Neutrophil extracellular traps: A central player in liver diseases

Abstract

Neutrophil extracellular traps (NETs) are known for their propensity to expel decondensed chromatin embedded with inflammatory proteins, which can entrap and kill pathogens. The destructive role of NETs in sterile liver inflammation has also been elucidated. Therefore, NETs appear to have both beneficial and pathogenic roles for varying diseases of the liver. In this review, we discuss recent findings related to NETs in inflammatory and fibrotic liver diseases as well as liver cancer. We provide a comprehensive overview of the underlying factors contributing to NET formation within the hepatic microenvironment, encompassing diverse pathological contexts. Moreover, we shed light on the multifaceted involvement of NETs in processes such as liver inflammation, fibrosis, and premalignant conditions. We also provide insights into the roles of NETs in providing a tumor-feeding niche for HCC survival and liver metastasis of other cancers. Finally, we will discuss potential therapies targeting NETs for future application in liver diseases. By reviewing these intricate interactions, we aim to enhance our understanding of the complex role played by NETs in hepatic pathophysiology.

KEY POINTS

1. NETs naturally protect the liver by entrapping and killing pathogens.

2. The pathologic role of excessive NET formation can exacerbate liver injury in various sterile inflammatory liver diseases.

3. NETs impair immune surveillance of premalignant hepatocytes.

4. NETs provide a pro-tumor environment for both primary and metastatic liver malignancies.

5. Neutrophil-modulating anticancer therapies have promising prospects in liver disease.

INTRODUCTION

Neutrophils are important immune phagocytes and play a central role in immune defense. Neutrophil extracellular traps (NETs) are fibrous web-like chromatin structures composed of decondensed DNA associated with histones and multiple granule proteins released by activated neutrophils during NETosis, which can occur through both lytic (suicidal) and non-lytic (vital) pathways.¹,²,³ First discovered in 2004, NETs are usually formed and possess antibacterial activity in a variety of infectious conditions, such as bacterial, parasitic, and fungal infections.⁴,⁵,⁶ NETs can help neutrophils immobilize and catch pathogens,⁷,⁸,⁹ which rely on the associated histones, proteolytic enzymes from granules, and enzymatic myeloperoxidase (MPO).¹,⁴

However, increasing evidence indicates that NET formation can also be found in nonpathogenic conditions, including but not limited to autoimmune disorders, diabetes, atherosclerosis, vasculitis, thrombosis, and carcinogenesis when dysregulated.¹⁰,¹¹ Under sterile conditions, NETs can be induced by IL-8,¹² immune complexes,¹³ crystals,¹⁴ or damage-associated molecular patterns (DAMPs), such as high mobility group box 1 (HMGB1).¹⁵ In contrast to the capacity to catch or kill pathogens, sterile-related NETs may be more harmful.¹⁶,¹⁷ NETs have been confirmed to damage tissues by directly killing epithelial and endothelial cells, which can contribute to the progression of tissue inflammation.¹⁸,¹⁹ Emerging evidence further emphasizes the protumorigenic role of NETs in various cancers,²⁰,²¹,²² which may be because they contribute to cell damage and regeneration. NETs have even been reported to favor tumor cell proliferation,²³ metastasis,²⁴,²⁵,²⁶,²⁷ immunosuppression,²⁸,²⁹ and cancer-associated thrombosis.³⁰,³¹ Furthermore, NETs can capture circulating tumor cells and promote their colonization.³²

Neutrophils and NETs have been reported to have protective effects in the pathogenesis of infectious liver diseases such as viral hepatitis.³³,³⁴ However, as a double-edged sword, excessive NET formation may play pathological roles in various sterile-related liver diseases, such as autoimmune hepatitis, ischemia-reperfusion liver injury (IRI), alcoholic liver disease (ALD), and metabolic dysfunction–associated steatotic liver disease (MASLD).³⁵,³⁶,³⁷ A typical sequence of chronic necro-inflammation, compensatory hepatocyte regeneration, liver fibrosis, and cirrhosis often precedes hepato-carcinogenesis.³⁸,³⁹ A disordered hepatic immune microenvironment is closely related to the initiation of hepatocellular carcinoma (HCC).⁴⁰,⁴¹ Accumulated NETs can lead to extensive hepatocyte damage and provide an immunosuppressive microenvironment for premalignant hepatocytes and cancer cell survival,⁴² which contribute to the initiation and development of HCC.³⁵ Reported as a novel biomarker to predict recurrence-free and overall survival,⁴³ NETs also correlate with the degree of liver dysfunction in patients with HCC. In addition, NETs contribute to the development of liver metastasis by facilitating the establishment of a premalignant niche for circulating cancer cells. In this review, we present the most recent research findings pertaining to NET formation and its involvement in premalignant conditions, particularly inflammatory and fibrotic conditions such as viral hepatitis, ALD, and MASLD. We further discuss how NETs contribute to the formation of a tumor-promoting niche that supports HCC progression and liver metastasis. By integrating recent findings from the past few years, this review provides an updated perspective on the multifaceted roles of NETs across the spectrum of liver diseases and highlights their potential as therapeutic targets.

