Neutrophil extracellular traps (NETs), first described in 2004, are released by neutrophils via an active process coined NETosis (1, 2). While first characterized for their role in combatting infectious organisms (1), these tangles of chromatin and antimicrobial proteins are now known to play a role in pathogenic autoimmunity and other sterile inflammatory states (3, 4). NETs may place organ systems at risk, including the vasculature (5–7), central nervous system (8), lungs (5), and kidneys (9, 10). Organ failure and thrombotic vessel occlusions are even possible (11–13). Neutrophils, as one of the first responders to inflammatory insults have long been known to interact with other cell types (especially platelets and endothelial cells) with implications for neutrophil recruitment, generation of reactive oxygen species (ROS), and phagocytosis. This cell-to-cell crosstalk may be mediated by either direct cell contact or soluble mediators. In this review, we will focus on the implications of crosstalk for NETosis. Relevant studies have characterized not only in vitro systems (typically with human cells) but also more complex murine models of disease. There is significant heterogeneity between studies, especially in terms of how NETosis is scored and the neutrophil pathways that are considered (which is probably not surprising as a canonical model of NETosis is still not established). Our goal is to highlight the similarities between studies and to point out the discrepancies that necessitate further research. Also, whenever possible, we will try to focus on the implications of these interactions for controlling infection and for regulating inflammation and end-organ damage.

Platelets are megakaryocyte-derived cell bodies that lack nuclei. They circulate in the bloodstream as well-established regulators of the hemostatic system (14). Platelets may be activated by the exposure of subendothelial matrix proteins, such as von Willebrand factor (vWF) and collagen, as might happen with mechanical vessel injury (15). Platelets recognize vWF via a glycoprotein receptor complex, glycoprotein Ib (GPIb)/IX/V (16), with the GPIb subunit playing a particularly key role (17). In parallel, collagen engages a different glycoprotein receptor, GPVI (18). Soluble plasma factors also activate platelets, including fibrinogen (via GPIIb/IIIa) (19) and thrombin (through protease-activated receptors or PARs) (20). When considering research studies, it is important to note that some studies may activate platelets with synthesized activators. An example is thrombin receptor activator peptide (TRAP), which acts as an agonist for all PARs (21), and the more specific TRAP-6, which binds specifically to PAR-1 (22).

These various activating signals lead to platelet aggregation and the release of copious amounts of preformed mediators from platelet granules, such as adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂) – with the potential for potent local effects and feedforward into further platelet activation (14, 17). Platelet factor 4 (PF4, also known as C–X–C motif ligand 4) is another mediator released by platelets. In addition to functioning as a chemokine for cells, such as neutrophils, PF4 binds and neutralizes negatively charged cell surface glycosaminoglycans, such as heparan sulfate, dermatan sulfate, and chondroitin sulfate, thereby mediating several downstream effects, including platelet aggregation (23). Another soluble mediator that will be discussed in this article is high-mobility group box 1 (HMGB1), a protein “alarmin”/cytokine released by activated platelets (24). Finally, proteins such as P-selectin may be either released locally, or expressed on the platelet surface, thereby regulating the local environment (25, 26). For example, P-selectin has been implicated in platelet aggregation under pulsatile shear stress conditions (27).

While platelets clearly play a key role in stemming blood loss in the event of vessel injury, they also have well-established immunomodulatory properties, potentially acting as sentinels of infectious and inflammatory events (28, 29). In particular, the innate immune receptors toll-like receptor 2 (TLR2) and TLR4 (for Gram-positive and Gram-negative organisms, respectively) are expressed on the platelet surface (30, 31). Activation of these receptors may lead to release of platelet granules (32), PF4 upregulation (33), GPIIb/IIIa conformational changes (34), and ultimately feed forward to thrombin generation (30). Having said that, some studies have found less potent responses. For example, exposure of platelets to triacylated lipoproteins (like Pam3CSK4, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) does not always lead to significant P-selectin release (35).

Platelets interact directly with neutrophils and thereby alter neutrophil function (17). Examples of ligand/receptor pairs that mediate direct platelet/neutrophil interactions include P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) (36, 37), intercellular adhesion molecule 2 (ICAM-2)/lymphocyte function-associated antigen (LFA-1) (38), and GPIb/macrophage-1 antigen (Mac-1) (17, 39). These interactions clearly support platelet adhesion to leukocytes (40, 41) and, in some cases, have been shown to be of fundamental importance for recruitment of neutrophils to sites of inflammatory insult (40). Furthermore, beyond traditional direct interaction, some molecules (such as GPIIb/IIIa) may be transferred from platelets to neutrophils via microparticles (MP), thereby regulating neutrophil function (an example being nuclear factor kappa B activation) (42).

