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

11 pags, 7 figsCLECSF8 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was funded by the Wellcome Trust, the National Research Foundation, the Deutscher Akademischer Austauschdienst, the University of Cape Town, the UK Research Council Basic Technology Initiative “Glycoar-rays” (GRS/79268), and the UK Medical Research Council. A. S. P is a fellowof the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal) and M. A. C. of the Consejo Superior de Investigaciones Cientificas, Programe “Junta para la Ampliación de Estudios” (JaeDoc/098/2011) cofinanced by the Fondo Social Europeo

The Induction of Inflammation by Dectin-1 In Vivo Is Dependent on Myeloid Cell Programming and the Progression of Phagocytosis

Dectin-1 is the archetypal signaling, non-Toll-like pattern recognition receptor that plays a protective role in immune defense to Candida albicans as the major leukocyte receptor for β-glucans. Dectin-1-deficiency is associated with impaired recruitment of inflammatory leukocytes and inflammatory mediator production at the site of infection. In this study, we have used mice to define the mechanisms that regulate the dectin-1-mediated inflammatory responses. Myeloid cell activation by dectin-1 is controlled by inherent cellular programming, with distinct macrophage and dendritic cell populations responding differentially to the engagement of this receptor. The inflammatory response is further modulated by the progression of the phagocytosis, with 'frustrated phagocytosis' resulting in dramatically augmented inflammatory responses. These studies demonstrate that dectin-1 in isolation is sufficient to drive a potent inflammatory response in a context-dependent manner. This has implications for the mechanism by which myeloid cells are activated during fungal infections and the processes involved in the therapeutic manipulation of the immune system via exogenous dectin-1 stimulation or blockade

The induction of inflammation by dectin-1 in vivo is dependent on myeloid cell programming and the progression of phagocytosis

Dectin-1 is the archetypal signaling, non-Toll-like pattern recognition receptor that plays a protective role in immune defense to Candida albicans as the major leukocyte receptor for β-glucans. Dectin-1-deficiency is associated with impaired recruitment of inflammatory leukocytes and inflammatory mediator production at the site of infection. In this study, we have used mice to define the mechanisms that regulate the dectin-1-mediated inflammatory responses. Myeloid cell activation by dectin-1 is controlled by inherent cellular programming, with distinct macrophage and dendritic cell populations responding differentially to the engagement of this receptor. The inflammatory response is further modulated by the progression of the phagocytosis, with 'frustrated phagocytosis' resulting in dramatically augmented inflammatory responses. These studies demonstrate that dectin-1 in isolation is sufficient to drive a potent inflammatory response in a context-dependent manner. This has implications for the mechanism by which myeloid cells are activated during fungal infections and the processes involved in the therapeutic manipulation of the immune system via exogenous dectin-1 stimulation or blockade

The Induction of Inflammation by Dectin-1 in Vivo Is Dependent on Myeloid Cell Programming and the Progression of Phagocytosis

Dectin-1 is the archetypal signaling, non-Toll-like pattern recognition receptor that plays a protective role in immune defense to Candida albicans as the major leukocyte receptor for β-glucans. Dectin-1-deficiency is associated with impaired recruitment of inflammatory leukocytes and inflammatory mediator production at the site of infection. In this study, we have used mice to define the mechanisms that regulate the dectin-1-mediated inflammatory responses. Myeloid cell activation by dectin-1 is controlled by inherent cellular programming, with distinct macrophage and dendritic cell populations responding differentially to the engagement of this receptor. The inflammatory response is further modulated by the progression of the phagocytosis, with frustrated phagocytosis resulting in dramatically augmented inflammatory responses. These studies demonstrate that dectin-1 in isolation is sufficient to drive a potent inflammatory response in a context-dependent manner. This has implications for the mechanism by which myeloid cells are activated during fungal infections and the processes involved in the therapeutic manipulation of the immune system via exogenous dectin-1 stimulation or blockade

Transfer of <i>N. brasiliensis</i> experienced B cells enhances immunity to N. brasiliensis independently of endogenous B cell populations.

<p><i>N. brasiliensis</i> infected μMT and BALB/c mice were re-infected with 500xL3 larvae and at day 5 post-secondary infection, the intestinal worm burdens was quantified (<b>A</b>). The possible role for IL-4Rα expressing B cells in boosting immunity independently of endogenous B cells was determined by transfer of B cells isolated from <i>N. brasiliensis</i> infected IL-4Rα^−/lox (WT B cells) or <i>MB</i>1^Cre IL-4Rα^−/lox (IL-4Rα^−/− B cells) into naïve μMT mice. These mice were then infected with 500xL3 <i>N. brasiliensis</i> and worm burdens quantified at day 5 post infection (<b>B</b>). Mediastinal lymph node CD3⁺CD4⁺ T cell populations IL-13 responses (<b>C</b>) were established by FACS staining. Results shown represent 2 independent experiments. n = 4–7 mice per group.</p

Rapid IL-Rα dependent B cell mediated protection against <i>N. brasiliensis</i> occurs in the lung.

