CLEC4E is a type 2 transmembrane receptor that is a member of the C-type lectin family of immune receptors and possesses a carbohydrate-recognition domain in its extracellular region. It was originally identified as a downstream transcriptional target of CCAAT/enhancer-binding protein beta in mouse macrophages and termed macrophage-inducible protein – Mincle (Matsumoto et al. 1999). The finding 10 years later that CLEC4E is a key receptor for the mycobacterial cell wall component, trehalose dimycolate, led to a surge in interest in the role of CLEC4E in controlling mycobacterial infection and in its ligands as potential vaccine adjuvants (Ishikawa et al. 2009). Furthermore, data has emerged implicating CLEC4E in the immune response to fungal infections including Candida and Malassezia and in sterile inflammation mediated by an endogenous ligand, SAP130 (Wells et al. 2008; Yamasaki et al. 2008, 2009). Crystallization of CLEC4E and refined ligand binding studies have led to a detailed model of ligand binding involving both sugar- and lipid-binding sites in CLEC4E (Furukawa et al. 2013).
CLEC4E Expression Pattern and Regulation of Expression
In humans, the CLEC4E gene is on chromosome 12 at the 12p13.31 locus and its 6 exon sequence encodes a 219 amino acid protein with a predicted 19 amino acid cytoplasmic domain and 20 amino acid transmembrane domain. In mice, CLEC4E is on chromosome 6 and encodes a 214 amino acid protein.
At the transcript level, mouse CLEC4E is expressed in bone marrow, lymph node, spleen, lung, and in healing corneas (Comelli et al. 2006; Saravanan et al. 2010). CLEC4E is transcribed in a wide range of leukocytes, including macrophages, neutrophils, dendritic cells, B cells, CD4+ T cells, CD8+ T cells, concanavalin A blasts, and M1 myeloblastic leukemia cells, as well as in brain microglia (Matsumoto et al. 1999; Flornes et al. 2004; McKimmie et al. 2006; Nakamura et al. 2006; Lee et al. 2012). At the protein level, mouse CLEC4E has been found predominantly on activated macrophages and CD11b+ Gr1+ neutrophils, present in the thymus of irradiated mice (Matsumoto et al. 1999; Wells et al. 2008; Yamasaki et al. 2008, 2009; Schoenen et al. 2010). Human CLEC4E is transcribed in LPS-stimulated THP1 monocytic leukemia cells and in bone marrow–derived mononuclear cells from rheumatoid arthritis patients (Matsumoto et al. 1999; Nakamura et al. 2006). Surface expression was detected on 13.7% of human peripheral B cells with higher expression on naive CD27−CD19+ compared to memory CD27+CD19+ B cells (Kawata et al. 2012). CLEC4E mRNA and protein was detected in human progenitor-derived mast cells (Ribbing et al. 2011). In guinea pigs, CLEC4E mRNA was detected in spleen, lymph nodes, and peritoneal macrophages, with surface expression detected on activated macrophages (Toyonaga et al. 2014).
CLEC4E expression is induced on macrophages by a variety of inflammatory stimuli, including lipopolysaccharide (LPS), TNF-α, interleukin (IL)-6, interferon-gamma (IFNγ), zymosan, mannan, β-glucan, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) (a bacterial peptidoglycan-derived small peptide), CpG oligodeoxyribonucleotides, curdlan, and mycobacterial glycolipids (Matsumoto et al. 1999; Guo et al. 2009; Schoenen et al. 2010, 2014; Toyonaga et al. 2014). Expression of CLEC4E is also induced by various infections, including Streptococcus pneumoniae, influenza A virus (Rosseau et al. 2007), Mycobacterium tuberculosis bacillus Calmette-Guérin (Khajoee et al. 2006), Semliki forest virus (McKimmie et al. 2006), C. albicans (Wells et al. 2008; Patin et al. 2016), and Helicobacter pylori (de Rivero Vaccari et al. 2015). At the protein level, CLEC4E has been shown to be upregulated by Malassezia fungal stimulation of thioglycollate-elicited peritoneal mouse macrophages (Yamasaki et al. 2009). Human CLEC4E is upregulated in bone marrow–derived mononuclear cells from rheumatoid arthritis patients compared with those from osteoarthritis patients (Nakamura et al. 2006). In mouse neutrophils, CLEC4E mRNA is upregulated by LPS and trehalose dimycolate (TDM) (Lee et al. 2012). In human peripheral B cells, surface expression was increased in response to a TLR9 ligand but not to LPS (Kawata et al. 2012). Stimulation of MCL, another receptor for TDM, caused mRNA level upregulation of CLEC4E through NFκb (Zhao et al. 2014) and increased protein expression of CLEC4E (Lobato-Pascual et al. 2013).
