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The pattern recognition receptors Toll-like receptor 2 (TLR2) and Dectin-1 play key roles in coordinating the responses of macrophages and dendritic cells (DC) to fungi. Induction of pro-inflammatory cytokines is instructed by signals from both TLR2 and Dectin-1. A recent report identified a role for CARD9 in innate anti-fungal responses, demonstrating CARD9-Bcl10-mediated activation of NF-κB and pro-inflammatory cytokine induction in murine bone marrow-derived DC (bmDC) stimulated via Dectin-1. We now report that Dectin-1-CARD9 signals fail to activate NF-κB and drive TNF-α induction in murine bone marrow-derived macrophages (bmM). However, priming of bmM with GM-CSF or IFN-γ permits Dectin-1-CARD9-mediated TNF-α induction. Analysis of other macrophage/DC populations revealed further variation in the ability of Dectin-1-CARD9 signaling to drive TNF-α production. Resident peritoneal cells and alveolar macrophages produce TNF-α upon Dectin-1 ligation, while thioglycollate-elicited peritoneal macrophages and Flt3L-derived DC do not. We present data demonstrating that CARD9 is recruited to phagosomes via its CARD domain where it enhances TLR-induced cytokine production even in cells in which Dectin-1 is insufficient to drive cytokine production. In such cells, Dectin-1, CARD9 and Bcl10 levels are not limiting, and data indicate that these cells express additional factors that restrict Dectin-1-CARD9 signaling for TNF-α induction.

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.

ABSTRACT

We assessed the role of Dectin-1 in the immune response to the pathogenic fungus Coccidioides, both in vitro and in vivo, using mice with a targeted mutation in Clec7a. Elicited peritoneal macrophages responded to formalin-killed spherules (FKS) and alkali-treated FKS by secreting proinflammatory cytokines in a Dectin-1- and β-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) secretion was independent of Dectin-1, while IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were largely but not entirely dependent on Dectin-1. After intranasal infection, Dectin-1−/− mice had lower concentrations of IL-12p70, gamma interferon (IFN-γ), IL-1β, and the Th17 cytokines IL-22, IL-23, and 17A in the lung lavage fluid, which may explain why they were significantly more susceptible to pulmonary coccidioidomycosis two weeks after infection. The Dectin-1 mutation was even more deleterious in (B6 × DBA/2)F2 mice, which are more resistant to coccidioidomycosis than B6 mice by virtue of protective genes from DBA/2, a genetically resistant strain. We also found that two susceptible strains of mice (B6 and BALB/c) expressed much less Dectin-1 in their lungs than did resistant DBA/2 mice. We conclude that Dectin-1 is necessary for resistance to Coccidioides immitis, that Dectin-1 promotes both Th1 and Th17 protective immune responses to this infection, and that there is a correlation between expression of Dectin-1 by the inflammatory infiltrate and resistance to coccidioidomycosis.

IMPORTANCE

Coccidioidomycosis is a fungal infection endemic in the southwestern United States and neighboring Mexico, causing ~150,000 lung infections in people and resulting in ~17,000 hospitalizations annually in California alone. Very little is known about innate immunity to this fungus. This paper shows that Dectin-1, the primary β-glucan receptor on myeloid cells, is required for resistance to this pathogen. Dectin-1 is part of the innate immune system, and it is needed to direct the acquired immune response toward into a pathway that will lead to macrophage activation. Lungs from infected mice lacking Dectin-1 had lower concentrations of Th1 and Th17 cytokines, two cytokine pathways that are very important for acquired T cell immunity to Coccidioides spp. This is the first demonstration that Dectin-1 is required for host resistance to a dimorphic, primary pathogenic fungus.