There is also a key role for platelet-released soluble mediators (ADP, TXA₂, etc.) in both perpetuating platelet–neutrophil interplay and activating neutrophils. As an example, ADP (which would presumably be platelet-derived in vivo) induces platelet–neutrophil complexes through a mechanism that may be dependent upon P-selectin, but not PSGL-1 (41). TXA₂ augments multiple neutrophil functions, including neutrophil adhesiveness (43), oxidative burst (44), and diapedesis (45). Platelet-derived HMGB1 can engage/activate neutrophil TLRs (46). Beyond TLRs, another well-recognized receptor for HMGB1 is the receptor for advanced glycation end products (RAGE), with engagement by HMGB1 leading to neutrophil recruitment and neutrophil-mediated tissue injury (47). PF4 interacts with neutrophil chondroitin sulfate (48) and (in the presence of co-stimulatory tumor necrosis factor alpha) mediates neutrophil granule release and surface adherence (49). PF4 has also been implicated in neutrophil chemotaxis (50). Neutrophil-activating peptide 2 (NAP-2) released from platelets can regulate neutrophil polarization and motility through CXCR1/2 (51). CCL5 (another chemokine released by platelets) may also play a role in neutrophil infiltration (52).

Platelets are far-and-away the most studied cellular regulators of NETosis. Most model systems have pointed to platelet activation as the first step. This is followed by platelet–neutrophil crosstalk, and ultimately regulation of neutrophil effector function. Studies have employed numerous platelet activators, including LPS, Pam3CSK4, thrombin, collagen, ADP, and TRAP-6 (53–55). These different strategies for activation, beyond anything else, make it challenging to compare studies side-by-side (Table 1).

Regarding in vitro studies, platelet–neutrophil interactions have been assessed under static conditions (53, 57), and also with the introduction of shear stress (53–56). It is worth noting that the methodology for quantifying NETosis has varied markedly across studies. Examples include cell-free DNA quantification (53, 55), myeloperoxidase-deoxyribonucleic acid (MPO-DNA) ELISA (5, 55, 60, 61), neutrophil elastase-DNA ELISA (53), neutrophil elastase concentration (57), or direct visualization of NETs by fluorescence microscopy (54). Microscopy samples have been scored by quantifying percent surface area of Sytox green staining (detects extracellular DNA) (54, 58), histone H2Ax percentage surface area (56), or citrullinated histone H3-positive cells per field (62).

We will first describe some notable in vivo studies in the field, which have focused on disease models (Table 2). We will then step through the various stages of platelet–neutrophil interplay, beginning with platelet activation and ending with NETosis (Figure 1).

Neutrophils develop in the bone marrow from myeloid precursors, reaching sites of infection or inflammation via the vasculature. This migration of neutrophils from the bloodstream to inflamed tissues is mediated by the interaction of adhesion molecules on the neutrophil surface with their respective ligands on the vascular endothelium. Details regarding this well-coordinated series of events arise from intravital microscopy studies in animals, as well as observations of patients with leukocyte adhesion deficiency (75). As an initial step, neutrophils leverage specific surface ligands in order to tether to P- and E-selectin molecules expressed on activated endothelial cells (selectin ligands potentially expressed on neutrophils include PSGL-1, E-selectin ligand 1, and CD44). Tethering of neutrophils is followed by their rolling along the endothelium (76–80). Rolling neutrophils develop membrane extensions at their rear end (tethers) and front (slings), which stabilize neutrophil rolling and allow the process to proceed despite the high shear stress of flowing blood (81). Subsequently, neutrophils firmly adhere to endothelial cells, mediated by the binding of neutrophil β₂ integrins (LFA-1 and Mac-1) to endothelial ligands such as intracellular adhesion molecule 1 (ICAM-1) and ICAM-2 (76, 78, 79, 82). β₂ integrins have two main states of activation: the first is an extended (but not open) form with low to intermediate affinity, and the second an extended and open form with high affinity (the form required for firm adhesion). Mechanisms and signaling pathways involved in these transitions have been delineated in great detail, and are reviewed elsewhere (82–84).

