CLEC5A is a type 2 transmembrane receptor originally identified by its ability to stabilize DAP (DNAX associating protein)-12 at the cell surface in myeloid cells (Bakker et al. 1999). It is emerging as a key component of the innate immune system; it activates macrophages, regulates osteoclastogenesis, and plays a role in inflammatory diseases including dengue virus-induced lethality, Japanese encephalitis-associated neuro-inflammation, and autoimmune arthritis (Bakker et al. 1999; Chen et al. 2008; Aoki et al. 2009; Joyce-Shaikh et al. 2010; Chen et al. 2012).
The predicted 161 amino acid extracellular sequence of CLEC5A contains a C-type lectin-like domain in the carboxy-terminal region (UniProt 2015). The N-terminal cytoplasmic tail is predicted to be only four amino acids in length and, thus, lacks a signaling motif (UniProt 2015). The putative 23 amino acid single pass transmembrane domain contains a conserved lysine residue. CLEC5A associates with DAP-12 by an interaction between this positively charged lysine and a negatively charged aspartate residue that is present in the transmembrane region of DAP-12 (Bakker et al. 1999). DAP-12 transduces activating signals in myeloid and natural killer cells and contains an immunoreceptor tyrosine activation motif (ITAM) in its cytosolic domain (Lanier 2009). Ligand binding by DAP-12-associated receptors in myeloid cells results in phosphorylation of the two key tyrosine residues within the ITAM motif by Src kinases. The two SH2 domains of Syk (Spleen tyrosine kinase) then bind to the phosphorylated ITAM, resulting in Syk activation (Aoki et al. 2003; Lanier 2009). DAP-12 has a very short extracellular domain and exists as a disulfide linked homodimer in association with CLEC5A or another member of the set of receptors with which it interacts (Lanier et al. 1998; Bakker et al. 1999).
The structure of the extracellular domain of human CLEC5A has been solved using X-ray crystallography to a resolution of 1.9 Å (Watson and O’Callaghan 2010; Watson et al. 2011). Although the overall formation was consistent with a C-type lectin-like molecule, several features were unique and suggested a mechanism for ligand binding. The long loop region of crystallized CLEC5A exhibited two conformations (Watson et al. 2011). In one conformation, an extra beta-strand was formed that would reduce loop flexibility and increase stability of the protein core (Watson et al. 2011). It was hypothesized that ligand binding (or stabilization by the crystal lattice) could cause this altered conformation, that is, in effect, a molecular switch. Computational analysis suggested this switch could enhance DAP12 binding and signal transduction (Watson et al. 2011). Similar to other C-type lectins, three disulfide bonds and noncovalent interactions maintained the core of CLEC5A. Using BRET CLEC5A was found to form nonconstitutive homodimers at the cell surface in transfected cells (Watson et al. 2011).
Orthologs of the CLEC5A gene have been identified in several species, including rat, mouse, dog, pig, cow, and chimpanzee (Bakker et al. 1999; Yim et al. 2001). In vitro studies have shown murine and porcine CLEC5A to be glycosylated (Yim et al. 2001; Aoki et al. 2009). Glycosylation is greater in murine neutrophils than macrophages, and these differences may influence ligand binding (Aoki et al. 2009).
A precise endogenous or exogenous ligand for CLEC5A has not been found, but human and mouse CLEC5A react directly with dengue virus (DV) strain 2 in a calcium-independent manner (Chen et al. 2008). This interaction can be inhibited in vitro by the monosaccharide fucose, suggesting that DV envelope protein glycans are important in mediating this interaction (Chen et al. 2008). Human CLEC5A also reacts with DV serotypes 1, 3, and 4 (Watson et al. 2011). Screening of a glycolipid array and oligosaccharide array, each with over 400 compounds, did not reveal significant binding (Watson et al. 2011). Both human and mouse CLEC5A bind to Japanese Encephalitis virus (by ELISA) and binding is absent from a splice variant lacking 23 amino acids from the stalk region (25aa in the mouse splice variant) (Chen et al. 2012).
