Sage Journals: Discover world-class research

Abstract

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box–1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

Keywords

blood coagulation disorders cats dogs heparin horses immune-mediated hemolytic anemia neutrophil extracellular traps NETs review sepsis thrombosis

The innate immune system actively participates in the initiation and propagation of thrombosis, through the activity of neutrophils, monocytes, and dendritic cells that can initiate and accelerate fibrin formation and activate platelets. Although thrombosis is typically thought of as pathologic, in some scenarios it may represent a beneficial physiological process that aids innate immunity and host defense against pathogen invasion. This protective process is now termed immunothrombosis^46,74 and is a proposed consequence of the conserved links between inflammation and coagulation.^119,120 Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of the genesis of immunothrombosis. While immunothrombosis may be protective in some settings, dysregulated or excessive clot formation likely contributes to morbidity and mortality in trauma, sepsis, heat stroke, and hemolytic anemia. Immunothrombosis may therefore represent a novel therapeutic target in these conditions, aided by distinct pathways and molecular mediators that separate immunothrombosis from normal hemostasis. Here, we discuss the cellular mechanisms that underpin neutrophil extracellular trap (NET) formation and immunothrombosis, appraise the methods for identification of NETs, review the existing veterinary literature, and suggest potential avenues for future therapeutic intervention.

Mechanisms of NET Formation

Neutrophils are the sentinel cells of innate immunity responsible for defending the host against invading microorganisms. Once recruited from the bloodstream to sites of infection, neutrophils may phagocytose, degranulate, and generate reactive oxygen species to effectively eliminate invading pathogens.¹⁴⁶ In response to certain stimuli, neutrophils can also enhance their antimicrobial activities by releasing NETs, composed of extracellular chromatin.¹⁸ Attached to these web-like scaffolds of cell-free DNA (cfDNA) are histones and neutrophil granule proteins like myeloperoxidase (MPO), lactoferrin, cathepsin G, neutrophil elastase (NE), and antimicrobial peptides such as defensins.^101,105,166 These components of NETs are crucial to their antimicrobial properties.

The process of NET formation, termed NETosis, is the outcome of a number of highly regulated cellular events induced by a variety of stimuli. The cellular orchestration of NETosis is dependent on the species and the stimulus, and it is currently the topic of intense investigation. NETs are released upon direct exposure to microorganisms or through recognition of pathogen-associated molecular patterns within the bloodstream or body compartments accessible via diapedesis. Ex vivo exposure of feline and equine neutrophils to Leishmania and Streptococcus equi or Streptococcus capitis, respectively, results in NETosis.^166,218 In vivo, in dogs with sepsis, NETs have been documented in the blood, in the lungs, and in abdominal and pleural effusion samples.^81,111 As part of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin initiates inflammation by activating neutrophils via the pattern recognition receptor, Toll-like receptor 4 (TLR4). In a model system, high-dose LPS stimulates canine neutrophils to undergo NETosis.¹¹² In comparison to those of dogs, equine neutrophils are extremely sensitive to LPS. Equine neutrophils undergo extensive NETosis when exposed to a fraction (1/100,000) of the LPS concentration required to induce NETosis in dogs. In addition to direct interactions with microorganisms and molecular patterns, cytokines such as interleukin (IL)–8, complement component C5a, or autoantibodies have also been shown to induce NETosis in human neutrophils.⁸⁹

Certain cellular events are essential for NETosis. After stimulation by the protein kinase C activator phorbal 12-myristate 13-acetate (PMA) or by LPS, canine neutrophils undergo sequential morphological changes including chromatin decondensation, loss of membrane integrity, and release of cfDNA (Fig. 1).¹¹² Human neutrophils undergo loss of the nuclear envelope and mixing of karyosomes and cytoplasmic granules prior to the release of cfDNA.⁴⁹ Similar to human and murine neutrophils, canine neutrophils undergo extensive NETosis in response to PMA.^81,112 Although the exact mechanism of PMA-induced NETosis remains unclear, PMA likely signals multiple intracellular pathways essential for mediating NETosis. First, PMA triggers the generation of reactive oxygen species by inducing calcium influx and activating NADPH oxidase. Reactive oxygen species generation must occur prior to the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation during NETosis.^80,90 In mice, the synergistic action of NE and MPO within the nucleus following translocation from the cytoplasm promotes chromatin decondensation.¹⁵⁶ Nuclear decondensation is also dependent on post-translational modification of histones, a group of positively-charged proteins responsible for hyper-condensing DNA into nucleosomes. Citrullination (also called deimination) of histones, catalyzed by the enzyme peptidylarginine deiminase 4 (PAD4), alters the electrostatic interactions between histones and DNA by decreasing the net positive charge of histones. This, in turn, causes chromatin decondensation facilitating the release of cfDNA.^105,167,217 In canine neutrophils, PAD4 inhibition by the chemical Cl-amidine blocks histone citrullination and prevents NETosis mediated by either LPS or PMA.¹¹²

Figure 1.

Fluorescence microscopy of live canine neutrophils demonstrating the sequential morphological changes seen during NETosis. Isolated living neutrophils, activated with PMA, were stained with either a cell-permeant nucleic acid dye (green) or cell-impermeant nucleic acid dye (red), and incubated for 30 (1a), 90 (1b), or 180 (1c) minutes prior to imaging. Only cell-free DNA or cells without an intact cell membrane are stained red. (1a) At 30 minutes, all neutrophils have an intact cell membrane with lobulated nuclei. (1b) By 90 minutes, chromatin within the cells are beginning to decondense (arrowhead). (1c) By 180 minutes, most neutrophils have lost their membrane integrity, and web-like cell-free DNA (arrows) are released.

Types of NETosis

Many of the early studies of NETosis relied heavily on electron microscopy or fluorescence microscopy to visualize neutrophils generating NETs. These techniques require fixation and immunolabeling and hence limit the potential physiologic relevance of the phenomena observed. As a result, NETosis was initially considered a type of cell death because expulsion of cfDNA and granule proteins requires disintegration of both the nuclear envelope and plasma membrane. This type of NETosis, described by some investigators as “suicidal” or “lytic,” occurs with PMA stimulation when cells can no longer maintain homeostasis following the event.²³⁰ Developments in intravital microscopy enabled investigators to observe NETosis in vivo and demonstrate that living neutrophils can maintain their functional capacities following NETosis. In human skin infections and in mouse sepsis models, direct encounters of neutrophils with Staphylococcus aureus and E. coli lead to release of cfDNA.^141,231 The remaining anuclear neutrophils maintain an intact cell membrane, perform chemotaxis, and phagocytize nearby bacteria.²³¹ This phenomenon, termed vital NETosis, has yet to be demonstrated in veterinary species.

Platelet-Neutrophil Interactions in NETosis

Platelets play key roles in innate immunity. Human, murine, and canine platelets express functional TLRs, allowing them to directly respond to pathogen-associated molecular patterns.^30,113,188 Activated platelets interact with neutrophils to facilitate NETosis.³⁰ In ex vivo systems, LPS-induced NETosis in human, canine, and murine neutrophils is limited,^112,118,160 but the response can be amplified by the presence of LPS-activated platelets, suggesting that platelet-neutrophil interactions promote and facilitate NETosis. In mouse endotoxemia models, platelet depletion significantly attenuates NET formation within liver sinusoids. Formation of platelet-neutrophil aggregates and NETosis in this model is dependent on platelet TLR4.³⁰ In addition to LPS, thromboxane and thrombin-mediated activation of platelets also support robust NETosis in humans and mice.²⁴

For platelet-mediated NETosis to occur, both cell types must synergistically interact via adhesive and soluble interactions. Platelet activation must first take place to initiate intracellular signaling cascades that induce secretion of soluble mediators, and upregulate and activate adhesion molecules. Platelet activation induced by agonists such as adenosine diphosphate and thrombin results in degranulation of alpha-granules with subsequent membrane surface P-selectin expression. Agonist-mediated activation also leads to inside-out signaling that alters the conformation of the extracellular domains of the α_IIbβ₃ integrin and, thereby, increases its affinity for ligands.

In mice, platelet-neutrophil interactions mediated by adhesive interactions between platelet P-selectin and its neutrophil receptor, P-selectin glycoprotein ligand–1 (PSGL-1), are essential for NETosis during sepsis.^48,187 Such findings, however, could not be replicated in humans, suggesting that P-selectin/PSGL-1–mediated NETosis may be species dependent.^22,139 It is known that equine peripheral blood mononuclear cells express PSGL-1 as sialylated homodimers. Several post-translational modifications and receptor dimerization are required for high-affinity P-selectin binding by equine PSGL-1.²²⁵ However, in contrast to human PSGL-1, sulfation of equine PSGL-1 is not required for P-selectin binding. Clearly, there are interspecies differences in the structure of these proteins that could influence their ability to mediate NET formation. In humans, neutrophils express the neutrophil integrin α_Mβ₂ (MAC-1), which binds to the platelet heterodimeric glycoprotein 1bα (GP1bα) via von Willebrand factor and can induce NETosis in vitro.¹⁶⁹ In mice, fibrinogen, which normally binds to its platelet receptor, α_IIbβ_3, also binds to neutrophil integrin MAC-1 to form an adhesive interaction between the 2 cell types (Fig. 2).

Figure 2.

A schematic diagram demonstrating the molecular mechanisms of platelet-neutrophil interaction involved in NETosis. Platelet-neutrophil interaction can be broadly divided into either adhesive or soluble interactions. Adhesive interactions: (A) Platelet activation induced by lipopolysaccharide (LPS) via toll-like receptor 4 (TLR4), or by thrombin, ADP, or arachidonic acid (AA) must take place for activation of the platelet integrin, α_IIbβ₃, and alpha-granule degranulation. (B) P-selectin on the alpha-granule membrane fuses with the platelet cell membrane upon degranulation and binds to the neutrophil receptor, P-selectin glycoprotein ligand-1 (PSGL-1). (C) The neutrophil integrin, MAC-1, once activated, binds indirectly to α_IIbβ₃ via fibrinogen and to glycoprotein 1bα (GP1bα) via von Willebrand factors (vWF). Soluble interactions: (D) Activated platelets secrete soluble mediators like high mobility group box–1 (HMGB-1), platelet factor 4 (PF4), and chemokine ligand CCL5. (E) PF4 and CCL5 form a heterodimer to initiate NETosis via the neutrophil G-protein coupled receptor (GPCR). (F) HMGB-1 initiates NETosis by binding to its neutrophil receptor, the receptor for advanced glycation end products (RAGE). Both types of interactions result in neutrophil chromatin decondensation (G) via histone citrullination by the enzyme, peptidylarginine deiminase 4 (PAD4), and eventual release of DNA extracellularly along with neutrophil granule proteins like myeloperoxidase (MPO) and neutrophil elastase (NE).

