Neutrophil Extracellular Traps and Liver Disease

Abstract

Neutrophil extracellular traps, or NETs, are heterogenous, filamentous structures which consist of extracellular DNA, granular proteins, and histones. NETs are extruded by a neutrophil in response to various stimuli. Although NETs were initially implicated in immune defense, subsequent studies have implicated NETs in a spectrum of disease processes, including autoimmune disease, thrombosis, and cancer. NETs also contribute to the pathogenesis of several common liver diseases, including alcohol-associated liver disease and portal hypertension. Although there is much interest in the therapeutic potential of NET inhibition, future clinical applications must be balanced against potential increased risk of infection.

Neutrophils are multifaceted cells with diverse functions in immunity including phagocytosis of pathogenic bacteria and implementation of innate and adaptive immunity. Along with their key role in immune defense, neutrophils have been implicated in the pathophysiology of several additional diseases through formation of neutrophil extracellular traps, or NETs. NETs are filamentous structures composed of extracellular DNA, histones, and granular proteins that are released by neutrophils.¹ NET formation is triggered by various extrinsic stimuli including bacterial products, immune complexes, and platelets. Since their description in 2004, NETs have been implicated in the pathophysiology of several disease processes including autoimmune diseases, cancer, and thrombosis.^2,3 NETs also contribute to the pathogenesis of several chronic liver diseases with pivotal roles in diseases as prevalent as alcohol-associated liver disease and portal hypertension (PHTN). The contribution of NETs to the pathophysiology of liver disease will be a focus of this review (Fig. 1).

Fig. 1.

Neutrophil extracellular traps have been implicated in the pathophysiology of several different liver diseases.

Neutrophil Extracellular Trap Function

Neutrophil extracellular traps were first identified in 2004 as an immune defense mechanism which allowed for the trapping and killing of pathogenic bacteria.¹ However, the specific role of NETs in immune defense is unclear and is difficult to define with certainty.⁴ Several NET components, including histones, possess bactericidal and antimicrobial properties.^1,5 The granular protein neutrophil elastase (NE) is also capable of degrading bacterial virulence factors.⁶ Studies suggest that the fibrous NET structure augments the bactericidal capacity of NETs by sequestering bacteria with a high local concentration of antimicrobial molecules.¹ This structure may also form a physical barrier to prevent dissemination of microbes.^7,8 However, this high local concentration of cytotoxic proteins can damage bystander cells and propagate proinflammatory responses.^9,10

Studies regarding the role of NETs in immune defense have yielded conflicting results which may be due in part to different techniques in analyzing NET formation and microbial killing.¹¹ Studies suggest that NETs are able to kill bacteria,¹ viruses,¹² parasites,¹³ and fungi.¹⁴ However, some microbes do not stimulate NET formation.¹¹ While both gram-negative and gram-positive bacteria are capable of inducing NET formation, the efficiency of NET induction varies with bacterial strains and location of infection.¹¹ NETs are capable of killing certain bacterial strains, such as Shigella flexneri,¹ but others have evolved mechanisms to evade and/or neutralize NETs, including certain Streptococcal and Staphylococcal strains.^15,16

Deficiency of NET formation and of certain NET components confers variable susceptibility to infection. NE deficient mice are more susceptible to infection with the gram-negative bacterium Klebsiella pnuemoniae.¹⁷ However, NE deficiency can also impair neutrophil phagocytosis and thus this susceptibility to Klebsiella infection may be due in part to decreased efficiency of phagocytosis. Peptidyl arginine deiminase IV (PAD4) is an enzyme which is thought to be critical in NET formation. Interestingly, deficiency of PAD4 does not increase bacteremia or mortality in a mouse model of polymicrobial sepsis.¹⁸ This suggests that NETs play a nuanced role in infection defense which is not limited to the killing of pathogens. The myeloid-related protein 14 (MRP14) is a neutrophil cytosolic protein which is extruded with NETs and which exerts antimicrobial properties.¹⁹ NETs from MRP14^‒/‒ mice permit increased dissemination of K. pneumoniae. This supports a likely role of NETs in suppressing bacterial dissemination.¹⁹

