Siglecs at the Host-Pathogen Interface.

Siglecs are sialic acid (Sia) recognizing immunoglobulin-like receptors expressed on the surface of all the major leukocyte lineages in mammals. Siglecs recognize ubiquitous Sia epitopes on various glycoconjugates in the cell glycocalyx and transduce signals to regulate immunological and inflammatory activities of these cells. The subset known as CD33-related Siglecs is principally inhibitory receptors that suppress leukocyte activation, and recent research has shown that a number of bacterial pathogens use Sia mimicry to engage these Siglecs as an immune evasion strategy. Conversely, Siglec-1 is a macrophage phagocytic receptor that engages GBS and other sialylated bacteria to promote effective phagocytosis and antigen presentation for the adaptive immune response, whereas certain viruses and parasites use Siglec-1 to gain entry to immune cells as a proximal step in the infectious process. Siglecs are positioned in crosstalk with other host innate immune sensing pathways to modulate the immune response to infection in complex ways. This chapter summarizes the current understanding of Siglecs at the host-pathogen interface, a field of study expanding in breadth and medical importance, and which provides potential targets for immune-based anti-infective strategies.

Precision glycocalyx editing as a strategy for cancer immunotherapy.

Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody-sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody-enzyme conjugates is therefore a promising avenue for cancer immune therapy.

The Role of Endothelial Dysfunction and Inflammation in Chronic Venous Disease.

Chronic venous disease is a potentially prevalent and debilitating condition affecting millions of individuals, mostly in Western world. Predisposing genetic and environmental factors contribute to its development. However, the main etiology remains to be elucidated. An extensive literature search was conducted in Medline using the following key words algorithm: ("Chronic venous disease" OR "Chronic venous insufficiency" OR "varicose veins") AND ("endothelial dysfunction" OR "inflammation"). Besides being a multifactorial disease, it is now recognized that the hallmark of chronic venous disease pathophysiology likely remains in inflammation, possibly triggered by sustained venous hypertension and valvular incompetence. Shear stress changes are directly sensed by endothelial cells, leading to its activation and subsequent recruitment of leukocytes and release of proinflammatory agents. Dysfunctional endothelium has a pivotal role perpetuating the inflammatory cascade, with consequent pathological venous changes and chronic venous disease worsening. Endothelial dysfunction may be the central player in the link between varicose veins and deep vein thrombosis. In this article, we aim to analyze the crucial role of endothelial activation in the persistent inflammatory cycle that characterizes chronic venous disease.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Lymph formation, composition and circulation: a proteomics perspective.

During the last 20 years a deeper understanding of the lymphatic circulatory system, lymph formation and composition has emerged. This review will examine the current knowledge on the organization of the lymphatic vascular tree, the formation of lymph from the extracellular fluid, lymph circulation and the lymph proteomic composition during physiological and pathological conditions. Formation of the lymph fluid is dependent on pressure gradients in the capillary beds and the composition of the endothelial cell glycocalyx, which acts as a molecular sieve. Fluid propulsion toward the draining node is dependent on the intrinsic pumping mechanism of the lymphangions and their unidirectional valves. The lymph 'omics' composition is dependent on the ultrafiltration of plasma proteins as well as proteins and molecules derived from the metabolic and catabolic activities of each parenchymal organ from which the lymph drains. Altogether, these new insights have brought about a new awareness of the importance of the lymphatic system in human physiology and pathology.

Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion.

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

Surface expression patterns of defined glycan antigens change during Schistosoma mansoni cercarial transformation and development of schistosomula.

During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcß1-4(Fucα1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcß1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galß1-4(Fucα1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets.

Glycocalyx-Mimicking Nanoparticles for Stimulation and Polarization of Macrophages via Specific Interactions.

Malignant tumors develop multiple mechanisms to impair and escape from antitumor immune responses, of which tumor-associated macrophages that often show immunosuppressive phenotype (M2), play a critical role in tumor-induced immunosuppression. Therefore, strategies that can reverse M2 phenotype and even enhance immune-stimulation function of macrophage would benefit tumor immunotherapy. In this paper, self-assembled glyco-nanoparticles (glyco-NPs), as artificial glycocalyx, have been found to be able to successfully induce the polarization of mouse primary peritoneal macrophages from M2 to inflammatory type (M1). The polarization change was evidenced by the decreased expression of cell surface signaling molecules CD206 and CD23, and the increased expression of CD86. Meanwhile, secretion of cytokines supported this polarization change as well. More importantly, this phenomenon is observed not only in vitro, but also in vivo. As far as we known, this is the first report about macrophage polarization being induced by synthetic nanomaterials. Moreover, preparation, characterization of these glyco-NPs and their interaction with the macrophages are also demonstrated.

Fasciola hepatica tegumental coat impairs mast cells' ability to drive Th1 immune responses.