CLEC4E expression is influenced by obesity. Coculture of RAW264 mouse macrophages with adipocytes increased CLEC4E mRNA expression and the saturated fatty acid, palmitate, increased both mRNA and protein expression (Ichioka et al. 2011). CLEC4E mRNA expression was increased in adipose tissue of both ob/ob genetically obese mice and diet-induced obesity (Ichioka et al. 2011).
Relatively little is known about the mechanisms of transcriptional regulation influencing CLEC4E expression. CLEC4E is a transcriptional target of myeloid differentiation transcription factors, CCAAT/enhancer binding protein beta (C/EBP-β), and interferon regulatory factor-8 (IRF-8) (Matsumoto et al. 1999; Tamura et al. 2005; Schoenen et al. 2014).
CLEC4E Ligands and Structure
CLEC4E is a C-type lectin with a carbohydrate-recognition domain that has conserved residues that play a role in calcium-dependent carbohydrate binding (Matsumoto et al. 1999; Flornes et al. 2004). CLEC4E was predicted to bind carbohydrate ligands, and in particular mannose-containing carbohydrates, since it contains an EPN (glutamic acid-proline-asparagine) motif, which is a well-characterized mannose-binding motif present in a number of other C-type lectins, including DC-SIGN (CD209), Dectin-2, and Langerin. Indeed, recombinant CLEC4E protein binds to a highly multivalent form of α-mannose in a Ca2+-dependent manner, but notably not to mannan (Yamasaki et al. 2009). In a plate-bound direct binding assay, human CLEC4E showed some evidence of binding to BSA-conjugated mannose, fucose, N-acetyl-glucosamine, Galactose, N-acetyl-galactosamine, and LewisX antigen (Ichioka et al. 2011).
CLEC4E is a receptor for the major mycobacterial glycolipid, trehalose-6,6′-dimycolate (TDM), in humans, mice, and guinea pigs (Ishikawa et al. 2009; Schoenen et al. 2010) (Toyonaga et al. 2014). TDM is an effective adjutant and potent immunostimulant causing TNFα release from macrophages and granuloma formation in vivo (Ryll et al. 2001). TDM is composed of a trehalose moiety (a disaccharide made up of two glucose units) and two long-chain α-branched, β-hydroxyl fatty acids, known as mycolic acids. Proteins with EPN mannose motifs are also able to bind structurally related sugars, such as glucose, and indeed CLEC4E recognition of M. bovis and M. tuberculosis is dependent upon the EPN motif of CLEC4E. Mutation experiments indicate that EPN motif is required for TDM binding (Furukawa et al. 2013). Neither trehalose alone nor purified mycolate alone stimulates CLEC4E-expressing cells (Ishikawa et al. 2009). In addition, the long, branched structure of mycolic acids is not essential for stimulatory activity since a synthetic analogue of TDM, trehalose dibehenate (TDB), which contains shorter fatty acid chains, is a potent stimulator of CLEC4E-expressing cells (Ishikawa et al. 2009). CLEC4E may, therefore, be specific for the ester linkage between the fatty acid and trehalose. Insertion of a polyethylene glycol moiety between the head group and acyl chain of trehalose esters reduces CLEC4E binding (Huber et al. 2016). The presence of two acyl chains is not required for signaling through CLEC4E since synthetic trehalose mono-esters with 22 or 26 carbon chain lengths trigger CLEC4E dependent NO and IL-6 release from mouse macrophages (Stocker et al. 2014). In another study, trehalose di-esters with acyl chains varying from 14–22 were more potent activators of CLEC4E than trehalose mono-esters (Huber et al. 2016). Glycerol monomycolate is a ligand for human but not for mouse CLEC4E (Hattori et al. 2014). A subsequent study suggested that it was predominantly the 20S-stereoisome that was responsible for signaling through CLEC4E (van der Peet et al. 2015). Synthetic analogues of corynebacterium glycolipids activate CLEC4E with similar potency to TDM (van der Peet et al. 2015). These include trehalose di-corynomycolic acid, trehalose mono- corynomycolic acid, and glucose mono-corynomycolic acid. By contrast, glucose 6-behenate did not signal through CLEC4E (van der Peet et al. 2015). Several synthetic analogues of another class of mycobacterial glycolipids, gentiobiosyl diglycerides, have shown a relatively weak ability to signal through mouse CLEC4E (Richardson et al. 2015). Of these compounds, one was shown to bind to mouse CLEC4E and stimulate BMDM (Richardson et al. 2015). This compound, iso-C 17b-glucosyldiglyceride was capable of signaling through human Mincle (Richardson et al. 2015).
