Neutrophil extracellular traps (NETs) are characterized as extracellular DNA fibers comprised of histone and cytoplasmic granule proteins. NETs were first described as a form of innate response against pathogen invasion, which can capture pathogens, degrade bacterial toxic factors, and kill bacteria. Additionally, NETs also provide a scaffold for protein and cell binding. Protein binding to NETs further activate the coagulation system which participates in thrombosis. In addition, NETs also can damage the tissues due to the proteins they carry. Many studies have suggested that the excessive formation of NETs may contribute to a range of diseases, including thrombosis, atherosclerosis, autoimmune diseases, and sepsis. In this review, we describe the structure and components of NETs, models of NET formation, and detection methods. We also discuss the molecular mechanism of NET formation and their disease relevance. Modulation of NET formation may provide a new route for the prevention and treatment of releated human diseases.
Neutrophils are the most abundant type of white blood cells in peripheral blood and participate in many physiological and pathological processes of the body [
NETs are extracellular traps mainly composed of DNA, and treatment with DNase can significantly disrupt the NETs structure [
There are two methods available for identifying NETs. One method involves staining secreted DNA with SYTOX Green nucleic acid dye, which can be further observed by fluorescence microscope, and fluorescence intensity can be detected with a microplate reader. This method is simple and direct, but only useful under select circumstances. The second method involves detecting the specific components of NETs, including DNA, citrullinated histone H3 (citH3), and MPO (or NE), by immunofluorescence. Therefore, in cells or tissues, the immunofluorescence detection of NETs is a combination of DNA+citH3+MPO/NE (Table
The detection targets and methods of NETs in cells, tissues, and blood.
Sample type | Detection target | Detection method | Advantage | Limitations |
---|---|---|---|---|
Cell or tissue | dsDNA | SYTOX Green nucleic acid dye | Simple and direct | Less specific |
DNA, citH3, and MPO (or NE) | Immunofluorescence | Specific and widely accepted | Qualitative rather than quantitative | |
Blood | dsDNA | SYTOX Green nucleic acid dye | Simple and easy | Less specific |
dsDNA, nucleosomes, MPO-DNA complexes (MPO or NE), and citH3 (citH4) | ELISA | Specific and widely accepted | — |
In peripheral blood, various components carried on NETs are analyzed, leading to large variations of reports on NET detection targets. Nucleosomes, dsDNA, MPO-DNA complexes, and citrullinated histone H4 (citH4) have been reported to be markers of cell death and NETs in vivo [
NETs are released by neutrophils in different forms depending on the specific assay employed and the stimuli used. At present, at least three NET formation pathways have been identified, namely, vesicle-mediated, cell lytic, and mitochondrial release (Table Vesicle-mediated NET releases: under external stimulation with Cell lytic: prototypical formation of NETs in response to PMA leads to cell lysis and requires more time (3–4 h). The nuclear membrane is enzymatically degraded upon external stimulation. Study reported that NE and MPO reportedly cooperate in nuclear lysis and chromatin decondensation in PMA-induced NET formation [ Mitochondrial DNA: currently, few studies have reported that neutrophils release mitochondrial DNA to form NETs in a reactive oxygen species- (ROS-) dependent manner, while the detailed molecular mechanisms of mitochondrial DNA release are still unclear [
Comparison of the three types of NET formation.
Type | Main process | Neutrophil survival status | Time |
---|---|---|---|
Vesicle-mediated | Nuclear envelope breakdown and vesicle formation | Alive | 5–60 min |
Cell lytic | Histone citrullination and cell membrane rupture | Dead | 180–240 min |
Mitochondrial DNA | Unclear | Alive | >35 min |
NETs, as a weapon for neutrophils to function, have received a lot of attention and, in recent years, have been the subject of intense investigation in the field of immunology. Detailed molecular mechanisms regulating NET formation have received significant attention, and some progress has been made. Multiple studies have reported that PAD4, which mediate histone citrullination, plays an important role in the formation of NET, to form NETs [
The mechanisms of NETs formation. Under the different stimulations, neutrophils release NETs via different signaling molecules.