Activated platelets also secrete soluble mediators capable of stimulating nearby neutrophils to produce NETs. Upon activation, human and mouse platelets upregulate and secrete high mobility group box–1 (HMGB-1), a damage-associated molecular pattern.^140,209,210 This protein binds to its neutrophil receptor, the receptor for advanced glycation end-products to facilitate NETosis.^139,209 In mouse septic peritonitis models, platelet-specific HMGB-1 knockout mice produce significantly fewer NETs, leading to bacteremia and an amplified systemic inflammatory response. This suggests that platelet-derived HMGB-1 is critical for bacterial clearance and survival in sepsis. The pathways downstream of receptor for advanced glycation end-products activation that lead to NETosis, however, remain to be determined.^{40,41,139,234} Other platelet-derived chemokines, like platelet factor 4 and RANTES (CCL5), can directly activate neutrophils to undergo adhesion. The heterodimerization of platelet-derived platelet factor 4 and CCL5 binding to neutrophil G-coupled protein receptors also enhances NETosis in mice (Fig. 2).¹⁶⁹

Role of NETs in Innate Immunity

NETs exert their antimicrobial activities by physically entrapping microorganisms, directly killing pathogens, or hindering their dissemination from the point of entry.¹⁸ The negatively charged surface of extracellular chromatin facilitates the binding of organisms like bacteria Candida albicans and Leishmania. Adhered microbes are then killed by high concentrations of antimicrobial proteins such as histones and cathepsin G. It was recently demonstrated that NETs derived from dogs with sepsis can bind directly to bacteria, suggesting that the active entrapment of bacteria by NETs may occur in dogs in vivo.^111,221 Microorganisms that evolve to dismantle the structural components of NETs are considered more virulent. For instance, Staphylococcus aureus degrades NETs by converting DNA to deoxyadenosine, which causes macrophage apoptosis, allowing the bacteria to escape from NETs and evade phagocytosis.¹⁹⁷ The respiratory pathogen Streptococcus pneumoniae can disseminate into the bloodstream by expressing endA, a virulence factor that degrades DNA.¹⁰

Immunothrombosis

Microvascular thrombosis induced by NETs is an important first line of defense as it prevents systemic dissemination of pathogens via the circulation. With their multifaceted ability to stimulate thrombus generation, NETs are key players in immunothrombosis.¹³³ The structural components of NETs can directly activate platelets, facilitate thrombus formation, and inhibit fibrinolysis and the natural anticoagulant pathways. In dogs, neutrophil-derived cfDNA decreases fibrinolysis, while NET proteins accelerate clot formation.⁸² Large fragments of double-stranded cfDNA measuring at 1.5-10 kbp collected from human septic patients strengthen clot formation by binding to factor XII and high-molecular-weight kininogen, both critical for activation of the contact pathway of coagulation.^53,176 NETs’ ability to trap and accumulate both circulating cells and soluble coagulation components is likely central to their thrombogenic abilities.¹⁵² The web-like structure of cfDNA fortifies thrombus formation by binding to circulating erythrocytes, platelets, and clotting factors, including tissue factor and fibrin.¹³³ Moreover, histones on NETs can activate platelets and increase thrombin generation via platelet TLR2 and TLR4, and histones induce platelets to release polyphosphates and expose phosphatidylserine and activated factor V.^152,179 These platelet changes in turn support the prothrombinase complex (FXa/FVa/Ca²⁺) assembly. Histones exhibit further prothrombotic effects by modulating the activation of the natural anticoagulant, protein C.^5,179 In vitro, cathepsin G and NE can further strengthen clots by degrading tissue factor pathway inhibitor bound on human endothelial cells.¹⁸⁹ Fibrin degradation is impeded by NETs as DNA forms a ternary complex with plasmin and fibrin, inhibiting plasmin-mediated fibrinolysis.¹⁵² Recent studies in DNAse-deficient mice suggest that NETs may also promote vascular occlusion independently of coagulation activation and fibrin clot formation.^86,152

NET-related thrombosis is an important component of pathogen control provided by NETs; as such, NETs highlight the intersection of blood coagulation and antimicrobial defense. When NETs trap tissue factor pathway inhibitor, which NET serine proteases subsequently inactivate, NETs promote extrinsic coagulation and arterial thrombosis in mice.¹³⁵ During systemic infection, the resulting arterial thrombi immobilize bacteria in the microvasculature, preventing pathogen spread. However, the same mechanism can result in undesired large vessel occlusion and thrombosis.

Acute infections, perhaps through NET generation, predispose patients to deep vein thrombosis, a major cause of cardiovascular death in humans.¹⁸³ In a murine venous stasis model, a link between NET-driven thrombosis and deep vein thrombosis development was established. In this model, venous stasis resulted in a rapid accumulation of neutrophils, NET formation, and subsequent activation of FXII.²¹¹ DNase1 administration reduced NETs and suppressed deep vein thrombosis growth, demonstrating the dependence of thrombosis on NETs.²¹¹ Tissue factor expressed by hematopoietic cells, platelets, and FXII, with which NETs co-localize, was also critical for thrombosis in this model.^152,211 Similarly, murine deep vein thrombosis models show reduced thrombus formation in PAD4-deficient mice that cannot generate NETs, again suggesting that NETs are integral to deep vein thrombosis.¹³¹ NETs have also been demonstrated in close association with von Willebrand factor in thrombi in murine and baboon deep vein thrombosis models.^51,133 NET markers are elevated in a multitude of human diseases associated with increased thrombotic risk, such as cancer and rheumatoid arthritis, and NET markers correlate with disease state in patients with thrombotic microangiopathies.^52,133 Interestingly, human patients with vascular occlusions secondary to severe bacterial infections have decreased plasma ability to degrade NETs ex vivo and form intravascular NET clots.⁸⁶

In addition to the deleterious effects of thrombus generation, NET components can be directly toxic to host tissues as well as microbes. NET histones, and to a lesser extent MPO, are cytotoxic to both endothelial and epithelial cells.¹⁷⁰ Many diseases and their animal models demonstrate the capacity of NETs to cause collateral host tissue damage. For example, in murine models of LPS-induced acute lung injury, NETs are generated and cause organ injury and local and systemic inflammation; NET degradation by DNase protects against lung injury.¹¹⁸ Circulating NET markers are also increased in the plasma of human patients with acute respiratory distress syndrome and correlate with disease severity and mortality.¹⁰⁴ Thus, the antimicrobial effects of NETs can cause significant bystander damage.

Biomarkers of NETosis

Multiple putative biomarkers have been investigated for the identification and quantification of NETosis, but presently there is no gold standard.^94,137 All of the available techniques have both strengths and weaknesses and distinct specificities for NETosis versus other mechanisms of DNA extrusion or passive release. It should be recognized that biomarkers that lack specificity for NETosis can still provide other valuable insights into pathogenesis. For instance, regardless of whether cfDNA is released by NETosis or other mechanisms, its procoagulant properties likely contribute to the morbidity and mortality of pathogenic immunothrombosis.⁵⁸

Cell-Free DNA (cfDNA)

Dyes capable of fluorescence when bound to nucleic acids are the most frequently used method for quantifying extracellular DNA in body fluids^{48,52,83,127,134,185} and for experiments using isolated neutrophils.^61,81 Total cfDNA provides a high-throughput, quantitative measure of DNA release applicable to a wide range of species.^29,155 However, measurement of cfDNA alone does not distinguish NETosis from double-stranded DNA derived from other sources. Extracellular DNA may derive from release of NET-like structures by non-neutrophils (eg, eosinophils, macrophages), from release during apoptotic or necrotic cell death, or from enucleation of maturing erythroid precursors.^{6,60,98,128,201} Extracellular DNA from apoptosis, necrosis, and erythroid maturation is not accompanied by cytoplasmic proteins. This may have pathophysiologic and therapeutic relevance as the presence of cytoplasmic proteins affects the properties of NETs, DNA, and histones.¹²⁸ Human clinical studies initially suggested that cfDNA likely originated from NETosis if there is a concurrent increase in DNA-histone complexes and neutrophil-derived proteins.^175,205 However, such correlations do not rule out release of cfDNA from endothelial cells or parenchymal cells, as demonstrated by investigations using bone marrow chimeric mice.²⁰⁴ More specialized approaches, such as determining the tissue of origin based on epigenetic signatures, have been used in human studies but have yet to be applied to companion animal samples.^98,190

Biomarker studies require careful design to ensure they reliably reflect in vivo cfDNA concentrations, including control of pre-analytical factors.⁴⁵ For example, plasma is preferred to serum, as serum can contain high concentrations of DNA released from leukocytes after sample collection.^45,103,193 The time between collection and processing,^20,227 centrifugation conditions (duration, relative centrifugal force),⁴⁵ and storage conditions^45,215 also influence DNA concentrations, although the effect of delayed processing may be minimized by use of tubes containing a stabilizing agent.¹⁵¹ Investigators must also consider if DNA extraction is required. DNA has been measured directly in serum²¹⁴ and plasma,^52,134,147 but autofluorescence¹⁸⁵ and interfering substances may introduce inaccuracy.^83,185 The most commonly used dyes are PicoGreen^{48,127,134,214} and Sytox Green.^147,185 Manufacturer’s instructions for the cell-viability dye Sytox Green do not list specific interfering substances, but an effect of growth medium is mentioned.¹⁶² This suggests that a complex matrix such as plasma may also contain interfering substances. For PicoGreen, the manufacturer lists numerous interfering substances, some of which are routinely present in plasma (eg, albumin, IgG).¹⁶¹ In addition, icteric and hemolytic interferences occur for both dyes.^83,185 To avoid interferences, DNA extraction can be performed before plasma DNA quantification.⁸³ However, extraction does not provide 100% recovery of DNA, and performance varies between different extraction protocols.^108,186,227

NET Components and NETosis Inducers

Increased concentrations or gene expression of inducers of NETosis (eg, IL-8, TNF-α, and HMGB1) is reported in veterinary patients with a variety of clinical conditions.^{56,62,70,200,202,212} This suggests NETosis may occur in various disease states, but it should be recognized that these markers are not specific for NETosis but contribute to numerous inflammatory and cell death pathways.^28,33,163 As discussed above, NETosis is an umbrella term for a variety of molecular pathways that result in DNA release,⁹⁴ so it is possible that NETs may be produced without concurrent increases in the concentrations of a particular inducer.⁷⁵

Concentrations or activity of individual NET components is increased in plasma and other body fluids in a number of clinical conditions.^{3,100,199,200} These include neutrophil granule proteins (eg, NE,^3,18 MPO^3,18,199), nuclear proteins (eg, histones,¹⁸ HMGB1^56,158), and DNA-histone complexes termed nucleosomes.^100,199 Similar to cfDNA or NET inducers, these markers are not specific for NETosis. For instance, NE and MPO are released during neutrophil degranulation,⁹⁶ while histones and HMGB1 are present in all nucleated cells and can be released during forms of cell death other than NETosis.^4,85,174

Citrullinated Histones

Citrullinated histones are purported to be highly specific markers for NETosis.^{100,118,194,195} This is based on evidence that knocking out or inhibiting PAD4 (the enzyme responsible for histone citrullination during NETosis) reduces NET formation.^14,110,217 However, involvement in NETosis does not necessarily imply specificity, since H3 citrullination has also been reported in neutrophils undergoing apoptosis, autophagy, and necrosis.¹⁶⁸ In addition, studies aiming to document NETosis in clinical conditions have used western blots¹⁰⁰ and ELISAs^195,196 to quantify citrullinated histones in body fluids, but these assays do not determine the cells from which the histones originated. A quantitative ELISA for citrullinated H3 is not currently available for use in veterinary species. The enzymes PAD2 and PAD4 responsible for histone citrullination are expressed in a variety of cell types,²¹⁶ and overexpression of PAD4 has been documented in cells other than neutrophils in cancer²⁵ and autoimmunity.¹³⁶ Citrullination at various residues of histone H3 has been cited as evidence of NETosis^100,195 but is also detectable in non-neutrophil cell types, including oligodendrocytes,¹³⁶ cultured fibroblast-like synoviocytes,¹⁴ and cancer cell lines.²⁷ The sensitivity of citrullinated histones for NETosis detection is also debated.⁹⁴ Investigations using PMA to trigger NETosis have produced conflicting results regarding H3 citrullination. Some studies suggest PAD4 activation and histone citrullination are essential for PMA-induced NETosis, while others have documented PMA induced-NETosis in the absence of citrullination.^94,148,149 Antibody performance likely contributes to these inconsistent results.¹⁴⁸ There are marked differences in the signals generated for western blots of the same neutrophil lysates using distinct anti-citrulline antibodies, to the extent that using an alternative antibody can lead to a different interpretation of the experiment.¹⁴⁸ These observations regarding antibody performance raise important issues regarding appropriate controls for measurement of NET markers in biological fluids. A widely used rabbit polyclonal anti-histone H3 antibody (abcam ab5103) shows weak reactivity with non-stimulated neutrophil lysates.¹⁴⁸ Anecdotally, longer incubation times are also reported to increase H3 citrullination in unstimulated neutrophils.⁹⁴ These are not necessarily problems for ex vivo experiments as signals can be compared between stimulated and control neutrophil lysates prepared after standardized incubation times and containing consistent concentrations of histones. However, such issues are more difficult to negate when comparing body fluids from different animals, which may contain different total histone H3 concentrations and histones originating from neutrophils at different stages of maturity.

