Loss of blood-brain barrier (BBB) integrity and neuroinflammation are central to the pathogenesis of central nervous system (CNS) pathologies. Neutrophils are the most abundant leukocyte population, which adhere to activated endothelium, transmigrate to tissues, and contribute to inflammatory processes. Neutrophil extracellular traps (NETs) produced by activated neutrophils are composed of a DNA scaffold on which various proteins are deposited (1). The release of NETs was traditionally related to neutrophil death (termed NETosis), but neutrophils also undergo vital and mitochondrial NET production by extruding their contents through blebbing from the cell membrane (2), remaining viable and retaining effector functions (Figure 1).

The production of different types of NETs is context-dependent, varying with the nature of the NET-inducing stimuli and disease process (3). For example, lytic NETosis is induced by infections (4), pro-inflammatory cytokines (5), damage-associated molecular patterns (DAMPs) (6, 7), activated platelets (8, 9), complement (10, 11), autoantibodies (12, 13), and immune complexes; vital NETs are mainly seen in infections (2, 14, 15); and mitochondrial NETs during infections and autoimmune diseases (16, 17). The molecular mechanisms governing NET production in each of these instances are beyond the scope of this review and are discussed elsewhere (18–20). Regulators of this heterogeneity of NETs and whether different types of NETs exert variable, context-specific effects (beneficial versus harmful) are outstanding questions in the field of NETs research (21).

Despite their apparent benefit in infectious disease (4), NETs cause significant collateral tissue damage (22). For example, cell-free DNA (16), histones (23–25), matrix metalloproteinase-9 (MMP-9) (26), and LL-37 (27), have all been implicated in various tissue-damaging effects, including direct cytotoxicity (28), thrombosis (29), and chronic inflammation (24). Several excellent reviews on NETs in defense and disease have been published (30–36). Recent studies have shown neutrophil recruitment in the CNS and NETs to contribute to various CNS pathologies. Table 1 summarizes seminal studies implicating NETs as pathogenic in brain CNS diseases. This review provides a concise summary of recent data on the role of NETs in various brain disorders.

Brain microvascular endothelial cells (BMECs) are connected by various adherens and tight junctions, forming the highly selective BBB. Neutrophils thus cannot readily cross the BBB and are rarely found in a healthy brain. However, BBB permeability can increase secondary to trauma, inflammation, ischemia, and degenerative changes. Pro-inflammatory cytokines released by activated astrocytes and microglia cells upregulate adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on BMECs, facilitating neutrophil adhesion (37). Subsequently, neutrophil-endothelial cell interactions without transmigration increase BBB permeability (Figure 2; 38).

Multiple mechanisms explain neutrophil adhesion-dependent BBB disruption. Firstly, neutrophil adhesion promotes blood flow stasis, leading to vascular obstruction; neutrophil depletion enhances CNS perfusion and decreases brain damage after stroke (39). Neutrophil adhesion to BMECs via the β₂ integrins LFA-1 and MAC-1 also activates neutrophils, increasing oxidative stress and NETosis (40). Activated neutrophils release neutrophil elastase (NE)—possibly within NETs—which disrupts adherens junction proteins VE-cadherin and β-catenin, increasing BBB permeability (41). Agaphelin—an NE inhibitor—reduces BBB permeability in stroke, decreasing infarct volume, improving neurologic function, and reducing mortality in mice (42). NET-associated MMP-9 in the cerebral microvessels degrades type IV collagen of the basal lamina to disrupt BBB integrity (43). Histones also increase BBB permeability, particularly in the hippocampus, by disrupting adherens and tight junctions (44).

Extracellular DNA, microbial DNA, and damaged intracellular self-DNA are major inducers of inflammation through cGMP-AMP synthase (cGAS)/STING-dependent type I IFN and pro-inflammatory cytokine production (45–48). DNA contained within NETs also activates cGAS and enhances type I IFN production and pro-inflammatory cytokine production (16, 49). Therefore, in the CNS, NETs may promote type I IFN and pro-inflammatory cytokine responses in microglia, which are known to express cGAS and contribute to CNS pathology (50–53). NET-induced microglial activation via cGAS has been noted in murine models of ischemic stroke (54) and tissue plasminogen activator (tPA)-induced intracerebral hemorrhage (55). A recent preprint study demonstrated NET-induced cGAS activation in microglia in TBI, associated with neuroinflammation and neurological deficits (56). Depleting neutrophils or inhibiting peptidyl-arginine demaniase-4 (PAD-4) significantly blunt the IFN response and improve neurologic deficits (54). Together, these findings indicate that NETs mediate BBB disruption, either directly via histones and various proteases or indirectly by augmenting type I IFN responses.

