Fungal melanin suppresses airway epithelial chemokine secretion through blockade of calcium fluxing.

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

Detailed characterisation of invasive aspergillosis in a murine model of X-linked chronic granulomatous disease shows new insights in infections caused by Aspergillus fumigatus versus Aspergillus nidulans.

Introduction: Invasive aspergillosis (IA) is the most prevalent infectious complication in patients with chronic granulomatous disease (CGD). Yet, understanding of fungal pathogenesis in the CGD host remains limited, particularly with regards to A. nidulans infection. Methods: We have used a murine model of X-linked CGD to investigate how the pathogenesis of IA varies between A. fumigatus and A. nidulans, comparing infection in both X-linked CGD (gp91-/-) mice and their parent C57BL/6 (WT) mice. A 14-colour flow cytometry panel was used to assess the cell dynamics over the course of infection, with parallel assessment of pulmonary cytokine production and lung histology. Results: We observed a lack of association between pulmonary pathology and infection outcome in gp91-/- mice, with no significant mortality in A. nidulans infected mice. An overwhelming and persistent neutrophil recruitment and IL-1 release in gp91-/- mice following both A. fumigatus and A. nidulans infection was observed, with divergent macrophage, dendritic cell and eosinophil responses and distinct cytokine profiles between the two infections. Conclusion: We have provided an in-depth characterisation of the immune response to pulmonary aspergillosis in an X-linked CGD murine model. This provides the first description of distinct pulmonary inflammatory environments in A. fumigatus and A. nidulans infection in X-linked CGD and identifies several new avenues for further research.

The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages.

Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex-3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Signalling through MyD88 drives surface expression of the mycobacterial receptors MCL (Clecsf8, Clec4d) and Mincle (Clec4e) following microbial stimulation.

The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this "two-step" model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity.

Role of Dectin-2 for host defense against systemic infection with Candida glabrata.

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2(-/-)) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2(-/-) mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2(-/-) macrophages and neutrophils. Neutrophils of Dectin-2(-/-) mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1.

Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein­coupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin­like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain­containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.

The C-type lectin receptor CLECSF8 (CLEC4D) is expressed by myeloid cells and triggers cellular activation through Syk kinase.

CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.

Pattern recognition: recent insights from Dectin-1.

The beta-glucan receptor Dectin-1 is an archetypical non-toll-like pattern recognition receptor expressed predominantly by myeloid cells, which can induce its own intracellular signalling and can mediate a variety of cellular responses, such as cytokine production. Recent identification of the components of these signalling pathways, such as Syk kinase, CARD9 and Raf-1, has provided novel insights into the molecular mechanisms underlying Dectin-1 function. Furthermore, a broader appreciation of the cellular responses mediated by this receptor and the effects of interactions with other receptors, including the TLRs, have greatly furthered our understanding of innate immunity and how this drives the development of adaptive immunity, particularly Th17 responses. Recent studies have highlighted the importance of Dectin-1 in anti-fungal immunity, in both mice and humans, and have suggested a possible involvement of this receptor in the control of mycobacterial infections.

LYVE-1-positive macrophages are present in normal murine eyes.

