Self and nonself recognition through C-type lectin receptor, Mincle

Abstract

Mincle (also called as Clec4e or Clecsf9) is a C-type lectin receptor expressed in activated macrophages. Recently, we have reported that Mincle transduces the activation signals through ITAM-containing adaptor protein, FcRγ and induces the secretion of inflammatory cytokines. Furthermore, we and other groups have identified that Mincle recognizes a wide variety of ligands such as damaged cells, fungus, yeast and mycobacteria. These results indicate that Mincle acts as a multi-task danger receptor for both self and nonself ligands. This review summarizes the recent discoveries about the ligands and immunological roles of Mincle.

Introduction

The CLR (C-type lectin receptor) is a family of Ca²⁺-dependent carbohydrate-binding lectins that contain at lease one C-type lectin-like domain (CTLD).¹ Recently, CLR was highlighted as a member of the innate immune receptor families, which contain TLRs (Toll-like receptors), NLRs (Nod-like receptors) and RLRs (RIG-I-like receptors). Some CLRs deliver their signals via an association with ITAM (immunoreceptor tyrosine-based activation motif)² bearing adaptors, which is used by acquired immune receptors, NK receptors and Fc receptors.³

Mincle (Macrophage-inducible C-type lectin) was originally identified as a transcriptional target of NF-IL6 (also called C/EBPβ) in macrophages.⁴ Mincle is a type II transmembrane protein mainly expressed in myeloid cells, such as macrophages and neutrophils and possesses a single carbohydrate-recognition domain (CRD) in the extracellular region. The expression level of Mincle in the steady-state condition is very low, however, it is strongly upregulated after exposure to various stimuli such as inflammatory cytokines and TLR ligands. Mincle is associated with an ITAM-containing Fc receptor γ chain (FcRγ) with a positively charged residue in the transmembrane region.⁵ The ligation of ITAM leads to a signaling cascade that begins with phosphorylation of ITAM tyrosine residues by Src-family kinases, followed by the recruitment and activation of Syk. Syk then activates a signaling cascade through CARD9, and this event leads to the induction of inflammatory cytokines such as TNFα, MIP-2 (CXCL2), KC (CXCL1) and IL-6. Therefore, Mincle may act as an inducible activating receptor for certain ligand(s).

Dead Cell Recognition by Mincle

To search for a specific ligand of Mincle, we first established a reporter cell line expressing Mincle, FcRγ and NFAT-GFP. When the cells were cultured alone without exchange of the medium, the number of GFP⁺ cells was gradually increased. During this culture period, increasing numbers of dead cells paralleled the increase in the GFP⁺ population, suggesting that a component derived from dead cells signals through Mincle. Indeed, we have identified SAP130 (spliceosome-associated protein 130) as a Mincle-binding protein.⁵ SAP130 is a component of the U2 snRNP, and is localized in the nucleus in normal live cells,⁶ and is released from necrotic cells, but not from live or early apoptotic cells. These results suggest that SAP130 released from necrotic cells may act as a danger signal through Mincle.

Whole-body irradiation or administration of dexamethazone induces massive cell death in the thymus, and consequently elicits transient infiltration of neutrophils into the thymus.^7,8 The administration of an anti-Mincle blocking antibody resulted in a considerable suppression of such neutrophil infiltration, thus suggesting that Mincle is a functional sensor for damaged cells in vivo.

It has been reported that other CLRs expressed on myeloid cells potentially bind to dead cells.⁹ MBL (mannose-binding lectin), Lox-1 and MGL-1 are involved in the phagocytotic clearance of dead cells.^10–13 Human Dectin-1 (CLEC7A), DNGR1 (CLEC9A) and DEC205 (CD205) were also reported to bind to apoptotic or necrotic cells.^14–16 In most cases, however, the nature of the ligand(s) derived from dead cells is still unknown.

Neutrophil infiltration induces acute inflammation and tissue damage, but in some cases, early, small-scale neutrophil-mediated tissue destruction eventually promotes tissue repair.¹⁷ It has also been reported that infiltrating neutrophils assist macrophage-mediated clearance of stress-induced dead cells in the thymus.¹⁸ Therefore, it is possible to speculate that Mincle may trigger the “beneficial” recruitment of inflammatory cells in response to tissue damage. However, the function of Mincle in the recovery process of tissue repair remains to be determined.

Fungus Recognition by Mincle

Some C-type lectin receptors directly recognize specific fungi.¹⁹ Dectin-1 recognizes β-glucans on the surface of a wide variety of fungal species, including S. cerevisiae, C. albicans, C. posadasii, P. carinii and A. fumigatus, and this recognition mediates fungal uptake and killing, and the production of inflammatory cytokines and chemokines.^20–24 FcRγ-coupled Dectin-2 recognizes C. albicans, M. audouinii and T. rubrum.^25,26 Other CLRs, such as MR (CD206), DC-SIGN, MBL, surfactant protein A (SP-A) and surfactant protein D (SP-D) have been implicated in antifungal immunity.^27–34

Wells et al. reported that Mincle recognized C. albicans and promoted the production of TNFα by macrophages.³⁵ In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. We established a Mincle reporter cell line and screened the cells for a response to 50 different kinds of fungi, including C. albicans. Three different strains of C. albicans were not recognized by Mincle in our reporter systems, although these strains differ from that used in the previous study. Therefore, Mincle may distinguish structural differences in the strain of C. albicans.

