Neutrophil granulocytes possess a panel of different mechanisms to combat invading pathogens. As first line of defense neutrophils can combat pathogens by phagocytosing them, by releasing antimicrobial peptides or reactive oxygen species (ROS) and by the release of neutrophil extracellular traps (NETs), a process first described in 2004 by Brinkmann et al. (1, 2). NET release can be induced by bacteria, viruses, fungi or non-physiological stimuli, such as ionophores and phorbol-myristate acetate (PMA) (3, 4). It seems to be that different pathways can be used to induce the formation of NET and even more than 13 years after the first description of NETs several mechanisms are controversially discussed and numerous open questions still need to be answered [excellently summarized in Boeltz et al. (5)]. What we know is that NET consists of a meshwork of decondensed DNA fibers, cytotoxic histones, and antimicrobial peptides and has the ability to catch and render invading pathogens harmless (3, 5, 6). Since NET contains several biomolecules, which are also cytotoxic for endogenous cells, in addition to the desired antimicrobial effects, NETs are associated with numerous pathologies (7–16). Therefore, an exaggerated release of NET has to be prevented, leading to the necessity of control mechanisms to regulate the formation of NETs.

Within the last few years, interest in glycans and how they modulate different functions has increased immensely (17–19). Since all of our cells are surrounded by a glycocalyx consisting of highly glycosylated proteins and lipids, it is obvious that glycan-dependent processes occur frequently (19, 20). Indeed, glycans have essential roles within various biological processes, such as cell proliferation, cell differentiation, the development of organs, and within the immune system (19, 21). In the field of immunology, glycans drive diverse mechanisms, ranging from the discrimination of the self and non-self, using for instance sialic acid-binding immunoglobulin-like lectins (Siglecs), to glycosaminoglycan (GAG)-mediated chemokine presentation (22).

GAGs are a number of long, unbranched polysaccharides composed of repeating disaccharide units, whereby, the repeating disaccharides consist of uronic acid or galactose and an amino sugar, which can be additionally modified. Prominent members of the GAG family are: hyaluronic acid, heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, and keratin sulfate (23). Besides their role in chemokine presentation, GAGs are also involved in several other biological processes, like cell signaling, angiogenesis (24), metastasis, tumor progression (25, 26) and coagulation (27, 28).

In addition to GAGs, sialylated glycans play an important role during immunological events. In mammals, glycans are frequently terminated by these sugar residues (29). Sialic acids are negatively charged and modulate both immunological processes and organ development (30–33). Sialylated structures can be recognized by Siglecs (34–36), which are transmembrane receptors expressed in different cells of vertebrates that can mainly either inhibit or activate the immune response (35, 37–39). Although Siglecs are meant to be a valuable tool to distinguish between self-associated molecular patterns (SAMPs) and pathogen-associated molecular patterns (PAMPs), several pathogens are already known to elude the immune system by mimicking host sialylation and, therefore, masking themselves as SAMPs.

This review is therefore focusing on the role of glycoconjugates of endogenous cells in controlling NET releases and impairing the negative outcome of NETs as well as on the role of glycoconjugates of pathogens that influence NET formation or that directly influence NET release and the biological activity of NETs.

An impaired NET release and missing NET clearance are, for instance, associated with the formation of a thrombus (7, 40, 41). Not only do the NET fibers serve as a scaffold for the formation of a thrombus. Furthermore, neutrophil elastase is released in the extracellular area and inhibits among others the anticoagulants antithrombin and tissue factor pathway inhibitor. In addition, extracellular histones are known to increase the thrombin generation, causing platelet activation and coagulation (40–43). Therefore, it is not surprising that an impaired NET release within circulation needs to be prevented.

Lizcano et al. investigated the reason why isolated neutrophils are more susceptible to undergoing NETosis than neutrophils within circulation (44). They determined that glycophorin A, a sialoglycoprotein that is located on the surface of erythrocytes, is a candidate that might be responsible for this effect. It is able to bind Siglec-9 on the surface of neutrophils, inhibiting neutrophil activation within circulation (Figure 1A). Remarkably, modification of the sialic acid on the surface of the erythrocytes prevents the outlined inhibitory effects (44). Interestingly, also cancer cells, which are characterized by hypersialylation, seem to use Siglec-5 and Siglec-9 to evade entrapment by activated neutrophils [excellently reviewed by Adams et al. (45) and Rodrigues and Macauley (46)].

