Introduction of the Role of Neutrophil Extracellular Traps in Stroke

Stroke elicits an extensive inflammatory response in the brain with the recruitment of circulating leukocytes. Among these, neutrophils are the most abundant white blood cells that are rapidly recruited to the site of injury providing an effective immune response. These neutrophils release their contents in the form of neutrophil extracellular traps (NETs), a process known as NETosis, which involves the release of DNA decorated with granular proteins and histones in response to infections.

NETs are a new regulator of neutrophils that can capture bacteria and release antimicrobial molecules to kill pathogens. They can also exacerbate some non-infectious diseases through autoimmune or inflammatory response. Recently, NETs have been recognized for their significant role in the pathological processes associated with stroke. NETs are highly prothrombotic and can contribute to local tissue damage by exerting toxic effects on surrounding cells. Notably, elevated NET markers have been strongly associated with poor outcomes in stroke patients, including increased morbidity, disability, and mortality. The increasing evidences suggest that NETs may be a potential target for stroke treatment. Inhibition of NETs formation or promotion of NETs degradation plays protective effects in stroke.

Fig.1. Neutrophil extracellular traps regulate ischemic stroke brain injury. (Denorme, et al., 2022)

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Physiological Functions of NETs

Structure of NETs

After activation, neutrophils flatten, and their nuclear lobules disappear within an hour, leading to chromatin decondensation. The nuclear membrane splits into vesicles, merging nucleoplasm and cytoplasm into clumps. As the cells condense and become round, the cytoplasmic membrane ruptures, releasing intracellular components that form NETs. NETs are negatively charged, web-like molecular structures mostly made up of DNA, histones and proteins such as neutrophil elastase, myeloperoxidase, high mobility group box-1 protein and Cathepsin G.

Basic Functions of NETs

NETs serve several key functions:

• Antibacterial Defense: The web-like structure of NETs, composed of DNA and proteins such as neutrophil elastase (NE) and myeloperoxidase (MPO), forms a physical barrier that hinders bacterial spread and enhances the concentration of antibacterial substances, effectively killing bacteria.
• Regulation of Inflammation: NET aggregation can reduce inflammation by degrading cytokines and chemokines, as well as interfering with the recruitment and activation of neutrophils.
• Impact of Degradation: When NETs are broken down, such as through treatment with exogenous DNase, there is an observed increase in bacterial presence, indicating their protective role.
• Antimicrobial Activity: NETs maintain their structure even when treated with excess cations or phosphatases, demonstrating that their negatively charged DNA can chelate cations and exert antimicrobial effects. Cationic histones also contribute to innate defense by interacting with negatively charged lipid membranes.

Despite these protective functions, excessive or inadequately cleared NETs can lead to various diseases. NETs can also exacerbate inflammation, cause vascular damage, trap tumor cells, and lead to thrombotic diseases, such as stroke. Therefore, further research is needed to enhance the protective effects of NETs while mitigating their harmful consequences.

NETosis

The formation of NETs is called NETosis, which occurs through three distinct mechanisms:

• Suicidal NETosis: This NADPH oxidase-dependent process occurs within 2-4 hours after neutrophil activation and involves cell death. When activated by PMA and IL-8, the NADPH oxidase complex is mobilized and produces reactive oxygen species (ROS) that promote decondensation of chromatin. The extracellular calcium also drives peptidyl arginine deaminase 4 (PAD4), which produces histone citrullination, further helping to decondensate chromatin. The nuclear membrane ruptures and releases nuclear cargoes that combine with cytoplasmic stuff to create NETs.
• Vital NETosis: This type occurs within 5-60 minutes, is independent of NADPH oxidase, and does not involve neutrophil death. Triggered by Toll-like receptors and complement proteins in response to pathogens, nuclear DNA fuses with the cytoplasm, leading to vesicle budding. Neutrophils can still engulf pathogens after releasing NETs.
• Mitochondrial NETosis: This mechanism occurs within 15 minutes and involves the release of mitochondrial DNA without neutrophil death, typically triggered by GM-CSF, LPS, or C5a.

Fig.2. The three mechanisms of NETs formation. (Zhao, et al., 2023)

The Role of NETs in Stroke Pathology

NETs Affect Inflammation and Thrombosis

Studies of neutrophils have revealed NETs as key to inflammation and thrombosis. NETs mediate thrombosis via prethrombotic factors including tissue factor (TF), factor XII (FXII), microparticles (MPs), von Willebrand factor (vWf), and fibrinogen. In thrombi, a network of fibrinogen, extracellular DNA, and vWF colocalizes; this is involved in various thrombotic disorders such as acute ischemic stroke (AIS).

