Janet A. Willment

Dectin-1 Mediates the Biological Effects of β-Glucans

The ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.

Dectin-1 is required for beta-glucan recognition and control of fungal infection.

β-Glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for β-glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for β-glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non–Toll-like pattern-recognition receptor required for the induction of protective immune responses.

Dectin-1 Is A Major β-Glucan Receptor On Macrophages

Zymosan is a β-glucan– and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti–Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan–dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

Human Dectin-1 Deficiency and Mucocutaneous Fungal Infections

Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages

We recently identified dectin-1 (βGR) as a major β-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate β-glucans. Using a novel mAb (2A11) raised against βGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mφ) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the βGR, but at lower levels. Alveolar Mφ, like inflammatory Mφ, exhibited the highest surface expression of βGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mφ expressed much lower levels of βGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mφ suggested the existence of an additional β-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mφ. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mφ mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mφ.

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

Recognition of microbial components by germ‐line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β‐glucan receptor, Dectin‐1, which collaborates through an undefined mechanism with Toll‐like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin‐1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP‐1α and MIP‐2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk‐ and MyD88‐coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes.

We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the “Dectin-1 cluster” of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3+ dendritic cells and on a small subset of CD14+CD16- monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.

The human β-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells

We identified the C‐type‐lectin‐like receptor, Dectin‐1, as the major receptor for fungal β‐glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform‐specific mAb, we show here that human Dectin‐1, the β‐glucan receptor (βGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin‐1 is not myeloid restricted. Both major functional βGR isoforms – βGR‐A and βGR‐B – were expressed by these cell populations in peripheral blood; however, only βGR‐B was significantly expressed on mature monocyte‐derived macrophages and immature DC, suggesting cell‐specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin‐window technique, demonstrated that Dectin‐1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other β‐glucan receptors on mature human macrophages, we have demonstrated that Dectin‐1 acted as the major β‐glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Dectin-1 Expression and Function Are Enhanced on Alternatively Activated and GM-CSF-Treated Macrophages and Are Negatively Regulated by IL-10, Dexamethasone, and Lipopolysaccharide

Dectin-1 is the major macrophage receptor for β-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mφ). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-γ, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-α, although some Mφ-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.

The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages.

We recently demonstrated that the β-glucan receptor Dectin-1 (βGR) was the major nonopsonic β-glucan receptor on macrophages (Mφ) for the yeast-derived particle zymosan. However, on resident peritoneal Mφ, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mφ mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mφ and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mφ. Both SIGNR1 and βGR cooperated in the nonopsonic recognition of zymosan by these Mφ. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mφ, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as βGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mφ expressing SIGNR1 resulted in TNF-α production. Treatment of RAW 264.7 Mφ expressing SIGNR1, which express low levels of βGR, with β-glucans had little effect on binding or TNF-α production, indicating that there was no absolute requirement for βGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mφ.