NET FORMATION IN THE LIVER

Neutrophils are abundant in peripheral blood but can be recruited rapidly into the liver under different conditions, interacting with several other adaptive immune cells to form the hepatic immune network.⁴⁴ In general, NET formation occurs when aggregated neutrophils recognize infectious pathogens or sterile stimuli through various surface receptors such as receptor for advanced glycosylation end product (RAGE), Fcgamma receptor (FcγR), and Toll-like receptors (TLRs).⁴⁵ The liver is continuously exposed to gut-derived pathogen-associated molecular patterns, particularly lipopolysaccharide (LPS),⁴⁶ through the portal circulation, which can trigger NET formation. Meanwhile, mechanisms for NET clearance also exist in the liver; for example, deoxyribonucleases (DNases) can degrade the DNA components of NETs.² In viral hepatitis, T cells produce C-X-C motif chemokine ligand 8 (CXCL8) to attract neutrophils into the liver.⁴⁷ These recruited neutrophils recognize the virus via TLRs and activate NET formation through the IL-1 receptor-associated kinase 4 (IRAK4) or receptor-interacting serine kinase (RIPK) pathway.¹⁶ A similar process occurs when the liver is exposed to bacteria and LPS.⁴⁸,⁴⁹ Studies also report that Schistosoma japonicum eggs can stimulate neutrophils to release mitochondrial DNA and pro-inflammatory cytokines and chemokines.⁵⁰ Inducers of NETs, such as endotoxin and bacterial DNA, were reported to be significantly increased after acute alcohol intake, leading to the spontaneous release of NETs in patients with alcoholic liver disease.⁴⁸

In addition to infection-related NET formation, sterile ligands of various receptors can also activate NET formation through different downstream effector proteins. MASLD ranges from simple steatosis to metabolic dysfunction–associated steatohepatitis (MASH), liver cirrhosis, and even HCC.⁵¹,⁵² Neutrophil infiltration has long been observed in human MASLD and is mainly recruited by CXCL1 and IL-8 of hepatocytes.⁵³ Meanwhile, hepatic NET levels have been reported to increase gradually as MASLD progresses. FFAs are considered to be stimuli for augmented NET formation in MASLD.³⁵ Cholesterol crystals, which accumulate in MASLD livers,⁵⁴ have been confirmed to be a strong inducer of NETs.¹⁴ Macrophages are reported to change their phenotype to an M4-like pro-inflammatory phenotype, recruiting neutrophils and inducing NET formation in MASH progression.⁵⁵ Furthermore, neutrophils can be stimulated by immune complexes or autoantibodies to form NETs. For example, in autoimmune liver diseases, anti-neutrophil cytoplasm antibodies (ANCAs) seem to be associated with a more severe course,⁵⁶,⁵⁷ which may be because they are the main stimuli of NET formation.⁵⁸,⁵⁹ A recent study also reported that patients with autoimmune hepatitis (AIH) had a higher median percentage of low-density granulocytes (LDGs), which are actively involved in the formation of NETs.⁶⁰ Moreover, immune complexes are confirmed to be inducers of NET formation in autoimmunity or sterile inflammation,¹³ which remains unclear in autoimmune liver disease.

During liver ischemia/reperfusion (I/R), IL-33 released from liver sinusoidal endothelial cells can drive neutrophil infiltration and NET formation via its receptor ST2.⁶¹ Meanwhile, our recent study also suggested that IL-17A is a central driver of neutrophil infiltration and NET formation after I/R.⁶² We also demonstrated that I/R-related membrane-nonpermeable superoxide functions can activate TLR4 signaling pathways, which subsequently activate NOX, leading to NET formation.⁶³ In addition, the stressed hepatocytes in liver I/R can release DAMPs, such as HMGB1, to recruit neutrophils and stimulate NET formation via TLRs.⁶⁴ Moreover, not only damaged hepatocytes but also malignant cells can secrete cytokines such as IL-8 to induce NETs in the liver.¹²,⁶⁵ Interestingly, platelets activated by histones in the NET structure can, in turn, directly stimulate NET formation via P-selectin (P-SEL)/P-selectin glycoprotein ligand-1 (PSGL-1), which may be related to cancer-associated thrombosis.⁶⁶ A study further revealed that the tumor microenvironment (TME)-induced metabolic switch toward glycolysis in infiltrated neutrophils promoted NET formation in a reactive oxygen species-dependent manner in HCC.⁶⁷ As shown above (Figure 1), NET formation in the liver can be triggered by infectious or sterile inducers, suggesting that neutrophils/NETs are central players in the immune network of the hepatic immune microenvironment.

FIGURE 1.

NET formation in the liver. Neutrophils are recruited to the liver by chemokines under various conditions. Together with in situ neutrophils, they can recognize infectious stimuli through surface receptors to form NETs. Several infectious stimuli such as bacteria, viruses, schistosome eggs, and the related LPS, stimulate NET formation via TLRs on the surface of neutrophils. In autoimmune conditions, the immune complex or autoantibodies can combine with the Fc receptor on the surface of neutrophils to form NETs. Surgical stress can induce liver sinusoidal endothelial cells to secrete IL-33 to stimulate NET formation via ST2 on neutrophils. FFAs and cholesterol crystals are considered to be able to induce NET formation in MASLD. DAMPs secreted from damaged hepatocytes and cytokines from cancer cells can enhance NET release. Platelets activated by the histones can, in turn, stimulate NET formation. Pro-inflammatory macrophages can recruit neutrophils and induce NET formation. Abbreviations: DAMPs, damage-associated molecular patterns; FFAs, Free fatty acids; IL-33, Interleukin-33; LPS, lipopolysaccharide; MASLD, Metabolic dysfunction-associated steatotic liver disease; NET, neutrophil extracellular trap; ST2, Suppressor of tumorigenicity 2; P-SEL, P-selectin; PSGL-1, P-selectin glycoprotein ligand-1; TLRs, Toll-like receptors.

