Neutrophils are the most abundant white blood cells of the immune system, serving as the ﬁrst line of defense against pathogens. Neutrophils execute phagocytosis, degranulation, and NETosis functions as part of the innate immune response to clear pathogens, recruit immune cells, and mediate tissue repair. When bacteria, viruses, injuries, or other threats are detected, neutrophils can produce neutrophil extracellular traps (NETs) — sticky webs made of long nucleosome strings that trap and immobilize pathogens to stop the threat from spreading around the body. NETs are composed of microbicidal components that include DNA, histones, and granule proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE).
NETs are released from neutrophils in a mechanism of cell death, termed NETosis, that is independent from apoptosis and necrosis. NETosis can be activated by the presence of pathogens or induced by immune components such as antibodies and cytokines. During NETosis, nuclear membranes are destructed, histones are modified, and chromatin is decondensated. The cell plasma membrane then ruptures leading to the release of NETs containing nucleosomes and bactericidal proteins into the extracellular space.
Recently, additional terms have been used in scientific literature in reference to NETosis. Lytic or suicidal NETosis describes the release of NETs caused by cell membrane lysis, which induces neutrophil death. Vital or survival NETosis occurs when chromosomal DNA is released through exocytosis of secretory vesicles, enabling neutrophil survival. Mitochondrial DNA (mtDNA) extracellular traps indicate the release of mtDNA instead of nuclear DNA during NETosis. The release of mtDNA ETs results in neutrophil viability. There is also a need to better indicate the cell that is releasing the extracellular trap because non-neutrophil cells can also generate extracellular traps. These terms are commonly used interchangeably in literature, indicating a need to standardize the terminology surrounding NETs and NETosis.
In addition to being a valuable ally in immune responses, imbalanced neutrophil activity can be destructive and contribute to mechanisms of tissue damage. Elevated levels of NETs are associated with poor patient outcomes in COVID-19, autoimmunity, sepsis, cancer, organ injury, and a range of other diseases. This highlights the need to investigate and consider the roles that NETosis-mediated damage may have on these disease states. Additionally, NET biomarkers can provide valuable insight into the mechanisms and severity of these diseases. Drug development is also underway to investigate the potential for therapies that target neutrophil activities.
Mechanisms of NETosis
NETosis is an innate immune response that protects and defends the body from pathogens. The first step of NETosis involves the recognition of pathogens. When pathogens such as bacteria or viruses enter the body, neutrophils are recruited to the site of infection via chemotaxis. After neutrophils reach the site of infection, they undergo a process of activation which alters the cell’s morphology and function. Neutrophil activation can include chromatin decondensation, which is the process of uncoiling the nuclear chromatin so that the DNA is accessible and the chromatin is available for NET formation. Next, decondensed chromatin and histones are ejected from the nucleus into the cytoplasm where they mix with granule proteins, such as NE and MPO, to form NETs. Following NET formation, NETs containing decondensed chromatin and antimicrobial components are expelled from the cell membrane into the extracellular matrix resulting in NETosis. Once in the extracellular space, NETs function to trap and immobilize pathogens due to the web-like nature of the decondensed chromatin while the antimicrobial proteins work towards killing the pathogens. In the final step of NETosis, NETs function as an immune signal to recruit additional immune cells to amplify the immune response at the site of infection. This final step is a critical tipping point where excessive or dysregulated NET release can cause tissue damage and contribute to inflammatory and thrombotic disease states.
Mechanisms of NET-mediated Damage
NET-mediated thrombosis, inflammation, tissue damage, or autoimmune responses can happen when the release of NETs is excessive or dysregulated. NET-mediated damage can occur through the following mechanisms:
Coagulation and thrombosis
- NETs function as scaffolds for platelets and adhesion factors, such as von Willebrand factor (vWF) and fibrinogen, to aggregate and adhere to. Next, interactions with platelets, vWF, and fibronectin promote blood coagulation and thrombus formation.