Elevated levels of TF generated by NETs are essential for the initiation and propagation of venous and arterial thrombi. Some studies indicate that FXIIa enhances the prethrombotic potential of NETs. Additionally, activated neutrophils produce a significant amount of neutrophil-derived MPs, which are rich in phosphatidylserine and can attract procoagulant factors. MPs can bind to NETs via histone-PS interactions, promoting thrombin production through the intrinsic clotting pathway.

The aggregation of NET-MP complexes also stimulates neutrophil recruitment in vivo. Moreover, the proinflammatory effects of this complex are mediated by high mobility group box 1 (HMGB1), which activates TLR2 and TLR4 signaling pathways. HMGB1 influences NET formation through interactions with TLR2, TLR4, and RAGE, relying on NADPH oxidase activity. Inhibiting HMGB1 may help reduce inflammatory damage associated with ANCA-induced NET formation.

NETs and Vascular Injury

NETs respond to endothelial damage and can change the anticoagulant activity of endothelial cells. Excited endothelial cells secrete cytokines and ROS that stimulate NETs; NETs stimulate endothelial cells via histones and defensins. Scientists have found that NETs induce interferon-β (IFN-β) via the STING pathway, which impedes vascular regeneration and remodeling after stroke. Inhibition of NET formation can enhance neurovascular regeneration and improve neurological function.

Furthermore, CitH3, a key component of NETs, disrupts intercellular adhesion and reorganizes the actin cytoskeleton, causing microvascular leakage. NETs have been linked to endothelial dysfunction in atherosclerosis and venous embolism, with studies showing that inhibiting PAD reduces atherosclerosis in mice.

NETs activate antigen-presenting cells, endothelial cells, and platelets, promoting inflammatory responses and endothelial dysfunction. They increase the expression of adhesion molecules and tissue factors in endothelial cells, accelerating thrombosis. While degrading the DNA backbone of NETs does not alleviate cytotoxic responses, inhibiting histones or MPO can reduce endothelial injury.

Fig.3. The pathological roles of NETs in stroke. (Zhao, et al., 2023)

NETs as Potential Biomarkers in Stroke

Biomarkers can enhance diagnostic accuracy, predict clinical outcomes, and monitor disease progression in stroke. An ideal stroke biomarker should be cost-effective, non-invasive, highly sensitive, and specific. Histological analyses show that thrombi from stroke patients contain numerous nucleated leukocytes and neutrophils, with NETs predominantly in the outer layers. Elevated NET levels indicate initial stages of venous thrombosis, correlating positively with the severity of stroke as measured by neurological function scores. NETs related components such as CitH3, MPO-DNA, and NE can be used as biomarkers for the diagnosis of stroke as well as to predict the severity and prognosis of stroke. The simultaneous detection of the components of NETs may have higher accuracy in stroke diagnosis and prognosis. The clinical application of NETs as biomarkers still faces some difficulties and requires further development, validation, and standardization.

Targeting NETs for The Treatment of Stroke

Inhibition of NETs Formation

NETs is closely linked to the generation of ROS and the activation of PAD4. PAD4 catalyzes the deamination or citrullination of histones, weakening their electrostatic binding to DNA and leading to chromatin decondensation. Meanwhile, ROS activates MPO, which triggers the activation and translocation of neutrophil elastase to the nucleus. NE then modifies histones, disrupting chromatin packaging. Therefore, targeting the inhibition of PAD4 and ROS generation is crucial for suppressing NET formation.

Degradation of NETs

Promoting the degradation of NETs is a potential strategy for stroke treatment. Studies have shown that DNase treatment reduces infarct volume and improves outcomes in stroke models by degrading NETs. Additionally, DNase accelerates the lysis of thrombi, and its combination with tPA can lower the required dose of tPA, reduce side effects, and extend the therapeutic time window.

While DNase effectively cleaves DNA in NETs, it does not significantly affect associated components like histones and NE, which can lead to inflammatory responses due to their release into the bloodstream. Research indicates that blocking histone-DNA complexes can inhibit NE expression and neuronal death. Activated protein C (APC) cleaves histones and mitigates their toxicity, with thrombomodulin-α (TM-α) promoting APC production that reduces histone-induced thrombin production and endothelial cell death. APC has a broad therapeutic window and aids in tissue repair, providing neuroprotective effects in ischemia and thrombosis-related diseases.

NE and MPO are both components of NETs and important enzymes of neutrophils, which play key roles in inflammation-related diseases. Inhibit the activities of NE or MPO decreases NETs-mediated inflammation.

1. Denorme, F., et al. (2022). Neutrophil extracellular traps regulate ischemic stroke brain injury. The Journal of clinical investigation, 132(10).
2. Zhao, Z., et al. (2023). Neutrophil extracellular traps: a novel target for the treatment of stroke. Pharmacology & Therapeutics, 241, 108328.