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The relevance of protein-glycan interactions in immunity has long been underestimated. Yet, the immune system possesses numerous classes of glycan-binding proteins, so-called lectins. Of specific interest is the group of myeloid C-type lectin receptors (CLRs) as they are mainly expressed by myeloid cells and play an important role in the initiation of an immune response. Myeloid CLRs represent a major group amongst pattern recognition receptors (PRRs), placing them at the center of the rapidly growing field of glycoimmunology. CLRs have evolved to encompass a wide range of structures and functions and to recognize a large number of glycans and many other ligands from different classes of biopolymers. This review aims at providing the reader with an overview of myeloid CLRs and selected ligands, while highlighting recent insights into CLR-ligand interactions. Subsequently, methodological approaches in CLR-ligand research will be presented. Finally, this review will discuss how CLR-ligand interactions culminate in immunological functions, how glycan mimicry favors immune escape by pathogens, and in which way immune responses can be affected by CLR-ligand interactions in the long term., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

Viral infections and reactivations are major causes of morbidity and mortality after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) as well as in patients with immunodeficiencies. Latent herpesviruses (e.g., cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6), lytic viruses (e.g., adenovirus), and polyomaviruses (e.g., BK virus, JC virus) can cause severe complications. Antiviral drugs form the mainstay of treatment for viral infections and reactivations after transplantation, but they have side effects and cannot achieve complete viral clearance without prior reconstitution of functional antiviral T-cell immunity. The aim of this study was to establish normal ranges for virus-specific T-cell (VST) frequencies in healthy donors. Such data are needed for better interpretation of VST frequencies observed in immunocompromised patients. Therefore, we measured the frequencies of VSTs against 23 viral protein-derived peptide pools from 11 clinically relevant human viruses in blood from healthy donors (n = 151). Specifically, we determined the VST frequencies by interferon-gamma enzyme-linked immunospot assay and classified their distribution according to age and gender to allow for a more specific evaluation and prediction of antiviral immune responses. The reference values established here provide an invaluable tool for immune response evaluation, intensity of therapeutic drugs and treatment decision-making in immunosuppressed patients. This data should make an important contribution to improving the assessment of immune responses in immunocompromised patients., (© 2022. The Author(s).)

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus endemic to Africa and the Arabian Peninsula, which causes diseases in humans and livestock. C-type lectin receptors (CLRs) represent a superfamily of pattern recognition receptors that were reported to interact with diverse viruses and contribute to antiviral immune responses but may also act as attachment factors or entry receptors in diverse species. Human DC-SIGN and L-SIGN are known to interact with RVFV and to facilitate viral host cell entry, but the roles of further host and vector CLRs are still unknown. In this study, we present a CLR-Fc fusion protein library to screen RVFV-CLR interaction in a cross-species approach and identified novel murine, ovine, and Aedes aegypti RVFV candidate receptors. Furthermore, cross-species CLR binding studies enabled observations of the differences and similarities in binding preferences of RVFV between mammalian CLR homologues, as well as more distant vector/host CLRs.

Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR -/- mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry. To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8 + T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8 + T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection., (© 2021. The Author(s).)

Multivalent ligand-protein interactions are a commonly employed approach by nature in many biological processes. Single glycan-protein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azide-alkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycan-lectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectin-glycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders., Competing Interests: FFL is named on a patent related to laser-based microarray synthesis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tsouka, Hoetzel, Mende, Heidepriem, Paris, Eickelmann, Seeberger, Lepenies and Loeffler.)

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella . Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris , host cells, and the microbiota.

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9 -/- and corresponding C57BL/6 wild-type control mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9 -/- mice showed an increased loss of neuronal nuclear protein + mature neurons and doublecortin + neuronal precursor cells and an increase in β-amyloid precursor protein + damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8 + T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface. IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts., (Copyright © 2021 Fabian et al.)