NETs and liver inflammation

As the first line of defense against infection, NETs can help hepatic neutrophils immobilize, capture, and kill invading pathogens in the liver. With hundreds of millions of cases estimated worldwide, chronic viral hepatitis is one of the major risk factors for HCC.⁶⁸,⁶⁹ NETs released by recruited neutrophils are regarded as the main players in antiviral immunity.⁵ However, a study indicated that NET release was decreased in patients with chronic hepatitis B virus (HBV) infection, and correlated negatively with hepatitis B surface antigen, hepatitis B e antigen, and hepatitis B core antibody levels.³⁴ Moreover, HBV C protein and HBV E protein might inhibit the release of NETs by decreasing reactive oxygen species production and autophagy. This suggests that impaired NETs function may promote viral escape from the immune system and provide a niche for chronic hepatitis virus infection. However, unlike chronic disease progression, NETs aggravated liver injury in fulminant viral hepatitis by promoting fibrin deposition and inflammation.⁷⁰ In HBV-related acute-on-chronic liver failure (ALF), circulating neutrophils display significantly impaired phagocytosis but an increased capacity to form NETs, which is closely related to poor outcomes in patients.⁷¹ Another recent study including a total of 676 patients with ALF also provides evidence that increased NET formation contributes to disease progression.⁷² The elevated plasma levels of MPO–DNA complexes in patients with ALF were further confirmed to be associated with poor outcomes.

In some other infectious conditions, impaired NET function also contributes to the progression of hepatic infection. Extracellular lipocalin-2 (LCN2) secreted by hepatocytes limits systemic bacterial infection, whereas neutrophils carry LCN2 protein to the infectious site and protect against bacterial infection through NETs.⁷³ A study confirmed that NET formation decreased at an early time point after LPS challenge in mice with alcohol binge.⁴⁸ However, in the efferocytosis phase, the clearance of NETs and related DAMPs by macrophages decreases as well. Dysfunctional NET function and efferocytosis after binge alcohol intake exacerbate liver injury associated with sepsis.⁴⁸,⁷⁴ In contrast, one study indicated that NET formation and platelet aggregation are the initiating factors of sepsis-related liver damage, and that extravasated platelet aggregation promoted by NET-facilitated detachment of sinusoidal endothelial cells is the origin of sepsis-induced liver dysfunction.⁷⁵ The phosphodiesterase III inhibitor, cilostazol (Ciz), which has a protective effect on liver sinusoidal endothelial cells and inhibits platelet aggregation, may prevent liver damage caused by NETs.⁷⁶ Interestingly, although NETs are abundant in the liver of patients with schistosomiasis, they do not kill schistosome eggs in vitro.⁵⁰ This evidence suggests that impaired NET function in the early phase of infection contributes to the establishment of chronic hepatitis or infection progression, but extensive NET formation leads to intensive hepatocyte damage, cell regeneration, and chronic hepatitis, which indicates that NETs are a double-edged sword in infectious hepatic liver injury.

The dark side of NETs is more evident in sterile hepatic inflammation, such as NALFD, autoimmune liver disease, or liver I/R. Several studies in humans and mice have confirmed a key role for neutrophils in MASLD.⁷⁷,⁷⁸ MASH is a progressive form of MASLD that slowly progresses toward cirrhosis and finally leads to the development of HCC.⁷⁹,⁸⁰ The levels of NET markers in the plasma of patients with MASH increased.⁸¹ Neutrophil infiltration and hepatic NETs help to form the protumorigenic inflammatory environment, and their elimination may reduce the progression of HCC in MASH.³⁵ Moreover, in MASH, increased intrahepatic platelet accumulation was confirmed to be correlated with NET formation in liver biopsies of patients with MASLD.⁸² Studies further indicate that NETs exert cytotoxic effects on endothelial cells,⁸¹,⁸³ converting them to a procoagulant and pro-inflammatory phenotype, which suggests the primary role of NETs in the hypercoagulable state of MASH patients.⁸⁴ LPS, a marker associated with NET formation was reported significantly higher in alcoholic hepatosteatosis and mice than in controls.⁴⁹ The liver-related symptoms of alcohol were alleviated in NETs knockout mice. In addition, emerging evidence suggests the role of NETs in the onset of autoimmune diseases as a contributor to disease flares.⁵⁹,⁸⁵,⁸⁶,⁸⁷ However, studies of NETs in autoimmune liver diseases such as autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cholangitis are lacking.⁸⁸ A recent study reported that patients with AIH had a higher median percentage of low-density granulocytes (LDGs) and LDGs expressing MPO (a specific marker of NETs), which may be a useful marker in the diagnosis of AIH.⁶⁰ NETs are also involved in hepatic I/R injury, promoting liver injury following I/R.⁶⁴,⁸⁹,⁹⁰ In sterile liver disease, NETs seem to be a foe rather than a friend, and inhibiting NET formation may prevent hepatocyte damage, regeneration, and subsequent liver fibrosis.

NETs and liver fibrosis

Liver fibrosis occurs in most types of chronic liver disease, such as viral or alcoholic hepatitis and MASLD.⁹¹ Advanced liver fibrosis results in cirrhosis, portal hypertension, and HCC.⁹²,⁹³ As a strong promoter of hepatocyte injury, excess NET formation contributes to the initiation of liver fibrosis and promotes the progression of fibrosis to liver cirrhosis (LC). NETs have been confirmed to be positively related to liver fibrosis and HCC in patients with MASH,³⁵ NET inhibition may act as an attractive treatment target for MASH-liver fibrosis.⁹⁴ Similarly, in patients with AIH, low-density granulocytes, which are actively involved in the formation of NETs, may be helpful in assessing liver fibrosis.⁶⁰ In addition, soluble egg antigens can trigger the release of NETs in patients with S. japonicum,⁵⁰ which may contribute to the related hepatocyte injury and liver fibrosis.

