NETosis in Surgery

Abstract

Objective:

To provide surgeons with an understanding of the latest research on NETosis, including the pathophysiology and treatment of conditions involving neutrophil extracellular traps (NETs) in the care of surgical patients.

Background:

A novel function of neutrophils, the formation of NETs, was described in 2004. Neutrophils form mesh-like structures of extruded decondensed chromatin, comprising DNA and histones decorated with bactericidal proteins. These NETs exert antimicrobial action by trapping microorganisms and preventing their wider dissemination through the body.

Results:

A narrative review of the existing literature describing NETosis was conducted, including NET pathophysiology, conditions related to NET formation, and treatments relevant to surgeons.

Conclusions:

In addition to its canonical antimicrobial function, NETosis can exacerbate inflammation, resulting in tissue damage and contributing to numerous diseases. NETs promote gallstone formation and acute pancreatitis, impair wound healing in the early postoperative period and in chronic wounds, and facilitate intravascular coagulation, cancer growth, and metastasis. Agents that target NET formation or removal have shown promising efficacy in treating these conditions, although large clinical trials are required to confirm these benefits.

Neutrophils comprise 50% to 75% of leukocytes in the peripheral blood.^1–3 Neutrophils are an essential part of the innate immune system and are the first line of defense against invasive microbial pathogens.^1–8 When the body is infected by a pathogen, neutrophils marginate, roll via endothelial (E)/platelet (P)/leukocyte (L)-selectin interactions with neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and tightly adhere to the vascular endothelium via neutrophil β2-integrin family binding with such endothelial ligands as intercellular adhesion molecule, glycosaminoglycans, or fibrin. Neutrophils can then transmigrate from the peripheral bloodstream to infection sites via pseudopodia formation, paracellular diapedesis, and chemotaxis. This is dependent on the reorganization of the neutrophil actin cytoskeleton and intracellular microtubule formation.^1,4

Rolling and P-selectin interactions are not required for neutrophil extravasation in pulmonary microvasculature or hepatic sinusoids.^1,4 Hepatic sinusoids contain fenestrated or discontinuous endothelium, with areas of direct exposure of extracellular matrix (ECM), which facilitates adhesion and extravasation. Constitutive neutrophil extravasation is a feature of lymphoid tissues containing high endothelial venules, including lymph nodes, lymphoid follicles, and omental milky spots. Extravasated, activated, or adherent neutrophils can engage and destroy pathogens using an arsenal of weapons, including phagocytosis, degranulation and release of cytotoxic enzymes, and oxidative burst. Oxidative burst involves activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with the generation of reactive oxygen species (ROS).^1–8 In 2004, a novel mechanism by which neutrophils attack pathogens was described: the formation of neutrophil extracellular traps (NETs).³

Apart from their canonical role of protecting against disseminated infection, NETs have subsequently been shown to have numerous pathological effects. These include prolonged wound healing, microcrystal diseases, systemic inflammatory response, venous and arterial thromboembolism, impaired immunosurveillance, and promotion of cancer metastases.^1–8 Postoperative inflammation, cytokine release, platelet and neutrophil activation, and NET formation may continue for a considerable time after major surgery and potentially be modified by perioperative interventions.^3,7 This narrative review aimed to examine the current pathophysiology, prevention, and potential treatment of NETosis in surgical conditions.

NEUTROPHIL EXTRACELLULAR TRAPS

NETs are extracellular mesh-like structures formed by extruded decondensed neutrophil chromatin, comprising double-stranded DNA and histones H1, H2A, H2B, H3, and H4.^1,2 This DNA mesh is decorated with bactericidal proteins, including neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, proteinase 3 (PR3), azurocidin, lactoferrin, gelatinase, lysozyme C, calprotectin, neutrophil defensins, and cathelicidins.^1–8 Other proteins and cells may also bind onto this sticky scaffold, including chemotaxins, growth factors, and fibrin.² NETs exert antimicrobial activity by forming a physical barrier to entrap microorganisms, immobilizing them, and preventing their wider dissemination throughout the body.^1,3,9 Furthermore, the NET structure facilitates a high local concentration of antimicrobial peptides and proteins at the site of infection,^1–8 including positively charged lysine-rich histones (H1, H2A, and H2B) and arginine-rich histones (H3 and H4). These histones bind negatively charged gram-negative bacterial cell wall lipopolysaccharides (LPSs) and teichoic acid and lipoteichoic acid in gram-positive bacteria. They also stimulate the release of proinflammatory cytokines and the recruitment of immune cells.^1–8

MECHANISM OF NETOSIS

NETs can arise via 2 pathways.² The predominant pathway is a neutrophil cell death program, termed lytic or suicidal NETosis.² This pathway is mainly driven by the generation of ROS from a NADPH oxidase (NOX)-dependent pathway, or mitochondrial-derived ROS (mROS), a NOX-independent pathway.⁵ The alternative pathway does not involve neutrophil death and is called vital NETosis^2,5 (Fig. 1).

FIGURE 1.

Activating stimuli and molecules involved in the 2 types of NETosis: Lytic NETosis (left) consists of a mechanism that effectively kills neutrophils, releasing the filamentous lattice composed of decondensed chromatin, histones, and lytic enzymes into the extracellular space. Known activators of lytic/suicidal NETosis are phorbol 12-myristate 13-acetate (PMA) and antibodies that bind to the Fc receptor (Fcγ-R), which leads to calcium-dependent activation of NADPH oxidase and the release of reactive oxygen species (ROS). ROS activate the PAD4 enzyme and its translocation from granules to the nucleus. Neutrophil Elastase (NE) and myeloperoxidase (MPO), and the combined action of PAD4, NE, and MPO results in citrullination of histones, particularly Histone 3 (cit-H3), and subsequent chromatin decondensation. The nuclear membrane of neutrophils breaks, and chromatin mixed with enzymes and histones is released first into the cytoplasm and then into the extracellular space, following the rupture of the cell membrane, forming Neutrophil Extracellular Traps (NETs). In vital NETosis (right), neutrophils remain intact, releasing the reticulum via a system of vesicles; the latter mechanism appears to be independent of NADPH oxidase activation. Microbial infections, especially from S. aureus, recognized by Toll-Like Receptor-2 (TLR2) or complement receptor and lipopolysaccharide (LPS)-activated platelets that bind TLR-4 are among the major activators of this second pathway. Reproduced from Ronchetti et al with permission.⁵

LYTIC/SUICIDAL NETOSIS

NOX-Dependent NET Formation

Of the various pathways that form NETs, NOX-dependent pathways are the best described.⁶ NOX2 is an enzyme complex located in neutrophil plasma and phagosome membranes.^1,6 Complex signaling pathways in neutrophils can result in the activation and assembly of NOX2, with the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/protein kinase C (PKC) pathway being a crucial upstream regulator of suicidal NETosis.¹ Stimulation of neutrophil surface membrane receptors activates second messenger signaling via phospholipase C and diacylglycerol (DAG), leading to the release of calcium ions from endoplasmic reticulum stores. The plant toxin phorbol 12-myristate 13-acetate (PMA) enters the neutrophil independently of receptors and simulates the effect of second messenger DAG signaling. The rise in cytoplasmic ionic calcium activates PKC. PKC phosphorylates NOX2 subunits (p47-phox) while suppressing apoptosis by activating anti-apoptotic B-cell lymphoma 2 family proteins, including myeloid leukemia 1 protein.^1,2 Another NOX-dependent NETosis pathway involves the activation of c-Jun N-terminal kinase by bacterial LPSs.¹ When NOX2 is phosphorylated and activated, neutrophils are driven toward suicidal NETosis rather than apoptosis.^1–6

Phosphorylated NOX2 reduces molecular oxygen by transferring electrons from NADPH across membranes.^1,6 This generates the superoxide radical (O₂ ^•−), which is rapidly converted into hydrogen peroxide.^1,6 Alternatively, ROS such as superoxides may be formed by electron leakage onto molecular oxygen from the electron transport chain during mitochondrial respiration powered by glycolysis-generated pyruvate.² NOX-derived ROS may be required for NETosis in contexts when mROS is insufficient (fungal infection, PMA stimulus).² ROS link upstream regulatory pathways and the cellular machinery driving NET formation.²

ROS, in turn, have multiple downstream effects.² ROS-mediated glutathionylation of actin and tubulin is necessary for cytoskeletal changes during NETosis. ROS mediate the release of serine proteases (NE, cathepsin G, PR3, neutrophil serine protease 4, and azurocidin) and the heme-containing MPO into the cytosol from azurophilic neutrophil granules, where they are normally sequestered in resting neutrophils.^1–8 Cytosolic NE binds to F-actin and degrades the intracellular actin cytoskeleton, which effectively blocks phagocytosis and commits the neutrophil to lytic NETosis.² NE then translocates to the nucleus via passive diffusion, a process which is inhibited by dectin-1–mediated phagocytosis of small pathogens.² NE partially cleaves nuclear histones, acting on lysine-rich and arginine-rich C-terminal histone tails.² NE, together with the binding of MPO and nuclear protein DEK, promotes chromatin depolymerization and swelling.^1,2,6 Neutrophil nuclear envelope and cell membrane permeability are compromised by the assembly of gasdermin D pores.² Gasdermin D pores exist in a positive feedback loop with NE: NE promotes the activation of gasdermin D, which in turn enhances NE release from neutrophil granules.² Permeability of the cell membrane increases, and it eventually ruptures, with decondensed DNA released into the extracellular milieu, forming NETs with antimicrobial action.⁵ Disruption of the cell membrane results in neutrophil death (Figs. 1, 2).