Toxocara canis and Toxocara cati , the worldwide occurring intestinal roundworms of canids and felids, represent an important public health threat due to various disease manifestations in humans. Host recognition of pathogens is mediated by pattern recognition receptors (PRRs). Myeloid C-type lectin receptors (CLRs) are PRRs and recognise carbohydrate structures of various pathogens. As Toxocara excretory-secretory products (TES) are predominantly composed of glycoconjugates, they represent suitable targets for CLRs. However, the range of host-derived CLRs recognising Toxocara spp. is still unknown. Using a CLR-hFc fusion protein library, T. canis and T. cati L3 somatic antigens (TSOM) were bound by a variety of CLRs in enzyme-linked immunosorbent assay (ELISA), while their TES products interacted with macrophage galactose-type lectin-1 (MGL-1). Two prominent candidate CLRs, MGL-1 and macrophage C-type lectin (MCL), were selected for further binding studies. Immunofluorescence microscopy revealed binding of MGL-1 to the oral aperture of L3. Immunoblot experiments identified distinct protein fractions representing potential ligands for MGL-1 and MCL. To evaluate how these interactions influence the host immune response, bone marrow-derived dendritic cell (BMDC) assays were performed, showing MCL-dependent T. cati -mediated cytokine production. In conclusion, MGL-1 and MCL are promising candidates for immune modulation during Toxocara infection, deserving further investigation in the future.

C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle -/- , CARD9 -/- or conditional Syk -/- mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly decreased. Importantly, this was accompanied by an altered CD4 + T cell priming capacity of Mincle -/- BMDCs resulting in an increased CD4 + T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Prado Acosta, Goyette-Desjardins, Scheffel, Dudeck, Ruland and Lepenies.)

Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

Tumor growth is accompanied with dramatic changes in the cellular glycome, such as the aberrant expression of complex branched N-glycans. However, the role of this protumoral N-glycan in immune evasion and whether its removal contributes to enhancement of immune recognition and to unleashing an antitumor immune response remain elusive. We demonstrated that branched N-glycans are used by colorectal cancer cells to escape immune recognition, instructing the creation of immunosuppressive networks through inhibition of IFNγ. The removal of this "glycan-mask" exposed immunogenic mannose glycans that potentiated immune recognition by DC-SIGN-expressing immune cells, resulting in an effective antitumor immune response. We revealed a glycoimmune checkpoint in colorectal cancer, highlighting the therapeutic efficacy of its deglycosylation to potentiate immune recognition and, thus, improving cancer immunotherapy., (©2020 American Association for Cancer Research.)

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after β-elimination showed glucose as the major component in the O- glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N -glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N -glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Malamud et al.)

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.

C-type lectins (CTLs), a superfamily of glycan-binding receptors, play a pivotal role in the host defense against pathogens and the maintenance of immune homeostasis of higher animals and humans. CTLs in innate immunity serve as pattern recognition receptors and often bind to glycan structures in damage- and pathogen-associated molecular patterns. While CTLs are found throughout the whole animal kingdom, their ligand specificities and downstream signaling have mainly been studied in humans and in model organisms such as mice. In this review, recent advancements in CTL research in veterinary species as well as potential applications of CTL targeting in veterinary medicine are outlined.

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell- and tissue-specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one-pot intein-mediated ligation (OPL) to obtain GPI-anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1 19 were prepared. Glypiation did not alter the structure of eGFP and MSP1 19 proteins in solution, but it induced a strong pro-inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