The parasitic worm Fasciola hepatica induces strong Th2 and T-regulatory immune responses while simultaneously suppressing Th1-driven immune responses to bystander microbial infections. It also prevents the initiation of Th1-mediated autoimmune disorders in mice through the suppression of Th17 and Th1 immune responses, and this can be mimicked by parasite-derived molecules. We have isolated F. hepatica tegumental coat Ag (FhTeg) and demonstrated its suppressive effect in vivo by directly targeting dendritic cells, impairing their ability to drive Th1 responses. Mast cells are critical in promoting Th1 protective immunity during bacterial infection and in driving Th1-mediated pathological conditions in autoimmune diseases. In this article, we show that FhTeg inhibits the ability of mast cells to drive the Th1 immune response by suppressing cytokine secretion (TNF-α, IL-6, IFN-γ, and IL-10) and ICAM1 expression in mast cells stimulated with LPS or heat-inactivated Bordetella pertussis Ag. These heat-inactivated B. pertussis Ag/LPS-stimulated mast cells fail to promote Th1 immune responses in CD4(+) T cells when pretreated with FhTeg, and a role for ICAM1 in this process was demonstrated. FhTeg suppresses the activation of transcription factors in the TLR signaling pathway, which explains the decrease in cytokine production and cell surface marker expression. We demonstrated that FhTeg suppresses MAPK and NF-κB activation and enhances SOCS3 expression, which could explain its negative effect on the TLR pathways. We conclude that FhTeg targets innate immune cells, inhibiting their ability to drive Th1 immune responses.

Modulation of heparan sulfate in the glomerular endothelial glycocalyx decreases leukocyte influx during experimental glomerulonephritis.

The glomerular endothelial glycocalyx is postulated to be an important modulator of permeability and inflammation. The glycocalyx consists of complex polysaccharides, the main functional constituent of which, heparan sulfate (HS), is synthesized and modified by multiple enzymes. The N-deacetylase-N-sulfotransferase (Ndst) enzymes initiate and dictate the modification process. Here we evaluated the effects of modulation of HS in the endothelial glycocalyx on albuminuria and glomerular leukocyte influx using mice deficient in endothelial and leukocyte Ndst1 (TEKCre+/Ndst1flox/flox). In these mice, glomerular expression of a specific HS domain was significantly decreased, whereas the expression of other HS domains was normal. In the endothelial glycocalyx, this specific HS structure was not associated with albuminuria or with changes in renal function. However, glomerular leukocyte influx was significantly reduced during antiglomerular basement membrane nephritis, which was associated with less glomerular injury and better renal function. In vitro decreased adhesion of wild-type and Ndst1-deficient granulocytes to Ndst1-silenced glomerular endothelial cells was found, accompanied by a decreased binding of chemokines and L-selectin. Thus, modulation of HS in the glomerular endothelial glycocalyx significantly reduced the inflammatory response in antiglomerular basement membrane nephritis.

Tumor glucose metabolism and the T cell glycocalyx: implication for T cell function.

The T cell is an immune cell subset highly effective in eliminating cancer cells. Cancer immunotherapy empowers T cells and occupies a solid position in cancer treatment. The response rate, however, remains relatively low (<30%). The efficacy of immunotherapy is highly dependent on T cell infiltration into the tumor microenvironment (TME) and the ability of these infiltrated T cells to sustain their function within the TME. A better understanding of the inhibitory impact of the TME on T cells is crucial to improve cancer immunotherapy. Tumor cells are well described for their switch into aerobic glycolysis (Warburg effect), resulting in high glucose consumption and a metabolically distinct TME. Conversely, glycosylation, a predominant posttranslational modification of proteins, also relies on glucose molecules. Proper glycosylation of T cell receptors influences the immunological synapse between T cells and tumor cells, thereby affecting T cell effector functions including their cytolytic and cytostatic activities. This review delves into the complex interplay between tumor glucose metabolism and the glycocalyx of T cells, shedding light on how the TME can induce alterations in the T cell glycocalyx, which can subsequently influence the T cell's ability to target and eliminate tumor cells.

Galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (DAMPs) and as receptors for pathogen-associated molecular patterns (PAMPs).

The glycocalyx is a glycan layer found on the surfaces of host cells as well as microorganisms and enveloped virus. Its thickness may easily exceed 50 nm. The glycocalyx does not only serve as a physical protective barrier but also contains various structurally different glycans, which provide cell- or microorganism-specific 'glycoinformation'. This information is decoded by host glycan-binding proteins, lectins. The roles of lectins in innate immunity are well established, as exemplified by collectins, dectin-1, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). These mammalian lectins are synthesized in the secretory pathway and presented on the cell surface to bind to specific glycan 'epitopes'. As they recognize non-self glycans presented by microorganisms, they can be considered as receptors for pathogen-associated molecular patterns (PAMPs), i.e. pattern recognition receptors (PRRs). One notable exception is the galectin family. Galectins are synthesized and stored in the cytoplasm, but upon infection-initiated tissue damage and/or following prolonged infection, cytosolic galectins are either passively released by dying cells or actively secreted by inflammatory activated cells through a non-classical pathway, the 'leaderless' secretory pathway. Once exported, galectins act as PRR, as well as immunomodulators (or cytokine-like modulators) in the innate response to some infectious diseases. As galectins are dominantly found in the lesions where pathogen-initiated tissue damage signals appear, this lectin family is also considered as potential damage-associated molecular pattern (DAMP) candidates that orchestrate innate immune responses alongside the PAMP system.