Other signaling pathways can also induce the formation of NETs. Douda et al. [
Marcos et al. [
In recent years, the role of NETs in thrombosis has attracted a lot of attention in both clinical and basic research. Studies have demonstrated that NETs can provide a scaffold for the binding of fibronectin, fibrinogen, vWF, and other protein components involved in thrombosis. NETs also trap red blood cells, promote platelet aggregation, and induce the formation of thrombi [
As mentioned above, NETs activate coagulation, indicating that they participate in thrombosis. Many histopathological studies have shown that NETs are involved in arterial thrombosis and venous thrombosis. Fuchs et al. [
The endothelium plays an important role in thrombosis, and many studies have demonstrated that NETs can induce endothelial dysfunction [
The above research suggests that NETs are involved in the formation and development of thrombosis (Figure
NETs participate in thrombosis. Under different stimuli, neutrophils release NETs. The proteins binding to NETs can activate the coagulation system and damage ECs. NETs also provide a scaffold for plasma protein and cell binding. Subsequently, NETs promote thrombosis formation.
Atherosclerosis is a chronic inflammatory process. Inflammation plays an important role in the occurrence and development of atherosclerotic plaques [
Many clinical studies have shown that plaque erosion can also cause acute coronary syndrome (ACS) due to vascular obstruction [
List of studies showing association between NETs and atherosclerosis.
Authors, reference | Study design | Main study findings |
---|---|---|
Warnatsch et al. [ | Using a mouse model | Cholesterol crystals triggered neutrophils to release NETs. NETs primed macrophages for cytokine production in atherosclerosis and blocking NETs formation significantly attenuated the plaque progression. |
Liu et al. [ | Using a mouse model | NETs are present in atherosclerotic lesions and are associated with the development of atherosclerosis. Specific deletion of PAD4 in the myeloid lineage diminished NET formation and significantly reduced atherosclerosis burden. |
Knight et al. [ | Using a mouse model | Pharmacological interventions that block NET formation via targeting PAD4 can reduce atherosclerosis burden and arterial thrombosis |
Franck et al. [ | Using a mouse model | NETs do not influence chronic experimental atherogenesis, but participate causally in acute thrombotic complications of intimal lesions that recapitulate features of superficial erosion. |
Quillard et al. [ | Analyzing 56 specimens of human carotid plaques. | NETs are present in human atherosclerotic plaques and associate with the number of luminal apoptotic ECs. |
Pertiwi et al. [ | Analyzing 12 thrombosed plaques obtained at autopsy from patients with acute MI. | NETs dominate numerically in early thrombosis and macrophage traps in late (organizing) thrombosis. |
Stakos et al. [ | Cell experiments (neutrophils obtained from patients with STEMI) | Neutrophils isolated from blood samples obtained by IRA have a higher ability to form NETs compared with those isolated from blood samples obtained by non-IRA. |
NET: neutrophil extracellular traps; PAD4: peptidylarginine deiminase 4; ECs: endothelial cells; MI: myocardial infarction; STEMI: ST-elevation myocardial infarction; IRA: infarct-related coronary arteries.
Autoimmune diseases are mainly caused by immune system disorders in which immune cells cannot distinguish self-antigens from foreign ones and the body produces autoantibodies or cytotoxic T cells against its own tissues and organs [
List of studies showing association between NETs and autoimmune diseases.