C-type lectin receptors (CTLRs) are pattern recognition receptors which are important constituents of the innate immunity. However, their role has mostly been studied in humans and in mouse models. To bridge the knowledge gap concerning CTLRs of veterinary relevant species, a novel ovine CTLR hFc-fusion protein library which allows in vitro ligand identification and pathogen binding studies has been established. Its utility was tested with known ligands of corresponding murine CTLRs in ELISA- and flow cytometry based binding studies. The ovine CTLR-hFc library was subsequently used in a proof-of-principle pathogen binding study with the ruminant pathogen Mycoplasma mycoides subsp. capri. Some ovine CTLRs, such as Dendritic Cell Immunoreceptor (DCIR, Clec4a), Macrophage C-Type Lectin (MCL, Clec4d) and Myeloid Inhibitory C-Type Lectin-Like Receptor (MICL, Clec12a) were identified as possible candidate receptors whose role in Mycoplasma recognition can now be unraveled in further studies. This study thus shows the utility of this novel ovine CTLR-hFc fusion protein library to screen for CTLR/pathogen interactions., Competing Interests: Declaration of Competing Interest No author has any competing interests or conflicts of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glycosylphosphatidylinositols (GPIs) are complex glycolipids present on the surfaces of Plasmodium parasites that may act as toxins during the progression of malaria. GPIs can activate the immune system during infection and induce the formation of anti-GPI antibodies that neutralize their activity. Therefore, an antitoxic vaccine based on GPI glycoconjugates may prevent malaria pathogenesis. To evaluate the role of three key modifications on Plasmodium GPI glycan in the activity of these glycolipids, we synthesized and investigated six structurally distinct GPI fragments from Plasmodium falciparum . The synthetic glycans were conjugated to the CRM197 carrier protein and were tested for immunogenicity and efficacy as antimalarial vaccine candidates in an experimental cerebral malaria model using C57BL/6JRj mice. Protection may be dependent on both the antibody and the cellular immune response to GPIs, and the elicited immune response depends on the orientation of the glycan, the number of mannoses in the structure, and the presence of the phosphoethanolamine and inositol units. This study provides insights into the epitopes in GPIs and contributes to the development of GPI-based antitoxin vaccine candidates against cerebral malaria.

Uptake of apoptotic cells (ACs) by dendritic cells (DCs) and induction of a tolerogenic DC phenotype is an important mechanism for establishing peripheral tolerance to self-antigens. The receptors involved and underlying signaling pathways are not fully understood. Here, we identify Dectin-1 as a crucial tolerogenic receptor binding with nanomolar affinity to the core domain of several annexins (annexin A1, A5, and A13) exposed on ACs. Annexins bind to Dectin-1 on a site distinct from the interaction site of pathogen-derived β-glucans. Subsequent tolerogenic signaling induces selective phosphorylation of spleen tyrosine kinase (SYK), causing activation of NADPH oxidase-2 and moderate production of reactive oxygen species. Thus, mice deficient for Dectin-1 develop autoimmune pathologies (autoantibodies and splenomegaly) and generate stronger immune responses (cytotoxic T cells) against ACs. Our data describe an important immunological checkpoint system and provide a link between immunosuppressive signals of ACs and maintenance of peripheral immune tolerance., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial surfaces are rich in glycoconjugates that are mainly present in their outer layers and are of great importance for their interaction with the host innate immune system. The innate immune system is the first barrier against infection and recognizes pathogens via conserved pattern recognition receptors (PRRs). Lectins expressed by innate immune cells represent an important class of PRRs characterized by their ability to recognize carbohydrates. Among lectins in innate immunity, there are three major classes including the galectins, siglecs, and C-type lectin receptors. These lectins may contribute to initial recognition of bacterial glycans, thus providing an early defence mechanism against bacterial infections, but they may also be exploited by bacteria to escape immune responses. In this review, we will first exemplify bacterial glycosylation systems; we will then describe modes of recognition of bacterial glycans by lectins in innate immunity and, finally, we will briefly highlight how bacteria have found ways to exploit these interactions to evade immune recognition., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Myeloid C-type lectin receptors (CLRs) are innate immune recognition molecules that bind to microorganisms via their carbohydrate recognition domains. In this study, we utilized a library of CLRs that recognize fungal mannans. We used this library to screen against Pneumocystis carinii (Pc) homogenates or purified Pc major surface glycoprotein (Msg) present on Pneumocystis . The results demonstrated that all of the mammalian CLR hFc-fusions tested displayed significant interaction/binding with Pc organisms, and furthermore to isolated Msg. Highest Pc organism and Msg binding activities were with CLR members Mincle, Dectin-2, DC-SIGN and MCL. An immunofluorescence assay with the respective CLR hFc-fusions against whole Pc life forms corroborated these findings. Although some of these CLRs have been implicated previously as important for Pneumocystis pathogenesis (Dectin-1/Dectin-2/Mincle), this is the first analysis of head-to-head comparison of known fungal mannan binding CLR-hFc fusions with Pc. Lastly, heat treatment resulted in reducted CLR binding.

Malaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8 + T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea . Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca 2+ -dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR-hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3, and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signaling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle -/- or CARD9 -/- mice were not capable to enhance OVA-specific response in CD4 + T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.

Alphaviruses and flaviviruses are important human pathogens that include Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV), which can cause diseases in humans ranging from arthralgia to hemorrhagic fevers and microcephaly. It was previously shown that treatment with surface layer (S-layer) protein, present on the bacterial cell-envelope of Lactobacillus acidophilus , is able to inhibit viral and bacterial infections by blocking the pathogen's interaction with DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a trans-membrane protein that is a C-type calcium-dependent lectin. DC-SIGN is known to act as an attachment factor for several viruses including alphaviruses and flaviviruses. In the present study, we used alphaviruses as a model system to dissect the mechanism of S-layer inhibition. We first evaluated the protective effect of S-layer using 3T3 cells, either wild type or stably expressing DC-SIGN, and infecting with the alphaviruses Semliki Forest virus (SFV) and CHIKV and the flaviviruses ZIKV and DENV. DC-SIGN expression significantly enhanced infection by all four viruses. Treatment of the cells with S-layer prior to infection decreased infectivity of all viruses only in cells expressing DC-SIGN. In vitro ELISA experiments showed a direct interaction between S-layer and DC-SIGN; however, confocal microscopy and flow cytometry demonstrated that S-layer binding to the cells was independent of DC-SIGN expression. S-layer protein prevented SFV binding and internalization in DC-SIGN-expressing cells but had no effect on virus binding to DC-SIGN-negative cells. Inhibition of virus binding occurred in a time-dependent manner, with a significant reduction of infection requiring at least a 30-min pre-incubation of S-layer with DC-SIGN-expressing cells. These results suggest that S-layer has a different mechanism of action compared to mannan, a common DC-SIGN-binding compound that has an immediate effect in blocking viral infection. This difference could reflect slower kinetics of S-layer binding to the DC-SIGN present at the plasma membrane (PM). Alternatively, the S-layer/DC-SIGN interaction may trigger the activation of signaling pathways that are required for the inhibition of viral infection. Together our results add important information relevant to the potential use of L. acidophilus S-layer protein as an antiviral therapy.

La Crosse virus (LACV) is a mosquito-transmitted arbovirus and the main cause of virus-mediated neurological diseases in children. To date, little is known about the role of C-type lectin receptors (CLRs)-an important class of pattern recognition receptors-in LACV recognition. DC-SIGN remains the only well-described CLR that recognizes LACV. In this study, we investigated the role of additional CLR/LACV interactions. To this end, we applied a flow-through chromatography method for the purification of LACV to perform an unbiased high-throughput screening of LACV with a CLR-hFc fusion protein library. Interestingly, the CARD9-associated CLRs Mincle, Dectin-1, and Dectin-2 were identified to strongly interact with LACV. Since CARD9 is a common adaptor protein for signaling via Mincle, Dectin-1, and Dectin-2, we performed LACV infection of Mincle -/- and CARD9 -/- DCs. Mincle -/- and CARD9 -/- DCs produced less amounts of proinflammatory cytokines, namely IL-6 and TNF-α, albeit no reduction of the LACV titer was observed. Together, novel CLR/LACV interactions were identified; however, the Mincle/CARD9 axis plays a limited role in early antiviral responses against LACV., Competing Interests: The authors declare no conflicts of interest.