Besides sialylated glycoconjugates, heparin, a well-known anticoagulant, inhibits neutrophil degranulation and aggregation in vitro (47). Furthermore, the influence of low molecular weight heparin, unfractionated heparin, O-desulfated heparin, hyaluronic acid, dextran sulfate, and poly-L-glutamic acid on neutrophil activation was investigated (48). The activation of neutrophils with different stimuli induced the release of neutrophil elastase. However, the application of the different stimuli in combination with low molecular weight heparin as well as dextran sulfate inhibited neutrophil activation and, therefore, the release of neutrophil elastase (Figure 1A) (48, 49). In contrast, the non-sulfated dextran and poly-L-glutamic acid showed no effect on neutrophil activation, leading to the assumption of a sulfate-dependent process. Furthermore, Xu et al. investigated the role of heparan sulfate in the biology of NETs (50). In heparan sulfate uronyl 2-O-sulgotransferase deficient mice less NET is formed after stimulation with group B streptococcus (GBS). Remarkably, when NET was treated with heparan lyase, its antimicrobial activity decreased (50). Thus, the formation and the activity of NETs seem to be modulated by heparan sulfate.

Besides heparan sulfate, other glycans are known to influence the biological activity of NETs. In this context, Brown et al. focused on neutrophil elastase and neutrophil-induced human bronchial epithelia cell detachment (48). Whilst hyaluronic acid had no effect, low-molecular-weight heparin, unfractionated heparin, O-desulfated heparin, and dextran sulfate significantly inhibited the neutrophil elastase-induced detachment (48). Furthermore, Fuchs et al. published that the treatment of NETs with heparin destroys their scaffold and prevents the formation of a thrombus (Figure 1B). Heparin has a high-charge-dependent affinity to histones (51, 52) and is able to release histones from chromatin fibers, therefore, destabilizing NETs (Figure 1B) (40). Since histones that are released during NETs are able to damage negatively charged cell membranes, histones are often described as antimicrobial peptides (AMPs) that are released during NETs alongside with other antimicrobial biomolecules like lactoferrin and neutrophil elastase (1, 6, 53–56). Unfortunately, these properties of all histones (H1, H2A, H2B, H3, and H4) are toxic not only for pathogens but also for endogenous cells (57–60). Within the plasma, the cytotoxicity of histones is reduced by the inter-alpha-inhibitor-protein (IAIP) associated with high molecular weight hyaluronic acid and chondroitin sulfate. IAIP as well as high molecular weight hyaluronic acid and chondroitin sulfate bind recombinant histone H4, contributing to reduced histone mediated cytotoxicity (Figure 1B) (61).

In addition to GAGs, such as hepain (52), another linear carbohydrate, polysialic acid (polySia), is a naturally occurring inhibitor of the cytotoxic effects of histones (59, 62, 63). Remarkably, polySia was detected in the plasma of different species, from fish to humankind, and may represent a natural buffer system for the inactivation of the cytotoxicity of extracellular histones in blood (Figure 1B) (64). PolySia influences histone-mediated cytotoxicity in a concentration as well as in a chain-length-dependent manner (65, 66). In line with that, polysialylated nanoparticles and in vitro polysialylated cervical mucins represent tools to counteract histone-mediated cytotoxicity during an exaggerated NET formation (65, 66). Interestingly, quite recently, Kühnle et al. (67, 68) published that polySia interacts with lactoferrin. Lactoferrin is known to inhibit a NET release by forming a “lactoferrin-shell” around the activated neutrophils (69). In vitro experiments suggested that the efficiency of lactoferrin in preventing the release of NETs was enhanced in the presence of polySia (67).

Thus, the presented examples show endogenous glycan-dependent ways to control the release of NETs in addition to decreasing their damaging effects, indicating, once more, the widespread function of glycosylation within the field of immunology. However, it has to be considered that a medal always has two sides. Some pathogens exploit the previously described mechanisms for their own purposes, as described in the next chapter.

This review gives a short summary concerning the impact of glycans to modulate the formation and activity of NET describing glycan dependent mechanisms of endogenous cells to prevent the activation of neutrophils or to inactivate cytotoxic molecules of NET, which are, however, also used as an escape strategy by distinct pathogens. All of the outlined examples show that glycans play a key role in the biology of NET and that they have great potential as a therapeutic tool in NETs-associated pathologies.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.