Background

Dectin-1 is a pattern recognition receptor (PRR) expressed by myeloid cells that specifically recognizes β-1,3 glucan, a polysaccharide and major component of the fungal cell wall. Upon activation, dectin-1 signaling converges, similar to NOD2, on the adaptor molecule CARD9 which is associated with inflammatory bowel disease (IBD). An early stop codon polymorphism (c.714T>G) in DECTIN-1 results in a loss-of-function (p.Y238X) and impaired cytokine responses, including TNF-α, interleukin (IL)-1β and IL-17 upon in vitro stimulation with Candida albicans or β-glucan. The aim of the present study was to test the hypothesis that the DECTIN-1 c.714T>G (p.Y238X) polymorphism is associated with lower disease susceptibility or severity in IBD and to investigate the level of dectin-1 expression in inflamed and non-inflamed colon tissue of IBD patients.

Methodology

Paraffin embedded tissue samples from non-inflamed and inflamed colon of IBD patients and from diverticulitis patients were immunohistochemically stained for dectin-1 and related to CD68 macrophage staining. Genomic DNA of IBD patients (778 patients with Crohn's disease and 759 patients with ulcerative colitis) and healthy controls (n = 772) was genotyped for the c.714T>G polymorphism and genotype-phenotype interactions were investigated.

Principal Findings

Increased expression of dectin-1 was observed in actively inflamed colon tissue, as compared to non-inflamed tissue of the same patients. Also an increase in dectin-1 expression was apparent in diverticulitis tissue. No statistically significant difference in DECTIN-1 c.714T>G allele frequencies was observed between IBD patients and healthy controls. Furthermore, no differences in clinical characteristics could be observed related to DECTIN-1 genotype, neither alone, nor stratified for NOD2 genotype.

Conclusions

Our data demonstrate that dectin-1 expression is elevated on macrophages, neutrophils, and other immune cells involved in the inflammatory reaction in IBD. The DECTIN-1 c.714T>G polymorphism however, is not a major susceptibility factor for developing IBD.

Objective

Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis.

Methods

The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan.

Results

Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis.

Conclusions

Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joints.

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.

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.

Introduction

Dectin-1, a pattern recognition receptor expressed by the innate immune system, is known to be a major receptor inducing Th17-type adaptive immune responses that have been demonstrated to mediate autoimmunity. In this study, dectin-1 mRNA and protein expression, as well as the recently characterized DECTIN-1 Y238X early stop codon polymorphism, were studied in relation to rheumatoid arthritis (RA) susceptibility and severity.

Methods

Dectin-1 mRNA expression was measured in synovial tissue specimens of RA, osteoarthritis (OA), and nonrheumatic patients. Dectin-1 protein expression and localization were assessed in RA synovial tissue specimens. Macrophages from individuals with different DECTIN-1 genotypes were examined for differences in cytokine responses on dectin-1 stimulation. Furthermore, clinical parameters of inflammation and bone destruction of 262 RA patients were correlated with the presence of the DECTIN-1 Y238X polymorphism.

Results

Evaluation of dectin-1 mRNA expression in synovial tissue biopsies revealed an increased expression in RA specimens, compared with biopsies from OA and nonrheumatic patients. Accordingly, dectin-1 protein expression in RA synovial tissue biopsies was moderate to high, especially on macrophage-like cells. Cytokine production capacity of macrophages bearing the DECTIN-1 Y238X polymorphism was demonstrated to be impaired on dectin-1 stimulation. However, the presence of the DECTIN-1 Y238X polymorphism was not associated with RA susceptibility or disease severity.

Conclusions

Although expression of dectin-1 was high in synovial tissue of RA patients, and reduced cytokine production was observed in macrophages of individuals bearing the DECTIN-1 Y238X polymorphism, loss of one functional allele of DECTIN-1 is not associated with either susceptibility to or severity of RA.

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the Toll-like receptor (TLR) family are not absolutely required for host defense against A. fumigatus in non-immunosuppressed hosts, suggesting that other pattern recognition receptors (PRRs) are involved. We show here that naive mice (i.e. not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1−/−) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1−/− mice demonstrated impaired interleukin (IL)-α, IL-1β, tumor necrosis factor (TNF)-α, CCL3/macrophage inflammatory protein (MIP)-α, CCL4/MIP-1β and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1−/− mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1−/− mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.