FIGURE 2.

Pathways and mechanisms regulating lytic NETosis. NETosis is triggered by microbial and endogenous stimuli via several activating molecules such as RAGE, PSGL1, TLR, fragment crystallizable-gamma immunoglobulin receptors (Fcγ-R) or sialic acid-binding immunoglobulin-type lectins (Siglec), among others. Activation of MAP kinase signaling induces ROS generation by the NADPH oxidase 2 (Nox2). Alternative ROS can be generated by mitochondria. ROS plays a central role in NETosis triggering NE release from the azurosome complex, a process aided by gasdermin D (GSDMD), which is activated by caspase-11 upon exposure to intracellular cytosolic bacteria. NE degrades F-actin and translocates to the nucleus where it will partially cleave histones promoting chromatin decondensation. Chromatin decondensation is also enhanced by the binding of cationic proteins like MPO or DEK and by PAD4-mediated histone citrullination. Phosphorylation of the lamin network drives its disassembly and the breakdowns of the nuclear envelope. High levels of ROS promote DNA damage triggering DNA repair via ATM and BRCA-1. NETosis also depends on cell cycle CDK4/6 and the duplication of centrosomes and autophagy. Inhibitory receptors such as Siglec-5,9 or SIRL1 block NEtosis. Phagocytic receptors like Dectin-1 inhibit NETosis in response to small microorganisms by sequestering NE to phagosomes. ATG7 indicates autophagy-related protein 7; ATM, ataxia-telangiectasia mutated; AZU, azurophilic granule; BRCA, BReast CAncer gene; CDK4/6, cyclin-dependent kinase 4/6; CG, cathepsin G; CR3, complement receptor 3; GSDMD, gasdermin D; IRAK, IL-1 receptor-associated kinase; MEK, MAPK/ERK kinase; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; RIPK1/3, receptor-interacting serine/threonine-protein kinase 1/3; Siglec-5,9, SIRL1, signal inhibitory receptor on leukocytes 1. Reproduced from Hidalgo et al with permission.²

NOX-Independent NET Formation

Suicidal NETosis can occur without the initial activation of NOX2. NOX-independent pathways are driven by extracellular Ca²⁺ influx, which can be stimulated by fungal ionophores (nigericin, ionomycin) and granulocyte-macrophage colony-stimulating factor (GM-CSF).⁸ In turn, calcium ions activate the small-conductance calcium-activated potassium 3 channel (SK3). SK3 mediates the production of mROS, which induces NET formation via peptidyl arginine deiminase 4 (PAD4).¹ Stimulated by calcium ion influx and mROS, PAD4 translocates from the cytosol to the nucleus, where it deiminates arginine residues to citrulline in H1, H3, and H4 histones, which reduces their positive charge.¹ Thus, histone citrullination weakens the electrostatic bond between histones and the negatively charged DNA backbone, causing chromatin decondensation and swelling, nuclear envelope rupture, and NETosis.¹ The role of PAD4 in NOX-dependent NETosis remains contentious.⁶

VITAL NETOSIS

A small subset of neutrophils may adopt an alternative pathway termed vital NETosis, in which vesicles containing chromatin and antimicrobial proteins are released into the extracellular space and assembled into NETs without neutrophil cell death.^1–8 This is a very rapid (5–60 min), tightly controlled process that is stimulated by bacterial infection.¹ However, the exact mechanism underlying this process remains unclear. It appears that bacteria such as Staphylococcus aureus, bacterial LPS, and LPS-activated platelets stimulate vital NETosis by toll-like receptor 2 (TLR2) and TLR4 activation via a NOX-independent pathway.^1,6 It has been proposed that vesicles are filled with nuclear DNA and exported through the intact cell membrane by exocytosis.² Alternatively, vital NETosis can occur when only mitochondrial DNA from neutrophils is released, which requires the formation of oxidized mitochondrial DNA and is NOX- dependent.^1,6,9 Vital NETosis allows neutrophils to maintain normal cellular functions as anuclear cytoplasts after NET release, including crawling, chemotaxis, phagocytosis, and oxidative burst activity.⁹ It is unclear how neutrophils survive and continue this antimicrobial activity after their enucleation.¹ (Fig. 1).

NET BREAKDOWN

Once formed, NETs are removed by macrophages.^1,6 Host plasma Deoxyribonuclease 1 (DNase1) degrades NETs, and complement component 1q (C1q) opsonizes the debris that is formed.^1,6 Subsequently, NETs are internalized by macrophages via a cytochalasin D-dependent endocytic process. NETs are further broken down in lysosomes.

TRIGGERS FOR NETOSIS

NET formation requires the activation of neutrophils either by bacterial toxins, hypoxia, or stimulation of plasma membrane receptors. Such neutrophil surface receptors include the following:

1. G-protein–coupled receptors (GPCR) that are chemoattractant/chemokine/complement receptors: platelet-activating factor receptor, complement component 5a receptor, CXC Motif Chemokine Receptor 1 and -2 (CXCR1/2), Chemokine (C-C motif) receptor 1 and -2 (CCR1/2);

2. Fragment crystallizable-gamma receptors (Fcγ-R) for IgG opsonized antigens or immune complexes;

3. Integrins (Lymphocyte function-associated antigen 1 (LFA-1) αLβ2 integrin, macrophage-1 antigen (Mac-1) αMβ2 integrin, very late activation antigen α4β1 integrin) and selectin ligands (L-selectin, PSGL-1);

4. Cytokine receptors: interleukin 1 (IL-1) receptor, GM-CSFR, interferon alpha receptor, tumor necrosis factor alpha (TNF-α) receptor;

5. Pattern recognition receptors in innate immunity that recognize pathogen-associated molecular patterns and host damage-associated molecular patterns (DAMPs): TLR, receptor for advanced glycation end-products (RAGE), formyl peptide receptors, transmembrane C-type lectin receptors, nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3), cytoplasmic nucleotide-binding and oligomerization domain–like (NOD-like) receptors, intracellular retinoic acid-inducible gene I-like receptors (RIG-I-like receptors).^1–8 (Fig. 2).

There is a diverse range of stimuli for NETosis, which can be both pathologic and physiological, including cytoplasmic calcium ion influx, ROS, signaling cascades (interleukin-1 receptor-associated kinase (IRAK), c-Jun N-terminal kinase, phosphoinositide 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin, and Raf-MEK-ERK-PKC), foreign or microbial pathogens, exosomes, oncogenes (mutant KRAS), proinflammatory cytokines (IL-1β, IL-8, TNF-α, and GM-CSF) and chemokines (IL-8, CXC ligand (CXCL)), microcrystals, or activated platelet-endothelial interactions.^1–8 The full range of factors that stimulate NET formation is incomplete, and many new triggers have been discovered, including those involved in sterile inflammation³ (Fig. 2).

