The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

Open Access funded by Wellcome Trust: Under a Creative Commons license Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. Acknowledgments We would like to thank S. Hardison, P. Redelinghuys, J. Taylor, C. Wallace, A. Richmond, S. Hadebe, A. Plato, F. Abbass, L. Fick, N. Allie, R. Wilkinson, K. Wilkinson, S. Cooper, D. Lang, and V. Kumar for reagents and assistance, and the animal facility staff for the care of our animals. This work was supported by the MRC (UK) and Wellcome Trust (G.D.B.); MRC (South Africa) and Sydney Brenner Fellowship (M.J.M.); Vici (M.G.N.), Vidi (R.v.C.), and Veni grants (T.S.P.) from the Netherlands Organization for Scientific Research; the Royal Netherlands Academy of Arts and Sciences (T.H.M.O.); EC FP7 projects (NEWTBVAC, ADITEC; T.H.M.O.); Carnegie Corporation and CIDRI (J.C.H.); and the University of Aberdeen (B.K.).Peer reviewedPublisher PD

MICL controls inflammation in rheumatoid arthritis

Acknowledgments We thank G Milne, D MacCallum, S Hardison, G Wilson, C Wallace, S Hadebe and A Richmond for assistance; H. El-Gabalawy for tissues and the animal facility staff for the care of our animals. Flow cytometry was undertaken in the Iain Fraser Cytometry Centre, University of Aberdeen. Funding: GDB was funded by the Wellcome Trust and MRC (UK). AA and CDB are supported by the Arthritis Research UK Tissue Engineering Centre (grant 19429). This study makes use of data generated by the Wellcome Trust Case Control Consortium. A full list of the investigators who contributed to the generation of the data is available from http://www.wtccc.org.uk, and was funded by the Wellcome Trust (076113). MJGF was funded by The Arthritis Society and the Canadian Arthritis Network and J-ML by a scholarship from the Canadian Arthritis Network.Peer reviewedPublisher PD

PPARγ Controls Dectin-1 Expression Required for Host Antifungal Defense against Candida albicans

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

Dectin-1: a role in antifungal defense and consequences of genetic polymorphisms in humans

The clinical relevance of fungal infections has increased dramatically in recent decades as a consequence of the rise of immunocompromised populations, and efforts to understand the underlying mechanisms of protective immunity have attracted renewed interest. Here we review Dectin-1, a pattern recognition receptor involved in antifungal immunity, and discuss recent discoveries of polymorphisms in the gene encoding this receptor which result in human disease

Maternal Influences on the Transmission of Leukocyte Gene Expression Profiles in Population Samples from Brisbane, Australia

Two gene expression profiling studies designed to identify maternal influences on development of the neonate immune system and to address the population structure of the leukocyte transcriptome were carried out in Brisbane, Australia. In the first study, a comparison of 19 leukocyte samples obtained from mothers in the last three weeks of pregnancy with 37 umbilical cord blood samples documented differential expression of 7,382 probes at a false discovery rate of 1%, representing approximately half of the expressed transcriptome. An even larger component of the variation involving 8,432 probes, notably enriched for Vitamin E and methotrexate-responsive genes, distinguished two sets of individuals, with perfect transmission of the two profile types between each of 16 mother-child pairs in the study. A minor profile of variation was found to distinguish the gene expression profiles of obese mothers and children of gestational diabetic mothers from those of children born to obese mothers. The second study was of adult leukocyte profiles from a cross-section of Red Cross blood donors sampled throughout Brisbane. The first two axes in this study are related to the third and fourth axes of variation in the first study and also reflect variation in the abundance of CD4 and CD8 transcripts. One of the profiles associated with the third axis is largely excluded from samples from the central portion of the city. Despite enrichment of insulin signaling and aspects of central metabolism among the differentially expressed genes, there was little correlation between leukocyte expression profiles and body mass index overall. Our data is consistent with the notion that maternal health and cytokine milieu directly impact gene expression in fetal tissues, but that there is likely to be a complex interplay between cultural, genetic, and other environmental factors in the programming of gene expression in leukocytes of newborn children

Scavenger receptors and β-glucan receptors participate in the recognition of yeasts by murine macrophages

Objectives: Numerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the $\beta$-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 $H_2O_2$ 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 $\beta$-glucan receptor which was distinct from dectin-1 and not linked to stimulation of $H_2O_2$ production. Ligands of $\beta$-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 $\beta$-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 $H_2O_2$ 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

A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion

Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells

C-Type Lectin in Chlamys farreri (CfLec-1) Mediating Immune Recognition and Opsonization

Background: C-type lectins are a superfamily of Ca 2+ dependent carbohydrate-recognition proteins that play significant diverse roles in nonself-recognition and clearance of invaders. Though they are well characterized in vertebrates, the study of the potential function and mechanism of C-type lectins in invertebrate immunity is still in its infancy. Methodology: A C-type lectin (CfLec-1) from scallop Chlamys farreri, a dominant cultured mollusk species in China, was selected to investigate its mRNA expression, localization and the possible functions in innate immunity in the present study. After scallop was stimulated by three typical PAMPs, the mRNA expression of CfLec-1 in hemocytes was poles apart. It was significantly up-regulated (p,0.01) after scallops were stimulated by LPS or b-glucan, but significantly down-regulated (p,0.01) after PGN stimulation. The binding ability of recombinant CfLec-1 (designated as rCfLec-1) towards eight PAMPs was investigated subsequently by PAMPs microarray, which revealed rCfLec-1 could bind LPS, PGN and mannan in vitro, indicating CfLec-1 served as a PRR involved in the pathogen recognition. Immunofluorescence assay with polyclonal antibody specific for CfLec-1 revealed that CfLec-1 was mainly located in the mantle and gill of the scallop. CfLec-1 could bind to the surface of scallop hemocytes and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-1 antibody. Meanwhile, rCfLec-1 could also enhance the phagocytic activity of scallop hemocytes against Escherichia coli

β-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells

The internalization of Aspergillus fumigatus into lung epithelial cells is a process that depends on host cell actin dynamics. The host membrane phosphatidylcholine cleavage driven by phospholipase D (PLD) is closely related to cellular actin dynamics. However, little is known about the impact of PLD on A. fumigatus internalization into lung epithelial cells. Here, we report that once germinated, A. fumigatus conidia were able to stimulate host PLD activity and internalize more efficiently in A549 cells without altering PLD expression. The internalization of A. fumigatus in A549 cells was suppressed by the downregulation of host cell PLD using chemical inhibitors or siRNA interference. The heat-killed swollen conidia, but not the resting conidia, were able to activate host PLD. Further, β-1,3-glucan, the core component of the conidial cell wall, stimulated host PLD activity. This PLD activation and conidia internalization were inhibited by anti-dectin-1 antibody. Indeed, dectin-1, a β-1,3-glucan receptor, was expressed in A549 cells, and its expression profile was not altered by conidial stimulation. Finally, host cell PLD1 and PLD2 accompanied A. fumigatus conidia during internalization. Our data indicate that host cell PLD activity induced by β-1,3-glucan on the surface of germinated conidia is important for the efficient internalization of A. fumigatus into A549 lung epithelial cells