Complexes of NET Components

Demonstration of DNA or histones complexed with neutrophil granule proteins (eg, NE or MPO) is generally considered specific for NETosis.¹³⁷ Three approaches that are commonly used to quantify DNA/granule protein complexes are ELISA, immunofluorescence, and flow cytometry. It should be noted, however, that extracellular interactions between negatively charged DNA and positively charged proteins such as NE and MPO might mimic NETs¹⁹⁴ and create false-positive results.⁸⁴

Sandwich ELISAs have been constructed with an anti-DNA and anti-myeloperoxidase antibody pair to quantify DNA/myeloperoxidase complexes in human and murine body fluids.^{24,91,134,214} The assays have yet to be fully validated, however, and presently there are no equivalent commercial kits. Creation of a robust ELISA for NET fragments is challenging, not least because NETs are highly variable in size. Large DNA fragments might physically block access of the anti-protein antibody to its substrate. This was proposed as the reason for erroneous results with a commercial anti-citrullinated histone/myeloperoxidase complex ELISA that was withdrawn from the market shortly after release (Cayman Chemical, personal communication). Exposing samples to brief DNase digestion before analysis to reduce the size of the DNA components might help,⁸⁸ but care must be taken to avoid overdigestion that may disrupt the NETs and produce false-negative results.

Immunofluorescence demonstrating spatial overlap of staining for DNA and protein components has been used to detect NETs in isolated neutrophil preparations,¹⁸ tissue sections,¹⁶ body fluid smears,^11,65,66,111 and in vivo.¹⁴¹ Quantification can be performed by manually counting NETs, but this is subjective and susceptible to error induced by microscopic field selection.³⁶ Automated or semi-automated alternatives are described and could improve objectivity and reduce the labor-intensive nature of manual quantification.^17,66,145 Further work is required to fully validate microscopy-based biomarkers for use in clinical samples and to investigate the possibility that cells lysed during blood smear production may mimic NETs.¹²¹

Several strategies are described for quantification of NETs in whole blood or isolated neutrophil preparations by flow cytometry, sometimes combined with image analysis.^{55,101,138,233} These focus on identification of early morphologic changes associated with NETosis²³³ or cell-associated NETs rather than circulating NET fragments.^55,101,138 Flow cytometry offers the potential to overcome the subjectivity that affects many immunostaining studies,^101,138 but information regarding the performance of flow cytometry as a biomarker of NETosis is currently limited.¹²⁵ For whole blood studies, the effects of pre-analytical factors (eg, time between collection and processing) have not been reported, and there is a lack of detailed descriptions of analytical performance criteria such as coefficients of variation.^55,101 Further work is needed to assess these various aspects of flow cytometric NETosis detection, but this area clearly holds future promise.

There is now a substantial body of literature describing measurements of NETosis biomarkers in a variety of sample types collected from various companion animal patient populations. The major limitation of this literature base, however, is the lack of specificity of many of these biomarkers for NETosis. As discussed above, there are multiple potential sources of biomarkers, such as cfDNA and histones, aside from neutrophils undergoing NETosis. In particular, apoptosis and necrosis likely contribute to the concentrations of HMGB-1, cfDNA, nucleosomes, and histones reported in the literature. Indeed, the cfDNA measured in patients with cancer may actually originate from the neoplastic cells themselves, potentially through apoptosis or cell necrosis.^8,9,102 In other scenarios, plasma cfDNA measured in companion animals may not even represent host DNA but could be present due to concurrent parasitemia due to Babesia spp., Leishmania, or filarial nematodes.¹

As such, currently the best evidence for NETosis in veterinary medicine likely comes from measurements of citrullinated histones and from imaging studies that document the presence of NETs in samples collected and handled so as to prevent ex vivo activation. Studies that have measured multiple biomarkers of NETosis are likely more robust. As previously noted, however, almost all potential NETosis biomarkers have overlapping origins that can confound interpretation of histone, nucleosome, and HMGB-1 measurements.

NETosis in Diseases of Animals

Infection

Some of the most compelling evidence for NETosis in companion animals relates to their role in antimicrobial and antiparasitic host defense. cfDNA and nucleosome concentrations have been investigated as prognostic biomarkers in canine sepsis.¹⁰⁷ A prospective study enrolled 45 dogs with sepsis, 10 dogs with nonseptic systemic inflammatory response syndrome, and 15 healthy controls. Biomarker concentrations were measured at admission, and patients were followed to hospital discharge. cfDNA and nucleosome concentrations were significantly higher in dogs with sepsis compared to healthy controls. cfDNA concentrations were also significantly higher in dogs with sepsis that had bacteremia compared to those with negative blood cultures, suggesting that this cfDNA may derive from intravascular NETosis. As such, the findings suggest that point-of-care cfDNA measurement might aid identification of bacteremia in dogs with signs of sepsis.

Canine neutrophils release NETs in vitro in response to E. coli lipopolysaccharide.¹¹² NETs were visualized by fluorescence microscopy of living neutrophils and using immunofluorescent microscopy for the NET components cfDNA, citrullinated histone H3, and myeloperoxidase. This process appears to be dependent on PAD4 because it was inhibited by the PAD4 inhibitor Cl-amidine. Compared to NETs induced by PMA, LPS stimulation resulted in smaller NETs with less extracellular citrullinated histone H3. In one case series of dogs with sepsis, NETs were identified using immunofluorescence microscopy in cytology samples of septic foci.¹¹¹ Samples of endotracheal tracheal wash, bronchoalveolar lavage fluid, and abdominal and pleural effusion collected from 3 dogs with sepsis and 2 other nonseptic disease processes were fixed, permeabilized, and stained for MPO, citrullinated H3, and cfDNA. NETs were identified based on co-localization of these components and quantified compared to the total number of neutrophils. NETs were identified in the fluid samples collected from all 3 septic dogs and in limited numbers from a dog with sterile chronic bronchitis.

A prospective study evaluated uteri removed from normal dogs during routine spay procedures and from dogs with pyometra.¹⁶⁴ Uterine contents were evaluated by bacterial culture and for identification of NETs by immunocytochemistry for histone, NE, or MPO; fluorescence microscopy; and scanning electron microscopy. The study identified NETs in uterine contents in all pyometra cases and could be seen entrapping bacteria. No bacteria and no NETs were identified in the control uteri.

A similar study by the same group evaluated the in vitro capacity of equine neutrophils to generate NETs by chemical activation or through ex vivo stimulation with bacteria associated with equine metritis, including Streptococcus equi zooepidemicus, E. coli, and Staphylococcus capitis.¹⁶⁶ Incubation of neutrophils with bacteria alone significantly increased NETosis compared to neutrophils treated only with PMA. Streptococcus equi zooepidemicus induced fewer NETs than did E. coli or Staphylococcus capitis. In addition, NETs were present in endometrial mucus samples taken from mares with bacterial endometritis. Although NETs may play protective roles in combating the bacteria that cause endometritis, certain components of NETs have the capacity to trigger endometrial fibroplasia that could lead to excessive collagen deposition and endometrial fibrosis. In an in vitro study, incubation of endometrial tissue explants with NET components including NE, cathepsin G, and MPO was associated with an increase in type I collagen protein expression.¹⁶⁵ It remains to be determined if this process can also occur in vivo and if it contributes to the pathogenesis of equine endometrial fibrosis. In a study evaluating the peritoneal fluid of women with endometriosis, NETs were present in 49% of patients, compared to only 18% of controls. In addition, when NETs were present, there were significantly more of them in the peritoneal fluid of women with endometriosis compared to controls.¹²

Feline neutrophils stimulated with protozoan parasites release structures comprising DNA and histones consistent with NETs.²¹⁸ Neutrophils from cats with symptomatic FeLV infections have significantly greater NET release by unstimulated neutrophils compared to FeLV^– and FeLV⁺ asymptomatic cats. This suggests that feline NET release can be modulated by viral infection and suggests the mechanism is overactivated in FeLV⁺ cats.

Although macrophages are the definitive host cells for Leishmania in dogs, neutrophils are the first cells to encounter the parasite following inoculation. Canine blood neutrophils isolated from healthy dogs were exposed to L. infantum promastigotes in vitro.¹⁵⁹ The neutrophils efficiently engulfed the parasite and generated intracellular superoxide radicals, but there was minimal release of NETs. This may suggest that NETosis is not an important host defense response against Leishmania or that the conditions necessary for efficient NETosis (eg, concurrent cell types such as platelets) were not present in the experimental system employed. Trypanosoma cruzi efficiently induced canine blood neutrophils to form NETs that were decorated with NE, as evaluated by cfDNA measurement and immunofluorescence microscopy.³⁵ Although T. cruzi can induce NET formation, the role of this host defense response in vivo is uncertain, because T. cruzi can circulate for years in dogs. The parasite can reside intracellularly in macrophages, which may reduce NET formation and help protect it from the host’s defenses.

Canine Immune-Mediated Hemolytic Anemia

Immune-mediated hemolytic anemia in dogs is strongly associated with thrombosis³⁷; hence, there is considerable interest in potential links between NETs and immunothrombosis in this condition. Nucleosomes (DNA-histone complexes) may also play physical roles in promoting and propagating clot formation, since they contribute to the physical clot structure and impair clot dissolution by inhibiting fibrinolysis.¹⁷⁸ cfDNA competes with plasmin on fibrin, which results in decreased clot lysis.⁴⁷ cfDNA can also suppress fibrinolysis by accelerating the inactivation of tissue plasminogen activator (tPA) by plasminogen-activator inhibitor-1 (PAI-1).¹¹⁷ Canine NETs have been demonstrated to increase the rate of thrombus formation and reduce the potential for fibrinolysis of any resulting blood clots.⁸² There is solid evidence that NETosis occurs in dogs with IMHA. Nucleosomes were measured in the serum of dogs with primary immune-mediated hemolytic anemia using a commercially available ELISA and were significantly higher in IMHA cases than controls.⁸¹ A subsequent prospective observational study of 28 dogs with IMHA and 20 healthy controls was conducted to assess the cfDNA concentrations as measured by PicoGreen fluorescence.⁸³ Concentrations of cfDNA were significantly higher in dogs with IMHA compared to controls, and high cfDNA concentrations were associated with increased odds of death. The origin of this cfDNA is uncertain, but it appears to result from increased release rather than decreased degradation, because DNase activity was not different between cases and controls. In a comparable study by a separate group, concentrations of cfDNA, nucleosomes, and citrullinated histone were measured in plasma samples from 35 dogs with IMHA and from 56 healthy controls.¹⁰⁰ cfDNA concentrations were above the reference interval in 17% of dogs with IMHA, while nucleosome concentrations were increased in almost all dogs with IMHA. Citrullinated histone H3 was detected in 84% of samples from dogs with IMHA.