- After platelets become activated, neutrophil-platelet interactions lead to more neutrophil activation, NET formation, and NETosis.
- The influx of NETs damages vascular endothelium, exposing tissue factor (TF). TF activates factor X (FX), driving a thrombin burst, fibrin formation, and activation of the extrinsic pathway of the coagulation cascade.
- NETs mechanistically activate platelets, which in turn stimulate NET release from neutrophils. This results in a positive feedback loop that drives coagulation, thrombosis, and inflammation.
Inflammation and tissue Damage
- The release of NETs activates macrophages, dendritic cells, and T cells. The arrival of leukocytes to the site of infection leads to the production of inflammatory cytokines, such as IL-1β and IL-18. Inflammatory cytokines can then trigger the release of more NETs in a positive feedback loop, ultimately resulting in an amplified inflammatory environment.
- Excessive levels of NETs can perpetuate local inflammation that impacts adjacent healthy tissue. Antimicrobial agents within NETs, such as histones, can be cytotoxic to healthy tissues.
- Immune responses are accelerated and amplified through complement pathway activation, which can lead to tissue-damaging levels of inflammation when dysregulated. Complement component 3 (C3) is particularly important during inflammatory processes, as indicated in studies analyzing a C3 knockout (C3 KO) mouse model. Neutrophil infiltration and NETs are dramatically decreased to barely detectable levels in C3 KO mice following ischemia reperfusion injury (IRI). This suggests that the complement C3 pathway is essential for neutrophil infiltration and NET formation after IRI.
- NETs can trigger autoimmunity by revealing phospholipids and autoantigens to the immune system. B cells are stimulated by NET components to produce autoantibodies against the exposed autoantigens. Autoantibodies typically seen in autoimmune diseases include anti-citrullinated protein antibodies (ACPAs), anti-MPO, anti-proteinase 3, and anti-dsDNA.
- Immune complexes formed between NET antigens and autoantibodies can be deposited in organs, such as the kidney, causing tissue damage and organ dysfunction.
- Neutrophil activation by autoantibodies can drive excessive NET release. For example, neutrophils, endothelial cells, and platelets are activated by antiphospholipid antibodies in antiphospholipid syndrome (APS). This alters the concentrations and activation of clotting factors von Willebrand factor (vWF), factor XII (FXII), along with others. Together this drives hypercoagulability that involves elevated levels of neutrophil recruitment/activation, NET release, and proinflammatory cytokine production during APS.
NETs in Autoimmune and Rheumatic Diseases
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by inflammation of small to medium sized blood vessels. Mechanistically, ANCA antibodies activate neutrophils, which in turn release NETs. NETs contain antigens, such as MPO, that are targeted by ANCA antibodies. This results in a positive feedback loop where NETs stimulate the production of ANCA antibodies, which in turn promote the release of more NETs. NETs also recruit immune cells, causing further inflammation of the blood vessels and tissue damage. Blood clot formation is promoted by NETs through the activation of platelets and coagulation cascades. These effects can extend to other organ systems, such as the kidney. Glomerulonephritis occurs when the small blood vessels of the kidney become inflamed. NETs have been identified in the glomeruli of kidney biopsies from patients with AAV. Elevated levels of circulating NETs have been documented in patients with AAV although the correlation of NET levels to disease activity is not known. It has been suggested that imbalanced NET clearance from the circulation may contribute to the pathogenicity of AAV.
Antiphospholipid syndrome (APS) is an autoimmune disorder that is caused by the immune system attacking the body’s healthy proteins. This can result in an increased risk of thrombosis and pregnancy complications. Hallmarks of APS include the presence of antiphospholipid antibodies, such as β2-glycoprotein 1, anticardiolipin, and lupus anticoagulant. Antiphospholipid antibodies activate the complement system, which recruits neutrophils. In individuals with APS, NETs undergo spontaneous release from neutrophils in the presence of autoantibodies, in a mechanism that correlates with autoantibody levels. NET functionality as a scaffold for platelet aggregation and activation increases the risk of pathological thrombotic events in APS. NETs may contribute to and exacerbate APS flare-ups due to their tissue damaging, pro-inflammatory, and pro-thrombotic properties.