Studies also suggest that NETs play a pivotal role in host defense against more sizeable pathogens such as fungal hyphae whose size precludes intracellular killing.²⁰ NETs seem sufficient to kill Candida albicans yeast and hyphae.^14,21 Several neutrophil proteins which regulate NET formation, such as myeloperoxidase (MPO) and calprotectin, contribute to antifungal defense.^22,23 Although neutrophils have not historically been considered important effector cells in antiviral defense, studies have identified NET release in response to infection with certain viruses, including human immunodeficiency virus (HIV) and respiratory syncytial virus.^12,24 NETs may trap HIV virions and decrease their infectivity.¹² In contrast, NETs do not contribute to immunity against the influenza virus,²⁵ further supporting strain-specific instigation of NET formation.

Neutrophil extracellular traps also contribute to the pathophysiology of several noninfectious diseases. NET formation results in the extrusion of intracellular proteins. Studies suggest that this exposure of intracellular proteins facilitates formation of the autoantibodies which characterize several autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).³ In addition, NETs promote thrombus formation through several different mechanisms. The fibrous NET structure serves as a scaffold which binds thrombus components, including platelets, red blood cells, fibrinogen, and fibronectin.²⁶ Histones propagate thrombus formation by triggering endothelial injury and thrombus formation. Finally, neutrophil serine proteases contained in NETs promote activation of both the intrinsic and extrinsic coagulation pathways.²⁷ These studies have intensified interest in NETs as a therapeutic target in a wide spectrum of diseases.

Neutrophil Extracellular Trap Detection

As heterogenous, acellular structures, NETs are challenging to detect and study. Neutrophils have a short half-life and are easily activated ex vivo. In vitro analyses of NET formation are subject to significant heterogeneity according to the concentration and duration of stimulus, methods of blood collection and neutrophil isolation, and culture conditions.²⁸ Furthermore, primary human neutrophils cannot undergo transfection to allow mechanistic intervention studies.⁹ These factors complicate the analysis of NETs and mechanisms of NET formation. In addition, NETs must be differentiated from cell-free DNA (cfDNA) originating independent of NET formation from necrosis, apoptosis, or tissue death.

Immunocytochemistry and immunohistochemistry are among the most common methods used to detect NETs.²⁸ Several methods permit NET detection real time in vivo, including live cell imaging,^29–32 intravital imaging,^10,33 and the use of DNA-intercalating dyes.^34,35 NETs can also be detected ex vivo in fluid secretions, serum, and tissue sections.^36,37 Many groups favor colocalization of at least three key NET components to accurately identify NETs in vivo: extracellular DNA, NE, and histones. SYTOX green is commonly used to detect extracellular DNA given its inability to penetrate live cells to stain intracellular DNA.³⁸ Detection of these three colocalized components helps to differentiate NETs from DNA released by dead or dying cells.³⁸

Flow cytometry methods employing antibodies against key NET components have been developed to augment in vivo analysis of NETs.³⁷ NETs in human sera have also been successfully detected with three-dimensional confocal laser scanning microscopy, which permits more sensitive detection of NETs.³⁹ The optimal means of NET detection remain an area of ongoing investigation and controversy.

Mechanisms of NET Formation

Neutrophil extracellular trap formation can occur via several distinct pathways and in response to multiple extrinsic stimuli.²⁸ The mechanisms of NET formation can be broadly categorized into lytic and nonlytic processes.⁹ Studies suggest that the activating stimulus determines the mechanism of NET formation.⁴⁰ During lytic NET formation, stimulation of neutrophils leads to nicotinamide adenine dinucleotide phosphate (NADPH) activation and production of reactive oxygen species (ROS) followed by the activation of PAD4 (Fig. 2). PAD4 converts arginine to citrulline on histones. This instigates loss of positive charges associated with arginine, disruption of the electrostatic charges between DNA and histones, and decondensation of chromatin.⁴¹ MPO-dependent translocation of NE into the nucleus further propagates chromatin unfolding.⁴² The nuclear membrane is disintegrated followed by the release of decondensed chromatin and granular proteins into the extracellular space. Neutrophils do not retain viability after lytic NET formation. Lytic NET formation independent of NADPH oxidase has been described in response to certain stimuli, including ionomycin and nicotine, which instead instigate mitochondrial ROS release.^43,44

Fig. 2.