Liver cirrhosis is a progressive process of liver fibrosis associated with impaired functions in adaptive and innate immune responses, which is also referred to as cirrhosis-associated immune dysfunction. A recent study including 95 patients with cirrhosis concluded that H3Cit-DNA and MPO-DNA, 2 specific markers of NETs, were significantly elevated in plasma from patients with cirrhosis compared with healthy controls and correlated with the degree of liver dysfunction.⁹⁵ NETs can also enhance procoagulant activity in LC patients with portal vein thrombosis.⁹⁶ NETs are also involved in fibrosis-related portal hypertension.⁹⁷ Furthermore, the presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis, which ultimately results in HCC.⁹⁸ In general, premalignant senescent hepatocytes can secrete chemokines and cytokines and are subject to immune-mediated clearance, which depends on an intact CD4⁺ Th1-cell–mediated adaptive immune response.⁹⁹,¹⁰⁰ However, our study indicates that NETs may interact with hepatic naïve CD4⁺T cells and balance the gene profile toward regulatory T cells rather than effector T cells, such as Th1 cells,⁴² which may impair the senescence surveillance and provide a premalignant niche for malignant cell survival in cirrhosis.

However, several studies also reported NETs as a protective factor in patients with decompensated cirrhosis, which is susceptible to infections and sepsis. A human study reported a significant decrease in the release capability of NETs as the level of LC in the patient increased,¹⁰¹ which demonstrated that LC patients with NET release deficiencies may have an increased rate of complications. Another study also provided evidence that patients with LC revealed a significant decrease in the release of NETs from the spontaneous bacterial peritonitis (SBP) group compared with controls, which suggests that impaired NET function is related to the pathogenesis of SBP in those patients.¹⁰² One study suggested that sepsis plasma can stimulate and increase NET formation in healthy neutrophils, but the phagocytic activity of neutrophils was significantly decreased in LC patients with sepsis.¹⁰³

As shown above, NETs, acting as a double-edged sword, kill invading pathogens in the liver while also contributing to the progression of chronic hepatitis, liver fibrosis, and carcinogenesis (Figure 2). The fate of NETs in the premalignant conditions of the liver varies as the disease stage changes. Impaired NETs may contribute to chronic liver infection, but excess NETs can cause intensive inflammatory liver injury, cell regeneration, and carcinogenesis. Dysfunctional NET function will also lead to severe complications, such as sepsis, in end-stage liver disease. NETs also contribute to impaired senescent surveillance. Therefore, in premalignant liver conditions, clinical therapy targeting NETs may largely rely on the disease stage of the patients and the most important clinical symptoms.

FIGURE 2.

The role of NETs in the premalignant liver. NETs act as a double-edged sword in the premalignant liver. Neutrophils protect against bacterial infection through NETs. Dysfunctional NET function and efferocytosis after binge alcohol intake exacerbate liver injury. NETs play protective roles in viral hepatitis infection, preventing the establishment of chronic hepatitis. However, NETs exacerbate liver injury in fulminant viral hepatitis (acute-on-chronic viral hepatitis) by promoting fibrin deposition and inflammation. Excessive NET formation and platelet aggregation are the initiating factors of sepsis-related liver damage. The dark side of NET formation is more obvious in the sterile hepatic inflammation, such as MASLD, alcoholic liver disease, autoimmune liver disease, or liver IRI, which may be related to the onset of chronic inflammation, liver fibrosis, cirrhosis, and carcinogenesis. Abbreviations: ALD, alcoholic liver disease; IRI, Ischemia–reperfusion injury; MASLD, Metabolic dysfunction-associated steatotic liver disease; HMGB1, high mobility group box 1; LPS, lipopolysaccharide; NETs, neutrophil extracellular traps; TLR4, Toll-like receptor 4.

NETS IN LIVER CANCER AND METASTASIS

The liver is the sixth most common site of primary cancer in humans, and HCC is the fourth leading cause of cancer-related death worldwide.¹⁰⁴,¹⁰⁵ Ninety percent of HCCs develop due to chronic liver inflammation, such as MASH, alcohol hepatitis, and viral hepatitis,¹⁰⁶,¹⁰⁷,¹⁰⁸ usually following the induction of fibrosis with or without subsequent cirrhosis.¹⁰⁹,¹¹⁰ The immune system can monitor abnormal hepatocytes and prevent them from becoming malignant, which is termed immune surveillance.¹¹¹ The initiation of HCC is closely combined with impaired or dysregulated immune surveillance.³⁸ Also, NETs can create a tumor-feeding niche, thereby facilitating liver metastasis of other cancers.¹¹²,¹¹³ Notably, the roles of NETs in immune surveillance and tumor progression may be stage-dependent and context-specific in liver cancer. During early inflammatory responses, NETs may participate in pathogen clearance and immune surveillance. However, persistent NET formation can exacerbate liver injury, sustain chronic inflammation, and reshape the TME.

NETs in the proliferation and metastasis of HCC

NET-releasing neutrophils were also reported to be positively correlated with the tumor metastasis potential of HCC patients and negatively associated with patient survival.⁶⁷ A human study including 82 patients with HCC revealed that H3Cit-DNA (a plasma marker of NET formation) was significantly elevated in plasma from patients with HCC compared with healthy controls,⁹⁵ which correlates with the degree of liver dysfunction in patients with HCC. Moreover, in patients with HCC who underwent surgery, a correlation between preoperative NETs levels and survival was reported,⁴³ which indicates that the preoperative NETs level as a biomarker can predict whether patients can benefit from closer follow-up because of the higher risk for recurrence. Furthermore, the poorer survival of patients with diabetic HCC than nondiabetic HCC has been reported to be related to diabetes-induced NETs.¹¹⁴ Emerging studies have suggested that NETs affect tumor progression and metastasis through multiple different mechanisms.