Dynamic factors controlling carrier anchoring on vascular cells.

This article reviews experimental and modeling methods for determining the critical roles played by the various factors that control nanocarrier drug delivery to vascular endothelial cells.

Changes in the mucosal barrier during acute and chronic Trichuris muris infection.

The intestinal mucosal barrier, part of the innate immune defence, is responsive to the external environment and changes in response to infection. There is disparate evidence for the epithelial and goblet cell products within the intrinsic barrier being part of a response to resolve infection. We comprehensively analysed the changes of mucosal glycoconjugates during acute and chronic infection by utilising the Trichuris muris (T. muris) model. Transcription factors, atonal homolog 1 (Math-1) and SAM pointed domain containing ETS transcription factor (Spdef) were activated during acute infection, which promoted stem cell fate towards a secretory cell phenotype. The thickness of the intermediate barrier, the carbohydrate-rich glycocalyx, composed of cell surface mucins increased with exposure to T. muris, with an increase in Muc4, Muc13 and Muc17. Overall, hypersecretion of glycoproteins into the extrinsic barrier (mediated by IL-13) via the gamma amino-butyric acid-α3 receptor (GABA-α3), was observed during acute infection. Furthermore, altered glycosylation was observed during acute and chronic infection; mucins were more highly charged during acute infection than during chronic infection. This study readdresses the changes within the mucosal barrier, in particular in the cell surface and secreted mucins during acute and chronic nematode infection.

The Epithelial Cell Glycocalyx in Ocular Surface Infection.

The glycocalyx is the main component of the transcellular barrier located at the interface between the ocular surface epithelia and the external environment. This barrier extends up to 500 nm from the plasma membrane and projects into the tear fluid bathing the surface of the eye. Under homeostatic conditions, defense molecules in the glycocalyx, such as transmembrane mucins, resist infection. However, many pathogenic microorganisms have evolved to exploit components of the glycocalyx in order to gain access to epithelial cells and consequently exert deleterious effects. This manuscript reviews the implications of the ocular surface epithelial glycocalyx to bacterial, viral, fungal and parasitic infection. Moreover, it presents some ongoing controversies surrounding the functional relevance of the epithelial glycocalyx to ocular infectious disease.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Interleukin 22 mitigates endothelial glycocalyx shedding after lipopolysaccharide injury.

BACKGROUND: The endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 µg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence. RESULTS: Exposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02). CONCLUSIONS: Interleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.

An Acquired and Endogenous Glycocalyx Forms a Bidirectional "Don't Eat" and "Don't Eat Me" Barrier to Phagocytosis.

Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis ("eat me" signals), while others avoid phagocytosis by displaying inhibitory ligands ("don't eat me" signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that the removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated. VIDEO ABSTRACT.

High molecular weight sodium hyaluronate improves survival of syndecan-1-deficient septic mice by inhibiting neutrophil migration.

METHODS: Sepsis was induced by cotton smoke inhalation followed by intranasal administration of Pseudomonas aeruginosa in female (> 6 months) Balb/c and syndecan-1 knockout mice. Survival of mice, lung capillary endothelial glycocalyx integrity, lung water content, and vascular hyper-permeability were determined with or without HMW-SH treatment in these mice. Effects of HMW-SH on endothelial permeability and neutrophil migration were tested in in vitro setting. RESULTS: In septic wildtype mice, we found a severely damaged pulmonary microvascular endothelial glycocalyx and elevated levels of shed syndecan-1 in the circulation. These changes were associated with significantly increased pulmonary vascular permeability. In septic syndecan-1 knockout mice, extravascular lung water content was higher, and early death was observed. The administration of HMW-SH significantly reduced mortality and lung water content in septic syndecan-1 knockout mice, but not in septic wildtype mice. In in vitro setting, HMW-SH inhibited neutrophil migration and reduced cultured endothelial cell permeability increases. However, these effects were reversed by the addition of recombinant syndecan-1 ectodomain. CONCLUSIONS: HMW-SH reduced lung tissue damage and mortality in the absence of syndecan-1 protein, possibly by reducing vascular hyper-permeability and neutrophil migration. Our results further suggest that increased shed syndecan-1 protein levels are linked with the inefficiency of HMW-SH in septic wildtype mice.