Elucidating the process of NET formation has led to the identification of potential therapeutic targets. Lytic NADPH-dependent NET formation is provoked by multiple stimuli, including binding of TLR4 and complement receptors by infectious stimuli, PMA, and binding of antibodies to Fc receptors.³ These stimuli enhance signaling via the PKC or Raf-MEK (MAPK/ERK kinase) pathways. This culminates in activation of NADPH oxidase and generation of ROS, including H₂O₂. ROS activate PAD4 which induces decondensation of chromatin. Nuclear translocation of NE and MPO augments chromatin decondensation. The process of lytic NET formation highlights potential therapeutic targets, including use of taurine or N-acetylcysteine to neutralize ROS. NE inhibitors such as sivelestat have been safely used in certain clinical settings and therefore have therapeutic potential. Trials of PAD4 inhibitors in SLE have not produced encouraging results thus far.^124,125 MAPK, mitogen-activated protein kinase; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; NE, neutrophil elastase; NET, neutrophil extracellular traps; PAD4, peptidyl argininesssss deiminase IV; PKC, protein kinase C; ROS, reactive oxygen species; SLE, systemic lupus erythematosus; TLR4, toll-like receptssorss 4.

In contrast, neutrophils remain viable and retain key functions, including chemotaxis and phagocytosis, following nonlytic NET formation.^33,34,45 Nonlytic NET formation has been identified after exposure to certain stimuli including platelet binding to neutrophils and engagement of complement receptors or toll-like receptors (TLRs) by infectious stimuli.^33,45–47 In nonlytic NET formation, PAD4 activation occurs independently of NADPH. This is followed by chromatin decondensation, extrusion of chromatin and embedded proteins via blebbing of the nuclear envelope, and resealing of the nuclear envelope.^3,48 While NETs containing entirely nuclear DNA have been reported following nonlytic NET formation,^34,45 Yousefi et al⁴⁹ reported release of mitochondrial DNA without nuclear DNA following nonlytic NET formation. In this study, mitochondrial DNA release was detected after short-term activation of toll-like receptors 4 (TLR4) or complement factor 5 (C5a) receptors and was dependent on ROS. This mode of NET formation generates NETs with altered composition, as NETs containing only mitochondrial DNA would lack histones.⁴⁰ This form of NET formation may represent a protective and physiologic response to certain stimuli.

Neutrophil Extracellular Traps in Liver Disease

Alcohol-Associated Liver Disease

Alcohol-associated liver disease (ALD) encompasses a clinical and histologic continuum which includes steatosis, alcoholic steatohepatitis, alcoholic hepatitis (AH), and cirrhosis.⁵⁰ The pathophysiology of ALD is complex and is impacted by genetic factors, oxidative stress, and inflammation. Neutrophil infiltration of the liver is known to correlate with mortality in AH.⁵¹ While neutrophils contribute to the pathogenesis and severity of ALD, they also constitute an important defense against infection which is a major cause of mortality in patients with ALD.^52–58 Thus, neutrophils play a complex role in the pathophysiology of ALD and AH. Recent studies found that neutrophils that have been exposed to alcohol demonstrate impaired NET formation in response to inflammatory or infectious stimuli.^59,60 The mechanism by which alcohol impairs NET formation is unclear but could be due to reduced production of ROS by neutrophils exposed to alcohol or impaired neutrophil reactivity in the setting of high circulating lipopolysaccharide (LPS) levels which prime neutrophils and impair their reactivity to further stimuli. This study also found that alcohol impaired the ability of macrophages to clear NETs from the liver and that this impaired clearance propagated hepatic inflammation and injury. Given the mortality associated with infection in ALD, the authors suggest that temporary neutrophil depletion constitutes a potential therapy to ameliorate liver injury.

