Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis

Journal of Thrombosis and HaemostasisVolume 6, Issue 3 p. 415-420 Free Access Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis A. C. MA, A. C. MA Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, CanadaSearch for more papers by this authorP. KUBES, P. KUBES Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, CanadaSearch for more papers by this author A. C. MA, A. C. MA Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, CanadaSearch for more papers by this authorP. KUBES, P. KUBES Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, CanadaSearch for more papers by this author First published: 10 December 2007 https://doi.org/10.1111/j.1538-7836.2007.02865.xCitations: 143 Paul Kubes, Institute of Infection, Immunity and Inflammation and Snyder Chair in Critical Care Research, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1.Tel.: +403 220 8558; fax: +403 283 1267; e-mail: pkubes@ucalgary.ca AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Introduction Sepsis is a severe illness caused by overwhelming infection of the bloodstream and affects more than 750 000 people in the US each year [1]. The American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) defines sepsis as systemic inflammatory response syndrome (SIRS) resulting from infection (bacterial, viral, fungal or parasitic) [2]. Increasing complications of sepsis can lead to severe sepsis, septic shock, and multiple organ dysfunction syndrome (MODS), and this often results in death. In fact, severe sepsis is a major cause of death in the intensive care unit (ICU) of hospitals, causing 215 000 deaths in the US annually [1]. Sepsis is a complex response involving many cell types and biological mediators. There is a growing body of evidence that both neutrophils and platelets are actively involved in sepsis, and together they cooperate to contribute to the inflammatory response. This review will briefly summarize some of the mechanisms of cooperativity between platelets and the immune system, focusing in part on a recently uncovered novel pathway for bacterial trapping in the vasculature. Platelets in innate immunity Platelets not only play an important role in hemostasis, but there is increasing evidence of their participation in the induction of inflammation and defense against infection [3, 4]. These anucleate megakaryocyte fragments rapidly localize to sites of injury and infection [5]. Platelets contain stores of cytokines and mediators within their α- and dense-granules that are released upon stimulation and contribute to the various stages of inflammation and immune progression [5]. In addition, platelets have a complex mode of action and are able to modulate and regulate the function of surrounding cells. Platelets are able to act on other cells through direct cell-cell contact mediated by adhesion molecules or by the release of various factors [6]. Platelets also contain antimicrobial proteins [7] and have the ability to bind and internalize bacteria and viruses through engulfing endosome-like vacuoles that fuse with the α-granules of the platelet and allow the granular proteins to have access to the pathogen [8]. Platelet Toll-like receptor 4 (TLR4) Toll-like receptors (TLRs) are pattern recognition receptors, which recognize pathogen associated molecular patterns (PAMPs). PAMPs are small molecular sequences unique to microorganisms that help in the ability of the host to recognize foreign pathogens [9]. Recently, both human and murine platelets, as well as megakaryocytes, have been shown to express the lipopolysaccharide (LPS) receptor, Toll-like receptor 4 (TLR4) [10-14]. However, the role for platelet TLR4 was not immediately apparent as LPS did not induce the hallmark features of platelet activation, namely aggregation or the expression of the common platelet activation marker P-selectin [12, 15]. One study demonstrated enhancement or priming of platelet aggregation with LPS, but the results were subtle [16]. Another group even concluded that platelet TLR4 has no functional role and is simply a vestigial remnant from megakaryocytes [12]. Much of this work was performed in vitro in various specific functional assays and the results were consistent with the view that platelet TLR4 does not contribute to any of the ‘mainstream’ platelet functions. In vivo during sepsis or in endotoxemia (in mice and humans), circulating platelet counts fall precipitously and the degree of thrombocytopenia correlates with the severity of the sepsis and the rate of mortality [17, 18]. In addition to inappropriate disseminated intravascular coagulation [19], platelet counts also decrease due to well-established migration into lungs and liver [20]. The rationale to explain why this migration occurs remains unclear. Andonegui et al. used adoptive transfer of wild-type platelets or TLR4-deficient platelets into wild-type LPS-treated mice to demonstrate that TLR4 on platelets was essential for platelet migration into lungs [10]. This was a striking observation when one considers that the mice were treated with LPS and many inflammatory mediators were generated, yet the platelets still