Asthma

Equine asthma, also known as recurrent airway obstruction, is a disease characterized by reversible airway obstruction, bronchoconstriction, and neutrophilic inflammation. Bronchoalveolar lavage fluid from horses with active recurrent airway obstruction contained NETs, while BAL fluid from horses with recurrent airway obstruction in remission did not.³¹ Interestingly, horses express 2 distinct secretoglobin family 1A members. These proteins are secreted by bronchiolar epithelial cells and are a major constituent of airway surface fluid. Their concentrations are reduced in human asthma and in equine recurrent airway obstruction. Ex vivo, recombinant secretoglobin 1A1A increases neutrophil oxidative burst and phagocytosis, reduces neutrophil chemotaxis, and inhibits NET formation. It is undetermined if these phenomena are mechanistically and causally related or if they represent epiphenomena in equine recurrent airway obstruction.

Low-density granulocytes are a distinct subset of proinflammatory cells that can be isolated from the peripheral blood mononuclear cell fractions of patients with immune-mediated and neoplastic diseases. These cells have an increased propensity for NETosis.²³ A study compared the low-density neutrophil profiles of horses with severe equine asthma to those of healthy controls.⁷³ The response profile of the low-density neutrophils in these animals was most consistent with a pro-inflammatory low-density granulocyte morphology. Specifically, these cells had increased expression of N-formylmethionine-leucyl-phenylalanine receptors and an increased capacity to produce NETs. However, there were no differences between the healthy and the asthmatic horses.

NETs within the lungs have been associated with the severity of airway obstruction and inflammation in humans with asthma. Similarly, NETosis was 2-fold higher in lavage fluid from the lungs of asthmatic horses compared to controls.²⁰⁶ Induced NETosis was enhanced in airway neutrophils from asthmatic horses relative to controls, but there was no difference in NETosis by blood neutrophils between healthy and asthmatic horses. Similarly, glucocorticoids did not affect NETosis by blood neutrophils but did diminish NETosis by airway neutrophils.

Toxicant Exposure

Two studies have evaluated the effect of environmental toxicants on NET formation by canine neutrophils. Exposure of canine neutrophils to sodium arsenite leads to formation of extracellular structures composed of DNA decorated with histones, NE, and MPO.²¹⁹ These structures were visualized by fluorescence confocal microscopy and appear consistent with previous descriptions of NETs. The mechanisms of NET formation in response to sodium arsenite are unknown at this time, however. Exposure of canine neutrophils to nickel nitrate hexahydrate (a common heavy metal used in batteries) also causes release of NETs based on fluorescence confocal microscopy observations.²²⁰ In addition, nickel exposure increased cfDNA release as quantified by a PicoGreen fluorescence assay. The mechanism of nickel-induced NET formation remains obscure, and the clinical significance of these findings is uncertain, particularly because environmental exposures of companion animals to nickel or arsenic are rare.

Trauma

A preliminary study assessed several techniques for measurement of cfDNA in canine plasma obtained from dogs with cancer, with sepsis, and following trauma.¹⁰⁸ Concentrations were also measured in samples from healthy controls. Compared to controls, plasma cfDNA concentrations were significantly increased in dogs with sepsis, trauma, and neoplasia. The findings were extended to a prospective case-control study in which plasma cfDNA and nucleosome concentrations were evaluated as prognostic biomarkers in canine trauma.¹⁰⁶ The study enrolled 49 dogs with moderate-severe trauma and followed them to hospital discharge. Concentrations of cfDNA and nucleosomes were positively correlated and were significantly higher in injured dogs compared to healthy controls. Only nucleosome concentration correlated with injury severity scores. Non-survivors had significantly higher nucleosome concentrations than survivors. Although cfDNA concentrations have been documented to be increased in dogs following trauma, the kinetics of this biomarker over time are unknown. A prospective study evaluated 3 groups of dogs undergoing ovariohysterectomy, cranial cruciate ligament repair, or hemilaminectomy and measured cfDNA concentrations repeatedly in the 72 hours following surgery.²²³ This study suggested that the biological half-life of plasma cfDNA is ∼6 hours. In dogs undergoing hemilaminectomy, cfDNA concentrations differed significantly between the time of admission and 6–12 hours after surgery. The authors suggested that the short half-life of cfDNA might be useful for assessment of acute severe tissue injury.

Other Conditions

A study compared the plasma cfDNA concentrations in a heterogeneous population of 97 dogs with various disease processes to those of 24 healthy dogs.²⁰ Plasma cfDNA was measured by fluorometry without prior DNA extraction. The diseased dog population had significantly higher cfDNA than clinically normal dogs and the concentration of cfDNA correlated with disease severity as assessed by American Society of Anesthesiologists status. Within the diseased population, non-survivors had significantly higher cfDNA concentrations than survivors. No attempt was made to identify the origin of the cfDNA measured; hence, the contribution of NETosis to these results cannot be determined. The authors concluded that measurement of canine cfDNA could be a useful nonspecific disease indicator and prognostic tool.

Strenuous exercise has been demonstrated to increase plasma cfDNA concentrations in dogs,⁷⁸ but cfDNA concentrations do not appear to be useful for identification of exertional rhabdomyolysis in racing sled dogs.³⁸ Dogs with gastric dilation and volvulus had significantly greater plasma cfDNA concentrations compared to controls, but cfDNA concentrations were not predictive of complications or of survival.²⁰⁰

In an experimental model in dogs, autologous blood clots were injected to induce pulmonary thromboembolism, and then plasma cfDNA concentrations were measured with a PicoGreen assay.²⁰³ Blood clot size correlated with induced pulmonary vascular resistance, pulmonary artery pressure, and plasma cfDNA concentrations. The cfDNA release was thrombus-dependent, because microsphere lung embolization did not alter plasma cfDNA concentrations. The same group also demonstrated that autologous blood clots allowed to undergo fibrinolysis release DNA in vitro.

Heat stroke in dogs is also associated with NETosis. In a prospective study, serum histones were measured at hospital admission in 16 dogs with heatstroke and compared to those of 7 healthy controls. Serum histone concentrations in dogs with heatstroke were significantly higher than in controls.¹⁹ Serum histone concentrations were also significantly higher among non-survivors and in those dogs with severe hemostatic derangements. The correlation between serum histones and coagulation abnormalities may reflect disease severity or might suggest a mechanistic link between histone release and hemostatic derangement. Further investigation of this finding is required. The correlation between histone concentrations and coagulation abnormalities is of interest, because histones may be amenable to pharmacologic manipulation.

NETs as Therapeutic Targets

Since the first description of NET formation in 2004,¹⁸ and the recognition of the thrombotic potential of these structures,^46,57 there has been an explosion of literature in this field. As understanding of the nature, mechanisms, and consequences of NETosis has grown, so has interest in therapeutic manipulation of this process. The complex and orchestrated nature of NETosis itself, and the multicomponent nature of the structures generated, provides numerous putative therapeutic targets. The major treatment strategies currently under investigation are summarized in Table 1, and the most important are discussed in detail below. To date, no studies of therapies targeting NETosis in companion animals have been reported.

Table 1.

Major Treatment Strategies Currently Under Investigation for the Management of Immunothrombosis.

Therapy	Proposed mechanism	Diseases	References
DNase I	Degrades extracellular DNA, breaks down NETs, reduces thrombotic potential	In vitro Endotoxemia Sepsis Ischemia/reperfusion injury Systemic lupus erythematosus Cystic fibrosis	2, 24, 30, 32, 34, 122, 123, 143, 169, 173, 180, 207, 208, 229
PAD4 inhibition	Prevents histone citrullination required for NETosis	In vitro Endotoxemia Sepsis Ischemia/reperfusion injury Systemic lupus erythematosus	92, 93
Anti-histone antibodies	Reduces histone cytotoxicity and thrombotic potentiation	Endotoxemia Sepsis Ischemia/reperfusion injury Pancreatitis	87, 144
Heparins	Scavenges extracellular histones and inhibits NETosis	In vitro Sepsis Histone infusion	50, 79, 126, 172, 213, 222
Antiplatelet agents	Prevents platelet activation and reduces platelet-neutrophil interactions that potentiate NETosis	In vitro Endotoxemia Sepsis	24, 99
Thrombomodulin	Unknown, inhibits NETosis	In vitro Sepsis	72, 181
Activated protein C	Unknown, inhibits NETosis	In vitro Sepsis	69
N-acetylcysteine	Antioxidant, reduces ROS and NETosis	Systemic lupus erythematosus	54, 97, 157
Metformin	Unknown, inhibits NETosis	Diabetes	142, 177
Complement inhibitors (C1 esterase inhibitor, anti-C5 antibodies)	Unknown, inhibits NETosis	Influenza Bacterial pneumonia	64, 224
Vitamin D	Unknown, reduces NETosis and endothelial dysfunction	Systemic lupus erythematosus	67
Antibiotics (azithromycin, chloramphenicol, gentamicin)	Unknown, inhibits NETosis	In vitro	21, 124
PGE₂	Unknown, potentially inhibits ROS generation or activity	In vitro Hematopoietic stem cell transplant	39, 182
Anti-inflammatory drugs	Unknown, inhibits NETosis	In vitro Sepsis	99, 232
Immunomodulatory drugs	Inhibits NETosis	In vitro	63
Antimalarials	Blocks dendritic cell processing of NETs through TLR9	Systemic lupus erythematosus	184

Abbreviations: NETs, neutrophil extracellular traps; NETosis, formation of NETs; PAD4, peptidylarginine deiminase 4; PGE₂, prostaglandin E₂; ROS, reactive oxygen species; TLR9, toll-like receptor 9.

While NETs and immunothrombosis may offer clinicians the potential to prevent and treat disease, it is clear that NETs are Janus-faced. Constrained, regulated, and controlled NETosis is likely protective, while excessive NETosis contributes to organ dysfunction and mortality through immunothrombosis. This poses substantial challenges for clinicians seeking to intervene therapeutically.¹⁷¹ For instance, treatment of murine Staphylococcus aureus skin infections with DNase, an enzyme that digests DNA, leads to systemic bacterial dissemination.²³⁰ Similarly, early treatment of septic mice with DNAse leads to deterioration in their condition, but late treatment reduces mortality.¹²³ A similar study also demonstrated that systemic treatment with recombinant DNase attenuated sepsis-induced organ dysfunction and improved survival in septic mice.³² Much work remains to be done identifying the safest and most effective treatment strategies and determining which patients will benefit.