Traumatic brain injury (TBI) carries a high mortality risk, and complications such as cognitive impairment, memory deficits, post-traumatic stress disorder, post-traumatic encephalopathy, and neuroinflammation in survivors are frequent (68, 69). Neutrophils adhere to cerebral vessels, infiltrate hypoxic brain tissue, and produce NETs in murine models of TBIs (70, 71), associated with worse TBI outcomes, including cerebral edema, cognitive deficits, and paroxysmal sympathetic hyperactivity (72, 73). Activation of toll-like receptor-4 (TLR4) on neutrophils activates NET formation after TBI, which correlates with higher intracranial pressure (ICP), suggesting that neutrophils cause cerebral edema by NET production (70). TLR4-knockout, the NET formation inhibitor Cl-amidine, and DNase-1 reduce NET formation in the brain post-TBI and better neurologic and behavioral outcomes (70). Together, these findings indicate that NET-targeted therapies may be beneficial in alleviating hypoxia and cerebral edema after TBI.

Neutrophils infiltrate the CNS following cerebral ischemia (74) and correlate positively with neuronal loss, infarct size, and cognitive dysfunction (75). Circulating neutrophils from ischemic stroke patients exhibit higher NET formation than healthy control neutrophils (76). Moreover, CNS-infiltrating neutrophils in ischemic stroke patients form NETs (77, 78). Higher serum cell-free DNA levels in acute ischemic stroke patients correlate with worse clinical outcomes using the modified Rankin scale, and lower serum DNase levels are found in patients who developed stroke-associated infections (79). Platelet-derived high-mobility group box-1 (HMGB1) is a major inducer of NET production in ischemic stroke, and HMGB1-depleted attenuates NET formation after stroke and betters neurologic outcomes (78, 80). Along similar lines, treating mice with neonatal NET-inhibitory factor (nNIF) ameliorates NET production and decreased infarct size (78). NETs may also protect thrombi from degradation by tPA (81), and adding DNase-1 to tPA regimens significantly accelerates ex vivo lysis of stroke thrombi (82). Furthermore, NETs can contribute to tPA-induced intracerebral hemorrhage and BBB disruption (discussed above) (55). Since BBB disruption underlies the narrow therapeutic window of tPA administration (83), whether inhibiting NETs delays BBB disruption and increases the therapeutic window of tPA administration warrants further investigation.

Neuropsychiatric manifestations of SLE (NPSLE) are commonly attributed to neuroinflammation; leukocyte infiltration precedes NPSLE-related cognitive deficits in mice (84). Neutrophils isolated from SLE patients display impaired phagocytosis, increased platelet aggregation, reactive oxygen species (ROS) production, and NET formation (85, 86). SLE skews the composition of the neutrophil compartment toward low-density granulocytes (LDGs), which readily produce NETs (87, 88). SLE patient plasma induces NET production by neutrophils isolated from healthy controls, indicating that autoantibodies and immune complexes in SLE plasma induce NET formation (16, 89). NETs released by LDGs upon stimulation by anti-ribonucleoprotein antibodies and immune complexes cause endothelial dysfunction—inducing apoptosis and impairing vasorelaxation—through components like MMP-2 (90). LDG NETs also contain various immunostimulatory proteins and autoantigens, like LL-37, IL-33, IL-17, and dsDNA, which can worsen inflammation and introduce neoepitopes, fostering an amplification of autoimmunity (91–93).

The degradation of NETs may be impaired in SLE, particularly during disease flares. Auto-antibodies against NET components—e.g., anti-dsDNA and anti-histone antibodies—are thought to protect NETs against degradation by circulating DNase (94). Furthermore, NET-activated C1q can directly inhibit DNase to prevent NET degradation (95). Genetic variations in DNase activity can also increase the risk of SLE. For example, individuals with DNASE1L3 gene mutations develop childhood SLE (96). These observations suggest that DNase-1 as a therapeutic strategy may not improve the clinical severity of SLE, as one study demonstrated in lupus nephritis patients (97).