Instead, we found that Mincle recognized Malassezia species such as M. pachydermatis, M. furfur and M. japonica. Malassezia, an obligatory lipophilic organism commonly found on human skin, is a causative fungus of skin diseases and fatal sepsis, including intravascular catheter-associated sepsis.^36–38 However, the specific receptor for this organism on host cells has not been identified. Malassezia-induced production of cytokines such as TNFα, MIP-2, KC and IL-10 was significantly impaired in Mincle-deficient macrophages in vitro. Cytokine production and neutrophil infiltration against Malassezia injection were also impaired in Mincle-deficient mice in vivo. These results suggest that Mincle plays a crucial role in immune response to Malassezia fungi. Although Malassezia species can live as commensal flora in normal skin, these fungi elicit an inflammatory response in the skin lesions of patients with atopic/eczema dermatitis syndrome and psoriasis.³⁶ Since Mincle expression is upregulated by several stresses, Mincle-induced inflammation may contribute to the regulation of dermatitis or sepsis elicited by Malassezia. The identification of a Mincle ligand in Malassezia will provide valuable information for the development of an effective therapy against Malassezia-related diseases.

Mincle Recognizes Mycobacterial Glycolipid TDM

Heat-killed Mycobacterium tuberculosis is widely used as a component of complete Freund's adjuvant (CFA), because it contains various immunostimulatory components. Among such components, cell wall glycolipid TDM (trehalose-6,6′-dimycolate), also known as cord factor, has been extensively studied for decades, as it possesses an effective adjuvant activity.³⁹ One class of the receptors implicated in TDM recognition is the TLRs. MARCO (macrophage receptor with collagenous structure) is also reported to be a tethering factor of TDM to the macrophage, and is thought to activate the TLR2 signaling pathway.⁴⁰ Another group reported that TDM activated macrophages and DCs via the Syk-Card9-Bcl10-Malt1 signaling axis rather than TLR-MyD88 pathway.⁴¹ Thus, the candidate receptor for TDM has been controversial.

Recently, we and another group have demonstrated that Mincle is the receptor for TDM.^42,43 TDM activates macrophages to produce nitric oxide and inflammatory cytokines such as TNFα and MIP-2, and this effect was completely suppressed in Mincle-deficient macrophages. The in vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and granuloma formation.^44,45 Granulomas are complex aggregates of immune cells, and are widely believed to constrain mycobacterial infection by physically surrounding the infecting bacteria.⁴⁶ No TDM-induced lung granuloma was formed in Mincle-deficient mice, suggesting that Mincle is a critical receptor for TDM-induced granuloma formation, most likely through the production of inflammatory cytokines/chemokines to recruit inflammatory cells.⁴⁷ Schoenen et al. demonstrated that Mincle is essential for generation of Th1/Th17 cellular immunity to subunit vaccination using TDB (torehalose dibehenate; a synthetic analogue of TDM) as an adjuvant.⁴³ These results suggest that Mincle may contribute to innate- and acquired-immunity against mycobacteria. Furthermore, the identification of the host receptor for TDM/TDB will provide valuable information related to the design of vaccine adjuvants against not only tuberculosis, but also other infectious diseases and cancers.

Conclusion

In recent years, many reports have shown that C-type lectin receptors are fundamental mediators of diverse immune interactions, especially in the recognition of various self and nonself ligands.^9,19,48,49 Mincle uniquely senses the “danger” derived from both damaged-self and non-self pathogen to induce inflammatory responses (Fig. 1). Recognition of these molecules by Mincle may lead to beneficial inflammation that promotes tissue repair or host defense responses.

Figure 1.

Mincle is a dual sensor for self and non-self ligands. Mincle recognizes SAP130 released from necrotic cells (damaged self). Invading pathogens (non-self), such as Mycobacterium and the pathogenic fungus, Malassezia, are also recognized by Mincle. TDM (torehalose dimycolate) is Mycobacterial molecule identified as a ligand of Mincle. Sensing of these self and non-self ligands by Mincle leads to transduction of activation signals through FcRγ (ITAM-containing adapter protein), thus inducing the inflammatory responses that possess both physiological advantages and potential risks.

From a pathological point of view, Mincle has been reported to be dramatically upregulated in patients with rheumatoid arthritis,⁵⁰ and rat chromosome 4q42 (which includes the gene encoding Mincle) has been linked to arthritis.⁵¹ These observations suggest that dysregulated expression of Mincle might elicit unwanted inflammation responsible for autoimmune or granulomatous diseases. The pathological risk, as well as the physiological advantage, of Mincle-mediated recognition of “danger” needs clarification, but is likely to provide information that could be useful for developing strategies against such diseases.

Abbreviations

CLR

C-type lectin receptor

FcRγ

Fc receptor common γ-chain

Mincle

macrophage-inducible C-type lectin

ITAM

immunoreceptor tyrosine-based activation motif