PATHOLOGICAL FACTORS

Microbial Pathogens

Pathogenic bacteria, viruses, fungi, and protozoa are known to stimulate NET formation, as well as microbial components (bacterial flagellin/LPS, Candida albicans hyphae) and bacterial formyl peptides.^1,2 These are recognized by pathogen-associated molecular patterns receptors, including formyl peptide receptor, C-type lectin receptor, TLR-2/4, and NOD-like receptors on neutrophils (Figs. 1, 2). Many microorganisms have developed mechanisms to overcome NETs,³ such as proteolytic enzymes capable of degrading NETs, including extracellular nucleases produced by S. aureus and Streptococcus species.³ Pathogens can also elude NETosis by producing extracellular capsules, such as Pseudomonas aeruginosa and Mycobacterium tuberculosis, or downregulating host inflammatory responses and neutrophil ROS generation (Group A Streptococcus).³

Autoimmune Diseases

Activation of estrogen membrane receptors on neutrophils by 17-β-estradiol, or Fcγ-receptors via immune complexes between IgG and opsonized antigens, have been found to promote NETosis. This is relevant to autoimmune conditions such as systemic lupus erythematosus (SLE) and rheumatoid arthritis and their sexual dimorphism.² When neutrophils are primed with type I interferon, immune complexes drive NET formation.² Furthermore, NETs activate the complement system in SLE through the production of C1q, which in turn decreases NET clearance. Auto-antibodies to cell-free (cf) double-stranded DNA, citrullinated proteins, or MPO/PR3 generated by NETosis are respectively implicated in SLE, rheumatoid arthritis, and anti-neutrophil cytoplasmic autoantibody vasculitis.²

Cytokines

NETs are involved in postoperative inflammation, systemic inflammatory response syndrome and end organ injury.^2,3 Proinflammatory cytokines such as IL-1β, IL-6, IL-8, IL-17A, TNF-α, and C-reactive protein (CRP) induce NETs via neutrophil membrane cytokine receptors and NOX-dependent pathways.² Interleukins (IL-8) increase cytoplasmic levels of ROS, mediated by nuclear factor kappa B (NF-κB) activation via the CXCR2-phosphoinositide 3-kinase-Protein Kinase B pathway and inducible nitric oxide synthase and cyclo-oxygenase 2 (COX2) induction by NF-κB.^2,3 Prostaglandin E2 can be a context-dependent anti-inflammatory mediator and inhibits NET production by increasing intracellular cyclic adenosine monophosphate.² Activated protein C (aPC) also inhibits NETosis via interactions with the endothelial cell protein C receptor and cleaves histones, which form the backbone of NETs.

Neutrophil Adhesion, Integrins, and Platelets

The dispensability of neutrophil adhesion and integrin activation in NET formation has been debated. Integrin activation and neutrophil adhesion appear dispensable for exogenous peroxide, calcium influx, or PMA-induced NETosis. However, it is required for viral, LPS, S. aureus, and activated platelet-induced NETosis.² Direct cell-cell interactions may also regulate NETosis, as attachment to vascular endothelium ligands via neutrophil β2-integrin receptors facilitates NETosis.² NET release can be achieved by TLR2 and TLR4 activation of platelets, which promotes the binding of platelet P-selectin to neutrophil PSGL-1. Activated platelets can thus relay signals such as the presence of LPSs to neutrophils, triggering NET release. Activated platelets also release soluble ligands, including CCL5, platelet factor 4 (PF4/CXCL4), and high mobility group box-1 (HMGB1). These can bind to neutrophil GPCRs and induce NETosis. Neutrophil activation involves the binding of neutrophil αMβ2 integrin (Mac-1) to its counter-receptor glycoprotein 1bα on platelets and is both proinflammatory and prothrombotic.²

Crystal Diseases

Endogenous inducers of sterile lytic NETosis include microcrystals such as those present in gout [monosodium urate (MSU)], pseudogout (calcium pyrophosphate dihydrate), cholelithiasis (cholesterol), atheromatous plaques (cholesterol), and pancreatitis (calcium carbonate).² Crystal diseases share a common pathway involving inadequate neutrophil phagocytosis, DAMPs recognition, and activation of NET formation. This is further discussed below.

HOST PHYSIOLOGICAL FACTORS

NET release is influenced by host factors, including patient age, gut microbiome, and circadian rhythm.² Not all neutrophils are equally predisposed to release NETs.² NET release varies across species, tissues, and within the same organism in different physiological states.² In the same physiological state, activation of human neutrophils results in NET formation in only 60% of the cells.² The reason for these differences in response is not fully understood. It is hypothesized that as neutrophils exit the peripheral bloodstream by crossing the basement membrane and circulating through tissues, they become primed, which is permissive for NETosis.²

Host Microenvironment and Homeostasis

Physiological conditions, including pH, O₂ content, hyperglycemia, and osmolarity, can modulate NET formation.² Moderately alkaline conditions cause increased NET formation after stimulation with PMA, uric acid microcrystals, or bacterial LPSs.¹¹ An increase in extracellular pH (>7.4) raises the intracellular concentration of calcium ions, enhancing the production of mROS, PAD4 citrullination of histone 3 (cit-H3), histone cleavage, and NOX-independent suicidal NETosis.^10,11 In contrast, a decrease in pH inhibits NETosis, possibly by inhibiting NE/MPO activity or glycolysis. The local oxygen concentration has been shown to influence NETosis.⁶ Stabilization of the hypoxia-inducible factor 1 (HIF-1α) protein, the major transcription factor that regulates adaptation to hypoxic conditions, promotes NETosis, whereas knockout of the HIF1A gene suppresses NETosis.⁶ Hyperglycemia or hyperosmotic stress stimulates NETosis by increasing neutrophil intracellular calcium ions.¹²

NETOSIS and PATHOLOGY

Along with its canonical antimicrobial functions, NETosis can exacerbate inflammation, resulting in tissue damage and contributing to numerous diseases.² Thus, a fine balance between NET formation and clearance is required to maintain homeostasis.² If this balance is disturbed by an abnormal increase in NET production or a decrease in NET clearance, pathological NETosis can result.²

CRYSTAL DISEASES

Cholelithiasis

Cholelithiasis is a common disease, with an estimated prevalence of 25% in European Caucasians and 60% to 70% in Native Americans over the age of 50.^13,14 Furthermore, cholelithiasis and its complications, including acute cholecystitis, common bile duct stones, cholangitis, and gallstone pancreatitis, result in substantial morbidity and mortality.^13,14

The mechanisms that lead to cholelithiasis development have been studied but remain incompletely elucidated.¹⁴ Bile is excreted by hepatic biliary canalicular cells and is composed of water, lipids, bilirubin conjugates, proteins, and electrolytes.¹⁴ The lipid component includes bile salts, phospholipids (>96% of which are mixed phosphatidylcholines), and cholesterol.¹⁴ Gallbladder luminal epithelial cells concentrate and acidify the bile to increase the solubility of cholesterol and calcium salts.¹³ When bile becomes super-saturated and its solubilization capacity is overwhelmed, calcium salts and cholesterol crystals precipitate, forming gallstones.^13,14 This is frequently the result of excessive cholesterol secretion and inadequate bile salt/phospholipid excretion from the liver. This is influenced by estrogen, obesity, hyperlipidemia, ethnicity, Western diet, female sex, multiparity, increasing age, and decreased hepatic cholesterol 7α-hydroxylase activity.^13,14

Muñoz et al¹³ demonstrated that NETs act as aggregation factors contributing to gallstone formation.¹³ After coming into contact with calcium salts or cholesterol crystals, aggregated NETs form, becoming the “glue” that binds the components of the developing gallstone together. The key mechanism of this process is neutrophil macropinocytosis of calcium and cholesterol crystals and ROS formation.¹³ Failure of neutrophils or macrophages to successfully complete phagocytosis of large cholesterol crystals stimulates NETosis.¹³ The successive lamination of cholesterol and NE then leads to gallstone development. The formation of aggregated NETs in developing gallstones was observed using fluorescence microscopy and revealed deposits of extracellular DNA (ecDNA), large ecDNA aggregates, and NE activity.¹³ In larger gallstones, microscopy showed ecDNA patches on the surface of the gallstones.¹³ The highest NE activity in the majority (88%) of gallstones analyzed was found at the gallstone surface (Fig. 3).¹³

FIGURE 3.

Composite macrophotograph of human gallstones, previously immersed in an aqueous propidium iodide solution, under oblique white light (left) and 488 nm light (right) illumination, respectively; extracellular DNA deposits appear in red color. Shown is 1 out of 3 independent experiments. Scale bar, 1 cm. Reproduced from Munoz et al with permission.¹³

The precise mechanism by which neutrophils enter the biliary system and initiate this process has not been fully described. However, in both animal models and humans, the formation of cholesterol gallstones is preceded by inflammation of the gallbladder wall, including edema and the presence of inflammatory cells.^12,13 Furthermore, bacteria have been implicated in the pathogenesis of both cholesterol and pigmented stones. A possible mechanism contributing to gallstone formation is the NLRP3 inflammasome.¹⁵ The NLRP3 inflammasome is triggered by cholesterol crystals in atherosclerosis and MSU crystals in gout, as well as other endogenous crystals (calcium oxalate, calcium pyrophosphate dihydrate, and cystine) and exogenous crystals (asbestos, silica, aluminum adjuvant, and titanium dioxide).^2,13,15

Cholesterol Crystals and NETosis

Duewell et al¹⁶ demonstrated that cholesterol crystals taken up by macrophages are incompletely degraded in phagosomes and transferred to lysosomes, where they induce the rupture of the phagolysosomal membrane. This results in the release of the lysosomal cysteine protease cathepsin B, which acts as a signal to prime and activate the NLRP3 inflammasome.¹⁵ Undegraded cholesterol crystals released upon phagolysosomal membrane rupture may also act as DAMPs sensed by pattern recognition receptors, resulting in activation of the innate immune system.¹⁵ Cholesterol crystals thus induce inflammation via “frustrated phagocytosis” and NLRP3 inflammasome activation, with IL-1β and IL-18 release.¹⁵ In turn, this inflammation attracts neutrophils to the site of cholesterol crystals, resulting in potent stimulation of neutrophils by interleukin-1 receptor-associated kinase and Receptor Interacting Protein Kinases (RIPK1–RIPK3)-mixed lineage kinase domain-like protein signaling and NET formation.²

