines, neutrophil infiltration and other inflammatory responses. Besides, Mincle can induce acquired immunity such as antigen-specific T-cell responses and antibody production as an adjuvant receptor. In this review, we will retrospectively sketch the discovery and study of Mincle, and outline some current work on this receptor. C-type lectin receptors, glycolipids, innate immunity, pathogens Introduction From its name (macrophage-inducible C-type lectin) and primary structure, one may suspect Mincle’s function as an inducible receptor for microbes. Indeed, the identification of its ligands and structure unveiled its critical role as an innate immune receptor. In this review, we will introduce the findings and their significance related to Mincle following the chronological order of several milestones. We begin by describing the identification of Mincle, and the mechanism of signaling transduction. Then we detail the findings of Mincle ligands, as well as its structure and mechanism of ligand recognition. In the later parts, we concentrate on the regulation and functions of Mincle, which lead to its potential applications in medicine. Identification of Mincle Mincle was firstly identified as a transcriptional target of NF-IL6 in macrophages by Akira’s group in 1999 (1). NF-IL6 (also called C/EBPβ) is a transcription factor that is activated by inflammatory stimuli and can induce the expression of various inflammatory genes (2). In a further screening for transcriptional targets using NF-IL6-deficient mice, Matsumoto et al. cloned Mincle as a lipopolysaccharide-induced NF-IL6-dependent molecule in macrophages. Subsequent analysis of the primary sequence suggested that Mincle is a type-II transmembrane molecule and has an extracellular carbohydrate-recognition domain (CRD), similar to C-type lectins such as macrophage C-type lectin (MCL) (3). Besides mouse Mincle, the authors also reported its homologue in humans, which has an overall identity of 67% and a similarity of 85% with mouse Mincle. Mincle is coupled with FcRγ It took nearly 10 years to make any progress in the study of Mincle after its identification. In 2008, our group firstly elucidated that Mincle couples with FcRγ and transduces an activation signal through the immunoreceptor tyrosine-based activation motif (ITAM) in FcRγ (4). The residue at position 42 (Arg42) in the transmembrane region of Mincle provides the positive charge that is needed in the interaction with FcRγ, which has a negatively charged residue. Taking advantage of an antibody that activates Mincle, we demonstrated that the ITAM in FcRγ is indispensable for activated Mincle to transduce downstream signaling, in which the kinases Syk and Erk are also activated, and finally lead to the expression of pro-inflammatory cytokines in a CARD9-dependent way. CARD9 is a key adaptor molecule downstream of ITAM signaling. Coupling with Bcl10 and Malt1, CARD9 mediates the cytokine expression in macrophages and is required for pathogen clearance (5–7). It is, therefore, possible that Mincle might also play a role in protective immunity against pathogens. Mincle ligands, from discovery to design Like ‘authentic’ pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), NOD-like receptors (NLRs) or RIG-I-like receptors (RLRs), a C-type lectin receptor (CLR) can recognize a series of ligands that have a specific ‘pattern’, and so can Mincle. In 2008, Mincle was found to have the capability of sensing endogenous and exogenous targets (4, 8, 9). After that, accumulated evidence showed that Mincle ligands exist in bacteria, fungi and mammals (Table 1). The first report related to the function of Mincle in the immune system was published in 2008 by Wells et al. (8). They found that the expression of Mincle in macrophages was up-regulated after exposure to live Candida albicans. Mincle-deficient mice infected with C. albicans showed a significantly higher fungal burden than wild-type (WT) mice did, suggesting that Mincle may play a role in the host defense against C. albicans, although the Mincle ligand in this fungus is unidentified even today. Table 1. Molecular candidates recognized by Mincle Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  b, bovine; h, human; m, mouse. View Large Table 1. Molecular candidates recognized by Mincle Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  b, bovine; h, human; m, mouse. View Large Recognition of dead cells derived from self The first Mincle ligand appeared in front of us by a mere chance. To screen the Mincle ligands, we established a reporter cell line expressing Mincle and FcRγ. When Mincle is activated by ligand, if any, it will activate the NFAT and lead to the expression of GFP, which was confirmed using a plate-coated antibody against Mincle. When we accidentally left the cultured reporter cells for several days without changing the medium, we found that the number of GFP-positive living cells increased with the number of dead cells, suggesting Mincle as a sensor for dead cells (4). Whole-body irradiation can induce significant thymocyte death followed by neutrophil infiltration into the thymus. As anti-Mincle mAb blocked this infiltration, Mincle may mediate dead-cell-induced inflammation. From dead cell lysates, we purified spliceosome-associated protein 130 (SAP-130) as a candidate that selectively bound to a Mincle–Ig fusion protein (4). However, the exact contribution of SAP-130 to dead-cell-induced inflammation is unclear, as this