The envelope protein (Env) is the only surface protein of the human immunodeficiency virus (HIV) and as such the exclusive target for protective antibody responses. Experimental evidences from mouse models suggest a modulating property of Env to steer antibody class switching towards the less effective antibody subclass IgG1 accompanied with strong TH2 helper responses. By simple physical linkage we were able to imprint this bias, exemplified by a low IgG2a/IgG1 ratio of antigen-specific antibodies, onto an unrelated antigen, namely the HIV capsid protein p24. Here, our results indicate the glycan moiety of Env as the responsible immune modulating activity. Firstly, in Card9 -/- mice lacking specific C-Type lectin responsiveness, DNA immunization significantly increased the IgG2a/IgG1 ratio for the Env-specific antibodies while the antibody response against the F-protein of the respiratory syncytial virus (RSV) serving as control antigen remained unchanged. Secondly, sequential shortening of the Env encoding sequence revealed the C2V3 domain as responsible for the strong IgG1 responses and TH2 cytokine production. Removing all potential N-glycosylation sites from the C2V3 domain by site-specific mutagenesis reversed the vaccine-induced immune response towards a Th1-dominated T-cell response and a balanced IgG2a/IgG1 ratio. Accordingly, the stretch of oligomannose glycans in the C2V3 domain of Env might mediate a specific uptake and/or signaling modus in antigen presenting cells by involving interaction with an as yet unknown C-type lectin receptor. Our results contribute to a deeper understanding of the impact of Env glycosylation on HIV antigen-specific immune responses, which will further support HIV vaccine development.

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a range of diseases, including fatal invasive infections. However, the mechanisms by which the innate immune system recognizes GAS are not well understood. We herein report that the C-type lectin receptor macrophage inducible C-type lectin (Mincle) recognizes GAS and initiates antibacterial immunity. Gene expression analysis of myeloid cells upon GAS stimulation revealed the contribution of the caspase recruitment domain-containing protein 9 (CARD9) pathway to the antibacterial responses. Among receptors signaling through CARD9, Mincle induced the production of inflammatory cytokines, inducible nitric oxide synthase, and reactive oxygen species upon recognition of the anchor of lipoteichoic acid, monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS infection, Mincle-deficient mice exhibited impaired production of proinflammatory cytokines, severe bacteremia, and rapid lethality. GAS also possesses another Mincle ligand, diglucosyldiacylglycerol; however, this glycolipid interfered with MGDG-induced activation. These results indicate that Mincle plays a central role in protective immunity against acute GAS infection., Competing Interests: The authors declare no conflict of interest.

Background: Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application., Methods: We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin., Results: We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system., Conclusions: L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin., General Significance: These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Understanding blood group antigen binding preferences for C-type lectin receptors holds promise for modulating immune responses, since several Gram-negative bacteria express blood group antigens as molecular mimicry to evade immune responses. Herein, we report the synthesis of ABO blood group antigen active tri and disaccharides to investigate the binding specificity with various C-type lectin receptors using glycan microarray. The results of binding preferences show that distinct glycosylation on the galactose and fucose motifs are key for C-type lectin receptor binding and that these interactions occur in a Ca 2+ -dependent fashion.

Carbohydrate-protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long-term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand-mediated interactions, we constructed a combinatorial library of mannose and galactose homo- and hetero-glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c + DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major -infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1 + DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1 + DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1 + DCs in adaptive immunity against L. major , we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania -specific CD4 + T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1 -/- BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies.

C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca 2+ -dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni ( C. jejuni ) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This "Methods" paper describes applications of CLR-Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the here-described applications of CLR-Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.

Correlated binary data arise in a large variety of biomedical research. In order to evaluate methods for their analysis, computer simulations of such data are often required. Existing methods can often not cover the full range of possible correlations between the variables or are not available as implemented software. We propose a genetic algorithm that approaches the desired correlation structure under a given marginal distribution. The procedure generates a large representative matrix from which the probabilities of individual observations can be derived or from which samples can be drawn directly. Our genetic algorithm is evaluated under different specified marginal frequencies and correlation structures, and is compared against two existing approaches. The evaluation checks the speed and precision of the approach as well as its suitability for generating also high-dimensional data. In an example of high-throughput glycan array data, we demonstrate the usability of our approach to simulate the power of global test procedures. An implementation of our own and two other methods were added to the R-package 'RepeatedHighDim'. The presented algorithm is not restricted to certain correlation structures. In contrast to existing methods it is also evaluated for high-dimensional data., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors., (Copyright © 2017 American Society for Microbiology.)