Pancreatitis and Pancreatic Ductal Stones

In addition to their role in gallstone formation, NETs contribute to acute pancreatitis and pancreatic ductal calcium carbonate stones in chronic pancreatitis.¹⁷ Pancreatic secretions contain alkaline bicarbonate ions and calcium carbonate crystals, which can strongly stimulate NET formation via PAD4 activation.^2,13 Pancreatic ductal occlusion caused by aggregated NETs can drive acute pancreatic inflammation.^2,13 In a murine model of severe acute pancreatitis, NETs induced trypsin activation, inflammation, and tissue injury in vivo.¹⁷ Cell-free histones derived from NETs disrupt the pancreatic acinar cell plasma membrane, resulting in acinar cell death. Interactions between activated platelets and NETs within the pancreas also cause thrombus formation and injury to the pancreatic microvasculature, leading to pancreatic necrosis. Systemic manifestations of severe acute pancreatitis associated with NETosis include acute lung injury, vascular damage, venous thrombosis, increased vascular permeability, and acute kidney injury. NETs also contribute to increased disease severity in patients with pancreatic ductal stones.¹⁷

Gout

Acute exacerbations of recurrent gout are common (17.2%–44.3%) after major surgery, related to fasting, systemic acidosis, blood transfusion, volume depletion, tissue hypoxia, or presurgical hyperuricemia. The risk of perioperative gout is especially increased in males, patients with obesity or diabetes mellitus, previous gout history, high-protein purine-rich diets, after abdominal, orthopedic or bariatric surgery, or perioperative cessation of colchicine gout prophylaxis.¹⁸ In acute gout, needle-like MSU microcrystals deposited in tissues and joints are incompletely phagocytosed by monocytes and neutrophils, causing activation of the NLRP3 inflammasome, production of IL-1β, and further neutrophil recruitment.^13,18 Neutrophils are stimulated by MSU crystals and macrophage-derived IL-1β to form aggregated NETs, which are known to be present in gouty tophi.¹³ While NETs contribute to tophi formation by densely packing MSU crystals with ecDNA, aggregated NETs can trap and cleave proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (CCL-2) in acute gout.^13,17 Thus, NETs can contribute to acute inflammation and tophi formation but also play a role in the resolution of inflammation in gout.^13,17

Treatments

Muñoz et al¹³ demonstrated that interfering with NET formation using neutrophil cytosolic factor-1/p47-phagocyte oxidase (Ncf1**/p47-phox)-deficient or PAD4-deficient mice models reduced both the prevalence and size of gallstones formed in the setting of a lithogenic diet. Pharmacological treatment of wild-type mice with a PAD4 inhibitor (GSK484) or the selective β1-adrenergic receptor antagonist metoprolol, which is known to stun neutrophils, also had this effect.¹³ Metoprolol and GSK484, in combination, completely abrogated gallstone progression.¹³ Colchicine inhibited microtubule-dependent neutrophil macropinocytosis of cholesterol crystals and prevented aggregated NET formation. Thus, NET inhibition was successful in preventing gallstone formation and growth.¹³ Inhibition of NETosis by colchicine treatment is also effective in other microcrystal diseases, including gout, pseudogout, and atherosclerosis. Colchicine binds to tubulin and inhibits microtubule assembly, which is required for neutrophil chemotaxis, adhesion, transmigration and recruitment, GPCR signaling, Ca²⁺ influx, NOX2 superoxide production, intracellular transport, nuclear chromatin swelling, organelle trafficking, and NETosis.^13,18 Acute perioperative gout can pose difficulties with arthritis severity, pain management, and patient immobilization. Colchicine is a useful intervention, due to its efficacy and the relative contraindications of perioperative systemic corticosteroid or nonsteroidal anti-inflammatory drug administration.¹⁸ PAD4 inhibition or DNase1 therapy was shown to decrease the severity of pancreatic and lung injury in murine models of severe acute pancreatitis.¹⁷

POSTOPERATIVE WOUND HEALING

Wound healing is an important concern of surgeons. Altered wound healing and complications can cause significant morbidity and mortality during the postoperative period.³ Wound healing is a coordinated physiological process that restores the skin barrier function. The phases of wound healing include hemostasis, inflammation, proliferation, and remodeling.¹⁹ Neutrophils play a crucial role in this process; during the inflammatory phase, they are recruited to the wound site and provide defense against microbial pathogens. However, numerous factors may affect normal wound healing. These include local factors, such as ischemia, infection, presence of a foreign body, and edema; and systemic factors, including diabetes mellitus, sepsis, medications, and obesity.^19–21 Although neutrophils provide crucial protection against infection, if inflammation is dysregulated by excessive NETs, delayed wound healing results.²

The deleterious effects of NETs are mediated through multiple mechanisms. NET-derived NE degrades essential wound proteins, including proteoglycans, collagen, and fibronectin, disrupting cell-cell interactions.^19–21 The presence of excessive NETs in wounds impairs angiogenesis and healing by promotion of endothelial-mesenchymal transition through the Merlin/Yes-associated protein (YAP)/HIPPO/mothers against decapentaplegic homolog 2 (SMAD2) pathway.¹⁹ NE present within NETs cleaves platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), also hindering angiogenesis.³ NETs may increase angiogenesis when in contact with platelets and endothelial cells; however, in chronic wounds, NETs can have an inhibitory effect.¹⁹ Furthermore, endothelial cell function is dysregulated by NET-derived mixed metalloproteinase 9 (MMP-9). Histones exert a local cytotoxic effect by integrating into the phospholipid bilayer of cell membranes, altering membrane permeability, which can result in calcium ion influx and subsequent cell death. Keratinocyte migration and proliferation are necessary for the restoration of the epithelial layer. However, high wound levels of NETs inhibit keratinocyte migration and delay wound healing.²¹ NETs can induce fibroblast activation and differentiation into myofibroblasts, thereby promoting wound fibrosis.¹⁹

Diabetes, Wound Healing, and NETosis

The impairment of wound healing by NETosis is common in patients with diabetes mellitus.² Diabetes mellitus primes neutrophils to form NETs.^2,12 A diabetic mouse model demonstrated higher levels of wound cit-H3, a biomarker of NET formation, and slower rates of wound healing.²¹ The mechanisms of increased NETosis in diabetes include hyperglycemia, glucotoxicity, increased cytokine/CRP priming of neutrophils, AGE formation via polyol and hexosamine pathways, neutrophil AGE-RAGE/NF-κB signaling and superoxide formation, de novo DAG synthesis, and PKC activation.¹² Furthermore, patients with diabetes are predisposed to NETosis due to elevated basal neutrophil calcium levels, which stimulates PAD4 activation. A preliminary study in surgical patients undergoing total joint arthroplasty showed obese patients with insulin-resistance had higher PAD4 expression at the surgical site than insulin-sensitive control patients.²⁰

Treatments

Currently, anti-NET treatments are being developed to promote wound healing.²¹ The most studied treatment is DNase1, which degrades NETs. In a murine study, the recombinant human DNase1 (Dornase alfa) reduced the wound area and enhanced re-epithelialization by 75%, with accelerated wound healing.²¹ By inhibiting NETosis, PAD4 knockdown achieved faster wound healing at 2 weeks compared with wild-type mice (80% vs 25% healed).²¹ A matrix of Thr-Asp-F-amidine tripeptide, a PAD4 inhibitor, with the standard wound healing materials alginate and gelatin was applied to wounds.²¹ Wounds treated in this manner showed accelerated re-epithelialization and improved healing compared to controls or wounds treated with alginate and gelatin alone.²¹ Intravenous treatment with Cl-amidine, which inhibits PAD4, also decreases wound area. In addition to its anti-hyperglycemic effect, metformin has been shown to inhibit the PKC-NOX pathway, ROS generation, and NET formation.^21,22 Therefore, metformin could have a positive effect on the promotion of wound healing and is an area that requires further investigation.