protein is essential for RNA splicing, a fundamental process in all living cells. Mincle recognizes pathogenic fungi Unlike the unexpected finding of the first Mincle ligand, the GFP-reporter cell line of Mincle was used in an intended screen of pathogenic fungus species. As a result, Mincle could specifically recognize only Malassezia species among a number of pathogenic fungus species (31). Interestingly, three C. albicans substrains did not activate cells through Mincle, although C. albicans was suggested to contain Mincle ligands (8, 9). To evaluate the function of Mincle in immune responses against Malassezia, we analyzed Mincle-deficient mice established by Akira’s group, and found that the production of inflammatory cytokines such as IL-6 and TNF as well as neutrophil infiltration at the injection site was significantly impaired in Mincle-deficient mice, indicating that Mincle mediates the host defense responses against Malassezia (31). Nevertheless, the identity of a Mincle ligand was still obscure at the time. Mincle recognizes mycobacterial glycolipids The breakthrough in exploring Mincle ligands was brought about by further bacterial study; we found that all three tested Mycobacterium species could be recognized by Mincle (10). Mycobacteria have long been known to possess unique lipid components with potent adjuvanticity (32). Using biochemical fractionation, we finally purified trehalose-6,6-dimycolate (TDM) as a Mincle ligand. TDM (initially called cord factor) is the most studied immunostimulatory lipid in mycobacteria and is one of the major components of complete Freund’s adjuvant. Although several candidate receptors were proposed, the identity of the TDM receptor remained elusive for a half century (33, 34). In 2009, Werninghaus et al. demonstrated that TDM signals through the FcRγ–Syk–CARD9 axis (35). Then, our group finally identified Mincle as a long-sought receptor of TDM (34). Mincle is essential for the recognition of TDM by macrophages and the subsequent activation of the mouse immune system, revealing that Mincle was the missing link between mycobacterial TDM and host immune responses (10, 36). A combination of sugar and lipid moieties appeared to be necessary for recognition by Mincle, as purified mycolic acid and soluble trehalose failed to activate Mincle (10). However, the structural basis for this recognition remained unclear until 4 years later (see below). Trehalose dibehenate (TDB), the synthetic analogue of TDM that was developed to replace TDM as adjuvant, showed a similar response to Mincle. Indeed, TDM and TDB augment strong T-cell responses and antibody production against immunized antigen, which disappeared in Mincle-deficient mice (36, 37). Other glycolipid ligands in pathogens Although Mincle has been known to recognize Malassezia since 2009, the Mincle ligands in Malassezia had remained unclear. As Malassezia uniquely requires lipid for its growth, we hypothesized that Malassezia may possess an unknown lipid ligand because it does not have TDM. We, therefore, purified the active fraction using the aqueous-organic solvents and chromatography that we established before (10). Four years after finding that Mincle recognizes Malassezia, the Mincle ligands in Malassezia were identified in 2013 as glyceroglycolipid and unique mannosyl fatty acids linked to mannitol (11). In addition, Behler-Janbeck, Takano et al. isolated glucosyl-diacylglycerol from Streptococcus pneumoniae as a ligand of Mincle (21). Thus, Mincle appears to recognize a similar but broad spectrum of amphiphilic glycolipids as ligands. The structures of this wide variety of ligands tell us the common signature of Mincle ligands (Fig. 1). Fig. 1. View largeDownload slide Structures of representative Mincle ligands. The typical Mincle ligands have the structure containing sugar and lipid moieties, through which they bind to the CRD of Mincle. These glycolipids are widely found in bacteria and fungi, mediating the recognition of pathogens by Mincle. Besides exogenous ligands, Mincle could also recognize some metabolites derived from our own body. Fig. 1. View largeDownload slide Structures of representative Mincle ligands. The typical Mincle ligands have the structure containing sugar and lipid moieties, through which they bind to the CRD of Mincle. These glycolipids are widely found in bacteria and fungi, mediating the recognition of pathogens by Mincle. Besides exogenous ligands, Mincle could also recognize some metabolites derived from our own body. How does Mincle recognize ligand? Structural analysis of the Mincle CRD revealed the mechanism by which Mincle recognizes the combination of sugar and lipid moieties (38–41). Like many other C-type CRDs, the Mincle CRD has a canonical sugar-binding site centered on Ca2+, which contains the EPN (Glu-Pro-Asn) motif and can bind one glucose residue of the trehalose headgroup. Next to this site, there is a second binding site for the other glucose residue. On the other side of the canonical glucose-binding site, there is a hydrophobic groove that can bind acyl groups, providing a docking site for the acyl chain that attaches to the first glucose residue. This mode of two binding sites for two glucose residues increases the affinity for trehalose binding compared with glucose binding (38). Extended studies from the same group showed that the binding affinity of Mincle ligands containing a hydrophobic moiety correlates with the length of the hydrophobic side chain (40, 42). Understanding these mechanisms promoted the discovery of Mincle ligand; in 2015, Jacobsen et al. discovered that brartemicin, a trehalose-derived metabolite of actinomycete, has a high affinity to Mincle (20, 41). Later, Decout et al. dissected the contribution of each moiety of ligand to its binding to Mincle (26). These studies enabled the rational design of a Mincle ligand, which could be used as adjuvant in vaccine development. Recognition of an intracellular metabolite, glucosylceramide In addition to microbes, glycolipids are also involved in biological processes in mammals. Considering that Mincle can recognize dead cells derived from self and can induce sterile inflammation (4), it is possible that some self-glycolipids could be released from damaged cells and act as Mincle ligands. We detected an active fraction in the culture supernatant of damaged cells and finally identified β-glucosylceramide (β-GlcCer) as an endogenous Mincle ligand (30). β-GlcCer is an intracellular metabolite in the ceramide pathway that is the first intermediate in the synthesis of a large family of glycosphingolipids from ceramide. The level of β-GlcCer is normally controlled by β-glucocerebrosidase (GBA1), which is a crucial enzyme in β-GlcCer degradation, and the homozygous mutation of human GBA1 leads to Gaucher disease due to the abnormal accumulation of β-GlcCer in cells (43–47). Patients suffer from systemic inflammation, but the causal relationship between this inflammation and β-GlcCer accumulation had not been elucidated clearly (48). A mouse model of Gaucher disease that has a mutant Gba1 gene showed exacerbated inflammation upon cell damage, which was reversed by further deletion of the Mincle allele (Gba1–/– × Mincle–/–) (30, 49). In addition, GBA1–/– dendritic cells (DCs) promoted acquired immune responses more potently than WT DCs did in a Mincle-dependent way, suggesting that β-GlcCer is an endogenous adjuvant exerting immunostimulatory activity through Mincle. Thus, the β-GlcCer–Mincle axis is the first discovered self-glycolipid–CLR pathway conserved in a wide variety of mammalian species. Endogenous cholesterol derivatives Besides glycolipids having fatty acids, steroid compounds were also found to be Mincle ligands. In 2015, our group observed that cholesterol crystals can specifically activate human Mincle, but not mouse Mincle (28). Cholesterol crystals exist in atherosclerotic plaques and induce inflammation and reportedly activate NLRP inflammasomes (50–52). Hence, human Mincle is the first identified direct sensor that provides ‘signal 1’ for IL-1β secretion during inflammasome activation. In 2017, Kostarnoy et al. reported that mouse Mincle recognizes cholesterol sulfate in the skin as a damage-associated ligand and mediates inflammatory responses in an allergic skin reaction (29). Currently, the structural basis of the recognition of these steroid compounds by Mincle remains unclear. However, this novel class of endogenous ligands may be involved in metabolic regulation in adipose tissue, tumors or the nerve system in physiological and pathological settings (53–56). The regulation of Mincle expression As its name suggests, Mincle is an inducible protein and is barely detectable in resting cells. We found that TDM stimulation itself can induce Mincle expression, implying that another TDM receptor is expressed in resting cells and induces Mincle expression upon TDM stimulation. Through sequence analysis of Mincle CRD, we identified another TDM receptor, MCL (Clec4d) (57), which is highly homologous to Mincle (1, 3). MCL is constitutively expressed in myeloid cells and could induce Mincle expression through FcRγ (57–59). In addition to transcriptional regulation, several reports then implied that MCL also controls Mincle expression post-translationally. In the absence of MCL, surface expression of Mincle is greatly impaired even after being exposed to stimuli that induce Mincle mRNA, such as TLR ligands. We and others revealed that MCL and Mincle form a heterodimer that is indispensable for the surface expression of both molecules (59–61). Thus, the legendary potency of TDM as an adjuvant may now be explained by the synergistic contribution of two CLRs, i.e. Mincle and MCL. Besides, heteromeric complexes of different CLRs may expand the limited diversity of the ligand spectrum recognized by invariant ‘germline-coded’ CLRs, and thus it is possible that some other unknown heteromeric CLR pairs are formed and expressed in our cells (61, 62). In vivo relevance of Mincle in health and diseases In 2012, the role of Mincle during mycobacterial infection was first addressed using animal models (63). Mincle-deficient mice showed a higher bacterial load after intratracheal infection with Mycobacterium bovis Bacillus Calmette–Guérin and had compromised innate immune responses in the lung. The protective effect of Mincle against mycobacteria was confirmed using a systemic infection model via intravenous injection (64). It is possible that multiple innate immune receptors including other CLRs that recognize mycobacteria (57, 62, 65, 66) may play some redundant roles, in particular in the in vivo setting, which may cause the different extent of the contribution of Mincle during infection (63, 64, 67, 68). Although Mincle was first identified as a ‘macrophage’-inducible C-type lectin, we now know that it may also be expressed in other cell types, such as DCs, neutrophils, B cells and even some subsets