needed TLR4 to move to the lungs. Another group found that mice lacking the TLR4 gene had fewer circulating platelets and decreased platelet turnover, as well as a reduction in thrombin-stimulated expression of P-selectin [21]. Others have also suggested that platelet TLR4 was responsible for the modulation of LPS-induced thrombocytopenia as well as tumor necrosis factor (TNF)-α production [11]. In addition, Semple and colleagues demonstrated that when platelet TLR4 binds LPS in the presence of anti-platelet antibodies, phagocytosis and destruction of platelets by human mononuclear cells was significantly enhanced [22]. Finally, platelets also bound to fibrinogen following LPS treatment in vitro [10, 14]. Clearly, platelet TLR4 was involved in platelet functions that included platelet adhesion, migration and destruction, and all of these factors could have contributed to the associated thrombocytopenia. Platelet-neutrophil cooperativity in sepsis There are countless ways a person can become septic, but regardless of the source of infection, once septicemic (bacteria and bacterial products in the blood), activated neutrophils become lodged primarily, but not exclusively, in the capillaries of the lungs and the sinusoids of the liver leading to lung and liver dysfunction and/or failure [23]. This has been viewed as an untoward effect of inappropriate neutrophil activation due to bacteremia or endotoxemia as a result of, for example, bacterial shedding of LPS [23]. The LPS activates the neutrophils, making them more rigid, and it has been argued that this leads to the inadvertent trapping of neutrophils in the lungs [24]. In other words, this could be viewed as a defense mechanism for pathogens to inappropriately activate and sequester neutrophils to sites remote to the infection. An alternative possibility is that the migration of neutrophils to lungs and liver is an active and coordinated mechanism of self-defense. Much like the neutrophils, platelets also migrate into the lungs and liver, but at a slightly delayed time [10]. When neutrophils are depleted prior to induction of endotoxemia, platelets no longer migrate to lungs and liver, suggesting that the neutrophils are essential for the platelet recruitment [10]. The platelets could either interact directly with the neutrophils or bind at sites made pro-adhesive by neutrophils. It is well accepted that heterotypic aggregation of platelets to neutrophils occurs both in vitro [25] and in whole blood [26] following stimulation with various molecules, including thrombin or ADP. When platelets become activated they become more spherical with arm-like protrusions that facilitate adhesion to the endothelium, exposed membranes, and leukocytes [6]. Patient studies have revealed increased platelet activation and adhesion to neutrophils and endothelium in septic patients [17, 27], and a similar observation has been noted in endotoxemic mice [10] and mice with acid-induced acute lung injury [28]. The evidence appears to favor platelets binding directly to neutrophils. However, when exposed to activated endothelium, or to activated neutrophils adhering to activated endothelium, platelets prefer to selectively bind to the neutrophils [15]. Indeed, visualization of the liver microvasculature revealed platelets adhering directly to neutrophils rather than endothelium [15]. Using a simple model to study platelet-neutrophil and platelet-endothelium interactions in vitro, platelets exposed to LPS bound much more avidly to immobilized neutrophils [15]. It remains unclear whether high concentrations of LPS, as used in the aforementioned study [15], ever exist in sepsis. However, plasma from septic patients, but not from healthy individuals, can induce platelet-neutrophil interactions as well [15]. The latter suggests that septic plasma has the needed constituents to induce platelet-neutrophil interactions. Interestingly, 40–50% of platelet binding to neutrophils was inhibitable by a TLR4 antagonist (eritoran), suggesting that platelets can be activated by TLR4 ligands in the septic milieu, but also that other mediators and/or bacterial products contribute to these events [15]. Although it is tempting to conclude that 50% is mediated by LPS, there are numerous other TLR4 ligands in the septic milieu, including high mobility group B1 (HMGB1) proteins, flagellin, fibronectin, fibrinogen, and heat-shock proteins that could also contribute to the pulmonary recruitment of platelets. This raises the possibility of an interplay between platelets and neutrophils in the fight against infection as a systematic innate immune response necessary for ultimate survival. Platelets activate neutrophils to trap bacteria Both platelets and neutrophils have the potential to trap microbial pathogens independently of each other [8, 29]. However, together platelet-neutrophil interactions induce transcellular synthesis and hyperactivation of neutrophils to produce increased pro-inflammatory molecules [30]. In a recent publication by Clark et al. [15], a novel mechanism of platelet-neutrophil interactions was observed leading to improved bacterial trapping (Fig. 1). This event is initiated with activated