DNase

NETs are primarily composed of extracellular DNA. This cfDNA is crucial for the capture of microbes but also plays a prominent role in immunothrombosis by restraining and co-localizing high-molecular-weight kininogen in proximity to the primary factors of the contact pathway of coagulation.¹⁵³ The helical structure of DNA is also important for contact pathway activation. Digestion of DNA by DNase abolishes these procoagulant effects.^59,191 Humans with acute thrombotic microangiopathies have increased plasma concentrations of NETosis biomarkers. These patients have deficient DNase activity, such that supplementation of plasma with recombinant human DNase I may restore endogenous NET-degradation activity in these patients.⁸⁶

There is considerable interest in the use of DNase I in the management of autoimmune conditions such as systemic lupus erythematosus.^7,130 Human patients in the 1950s and 1960s were given the compound on an experimental basis with anecdotal temporary improvement in their conditions. Development of recombinant human DNase I in the 1990s enabled management of sputum viscosity in patients with cystic fibrosis.¹⁸⁰ Studies in mouse models of systemic lupus erythematosus suggest that recombinant DNase I may improve kidney histologic lesions and prolong survival,^122,207 but these effects are not consistent.²⁰⁸ A phase Ib, randomized, blinded placebo-controlled trial of 17 patients with systemic lupus erythematosus nephritis suggested that recombinant DNase I is safe, achieves appreciable activity after IV administration, and was not associated with the development of neutralizing antibodies. No beneficial effects on markers of the disease process were discernable, however.³⁴

Treatment of mice subjected to ischemia-reperfusion injury with DNase I (during ischemia and reperfusion periods) significantly reduced NETosis, inflammatory cell infiltration, and thrombin-antithrombin complex generation and improved perfusion in post-ischemic muscle. This DNase treatment did not alter skeletal muscle fiber injury or concentrations of proinflammatory molecules, however.²

Treatment of patients with suspected sepsis is an attractive possibility, but using DNase carries risk because the cause of the inflammatory response seen clinically is not always readily apparent, and it is possible to mistake the nature of the pathogen. NETs may be essential for defense against fungal pathogens, and inadvertent DNase intervention might cause a deterioration in the patient’s condition.¹⁵ DNase treatment of sepsis is also complicated by temporal effects. Studies to date have used mouse cecal-ligation puncture models and suggest that early therapy is deleterious, while late treatment reduces organ damage and bacterial dissemination.¹²³ Concurrent treatment with DNase and antibiotics resulted in improved survival, reduced bacteremia, and organ dysfunction in a separate murine study, however,³² which might indicate that combined therapies may limit any detrimental effects of the DNase therapy and potentiate the benefits.

Experimental studies have linked NETosis to acute lung injury,¹⁷³ a phenomenon that is likely dependent on platelet-neutrophil interactions.²⁴ DNase therapy in disorders like transfusion-related acute lung injury may be beneficial.¹⁹⁸ Results from murine studies of DNase therapy in lung injury are not consistent, however, with 2 studies evaluating DNase therapy in models of ventilator-induced lung injury demonstrating varying levels of benefit from this intervention.^169,229 These disparities may result from different disease severities at the time of intervention, particularly if DNase therapy alters aspects of neutrophil biology other than NETs, such as inhibiting cell recruitment. The tissue location in which NETosis is occurring (eg, blood vs lung vs liver, etc) may influence the outcome of therapy by allowing the spread of organisms in one area, while reducing thrombotic complications in another.^30,143

PAD4 Inhibition

The enzyme PAD4 is a key therapeutic target for diminishing NETosis because of its essential role in catalyzing the histone citrullination crucial for NET formation.¹⁰⁹ Sepsis models utilizing PAD4-knockout mice demonstrated that PAD4 deficiency improves survival and decreases the severity of organ dysfunction without exacerbating bacteremia.^13,132 In a separate murine study of lethal endotoxemia, LPS-induced NETosis was inhibited by treatment with the PAD2/PAD4 inhibitor YW3-56.¹¹⁶ This therapy also reduced pulmonary vascular leakage and diminished the resulting lung injury and translated into a survival benefit. Systemic treatment of mice with inhibitors of PAD4 improves survival in sepsis models¹¹⁵ and protects hepatocytes from liver ischemia-reperfusion injury.⁷⁷ PAD4 is also an important target in autoimmune disease. In mouse models of SLE, inhibition of PAD4 with the drug BB-Cl-amidine decreased autoimmune responses, protected against vascular damage, decreased proteinuria, and reduced skin and endothelial dysfunction.^92,93 The same PAD4 inhibitor also reduced mortality in a murine sepsis model.¹⁴ Despite all these potential benefits, the long-term physiologic consequences of systemic PAD4 inhibition are unknown. As such, therapeutic strategies aimed at PAD4 may have unintended consequences, and safe therapies targeting this enzyme may be difficult to develop.

Anti-Histone Antibodies

In the extracellular environment, histones are pro-inflammatory, cytotoxic, and prothrombotic.¹⁹² Histones facilitate thrombus formation in both platelet-dependent and platelet-independent manners⁵⁹ and impair clot dissolution by inhibiting fibrinolysis.¹⁷⁸ Histones are key components of NETs, making them a rational potential target for therapeutic interventions. Multiple strategies have been used to reduce the cytotoxicity, organ dysfunction, and mortality that can arise from increased concentrations of histones in tissues or in circulation,¹⁹² but several histone subtypes exist. This complicates therapeutic intervention, particularly because histone subtype varies with the disease process. For instance, antibodies against histone H3 may reduce mortality in mouse models of pancreatitis,⁸⁷ while in mouse models of hepatic ischemia/reperfusion injury, histone neutralization using antibodies against both histones H3 and H4 significantly protects against injury.⁷⁶ In a histone-induced acute kidney injury model, antibodies against histone H2A and H4 suppressed renal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function.⁴ The same antibodies also reduced mortality in lipopolysaccharide, tumor necrosis factor or cecal-ligation, and puncture models of sepsis in mice.²²⁶ Separately, antibodies against H1 reduce the mortality of mice in LPS models of sepsis,⁹⁵ and anti-H3 antibodies also significantly improve survival in mouse cecal-ligation and puncture sepsis models.¹¹⁵ As such, anti-histone antibody therapies will likely need to be targeted to individual disorders, and this may also be species-specific. At this time, anti-histone antibody therapies have been deployed only in mice; hence, it is uncertain if such therapies will generalize to humans or to companion animals.

Heparins and Antiplatelet Agents

Antithrombotic therapies may be of value in the management of excessive NETosis and immunothrombosis.¹¹⁴ Potential therapeutic strategies include the use of unfractionated heparin, low-molecular-weight heparins, and antiplatelet agents to target distinct aspects of immunothrombosis pathogenesis.^126,172 Extracellular histones released during NETosis can be scavenged by heparin and may prevent histone interactions with tissues and with platelets.⁵⁰ These effects are noteworthy, because treatment of dogs with individually dose-adjusted heparin improves survival in IMHA,⁷¹ a disease in which NETosis has been repeatedly demonstrated.^81,83,100 Heparin, through unknown mechanisms, has been shown to inhibit NETosis in human neutrophils ex vivo.¹²⁶ Unfractionated heparin improves survival in mouse cecal-ligation and puncture models of sepsis better than antibiotics alone, while the combination of antimicrobial drug therapy and heparin was superior to either.²¹³ In rat models of histone-induced organ dysfunction, both unfractionated heparin and low-molecular-weight heparins reduced histone-induced leukopenia and thrombocytopenia, diminished organ dysfunction, and improved survival.⁷⁹ Non-anticoagulant heparin, which binds to extracellular histones but has minimal affinity for antithrombin, has also been shown to reduce histone-mediated cytotoxicity, attenuate endotoxin-mediated lung injury, and improve survival in septic mice.²²²

Pharmaceutical activated protein C is no longer available for use in humans¹²⁹; however, interest remains in determining how the drug affects NETosis. In human sepsis, plasma histone concentrations are inversely related to endogenous aPC activity,⁴⁴ and several studies suggest that aPC reduces the toxicity of histones^179,226 and inhibits NETosis.⁶⁹ Similarly, thrombomodulin attenuates histone-induced thrombin generation and endothelial cell death in vitro, likely through its ability to activate protein C.¹⁵⁴ Thrombomodulin also reduces NETosis and blunts the prothrombotic state resulting from cecal-ligation and puncture-induced peritonitis in rats.⁷² Recombinant human thrombomodulin is used successfully in humans with sepsis-induced DIC in Japan,^68,228 and Phase III clinical trials are ongoing in the USA (NCT01598831, NCT03517501). If these prove successful, then some of the benefits may derive from inhibition of immunothrombosis.

Platelet activation and platelet-neutrophil interaction are crucial for NETosis in vivo. Thus, antiplatelet therapy may attenuate NETosis and reduce the potential consequences of excessive immunothrombosis. Endotoxin-mediated platelet activation is associated with increased thromboxane A₂ generation.¹⁵⁰ This is noteworthy because pretreatment of mice with aspirin decreases NETosis and lung injury following endotoxemia.²⁴ Similarly, endotoxin-mediated platelet activation is dependent on ADP in canine and equine platelets,¹¹² which suggests that clopidogrel therapy might attenuate platelet-neutrophil interaction and NETosis. In humans with sepsis, prior antiplatelet therapy is associated with improved outcome.^26,43 This finding prompted initiation of a clinical trial investigating aspirin therapy in people with sepsis,⁴² which completed the enrollment of 17,000 patients in June 2018. Publication of this trial is still pending. Platelet inhibition in sepsis may have unexpected effects, however. In a murine peritonitis model, pretreatment of mice with aspirin resulted in suppression of NETosis coupled with increased bacteremia.⁹⁹ As with other therapies to regulate NETosis, timing of therapy, as well as type and location of infection, may alter the impact of antiplatelet therapy on outcomes in humans and animals with sepsis.

Conclusions

The processes of NETosis and immunothrombosis are complex, orchestrated, and integral components of the innate immune response, but when misdirected or in excess, these processes can be harmful to the host. Detecting NET components such as cfDNA or histones is straightforward, but accurately and definitively determining the origin of these components is much more difficult. Developing ways to enhance differentiation of NETosis from apoptosis and necrosis will be crucial to the continued development of this field and will underpin the use of therapeutic interventions. The study of NETs and immunothrombosis holds much future promise both diagnostically and therapeutically, but a considerable amount of work remains to be done in this arena.

Footnotes

Author Contribution

Robert Goggs, Unity Jeffery, Dana N. LeVine, Ronald H. L. Li All authors contributed equally to this manuscript

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Robert Goggs

References

Akter

Nakao

Imasato

, et al. Potential of cell-free DNA as a screening marker for parasite infections in dog [published online May 2018]. Genomics. doi:10.1016/j.ygeno.2018.05.020.

Albadawi

Oklu

Raacke Malley

, et al. Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice. J Vasc Surg. 2016;64(2):484–493.

Aldabbous

Abdul-Salam

McKinnon

, et al. Neutrophil extracellular traps promote angiogenesis: evidence from vascular pathology in pulmonary hypertension. Arterioscler Thromb Vasc Biol. 2016;36(10):2078–2087.

Allam

Scherbaum

Darisipudi

, et al. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol. 2012;23(8):1375–1388.

Ammollo

Semeraro

, et al. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost. 2011;9(9):1795–1803.

Aucamp

Bronkhorst

Badenhorst

CPS

, et al. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev Camb Philos Soc. 2018;93(3):1649–1683.

Barnado

Crofford

Oates

. At the bedside: neutrophil extracellular traps (NETs) as targets for biomarkers and therapies in autoimmune diseases. J Leukoc Biol. 2016;99(2):265–278.

Beck

Hennecke

Bornemann-Kolatzki

, et al. Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA. Plos One. 2013;8(9):e75485.

Beffagna

Sammarco

Bedin

, et al. Circulating cell-free DNA in dogs with mammary tumors: short and long fragments and integrity index. PLoS One. 2017;12(1): e0169454.

10.

Beiter

Wartha

Albiger

, et al. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol. 2006;16(4):401–407.

11.

Beiter

Fragasso

Hudemann

, et al. Neutrophils release extracellular DNA traps in response to exercise. J Appl Physiol. 2014;117(3):325–333.

12.

Berkes

Oehmke

Tinneberg

, et al. Association of neutrophil extracellular traps with endometriosis-related chronic inflammation. Eur J Obstet Gynecol Reprod Biol. 2014;183;193–200.

13.

Biron

Chung

Chen

, et al. PAD4 deficiency leads to decreased organ dysfunction and improved survival in a dual insult model of hemorrhagic shock and sepsis. J Immunol. 2018;200(5):1817–1828.

14.

Biron

Chung

O’Brien

, et al. Cl-amidine prevents histone 3 citrullination and neutrophil extracellular trap formation, and improves survival in a murine sepsis model. J Innate Immun. 2017;9(1):22–32.