Rolling and adhesion may be followed by transmigration, when neutrophils pass between endothelial cells (paracellular) or through endothelial cells (transcellular). While many details remain to be determined, the paracellular process is more prevalent, occurring perhaps 90% of the time (76, 83, 85) and favored by neutrophils expressing Mac-1 (86, 87). By contrast, the transcellular route may be favored by increased endothelial expression of ICAM-1 (88) or by activation of endothelial cells by neutrophils through annexin A1 secretion (89). Beyond the above, adhesion molecules involved in the transmigration process include platelet endothelial cell adhesion molecule 1 (PECAM-1), CD99, ICAM-2, junctional adhesion molecules (JAMs), and cadherins (90). The roles of these adhesion molecules have primarily been demonstrated in mouse models wherein their deletion results in inhibition of transmigration and reduced accumulation of neutrophils in tissues (83, 85, 91).

Within inflamed tissues, neutrophils home via chemokine gradients. Interestingly, recent studies have demonstrated that neutrophils are able to undergo a “reverse transmigration” process such that tissue neutrophils may migrate back to the vascular lumen. Studies in mice have demonstrated that downregulation of JAM-C by neutrophil elastase plays a key role in the process (92). At present, the functional significance of reverse transmigration is not entirely clear. One idea is that the reverse transmigration has a significant downside, as it may contribute to dissemination of a local immune response into a systemic inflammatory phenomenon (93). Alternatively, it may play a role in dampening immune response as observed in zebrafish (94) and, we speculate patients with systemic inflammation (95).

Circulating neutrophils tend to be quiescent in nature, with their activation tightly linked to migration from circulation to tissue. Neutrophil activation can be thought of as a two-step process whereby exposure to one stimulus (priming) ensures a maximum response to a second. So, rolling and adhesion of neutrophils on the endothelium may initiate their activation, but full effector functions only become available to neutrophils once they encounter certain pro-inflammatory chemokines/cytokines or pathogen-derived ligands that can activate other receptors (G protein-coupled receptors and innate pattern-recognition receptors as classic examples). Neutrophils can then rapidly undergo degranulation, activation of their NADPH oxidase pathway for free radical generation, phagocytosis, and even NETosis (96–98). An example comes from studies of P-selectin overexpressing mice in which neutrophils seem to be sensitized to NETosis by excess P-selectin exposure, but do not actually release NETs unless confronted with a second stimulus (59).

Netting neutrophils externalize not just chromatin but also a variety of antimicrobial peptides and proteases that target pathogens. Recent work has demonstrated that these mediators of host defense may also promote tissue damage (12). NETs induce endothelial cell death in a dose-dependent and partially DNA-independent manner (99). Rather than DNA, associated histones and to some extent myeloperoxidase may be most responsible for NET-mediated endothelial cytotoxicity (99). Another study demonstrated the externalization of matrix metalloproteinase-9 (MMP-9) and MMP-25 along with NETs. This externalized MMP-9 activates pro-MMP-2 produced by the endothelium, resulting in cytotoxicity and vessel dysfunction (100).

An interesting in vitro study investigated the implications of co-culture of activated endothelial cells with neutrophils (101). The result was not just increased NETosis by neutrophils, but also increased endothelial cell death (101). The death was attributable to increased IL-8 production by the endothelial cells themselves (101). One can imagine a scenario in vivo in which activated endothelial cells induce NETosis, followed by endothelial cytotoxicity and potentially the release of mediators that feed forward into more NETosis.

It should also be noted that although endothelial cells have not been the explicit focus of most NETs studies, they almost surely play a prominent role in vivo, either through direct regulation of neutrophil activity, or through modulation of other cellular elements, such as platelets (Figure 1). As an example, in the aforementioned sepsis model, liver sinusoids support neutrophil adhesion even in the absence of platelets, perhaps providing certain activating signals to the neutrophils that prime them for subsequent platelet capture (56). One might also point to the TRALI model (5). There, GPIIb/IIIa plays a key role in NETosis beyond anything that has been seen in vitro (53–55) – raising the question of whether additional synergistic signals may emanate from the endothelium in vivo (5). Finally, although studies focusing on platelet–neutrophil interactions in vitro have suggested contradictory roles for P-selectin (53–55), it is worth noting that P-selectin is also present on endothelial cells, which may help explain its more clear-cut role in vivo (59). We expect to see much more on this front in the coming years.