Authors, reference | Study design | Main study findings |
---|---|---|
Kessenbrock et al. [ | Cell experiments (neutrophils isolated from human peripheral blood) and analyzing 15 kidney needle biopsies from SVV patients with glomerulonephritis. | NETs are released by ANCA-stimulated neutrophils and contain the targeted autoantigens PR3 and MPO. NETs were prominent in specimens with strong neutrophil infiltration. |
Sangaletti et al. [ | Using a mouse model | Myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity. NET intermingling with myeloid DC positive for neutrophil MPO in MPO-ANCA-associated microscopic polyangiitis. |
Kusunoki et al. [ | Using a mouse model | PAD inhibitor suppresses NETs formation and MPO-ANCA production in mouse models with MPO-ANCA production. |
Tang et al. [ | Cell experiments (neutrophils isolated from human peripheral blood) and analyzing 6 kidney needle biopsies from AVV patients. | Enhanced NET formation, which contains LAMP-2, was observed in kidney biopsies and neutrophils from AAV patients. Anti-LAMP-2 antibody can further promote NETs formation. |
Garcia-Romo et al. [ | Analyzing the human neutrophils. | Mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived antiribonucleoprotein antibodies, releasing NETs. SLE NETs facilitate the uptake and recognition of mammalian DNA by pDCs and activate pDCs to produce high levels of IFN- |
Guo et al. [ | Analyzing the human neutrophils. | Neutrophils derived from SLE patients with decreased RIPK1 expression are more likely to form NETs, and RIPK1 inhibitor can greatly increase NETs formation. |
Carmona-Rivera et al. [ | Cell experiments. | MMP-9 is externalized during NET formation, and MMP-9 induces endothelial dysfunction by activating MMP-2. Inhibition of MMP-2 activation can restore endothelium-dependent function and decreased NET-induced vascular cytotoxicity. |
Hakkim et al. [ | Analysis of sera from 61 unrelated patients with SLE, 54 healthy controls, 30 RA patients, and 4 patients with IgA nephropathy. | A subset of SLE patients’ sera DNase I inhibitors or anti-NET antibodies prevented DNase1 access to NETs. |
Knight et al. [ | Using a mouse model | Neutrophils in SLE mouse model have a significantly higher ability to release NETs compared with controls. PAD inhibition can reduce NET formation, protecting against lupus-related damage to the vasculature, kidneys, and skin. |
Campbell et al. [ | Using a mouse model | NETs does not contribute to SLE in Nox2-deficient lupus-prone mice. |
Spengler et al. [ | Analysis of synovial fluid from patients with RA, patients with osteoarthritis, and patients with psoriatic arthritis. | Extracellular DNA levels were significantly higher in RA patient than in OA patients, and correlated with neutrophil concentrations and PAD activity in RA. PAD2 and PAD4 were attached to NETs and also freely diffused in the supernatant. |
Khandpur et al. [ | Experiments using neutrophils, sera, and synovial fluid obtained from RA patients, healthy controls, and patients with osteoarthritis. | Neutrophils from RA patients display a significantly enhanced capacity to form NETs, and NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in RA. |
Seri et al. [ | Using a mouse model | Deletion of the PAD4 gene reduces the severity of arthritis induced by recombinant human glucose-6-phosphate isomerase. |
Rohrbach et al. [ | Using a mouse model | PAD4 deficiency did not affect the severity of arthritis in the K/BxN murine. |
Mor-Vaknin et al. [ | Using a mouse model | DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form NETs. |
Perez-Sanchez et al. [ | Experiments using neutrophils and plasma obtained from RA patients and healthy controls. | NETs was found increased in RA patients. Inhibition of NETs extrusion can further abridge the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. |
Sur Chowdhury et al. [ | Human study. | Neutrophils from RA cases exhibited increased spontaneous NET formation, and NETs-derived in RA serum products demonstrated diagnostic potential. |
Wang et al. [ | Analysis of serum from 74 RA patients and 50 healthy controls. | RA patients exhibited significantly higher levels of MPO-DNA complexes, and these levels were associated with increased neutrophil counts and positivity for rheumatoid factor and anticitrullinated protein/peptide antibodies. |
NET: neutrophil extracellular traps; ANCA: antineutrophil cytoplasmic antibody; SVV: small-vessel vasculitis; PR3: proteinase 3; MPO: myeloperoxidase; DCs: dendritic cells; PAD: peptidylarginine deiminase; LAMP-2: lysosomal-associated membrane protein-2; pDCs: plasmacytoid dendritic cells; IFN: interferon; AAV: ANCA-associated vasculitis; RIPK1: receptor interacting serine/threonine kinase 1; SLE: systemic lupus erythematosus RA: rheumatoid arthritis; MMP: matrix metalloproteinase; OA: osteoarthritis; JIA: juvenile idiopathic arthritis.
Antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitis (AAV) is a group of diseases, characterized by the destruction and inflammation of small vessels. Anti-MPO and proteinase 3 (PR3) antibodies are the two common ANCAs [
Systemic lupus erythematosus (SLE) is a chronic disease that causes inflammation in connective tissues and is characterized by the production of antibodies to autologous dsDNA [
Rheumatoid arthritis (RA) is a long-term chronic inflammatory process and mainly affects joints. Citrullinated proteins are the most important target antigens in the pathogenesis of RA [
Sepsis is a life-threatening bloodstream infection that is accompanied by systemic inflammation and can cause multiple organ dysfunction. Neutrophils, the most abundant inflammatory cells in peripheral blood, are rapidly recruited and infiltrate into organs during the process of sepsis [
Studies showing an association between NETs and sepsis.
Authors, reference | Study design | Main study findings |
---|---|---|
Brinkmann et al. [ | Cell experiments. | Upon activation, neutrophils release NETs that bind Gram-positive and Gram-negative bacteria. NETs further degrade virulence factors and kill bacteria. |
Yang et al. [ | Analyzing neutrophils, platelets and plasma obtained from sepsis patients, nonsepsis patients and healthy controls. | Neutrophils from septic patients had significantly enhanced NETs releasing. NETs further promote hypercoagulability in patients with sepsis. |
Lefrancais et al. [ | Human and mouse study. | They detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome from pneumonia or sepsis. Increased plasma NETs were associated with ARDS severity and mortality in humans. |
Tanaka et al. [ | Using a mouse model | In septic mice, NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. |
Chen et al. [ | Using a mouse model. | NETs induce M |
McDonald et al. [ | Using a mouse model. | NETs were critical for the development of sepsis-induced intravascular coagulation in mice. Inhibition of NET-induced coagulation can markedly improve microvascular perfusion and attenuate the end-organ damage in septic mice. |
Biron et al. [ | Using a mouse model. | Cl-Amidine (PAD4 inhibitor) treatment prior to cecal ligation and puncture improves overall survival in sepsis. |
McDonald et al. [ | Using a mouse model. | NET release increases bacterial trapping, and blocking NET formation reduces the capture of circulating bacteria during sepsis, resulting in increased dissemination to distant organs. |
Meng et al. [ | Using a mouse model. | They found that depletion of NETs by rhDNase administration can impede the early immune response and aggravates the pathology that follows polymicrobial sepsis. |
Czaikoski et al. [ | Using a mouse model. | Degradation of NETs by rhDNase treatment did not prevent organ damage during polymicrobial sepsis, while rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. |
NET: neutrophil extracellular traps; ARDS: acute respiratory distress syndrome; M
In recent years, research on NETs has attracted much attention in the field of immunology. The progress made give us a greater understanding of their role in immune diseases. Here, our focus has mainly been on three aspects: methods for detecting NETs, molecular mechanisms of NET formation, and the correlation between NET formation and disease. In this review, we summarized the detection targets of NETs in the blood, cells, and tissues based on previous reports and our own work. Plasma dsDNA, nucleosomes, MPO-DNA complexes (MPO or NE), and citH3 (citH4) are in vivo markers of NETs. In cells or tissues, the detection of NETs is a combination of dsDNA, citH3, and MPO (NE) by immunofluorescence. However, our suggestions are only for researchers’ reference owing to lack of a unified NETs detection standard, and further work is required to establish a guideline for NETs detection [
The authors declare that they have no conflict of interest.
Tiewei Li, Zhengyan Zhang, and Xiaojuan Li have contributed equally to this work and should be considered co-first authors.
This work was supported by the Medical Science and Technology Project of Henan Province (2018020698) and Key Research, Development and Promotion Projects of Henan Province (202102310132). We also thank LetPub (