Kawasaki disease (KD) is an acute, multisystem vasculitis syndrome that is characterized by fever and systemic swelling and inflammation of blood vessels. KD occurs most commonly in children and is treated by intravenous immunoglobulin (IVIG) therapy and aspirin. The exact cause of KD is not known; however, it has been proposed that an initial infection triggers an exaggerated immune response, which includes neutrophils. Studies indicate that neutrophils from individuals with KD are more likely to form NETs than healthy controls. Spontaneous NET formation has also been reported in individuals with acute KD, which suggests that circulating neutrophils are primed to undergo NETosis in KD. Elevated levels of cell-free DNA and NE-DNA complexes have been observed during acute KD. Together this research points to the involvement of dysregulated NET formation in KD and the consideration of NETs as a therapeutic target for KD.
Rheumatoid arthritis (RA) is characterized by the immune system attacking the synovium lining of the joints, causing chronic inflammation, damage to the joints, and bone destruction. RA is characterized by the presence of autoantibodies against post-translationally modified proteins, such as anti-citrullinated protein antibodies (ACPA). Mechanistically, NETs expose citrullinated antigens, which can then be bound by ACPA to form pro-inflammatory immune complexes. In fact, the key NET component citrullinated-H3 (CitH3) significantly associates with RA disease activity and is a candidate biomarker to assess RA. This illuminates the important and pivotal role that NETs have in RA disease progression.
As is common with most autoimmune diseases, NETs can promote inflammation and endothelial dysfunction through the recruitment and activation of immune cells. Elevated levels of MPO at sites of inflammation, such as the joint synovial fluid, support the role of NETs in RA pathogenesis. Furthermore, circulating levels of MPO-DNA complexes, and cell-free nucleosome levels are elevated in the serum of RA individuals. This suggests that NET components could function as biomarkers of RA disease activity and should be given consideration as therapeutic targets to treat RA.
Systemic lupus erythematosus
Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by widespread inflammation that can damage many organ systems. A hallmark of SLE is the presence of anti-dsDNA autoantibodies, likely resulting from the exposure of dsDNA by NETs. The interaction between autoantigens and autoantibodies can form immune complexes which trigger inflammation and tissue damage. The mechanisms of NET activity contribute to the pathogenesis, particularly when NET clearance is impaired. Studies have shown that NET degradation is impaired in individuals with SLE in a trend that correlates with SLE-associated autoantibody levels. NETs promote vascular endothelial damage, which contributes to the increased risk of cardiovascular disease associated with SLE. A severe complication of SLE is lupus nephritis, which is the accumulation of NETs and associated immune complexes in the kidneys leading to tissue damage and loss of kidney function. NETs are a key contributor to the damaging effects of sustained autoinflammation observed in SLE.
Type 1 Diabetes
Type 1 diabetes (T1D) is an autoimmune disease where the immune system targets and destroys the pancreatic beta cells which produce insulin. In addition to T cell involvement, recent studies suggest that neutrophils can also engage in T1D disease progression. Human T1D pancreatic samples indicate that neutrophils infiltrate the pancreas and undergo NETosis. Circulating levels of NET granule components NE, Proteinase-3, and MPO are elevated in T1D and correlate with autoantibody levels. PAD4 drives NET formation via histone citrullination. PAD4 levels are elevated in neutrophils of individuals with T1D, suggesting that neutrophils are primed to undergo NETosis upon activation. NET formation is stimulated by hyperglycemia outcomes of T1D. NETosis also occurs prior to the onset of T1D as indicated by the appearance of NET products in the pre-T1D pancreas. Extracellular histones damage pancreatic islets in vitro, suggesting that NET components may contribute to islet damage in T1D. Despite these insights, the mechanisms surrounding how neutrophils are trafficked to the pancreas and the impact that NET components have on the pathogenesis of T1D is not well understood.