Macrophages are phagocytic cells derived from monocytes and play a key role in immunity. CLEC5A has a role in macrophage activation (Bakker et al. 1999). When the mouse macrophage cell line J774 is transfected with CLEC5A, cross-linking of CLEC5A/DAP-12 complexes results in calcium mobilization (Bakker et al. 1999). Signaling through CLEC5A in murine myeloid 32Dcl3 cells upregulates expression of the leukocyte adhesion molecule CD11b and of cytokines, including RANTES (regulated upon activation normal T-cell expressed and presumably secreted), IP-10 (Interferon-gamma-induced protein 10), and MDC (macrophage-derived chemokine) (Aoki et al. 2009). Stimulation of Toll-like receptor 4 by lipopolysaccharide had a synergistic effect with CLEC5A on the expression of RANTES and MDC (Aoki et al. 2009). IL-23 stimulation increases CLEC5A binding to DAP12 and DAP10 in human PBMC (peripheral blood mononuclear cells) as measured by immunoprecipitation (Shin et al. 2015). CLEC5A interacts with DV causing DAP-12 phosphorylation, triggering tumor necrosis factor (TNF)-α production (Chen et al. 2008). Conversely, knockdown of CLEC5A suppresses the release of TNF-α, IL-6, IL-8, macrophage inflammatory protein (MIP), and IP-10 in response to DV (Chen et al. 2008). Signaling through CLEC5A in GM-CSF differentiated macrophages contributes to NLRP3 inflammasome activation and secretion of IL-1β and IL-18 (Wu et al. 2013). In a mouse model of lethal shock, CLEC5A activation on myeloid cells causes nitric oxide release, contributing to disease severity (Cheung et al. 2011). Overall, signaling through CLEC5A in myeloid cells triggers the release of inflammatory mediators contributing to inflammation.
CLEC5A also has a role in bone metabolism. Bone homeostasis depends on the opposing action of osteoblasts of mesenchymal origin that promote bone-matrix formation and osteoclasts derived from myeloid precursor cells that enhance bone resorption. Stimulation of CLEC5A enhances osteoclastogenesis from murine bone marrow-derived monocytes by signaling through DAP-10 and DAP-12 (Inui et al. 2009; Joyce-Shaikh et al. 2010). DV infection of monocyte-derived osteoclasts triggers TNF-α and IL-6 release that is abrogated by a CLEC5A blocking antibody (Huang et al. 2016). CLEC5A knockout mice showed less osteolytic activity in response to DV infection (Huang et al. 2016).
General Expression Pattern
In humans, CLEC5A is expressed in peripheral blood monocytes and in the monocyte/macrophage cell lines U937 and MonoMac6 but not in cells of non-myeloid origin (Bakker et al. 1999). Surface expression of CLEC5A was detected on human peripheral blood cells expressing either CD66 or CD14+ (Chen et al. 2008). In another study CLEC5A expression by flow cytometry with gating for both CLEC5A and DAP12 in PBMCs was found to be mainly confined to CD16+ cells rather than CD14+ cells (Shin et al. 2015). CLEC5A is expressed on porcine monocytes and pulmonary alveolar macrophages but not lymphocytes or polymorphonuclear granulocytes (Yim et al. 2001). In mice, CLEC5A expression has been detected on granulocytes (CD11b+ Ly6Ghigh) and monocytes (CD11b+ Ly6Glow) from bone marrow and peripheral blood, on peritoneal macrophages, thioglycollate-elicited neutrophils, and osteoclasts (Aoki et al. 2009; Inui et al. 2009; Joyce-Shaikh et al. 2010). In humans and mice, the main tissue sites of expression are in bone marrow, synovium/joint, and lung (Joyce-Shaikh et al. 2010). In inflamed pannus from patients with rheumatoid arthritis, CLEC5A was expressed in CD68+ macrophages (Joyce-Shaikh et al. 2010).