PURPOSE: A functioning lymphatic system is necessary not only to permit the organism to mount a rapid and effective immune response but, even more so, to maintain tissue fluid homeostasis. However, no clear evidence of lymphatic vessels draining intraocular and orbital tissues--retina, choroid, sclera, and extraocular muscles--exists. METHODS: Ocular tissue flatmounts from normal or enhanced green fluorescence protein (EGFP) chimeric mice were immunostained for lymphatic endothelium hyaluronan receptor (LYVE-1, a routinely used lymphatic endothelial marker), podoplanin, Flt4/VEGFR3, Sca-1, CD11b, or F4/80 and were observed by confocal microscopy. Single-cell suspensions from ocular tissues were also prepared and were analyzed by flow cytometry. RESULTS: Lymphatic vessels were detected in the posterior regions of the extraocular muscles and the connective tissues of the extraocular muscle cones in the normal mouse. No typical lymphatic vessels were found within the eye. A large population of LYVE-1(+) nonendothelial cells, distributed as single cells, was detected in all ocular tissues except the central cornea. These cells also express another lymphatic endothelial cell marker, Flt4/VEGFR3, but not podoplanin, and they have hyaluronan-binding ability. Bone marrow chimerism studies indicated that the LYVE-1(+) cell populations are bone marrow derived and have a slow turnover in ocular tissues (3-6 months). Phenotype analysis revealed that nonendothelial LYVE-1(+)cells in the sclera, choroid, and iris included CD11b(+)F4/80(+) macrophages, CD11b(+)F4/80(-) macrophages, and CD11b(-)F4/80(-) bone marrow-derived cells. All LYVE-1(+) cells in the retina were CD11b(+)F4/80(+) macrophages. Cells in the limbus and the iris root also express Sca-1, suggesting that they are hematopoietic lymphatic vessel progenitor cells. CONCLUSIONS: These observations suggest that a lymphatic system exists for the transport of immune cells and fluids from the posterior segment of the eye, that ocular tissues are rich in bone marrow-derived LYVE-1(+) macrophages under normal physiological conditions, and that a subpopulation of these cells may represent resident precursor cells necessary for the de novo formation of ocular/orbital lymphatic vessels in pathologic conditions.

Ly49B is expressed on multiple subpopulations of myeloid cells.

Using a novel mAb specific for mouse Ly49B, we report here that Ly49B, the last remaining member of the C57 Ly49 family to be characterized, is expressed at low levels on approximately 1.5% of spleen cells, none which are NK cells or T cells but which instead belong to several distinct subpopulations of myeloid cells defined by expression of CD11b and different levels of Gr1. Much larger proportions of bone marrow and peritoneal cells expressed Ly49B, all being CD11b+ and comprising multiple subpopulations defined by light scatter, F4/80, and Gr1 expression. Costaining for Ly49Q, also expressed on myeloid cells, revealed that Ly49B and Ly49Q were most strongly expressed on nonoverlapping subpopulations, Ly49Q(high) cells being mostly B220+CD4+ and/or CD8+, Ly49B+ cells lacking these markers. Myeloid populations that developed from bone marrow progenitors in vitro frequently coexpressed both Ly49B and Ly49Q, and Ly49B expression could be up-regulated by LPS, alpha-IFN, and gamma-IFN, often independently of Ly49Q. PCR analysis revealed that cultured NK cells and T cells contained Ly49B transcripts, and Ly49B expression could be detected on NK cells cultured in IL-12 plus IL-18, and on an immature NK cell line. Immunohistochemical studies showed that Ly49B expression in tissues overlapped with but was distinct from that of all other myeloid molecules examined, being particularly prominent in the lamina propria and dome of Peyer's patches, implicating an important role of Ly49B in gut immunobiology. In transfected cells, Ly49B was found to associate with SHP-1, SHP-2, and SHIP in a manner strongly regulated by intracellular phosphorylation events.

Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.

The human beta-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 beta-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 beta-glucan receptor (betaGR), 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 betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-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 beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-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 beta-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 (Mphi). 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-gamma, 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-alpha, although some Mphi-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 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 (betaGR) as a major beta-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate beta-glucans. Using a novel mAb (2A11) raised against betaGR, 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 (Mphi) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the betaGR, but at lower levels. Alveolar Mphi, like inflammatory Mphi, exhibited the highest surface expression of betaGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mphi expressed much lower levels of betaGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mphi suggested the existence of an additional beta-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mphi. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mphi 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 Mphi.

Dectin-1 is a major beta-glucan receptor on macrophages.

Zymosan is a beta-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 beta-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 beta-glucan receptor mediating this activity. To address the role of the recently described beta-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 beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte beta-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 beta-glucans for therapeutic drug design.