NETs were found to be increased in human HCC tumor tissues compared with paired nontumor liver tissues,⁶⁷ which suggests the central role of NETs in tumor proliferation. Our study provides evidence that NET inhibition reduces the progression of MASH to HCC in a clinically relevant mouse model of MASH.³⁵ The formation of NETs upon chronic alcohol use, which is induced by gut-derived LPS, promotes alcoholic hepatosteatosis and subsequent HCC.⁴⁹ Different studies have confirmed that NET depletion can inhibit tumor growth in mice.⁶⁷,¹¹⁴ Meanwhile, NETs in the TME of HCC seem to be highly pro-inflammatory and prometastatic. The invasion ability of HCC cells was enhanced by coculture with HCC neutrophils via NETs, while neutrophils from healthy donors inhibited the invasion ability.¹¹⁵ Further mechanistic investigation revealed that NET-associated cathepsin G (cG) promoted HCC metastasis in vitro as well as in vivo.¹¹⁵ Other evidence suggests that NETs can induce the migration of cancer cells and downregulate the tight junction molecules on adjacent endothelial cells, which subsequently facilitate tumor metastasis.⁶⁷ A recent study revealed that neutrophils in patients with HCC contained high levels of mitoROS, which means that related NETs were enriched in oxidized mitDNA.¹¹⁶ In vitro, the oxidized mtDNA bound to NETs can trigger HepG2 cells to secrete metastasis-promoting inflammatory cytokines and chemokines. Furthermore, NETs can trap HCC cells and activate Toll-like receptor (TLR) 4/9-COX signaling, which subsequently induces cell-death resistance and enhances their invasiveness.¹¹⁷

NETs and immune surveillance

The liver has a strong ability to repair itself after acute damage. The immune system will recognize and clear abnormal hepatocytes (including premalignant hepatocytes and cancer cells), which is termed immune surveillance.¹¹¹,¹¹⁸ However, in chronic necro-inflammation, cell death, compensatory regeneration, and disordered immune response together lead to the survival of abnormal or malignant hepatocytes, which contribute to the initiation or development of HCC.¹¹⁹,¹²⁰ Exhausted or ineffective adaptive immune cells provide a chance for malignant cells to escape surveillance and survive.¹²¹,¹²² An intact CD4⁺ T-cell (Th1)-mediated adaptive immune response in cooperation with monocytes can complete the clearance of premalignant senescent hepatocytes.⁹⁹ Cytotoxic CD4⁺ and CD8⁺ T cells are also needed for the recognition and clearance of malignant hepatocytes.³⁸ Recently, our results suggested that NETs can link innate and adaptive immunity by promoting regulatory T cells (Tregs) differentiation via metabolic reprogramming of naïve CD4⁺ T cells in MASH,⁴² which results in the impaired function of Th1 cells and provides an immunosuppressive environment for HCC initiation. NETs can impact the gene expression profiles in naïve CD4⁺ T cells and tend them toward Tregs rather than effector T cells (Teffs). Blockade of NETs in vivo using PAD4^−/− mice or DNase I treatment reduces the activity of Tregs and promotes the function of cytotoxic CD4⁺ and CD8⁺ T cells, therefore preventing HCC initiation and development. Moreover, exhausted cytotoxic T cells were also found in the NETs-rich hepatic tumor microenvironment.²⁸ In vitro, NETs have been confirmed to contain the immunosuppressive ligand PD-L1, which can induce the exhaustion and dysfunction of CD4⁺ and CD8⁺ T cells.²⁸ Mechanistically, NETs affect the exhaustion of T-cell responses through metabolic reprogramming. Interestingly, NETs have also been reported to be able to coat tumor cells to build a physical barrier, shielding them from cytotoxic T-lymphocytes and NK-cell–mediated killing.¹²³,¹²⁴ Excessive NETs may be a barrier to the success of adoptive NK-cell therapy in patients with HCC.¹²⁵ This evidence emphasizes the important role of NETs in immune surveillance, which matters not only in the phase of the premalignant niche but also in the tumor microenvironment of HCC (Figure 3).

FIGURE 3.

The role of NETs in HCC and liver metastasis. NETs are involved in immune surveillance, contributing to the immunosuppressive microenvironment for malignant cell survival. NETs favor tumor cell proliferation, metastasis, and cancer-associated thrombosis of HCC. NET formation in the premetastatic liver is important for the onset of liver metastasis. NETs can act as the “fishing net” to capture the passing cancer cells into the liver. Abbreviations: HCC, hepatocellular carcinoma; IL-10, Interleukin-10; NETs, neutrophil extracellular traps; PD-1, Programmed cell death protein 1; PD-L1, Programmed death-ligand 1; PVTT, portal vein tumor thrombosis; Teff, effector T cell; Treg, regulatory T cell.

NETs in HCC-associated thrombosis

NETs can contribute to thrombus formation by acting as scaffolds for platelet adhesion and aggregation and enhancing coagulation.¹²⁶,¹²⁷,¹²⁸ Recent studies suggest that NET components enhance coagulation by both activating the intrinsic pathway and degrading an inhibitor of the extrinsic pathway, leading to cancer-associated thrombosis.¹²⁹,¹³⁰ NETs were found in thrombi from cancer patients.¹³¹,¹³² Similarly, an increase in the release of NETs has been reported in patients with HCC, particularly those with portal vein tumor thrombosis (PVTT).¹¹⁵,¹¹⁷ In a human study including 177 patients with HCC (77 patients suffered from PVTT), NET formation markers and contact system activation markers were indicated to be significant thrombotic risk factors in patients with HCC.¹³³ A mechanistic study revealed that the negatively charged histone–DNA complex can activate coagulation factor XII, which initiates contact system activation and promotes venous thrombus formation. In addition, histones can increase thrombin generation in a platelet-dependent manner, and the platelets activated by NETs, in turn, promote the pathological thrombus formation.¹³⁴,¹³⁵,¹³⁶,¹³⁷