β(1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal β(1,3)-glucans and induce innate immune responses. The importance of β-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of β-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of β-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-α. In contrast, stimulation of Th1-type cytokines, such as interferon-γ, by C. albicans was independent of the recognition of β-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.

We recently showed that IL-13 or peroxisome proliferator activated receptor γ (PPARγ) ligands attenuate Candida albicans colonization of the gastrointestinal tract. Here, using a macrophage-specific Dectin-1 deficient mice model, we demonstrate that Dectin-1 is essential to control fungal gastrointestinal infection by PPARγ ligands. We also show that the phagocytosis of yeast and the release of reactive oxygen intermediates in response to Candida albicans challenge are impaired in macrophages from Dectin-1 deficient mice treated with PPARγ ligands or IL-13. Although the Mannose Receptor is not sufficient to trigger antifungal functions during the alternative activation of macrophages, our data establish the involvement of the Mannose Receptor in the initial recognition of non-opsonized Candida albicans by macrophages. We also demonstrate for the first time that the modulation of Dectin-1 expression by IL-13 involves the PPARγ signaling pathway. These findings are consistent with a crucial role for PPARγ in the alternative activation of macrophages by Th2 cytokines. Altogether these data suggest that PPARγ ligands may be of therapeutic value in esophageal and gastrointestinal candidiasis in patients severely immunocompromised or with metabolic diseases in whom the prevalence of candidiasis is considerable.

Author Summary

Since the early 1980s, Candida albicans has emerged as major cause of human disease, especially among immunocompromised individuals and those with metabolic dysfunction. The main host defense mechanisms against this yeast are engulfment and the production of reactive oxygen molecules by macrophages through Dectin-1 and the Mannose Receptor, two macrophage receptors for Candida albicans cell wall sugars. However, the contribution of these two receptors remains unclear. In our animal experiments, the lack of Dectin-1 in macrophages renders the animals more susceptible to gastrointestinal infection with Candida albicans, demonstrating the essential role of Dectin-1 in antifungal defense. In addition, our experiments established that the interaction between Dectin-1 and Mannose Receptor is important to orchestrate the host antifungal defense. Thus, Candida albicans clearance would be improved by Dectin-1 and Mannose Receptor up-regulation. Interestingly, we had established that the expression of these two receptors was increased by IL-13 through the activation of the nuclear receptor PPARγ, suggesting that PPARγ could be a therapeutic target to eliminate fungal infection. This paper, which highlights a new area of application of PPARγ ligands in infectious diseases, hence heralds the emergence of a new therapeutic strategy against fungal infection in severely immunocompromised patients or those with metabolic diseases.

Human infections involving yeast of the genus Candida often occur in the presence of bacteria, and, as such, it is important to understand how these bacteria influence innate host immunity towards Candida. Dectin-1 is a cell receptor of macrophages for Candida albicans recognition. The aim of this study was to examine dectin-1 expression by monocytes after stimulation with bacterial lipopolysaccharide (LPS), followed by heat-killed C. albicans (HKC). Freshly isolated human peripheral blood monocytes (PBMCs) and human monocytes cell line (THP-1) cells expressed low levels of dectin-1. Stimulation with LPS and GM-CSF/IL-4 was found to increase dectin-1 expression in both CD14+ human PBMC and THP-1 cells. Enhanced dectin-1 expression resulted in increased phagocytosis of Candida. When THP-1 cells were challenged only with HKC, detectable levels of IL-23 were not evident. However, challenge by LPS followed by varying concentrations of HKC resulted in increased IL-23 expression by THP-1 cells in HKC dose-dependent manner. Increased expression of IL-17 by PBMC also occurred after stimulation with Candida and LPS. In conclusion, bacterial LPS induces an enhanced immune response to Candida by immune cells, and this occurs through increasing dectin-1 expression.