NETs in Infectious Disease
The COVID-19 respiratory condition is an infectious disease that can manifest in severe cases as acute respiratory distress syndrome (ARDS), organ dysfunction, thromboinflammation, and death. Clinical studies suggest that circulating nucleosome levels are highly elevated in the plasma of severely affected COVID-19 patients compared to healthy controls, which could contribute to organ damage and mortality. Further investigation into the utility of circulating nucleosomes levels as a biomarker of COVID-19 disease severity should be considered along with the potential to develop therapeutics targeting NETs to reduce the disease severity of COVID-19.
Sepsis occurs when an infection triggers dangerous levels of inflammatory responses throughout the body. In sepsis, the immune system damages healthy tissues and can activate the coagulation system. This can lead to thrombosis, tissue damage, organ failure, and death. NETs can be beneficial to defend against pathogens, yet when dysregulated, NETs can be damaging and contribute to sepsis severity. Elevated levels of NETs have been observed in individuals with sepsis, suggesting a dysregulation of NET release and clearance. Excessive NET release in blood vessels can create microvascular obstruction, impeding blood flow and exacerbating sepsis conditions. Increased NET formation associate with sepsis-induced disseminated intravascular coagulation (DIC) suggesting that NETosis could be a link between inflammation and thrombosis. Studies analyzing the therapeutic removal of NETs from the blood in a porcine model of sepsis led to improved outcomes. This illustrates the potential for NETs to be a therapeutic target to protect against coagulopathy in septic shock and sepsis.
NETs in Hematology and Oncology
Coagulation and thrombosis
In addition to preventing the spread of pathogens, NET components contribute to the activation of the coagulation pathway and thrombosis. NETs contain DNA and histones that provide a scaffold for platelets, erythrocytes, fibrinogen, von Willebrand factor (vWF), and platelet adhesion factors to adhere to. NETs can also activate platelets through interactions with fibronectin and vWF, leading to platelet aggregation and the formation of blood clots. In perpetuation of these mechanisms, studies suggest that activated platelets promote the release of NETs, creating a reciprocal relationship between NETosis and thrombosis. NET release can damage the vascular endothelium, triggering the expression of tissue factor (TF), which functions to initiate the coagulation cascade. Together, this causes dysregulation of pro-coagulant and anti-coagulant factors, resulting in a pro-thrombotic state. Finally, histones released by NETs interfere with fibrinolysis, the process of breaking down blood clots. Extracellular histones mediate changes to fibrin, making it resistant to fibrinolysis. NETs play important roles in coagulation and blood clot formation that can have dangerous outcomes when dysregulated.
Immunothrombosis is the formation of localized blood clots in response to infection, tissue injury, or inflammation. The release of NETs at the sight of injury or infection functions to defend against invading pathogens. However, NETs also have pro-thrombotic properties that can lead to complications when NET release and clearance are dysregulated. NETs lead to the formation of blood clots through mediating the activation of platelets and triggering of the clotting cascade. Excessive NET release can promote thrombotic conditions, autoimmune diseases, and other inflammatory and clotting disorders. The described impact of NETs on immunothrombosis can also apply to aberrant immune function and thrombosis seen in individuals with sepsis, antiphospholipid syndrome, or lupus.
The complement system is a key facilitator of innate immunity and activator of coagulation pathways. Complement proteins function to recruit immune cells to the site of tissue damage and then stimulate the coagulation cascade via exposed tissue factor (TF) and activated endothelial cells. There is cross-regulation and cross-activation between the complement system and the coagulation system, creating a delicate balance regulating health. This is where NETs come into the picture because complement component 3 (C3) is essential for NET formation, as described in C3 KO mice. This supports the notion that the complement system is necessary for NET generation which in turn contributes to the immune and thrombotic functionalities of the complement pathway.