Portal Hypertension

Chronic liver disease which progresses to fibrosis and cirrhosis ultimately culminates in PHTN. PHTN accounts for significant morbidity and mortality in patients with chronic liver disease as it can be complicated by gastroesophageal varices, ascites, hepatic encephalopathy, and hepatorenal syndrome.⁶¹ We recently implicated NETs in the pathogenesis of PHTN in chronic liver disease secondary to congestive hepatopathy (CH) and cholestasis.⁶² We found that liver sinusoidal endothelial cells (LSECs) secrete the neutrophil chemotactic chemokine (C-X-C motif) ligand 1 (CXCL1) in response to mechanical stretch imposed by congestion. Using intravital imaging, we visualized early infiltration of neutrophils and platelets in a murine model of CH which entails partial ligation of the inferior vena cava (pIVCL). NETs were seen lining liver sinusoids 6 weeks after pIVCL and were spatially associated with fibrin, suggesting that they instigate formation of microvascular thrombosis in chronicliver disease. Genetic inhibition of NETs significantly abrogated PHTN in murine models of CH and cholestatic liver disease. Treatment with the NE inhibitor sivelestat similarly led to a significant decrease in portal pressures in two murine models of chronic liver disease. We found that NETs propagate PHTN by promoting formation of sinusoidal microvascular thrombosis which we suspect drives PHTN through volume and pressure impact in the liver sinusoids. The efficacy of sivelestat in reducing portal pressures in different models of chronic liver disease suggests that this constitutes a potential therapeutic modality to alleviate PHTN and prevent its complications.

Sepsis

The liver plays a formidable role in immune defense through the complementary actions of neutrophils and Kupffer cells. Studies suggest that a large proportion of circulating bacteria are efficiently sequestered within the liver.^63,64 Kupffer cells can capture bacteria under flow conditions by virtue of the complement receptor of immunoglobulin superfamily of receptors. Although neutrophils cannot directly capture bacteria, their ability to capture circulating pathogens via NET formation is critical in the defense against infection.⁴⁷ NET formation within the liver seems to exceed NET formation in other organs, including skin and lung.^10,33 This may be due in part to the heightened capacity of LSECs to bind neutrophils during endotoxemia. Stimulation of the endothelial TLR4 receptor by LPS facilitates binding of neutrophil CD44 (Cluster of Differentiation 44) to activated hyaluronan and thereby promotes adhesion of neutrophils within liver sinusoids.⁶⁵ Furthermore, while other organs are largely unable to retain NETs, the liver may be especially equipped to retain NETs by virtue of expression of VWF (Von Willebrand Factor) in liver sinusoids. Although host DNases degrade circulating NETs in both sterile and infectious conditions,⁶⁶ VWF anchors NETs to LSECs through its ability to bind histones¹⁰ and thus may prolong their retention and action in liver sinusoids.

While NET formation within the liver is critical in fighting infection, NETs may also be responsible for resident cell damage during infection and sepsis. Several NET components, including NE and histones, have cytotoxic properties which can damage epithelial and endothelial cells.^1,67–70 A recent study utilized intravital microscopy to visualize architectural disruption and focal necrosis within the liver following MRSA (methicillin-resistant Staphylococcus aureus) infection.¹⁰ Liver damage was significantly attenuated through genetic inhibition of NET formation, suggesting that NETs, as opposed to invading pathogens, are responsible for liver damage in the setting of infection.¹⁰ The authors attribute much of the bystander tissue damage to NE which remains proteolytically active after NET formation within the liver vasculature. The NE inhibitor sivelestat also significantly ameliorated liver damage after MRSA infection. The potential benefits of inhibiting NET formation to mitigate liver damage in the setting of infection must be weighed against risks of propagating infection given the key role of NETs in immune defense.