platelets adhering to immobilized neutrophils in a model of endotoxemia and sepsis [15]. Using an in vitro flow chamber assay, LPS-stimulated platelets were perfused across immobilized neutrophils and the platelets bound to the neutrophils, thereby activating them in a very profound manner. Interestingly, the platelets were an active participant as platelet fixation resulted in no platelet-neutrophil interactions [15]. This would suggest that this platelet-neutrophil interaction is dependent on LPS-induced platelet activation. In addition to releasing many of their granules, the activated neutrophils with platelets bound to them were seen to release their DNA, which seemed to contribute to the trapping of bacteria. These structures were reminiscent of the neutrophil extracellular traps (NETs) that were originally identified by Brinkman et al. [31] and are reviewed in more detail below. Figure 1Open in figure viewerPowerPoint Novel model of bacterial trapping in the microvasculature. (A) Prior to detection of bacteria. Inactivated neutrophils and platelets are circulating through the microvasculature. The presence of E. coli leads to TLR4 activation. (B) Neutrophils detect LPS and are recruited to the endothelium lining the microvasculature. TLR4-activated platelets are then recruited to the adherent neutrophils, where they bind to the immobilized neutrophils. (C) This leads to robust neutrophil activation and NET formation. A greater number of E. coli are now trapped within the microvasculature by the NETs, where they can be killed and cleared. Plasma from septic patients could also induce platelet-neutrophil interactions and NET formation. Compared with LPS alone, the formation of NETs was greatly increased when LPS stimulated platelets bound adherent neutrophils [15]. This latter mechanism of NET formation occurred very rapidly (in minutes) and appeared not to involve death of the neutrophils. Neutrophils anchored the NETs by remaining attached to the substratum and continued to restrict dyes that are only permeable in dead cells. Although loss of nuclei would normally be associated with cell death, in the case of neutrophils, removal of nuclei to make ‘cytoplasts’ was a common approach to study non-nuclear events in neutrophils, including chemotaxis and oxidant production [32]. Importantly, the NETs were able to withstand a physiological shear in the flow chamber (0.5 dyne cm−2) that might be expected in sinusoids and capillaries of the lungs and liver. When GFP-expressing Escherichia coli (E. coli) were perfused across the neutrophils that had formed NETs, these NETs significantly enhanced the trapping of bacteria compared with untreated and LPS-stimulated neutrophils alone, which only trap bacteria through phagocytosis [15]. Importantly, DNase treatment of the NETs resulted in degradation of these NETs [33], and also significantly decreased trapping of bacteria [15]. The NETs were also produced in vivo. Using in vivo imaging, it appeared that the liver sinusoids and lung capillaries where platelets bound neutrophils were hot spots for bacterial trapping and removal of platelets or neutrophils impaired bacterial clearance [15]. Intracellular and extracellular killing by neutrophils Neutrophils play an important role in the first line of defense against invading microbial pathogens [29]. Neutrophils are terminally differentiated cells with a short life span and are the first immune cells recruited to a site of inflammation from the bloodstream [34, 35]. Equipped with a full arsenal of antimicrobial proteins at their disposal, neutrophils have a well-described role in phagocytic uptake and intracellular killing [29, 36]. When a neutrophil encounters a microbe, it engulfs the microbe into a phagosome, which then fuses with intracellular granules forming a phagolysosome. Within the phagolysosome the microbe is destroyed by a combination of oxidative (reactive oxygen species; ROS) and non-oxidative (enzymes, proteases, and antimicrobial peptides) mechanisms [29, 36]. Although intracellular generation and release of oxidants and proteases into phagolysosomes is unlikely to cause extracellular damage, inflammation is often associated with damage and destruction of ‘innocent’ parenchymal cells in proximity to neutrophils. In 2004, neutrophils were described to have a novel extracellular killing function where, upon activation by phorbol myristate acetate (PMA) and interleukin-8 (IL-8), neutrophils released their granular (peptides and enzymes) and nuclear (chromatin DNA and histones) constituents, which combined to form neutrophil extracellular traps (NETs) [31]. DNA was the major structural component of the NETs with granule proteins from azurophilic, specific, and gelatinase granules attached to this DNA backbone [33]. Using a high-resolution scanning electron microscope, Brinkmann et al. observed stretches of DNA and globular protein domains with a diameter between 15 and 17 nm and 25 nm, respectively, which could aggregate into larger threads with a diameter of 50 nm [31]. These NETs, or web-like DNA structures, were very effective at trapping bacteria in vitro under static conditions [31]. In the flow chamber