of T cells (64, 67, 69–71). Thus, the role of Mincle in these particular cells should be taken into account in various stages of infection. In addition to mycobacteria, the protective role of Mincle against other bacterial and fungal infection has been reported, such as Klebsiella pneumoniae, Streptococcus pneumoniae, Fonsecaea pedrosoi and Pneumocystis (13,15, 21, 24). Irrespective of the identification of Mincle as a TDM receptor, TDM and TDB have been known to promote potent acquired immune responses as adjuvants (35–37, 57, 72, 73), suggesting a critical role of Mincle in acquired as well as innate immunity. Several studies reported that Mincle and MCL are necessary for the responses of Th1 cells, Th17 cells or both (36, 37, 57, 72). Although the precise mechanism by which the same CLR induces different Th cell responses has not been elucidated, the intensity of signaling through ITAMs may determine Th cell orientation (27, 74, 75). Conclusion In recent years, CLRs were found to play a very important role in immune responses, because of their characteristic specificity and diversity. Among them, Mincle was shown to be a crucial PRR, serving in pathogen elimination and immune homeostasis, by sensing pathogens including bacteria, fungi and parasites as well as endogenous ligands (Table 1). Interestingly, Mincle from different species have different affinity in binding some specific ligands (12, 28). It would be tempting to speculate that Mincle evolved with the species to flexibly respond to environmental stresses. In addition to being a direct receptor of vaccine adjuvants, Mincle may also be a provocative molecule as a target for antibody-mediated therapy for diseases in which Mincle is inappropriately activated (53–55, 76–78). As the study of Mincle goes deeper, it becomes more clear that Mincle is a critical and resourceful receptor in our immune system (Fig. 2). As a promising therapeutic target, Mincle still has various obscure properties that need to be investigated further in the future. Fig. 2. View largeDownload slide Various immune responses triggered by Mincle. Mincle expressed on myeloid cells can sense ligands come from pathogens and damaged self. Myeloid cells are activated upon ligand binding, leading to the secretion of cytokines as well as antigen presentation by MHC class II, and consequently activate adaptive immunity. On the other hand, activated myeloid cells could also secrete some effectors. Through these responses, Mincle functions in host defense (13, 15, 21, 24, 63, 64) and of dead tissue clearance (79, 80). These procedures are precisely controlled in healthy immune system; however, the overreaction of any step would induce harmful responses to our body, generating disorders such as inflammatory diseases (29, 30, 53, 55, 77, 78), autoimmune (81) or tissue injury (82, 83). Fig. 2. View largeDownload slide Various immune responses triggered by Mincle. Mincle expressed on myeloid cells can sense ligands come from pathogens and damaged self. Myeloid cells are activated upon ligand binding, leading to the secretion of cytokines as well as antigen presentation by MHC class II, and consequently activate adaptive immunity. On the other hand, activated myeloid cells could also secrete some effectors. Through these responses, Mincle functions in host defense (13, 15, 21, 24, 63, 64) and of dead tissue clearance (79, 80). These procedures are precisely controlled in healthy immune system; however, the overreaction of any step would induce harmful responses to our body, generating disorders such as inflammatory diseases (29, 30, 53, 55, 77, 78), autoimmune (81) or tissue injury (82, 83). Conflicts of interest statement: The authors declared no conflicts of interest. References 1 Matsumoto, M., Tanaka, T., Kaisho, T.et al.   1999. A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages. J. Immunol . 163: 5039. Google Scholar PubMed  2 Tanaka, T., Akira, S., Yoshida, K.et al.   1995. 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Mincle: 20 years of a versatile sensor of insults

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Abstract

Abstract Macrophage-inducible C-type lectin, better known as Mincle, is a member of the C-type lectin receptor family and is encoded by Clec4e. Mincle was an orphan receptor for a long time after having been discovered as a lipopolysaccharide-induced protein, yet later an adjuvant glycolipid in mycobacteria—trehalose dimycolate—was identified as a ligand. Ligands for Mincle were also found existing in bacteria, fungi and even mammals. When confronted with foreign elements, Mincle can recognize characteristic pathogen-associated molecular patterns, mostly glycolipids, from Mycobacterium tuberculosis and other pathogens, and thus induce immune responses against infection. To maintain self-homeostasis, Mincle can recognize lipid-based damage-associated molecular patterns, thereby monitoring the internal environment. The mechanism by which Mincle functions in the immune system is also becoming more clear along with the identification of its ligands. Being expressed widely on antigen-presenting cells, Mincle activation leads to the production of cytokines and chemokines, neutrophil infiltration and other inflammatory responses. Besides, Mincle can induce acquired immunity such as antigen-specific T-cell responses and antibody production as an adjuvant receptor. In this review, we will retrospectively sketch the discovery and study of Mincle, and