<p>MB^creIL-4Rα^−/lox and IL-4Rα^−/lox mice were infected for 1 day with N. brasiliensis before spleen B cells were isolated and transferred into naive wild type mice (<b>As in </b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003662#ppat-1003662-g004" target="_blank"><b>Figure 4C</b></a>). These were infected with 500xL3 N. brasiliensis and intestinal worm burdens were quantified at day 5 post infection (<b>A</b>). Lung CD3⁺CD4⁺ and CD3⁺CD4⁺CD44^hi T cell populations were analysed by FACS staining (<b>B</b>). Lung CD4⁺ T cell (<b>C</b>) and mediastinal lymph node CD4⁺ T cell and B220⁺ B cell population (<b>D</b>) IL-13 responses were established by intracellular FACS staining. Data is representative of 2 independent experiments. n = 4–6 mice per group.</p

B cell MyD88 expression dependent protection against <i>N. brasiliensis</i> infection.

<p>B cells were isolated from WT or MyD88^−/− mice 24 hours post <i>N. brasiliensis</i> infection and adoptively transferred into naive WT mice (<b>As in </b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003662#ppat-1003662-g004" target="_blank"><b>Figure 4C</b></a>). 24 hours later these mice were infected with 500xL3 <i>N. brasiliensis</i> and subsequently killed 5 days post infection and worm burdens were counted (<b>A</b>). The mediastinal lymph node CD4⁺ T-cell IL-13 response was established (<b>B</b>). B cells were isolated from naive C57/BL6, MyD88^−/− or MHCII^−/− mice and pulsed with <i>N. brasiliensis</i> antigen overnight. These were then washed and transferred into naive C57/BL6 mice 24 h prior to infection (<b>C</b>). D5 PI intestinal worm counts are shown (<b>D & F</b>). The mediastinal lymph node CD4⁺ T-cell IL-13 response was established (<b>E</b>). Data is representative of 2 experiments, n = 5–7 mice per group.</p

B cell MHCII antigen presentation mediates optimal immunity to <i>N. brasiliensis</i>.

<p>Surface expression of CD28 and TCR on CD4⁺ T cells and CD86 and MHCII on B cells in naive (<b>A</b>) and <i>N. brasiliensis</i> re-infected (<b>B</b>) IL-4Rα^−/lox mice and <i>MB1</i>^CreIL-4Rα^−/lox mice was established by FACS analysis. Histograms: filled gray: isotype control, thin line: IL-4Rα^−/lox, thick black line: <i>MB</i>1^CreIL-4Rα^−/lox. Contributions by MHCII dependent antigen presentation were demonstrated by isolating WT or MHCII^−/− B cells from naive or infected mice then adoptively transferring into naive C57BL/6 mice (<b>C</b>). Mice were then infected with 500xL3 <i>N. brasiliensis</i> larvae and worm burdens were established at day 5 post infection (<b>D</b>). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4⁺ T-cell and B220⁺ B cell populations (<b>E</b>). MHC dependent antigen presentation was confirmed by isolating WT and BALB/b B cells from naive or infected mice adoptively transferring into naive BALB/c mice. Mice were then infected with 500xL3 <i>N. brasiliensis</i> larvae and worm burdens were established at day 5 post infection (<b>F</b>). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4⁺ T-cell and B220⁺ B cell populations (<b>G</b>). Data is representative of 2 independent experiments. n = 4–6 mice per group.</p

Protective immunity to <i>N. brasiliensis</i> re-infection is IL-13 and IL-4Rα dependent.

<p>IL-4Rα^−/− and IL-4Rα^−/lox mice were infected for 5 or 7 days post-secondary <i>N. brasiliensis</i> infection (<b>A</b>). Intestinal worm burdens were then quantified (<b>B</b>). Pulmonary mucus production was established by PAS staining (<b>C</b>). Serum Antibody titers of <i>N. brasiliensis</i> specific IgG1 were determined by ELISA (<b>D</b>). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4⁺ T-cell and B220⁺ B-cell populations (<b>E</b>). IL-4^−/−, IL-13^−/− and IL-4Rα^−/lox mice were infected for 5 days post-secondary <i>N. brasiliensis</i> infection and intestinal worm burdens were then quantified (<b>F</b>). Pulmonary mucus production was established by PAS staining (<b>G</b>). Serum Antibody titers of <i>N. brasiliensis</i> specific IgG1 were determined by ELISA (<b>H</b>). Data is representative of 3–4 independent experiments. n = 4–6 mice per group.</p