NETs have several key roles that contribute to cancer pathogenesis. The presence of elevated, circulating NETs can affect cancer disease progression, metastatic reach, and poor outcomes. Some cancers show elevated levels of NETs during advanced cancer stages in correlation with disease severity, as observed with lung and pancreatic cancers. This could be due to cancer cells mechanistically inducing NET release from neutrophils. This is beneficial to cancer cells because elevated levels of NETs in the tumor microenvironment promote inflammation and help to shape the microenvironment to be conducive to tumor growth. Additionally, studies have suggested that NETs entrap circulating tumor cells (CTCs) to promote metastatic dissemination. NETs can also reactivate dormant cancer cells, promoting metastatic cancer relapse, and increasing risks of thrombosis.
Cancer-associated thrombosis is a leading cause of mortality in cancer patients. Thus, blood clots can function as a first indicator of cancer and can be associated with poor prognosis. The relationship of NETs with coagulation is not lost on tumors. Circulating levels of NETs correlate with elevated levels of hypercoagulability markers. The prothrombotic state induced by NETs appears to be DNA-dependent. Animal model studies show that pre-treatment with DNAse prevents thrombosis, while treatment of whole blood with cell-free DNA induces thrombin generation. This data suggests that NETs can stimulate the formation of cancer-associated thrombosis in tumors.
Circulating nucleosomes can also be derived from cancer cells and thus contain valuable information about the cancer cells from which they came. Epigenetic information contained within a cancer cell’s nucleosomes, such as histone modifications, could provide insight into cancer screens, diagnosis, treatment, response to treatment, and disease progression. Oncology studies are underway to investigate the utility of nucleosome detection technologies during the research of cancers including lung cancer, colorectal cancer, non-Hodgkin lymphoma, and prostate cancer.
NETs in Organ System Diseases
Kidney diseases such as acute kidney injury (AKI), ischemia-reperfusion injury (kidney transplants/surgeries), and hemolytic uremic syndrome (HUS) show elevated levels of circulating and local NETs that may contribute to kidney damage. Chronic kidney disease (CKD) and autoimmune diseases, such as lupus nephritis, may also involve aberrant NET release. In the kidney, NET release can lead to chronic inflammation, thrombosis, and tissue damage. The imbalanced production and clearance of NETs can cause glomerular injury and epithelial tubule cell death which drives renal failure. Moreover, epithelial tubular cell damage initiates a pro-inflammatory cycle that causes neutrophils to release NETs. The influx of NETs then causes additional damage to epithelial cells, perpetuating the pro-inflammatory cycle. Aberrant release and clearances of NETs can lead to kidney tissue damage, sustained hypoxia, and an increased potential for remote organ dysfunction. This makes the detection of NETs an interesting and important biomarker to consider during kidney disease research.
NETs impact the pathogenesis of liver disease, particularly in regard to inflammation and tissue injury. Liver diseases that involve inflammation such as viral hepatitis, autoimmune hepatitis, alcohol-associated liver disease (ALD), and non-alcoholic steatohepatitis (NASH), can be mediated by dysregulated NET release. Excessive NETs in the liver can perpetuate liver inflammation, promote fibrosis, and contribute to liver damage. For example, the frequent occurrence of leaky gut in alcohol-associated hepatitis (AH) causes an increase of bacterial load in the enterohepatic circulation (EHC), which primes and activates neutrophils prior to accumulating in the liver. Neutrophil counts then become elevated in the circulation. Inflammatory cytokines facilitate the recruitment of neutrophils to the hepatic tissues of individuals with AH. Neutrophils are predisposed to undergo spontaneous NETosis which is accompanied by direct induction of NETosis by alcohol. Together this drives elevated levels of NETs in acute AH and following binge-drinking, contributing to liver injury and sepsis.