There is evidence suggesting that CLEC4E is a receptor for Candida albicans and Malassezia fungal species. Mouse CLEC4E binds to a soluble component of heat-killed Candida albicans and Saccharomyces cerevisiae (Bugarcic et al. 2008; Wells et al. 2008). Recognition of Malassezia is dependent upon the EPN motif of CLEC4E (Yamasaki et al. 2009). Two glycolipids from Malassezia, termed 44-1 and 44-2 have been identified as specific ligands for CLEC4E (Ishikawa et al. 2013).
Mouse CLEC4E is able to bind to Streptococcus pneumonia but does not have a significant role in the immune response to this pathogen (Rabes et al. 2015). Bovine CLEC4E binds to Brartemicin, a natural product of Nonomuraea sp., that contains a doubly esterified α,α-trehalose core structure similar to TDM (Jacobsen et al. 2015). Brartemicin, and several analogues that also bind CLEC4E have potent antitumor activity (Jacobsen et al. 2015).
Two endogenous ligands for CLEC4E have been identified (Yamasaki et al. 2008; Kiyotake et al. 2015). CLEC4E binds to spliceosome-associated protein 130 (SAP130) in a carbohydrate-independent manner (Yamasaki et al. 2008). SAP130 is normally located in the nucleus as a component of the SF3b complex, which forms part of the U2 snRNP. U2 snRNP is involved in the assembly of spliceosomes. It is proposed that during cell death, SAP130 is released into the extracellular environment. Cholesterol crystals bind to and activate human CLEC4E, but not mouse CLEC4E which lacks a cholesterol recognition/interaction amino acid consensus sequence (Kiyotake et al. 2015). Related plant sterol sitosterol and cholesterol metabolism intermediary, desmosterol, also activate CLEC4E signaling (Kiyotake et al. 2015).
The structures of the extracellular CRD of human and bovine CLEC4E have been solved using crystallography (Feinberg et al. 2013; Furukawa et al. 2013). Together with other ligand binding studies, a detailed model has emerged of the mechanism by which CLEC4E binds to its canonical ligand, TDM. One glucose residue binds to the principle Ca2+-binding site and a second glucose binds to a secondary site (Jegouzo et al. 2014). The mycolyl chains can then bind to hydrophobic grooves adjacent to the sugar binding sites (Furukawa et al. 2013; Jegouzo et al. 2014). Residues within the CRD that are crucial for ligand binding are conserved across 62 mammalian species at the primary and secondary sugar binding sites (Rambaruth et al. 2015). There is also conservation of hydrophobic residues that may contribute to the site of acyl chain binding (Rambaruth et al. 2015).
CLEC4E Signaling and Regulation of Activity
Efficient cell-surface expression of CLEC4E on bone marrow–derived macrophages in response to LPS stimulation requires the expression of FcεRIγ (Yamasaki et al. 2008). FcεRIγ is essential for CLEC4E-mediated signaling. Stimulation of thioglycollate-elicited peritoneal macrophages or bone marrow–derived macrophages with monoclonal antibodies to CLEC4E (Yamasaki et al. 2008), or with fungal species (Wells et al. 2008; Yamasaki et al. 2009), induces the production of pro-inflammatory cytokines and chemokines in an FcεRIγ-dependent manner (Yamasaki et al. 2008). Furthermore, inflammatory cytokine production, lung swelling, and the formation of granulomas in response to TDM stimulation in vivo are dependent upon FcεRIγ (Ishikawa et al. 2009). FcεRIγ is also essential for the adjuvant properties of TDB (Schoenen et al. 2010). Cross-linking of CLEC4E induces phosphorylation of the kinases Syk and extracellular signal-regulated kinase (Erk) in peritoneal macrophages (Yamasaki et al. 2008). The adaptor molecule CARD9 has an important role in the downstream signaling, resulting in chemokine production (Yamasaki et al. 2008). The CLEC4E cytoplasmic domain contains two putative phosphorylation sites, Ser 3 and Thr 12 (Matsumoto et al. 1999).