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcRγ-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle -/- ) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle -/- mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle -/- mice. Of note, the P. murina -infected Mincle -/- mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Glycosylation is an integral post-translational modification present in more than half of all eukaryotic proteins. It affects key protein functions, including folding, stability, and immunogenicity. Glycoengineering approaches, such as the use of bacterial N-glycosylation systems, or expression systems, including yeasts, insect cells, and mammalian cells, have enabled access to defined and homogenous glycoproteins. Given that glycan structures on proteins can be recognized by host lectin receptors, they may facilitate cell-specific targeting and immune modulation. Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are attractive targets to shape immune responses. Multivalent glycan display on nanoparticles, liposomes, or dendrimers has successfully enabled CLR targeting. In this review, we discuss novel strategies to access defined glycan structures and highlight CLR targeting approaches for immune modulation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sterile (noninfected) inflammation underlies the pathogenesis of many widespread diseases, such as allergies and autoimmune diseases. The evolutionarily conserved innate immune system is considered to play a key role in tissue injury recognition and the subsequent development of sterile inflammation; however, the underlying molecular mechanisms are not yet completely understood. Here, we show that cholesterol sulfate, a molecule present in relatively high concentrations in the epithelial layer of barrier tissues, is selectively recognized by Mincle (Clec4e), a C-type lectin receptor of the innate immune system that is strongly up-regulated in response to skin damage. Mincle activation by cholesterol sulfate causes the secretion of a range of proinflammatory mediators, and s.c. injection of cholesterol sulfate results in a Mincle-mediated induction of a severe local inflammatory response. In addition, our study reveals a role of Mincle as a driving component in the pathogenesis of allergic skin inflammation. In a well-established model of allergic contact dermatitis, the absence of Mincle leads to a significant suppression of the magnitude of the skin inflammatory response as assessed by changes in ear thickness, myeloid cell infiltration, and cytokine and chemokine secretion. Taken together, our results provide a deeper understanding of the fundamental mechanisms underlying sterile inflammation.

Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into host cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.

Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here, we show that keratan sulfate (KS), one of the major glycosaminoglycans produced in the small airway, decreased in lungs of cigarette smoke-exposed mice. To confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO 3 - -6]Galβ1-4[SO 3 - -6]GlcNAc) was administered to two murine models: elastase-induced-emphysema and LPS-induced exacerbation of a cigarette smoke-induced emphysema. Histological and microcomputed tomography analyses revealed that, in the mouse elastase-induced emphysema model, administration of L4 attenuated alveolar destruction. Treatment with L4 significantly reduced neutrophil influx, as well as the levels of inflammatory cytokines, tissue-degrading enzymes (matrix metalloproteinases), and myeloperoxidase in bronchoalveolar lavage fluid, suggesting that L4 suppressed inflammation in the lung. L4 consistently blocked the chemotactic migration of neutrophils in vitro. Moreover, in the case of the exacerbation model, L4 inhibited inflammatory cell accumulation to the same extent as that of dexamethasone. Taken together, L4 represents one of the potential glycan-based drugs for the treatment of COPD through its inhibitory action against inflammation., (Copyright © 2017 the American Physiological Society.)

C-type lectins (CTLs) represent the most complex family of animal/human lectins that comprises 17 different groups. During evolution, CTLs have developed by diversification to cover a broad range of glycan ligands. However, ligand binding by CTLs is not necessarily restricted to glycans as some CTLs also bind to proteins, lipids, inorganic molecules, or ice crystals. CTLs share a common fold that harbors a Ca 2+ for contact to the sugar and about 18 invariant residues in a phylogenetically conserved pattern. In vertebrates, CTLs have numerous functions, including serum glycoprotein homeostasis, pathogen sensing, and the initiation of immune responses. Myeloid CTLs in innate immunity are mainly expressed by antigen-presenting cells and play a prominent role in the recognition of a variety of pathogens such as fungi, bacteria, viruses, and parasites. However, myeloid CTLs such as the macrophage inducible CTL (Mincle) or Clec-9a may also bind to self-antigens and thus contribute to immune homeostasis. While some CTLs induce pro-inflammatory responses and thereby lead to activation of adaptive immune responses, other CTLs act as inhibitory receptors and dampen cellular functions. Since CTLs are key players in pathogen recognition and innate immunity, targeting CTLs may be a promising strategy for cell-specific delivery of drugs or vaccine antigens and to modulate immune responses.