outline some current work on this receptor. C-type lectin receptors, glycolipids, innate immunity, pathogens Introduction From its name (macrophage-inducible C-type lectin) and primary structure, one may suspect Mincle’s function as an inducible receptor for microbes. Indeed, the identification of its ligands and structure unveiled its critical role as an innate immune receptor. In this review, we will introduce the findings and their significance related to Mincle following the chronological order of several milestones. We begin by describing the identification of Mincle, and the mechanism of signaling transduction. Then we detail the findings of Mincle ligands, as well as its structure and mechanism of ligand recognition. In the later parts, we concentrate on the regulation and functions of Mincle, which lead to its potential applications in medicine. Identification of Mincle Mincle was firstly identified as a transcriptional target of NF-IL6 in macrophages by Akira’s group in 1999 (1). NF-IL6 (also called C/EBPβ) is a transcription factor that is activated by inflammatory stimuli and can induce the expression of various inflammatory genes (2). In a further screening for transcriptional targets using NF-IL6-deficient mice, Matsumoto et al. cloned Mincle as a lipopolysaccharide-induced NF-IL6-dependent molecule in macrophages. Subsequent analysis of the primary sequence suggested that Mincle is a type-II transmembrane molecule and has an extracellular carbohydrate-recognition domain (CRD), similar to C-type lectins such as macrophage C-type lectin (MCL) (3). Besides mouse Mincle, the authors also reported its homologue in humans, which has an overall identity of 67% and a similarity of 85% with mouse Mincle. Mincle is coupled with FcRγ It took nearly 10 years to make any progress in the study of Mincle after its identification. In 2008, our group firstly elucidated that Mincle couples with FcRγ and transduces an activation signal through the immunoreceptor tyrosine-based activation motif (ITAM) in FcRγ (4). The residue at position 42 (Arg42) in the transmembrane region of Mincle provides the positive charge that is needed in the interaction with FcRγ, which has a negatively charged residue. Taking advantage of an antibody that activates Mincle, we demonstrated that the ITAM in FcRγ is indispensable for activated Mincle to transduce downstream signaling, in which the kinases Syk and Erk are also activated, and finally lead to the expression of pro-inflammatory cytokines in a CARD9-dependent way. CARD9 is a key adaptor molecule downstream of ITAM signaling. Coupling with Bcl10 and Malt1, CARD9 mediates the cytokine expression in macrophages and is required for pathogen clearance (5–7). It is, therefore, possible that Mincle might also play a role in protective immunity against pathogens. Mincle ligands, from discovery to design Like ‘authentic’ pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), NOD-like receptors (NLRs) or RIG-I-like receptors (RLRs), a C-type lectin receptor (CLR) can recognize a series of ligands that have a specific ‘pattern’, and so can Mincle. In 2008, Mincle was found to have the capability of sensing endogenous and exogenous targets (4, 8, 9). After that, accumulated evidence showed that Mincle ligands exist in bacteria, fungi and mammals (Table 1). The first report related to the function of Mincle in the immune system was published in 2008 by Wells et al. (8). They found that the expression of Mincle in macrophages was up-regulated after exposure to live Candida albicans. Mincle-deficient mice infected with C. albicans showed a significantly higher fungal burden than wild-type (WT) mice did, suggesting that Mincle may play a role in the host defense against C. albicans, although the Mincle ligand in this fungus is unidentified even today. Table 1. Molecular candidates recognized by Mincle Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  b, bovine; h, human; m, mouse. View Large Table 1. Molecular candidates recognized by Mincle Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  Self/ non-self  Ligand name  Property  Source  Reaction  Ref.  Non-self  TDM  Glycolipid  Mycobacteria  m, h  (10)  TDB  Glycolipid  Synthesized  m, h  (10)  Glyceroglycolipid  Glycolipid  Malassezia  m  (11)  Mannosyl fatty acids linking mannitol  Glycolipid  Malassezia  m  (11)  Glycerol monomycolate (GroMM)  Glycolipid  Mycobacteria  h  (12)  Unknown  Unknown  Candida albicans  m, h  (8, 9)  Unknown  Unknown  Fonsecaea pedrosoi  m  (13)  Unknown  Unknown  Fonsecaea monophora  h  (14)  Unknown  Unknown  Klebsiella pneumoniae  m  (15)  Corynomycolic acid- containing glycolipids, i.e. trehalose dicorynomycolate  Glycolipid  Synthesized  m, h  (16, 17)  Corynebacteria  m  (18)  β-Gentiobiosyl diacylglycerides  Glycolipid  Mycobacteria, synthesized  m  (19)  β-Glucosyl diglyceride  Glycolipid  Synthesized  m, h  (19)  Brartemicin  Glycolipid  Nonomuraea sp.  h, b  (20)  α-Glucosyl- diacylglycerol  Glycolipid  Streptococcus pneumoniae  m, h  (21)  Lactobacillus plantarum, synthesized  m, h  (22)  Unknown  Glycoprotein  Tannerella forsythia  h  (23)  Unknown  Glycoprotein  Pneumocystis  m  (24)  Unknown  Unknown  Leishmania  m, h  (25)  Glucose 2-tetradecyloctadecanoate (GlcC14C18)  Glycolipid  Synthesized  m, h  (26)  Mannose 2-tetradecyloctadecanoate (ManC14C18)  Glycolipid  Synthesized  m, h  (26)  Glucose monomycolate (GMM)  Glycolipid  Mycobacteria  m, h  (26)  Arabinose monomycolates (AraMM)  