15.

Branzk

Lubojemska

Hardison

, et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol. 2014;15(11):1017–1025.

16.

Brinkmann

Abu Abed

Goosmann

, et al. Immunodetection of NETs in paraffin-embedded tissue. Front Immunol. 2016;7:513–513.

17.

Brinkmann

Goosmann

Kühn

, et al. Automatic quantification of in vitro NET formation. Front Immunol. 2013;3:413.

18.

Brinkmann

Reichard

Goosmann

, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532–1535.

19.

Bruchim

Ginsburg

Segev

, et al. Serum histones as biomarkers of the severity of heatstroke in dogs. Cell Stress Chaperones. 2017;22(6):903–910.

20.

Burnett

Cave

Gedye

, et al. Investigation of cell-free DNA in canine plasma and its relation to disease. Vet Q. 2016;36(3):122–129.

21.

Bystrzycka

Manda-Handzlik

Sieczkowska

, et al. Azithromycin and chloramphenicol diminish neutrophil extracellular traps (NETs) release. Int J Mol Sci. 2017;18(12):E2666.

22.

Carestia

Kaufman

Rivadeneyra

, et al. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets. J Leukoc Biol. 2016;99(1):153–162.

23.

Carmona-Rivera

Kaplan

. Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity. Semin Immunopathol. 2013;35(4):455–463.

24.

Caudrillier

Kessenbrock

Gilliss

, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest. 2012;122(7):2661–2671.

25.

Chang

Han

Pang

, et al. Increased PADI4 expression in blood and tissues of patients with malignant tumors. BMC Cancer. 2009;9:40.

26.

Chen

Janz

Bastarache

, et al. Prehospital aspirin use is associated with reduced risk of acute respiratory distress syndrome in critically ill patients: a propensity-adjusted analysis. Crit Care Med. 2015;43(4):801–807.

27.

Cherrington

Morency

Struble

, et al. Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones. Plos One. 2010;5(7):e11768.

28.

Chu

W-M

. Tumor necrosis factor. Cancer Letters. 2013;328(2):222–225.

29.

Chuammitri

Ostojic

Andreasen

, et al. Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils. Vet Immunol Immunopathol. 2009;129(1-2):126–131.

30.

Clark

Tavener

, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007;13(4):463–469.

31.

Cote

Clark

Viel

, et al. Secretoglobin 1A1 and 1A1A differentially regulate neutrophil reactive oxygen species production, phagocytosis and extracellular trap formation. Plos One. 2014;9(4):e96217.

32.

Czaikoski

Mota

Nascimento

, et al. Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis. Plos One. 2016;11(2):e0148142.

33.

David

Dominguez

Hamilton

, et al. The IL-8/IL-8 R axis: a double agent in tumor immune resistance. Vaccines (Basel). 2016;4(3):E22.

34.

Davis

Jr Manzi

Yarboro

, et al. Recombinant human Dnase I (rhDNase) in patients with lupus nephritis. Lupus. 1999;8(1):68–76.

35.

de Buhr

Bonilla

Jimenez-Soto

, et al. Extracellular trap formation in response to trypanosoma cruzi infection in granulocytes isolated from dogs and common opossums, natural reservoir hosts. Front Microbiol. 2018;9:966.

36.

de Buhr

von Köckritz-Blickwede

. How neutrophil extracellular traps become visible. J Immunol Res. 2016;2016:4604713.

37.

deLaforcade

Bacek

Blais

, et al. Consensus on the rational use of antithrombotics in veterinary critical care (CURATIVE): domain 1—Defining populations at risk. J Vet Emerg Crit Care. 2019;29(1):37–48.

38.

Devall

Goggs

Hansen

, et al. Serum myoglobin, creatine kinase, and cell-free DNA in endurance sled dogs and sled dogs with clinical rhabdomyolysis. J Vet Emerg Crit Care. 2018;28(4):310–316.

39.

Domingo-Gonzalez

Martinez-Colon

Smith

, et al. Inhibition of neutrophil extracellular trap formation after stem cell transplant by prostaglandin E2. Am J Respir Crit Care Med. 2016;193(2):186–197.

40.

Dyer

Chen

Haldeman

, et al. Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA. Sci Rep. 2018;8(1):2068.

41.

Dyer

Chen

Neal

, et al. HMGB1 expression on platelet-derived microparticles promotes deep vein thrombosis. J Am Coll Surg. 2016;223(4):S153.

42.

Eisen

Moore

Leder

, et al. AspiriN to inhibit SEPSIS (ANTISEPSIS) randomised controlled trial protocol. BMJ Open. 2017;7(1):e013636.

43.

Eisen

Reid

McBryde

. Acetyl salicylic acid usage and mortality in critically ill patients with the systemic inflammatory response syndrome and sepsis. Crit Care Med. 2012;40(6):1761–1767.

44.

Ekaney

Otto

Sossdorf

, et al. Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation. Crit Care. 2014;18(5):543.

45.

El Messaoudi

Rolet

Mouliere

, et al. Circulating cell free DNA: preanalytical considerations. Clinica Chimica Acta. 2013;424:222–230.

46.

Engelmann

Massberg

. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol. 2013;13(1):34–45.

47.

Esmon

. Molecular circuits in thrombosis and inflammation. Thromb Haemost. 2013;109(3):416–420.

48.

Etulain

Martinod

Wong

, et al. P-selectin promotes neutrophil extracellular trap formation in mice. Blood. 2015;126(2):242–246.

49.

Fuchs

Abed

Goosmann

, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 2007;176(2):231–241.

50.

Fuchs

Bhandari

Wagner

. Histones induce rapid and profound thrombocytopenia in mice. Blood. 2011;118(13):3708–3714.

51.

Fuchs

Brill

Duerschmied

, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107(36):15880–15885.

52.

Fuchs

Kremer Hovinga

Schatzberg

, et al. Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies. Blood. 2012;120(6):1157–1164.

53.

Gansler

Jaax

Leiting

, et al. Structural requirements for the procoagulant activity of nucleic acids. Plos One. 2012;7(11):e50399.

54.

Garcia

Francis

Dawood

, et al. Attention deficit and hyperactivity disorder scores are elevated and respond to N-acetylcysteine treatment in patients with systemic lupus erythematosus. Arthritis Rheum. 2013;65(5):1313–1318.

55.

Gavillet

Martinod

Renella

, et al. Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples. Am J Hematol. 2015;90(12):1155–1158.

56.

Goggs

Letendre

. High mobility group box-1 and pro-inflammatory cytokines are increased in dogs after trauma but do not predict survival. Front Vet Sci. 2018;5:179.

57.

Gould

Lysov

Liaw

. Extracellular DNA and histones: double-edged swords in immunothrombosis. J Thromb Haemost. 2015;13(suppl 1):S82–S91.

58.

Gould

Stafford

, et al. Cell-free DNA modulates clot structure and impairs fibrinolysis in sepsis. Arterioscler Thromb Vasc Biol. 2015;35(12):2544–2553.

59.

Gould

Swystun

, et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol. 2014;34(9):1977–1984.

60.

Granger

Faille

Marani

, et al. Human blood monocytes are able to form extracellular traps. J Leukoc Biol. 2017;102(3):775–781.

61.

Gray

Lucas

MacKellar

, et al. Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps. J Inflamm. 2013;10(1):12.

62.

Gruen

Messenger

Thomson

, et al. Evaluation of serum cytokines in cats with and without degenerative joint disease and associated pain. Vet Immunol Immunopathol. 2017;183:49–59.

63.

Gupta

Giaglis

Hasler

, et al. Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. Plos One. 2014;9(5):e97088.

64.

Hair

Enos

Krishna

, et al. Inhibition of immune complex complement activation and neutrophil extracellular trap formation by peptide inhibitor of complement C1. Front Immunol. 2018;9:558.

65.

Hamaguchi

Hirose

Akeda

, et al. Identification of neutrophil extracellular traps in the blood of patients with systemic inflammatory response syndrome. J Int Med Res. 2013;41(1):162–168.

66.

Hamaguchi

Hirose

Matsumoto

, et al. Neutrophil extracellular traps in bronchial aspirates: a quantitative analysis. Eur Resp J. 2014;43(6):1709–1718.

67.

Handono

Sidarta

Pradana

, et al. Vitamin D prevents endothelial damage induced by increased neutrophil extracellular traps formation in patients with systemic lupus erythematosus. Acta Med Indones. 2014;46(3):189–198.

68.

Hayakawa

Yamakawa

Saito

, et al. Recombinant human soluble thrombomodulin and mortality in sepsis-induced disseminated intravascular coagulation. A multicentre retrospective study. Thromb Haemost. 2016;115(6):1157–1166.

69.

Healy

Puy

Fernandez

, et al. Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo. J Biol Chem. 2017;292(21):8616–8629.

70.

Hegemann

Wondimu

Kohn

, et al. Cytokine profile in canine immune-mediated polyarthritis and osteoarthritis. Vet Comp Orthop Traumatol. 2005;18(2):67–72.

71.

Helmond

Polzin

Armstrong

, et al. Treatment of immune-mediated hemolytic anemia with individually adjusted heparin dosing in dogs. J Vet Intern Med. 2010;24(3):597–605.

72.

Helms

Clere-Jehl

Bianchini

, et al. Thrombomodulin favors leukocyte microvesicle fibrinolytic activity, reduces NETosis and prevents septic shock-induced coagulopathy in rats. Ann Intensive Care. 2017;7(1):118.

73.

Herteman

Vargas

Lavoie

. Characterization of circulating low-density neutrophils intrinsic properties in healthy and asthmatic horses. Sci Rep. 2017;7(1):7743.

74.

Hickey

Kubes

. Intravascular immunity: the host-pathogen encounter in blood vessels. Nat Rev Immunol. 2009;9(5):364–375.

75.

Hirose

Hamaguchi

Matsumoto

, et al. Presence of neutrophil extracellular traps and citrullinated histone H3 in the bloodstream of critically ill patients. Plos One. 2014;9(11):e111755.

76.

Huang

Evankovich

Yan

, et al. Endogenous histones function as alarmins in sterile inflammatory liver injury through toll-like receptor 9 in mice. Hepatology. 2011;54(3):999–1008.

77.

Huang

Tohme

Al-Khafaji

, et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology. 2015;62(2):600–614.

78.

Hunt

Cave

Bridges

, et al. Plasma NT-proBNP and cell-free DNA concentrations after prolonged strenuous exercise in working farm dogs. J Vet Intern Med. 2018;32(1):135–141.

79.

Iba

Hashiguchi

Nagaoka

, et al. Heparins attenuated histone-mediated cytotoxicity in vitro and improved the survival in a rat model of histone-induced organ dysfunction. Intensive Care Med Exp. 2015;3(1):36.

80.

Itakura

McCarty

. Pivotal role for the mTOR pathway in the formation of neutrophil extracellular traps via regulation of autophagy. Am J Physiol Cell Physiol. 2013;305(3):C348–C354.

81.

Jeffery

Kimura

Gray

, et al. Dogs cast NETs too: canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia. Vet Immunol Immunopathol. 2015;168(3–4):262–268.

82.

Jeffery

LeVine

. Canine neutrophil extracellular traps enhance clot formation and delay lysis. Vet Pathol. 2018;55(1):116–123.

83.

Jeffery

Ruterbories

Hanel

, et al. Cell-free DNA and DNase activity in dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2017;31(5):1441–1450.

84.

Jethwa

Nachman

Falk

, et al. False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies. Clin Exp Immunol. 2000;121(3):544–550.

85.

Jiang

Reich

CF III

Pisetsky

. Role of macrophages in the generation of circulating blood nucleosomes from dead and dying cells. Blood. 2003;102(6):2243–2250.