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs., Competing Interests: The authors declare no conflict of interest.

Multivalent glycodendrimers make promising tools to tackle the basic and translational research in the field of carbohydrate-mediated interactions. Despite advances in glycodendrimers and glycopolymers, the multivalent probes available to date are still far from being ideal biological mimics. This work demonstrates the inherent chirality of glycodendrimers to be one of the promising factors to generate different spatial carbohydrate micro-environments to modulate specific carbohydrate-protein interactions. By exploiting the host-guest strategy, chiral Ru(ii) complexes (Δ and Λ) and mannose capped β-cyclodextrin (β-CD), we generated a library of homologous metallo-glycodendrimers (MGDs) with sizes of 50-70 nm. These nanoclusters can enantioselectively bind to specific C-type lectins and displayed selectivity in cellular uptake. We also discovered their potential clathrin-mediated endocytotic pathway in DC-SIGN and SIGNR3-transfected cell lines. Finally, in vivo biodistribution and sequestration of MGDs was determined to understand the role of chirality mediated spatial arrangement in carbohydrate-mediated interactions.

Targeting antigens to dendritic cell subsets is a promising strategy to enhance the efficacy of vaccines. C-type lectin receptors (CLRs) expressed by dendritic cells are particularly attractive candidates since CLR engagement may promote cell uptake and may further stimulate antigen presentation and subsequent T cell activation. While most previous approaches have involved antibody-mediated CLR-targeting, glycan-based CLR targeting has become more and more attractive in recent years. In the present study, we show that small structural glycan modifications may markedly influence CLR recognition, dendritic cell targeting, and subsequent T cell activation. A biantennary N-glycan (G0) and its analogous O-2 core xylosylated N-glycan (XG0) were synthesized, covalently conjugated to the model antigen ovalbumin, and analyzed for binding to a set of murine CLR-Fc fusion proteins using lectin microarray. To evaluate whether the differential binding of G0 and XG0 to CLRs impacted dendritic cell targeting, uptake studies using murine dendritic cells were performed. Finally, effects of the ovalbumin glycoconjugates on T cell activation were measured in a dendritic cell/T cell cocultivation assay. Our results highlight the utility of glycan-based dendritic cell targeting and demonstrate that small structural differences may have a major impact on dendritic cell targeting efficacy.

The T-cell adjuvanticity of mycobacterial cord factor trehalose 6,6'-dimycolate (TDM) is well established. The identification of the C-type lectin Mincle on innate immune cells as the receptor for TDM and its synthetic analogue trehalose 6,6'-dibehenate (TDB) has raised interest in development of synthetic Mincle ligands as novel adjuvants. Trehalose mono- (TMXs) and diesters (TDXs) with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), and myristate (M)] were tested. Upon stimulation of murine macrophages, G-CSF secretion and NO production were strongly augmented by all TDXs tested, in a wide concentration range. In contrast, the TMXs triggered macrophage activation only at high concentrations. Macrophage activation by all TDXs required Mincle, but was independent of MyD88. The superior capacity of TDXs for activating macrophages was paralleled by direct binding of TDXs, but not of TMXs, to a Mincle-Fc fusion protein. Insertion of a short polyethylene glycol between the sugar and acyl chain in TDS reduced Mincle-binding and macrophage activation. Immunization of mice with cationic liposomes containing the analogues demonstrated the superior adjuvant activity of trehalose diesters. Overall, immune activation in vitro and in vivo by trehalose esters of simple fatty acids requires two acyl chains of length and involves Mincle., (© The Author(s) 2016.)