CHRONIC WOUNDS

In addition to postoperative wounds, NETosis also affects chronic wounds and secondary intention healing, including bacterial biofilm formation. Extracellular DNA is a major component of bacterial biofilms.^23–25 Such ecDNA can originate from neutrophils via NETosis or neutrophil necrosis or from bacteria.²⁴ In vitro, ecDNA acts as a structural component of bacterial biofilm extracellular polymeric substance^23,24 as well as a diffusion barrier that increases the resistance of bacteria to cationic antibiotics (aminoglycosides and colistin).²⁵ A recent in vivo study of Pseudomonas aeruginosa biofilms showed that host-derived ecDNA acted as a protective shell overlying the biofilm but was not found inside the biofilm.²⁴ It was thought that because the neutrophil-derived ecDNA on P. aeruginosa biofilms in vivo contained H3 but not cit-H3, it was derived from neutrophil necrosis rather than NETosis.²⁴ Not all NETs contain cit-H3, and NETosis can occur in the absence of histone citrullination. Thus, further studies of other bacterial in vivo biofilms are required.^2,8,23

Treatments

Recombinant DNase1 has been investigated as a potential treatment for chronic wounds and biofilms, including pleural empyema, surgical wounds, and diabetic foot ulcers.^2,3 The use of maggot therapy with anti-NET/DNase properties should also be explored. Topical maggot therapy facilitates debridement of necrotic, gangrenous, or infected tissues, has antimicrobial effects, and stimulates wound healing.^26,27 The debridement of both acute and chronic wounds is an important aspect of wound management.^26,27 Debridement takes different forms, including surgical, biological (eg., maggot therapy), mechanical (eg, wet-to-dry dressing changes, negative pressure wound therapy), enzymatic (eg, streptokinase in hydrogel), and autolytic (eg, manuka honey).^26,27 Debridement facilitates wound healing by the direct removal of all devitalized tissue, reduction of bacterial burden and biofilm, and stimulation of granulation tissue. However, biofilm recurrence after surgical debridement can occur within 3 days.²⁷

Maggot debridement therapy (MDT) for infected or chronic wounds has been used since antiquity.^26,27 The larvae of the green bottle blowfly Lucilia sericata are used in sterile medicinal MDT, which involves multiple mechanisms.^26,27 Firstly, maggots scrape the wound base as the larvae move across the surface of the wound, loosening and feeding on non-viable tissue and bacteria.²⁷ Maggots secrete digestive enzymes that liquefy necrotic tissue and slough in the wound, including a wide range of MMPs and serine proteases.²⁷ Maggots not only digest bacteria in a wound but secrete and excrete bactericidal compounds with activity against aerobic and anaerobic gram-positive bacteria and some gram-negative bacteria.²⁷ Maggots also release DNases, which can effectively degrade both microbial and human DNA within necrotic debris, potentially explaining their ability to remove biofilms and prevent biofilm reformation.^26,27 This disruption of the bacterial biofilm is synergistic with systemic antibacterial therapy, as neutrophil-derived ecDNA is used to form a capsule around the biofilm, preventing antibiotic penetration.²⁶ MDT has been shown to be superior to conventional treatments for chronic wounds in comparative studies (including 6 RCTs), with faster healing, longer antibiotic-free periods, and lower amputation risk (Fig. 4).²⁷

FIGURE 4.

Maggot therapy with free-range larvae. Diabetic foot ulcers during and after MDT. Photographs by Parizad et al.²⁷

THROMBOSIS

NETs interact with platelets, complement (C5), and tissue factor (TF) and activate both the intrinsic and extrinsic coagulation cascades to promote venous thromboembolism (VTE) and arterial thrombosis and occlusion.^{5,9,22,28–36} (Fig. 5) Risk factors for VTE include malignancy, major surgery, hypoxia, obesity, pregnancy, infection, inflammation, and trauma-all of which involve NETosis.^5,9,22,29 NETs also generate ROS and endothelial dysfunction, LDL oxidation and atherosclerotic plaque instability.¹⁵ Examples of NET-related arterial thrombosis include acute coronary syndrome and acute myocardial infarction, acute ischemic stroke, abdominal aortic aneurysm, peripheral vascular disease, and thrombotic microangiopathies (disseminated intravascular coagulation, thrombotic thrombocytopenic purpura).²⁹ NETs provide a scaffold upon which platelets, fibrin, and erythrocytes can aggregate.²⁹ In malignancy, ecDNA present in NETs increases platelet prothrombotic function by enhancing tumor cell-induced platelet aggregation. Arterial and venous thrombi related to systemic NETosis are common, particularly in patients with gastric and pancreatic cancers.^22,28 NETosis activates platelets via cit-H3, NE, and MPO, causing platelets to display granular P-selectin on their surface.³⁰ The neutrophil PSGL-1 receptor interacts with platelet P-selectin, which further promotes NET formation and the development of a positive feedback loop between platelet activation and NETs.⁵ NETs also promote phosphatidylserine exposure on the surface of platelets, catalyzing the coagulation cascade. Further platelet aggregation is promoted, resulting in thrombus formation and propagation.^3,31 This feed-forward effect of progressive platelet activation and NET formation is involved in the pathogenesis of transfusion related acute lung injury (TRALI).³¹

FIGURE 5.

Multiple roles of NETs in tumor progression and metastasis. Neutrophils are mobilized from bone marrow, enter into the circulation and migrate towards proangiogenic and proinflammatory gradients. Neutrophils are recruited to the primary tumor site through various cytokines and chemokines such as CXCL1, IL6, or CCL3, ultimately leading to neutrophil activation and NET release. Cancer cell–derived exRNA can also induce NETs which in turn amplify the release of exRNA. In growing tumors, NETs enhance cancer progression by enhancing thrombin activity, increasing the expression of stem cell markers and inflammatory chemokines and cytokines and promoting epithelial-mesenchymal transition. NET formation is also enhanced by the uptake of exosomes transporting oncogenic mutations to the tumor sites. NETs regulate cancer cell migration and tumor growth by directly interacting with T cells, inducing the exhaustion of cytotoxic T cells and differentiation of naive T cells into regulatory T cells, thereby promoting an immunosuppressive environment. During their transit in the circulatory system, cancer cells are captured by the chromatin web network of NETs and this physical and functional interaction provides shielding, protecting cancer cells from cytotoxic effects of immune cells. NETs also provide an “anchor” to the cancer cells, facilitating their adhesion and extravasation into the secondary tumor sites to form distant metastasis. The CCDC25 is expressed by cancer cells and can serve as a NET-DNA receptor that senses NETs and recruits invasive cancer cells to the metastatic sites. During inflammation, NETs can activate dormant tumor cells and stimulate them to migrate and form metastasis by cleaving basement membrane components (laminins). NETs also induce thromboinflammation leading to ischemia and injury in organs, such as the heart and kidney. G-CSF predisposes circulating neutrophils to form NETs through the recruitment of blood platelets. Interactions between platelets and neutrophils play an important role in cancer progression and metastasis by inducing platelet activation and NETosis and consequently enhancing tumor-associated coagulation and thrombosis. Reproduced from Mamtimin et al with permission.³⁶ CCDC25 indicate coiled-coil domain containing 25 protein.

NETosis and the Procoagulant State

NETs provide a backbone on which platelet-derived, neutrophil-derived, or tumor-derived extracellular vesicles, such as exosomes and microparticles, can be captured.^29,31 Exosomes adhere to NETs in vitro, where they have a dose-dependent procoagulant effect, while microparticles express TF. Tissue factor pathway inhibitors are cleaved by NET-bound NE, enabling TF to promote the extrinsic coagulation pathway by activating factor VII and thence factor X. NETs also activate the intrinsic coagulation pathway via autoactivation of factor XII by the negatively charged histone-DNA complexes in NETs; and the common coagulation pathway by NET-derived NE digesting antithrombin III, leading to thrombin formation.^5,9,22,29 Factor XIIa levels were significantly correlated with NET histone–DNA complexes in patients with hepatocellular carcinoma.³⁰ NETosis in the peripheral blood of colorectal cancer patients (≥stage II) significantly shortened the activated partial thromboplastin time and led to a procoagulant interaction between platelets, neutrophils, and endothelial cells.³¹

Inhibition and Treatment of NET-induced Thrombosis

Given the multiple pathways by which NETs induce deep venous thrombosis and arterial thrombosis, prevention and treatment of NET-related thrombus formation remains challenging³ (Fig. 6A, B). Antiplatelet agents, such as aspirin (COX/thromboxane A2 inhibition), clopidogrel or ticagrelor (P2Y12 receptor antagonists), dipyridamole (phosphodiesterase 3 inhibitor), and crizanlizumab (anti-P-selectin antibodies), can interfere with platelet-neutrophil-endothelial interactions and potentially prevent neutrophil recruitment and NET formation.³² (Fig. 6B) CXCR1/2 inhibitors can block the binding of neutrophil CXCR with endothelial-bound or circulating chemokine ligands such as CXCL8, preventing neutrophil activation and adherence to endothelium.³² However, anticoagulant therapy for NET-induced thrombosis is not universally effective and use of potent fibrinolytic agents such as tissue plasminogen activator is limited by interactions with NET histones/ecDNA-bound NE or bleeding risks.^22,32 Current standard therapies, with the exception of heparin, lack efficacy in NET-induced thrombosis, and combined DNA degradation and fibrinolysis may be required^3,29,32 (Fig. 6A, B).