CLEC4E appears to form heterodimeric complexes with MCL, another C-type lectin that recognizes TDM (Lobato-Pascual et al. 2013). MCL expression aids the surface localization of CLEC4E (Miyake et al. 2015). CLEC4E is not known to be involved in the internalization of ligands and does not play a role in the uptake of trehalose-related glycolipids (Kodar et al. 2015).
Role of CLEC4E Signaling in Disease
CLEC4E is a macrophage receptor for a number of pathogens, including Candida albicans, several species of Malassezia, and mycobacteria (Bugarcic et al. 2008; Wells et al. 2008; Ishikawa et al. 2009; Yamasaki et al. 2009; Schoenen et al. 2010). Signaling through CLEC4E contributes to inflammatory responses by inducing the production of cytokines and chemokines, including TNF-α, IL6, CXCL1 (KC), and CXCL2 (MIP-2) (Wells et al. 2008; Yamasaki et al. 2008; Ishikawa et al. 2009). CLEC4E may, however, have a more complex immunomodulatory role and induces secretion of the anti-inflammatory cytokine IL-10 in macrophages (Patin et al. 2016). In another study, CLEC4E signaling led to NO production, via upregulation of iNOS, that inhibited the NLRP3 inflammasome (Lee et al. 2016). CLEC4E-deficient mouse splenocytes are also hyper-responsive to TLR4 ligands (Greco et al. 2016).
The precise role and requirement of CLEC4E in the control of in vivo mycobacterial infection remains unclear. Overall, CLEC4E knockout mice are capable of mounting an efficient granulomatous immune response and control mycobacterial infection similarly to wild-type mice (Heitmann et al. 2013). Other studies show potential deleterious effects; CLEC4E-deficient mice have a higher burden of splenic infection when infected intravenously with BCG (Behler et al. 2015). Mice lacking CLEC4E have reduced lung inflammation and granuloma formation in response to TDM (Ishikawa et al. 2009; Lee et al. 2012). In this study, CLEC4E-deficient mice developed a higher infection load in response to M. tuberculosis infection, but maintained similar lung granuloma formation compared with wild-type mice (Lee et al. 2012). CLEC4E-deficient mice had normal TNFα production in response to mycobacterial infection, whereas partial inhibition of IL-6 and MIP-2 production was observed, suggesting other pathways by which whole mycobacteria activate inflammatory responses (Ishikawa et al. 2009; Schoenen et al. 2010).
Both TDM and TDB are effective adjuvants which influence Th1 and Th17 responses. CLEC4E plays a key role in the adjuvant effect of TDB in subunit vaccination with M. tuberculosis fusion protein Ag85B-ESAT6 (H1); the absence of CLEC4E reduces cellularity in the draining lymph node and significantly impairs IFNγ and IL-17 production by lymph node cells (Schoenen et al. 2010).
CLEC4E is involved in the recognition and response to Candida and Malassezia infection (Wells et al. 2008; Yamasaki et al. 2009). Absence or inhibition of CLEC4E reduces cytokine and chemokine production in response to challenge with Candida albicans and Malassezia species (Wells et al. 2008; Yamasaki et al. 2009). CLEC4E localizes to the phagocytic cup that forms around yeast particles after exposure to C. albicans (Wells et al. 2008). However, it is not involved in the phagocytosis of C. albicans (Wells et al. 2008). CLEC4E contributes to divergent functions of human blood monocytes and neutrophils since CLEC4E expression is associated with reduced phagocytosis and yeast Candida killing in monocytes but increased phagocytosis and Candida killing in neutrophils (Vijayan et al. 2012).
The fungal pathogen Fonsecaea pedrosoi infection causes a chronic skin infection and is recognized by CLEC4E and another lectin Dectin-2. Unlike Dectin-2 stimulation, CLEC4E activation inhibited the development of Th17 polarization of T cells and suppressed antifungal immunity mediated by another CLR, dectin-1 (Wevers et al. 2014; Wuthrich et al. 2015). In response to Klebsiella pneumonia infection, neutrophils have deficient extracellular trap formation and phagocytosis in CLEC4E-deficient mice, although the precise role of CLEC4E in this context remains unknown (Sharma et al. 2014).