86.

Jimenez-Alcazar

Napirei

Panda

, et al. Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies. J Thromb Haemost. 2015;13(5):732–742.

87.

Kang

Zhang

Hou

, et al. Intracellular HMGB1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice. Gastroenterology. 2014;146(4):1097–1107.

88.

Kano

H HM

Tsuda

Noguchi

, et al. Sandwich ELISA for circulating myeloperoxidase- and neutrophil elastase-DNA complexes released from neutrophil extracellular traps. Adv Techniq Biol Med. 2016;5(1):196.

89.

Keshari

Jyoti

Dubey

, et al. Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition. Plos One. 2012;7(10):e48111.

90.

Keshari

Verma

Barthwal

, et al. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils. J Cell Biochem. 2013;114(3):532–540.

91.

Kessenbrock

Krumbholz

Schonermarck

, et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 2009;15(6):623–625.

92.

Knight

Subramanian

O’Dell

, et al. Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann Rheum Dis. 2015;74(12):2199–2206.

93.

Knight

Zhao

Luo

, et al. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus. J Clin Invest. 2013;123(7):2981–2993.

94.

Konig

Andrade

. A critical reappraisal of neutrophil extracellular traps and netosis mimics based on differential requirements for protein citrullination. Front Immunol. 2016;7:461.

95.

Kusano

Chiang

Inomata

, et al. A novel anti-histone H1 monoclonal antibody, SSV monoclonal antibody, improves lung injury and survival in a mouse model of lipopolysaccharide-induced sepsis-like syndrome. Biomed Res Int. 2015;2015:491649.

96.

Lacy

. Mechanisms of degranulation in neutrophils. Allergy Asthma Clin Immunol. 2006;2(3):98–108.

97.

Lai

Hanczko

Bonilla

, et al. N-acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 2012;64(9):2937–2946.

98.

Lam

WKJ

Gai

Sun

, et al. DNA of erythroid origin is present in human plasma and informs the types of anemia. Clin Chem. 2017;63(10):1614–1623.

99.

Lapponi

Carestia

Landoni

, et al. Regulation of neutrophil extracellular trap formation by anti-inflammatory drugs. J Pharmacol Exp Ther. 2013;345(3):430–437.

100.

Lawson

Smith

O’Brien

, et al. Neutrophil extracellular traps in plasma from dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2018;32(1):128–134.

101.

Lee

Cavanaugh

Leung

, et al. Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis. Int J Lab Hematol. 2018;40(4):392–399.

102.

Lee

Shin

Kim

, et al. Methylation of LINE-1 in cell-free DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors. Sci Rep. 2019;9(1):175.

103.

Lee

Montalvo

Chrebtow

, et al. Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion. 2001;41(2):276–282.

104.

Lefrancais

Mallavia

Zhuo

, et al. Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury. JCI Insight. 2018;3(3):98178.

105.

Leshner

Wang

Lewis

, et al. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol. 2012;3:307.

106.

Letendre

Goggs

. Concentrations of plasma nucleosomes but not cell-free DNA are prognostic in dogs following trauma. Front Vet Sci. 2018;5:180.

107.

Letendre

Goggs

. Determining prognosis in canine sepsis by bedside measurement of cell-free DNA and nucleosomes. J Vet Emerg Crit Care. 2018;28(6):503–511.

108.

Letendre

Goggs

. Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia. J Vet Emerg Crit Care. 2017;27(3):307–314.

109.

Lewis

Liddle

Coote

, et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol. 2015;11(3):189–191.

110.

Lindberg

, et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010;207(9):1853–1862.

111.

RHL

Johnson

Kohen

, et al. A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy. BMC Vet Res. 2018;14(1):210.

112.

RHL

Tablin

. Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase. Vet Immunol Immunopathol. 2017;193–194:29–37.

113.

RHL

Nguyen

Tablin

. Dog platelets express functional TLR4: platelet agonists upregulate platelet surface TLR4 and facilitate LPS-induced platelet activation in dogs (abstract). J Vet Emerg Crit Care. 2017;26(suppl 1):S6.

114.

. The role of heparin in sepsis: much more than just an anticoagulant. Br J Haematol. 2017;179(3):389–398.

115.

Liu

, et al. Citrullinated histone H3: a novel target for the treatment of sepsis. Surgery. 2014;156(2):229–234.

116.

Liang

Pan

Alam

, et al. Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia. Eur J Pharmacol. 2018;833:432–440.

117.

Liaw

Ito

Iba

, et al. DAMP and DIC: the role of extracellular DNA and DNA-binding proteins in the pathogenesis of DIC. Blood Rev. 2016;30(4):257–261.

118.

Liu

Pan

, et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Sci Rep. 2016;6:37252.

119.

Loof

Morgelin

Johansson

, et al. Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense. Blood. 2011;118(9):2589–2598.

120.

Loof

Schmidt

Herwald

, et al.

Coagulation systems of invertebrates and vertebrates and their roles in innate immunity: the same side of two coins?

J Innate Immun. 2011;3(1):34–40.

121.

Lynch

. Peripheral blood smear. In: Walker

Hall

Hurst

, eds. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd ed. Boston: Butterworths; 1990.

122.

Macanovic

Sinicropi

Shak

, et al. The treatment of systemic lupus erythematosus (SLE) in NZB/W F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone. Clin Exp Immunol. 1996;106(2):243–252.

123.

Mai

Khan

Dwivedi

, et al. Delayed but not early treatment with dnase reduces organ damage and improves outcome in a murine model of sepsis. Shock. 2015;44(2):166–172.

124.

Manda-Handzlik

Bystrzycka

Sieczkowska

, et al. Antibiotics modulate the ability of neutrophils to release neutrophil extracellular traps. Adv Exp Med Biol. 2017;944:47–52.

125.

Manda-Handzlik

Ostafin

Bystrzycka

, et al. Flow cytometric quantification of neutrophil extracellular traps: limitations of the methodological approach. Am J Hematol. 2016;91(3):E9–E10.

126.

Manfredi

Rovere-Querini

D’Angelo

, et al. Low molecular weight heparins prevent the induction of autophagy of activated neutrophils and the formation of neutrophil extracellular traps. Pharmacol Res. 2017;123:146–156.

127.

Margraf

Logters

Reipen

, et al. Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock. 2008;30(4):352–358.

128.

Marsman

Zeerleder

Luken

. Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation. Cell Death Dis. 2016;7(12):e2518.

129.

Marti-Carvajal

Sola

Gluud

, et al. Human recombinant protein C for severe sepsis and septic shock in adult and paediatric patients. Cochrane Database Syst Rev. 2012;12:CD004388.

130.

Martinez Valle

Balada

Ordi-Ros

, et al. DNase 1 and systemic lupus erythematosus. Autoimmun Rev. 2008;7(5):359–363.

131.

Martinod

Demers

Fuchs

, et al. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013;110(21):8674–8679.

132.

Martinod

Fuchs

Zitomersky

, et al. PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock. Blood. 2015;125(12):1948–1956.

133.

Martinod

Wagner

. Thrombosis: tangled up in NETs. Blood. 2014;123(18):2768–2776.

134.

Maruchi

Tsuda

Mori

, et al. Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock. Crit Care. 2018;22(1):176–176.

135.

Massberg

Grahl

von Bruehl

, et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med. 2010;16(8):887–896.

136.

Mastronardi

Wood

Mei

, et al. Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation. J Neurosci. 2006;26(44):11387–11396.

137.

Masuda

Nakazawa

Shida

, et al. NETosis markers: quest for specific, objective, and quantitative markers. Clin Chim Acta. 2016;459:89–93.

138.

Masuda

Shimizu

Matsuo

, et al. Measurement of NET formation in vitro and in vivo by flow cytometry. Cytometry A. 2017;91(8):822–829.

139.

Maugeri

Campana

Gavina

, et al. Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Haemost. 2014;12(12):2074–2088.

140.

Maugeri

Franchini

Campana

, et al. Circulating platelets as a source of the damage-associated molecular pattern HMGB1 in patients with systemic sclerosis. Autoimmunity. 2012;45(8):584–587.

141.

McDonald

Urrutia

Yipp

, et al. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Host Microbe. 2012;12(3):324–333.

142.

Menegazzo

Scattolini

Cappellari

, et al. The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo. Acta Diabetol. 2018;55(6):593–601.

143.

Meng

Paunel-Gorgulu

Flohe

, et al. Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice. Crit Care. 2012;16(4):R137.

144.

Merza

Rahman

Zhang

, et al. Human thrombin-derived host defense peptides inhibit neutrophil recruitment and tissue injury in severe acute pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2014;307(9):G914–G921.

145.

Mohanty

Sørensen

Nordenfelt

. NETQUANT: automated quantification of neutrophil extracellular traps. Front Immunol. 2018;8:1999.

146.

Nathan

. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol. 2006;6(3):173–182.

147.

Naumann

Hazeldine

Dinsdale

, et al. Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: a prospective observational study. Plos One. 2017;12(12):e0189870.

148.

Neeli

Radic

. Current challenges and limitations in antibody-based detection of citrullinated histones. Front Immunol. 2016;7:528.

149.

Neeli

Radic

. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release. Front Immunol. 2013;4:38.

150.

Nocella

Carnevale

Bartimoccia

, et al. Lipopolysaccharide as trigger of platelet aggregation via eicosanoid over-production. Thromb Haemost. 2017;117(8):1558–1570.

151.

Norton

Lechner

Williams

, et al. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin Biochem. 2013;46(15):1561–1565.

152.

Noubouossie

Reeves

Strahl

, et al. Neutrophils: back in the thrombosis spotlight. Blood. 2019;133(20):2186–2197.

153.

Oehmcke

Morgelin

Herwald

. Activation of the human contact system on neutrophil extracellular traps. J Innate Immun. 2009;1(3):225–230.

154.

Osada

Minami

Arioka

, et al. Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation. Thromb Res. 2017;160:51–57.

155.

Palic

Ostojic

Andreasen

, et al. Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils. Dev Comp Immunol. 2007;31(8):805–816.

156.

Papayannopoulos

Metzler

Hakkim

, et al. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol. 2010;191(3):677–691.

157.

Patel

Kumar

Jyoti

, et al. Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation. Nitric Oxide. 2010;22(3):226–234.

158.

Peng

Liu

Ojcius

, et al. Mineral particles stimulate innate immunity through neutrophil extracellular traps containing HMGB1. Sci Rep. 2017;7(1):16628.

159.

Pereira

Valerio-Bolas

Santos-Mateus

, et al. Canine neutrophils activate effector mechanisms in response to Leishmania infantum. Vet Parasitol. 2017;248:10–20.

160.

Pieterse

Rother

Yanginlar

, et al. Neutrophils discriminate between lipopolysaccharides of different bacterial sources and selectively release neutrophil extracellular traps. Front Immunol. 2016;7:484.

161.

Quant-iT™ PicoGreen ® dsDNA reagent and kits. Invitrogen Molecular Probes, Life Technologies Corp., Carlsbad, CA, USA; 2008.

162.

SYTOX® green nucleic acid stain. Invitrogen Molecular Probes, Life Technologies Corp., Carlsbad, CA, USA; 2006.

163.

Raucci

Palumbo

Bianchi

. HMGB1: a signal of necrosis. Autoimmunity. 2007;40(4):285–289.

164.

Rebordao

Alexandre-Pires

Carreira

, et al. Bacteria causing pyometra in bitch and queen induce neutrophil extracellular traps. Vet Immunol Immunopathol. 2017;19:8–12.

165.