FIGURE 6.

A, Therapies that mechanistically inhibit or prevent NETosis or neutralize NDP toxicities. Many agents have been developed to interfere with the intracellular processes that result in NETosis. Cl-amidine, GSK199, GSK484, and BMS-P5 all inhibit the activity of peptidyl arginine deiminase 4, preventing chromatin decondensation that precedes NET release. The antibody CIT-013 binds to histones to similarly inhibit chromatin decompaction. LDC7559 and disulfiram both interfere with the activity of the pore forming protein gasdermin D that potentiates NETosis by allowing myeloperoxidase and neutrophil elastase to enter the nucleus, where they can cleave histones to decondense chromatin. N-acetylcysteine, metformin, and colchicine are all thought to act by decreasing reactive oxygen species production, which promotes NETosis. NET modification. Several agents have been developed to decrease the toxicity of NETs after their release. Nucleases, including DNase1 and DNase1L3, digest NETs and accelerate their removal from the circulation. However, this strategy may liberate captured bacteria and NET degradation products such as cell-free (cf)DNA, histones, and neutrophil serine proteases. cfDNA scavengers have been developed to neutralize the inflammatory effects of the cleaved DNA. Recombinant human thrombomodulin, activated protein C, and heparin neutralize histone toxicity. Sivelestat and prolastin inhibit the activity of neutrophil elastase, whereas PF-1355 inhibits myeloperoxidase activity. Finally, PF4 compacts NETs and prevents their digestion, limiting the systemic release of NDPs, while enhancing the capture of bacteria. Deglycosylated KKO, an antibody directed against complexes of PF4 and polyanions, enhances these effects and improves outcomes in murine sepsis models. aPC, activated protein C; ART-123, recombinant human thrombomodulin; cfDNA, cell-free DNA; DG-KKO, deglycosylated KKO; DNase, deoxyribonuclease; GSDMD, gasdermin D; MPO, myeloperoxidase; NAC, N-acetylcysteine; NDP, NET degradation product; NE, neutrophil elastase; NET, neutrophil extracellular trap; PAD4, peptidyl arginine deiminase 4; PF4, platelet factor 4; ROS, reactive oxygen species. Reproduced from Ngo et al with permission.³² B, Interventions that prevent neutrophil activation or recruitment to inhibit NETosis. Neutrophils can be stimulated to release NETs through multiple pathways, including communication with activated platelets, exposure to inflammatory cytokines, interaction with the complement system, and recruitment to sites of endothelial inflammation or injury. Multiple therapies that block platelet activation have been shown to reduce NET release, including aspirin, which irreversibly inhibits COX enzymes to prevent thromboxane A2 (TXA2) production; clopidogrel and ticagrelor, which block the platelet P2Y12 receptor; and dipyridamole, which increases adenosine that binds to A2A receptors to stimulate the production of cyclic adenosine monophosphate (cAMP), which prevents the activation of both platelets and neutrophils. The anti-P-selectin antibody crizanlizumab also inhibits neutrophil-platelet interactions and selectin-mediated neutrophil endothelial rolling; however, further studies are required to determine if it prevents NETosis. The cytokine-neutralizing antibodies anakinra, canakinumab, tocilizumab, and infliximab have been shown to reduce NET release in autoimmune disorders; however, more work is required to determine if they act directly on neutrophils or by modifying the inflammatory microenvironment. Hydroxychloroquine also interferes with neutrophil activation and NET release, potentially by preventing autophagy or inhibiting toll-like receptor 9 (TLR9). The humanized monoclonal antibody eculizumab inhibits cleavage of complement component C5, preventing the production of anaphylatoxin C5a, which stimulates neutrophil chemotaxis and NETosis. It also stops the assembly of the membrane attack complex (MAC), which can trigger NET release. The chemokine CXCR 2 inhibitors danirixin and navarixin, and the CXCR1/2 inhibitors ladarixin and reparixin prevent neutrophil activation by chemokines, including IL-8, CXCL5, and CXCL7, and have been shown to decrease neutrophil recruitment and NETosis in the lungs. AA, arachidonic acid; GP1bα, glycoprotein 1bα; PSGL-1, P-selectin glycoprotein 1. Reproduced from Ngo et al with permission.³²

Heparin can be used to combat NET-induced thrombus formation, although its effect is dose related.³⁷ Heparin can hinder NET formation and also target existing NETs by inhibiting inflammation, cytokine release, coagulation (anti-Xa and anti-IIa), complement and platelet activation, HMGB1-RAGE interactions, and neutrophil adhesion.²² Heparin inhibits the NE and cathepsin G present in NETs.²² Heparin also limits the activation and degranulation of platelets by inhibiting the binding of the von Willebrand factor/factor VIII complex as well as TF-carrying microparticles binding to NETs.^22,33 Heparin binds to histones, assisting host DNases in degrading NETs and preventing end-organ damage from histone toxicity³² (Fig. 6A). Unfractionated heparin inhibits neutrophil rolling and firm adhesion to vascular endothelium by binding to P/L-selectin and MAC-1 more potently than low molecular weight heparin (LMWH).³³ By interacting with extracellular protein ligands, heparin and LMWH are anti-angiogenic by binding VEGF and FGF and antineoplastic by inhibiting circulating tumor cell adhesion and implantation.³³ Extended DVT prophylaxis for 4 weeks after major abdominal or pelvic cancer surgery with LMWH is advocated because of the continued risk of proximal DVT and pulmonary embolus after patients are discharged from the hospital.²⁸ NETosis directly contributes to DVT development and propagation and is a crucial component of pulmonary VTE at thrombus retrieval or autopsy.^29,30

Colchicine blocks microtubule polymerization and prevents NET formation by inhibiting neutrophil adhesion, neutrophil-platelet interactions, NE release, neutrophil organelle/granule transport and exocytosis, neutrophil superoxide production, and the intracellular assembly of the NLRP3 inflammasome. These pathways are involved in progressive endothelial inflammation and occlusive arterial thrombosis (thromboinflammation). This is because oxidized LDL, cholesterol crystals, and foamy macrophages in sterile atheromatous plaques trigger neutrophil recruitment, NETosis, and the NLRP3 inflammasome. In addition to inhibiting inflammation caused by MSU crystals in acute gout, colchicine has been shown to be effective (and United States Food and Drug Administration approved (US FDA)) in reducing the risk of acute coronary syndrome, myocardial infarction, stent thrombosis, ischemic stroke, urgent coronary revascularization, and cardiovascular death in high-risk patients.^15,32 (Fig. 6A, B)

DNase1 has been shown to improve the procoagulant state in colorectal cancer and resolve NETosis-related thrombosis in both in vitro and in vivo models.³² However, systemic DNase1 monotherapy should be used with caution because of the risk of releasing previously sequestered microbial pathogens or NET degradation products (NDPs), including short fragment DNA, histones, and neutrophil serine proteases.³² The release of NDPs may be cytotoxic to the vascular endothelium and increase further thrombin generation.³² Thus, concomitant treatment to neutralize NDPs rather than simple NET degradation is suggested, with agents such as heparin, NE inhibitors (Sivelestat), MPO inhibitors (PF-1355), activated protein C, cfDNA sequestration (cationic nucleic acid binding polymers), or plasmaphoresis (CytoSorb)³² (Figs. 6A, B). Non-anticoagulating heparins (sevuparin) to neutralize histone toxicity without increasing the risk of bleeding may be a useful adjunct to DNase therapies in the future.³²

CANCER

Tumor cell promotion of local or remote NETosis is a phenomenon in multiple malignancies. It is correlated with higher tumor stage in breast cancer,² ovarian cancer,³⁴ colon cancer,³⁵ esophageal/gastric adenocarcinoma,³⁵ and lung cancer.³⁵ Tumor cells release chemokines, including IL-8 and GM-CSF, which attract neutrophils to the primary site of the tumor.⁵ Neutrophils are then induced to undergo NETosis. The mechanism may include a hypoxic tumor microenvironment with increased expression of HIF-1α or exosomal transfer of mutant KRAS to neutrophils.^5–7

NETosis and Tumor Metabolism

NETosis affects the metabolism of tumor cells and promotes tumor growth. NETosis in murine neutrophils can be induced by incubation with murine colon adenocarcinoma cells.² The presence of NETs in the tumor microenvironment is associated with increased mitochondrial function.² NET-associated NE can increase mitochondrial biogenesis via the activation of TLR-4 in cancer cells and subsequent peroxisome proliferator-activated receptor-gamma coactivator upregulation. NET-mediated TLR-9 signaling can also activate cancer proliferation and accelerate tumor growth, as demonstrated in colorectal cancers (CRCs).²