We have re-investigated the role of the complement system and the non-opsonic pattern recognition receptors dectin-1 and dectin-2 in the recognition of fungal particles by inflammatory neutrophils, monocytes and macrophages. We have used in vivo and ex vivo models to study the recognition and response of these cells: i) We confirm previous observations regarding the importance of complement to neutrophil but not monocytic responses; ii) We show that dectin-1 is important for driving inflammatory cell recruitment to fungal stimuli and that it biases the immediate inflammatory response to one that favors neutrophil over monocyte recruitment; iii) We show that dectin-2 contributes to the physical recognition of fungal particles by inflammatory monocytes/macrophages, but is also expressed on neutrophils, where we show it has the potential to contribute to cellular activation; iv) Additionally, we show that serum-opsonization has the potential to interfere with non-opsonic recognition of fungal particles by dectin-1 and dectin-2, presumably through masking of ligands. Collectively these roles are consistent with previously described roles of dectin-1 and dectin-2 in driving inflammatory and adaptive immune responses and complement in containing fungal burdens. This study emphasizes the importance of heterogeneity of receptor expression across myeloid cell subsets in protective immune responses.

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.

Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for β-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif–like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G–opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor κB by β-glucan–containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor α. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 is a pattern recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular anti-microbial activity, including phagocytosis and production of reactive oxygen species1, 2. In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 are excluded (Supplementary Figure 1). The “phagocytic synapse” now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular anti-microbial responses only when they are required.

CLEC-2 is a member of the ‘Dectin-1 cluster’ of C-type lectin-like receptors and was originally thought to be restricted to platelets. Here we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone-marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of antibody-coated beads and the production of proinflammatory cytokines, including TNFα, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of Dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like Dectin-1, CLEC-2 can recruit the signalling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.

Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as β-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall β-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IκB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in β-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

Background

It is believed that inflammatory bowel diseases (IBD) result from an imbalance in the intestinal immune response towards the luminal microbiome. Dectin-1 is a widely expressed pattern recognition receptor that recognizes fungi and upon recognition it mediates cytokine responses and skewing of the adaptive immune system. Hence, dectin-1 may be involved in the pathogenesis of IBD.

Methods

We assessed the responses of dectin-1 deficient macrophages to the intestinal microbiota and determined the course of acute DSS and chronic Helicobacter hepaticus induced colitis in dectin-1 deficient mice.

Results

We show that the mouse intestinal microbiota contains fungi and the cytokine responses towards this microbiota were significantly reduced in dectin-1 deficient macrophages. However, in two different colitis models no significant differences in the course of inflammation were found in dectin-1 deficient mice compared to wild type mice.

Conclusions

Together our data suggest that, although at the immune cell level there is a difference in response towards the intestinal flora in dectin-1 deficient macrophages, during intestinal inflammation this response seems to be redundant since dectin-1 deficiency in mice does not affect intestinal inflammation in experimental colitis.

Objectives

Numerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the β-glucan receptor dectin-1. The role of scavenger receptors (SRs) in anti-fungal immunity is not well characterized.

Methods

We studied uptake of unopsonized Saccharomycetes cerevisiae (zymosan) and live Candida albicans yeasts as well as zymosan-stimulated H2O2 production in J774 macrophage-like cells and peritoneal exudate macrophages (PEMs). The role of different receptors was assessed with the use of competitive ligands, transfected cells and receptor-deficient macrophages.

Results

The uptake of zymosan by untreated J774 cells was mediated approximately half by SRs and half by a β-glucan receptor which was distinct from dectin-1 and not linked to stimulation of H2O2 production. Ligands of β-glucan receptors and of SRs also inhibited uptake of C. albicans by macrophages (J774 cells and PEMs). In macrophages pretreated with a CpG motif-containing oligodeoxynucleotide (CpG-ODN) the relative contribution of SRs to yeast uptake increased and that of β-glucan receptors decreased. Whereas the class A SR MARCO participated in the uptake of both zymosan and C. albicans by CpG-ODN-pretreated, but not untreated macrophages, the related receptor SR-A/CD204 was involved in the uptake of zymosan, but not of C. albicans. The reduction of zymosan-stimulated H2O2 production observed in DS-pretreated J774 cells and in class A SRs-deficient PEMs suggest that class A SRs mediate part of this process.