Ischemia-Reperfusion Injury

In addition to their role in infectious diseases detailed earlier, NETs have been implicated in several forms of sterile injury, including ischemia-reperfusion (I/R) injury. Liver I/R can occur in the setting of liver surgery, trauma, or hypovolemia. I/R injury is of particular concern in the setting of liver transplantation as it increases the risk for impaired early graft function and primary nonfunction.⁷¹ Liver I/R has also been implicated in systemic injury, including acute kidney injury and injury to the small intestine.^72,73 The pathogenesis of cell damage following I/R is complex and involves multiple cell types. An ischemic insult induces damage to hepatocytes and LSECs which can culminate in cell death. LSEC injury leads to upregulation of P-selectin and enhanced platelet adhesion which impairs sinusoidal blood flow and exacerbates ischemia.⁷⁴ This is followed by a robust inflammatory response which accompanies reperfusion and which propagates damage.⁷⁵ Neutrophils infiltrate early after ischemia and release ROS and proinflammatory cytokines and chemokines.⁷¹ Recent studies also implicate NETs in the pathogenesis of I/R. Damage associated molecular patterns such as histones and high mobility group box 1 protein are released from damaged hepatocytes and induce NET formation by activating neutrophil TLR4 and TLR9.⁷⁶ Liver injury following I/R was decreased in mice with neutrophil depletion or genetic knockout of TLR4. Another recent study implicates neutrophil NADPH-mediated superoxide formation in the generation of NETs following I/R. Mice pretreated with allopurinol and N-acetylcysteine to decrease circulating superoxide levels decreased NET formation and liver injury following I/R.⁷⁷ While neutrophil depletion carries infectious risks in patients undergoing liver surgery or transplantation, these results suggest that antioxidant treatment can protect against liver I/R by attenuating NET formation.

Liver Transplantation

Neutrophil extracellular traps have been implicated in several complications following solid organ transplantation. Recent studies in lung transplantation suggest that NETs stimulate release of inflammatory cytokines by alveolar macrophages. These cytokines promote CD4+ T helper cell differentiation and increase risk for allograft rejection.⁷⁸ NETs also contribute to the pathogenesis of primary graft dysfunction following lung transplantation through cytotoxic effects on pulmonary epithelial and endothelial cells, microvascular occlusion and ischemia, and perpetuation of inflammation posttransplant.⁷⁹

It has been hypothesized that NETs also contribute to cell death and dysregulated coagulation during liver transplantation. A recent study compared serum markers of NET formation in patients undergoing liver transplantation compared with healthy controls.⁸⁰ The indications for liver transplantation in this patient cohort included several forms of chronic liver disease, including alcoholic liver disease, nonalcoholic steatohepatitis (NASH), and primary sclerosing cholangitis. The authors found significantly elevated levels of circulating nucleosomes and cfDNA during liver transplantation. Levels of MPO-DNA complexes were elevated at the start of transplantation and increased during the anhepatic phase of transplantation. The author attributed the high baseline level of MPO-DNA to ongoing formation of NETs in the setting of chronic liver disease. However, it is unclear if elevated MPO-DNA levels noted during the anhepatic phase of transplantation are due to decreased clearance from the liver as opposed to increased NET formation during transplantation. The concentrations of cfDNA and nucleosomes, but not MPO-DNA, demonstrated significant correlation with markers of coagulation including thrombin-antithrombin complex levels and prothrombin fragments. While NET formation was detected prior to and during transplantation, markers of NET formation did not correlate significantly with markers of activated coagulation. The authors concluded that cfDNA and nucleosomes likely originated from cell necrosis and cell death sustained during surgery and that these molecules subsequently drive systemic activation of coagulation. The role of NETs in liver transplantation requires further evaluation, and NET inhibition has not yet been studied in the context of liver transplantation. However, the therapeutic potential of NET inhibition in transplantation must be balanced against the potential risks of infection in immunosuppressed hosts.

Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) has a worldwide prevalence which ranges from 5 to 46% and is the most prevalent chronic liver disease in the United States.^81–83 NAFLD encompasses two distinct histopathologic categories: isolated steatosis and NASH which manifests evidence of liver injury including inflammatory changes, hepatocyte injury and cell death, and fibrosis.⁸⁴ The pathogenesis of NAFLD is complex and is influenced by nutritional, genetic, metabolic, and inflammatory factors.^84,85

Nonalcoholic fatty liver disease is influenced by peripheral metabolic signals including visceral fat and insulin resistance.^86,87 Insulin resistance has been strongly associated with NASH, potentially through modulation of lipid metabolism which increases delivery of FFA (free fatty acids) to the liver.⁸⁸ Recent studies implicate NE in the pathogenesis of insulin resistance and obesity.^89,90 Serum from obese mice and human subjects demonstrate increased NE activity and decreased levels of the NE inhibitor α-1-antitrypsin. Similarly, mice with genetic deletion of NE have higher hepatic and adipose insulin sensitivity than wild type mice. NE promotes hepatic insulin resistance by degrading hepatocyte insulin receptor substrate 1. This in turn mitigates insulin signaling, leads to higher glucose production, and propagates cellular insulin resistance. Although the impact of NETs on insulin resistance and the metabolic syndrome has not been studied, these results implicate NET components in the pathogenesis of NASH through peripheral mechanisms. NETs may also promote development of hepatocellular carcinoma (HCC) in patients with NASH which is detailed below.

Cancer

Neutrophils are thought to comprise a significant proportion of immune cells that infiltrate and surround many solid tumors.^91,92 However, studies have produced conflicting results regarding the role of neutrosphils in the progression of cancer. This is due in part to the existence of two distinct neutrophil populations with opposing roles in the pathogenesis of cancer: antitumor N1 neutrophils and pro-tumorigenic N2 neutrophils. N1 neutrophils defend against cancer progression through several mechanisms, including production of ROS⁹³ and modulation of adaptive immune responses.⁹⁴ In contrast, N2 neutrophils can promote cancer development by facilitating angiogenesis,⁹⁵ inhibiting T-cell effector functions,⁹³ and producing tumorigenic cytokines.^9,92

The impact of NETs on cancer pathogenesis may be similarly context-dependent and impacted by the population of involved neutrophils, as both N1 and N2 neutrophils can form NETs.⁹ Cancer cells secrete factors, including granulocyte colony-stimulating factor (G-CSF)⁹⁶ and IL-8,⁹⁷ which promote NET formation. A subset of neutrophils (CD16^highCD62L^low) with enhanced capacity for NET formation was found to be increased in patients with head and neck squamous cell carcinoma.⁹⁷ This study found increased survival among patients with high population of CD16^highCD62L^low cells, suggesting a protective role for NET formation. The authors found that this population of neutrophils inhibited tumor cell migration although the role of NETs in the tumor immune response requires further clarification.

Several studies identify a pro-tumorigenic role of NETs in different cancers, including Ewing sarcoma, lung cancer, and pancreatic cancer.^92,98–100 Several NET components may promote tumorigenesis. A recent study found that NE and matrix metalloproteinase-9 (MMP-9) associated with NETs incite dormant cancer cells in mouse models of lung cancer.¹⁰¹ The authors found that NE and MMP-9 induced proteolytic remodeling of the extracellular matrix protein laminin. This processing event exposes an epitope of laminin which triggers an integrin β−1 signaling pathway and activation of dormant cancer cells. NE also promotes proliferation of adenocarcinoma cells by entering tumor cell endosomes and enhancing tumor cell signaling.^102,103 NE deficiency successfully decreased tumor burden in mice, suggesting that NE constitutes a potential therapeutic starget to decrease cancer progression.¹⁰²