NETosis may also induce other characteristic properties of cancer cells. NETs have been shown to contain MMP-9, which degrades the ECM and releases sequestered VEGF, thereby promoting neoangiogenesis.² Neoangiogenesis may also occur after the primary tumor has been removed, leading to the growth of distant metastases due to the loss of angiogenesis inhibitors (endostatin/angiostatin) emanating from the primary (resected) tumor.⁷

NETosis and Cancer Metastasis

NETs also facilitate metastasis. The process by which tumor cells leave the primary site to form a secondary lesion in a distant organ includes a series of events:

1. Local invasion facilitated by local breakdown of the ECM,

2. Penetration into the vasculature (intravasation),

3. Evasion of host immunosurveillance,

4. Circulatory arrest in distant microvasculature,

5. Epithelial–mesenchymal transition (EMT) and extravasation into parenchyma at the metastatic site.⁹

NETs have been shown to contribute to all phases of this metastatic process (Fig. 5).³⁶ NE and MMP-9 derived from NETosis can also reactivate dormant cancer cells by remodeling ECM laminin, thereby stimulating integrin α3β1 and focal adhesion kinase/ERK/myosin regulatory light chain kinase/Yes-associated protein signaling cascades.^2,36

NETs facilitate local invasion by degrading the ECM.³⁶ Enzymes bound to the NET-DNA scaffold, including NE, MMP-9, and cathepsin G, cleave laminin, an important component of ECM. NETs then promote tumor cell intravasation into the bloodstream by increasing permeability between endothelial cells.⁹ NET-associated NE disrupts the tight junctions between adjacent endothelial cells by promoting nuclear translocation of junctional β-catenin, with subsequent downregulation of vascular endothelial-cadherin.² (Fig. 5).

NETosis and Immunosurveillance

Elevated tumor-infiltrating neutrophils (TINs) or blood neutrophil-to-lymphocyte ratio (NLR) are associated with poor prognosis in cancer.^38,39 As cancer cells circulate in the bloodstream, they may be protected by NETs, which create a physical barrier against vascular shear stress as well as the host immune system.⁹ Circulating tumor cells frequently downregulate their surface expression of major histocompatibility complex antigens and are thereby recognized by NK cells through the “missing self” mechanism.³⁸ However, cancer cells secrete CXCR1 and CXCR2 ligands [CXCL1, CXCL2, and CXCL8 (IL-8)], which recruit neutrophils and induce NETs to surround tumor cells in both murine models and humans.³⁹ The surrounding NETs coat the malignant cells with ecDNA, platelets, and fibrin, obstructing CD8⁺ T and NK cell immunosurveillance and promoting Treg cell differentiation.^36,39 This may occur at the primary tumor site or remotely in the circulation (Fig. 5).

NETosis, Platelets, and Cancer Metastasis

Interactions between platelets, NETs, and tumor cells facilitate metastasis.^2,38 In addition to creating a physical barrier as described above, platelets can transfer major histocompatibility complex class I self-antigens to tumor cells, enabling evasion of NK cell surveillance, a process known as trogocytosis.³⁸ Activated platelets further dampen NK cell antitumor activity by releasing proteases that cleave NKG2D ligands from the surface of the tumor cells.³⁸ In addition, the platelet cloak forms a local microenvironment with a high concentration of signals that promote EMT.⁴⁰ In particular, platelet–tumor cell interactions trigger the secretion of TGF-β and NF-κB.⁴⁰ These act synergistically to induce rapid EMT. Transmembrane protein glycoprotein VI facilitates EMT by promoting PDGF secretion. In colon cancer cells, binding of glycoprotein VI to galectin 3 on the surface of tumor cells can induce platelet activation and PDGF release.⁴⁰

NETosis and the Premetastatic Niche

Cancers orchestrate a permissive microenvironment for metastasis before colonization and show organotropism for specific sites, including the bone, lung, liver, brain, or peritoneum: the premetastatic niche.³⁴ This is achieved by the remote recruitment of neutrophils and generation of NETs by cancer-derived chemokines, exosomes, and inflammatory cytokines.^34,36 NETosis may also be permissive in the hypoxic, nutrient-poor tumor microenvironment of the peritoneal cavity and allow evasion of anoikis or activation of quiescent cancer cells.³⁴ The mechanism by which NETs promote metastasis at distant organ sites is 2-fold. First, NETs capture tumor cells within the microvasculature and coelom.²⁴ This has been demonstrated in vivo in the hepatic sinusoids and pulmonary microvasculature and in the omentum.^2,24,40 In addition to acting as a physical trap for metastatic cells, NETs act as powerful chemotactic factors for cancer cells, attracting them to the premetastatic niche.⁹ In both mice and humans, NETs can be sensed by the cancer cell transmembrane receptor coiled-coil domain containing 25 protein (CCDC25).² Activation of this NET-DNA receptor induces cancer cell motility toward NETs, creating a metastatic niche for locoregional and distant metastasis.² This is relevant after resectional surgery for cancer, as NETs are associated with cancer progression in the context of sterile inflammation or postoperative sepsis and the peritoneal or systemic inflammation induced by neutrophils, platelets, and cytokines/CRP.^3,7,37,41 For example, intrabdominal sepsis promoted NETosis in ascitic fluid and peripheral blood and enhanced peritoneal and hepatic metastases in in vitro and in vivo studies of gastric cancer³⁷ (Fig. 5).

NETosis and Cancer Surgery

Surgical resection of cancers can increase the release of circulating tumor cells and also promote a systemic inflammatory response, neutrophil priming, and NET formation.^3,7 The postoperative period is thus a dangerous time for cancer progression due to NET-induced promotion of micrometastases, reactivation of dormant cancer cells and impaired host immunosurveillance, as well as the postponement of cytotoxic therapies.^3,7,36,41 Increased postoperative serum levels of NET MPO-DNA in patients after major liver resection for metastatic CRC is associated with a greater than 4-fold decrease in disease-free survival.⁴¹ Widespread postoperative intrahepatic NETosis is related to intraoperative surgical vascular occlusion (Pringle maneuver), hepatic ischemia/reperfusion (I/R), and hypoxia which promote hepatic CRC micrometastatic growth.⁴¹ In a similar murine metastatic colorectal cancer model of 70% segmental hepatic I/R for 1 hour, intrahepatic NETosis in the ischemic segments persisted for 3 weeks after surgery and induced metastatic CRC growth and a 12-fold increase in tumor-associated neutrophils. Perioperative intraperitoneal injections of DNase1 or PAD4 inhibitors resulted in a 68% and 73% respective decrease in the growth of metastatic tumors, with attenuated postoperative hepatic cit-H3 and circulating NET MPO-DNA levels at 3 weeks.⁴¹ Tumor proliferation and angiogenesis were also significantly decreased by NET inhibition. Importantly, the presence of intrahepatic NETosis did not persist at 3 weeks after hepatic I/R in the absence of tumor. Intratumoral hypoxia has been shown to be associated with HIF-1α activation, which promotes TINs, NETosis, and cit-H3 release.⁴¹ Extracellular release of the nuclear DNA binding protein HMGB1 from NETs after hepatic I/R and activation of DAMPs receptors (TLR-9/RAGE) promotes tumor progression and is associated with reduced patient survival after liver resection for metastatic colorectal cancer⁴¹ (Fig. 2). Within a NET-rich tumor metastatic niche, neutrophil programmed death ligand-1 (PDL-1) expression in NETs has been shown to exhaust T cells. In a murine model of hepatic metastases from CRC, administration of anti-PDL-1 antibody immediately before and after liver ischemia and reperfusion was shown to reduce T cell exhaustion, as evidenced by increased T cell cytokine levels and metabolic function, and reduced tumor size³⁶ (Fig. 5).

NETosis and Peritoneal Metastasis

In a murine intraperitoneal ovarian cancer model and in female patients with early high-grade serous ovarian cancer (HGSOC), NET formation in the omentum is related to neutrophil recruitment from high endothelial venules in omental milky spots.³⁴ NETs form a premetastatic niche for subsequent ovarian cancer metastases.³⁴ Tropism of metastatic cancer cells to the omentum is related to the release of ovarian cancer-derived chemokines IL-8, GM-CSF, CXCL1, and CXCL2, which remotely promote omental neutrophil chemotaxis, extravasation, and NETosis.³⁴ Neutrophil recruitment was not observed in other adipocyte-rich sites in the peritoneum, including perirenal, gonadal, and mesenteric fat, even though ovarian cancers are known to utilize lipids derived from adipocytes for growth and proliferation.³⁴ NETosis was also found in the ascitic peritoneal fluid from women with advanced HGSOC and could be decreased by systemic neoadjuvant chemotherapy and potentially by intraperitoneal chemotherapy.³⁴ Presumably, this is due to chemotherapy cytotoxicity in HGSOC cells, but may also be related to the neutropenia associated with systemic taxane/platinum-based chemotherapy.³⁴ This raises the question of the routine use of GM-CSF in chemotherapy-induced bone marrow suppression, as GM-CSF may promote NETosis and tumor progression.⁴²

NETosis Treatments and Cancer

Anti-NET therapies have the potential to diminish tumor EMT and metastasis.^{34,35,37,41,43–75} Some are already US FDA-approved for medical conditions, such as Dornase alfa for cystic fibrosis, SLE, or COVID-19–related acute respiratory distress syndrome.^3,54 However, large clinical trials are required before US FDA approval for oncological indications^43–75 (Table 1).