Unregulated cell death contributes to sterile inflammation and CLEC4E is a receptor for SAP130 that is released from dying cells. In vivo mouse CLEC4E has a role in neutrophil recruitment to sites of thymocyte necrosis in the thymus caused by whole-body irradiation (Yamasaki et al. 2008). Blockade of CLEC4E-mediated MIP-2 and TNF-α production from macrophages by injection of a monoclonal antibody to CLEC4E significantly inhibited the migration of neutrophils to sites of substantial cell death in vivo (Yamasaki et al. 2008). The exact role of CLEC4E in response to cell death remains unclear since a subsequent study using genetic deletion of CLEC4E did not show an effect of CLEC4E on cell-death associated inflammation (Kataoka et al. 2014). CLEC4E may have a role in autoimmune disease as its expression is considerably upregulated in patients with rheumatoid arthritis (Nakamura et al. 2006).
CLEC4E is expressed in immune, endothelial, and neuronal cells of human brain after an ischemic stroke (Suzuki et al. 2013). Mice deficient in CLEC4E have attenuated neuro-inflammation in response to vascular injury and traumatic brain injury (de Rivero Vaccari et al. 2015; He et al. 2015). CLEC4E-deficient mice have reduced inflammatory responses and reduced size of ischemic lesions after transient middle cerebral artery occlusion (Arumugam et al. 2016). CLEC4E has a potential role in obesity-induced adipose tissue fibrosis since it is upregulated at the mRNA level in adipose-associated macrophages in obese mice (Tanaka et al. 2014). CLEC4E-deficient mice have abrogated adipose tissue interstitial fibrosis and better glycemic control (Tanaka et al. 2014).
Oncogenic effects of CLEC4E signaling have been demonstrated in a mouse model of pancreatic cancer. CLEC4E expressing immune cells infiltrated pancreatic tumor and continuous in vivo activation of CLEC4E using TDB stimulated tumor growth (Seifert et al. 2016). Inoculation of pancreatic adenocarcinoma tumors with SAP130 accelerated tumor growth, but this effect was not seen in CLEC4E-deficient mice (Seifert et al. 2016). CLEC4E deletion lead to greater activated T cell infiltration of tumors and this was associated with retardation of tumor growth (Seifert et al. 2016).
CLEC4E Knockout Models and Human Mincle Genetics
Knockout mouse models of CLEC4E have been generated (Wells et al. 2008; Yamasaki et al. 2009). In terms of total white blood cell counts and subpopulations of the thymus, spleen, lymph node, and peritoneal cells, no significant phenotype is observed. Bone marrow macrophages show normal differentiation, morphology, and number. CLEC4E is therefore not required for the development of the hematopoietic lineage cells, including the differentiation of macrophages. Immune functions, as measured by delayed-type hypersensitivity responses and immunoglobulin production, are also unaffected by CLEC4E deficiency (Wells et al. 2008). Although CLEC4E-knockout mice do not show any gross anatomical phenotype, they seem to show evidence of abnormal heart valves, with accumulation of higher levels of extracellular matrix compared to heart valves in wild-type mice. This suggests a role for CLEC4E in heart valve development (Wells et al. 2008).
In humans, a case-control study found no association between the genotype of four SNPs in the CLEC4E gene with susceptibility to tuberculosis (Bowker et al. 2016). In a major study of human tissue expression quantitative loci (www.gtexportal.org/home/), 130 associations between genotype at certain SNPs and CLEC4E expression have been found (tissues include whole blood, lung, tibial nerve, subcutaneous adipose tissue, tibial artery, and thyroid). No associations for CLEC4E and disease have been found in genome-wide association studies (www.ebi.ac.uk/gwas/home).
CLEC4E is an important pathogen recognition receptor of the C-type lectin family. It is involved in the recognition of fungal species and mycobacteria in a carbohydrate-dependent manner. There is also evidence indicating that CLEC4E recognizes an endogenous protein ligand, SAP130, in a carbohydrate-independent manner. CLEC4E is predominantly expressed on activated macrophages and associates with the FcεRIγ signaling adaptor. Ligand binding to CLEC4E triggers a signaling cascade through FcεRIγ, Syk, and CARD9, which results in the production of inflammatory cytokines and chemokines. CLEC4E mediates the adjuvant properties of the trehalose glycolipid, TDB. This is an important finding as it provides a target for the design of synthetic adjuvants for effective vaccination strategies against infectious diseases and cancer.
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