NETs in liver metastasis

In the hepatic microenvironment, NETs also play a central role in establishing the premalignant niche for circulating cancer cells (Figure 3). Elevated NET levels have been confirmed to be associated with liver metastasis of various cancers (Table 1). The most common site of colorectal cancer (CRC) metastasis is the liver.¹¹²,¹¹³ A study showed that in a liver sterile inflammation setting, murine neutrophils can exit the inflammatory environment.¹⁵⁰,¹⁵¹ Then, NET-formed neutrophils in blood vessels can further capture passing cancer cells.³⁶ A recent investigation revealed that NET formation in premetastatic liver is more important for the onset of liver metastasis compared with the primary tumor.¹⁵² Serotonin (5-hydroxytryptamine, 5-HT) released by neuroendocrine tumor cells has been shown to facilitate liver metastasis through inducing NET formation.¹⁴⁹ Therefore, NETs may be a key metastatic initiation factor. Except for the fact that NETs can promote the motility of cancer cells,³²,¹⁵³ hepatic NETs can act as the “fishing net” to capture the passing cancer cells into the liver³⁶,⁶⁵,⁸¹,¹⁴¹ and shield them from cytotoxic lymphocytes.²⁵ In mice subjected to liver I/R, circulating platelet activation and platelet-neutrophil aggregates were reported to be significantly increased, which results in the capture of circulating tumor cells³⁶ and immunothrombosis.¹³⁷ A study further revealed that β1 integrin expressed on NETs participates in the adhesion of tumor cells to liver sinusoids.¹⁴¹ NET-associated carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has also been shown to stimulate the relocation of CRC cells to the liver both in vitro and in vivo.¹²⁴ These findings may explain the fact that MASLD, in which NETs aggregate in the liver, can enhance the liver metastasis of CRCs.¹⁵⁴,¹⁵⁵,¹⁵⁶

TABLE 1.

The role of NETs in liver metastasis of various cancers

Cancer	Experimental type	The origin of NETs	The mechanism	Year
Colorectal cancer	Patients and a mouse model (cell line MC38)	Surgical stress	Trigger HMGB1 release and activate TLR9 pathways in CRC cells	2016138
	Mouse model (cell lines MC38 and CT26)	Surgical stress	Interact with platelets through TLR4-ERK5 to capture CTCs	202136
	Patients and mouse model (cell lines MC38 and HCT116)	Solid tumors recruit neutrophils in the TME	Increase the migratory ability of CRC cells through mitochondrial respiration	201923
	Mouse model (cell lines HT-29 and MC38)	Tumor-related/PMA-stimulated induction	CEACAM1 on NETs promotes CRC cells' adherence to liver sinusoids	2020124
	Patients and a mouse model (cell line MC38)	Tumorous IL-8–induced NET formation	Trap and enhance the metastatic capacity of CTCs	202065
	Mouse model (cell line MC38)	Tumor-related induction	Promote adhesion of CTCs to the liver	2019139
	Patients and a mouse model (cell line HCT116)	Hepatic infiltrating neutrophils	Bind to CCD25 on CTCs	202032
	Patients and a mouse model (cell line HCT116)	LPS	—	2021140
Lung cancer	Mouse model (cell lines: A549 and H59)	CLP-induced polymicrobial abdominal sepsis	Trap and promote CTC cell adhesion in hepatic sinusoids	201381
			Adhere CTC cells through the expression of β1 integrin	2017141
	Mouse model (cell line H59)	Tumor-related induction	Promote adhesion of CTCs to the liver	2019139
Breast cancer	Patients and mouse model (cell lines: MDA-MB-231, MCF-7, 4T1, and E0771)	Infiltrating neutrophils in the liver	Bind to CCDC25 on CTCs and activate the ILK-β-parvin pathway to enhance cell motility	202032
	Mouse model (cell line: 4T1)	CXCR4hiCD62llo aged neutrophils	—	2021142
	Mouse model (cell line: 4T1)	Immature low-density neutrophils	—	2019143
Gastric cancer	Patients and mouse model (cell line: AGS)	GC-related NET formation	Induce the EMT of GC cells	2021144
	Patients and mouse model (cell lines: MKN-45 and MGC-803)	Postoperative abdominal infection	Modulate EMT in GC cells and trap GC cells	2022145
Pancreatic cancer	Patients and mouse model (cell line: MDA-PATC)	Recruited neutrophils in primary and metastatic foci	Enhance PC cells' invasion	2021146
	Patients and mouse model (KrasLSL-G12D, PDX1-Cre and Trp53R172H)	PC cells-induced formation	Activate cancer-associated fibroblasts	2020147
	Mouse model (cell lines: PANC-1 and MIAPaCa-2)	PC cells-related formation	Promote EMT of PC cells	2021148
Melanoma	Mouse model (cell line: B16LS9)	Aged neutrophils in the liver	—	2021142
Neuroendocrine cancer	In vitro and in mouse models of neuroendocrine cancers (NEPC, SCLC, MTC)	5-HT secreted from neuroendocrine tumor cells induced the formation	5-HT induces H3Q5ser and PAD4-dependent histone citrullination	2025149

Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); CCDC25, coiled-coil domain containing 25; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1; CLP, cecal ligation and puncture; CRC, colorectal cancer; CTC, circulating cancer cell; CXCR4, C-X-C motif chemokine receptor 4; EMT, epithelial–mesenchymal transition; ERK5, extracellular signal-regulated kinase 5; GC, gastric cancer; H3Q5ser, histone serotonylation; IL-8, interleukin 8; ILK, integrin-linked kinase; LPS, lipopolysaccharide; MTC, medullary thyroid cancer; NEi, NE (neutrophil elastase) inhibitor; NEPC, neuroendocrine prostate cancer; PAD4, peptidylarginine deiminase 4; PC, pancreatic cancer; SCLC, small-cell lung cancer; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9; TME, tumor microenvironment.