TABLE 1.

Agents Targeting NETs, Their Mechanisms and Potential Uses in Cancer Therapies

Target	Mechanism	Agent	Disease	References
NET components
DNA	DNA degradation	DNase1	TN breast cancer Gastric Cancer CRC liver metastases PDAC Diffuse large B-cell lymphoma PDAC liver metastases	43 44 41,45,56 47 48 49
NE	NE inhibitor	GW311616 Sivelestat	Leukemia Lung Ca/CRC liver metastasis	50 35,51
MPO	MPO inhibitor	Heparin, 4-ABAH	Lung cancer	52
PAD4	PAD4 inhibitor	GSK484, Cl-amidine	PDAC, melanoma Ovarian HGSOC	53 34
CEACAM1	CEACAM inhibitor	Monoclonal antibody 5F4	Colon cancer	46
Enzymes
NADPH oxidase	NOX inhibitor	Apocynin DPI	PDAC —	49 57
Cathepsin C	Cathepsin C inhibitor	AZD7986	Lung metastasis in breast cancer	58
Chemokines
CXCR1	CXCR1 inhibitor	SX-682, reparixin	HNSCC Breast cancer	59 60
CXCR2	CXCR2 inhibitor	SX-682, reparixin, SB225002	HNSCC Breast cancer PDAC	59 60 61,62
IL-8 (CXCL8)	IL-8 neutralization	anti-IL-8 Ab	CRC	75
IL-17	IL17-blockade + checkpoint inhibitor	anti-IL17/IL17R/PD-1 antibodies	PDAC	63
Cytokine/DAMP receptors
TLR4	TLR4 inhibitor	TAK-242	Colorectal cancer	64
TLR4/9	TLR4/9-COX2 inhibitor	Hydroxychloroquine-aspirin	HCC	65
TβRI	TGF-β RI inhibitor	SB525334 LY 2157299	PDAC Gastric cancer	66 37
Miscellaneous
ACE, NOX, MMP-9	ACE inhibition 5-FU sensitivity	Enalapril	CRC	67

ACE indicates angiotensin-converting enzyme; 5-FU, 5-Fluorouracil; HCC, hepatocellular carcinoma; HGSOC, high grade serous ovarian cancer; HNSCC, head and neck squamous cell cancer; PDAC, Pancreatic ductal adenocarcinoma.

Adapted from Hu et al and Chen et al.^68,69

Degradation of NETS by DNase1 therapy has shown efficacy in preclinical cancer studies^41,43–49 (Fig. 6A). Intraperitoneal administration of DNase1 suppressed the development of gross liver metastases and growth of established liver micrometastases associated with intrahepatic NETosis and surgical stress in a murine metastatic colorectal cancer model.⁴¹ In a mouse model of triple-negative breast cancer, DNase1 treatment via intraperitoneal injection of DNase1-coated nanoparticles successfully degraded NETs and subsequently reduced metastases.⁴³ Gastric cancer cells treated with PAD4 or DNase1 showed decreased in vitro tumor cell migration and EMT.⁴⁴

Cancer metastasis can be disrupted through the inhibition of Cathepsin C, NOX, PAD4, NE, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), TLR4/9-COX2, transforming growth factor beta receptor I (TβRI) signaling, IL-17, CXCR, or angiotensin-converting enzyme, which are molecules involved in NETosis^43–73 (Table 1). AZD7986, a Cathepsin C inhibitor, effectively suppressed circulating pulmonary NETs and decreased lung metastases of breast cancer in a mouse model.⁵⁸ However, it had no effect on primary tumor growth.⁵⁸ Inhibition of PAD4 by Cl-amidine or GSK484 decreased omental metastases in a murine model of HGSOC peritoneal metastasis.³⁴ In a PAD4-defective mouse model, inhibition of NETosis with DNase or NE inhibitors reduced spontaneous lung and liver metastasis in lung cancer.³⁵ CEACAM1 is abundant in NETs, and blockade of CEACAM by the monoclonal antibody 5F4, or inhibition of NETosis by the NE inhibitor sivelestat or DNase1 was shown to inhibit the adhesion of circulating tumor cells in hepatic sinusoids and decrease metastasis of CRC in vivo ^35,46 (Fig. 6). Targeting TLR4/9-COX2 signaling with chloroquine/aspirin can decrease NET-enabled hepatocellular carcinoma metastatic activity in vivo, and was more effective in combination with DNase1.⁷² In a mouse model of hepatic ischemia-reperfusion, hydroxychloroquine successfully inhibited NET formation via inhibition of TLR-9, ROS, and PAD4⁶⁵ (Fig. 6B). IL-17 may also be a useful therapeutic target, with studies suggesting the neutralization of IL-17 could inhibit tumor cell growth and prevent metastasis.^63,73

There is potential for developing anti-NET therapies that facilitate cancer immunotherapy or cytotoxic chemotherapy.⁵⁶ CEACAM1 expression is associated with T-cell exhaustion and resistance to tumor-infiltrating lymphocyte immunotherapy in melanoma.⁵⁵ Therefore, targeting CEACAM1 may improve immunotherapy efficacy for melanoma. Degradation of NETs with DNase1 was shown to reverse anti-PD-1 antibody monotherapy resistance in a murine MSI-high CRC model.⁷⁴ Combination therapy (DNase1 + immune checkpoint inhibitor) restored CD8⁺ T cell infiltration and cytotoxicity, significantly decreased tumor volume and TINs, and markedly improved survival compared to control or individual monotherapy groups.⁷⁴

The CXCR1 and CXCR2 pathways, which stimulate neutrophil chemotaxis and NETosis, are potential anticancer therapy targets (Fig. 6B). They can be blocked by pertussis toxin (Reparixin), which inhibits GPCR G-unit.³⁹ Administration of SX-682, an inhibitor of IL-8 receptors CXCR1 and CXCR2, enhanced the effect of NK cell therapy in a murine in vivo model of head and neck squamous cell carcinoma.⁵⁹ Furthermore, NETs induce tumors to release IL-8, which increases neutrophil recruitment, NETosis, tumor cell proliferation, and liver metastases in CRC. Neutralizing IL-8 antibodies block this feed-forward loop of IL-8/CXCR2/NETosis/CRC liver metastasis⁷⁵ (Fig. 6B). In a murine model, the angiotensin converting enzyme (ACE) inhibitor enalapril resensitized CRC to 5-Fluorouracil through inhibition of NF-κB/STAT3, NOX, and MMP-9. Combined enalapril/5-Fluorouracil, but not the individual monotherapies, synergistically reduced EMT and lymphatic, peritoneal, and hepatic CRC metastatic disease.⁶⁷ Inhibition of NETosis in vivo is inferred, since ACE or angiotensin II type 1 receptor (AT1R) inhibition significantly decreases neutrophil ROS, NOX activity and NETosis^10,12 (Table 1).

CONCLUSIONS

The pathophysiology of NETosis is of importance to surgeons, as NET formation is closely related to postoperative complications and cancer recurrence after surgery. NET ecDNA filaments decorated with histones and enzymes act as aggregation factors in cholelithiasis, pancreatitis, gout, and postoperative thrombosis. Excessive NETosis impairs wound healing in both the early postoperative period and in chronic wounds, particularly in patients with diabetes. Elevated levels of TINs, neutrophil-to-lymphocyte-ratio or NETs are associated with poor cancer prognosis. The perioperative period is characterized by ROS generation, cytokine release, tissue hypoxia and the activation of platelets, coagulation cascades, and neutrophils with NET formation. NETs enhance tumor growth, tumor-associated thrombosis, PDL-1 expression, T-cell exhaustion, evasion of host immunosurveillance, and facilitate metastatic organotropism in multiple cancer types, including colorectal, gastric, pancreatic, breast, ovarian, and lung cancers. Cholesterol crystals stimulate NETosis and thromboinflammation in atheromatous plaques. Currently, treatments targeting NETs are being investigated, including NET prevention, degradation or stabilization, neutralization of NDP, and scavenging of cfDNA. Such treatments include the use of recombinant human DNase1 or maggot DNase, PAD4 inhibitors, ROS scavengers, neutrophil protease inhibitors, heparins, and targeting of neutrophils, platelets, chemokines, and cytokines. Perioperative inhibition of NETosis without interfering with canonical antimicrobial protection requires further clinical research but has the potential to enhance recovery after surgery and oncological outcomes.