Glycolipid  Synthesized  m  (27)  Self  SAP-130  Protein  Mammal  m  (4)  Cholesterol crystal  Cholesterol  Mammal  h  (28)  Cholesterol sulfate  Cholesterol  Mammal  m  (29)  β-GlcCer  Glycolipid  Mammal  m, h  (30)  b, bovine; h, human; m, mouse. View Large Recognition of dead cells derived from self The first Mincle ligand appeared in front of us by a mere chance. To screen the Mincle ligands, we established a reporter cell line expressing Mincle and FcRγ. When Mincle is activated by ligand, if any, it will activate the NFAT and lead to the expression of GFP, which was confirmed using a plate-coated antibody against Mincle. When we accidentally left the cultured reporter cells for several days without changing the medium, we found that the number of GFP-positive living cells increased with the number of dead cells, suggesting Mincle as a sensor for dead cells (4). Whole-body irradiation can induce significant thymocyte death followed by neutrophil infiltration into the thymus. As anti-Mincle mAb blocked this infiltration, Mincle may mediate dead-cell-induced inflammation. From dead cell lysates, we purified spliceosome-associated protein 130 (SAP-130) as a candidate that selectively bound to a Mincle–Ig fusion protein (4). However, the exact contribution of SAP-130 to dead-cell-induced inflammation is unclear, as this protein is essential for RNA splicing, a fundamental process in all living cells. Mincle recognizes pathogenic fungi Unlike the unexpected finding of the first Mincle ligand, the GFP-reporter cell line of Mincle was used in an intended screen of pathogenic fungus species. As a result, Mincle could specifically recognize only Malassezia species among a number of pathogenic fungus species (31). Interestingly, three C. albicans substrains did not activate cells through Mincle, although C. albicans was suggested to contain Mincle ligands (8, 9). To evaluate the function of Mincle in immune responses against Malassezia, we analyzed Mincle-deficient mice established by Akira’s group, and found that the production of inflammatory cytokines such as IL-6 and TNF as well as neutrophil infiltration at the injection site was significantly impaired in Mincle-deficient mice, indicating that Mincle mediates the host defense responses against Malassezia (31). Nevertheless, the identity of a Mincle ligand was still obscure at the time. Mincle recognizes mycobacterial glycolipids The breakthrough in exploring Mincle ligands was brought about by further bacterial study; we found that all three tested Mycobacterium species could be recognized by Mincle (10). Mycobacteria have long been known to possess unique lipid components with potent adjuvanticity (32). Using biochemical fractionation, we finally purified trehalose-6,6-dimycolate (TDM) as a Mincle ligand. TDM (initially called cord factor) is the most studied immunostimulatory lipid in mycobacteria and is one of the major components of complete Freund’s adjuvant. Although several candidate receptors were proposed, the identity of the TDM receptor remained elusive for a half century (33, 34). In 2009, Werninghaus et al. demonstrated that TDM signals through the FcRγ–Syk–CARD9 axis (35). Then, our group finally identified Mincle as a long-sought receptor of TDM (34). Mincle is essential for the recognition of TDM by macrophages and the subsequent activation of the mouse immune system, revealing that Mincle was the missing link between mycobacterial TDM and host immune responses (10, 36). A combination of sugar and lipid moieties appeared to be necessary for recognition by Mincle, as purified mycolic acid and soluble trehalose failed to activate Mincle (10). However, the structural basis for this recognition remained unclear until 4 years later (see below). Trehalose dibehenate (TDB), the synthetic analogue of TDM that was developed to replace TDM as adjuvant, showed a similar response to Mincle. Indeed, TDM and TDB augment strong T-cell responses and antibody production against immunized antigen, which disappeared in Mincle-deficient mice (36, 37). Other glycolipid ligands in pathogens Although Mincle has been known to recognize Malassezia since 2009, the Mincle ligands in Malassezia had remained unclear. As Malassezia uniquely requires lipid for its growth, we hypothesized that Malassezia may possess an unknown lipid ligand because it does not have TDM. We, therefore, purified the active fraction using the aqueous-organic solvents and chromatography that we established before (10). Four years after finding that Mincle recognizes Malassezia, the Mincle ligands in Malassezia were identified in 2013 as glyceroglycolipid and unique mannosyl fatty acids linked to mannitol (11). In addition, Behler-Janbeck, Takano et al. isolated glucosyl-diacylglycerol from Streptococcus pneumoniae as a ligand of Mincle (21). Thus, Mincle appears to recognize a similar but broad spectrum of amphiphilic glycolipids as ligands. The structures of this wide variety of ligands tell us the common signature of Mincle ligands (Fig. 1). Fig. 1. View largeDownload slide Structures of representative Mincle ligands. The typical Mincle ligands have the structure containing sugar and lipid moieties, through which they bind to the CRD of Mincle. These glycolipids are widely found in bacteria and fungi, mediating the recognition of pathogens by Mincle. Besides exogenous ligands, Mincle could also recognize some metabolites derived from our own body. Fig. 1. View largeDownload slide