Liver Metastases

Several studies suggest that neutrophils promote metastatic spread of tumors to the liver. A high neutrophil to lymphocyte count has been identified as a poor prognostic indicator in patients undergoing hepatectomy for HCC¹⁰⁴ and colorectal liver metastases.¹⁰⁵ NET formation induced by postsurgical infection has been shown to promote formation of hepatic metastases in a mouse model of lung carcinoma.⁹⁹ NETs have similarly been found to promote progression of liver metastases following surgical stress in models of colorectal cancer.¹⁰⁶

Neutrophil extracellular traps promote metastatic spread through several mechanisms. NETs trap circulating tumor cells via interaction with extracellular DNA and also promote early adhesion, migration, and invasion of cancer cells within the hepatic microcirculation. Intravascular NET formation can compromise endothelial integrity and thus promote extravasation of tumor cells from the circulation.^9,107 Systemic inflammation such as postsurgical stress or infection can also induce activation of platelets which can bind NETs and synergistically promote the adhesion and invasion of tumor cells.¹⁰⁸

Hepatocellular Carcinoma

Nonalcoholic steatohepatitis increases the risk of HCC even among patients without cirrhosis.^83,109,110 Neutrophils are among the inflammatory cells which infiltrate the liver in NASH,¹¹¹ and a recent study by van der Windt et al implicates NETs in the pathogenesis of HCC in the setting of NASH.¹¹² The authors employed murine model of neonatal streptozocin treatment and a high-fat diet which induces NASH and ultimately culminates in HCC at 20 weeks.¹¹³ The authors found that neutrophil recruitment and NET formation occur early in the pathogenesis of NASH. They suggest that exposure of neutrophils to free fatty acids induces NET formation. Shortly after NET formation, the authors observe infiltration of a population of macrophages derived from monocytes which are the key effector cells and sources of inflammatory cytokines in NASH.¹¹⁴ Genetic and pharmacologic inhibition of NET formation resulted in a variable impact on the NASH activity. However, NET inhibition significantly reduced the infiltration of monocyte-derived macrophages, leading the authors to conclude that NETs instigate macrophage infiltration. Inhibition of NET formation also led to a significant reduction in the size and numbers of HCCs formed, suggesting a pro-tumorigenic role in the setting of NASH.

Cancer-Associated Thrombosis and Portal Vein Thrombosis

The thrombogenic properties of NETs are well established in a variety of disease settings, including deep vein thrombosis and the antiphospholipid syndrome. NETs have also been implicated in the pathogenesis of cancer-associated thrombosis, which constitutes the second leading cause of death in cancer patients.^115,116 Studies suggest that neutrophils from mice with tumors are more likely to form NETs due to elevated circulating levels of G-CSF.¹¹⁷

Hepatocellular carcinoma predisposes to thrombotic complications, including portal vein thrombosis (PVT) and Budd–Chiari syndrome. The reported incidence of PVT in patients with HCC ranges from 20 to 65%, and studies suggest that PVT constitutes a poor prognostic indicator.^118–121 An elevated neutrophil-to-lymphocyte ratio portends poorer disease-free and overall survival among patients with HCC and PVT undergoing hepatectomy for HCC.¹²² Recent studies identified increased markers of NET formation and contact system activation in serum from patients with HCC, when compared with healthy controls.¹²³ Although these studies do not establish a causative role, they do suggest a potential role for neutrophils and NETs in the pathogenesis of PVT associated with HCC.

Conclusion

Evidence is accumulating that NETs contribute to the pathogenesis of several forms of chronic liver diseases. This heightens the need for the development of techniques to facilitate and standardize NET detection. The study of NETs is limited in part by the difficulty of NET visualization and lack of specificity of several NET markers, such as cfDNA. Neutrophils are easily activated in vitro; as a result, the in vitro analysis of NETs requires specific, standardized culture conditions and is prone to artifact.²⁸

The study of NETs remains a relatively new field with several areas which require further exploration, including context and disease-specific mechanisms of NET formation; impact of NET components on neighboring cells; and optimal means of NET inhibition. Their potential as a therapeutic target requires further study given the pivotal role that neutrophils and NETs play in immune defense. This is especially pertinent in the setting of liver disease as patients with chronic liver disease are at increased risk for infection.