POTENTIAL THERAPIES TARGETING NETs

Given the destructive roles in the carcinogenesis and development of HCC, NETs are quickly becoming a novel target for therapeutics in the prevention and management of HCC (Table 2). The blockade of other tumor-associated neutrophil functions has been involved in several clinical trials and preclinical research (NCT05012501, NCT07207304, and NCT05909319).¹⁶⁵,¹⁶⁶,¹⁶⁷ However, as we know, the role of NETs in capturing and clearing pathogens, inappropriate NET clearance may have other unwanted effects on the immune system.¹⁶⁸ Therefore, when and how to inhibit NETs to prevent the progression of liver disease and HCC development remain urgent issues.

TABLE 2.

Potential anti-NET therapeutics in HCC and liver metastasis

Disease	Animal model	Intervention to inhibit NETs	Clinical significance	Year
Liver inflammatory condition
Liver IRI	Rat liver I/R model	Thrombomodulin	Reduce apoptosis of hepatocytes	2022157
	Mouse liver I/R model	Allopurinol and N-acetylcysteine	Ameliorate liver inflammation	201663
		Hydroxychloroquine	Prevent hepatic injury	2020158
		Anti-IL17A antibody or DNase I	Reduce hepatic damage	201962
		rAAV8 encoding mouse TIMP-1	Limit the NET-mediated cytotoxicity	2018159
		IL-33 and ST2 KO	Reduce inflammatory cascades	201761
		DNase I or PAD4 KO	Reduce immunothrombosis and organ injury	2020137
	Liver I/R and metastasis mouse model	Exercise	Protect the liver from injury and metastasis	202189
MASH-liver IRI	MCD plus liver I/R mouse model	Supplementary histidine-rich glycoprotein	Minimize liver damage in steatotic liver	2021160
FVH	FVH mouse model	DNase I	Reduce fibrin deposition and hepatitis	202270
ACLF	Neutrophil from ACLF patients	Glycolysis inhibitor: 2-deoxy-glucose	Reverse neutrophil dysfunction; ameliorate hepatitis	202171
ALD	ALD mouse model	Depletion of neutrophils	Ameliorate sepsis-related liver injury	201848
HCC initiation and development
ALD-HCC	ALD-HCC mouse model	Removal of intestinal bacteria or TLR4 KO	Decrease the formation of NETs and HCC initiation in ALD	202249
MASH-HCC	STAM and DEN-HFCD mouse model	DNase I or PAD4 KO	Reduce HCC initiation in MASH	202142 201835
HCC	HCC mouse model (Hepa1-6 cells)	DNase I + aspirin/hydroxychloroquine	Prevent HCC metastasis	2020117
		DNase I or GSK484 (PAD4 inhibitor)	Inhibit tumor growth in vivo	202267
		A biocompatible hydrogel with NETs lyase	Inhibit post-surgical HCC recurrence	2022125
Liver metastasis
Colorectal cancer	CRC liver metastasis mouse model	AAV-mediated DNase I liver gene transfer	Inhibit liver metastasis	2020161
	Orthotopic murine model of CRC	A nanoplatform for efficient loading and release of DNase I	Sensitize CRC to immune therapy; reduce liver metastasis	2022162
Breast cancer	Breast cancer mouse model (4T1 and EMT-6 cell lines)	A doxorubicin-loaded micellar low-molecular-weight-heparin-astaxanthin nanoparticle	Inhibit liver metastasis	2021163
Pancreatic cancer	PC mouse model (PANC-1 cell line)	Thrombomodulin	Degrade HMGB; reduce liver metastasis	2021148
	Spontaneous PDAC mouse model	DNase I	Suppress liver micrometastasis	2020147
Melanoma	B16 mouse model	Pulmozyme	Reduce liver metastasis	2021164

Abbreviations: ACLF, acute-on-chronic liver failure; ALD, alcoholic liver disease; CRC, colorectal cancer; DEN-HFCD, high-fat diet + diethylnitrosamine; DNase I, deoxyribonuclease I; EMT, epithelial–mesenchymal transition; FVH, fulminant viral hepatitis; HCC, hepatocellular carcinoma; HMGB1, high mobility group box 1; IL-17A, interleukin 17A; I/R, ischemia-reperfusion; IRI, ischemia-reperfusion injury; KO, knockout; MASH, metabolic dysfunction–associated steatohepatitis; MCD, methionine- and choline-deficient diet; PAD4, peptidylarginine deiminase 4; PC, pancreatic cancer; PDAC, pancreatic ductal adenocarcinoma; rAAV, recombinant adeno-associated virus; ST2, suppression of tumorigenicity 2; STAM, stelic animal model; TIMP-1, TIMP metallopeptidase inhibitor 1; TLR4, Toll-like receptor 4.