Dendritic cells (DCs) are best known for their role as professional antigen-presenting cells, bridging the gap between innate and adaptive immunity. In recent years, the intersection of neutrophils/NETosis and DCs has been increasingly considered. First, neutrophils are well established to play a role in the recruitment of DCs to sites of inflammation, and promote maturation of DCs via secretion of a variety of soluble mediators, such as CCL3, CCL4, CCL5 (RANTES), CCL20, tumor necrosis factor α, α-defensins, and cathelicidins (102–106). At the same time, in vivo immunization studies have demonstrated that neutrophils can dampen immune responses by competing for antigen with DCs and limiting contact between T cells and DCs (107). So, at least in some contexts, vaccination responses may improve with temporary depletion of neutrophils. In other contexts, NETs seem to do the opposite, quite specifically transferring antigens to DCs, and thereby initiating autoimmune disorders, such as small vessel vasculitis (108).

With further implications for autoimmunity and sterile inflammation, NETs activate plasmacytoid DCs in lupus and atherosclerosis via TLR9. Activated plasmacytoid DCs produce interferons, which in turn prime neutrophils for more NETosis (thereby setting up a positive feedback loop) (109, 110).

Again pointing to different roles in different contexts, DCs may sometimes downregulate NETosis. This has been described in the specific context of human immunodeficiency virus (HIV), which acts through CD209 on DCs to produce interleukin 10 (IL-10). IL-10 then inhibits HIV/TLR7-mediated NETosis (111). Demonstrating at least some specificity, PMA-induced NETosis is not suppressed by IL-10 (111).

MP are small, cell membrane-derived vesicles (112). MP from endothelial cells (113, 114), platelets (115), and red blood cells (116) have all been implicated in activating neutrophils. Furthermore, both platelet-derived (115) and red blood cell-derived (116) MP induce Mac-1 expression on neutrophils and stimulate neutrophil phagocytic activity (115, 116). The role of MP in promoting NETosis was also demonstrated in a paper focusing on preeclampsia, in which placenta syncytiotrophoblast-derived MP seem to promote NETosis (117). In inflammatory bowel disease, MP also appear to activate NETosis (118).

While NETs play a critical role in host defense, excessive formation or persistence of NETs may lead to adverse effects. Thus, clearance of NETs is an important physiological process that helps minimize excessive presentation of both toxic products and potential self-antigens. Degradation of NETs by serum DNase is one mechanism by which NETs are cleared, with impairment of this process leading to a lupus-like syndrome in mice (119). Interestingly, inadequate DNase activity has also been detected in the blood of patients with both lupus (119–121) and autoimmune vasculitis (122). Beyond the enzymatic activity of DNase, macrophages also play a role in the clearance of NETs. DNase processing of NETs prepares them for engulfment by macrophages, with the process further facilitated by the opsonization of NETs by complement C1q (123). Though this process was initially thought to be immunologically silent, recent in vitro studies have demonstrated a potentially complicated response that depends upon macrophage polarization (124). The authors show that M2 macrophages induce a pro-inflammatory response when exposed to NETs (including the release of a variety of pro-inflammatory cytokines/chemokines). By contrast, M1 macrophages initially undergo cell death that leads to their own nuclear decondensation and DNA release. Interestingly, over time, M1 macrophages then degrade this macrophage-derived DNA in a caspase-activated DNase-dependent manner (124). The full implications of this interplay remain unclear in vivo (and in disease states) and will hopefully be elucidated by future studies.

This is a field in which much remains to be defined, as is especially highlighted by the various studies of platelet-induced NETosis. Studies in different systems and by different investigators have revealed surprisingly little mechanistic consensus, which probably points to an involvement of multiple pathways, thereby allowing certain aspects to be revealed by different groups. An obvious barrier is that platelet activation is achieved through different methodology in each study. It would be very interesting to see one group (or preferable a number of groups) take a systematic approach to this question, asking how the method of stimulation influences the specifics of platelet–neutrophil crosstalk. Given the highly regulated crosstalk that exists between the endothelium and neutrophils, endothelial cells surely play an important role in regulating NETosis in vivo – although relatively few studies have specifically probed that role. Studies should, therefore, increasingly consider the triumvirate of neutrophils, platelets, and the endothelium when exploring NETosis, especially in disease states.

NK and GS wrote the review. JK edited and approved the final version for publication.

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.