Structures of representative Mincle ligands. The typical Mincle ligands have the structure containing sugar and lipid moieties, through which they bind to the CRD of Mincle. These glycolipids are widely found in bacteria and fungi, mediating the recognition of pathogens by Mincle. Besides exogenous ligands, Mincle could also recognize some metabolites derived from our own body. How does Mincle recognize ligand? Structural analysis of the Mincle CRD revealed the mechanism by which Mincle recognizes the combination of sugar and lipid moieties (38–41). Like many other C-type CRDs, the Mincle CRD has a canonical sugar-binding site centered on Ca2+, which contains the EPN (Glu-Pro-Asn) motif and can bind one glucose residue of the trehalose headgroup. Next to this site, there is a second binding site for the other glucose residue. On the other side of the canonical glucose-binding site, there is a hydrophobic groove that can bind acyl groups, providing a docking site for the acyl chain that attaches to the first glucose residue. This mode of two binding sites for two glucose residues increases the affinity for trehalose binding compared with glucose binding (38). Extended studies from the same group showed that the binding affinity of Mincle ligands containing a hydrophobic moiety correlates with the length of the hydrophobic side chain (40, 42). Understanding these mechanisms promoted the discovery of Mincle ligand; in 2015, Jacobsen et al. discovered that brartemicin, a trehalose-derived metabolite of actinomycete, has a high affinity to Mincle (20, 41). Later, Decout et al. dissected the contribution of each moiety of ligand to its binding to Mincle (26). These studies enabled the rational design of a Mincle ligand, which could be used as adjuvant in vaccine development. Recognition of an intracellular metabolite, glucosylceramide In addition to microbes, glycolipids are also involved in biological processes in mammals. Considering that Mincle can recognize dead cells derived from self and can induce sterile inflammation (4), it is possible that some self-glycolipids could be released from damaged cells and act as Mincle ligands. We detected an active fraction in the culture supernatant of damaged cells and finally identified β-glucosylceramide (β-GlcCer) as an endogenous Mincle ligand (30). β-GlcCer is an intracellular metabolite in the ceramide pathway that is the first intermediate in the synthesis of a large family of glycosphingolipids from ceramide. The level of β-GlcCer is normally controlled by β-glucocerebrosidase (GBA1), which is a crucial enzyme in β-GlcCer degradation, and the homozygous mutation of human GBA1 leads to Gaucher disease due to the abnormal accumulation of β-GlcCer in cells (43–47). Patients suffer from systemic inflammation, but the causal relationship between this inflammation and β-GlcCer accumulation had not been elucidated clearly (48). A mouse model of Gaucher disease that has a mutant Gba1 gene showed exacerbated inflammation upon cell damage, which was reversed by further deletion of the Mincle allele (Gba1–/– × Mincle–/–) (30, 49). In addition, GBA1–/– dendritic cells (DCs) promoted acquired immune responses more potently than WT DCs did in a Mincle-dependent way, suggesting that β-GlcCer is an endogenous adjuvant exerting immunostimulatory activity through Mincle. Thus, the β-GlcCer–Mincle axis is the first discovered self-glycolipid–CLR pathway conserved in a wide variety of mammalian species. Endogenous cholesterol derivatives Besides glycolipids having fatty acids, steroid compounds were also found to be Mincle ligands. In 2015, our group observed that cholesterol crystals can specifically activate human Mincle, but not mouse Mincle (28). Cholesterol crystals exist in atherosclerotic plaques and induce inflammation and reportedly activate NLRP inflammasomes (50–52). Hence, human Mincle is the first identified direct sensor that provides ‘signal 1’ for IL-1β secretion during inflammasome activation. In 2017, Kostarnoy et al. reported that mouse Mincle recognizes cholesterol sulfate in the skin as a damage-associated ligand and mediates inflammatory responses in an allergic skin reaction (29). Currently, the structural basis of the recognition of these steroid compounds by Mincle remains unclear. However, this novel class of endogenous ligands may be involved in metabolic regulation in adipose tissue, tumors or the nerve system in physiological and pathological settings (53–56). The regulation of Mincle expression As its name suggests, Mincle is an inducible protein and is barely detectable in resting cells. We found that TDM stimulation itself can induce Mincle expression, implying that another TDM receptor is expressed in resting cells and induces Mincle expression upon TDM stimulation. Through sequence analysis of Mincle CRD, we identified another TDM receptor, MCL (Clec4d) (57), which is highly homologous to Mincle (1, 3). MCL is constitutively expressed in myeloid cells and could induce Mincle expression through FcRγ (57–59). In addition to transcriptional regulation, several reports then implied that MCL also controls Mincle expression post-translationally. In the absence of MCL, surface expression of Mincle is greatly impaired even after being exposed to stimuli that induce Mincle mRNA, such as TLR ligands. We and others revealed that MCL and Mincle form a heterodimer that is indispensable for the surface expression of both molecules (59–61). Thus, the legendary