Rebordao

Amaral

Lukasik

, et al. Constituents of neutrophil extracellular traps induce in vitro collagen formation in mare endometrium. Theriogenology. 2018;113:8–18.

166.

Rebordao

Carneiro

Alexandre-Pires

, et al. Neutrophil extracellular traps formation by bacteria causing endometritis in the mare. J Reprod Immunol. 2014;106:41–49.

167.

Rohrbach

Slade

Thompson

, et al. Activation of PAD4 in NET formation. Front Immunol. 2012;3:360.

168.

Romero

Fert-Bober

Nigrovic

, et al. Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis. Sci Transl Med. 2013;5(209):209ra150.

169.

Rossaint

Herter

Van Aken

, et al. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation. Blood. 2014;123(16):2573–2584.

170.

Saffarzadeh

Juenemann

Queisser

, et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. Plos One. 2012;7(2):e32366.

171.

Saffarzadeh

Preissner

. Fighting against the dark side of neutrophil extracellular traps in disease: manoeuvres for host protection. Curr Opin Hematol. 2013;20(1):3–9.

172.

Sanchez

. Low molecular weight heparins: a new tool to disetangle from the NETs. Pharmacol Res. 2017;123:157.

173.

Sayah

Mallavia

Liu

, et al. Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med. 2015;191(4):455–463.

174.

Scaffidi

Misteli

Bianchi

. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 2002;418(6894):191–195.

175.

Schimmel

Nur

Biemond

, et al. Nucleosomes and neutrophil activation in sickle cell disease painful crisis. Haematologica. 2013;98(11):1797–1803.

176.

Schmaier

McCrae

. The plasma kallikrein-kinin system: its evolution from contact activation. J Thromb Haemost. 2007;5(12):2323–2329.

177.

Schuiveling

Vazirpanah

Radstake

, et al.

Metformin, a new era for an old drug in the treatment of immune mediated disease?

Curr Drug Targets. 2018;19(8):945–959.

178.

Schulz

Engelmann

Massberg

. Crossroads of coagulation and innate immunity: the case of deep vein thrombosis. J Thromb Haemost. 2013;11(suppl 1):233–241.

179.

Semeraro

Ammollo

Morrissey

, et al. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood. 2011;118(7):1952–1961.

180.

Shak

Capon

Hellmiss

, et al. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci U S A. 1990;87(23):9188–9192.

181.

Shimomura

Suga

Kuriyama

, et al. Recombinant human thrombomodulin inhibits neutrophil extracellular trap formation in vitro. J Intensive Care. 2016;4:48.

182.

Shishikura

Horiuchi

Sakata

, et al. Prostaglandin E2 inhibits neutrophil extracellular trap formation through production of cyclic AMP. Br J Pharmacol. 2016;173(2):319–331.

183.

Smeeth

Cook

Thomas

, et al. Risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting. Lancet. 2006;367(9516):1075–1079.

184.

Smith

Vivekanandan-Giri

Tang

, et al. Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus. Arthritis Rheumatol. 2014;66(9):2532–2544.

185.

Smith

Lawson

McMichael

, et al. Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis. Vet Clin Pathol. 2017;46(2):278–286.

186.

Sorber

Zwaenepoel

Deschoolmeester

, et al. A comparison of cell-free DNA isolation kits: isolation and quantification of cell-free DNA in plasma. J Mol Diagn. 2017;19(1):162–168.

187.

Sreeramkumar

Adrover

Ballesteros

, et al. Neutrophils scan for activated platelets to initiate inflammation. Science. 2014;346(6214):1234–1238.

188.

Stahl

Svensson

Morgelin

, et al. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome. Blood. 2006;108(1):167–176.

189.

Steppich

Seitz

Busch

, et al. Modulation of tissue factor and tissue factor pathway inhibitor-1 by neutrophil proteases. Thromb Haemost. 2008;100(6):1068–1075.

190.

Sun

Jiang

Chan

, et al. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proc Natl Acad Sci U S A. 2015;112(40):E5503–E5512.

191.

Swystun

Mukherjee

Liaw

. Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus. J Thromb Haemost. 2011;9(11):2313–2321.

192.

Szatmary

Huang

Criddle

, et al. Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders. J Cell Mol Med. 2018;22(10):4617–4629.

193.

Taback

O’Day

Hoon

DSB

. Quantification of circulating DNA in the plasma and serum of cancer patients. Ann N Y Acad Sci. 2004;1022(1):17–24.

194.

Thalin

Daleskog

Goransson

, et al. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma. Immunol Res. 2017;65(3):706–712.

195.

Thalin

Demers

Blomgren

, et al. NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation. Thromb Res. 2016;139:56–64.

196.

Thalin

Lundstrom

Seignez

, et al. Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer. Plos One. 2018;13(1):e0191231.

197.

Thammavongsa

Missiakas

Schneewind

. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science. 2013;342(6160):863–866.

198.

Thomas

Carbo

Curtis

, et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood. 2012;119(26):6335–6343.

199.

Tripodi

Ammollo

Semeraro

, et al. Hypercoagulability in patients with Cushing disease detected by thrombin generation assay is associated with increased levels of neutrophil extracellular trap-related factors. Endocrine. 2017;56(2):298–307.

200.

Troia

Giunti

Calipa

, et al. Cell-free DNA, high-mobility group box-1, and procalcitonin concentrations in dogs with gastric dilatation-volvulus syndrome. Front Vet Sci. 2018;5:67.

201.

Ueki

Melo

Ghiran

, et al. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans. Blood. 2013;121(11):2074–2083.

202.

Uhrikova

Rauserova-Lexmaulova

Rehakova

, et al. C-reactive protein and high mobility group box 1 in dogs with gastric dilatation and volvulus. J Vet Emerg Crit Care. 2015;25(4):488–494.

203.

Uzuelli

Dias-Junior

Izidoro-Toledo

, et al. Circulating cell-free DNA levels in plasma increase with severity in experimental acute pulmonary thromboembolism. Clin Chim Acta. 2009;409(1-2):112–116.

204.

van der Meer

Kroeze

Hoogendijk

, et al. Systemic inflammation induces release of cell-free DNA from hematopoietic and parenchymal cells in mice and humans. Blood Adv. 2019;3(5):724–728.

205.

van Montfoort

Stephan

Lauw

, et al. Circulating nucleosomes and neutrophil activation as risk factors for deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2013;33(1):147–151.

206.

Vargas

Boivin

Cano

, et al. Neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma. Respir Res. 2017;18(1):207.

207.

Venkatesh

Yoshifuji

Kawabata

, et al. Antigen is required for maturation and activation of pathogenic anti-DNA antibodies and systemic inflammation. J Immunol. 2011;186(9):5304–5312.

208.

Verthelyi

Dybdal

Elias

, et al. DNAse treatment does not improve the survival of lupus prone (NZB x NZW)F1 mice. Lupus. 1998;7(4):223–230.

209.

Vogel

Bodenstein

Chen

, et al. Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest. 2015;125(12):4638–4654.

210.

Vogel

Rath

Borst

, et al. Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes. Biochem Biophys Res Commun. 2016;478(1):143–148.

211.

von Bruhl

Stark

Steinhart

, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012;209(4):819–835.

212.

Voorwald

Marchi

Villacis

, et al. Molecular expression profile reveals potential biomarkers and therapeutic targets in canine endometrial lesions. Plos One. 2015;10(7):e0133894.

213.

Wang

Zhang

, et al. Heparin defends against the toxicity of circulating histones in sepsis. Front Bioscis. 2015;20:1259–1270.

214.

Wang

Sha

, et al. Circulating level of neutrophil extracellular traps is not a useful biomarker for assessing disease activity in antineutrophil cytoplasmic antibody-associated vasculitis. Plos One. 2016;11(2):e0148197.

215.

Wang

Cai

Brady

, et al. Real-time PCR evaluation of cell-free DNA subjected to various storage and shipping conditions. Genet Mol Res. 2015;14(4):12797–12804.

216.

Wang

. Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis. Biochim Biophys Acta. 2013;1829(10):1126–1135.

217.

Wang

Stadler

, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009;184(2):205–213.

218.

Wardini

Guimaraes-Costa

, et al. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol. 2010;91(pt 1):259–264.

219.

Wei

Zhang

Wang

, et al. The formation of canine neutrophil extracellular traps induced by sodium arsenic in polymorphonuclear neutrophils. Chemosphere. 2018;196:297–302.

220.

Wei

Zhang

Wang

, et al. Nickel (II) nitrate hexahydrate triggered canine neutrophil extracellular traps release in vitro. Chemosphere. 2018;208:117–121.

221.

Weinrauch

Drujan

Shapiro

, et al. Neutrophil elastase targets virulence factors of enterobacteria. Nature. 2002;417(6884):91–94.

222.

Wildhagen

Garcia de Frutos

Reutelingsperger

, et al. Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood. 2014;123(7):1098–1101.

223.

Wilson

Burchell

Worth

, et al. Kinetics of plasma cell-free DNA and creatine kinase in a canine model of tissue injury. J Vet Intern Med. 2018;32(1):157–164.

224.

Wygrecka

Kosanovic

Wujak

, et al. Antihistone properties of C1 esterase inhibitor protect against lung injury. Am J Respir Crit Care Med. 2017;196(2):186–199.

225.

Cai

Suresh

, et al. Equine PSGL-1 modifications required for P-selectin binding. Vet Immunol Immunopathol. 2009;131(1–2):33–43.

226.

Zhang

Pelayo

, et al. Extracellular histones are major mediators of death in sepsis. Nat Med. 2009;15(11):1318–1321.

227.

Xue

Teare

Holen

, et al. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta. 2009;404(2):100–104.

228.

Yamakawa

Aihara

Ogura

, et al. Recombinant human soluble thrombomodulin in severe sepsis: a systematic review and meta-analysis. J Thromb Haemost. 2015;13(4):508–519.

229.

Yildiz

Palaniyar

Otulakowski

, et al. Mechanical ventilation induces neutrophil extracellular trap formation. Anesthesiology. 2015;122(4):864–875.

230.

Yipp

Kubes

. NETosis: how vital is it? Blood. 2013;122(16):2784–2794.

231.

Yipp

Petri

Salina

, et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med. 2012;18(9):1386–1393.

232.

Zawrotniak

Kozik

Rapala-Kozik

. Selected mucolytic, anti-inflammatory and cardiovascular drugs change the ability of neutrophils to form extracellular traps (NETs). Acta Biochim Pol. 2015;62(3):465–473.

233.

Zhao

Fogg

Kaplan

. A novel image-based quantitative method for the characterization of NETosis. J Immunol Methods. 2015;423:104–110.

234.

Zhou

Deng

Liu

, et al. Platelet HMGB1 is required for efficient bacterial clearance in intra-abdominal bacterial sepsis in mice. Blood Advances. 2018;2(6):638–648.

Neutrophil-Extracellular Traps,Cell-Free DNA,and Immunothrombosis in Companion Animals: A Review

Abstract

Keywords

Mechanisms of NET Formation

Types of NETosis

Platelet-Neutrophil Interactions in NETosis

Role of NETs in Innate Immunity

Immunothrombosis

Biomarkers of NETosis

Cell-Free DNA (cfDNA)

NET Components and NETosis Inducers

Citrullinated Histones

Complexes of NET Components

NETosis in Diseases of Animals

Infection

Canine Immune-Mediated Hemolytic Anemia

Asthma

Toxicant Exposure

Trauma

Other Conditions

NETs as Therapeutic Targets

DNase

PAD4 Inhibition

Anti-Histone Antibodies

Heparins and Antiplatelet Agents

Conclusions

Footnotes

Author Contribution

Declaration of Conflicting Interests

Funding

ORCID iD

References