In the context of NASH, studies have suggested that NETs promote the progression of hepatocellular carcinoma (HCC). Therefore, it has been proposed to leverage this knowledge while monitoring individual response to treatment of HCC. Early studies have shown benefit in analyzing circulating levels of histone H3, H3.1 variants, and H3 epigenetic modifications as biomarkers to monitor response to treatment in HCC. Dysregulated NETs have substantial impacts on liver function particularly during liver disease. Opportunities are being explored to modulate NET formation and develop therapeutic strategies that target NETs in liver diseases.
Following lung injury, neutrophils are recruited to the site of pulmonary damage and release NETs in a mechanism of immune response. However, when NETs are released in a dysregulated manner, inflammation and tissue damage can contribute to lung fibrosis, microvascular thrombosis, and exacerbation of symptoms. Lung diseases that have NET implications include pulmonary infectious diseases, chronic obstructive pulmonary disease (COPD), severe asthma, acute respiratory distress syndrome (ARDS), lung cancer, and pulmonary arterial hypertension (PAH). COPD is a progressive lung disease characterized by neutrophilic airway inflammation. Following NET release, elevated levels of NETosis exacerbates airway inflammation in COPD in a manner that is linked to COPD disease severity. Acute respiratory distress syndrome (ARDS) occurs when fluid accumulates in the lungs. NETs are elevated in bronchial aspirates from patients with ARDS and correlate with ARDS severity, presumably through NET-induced tissue damage and inflammation. In PAH, a positive feedback loop has been suggested to occur where NETs activate platelets, which in turn induce NETosis through endothelial cells, platelets, and neutrophil signaling. Given the mechanism of NETs activity in lung disease pathogenesis, the development of NET-targeted therapeutic strategies are important to consider.
NETs as Potential Therapeutic Targets
Due to the involvement of dysregulated neutrophils in organ injury and diseases, NETs and their components are gaining attention as potential therapeutic targets for drug development. The treatment of autoimmune disorders, liver transplantation, and COVID-19 are some of the areas being considered for therapeutics that target neutrophil activity. Much of these therapies are focused on targeting the recruitment of neutrophils, the activation of neutrophils, and the reduction/clearance of NETs.
In addition to antimicrobial and anti-inflammatory therapies, two major research strategies are underway to explore potential opportunities to modulate the formation of NETs and facilitate NET degradation:
- Prevent or reduce NET formation.
- The NET component neutrophil elastase (NE) degrades extracellular matrix components, activates immune cells, and indirectly drives the production of inflammatory cytokines. Thus, NE inhibition could prevent NET formation and reduce inflammation caused by excessive NETs.
- Gasdermin D (GSDMD) mediates pyroptosis and the cytolysis of NETs. GSDMD inhibitors abrogate NET formation and studies have shown promise in using GSDMD inhibitors to treat sepsis and autoimmune diseases.
- PAD4 induces citrullination of histones, which opens up chromatin structure during DNA decondensation. PAD4 inhibitors have potential to block NETosis in order to improve the treatment of autoimmune and thromboinflammatory disorders.
- Activated neutrophils undergo NETosis to release NETs into the extracellular space, which cause NET-induced injury. Activated neutrophil inhibitors are being explored to inhibit the function of activated neutrophils. For example, hydroxychloroquine has been suggested to inhibit NETosis of activated neutrophils by blocking the expression of PAD4 and other components necessary to form NETs.
- Promote NET clearance.
- DNase1 and DNase1L3 clear NETs to prevent NET accumulation. Studies in mice have shown that treatment with DNase1 reduces SLE disease severity.
- Treatments that remove or block the production of anti-DNase autoantibodies, which inhibit DNase activity, could be beneficial to SLE treatment.
It is important to highlight that high-quality diagnostics are needed to mechanistically monitor the effectiveness of novel therapeutics targeting NETs. Early studies have shown that circulating levels of modified histone H3 can serve as a biomarker to monitor response to treatment in hepatocellular carcinoma (HCC). Additional NETosis biomarkers to consider include levels of cell-free DNA, nucleosomes, neutrophil granule proteins, and citrullinated histones.