Multicellular organisms require cell-to-cell communication to maintain homeostasis and thrive. For cells to communicate, a network of filamentous, actin-rich tunneling nanotubes (TNTs) plays a pivotal role in facilitating efficient cell-to-cell communication by connecting the cytoplasm of adjacent or distant cells. Substantial documentation indicates that diverse cell types employ TNTs in a sophisticated and intricately organized fashion for both long and short-distance communication. Paradoxically, several pathogens, including viruses, exploit the structural integrity of TNTs to facilitate viral entry and rapid cell-to-cell spread. These pathogens utilize a \"surfing\" mechanism or intracellular transport along TNTs to bypass high-traffic cellular regions and evade immune surveillance and neutralization. Although TNTs are present across various cell types in healthy tissue, their magnitude is increased in the presence of viruses. This heightened induction significantly amplifies the role of TNTs in exacerbating disease manifestations, severity, and subsequent complications. Despite significant advancements in TNT research within the realm of infectious diseases, further studies are imperative to gain a precise understanding of TNTs\' roles in diverse pathological conditions. Such investigations are essential for the development of novel therapeutic strategies aimed at leveraging TNT-associated mechanisms for clinical applications. In this chapter, we emphasize the significance of TNTs in the life cycle of viruses, showcasing the potential for a targeted approach to impede virus-host cell interactions during the initial stages of viral infections. This approach holds promise for intervention and prevention strategies.

The Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer\'s disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2\'s binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including Surface Plasmon Resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2\'s robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer\'s disease.

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/- bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/- chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology.

The endothelial glycocalyx is damaged in postcardiac arrest syndrome (PCAS), but the prognostic value is unknown. We aimed to observe the expression and prognostic value of glycocalyx shedding products, including syndecan-1 (SDC-1), hyaluronan (HA), and heparan sulfate (HS) in PCAS. Data on clinical and 28-day outcomes of seventy-one consecutive patients with out-of-hospital cardiac arrest (OHCA) after the return of spontaneous circulation (ROSC) were collected. SDC-1, HA, and HS were measured on days 0, 1, and 3 after ROSC. Thirty healthy individuals were controls. Glycocalyx shedding was observed in human umbilical vein endothelial cells (HUVECs) stimulated during hypoxia and reoxygenation in vitro. Within 4 h of ROSC, SDC-1 and HA levels, significantly increased. In the 28-day non-survivors, HA levels showed a gradual upward trend, SDC-1 remained at a high level, and HS levels first increased, then decreased. Kaplan-Meier curves and binary logistic regression analysis showed the prognostic value of SDC-1 levels on days 0, 1, and 3, HA levels on days 1 and 3, and HS levels on day 1. Only HS levels on day 1 showed a prognostic value for 28-day neurological outcomes. SDC-1 and HA levels were positively correlated with the no-flow time. In vitro, HUVECs showed shedding of SDC-1 and HS during a prolonged duration of hypoxia. After ROSC, SDC-1, HA, and HS levels may predict the 28-day survival after PCAS, and HS levels are associated with functional outcomes.

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BACKGROUND: Alzheimer\'s disease (AD) is characterized by accumulation of amyloid beta (Aβ) and hyperphosphorylated tau (Tau-P) in the brain. Aβ enhances the activity of kinases involved in the formation of Tau-P. Phosphorylation at Thr 181 determines the propagation of multiple tau phosphorylations. Aβ is derived from the amyloid precursor protein (APP). Cleavage of APP by β-secretase also initiates release of heparan sulfate (HS) from the proteoglycan glypican-1 (GPC1).
OBJECTIVE: In this study, we have explored possible connections between GPC1 expression, HS release, APP processing and Tau-P formation in human neural stem cells.
METHODS: GPC1 formation was suppressed by using CRISPR/Cas9 and increased by using a vector encoding GPC1. HS release from GPC1 was increased by growing cells in medium containing Arg and ascorbate. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies/antisera recognizing Aβ, GPC1, HS released from GPC1, total Tau, and Tau phosphorylated at Thr-181, 217 or 231. The latter have been used as blood biomarkers for AD.
RESULTS: Suppression of GPC1 expression resulted in increased phosphorylation at Thr 181 and Thr 217. When GPC1 was overexpressed, phosphorylation at Thr 217 decreased. Stimulation of HS release from GPC1 diminished tau phosphorylation at all of the three Thr positions, while expression of GPC1 was unaffected. Simultaneous stimulation of HS release and APP processing by the cytokine TNF-α also suppressed tau phosphorylation.
CONCLUSIONS: The increased release of GPC1-derived HS may interfere with Aβ formation and/or Aβ interaction with tau.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

A large number of bacterial pathogens bind to host extracellular matrix (ECM) components. For example, many Gram-negative and Gram-positive pathogens express binding proteins for fibronectin (FN) on their cell surface. Mutagenesis studies of bacterial FN-binding proteins have demonstrated their importance in pathogenesis in preclinical animal models. However, means to draw on these findings to design therapeutic approaches that specifically target FN-bacteria interactions have not been successful because bacterial pathogens can elaborate several FN-binding proteins and also because FN is an essential protein and likely a nondruggable target. Here we report that select heparan compounds potently inhibit Streptococcus pneumoniae infection of injured corneas in mice. Using intact heparan sulfate (HS) and heparin (HP), heparinase-digested fragments of HS, HP oligosaccharides, and chemically or chemoenzymatically modified heparan compounds, we found that inhibition of S. pneumoniae corneal infection by heparan compounds is not mediated by simple charge effects but by a selective sulfate group. Removal of 2-O-sulfates significantly inhibited the ability of HP to inhibit S. pneumoniae corneal infection, whereas the addition of 2-O-sulfates to heparosan (H) significantly increased H\'s ability to inhibit bacterial corneal infection. Proximity ligation assays indicated that S. pneumoniae attaches directly to FN fibrils in the corneal epithelial ECM and that HS and HP specifically inhibit this binding interaction in a 2-O-sulfate-dependent manner. These data suggest that heparan compounds containing 2-O-sulfate groups protect against S. pneumoniae corneal infection by inhibiting bacterial attachment to FN fibrils in the subepithelial ECM of injured corneas. Moreover, 2-O-sulfated heparan compounds significantly inhibited corneal infection in immunocompromised hosts, by a clinical keratitis isolate of S. pneumoniae, and also when topically administered in a therapeutic manner. These findings suggest that the administration of nonanticoagulant 2-O-sulfated heparan compounds may represent a plausible approach to the treatment of S. pneumoniae keratitis.

UNASSIGNED: Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa.
UNASSIGNED: We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays.
UNASSIGNED: Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.