system under flow conditions, the NETs formed even larger structures, hundreds of nanometers in length and width [15]. The mechanism of NET release is not clear in terms of whether it is an active and controlled process and/or whether it is an early event in neutrophil programmed cell death. It has been demonstrated by some groups that these NETs are released within 5–10 min after activation of the neutrophils [15, 31, 37], which is too quick for apoptosis to occur. Brinkmann et al. have provided evidence that NETs are formed actively and are not the result of leakage during cellular disintegration [31]. In a much more systematic assessment, this same group reported that NETs do not form during true apoptotic or necrotic cell death [38]. Rather, over the period of 2–4 h, oxidative stress in neutrophils caused disintegration of the nuclear membrane so that the nuclear material distributes throughout the cytoplasm. However, cellular integrity remains intact. Eventually, the DNA is extruded from the cell and at this stage impermeant molecules can enter the cell, suggesting a breach of the cell membrane. It is thought that the cell dies, but then the NETs capture and kill bacteria even after the death of the neutrophil [38]. Whether one or more than one mechanism of release exists, these NETs have been consistently shown to bind and kill both Gram-negative and Gram-positive bacteria [31] as well as pathogenic yeast [39]. NET trapping and killing The NETs contain histones H1, H2A, H2B, H3 and H4, as well as granule proteins, including neutrophil elastase, myeloperoxidase, and bactericidal permeability increasing protein (BPI), which line the DNA backbone [31]. Once bound to these NETs, the associated histones and antimicrobial proteins degrade virulence factors and kill pathogens [33]. When one considers mechanisms by which the immune system walls off bacteria, NETs would certainly be potential candidates due to their sticky nature and immobilizing properties at sites of infection. NETs not only trap and prevent the spread of pathogens from the initial site of infection, but by being bound by all the noxious stimuli they also act to concentrate the antimicrobial proteins to the site of infection [40]. To this point, NETs have been shown to trap various types of pathogens. They interact with both Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, and Group A streptococci) [31, 37, 41-43] and Gram-negative (Salmonella typhimurium, Shigella flexneri, and E. coli) bacteria [15, 31], as well as both hyphae and yeast forms of Candida albicans [39]. The S. aureus virulence factor α toxin and the S. flexneri virulence factor IpaB were both degraded when these bacteria were trapped in NETs, and NETs were able to kill these bacteria, as well as S. typhimurium, through BPI- and histone-mediated killing [31]. Similarly, C. albicans was also killed by NETs, but granule proteins and not histones were involved in this mechanism of NET-mediated killing [39]. Evading NETs In contrast to those pathogens susceptible to NET-mediated killing, both Group A streptococci [37, 43] and S. pneumoniae [41, 42] were relatively resistant to killing by NETs. These bacteria have developed mechanisms of escaping NETs, such as expression of DNases that work to degrade the DNA scaffold of the NETs, or changing their surface charge to electrochemically repel NETs [40]. All strains of Group A streptococci produce at least one extracellular DNase, which has been shown to be important in its virulence of Group A streptococci by degrading NETs, thus accounting for Group A streptococci resistance to NET-mediated killing [37, 43]. Beiter et al. showed that the expression of the surface endonuclease, EndA, on S. pneumoniae could degrade the DNA backbone of NETs, allowing for their escape, thereby promoting their spread through the airway and into the blood [41]. In addition, Wartha et al. found an alternative mechanism by which S. pneumoniae evade NETs, whereby the Gram-positive bacteria incorporate a positive charge to its surface to repel the positive charge of antimicrobial proteins that are bound to the NETs [42]. A combination of the expression of a polysaccharide capsule and lipoteichoic acid D-alanylation is responsible for the positive charge of the surface of S. pneumoniae [42]. The damaging effects of NETs Although NETs have been demonstrated to have beneficial effects in enhancing bacterial trapping, the formation of NETs also occurs at the expense of injury to the host. Although adhesion of LPS-activated neutrophils to endothelium caused no damage, when LPS-treated platelets activated neutrophils to release their NETs, there was damage to the underlying endothelium in vitro and liver damage in vivo [15]. Presumably, exposure to the proteases and granular proteins in the extracellular environment via NETs was responsible for the damage, but this awaits confirmation. Depletion of either neutrophils or platelets is sufficient to reduce damage, suggesting that the neutrophil is dependent on the platelet for activation and NET formation and inadvertent hepatotoxicity in this setting [15]. Unresolved issues Circulating platelet-neutrophil complexes have