potency of TDM as an adjuvant may now be explained by the synergistic contribution of two CLRs, i.e. Mincle and MCL. Besides, heteromeric complexes of different CLRs may expand the limited diversity of the ligand spectrum recognized by invariant ‘germline-coded’ CLRs, and thus it is possible that some other unknown heteromeric CLR pairs are formed and expressed in our cells (61, 62). In vivo relevance of Mincle in health and diseases In 2012, the role of Mincle during mycobacterial infection was first addressed using animal models (63). Mincle-deficient mice showed a higher bacterial load after intratracheal infection with Mycobacterium bovis Bacillus Calmette–Guérin and had compromised innate immune responses in the lung. The protective effect of Mincle against mycobacteria was confirmed using a systemic infection model via intravenous injection (64). It is possible that multiple innate immune receptors including other CLRs that recognize mycobacteria (57, 62, 65, 66) may play some redundant roles, in particular in the in vivo setting, which may cause the different extent of the contribution of Mincle during infection (63, 64, 67, 68). Although Mincle was first identified as a ‘macrophage’-inducible C-type lectin, we now know that it may also be expressed in other cell types, such as DCs, neutrophils, B cells and even some subsets of T cells (64, 67, 69–71). Thus, the role of Mincle in these particular cells should be taken into account in various stages of infection. In addition to mycobacteria, the protective role of Mincle against other bacterial and fungal infection has been reported, such as Klebsiella pneumoniae, Streptococcus pneumoniae, Fonsecaea pedrosoi and Pneumocystis (13,15, 21, 24). Irrespective of the identification of Mincle as a TDM receptor, TDM and TDB have been known to promote potent acquired immune responses as adjuvants (35–37, 57, 72, 73), suggesting a critical role of Mincle in acquired as well as innate immunity. Several studies reported that Mincle and MCL are necessary for the responses of Th1 cells, Th17 cells or both (36, 37, 57, 72). Although the precise mechanism by which the same CLR induces different Th cell responses has not been elucidated, the intensity of signaling through ITAMs may determine Th cell orientation (27, 74, 75). Conclusion In recent years, CLRs were found to play a very important role in immune responses, because of their characteristic specificity and diversity. Among them, Mincle was shown to be a crucial PRR, serving in pathogen elimination and immune homeostasis, by sensing pathogens including bacteria, fungi and parasites as well as endogenous ligands (Table 1). Interestingly, Mincle from different species have different affinity in binding some specific ligands (12, 28). It would be tempting to speculate that Mincle evolved with the species to flexibly respond to environmental stresses. In addition to being a direct receptor of vaccine adjuvants, Mincle may also be a provocative molecule as a target for antibody-mediated therapy for diseases in which Mincle is inappropriately activated (53–55, 76–78). As the study of Mincle goes deeper, it becomes more clear that Mincle is a critical and resourceful receptor in our immune system (Fig. 2). As a promising therapeutic target, Mincle still has various obscure properties that need to be investigated further in the future. Fig. 2. View largeDownload slide Various immune responses triggered by Mincle. Mincle expressed on myeloid cells can sense ligands come from pathogens and damaged self. Myeloid cells are activated upon ligand binding, leading to the secretion of cytokines as well as antigen presentation by MHC class II, and consequently activate adaptive immunity. On the other hand, activated myeloid cells could also secrete some effectors. Through these responses, Mincle functions in host defense (13, 15, 21, 24, 63, 64) and of dead tissue clearance (79, 80). These procedures are precisely controlled in healthy immune system; however, the overreaction of any step would induce harmful responses to our body, generating disorders such as inflammatory diseases (29, 30, 53, 55, 77, 78), autoimmune (81) or tissue injury (82, 83). Fig. 2. View largeDownload slide Various immune responses triggered by Mincle. Mincle expressed on myeloid cells can sense ligands come from pathogens and damaged self. Myeloid cells are activated upon ligand binding, leading to the secretion of cytokines as well as antigen presentation by MHC class II, and consequently activate adaptive immunity. On the other hand, activated myeloid cells could also secrete some effectors. Through these responses, Mincle functions in host defense (13, 15, 21, 24, 63, 64) and of dead tissue clearance (79, 80). These procedures are precisely controlled in healthy immune system; however, the overreaction of any step would induce harmful responses to our body, generating disorders such as inflammatory diseases (29, 30, 53, 55, 77, 78), autoimmune (81) or tissue injury (82, 83). Conflicts of interest statement: The authors declared no conflicts of interest. References 1 Matsumoto, M., Tanaka, T., Kaisho, T.et al.   1999. A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages. J. Immunol . 163: 5039. Google Scholar PubMed  2 Tanaka, T., Akira, S., Yoshida, K.et al.   1995. 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International ImmunologyOxford University Press

Published: May 2, 2018

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