Response to Differentiation
CLEC5A expression is affected by cell ontogeny. CLEC5A expression is higher in peripheral blood-derived CD14+ mature monocytes than in undifferentiated CD34+ bone marrow cells (Gingras et al. 2002). Expression is lower in fetal tissue compared to adult tissue and in malignant cells compared to normal cells (Gingras et al. 2002). Differentiation of myeloid precursor 32Dcl3 cells into neutrophils induces CLEC5A expression (Aoki et al. 2009). CLEC5A induction during differentiation of myeloid precursor cell line HL60 was in part dependent on expression of the myeloid master transcription factor PU.1 (Batliner et al. 2011). PU.1 binds to the promoter of CLEC5A in U937 cells inducing a transcriptional response (Batliner et al. 2011). Human monocyte-derived osteoclasts express surface levels of CLEC5A similar to that of macrophages (Huang et al. 2016). CLEC5A mRNA and protein levels were upregulated on peripheral blood mononuclear cells after 8 days of treatment with RANKL or IL-23 compared to M-CSF alone (Shin et al. 2015). CLEC5A cell surface and mRNA expression was higher in GM-CSF-derived compared to M-CSF-derived human (Wu et al. 2013; Gonzalez-Dominguez et al. 2015). However, others have reported a reduction in mRNA CLEC5A expression when monocytes are differentiated into a dendritic phenotype by stimulation with GM-CSF and IL-4 (Bakker et al. 1999).
Response to Inflammatory Stimuli
CLEC5A expression on monocytes is increased by IL-1β, TNF-α, and TGF-1β and suppressed by IL-10 (Gonzalez-Dominguez et al. 2015). Surface expression of murine CLEC5A is upregulated during pulmonary mycobacterial infection and correlates with upregulation of the type 1 cytokines TNF-α and IFN-γ (Aoki et al. 2004). However, whereas TNF-α is critical for induction of CLEC5A during murine pulmonary mycobacterial infection, IFN-γ suppressed CLEC5A expression (Aoki et al. 2004). Cigarette smoke increases surface expression of CLEC5A in alveolar macrophages from mice and humans (Wortham et al. 2016). In inflammatory bowel disease, there is an increased number of CLEC5A expressing macrophages in diseased compared with healthy intestine (Gonzalez-Dominguez et al. 2015). CLEC5A has been suggested as a marker of inflammatory tissue macrophages (Gonzalez-Dominguez et al. 2015).
Genetics and Splice Variants
The human CLEC5A gene is on chromosome 7 and encodes a 3524 base pair mRNA (NM_013252.2) (Maglott et al. 2007). The mRNA includes a 567 nucleotide open reading frame that codes for a 188 amino acid protein (Uniprot identifier: Q9NY25–1). The mouse CLEC5A gene is located on chromosome 6 and the 573 nucleotide open reading frame encodes a 190 amino acid protein (Uniprot identifier: Q9R007). Data from large-scale sequencing projects indicates the presence of multiple single nucleotide polymorphisms in the coding sequence of CLEC5A, but the majority of these have not been subjected to further experimentation (Genomes Project et al. 2012).
CLEC5A exists as alternatively spliced variants in mice and pigs, with the noncanonical isoforms lacking 25 and 19 amino acids in their extracellular domains, respectively (Bakker et al. 1999; Yim et al. 2001). The significance of these alternatively spliced transcripts is unknown.
CLEC5A−/− mice display a dysregulated bone phenotype with irregular trabecular bone connectivity (Joyce-Shaikh et al. 2010). DAP-10−/− mice display mild osteopetrosis and a reduction in the number of osteoclasts (Inui et al. 2009). DAP-12-deficient mice also display altered T-cell activity due to impaired antigen priming (Bakker et al. 2000). They have impaired osteoclast development and function and macrophages that are hyperresponsive to in vitro exposure to TLR ligands (Lanier 2008, 2009). In humans, lack of DAP-12 results in Nasu-Hakola disease, characterized by presenile dementia and bone cysts (Aoki et al. 2003). However, it is important to note that DAP-12 interacts with a multitude of cell membrane receptors, so this effect is not necessarily mediated by CLEC5A.
A case-control study examining the effect of 4 SNPs in CLEC5A exons or the 5’UTR found no association between markers of Kawasaki disease and response to treatment in a Taiwanese population (Yang et al. 2012). The rs1285933 SNP is associated with both expression level of CLEC5A in monocytes (expression quantitative trait locus) and shows borderline significance for association with severity of DV infection in a Brazilian pediatric population (TT genotype increased severity) (Zeller et al. 2010; Xavier-Carvalho et al. 2013).