been identified in septic blood and represent a subset of activated neutrophils that have an increased ability to adhere, phagocytose, and produce superoxide [44]. The binding of the platelet to the neutrophil in the circulation is via P-selectin (CD62P), which is an adhesion molecule that is released from the α-granules of platelets activated with thrombin and other mediators and expressed on the membrane surface. The binding partner in this regard is P-selectin glycoprotein ligand-1 (PSGL-1) found on neutrophils and other leukocytes. Another way that platelets can be activated to express P-selectin, is through contact with the substratum, including collagen and fibrinogen, and binding to this substratum [45]. Adherent platelets expressing P-selectin then mediate the initial tether of circulating neutrophils via PSGL-1, leading to rolling of neutrophils on the platelet monolayer [46, 47]. By contrast, platelets binding to immobilized neutrophils is an entirely different process. Platelet P-selectin is not expressed following LPS stimulation and not surprisingly, P-selectin does not play a role in the platelet binding to immobilized neutrophils (A. C. Ma and P. Kubes, unpublished observations). In vivo, however, some studies have reported increased neutrophil-platelet interactions via P-selectin, but whether these were platelets binding to already adherent neutrophils or whether these were neutrophil-platelet interactions in the circulation that then adhered to vessels remains unclear [28, 48, 49]. The subsequent sustained adhesion of neutrophils on platelet monolayers is integrin-dependent through Mac-1 (CD11b/CD18) binding directly to GPIb [50] or indirectly to GPIIb/IIIa through fibrinogen [51]. Firm adhesion of neutrophils to immobilized platelets can also occur through platelet intracellular adhesion molecule (ICAM)-2 with LFA-1 (CDlla/CD18) [52]. However, no data to suggest integrins mediate platelet binding to adherent neutrophils exist. Both contact-dependent and soluble factors have been postulated to contribute to neutrophil-mediated activation by platelets [44]. The firm adhesion is mediated by various activating molecules made by platelets including platelet activating factor (PAF) and β-thromboglobulin, all of which cause activation of the neutrophils. Identifying how the platelets bind to the adherent neutrophils remains a major gap in our knowledge and could provide an important new therapeutic avenue. Moreover, the platelet-derived mediator that signals neutrophils to subsequently release NETs remains unelucidated. Finally, the pathology induced by NETs needs further investigation. It has been suggested that NETs could impede the immune response by creating a barrier and preventing the recruitment of more white blood cells, as well as interfering with the cilia and hindering proper mechanical clearance of the airways [53]. NET formation has been seen in cystic fibrosis patients (Dr Francis Green, personal communication), but its role remains unclear. It is intriguing that DNase is issued to help clear lungs in this setting. NETs may also contribute to the development of autoimmune diseases, such as systemic lupus erythematosus (SLE), because of leakage of the host’s own nucleic acids [33]. In fact, removal of NETs must also be a well-regulated process. Indeed, endogenous DNases are found in blood and the DNaseI-deficient mouse develops an SLE-like syndrome, characterized primarily by the presence of anti-nuclear antibodies and signs of glomerulonephritis [54]. Conclusion Both neutrophils and platelets possess all the machinery needed to interact with one another and have both been shown to be recruited to the microvasculature of the liver and lung in models of endotoxemia and sepsis. NETs, an extracellular mechanism for microbial trapping and killing by neutrophils, are also emerging as an important part of the innate immune system in the defense against invading pathogens and recently it has been shown that platelets can mediate this mechanism. In our working model, platelet TLR4 functions as a barometer for systemic infection, binding avidly to sequestered neutrophils during septicemia and endotoxemia in the capillaries of lungs and liver (and perhaps elsewhere). This leads to the rapid formation of NETs that maintain their integrity under flow conditions and trap bacteria in the circulation but also cause local damage to host vasculature. Clearly this topic requires further attention, as it may one day be directly linked to our understanding of the fundamental issue of whether NET formation in sepsis is beneficial or detrimental, particularly now that antibiotics and other forms of support can replace killing mechanisms that inadvertently injure endogenous cells. Acknowledgements This work is supported by grants from Canadian Institutes of Health (CIHR) and a CIHR group grant. A.C. Ma is an Alberta Heritage Foundation for Medical Research (AHFMR) student. P. Kubes is an AHFMR Scientist, a Canadian Research Chair recipient and the Snydor Chair in Critical Care Medicine. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001; 29: 1303– 10. 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