Conclusions

Our results revealed that SRs belong to a redundant system of receptors for yeasts. Binding of yeasts to different receptors in resting versus CpG-ODN-pre-exposed macrophages may differentially affect polarization of adaptive immune responses.

Candida albicans is a medically important pathogen, and recognition by innate immune cells is critical for its clearance. Although a number of pattern recognition receptors have been shown to be involved in recognition and phagocytosis of this fungus, the relative role of these receptors has not been formally examined. In this paper, we have investigated the contribution of the mannose receptor, Dectin-1, and complement receptor 3; and we have demonstrated that Dectin-1 is the main non-opsonic receptor involved in fungal uptake. However, both Dectin-1 and complement receptor 3 were found to accumulate at the site of uptake, while mannose receptor accumulated on C. albicans phagosomes at later stages. These results suggest a potential role for MR in phagosome sampling; and, accordingly, MR deficiency led to a reduction in TNF-α and MCP-1 production in response to C. albicans uptake. Our data suggest that pattern recognition receptors sample the fungal phagosome in a sequential fashion.

Author Summary

Infection by Candida albicans has increased as a result of immunosuppression associated with AIDS and organ transplantation. We assessed the role of three pattern recognition receptors, namely Dectin-1 (a beta glucan receptor), the type 3 complement receptor (CR3), and the mannose receptor, in mediating uptake of this fungus. These receptors are known to recognize structures on the C. albicans cell wall, but their exact contribution to binding and uptake is still unclear. We show that only Dectin-1 plays a major role in binding and uptake of C. albicans. Furthermore, we are the first to find that these receptors sample the internalized particle in a sequential manner; intracellular mannose receptor is recruited later and is involved in secretion of immune modulators. These findings provide a better understanding of the innate immune mechanisms involved in protection against this medically important fungal pathogen.

The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dectin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dectin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

C-type lectins are a diverse family of proteins which recognize a wide range of ligands. This review focuses on the Dectin-2 family of C-type lectins that includes Dectin-2, BDCA-2, DCIR, DCAR, Clecsf8 and Mincle whose genes are clustered in the telomeric region of the NK-gene cluster on mouse chromosome 6 and human chromosome 12. These type II receptors are expressed on myeloid and non-myeloid cells and contain a single extracellular carbohydrate recognition domain and have diverse functions in both immunity and homeostasis. DCIR is the only member of the family which contains a cytoplasmic signalling motif and has been shown to act as an inhibitory receptor, while BDCA-2, Dectin-2, DCAR and Mincle all associate with FcRγ chain to induce cellular activation, including phagocytosis and cytokine production. Dectin-2 and Mincle have been shown to act as pattern recognition receptors for fungi, while DCIR acts as an attachment factor for HIV. In addition to pathogen recognition, DCIR has been shown to be pivotal in preventing autoimmune disease by controlling dendritic cell proliferation, whereas Mincle recognizes a nuclear protein released by necrotic cells. Here we review each of these receptors in detail describing their expression, ligand recognition, signalling and known physiological functions.

The therapeutic benefits of fungal β-glucans have been demonstrated as immuno-stimulating agents. In this study, we aimed to explore the mechanisms used by yeast β-glucan-rich particles to activate murine resident macrophages for cytokine secretion. We demonstrated that resident macrophages were effectively activated by whole yeast β-glucan particles (WGPs), such as with the up-regulation of co-stimulatory molecules and the secretion of cytokines. The binding ability of WGPs and the levels of cytokine secretion in resident macrophages were significantly inhibited by soluble yeast β-glucan but not by blockade of zymosan glucan receptor dectin-1. In addition, WGP-stimulated cytokine secretion was partially dependent on the MyD-88 pathway but was not significantly affected in CR3-deficient (CR3−/−) mice. Furthermore, we showed that Syk kinase was recruited upon WGP stimulation and was required for the production of cytokines. Taken together, these observations suggest that β-glucan recognition is necessary but not sufficient to induce inflammatory response on resident macrophages. In addition, β-glucan particles may use differential mechanisms for cytokine secretion in resident macrophages that may modulate both innate and adaptive immunity.