Function in Disease
A role for CLEC5A is evident in the immune response and pathogenesis of several infections and in inflammatory arthritis. Infection of macrophages with DV that contains a ligand for CLEC5A caused phosphorylation of DAP-12, but knockdown of CLEC5A did not reduce viral replication, suggesting that the interaction is important in the inflammatory changes associated with the disease rather than the mechanisms of cellular infection (Chen et al. 2008). In vivo blockade of the DV-CLEC5A interaction with an antagonistic anti-CLEC5A antibody reduced lethality of DV infection in STAT−/− mice with decreased plasma leakage and hemorrhage, but did not suppress viral replication (Chen et al. 2008). CLEC5A also binds to another flavivirus, Japanese encephalitis virus (Chen et al. 2012). Mouse infection was associated with increased expression of CLEC5A in brain and splenic tissue and was associated with increased IFN-γ, TNF-α, and IL-1α production (Gupta et al. 2010). Administration of a CLEC5A blocking antibody did not prevent neuronal infection but reduced neuro-inflammation and lethality (Chen et al. 2012). Mouse pulmonary mycobacterial infection induced CLEC5A expression, suggesting a role in mycobacterial immunity (Aoki et al. 2004).
CLEC5A has recently been shown to have an important role in autoimmune-mediated synovial injury and bone erosion. In a mouse model of inflammatory arthritis models, CLEC5A stimulation increased mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-17, TNF-α), bone remodeling genes (receptor activator of NF-κB ligand, Tartrate-resistant acid phosphatase, matrix metallopeptidase 9, ATPV0D2), and myeloid associated genes (CXCL1, CD11b, DAP-12, RANK) (Joyce-Shaikh et al. 2010). Activation of the CLEC5A pathway was associated with a significant increase in the clinical severity, including bone destruction (Joyce-Shaikh et al. 2010). The therapeutic potential of this pathway was demonstrated by improvement in the clinical course by intravenous administration of a CLEC5A-Fc fusion protein. The Fc binding site was mutated to attenuate antibody binding, and the fusion protein presumably bound to and blocked an unknown ligand for CLEC5A. Similar results were achieved by deleting the CLEC5A gene in mice (Joyce-Shaikh et al. 2010). The PBMC of patients with active rheumatoid arthritis expressed higher levels of CLEC5A (surface expression and mRNA) and exhibited greater incremental increases in CLEC5A expression in response to TNF-α or IL-1β. Expression levels on monocytes and PBMCs were positively correlated with markers of rheumatoid arthritis severity and inflammatory cytokines (Chen et al. 2014).
Chronic obstructive pulmonary disease is largely caused by cigarette smoking. A role for CLEC5A in COPD progression was suggested by a significant increase in CLEC5A expressing macrophages from smokers and abrogated inflammatory responses in a mouse model of COPD with CLEC5A-deficient mice (Wortham et al. 2016). Coronary artery disease risk is associated with common haplotypes at the 9p21.3 locus (Consortium et al. 2013). Macrophages from patients with a previous myocardial infarction had 1.5 fold greater mRNA expression of CLEC5A in patients with the 9p21.3 risk haplotype (Zollbrecht et al. 2013). It was speculated that CLEC5A-mediated inflammatory responses may contribute to atherosclerosis. Finally, a study of kidney transplant biopsy specimens with borderline changes for rejection showed a greater risk of subsequent deteriorating renal function from patients with higher levels of CLEC5A mRNA in biopsy tissue (Hruba et al. 2015).
CLEC5A is a C-type lectin that activates macrophages and osteoclasts by signaling through DAP-12 and DAP-10. CLEC5A binds to Dengue and Japanese encephalitis virus, but specific viral ligands remain unknown. There are no known endogenous ligands. CLEC5A has a role in the pathogenesis of inflammatory arthritis in a mouse model and contributes to dengue virus and Japanese encephalitis virus-induced lethality. Future research objectives include characterization of endogenous and pathogen ligands, understanding the cell surface events that lead to signaling through DAP-12, assessing its role in other TNF-α-mediated diseases such as inflammatory bowel disease, and exploring the therapeutic potential of CLEC5A blockade.
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