For example, improving NET function can prevent HBV from escaping the immune system and causing chronic infection,³⁴ but inhibiting NETs may be a new strategy for the treatment of fulminant viral hepatitis.⁷⁰ In ACLF patients, glycolysis inhibition using 2-deoxy-glucose can reverse neutrophil dysfunction and inhibit NET formation.⁷¹ Consistent with these findings, clinical studies have begun to investigate NET-related biomarkers in ACLF to evaluate their diagnostic and prognostic value (NCT05909319). Excessive NETs contribute to the pathogenesis of ALD, but NET formation is impaired in response to infectious stimuli in ALD, in which the depletion of neutrophils before binge alcohol ameliorates LPS-induced systemic inflammation and liver injury.⁴⁸,⁷⁴ Moreover, in the MASH background, supplementary histidine-rich glycoprotein can prevent hepatic IRI in the liver by decreasing neutrophil infiltration and NET formation.¹⁶⁰

In addition, increasing evidence suggests that targeting NETs and the related pathways might be a novel therapeutic strategy for hepatic I/R treatment.¹⁶⁹ Therapeutic targeting of IL-33/ST2 and subsequent NET induction might extend novel strategies to minimize liver damage associated with sterile inflammation.⁶¹ Inhibiting NETs using an anti-IL17A antibody or DNase I can significantly disassemble the inflammatory injury during liver I/R.⁶² However, exercise can protect the liver from IRI and metastasis by decreasing neutrophil infiltration and NET formation.⁸⁹ The protective effect of thrombomodulin on hepatic injury during IRI via inhibition of NETs has also been reported.¹⁵⁷ Allopurinol and N-acetylcysteine can decrease superoxide-induced NETs in hepatic pathophysiologic settings in liver I/R.⁶³ Hydroxychloroquine inhibiting NET formation effectively alleviates hepatic inflammation.¹⁵⁸ Furthermore, recombinant adeno-associated virus type 8 vectors (rAAV8) encoding mouse metalloproteinases-1 (TIMP-1), the physiological inhibitor of matrix metalloproteinase-9 (MMP-9), were reported as a novel strategy to limit the impact of NET-mediated cytotoxicity in hepatic I/R.¹⁵⁹ It is worth noting that NETs can cause systemic immunothrombosis and distant organ injury following a local inflammatory insult with liver I/R, and immune-thrombi and organ damage were dramatically decreased when NETs were inhibited.¹³⁷ These findings suggest that we may give attention to NET intervention depending on the phase and severity of liver disease.

As the disease progresses, targeting NET exhaustion exerts more effective therapeutic effects. A clinical study in patients with liver cirrhosis has investigated whether NET-associated markers, including myeloperoxidase, neutrophil elastase, and citH3, are correlated with procoagulant activity and can predict the development of portal vein thrombosis (NCT05012501). Our recent study confirmed that NET inhibition through DNase I or PAD4^−/− mice can reduce HCC initiation in MASH livers.³⁵,⁴² We also found that combining anti-Treg therapy with NET inhibition may be more effective in preventing carcinogenesis in MASH.⁴² In alcoholic liver disease-related HCC, removal of intestinal bacteria with antibiotics or TLR4 KO can lead to a decrease in NETs and subsequent HCC development.⁴⁹ Similarly, another study confirmed that NET depletion using DNase I or GSK484 (PAD4 inhibitor) can inhibit tumor growth in an HCC mouse model established with the Hepa1-6 cell line.⁶⁷ Moreover, a recent study confirmed that a combined strategy of DNase I plus the anti-inflammatory drugs aspirin/hydroxychloroquine effectively impaired HCC metastasis in a mouse model established with the Hepa1-6 liver cancer cell line.¹¹⁷ The study also reveals that targeting NETs rather than neutrophils themselves can be a practice strategy against HCC metastasis. This approach may provide greater specificity by preserving the antimicrobial functions of neutrophils while limiting NET-mediated inflammation and tumor-promoting effects.

It is worth noting that post-resection recurrence in patients with HCC remains an intractable issue. Recently, a biocompatible hydrogel with a tumor acidity neutralizer (mesoporous bioactive glass nanoparticles) and NET lyase (DNase I) has been reported to enhance NK infusion to prevent post-resection HCC recurrence without systemic toxicity.¹²⁵ This “gel” reshapes the tumor immune microenvironment and removes NETs from tumor cells, leading to maximal lymphocytotoxicity in the TME of HCC. A prospective randomized clinical trial is currently evaluating whether different anesthetic strategies (total intravenous anesthesia vs. inhalational anesthesia), with or without perioperative lidocaine, influence NET formation and clinical outcomes in patients undergoing surgery for HCC or hepatic metastases (NCT07207304).

Targeting hepatic NETs in the premalignant niche represents a novel therapeutic strategy for liver metastasis.¹⁴⁷,¹⁴⁸,¹⁵³,¹⁶⁴ Our recent study demonstrated that an adeno-associated virus (AAV) gene therapy vector specifically expresses DNase I in the liver.¹⁶¹ Following a single intravenous injection, liver metastasis in a mouse model of CRC was significantly suppressed. A recent study reported a nanoplatform for the efficient loading and photoregulated release of DNase I, which can help break the ”NET-mediated physical barrier,” thereby increasing the contact of immune cytotoxic cells with tumor cells and decreasing liver metastasis.¹⁶² A doxorubicin-loaded micellar low-molecular-weight-heparin-astaxanthin nanoparticle has been reported to decrease the formation of NETs,¹⁶³ which provides a promising therapeutic strategy for breast cancer and its liver metastasis.

CONCLUSION

NETs appear to play a double-edged sword in various liver diseases. Impaired NETs lead to the escape and survival of pathogens such as the hepatitis virus, which contribute to the progression of infection and the establishment of chronic hepatitis. However, excessive NET formation exacerbates liver injury at the later stage of viral hepatitis. The negative sides of NETs stand out even more in sterile hepatitis such as ALD, MASLD, and liver IRI. Hepatic NETs also participate in providing the prometastatic niche in the liver for various cancers, such as colorectal cancer and breast cancer. Targeting NETs as a potential clinical therapy for HCC and liver metastasis is promising but still faces some challenges, such as treatment timing and management, which may depend on the disease phase or the heterogeneity of patients.