Serum NET levels have not yet been studied as prognostic markers in NPSLE, nor has the effect of inhibiting neutrophil CNS trafficking or lowering serum and CSF NET markers on cognitive function been tested experimentally. The fundamental importance of the type I IFN response in SLE is well-established. Type I IFNs have indeed been linked to NPSLE (93, 98), but mechanistic links between type I IFNs and NPSLE manifestations are lacking. Neutrophils are perhaps major downstream mediators of the type I IFN response in SLE. They exhibit the highest expression of interferon-stimulated genes (ISGs) out of the myeloid cells (99). Type I IFNs induce mitochondrial NET formation by LDGs, and mitochondrial DNA within LDG NETs can activate cGAS-STING signaling in plasmacytoid dendritic cells to drive type I IFN production (16, 100). Therefore, whether NETs—cGAS/STING axis increases BBB permeability and neuroinflammation in NPSLE is an important question for future research.

Glioma tumor cells produce granulocyte colony-stimulating factor that drives granulopoiesis in the bone marrow (133, 134). Consequently, neutrophilia and an elevated neutrophil-to-lymphocyte ratio are commonly seen in glioma patients (135), are more pronounced in high-grade (IV) tumors, and confer a poor prognosis (136). In agreement with these findings, depleting neutrophils by using an anti-Ly6G monoclonal antibody prolongs survival in mice with gliomas (137). Glioma cells also produce IL-8 that recruits neutrophils, termed tumor-associated neutrophils (TANs) (138). IL-8 induces NETosis in TANs through PI3K-signaling and ROS production. Indeed, TANs and NETs have been visualized within glioma lesions (138). NET components such as HMGB1 bind RAGE receptors on the surface of glioma tumor cells and stimulate proliferation, invasion, and IL-8 production in vitro (138). These findings suggest that a self-reinforcing NETs/IL-8 axis may collectively amplify tumor inflammation in glioma. In glioblastoma multiforme (GBM), TANs stimulate GBM tumor cell proliferation and epithelial-to-mesenchymal transition through S100A4 (139). NETs also contribute to a hypercoagulable state in high-grade glioma by inducing endothelial cell dysfunction, alleviated by DNase1 + protein C treatment in vitro (140). Given these findings, exploring specific pro-tumorigenic NET components in glioma may reveal novel disease markers and therapeutic targets in glioma. However, it is important to mention that neutrophils also exert numerous anti-tumor functions by killing tumor cells and T enhancing anti-tumor -cell responses (141). Therefore, the dilemma of inhibiting NETs and inadvertently attenuating beneficial—in this case, tumor suppressive—neutrophil functions must be carefully considered. Only rigorous mechanistic analyses can dissect beneficial versus detrimental effects of NETs in the context of cancer. We refer readers to in-depth reviews on neutrophils, NETs and cancer for more information (142–147).

Considerable data exist on TANs and NETs as mediators of chemotherapy, immunotherapy, and radiotherapy resistance (148–150). In glioma, NET-derived S100A4 mediates resistance to anti-VEGF therapy, whereas inhibition of S100A4 enhances response to treatment (139). Silencing S100A4 gene expression in endothelial cells by S100A4 small-interfering RNA induces an anti-angiogenic gene signature, and administering S100A4 siRNA into human prostate cancer xenografts significantly decreased tumor vascularity and inhibited tumor growth (151). These studies have set the stage for S100A4-targeted anti-cancer therapies. Since S100A4 may be derived from NETs, future research must ascertain whether inhibiting NET production normalizes the tumor vasculature and constitutes a potential anti-angiogenic cancer treatment strategy.

Single-cell technologies have revealed that the neutrophil population comprises functionally distinct subsets. Understanding how the neutrophil population is skewed in CNS diseases is essential to identifying context-dependent neutrophil transcriptomic alterations and novel disease-specific therapeutic strategies. Therefore, the composition and function of NETs may also vary in different CNS diseases; lytic NETs, vital NETs, and mitochondrial NETs are released in response to distinct stimuli and have varying compositions. However, studies investigating NETs in CNS disease thus far do not account for NET heterogeneity; the specific type of NETs seen are rarely reported. Therefore, if NETs are to be considered a future therapeutic target in CNS disease, they need to be studied in greater resolution. Future studies into the topic may reveal novel disease markers and cause-specific therapeutic targets to improve the care of patients suffering from CNS diseases. Interestingly, while the activation of canonical and non-canonical inflammasome pathways was previously linked to NETosis (152), a recent study showed that inflammasome-related NET formation does not require cell death (153). Therefore, the field of NETs research is wide open; new research will likely reveal new NET-inducing stimuli, important signaling pathways worth targeting to mitigate NET formation, new NET components, and yet unknown beneficial and harmful effects.

AS and AY: conceptualization. AS, ANE, GA, MA-R, SAR, and KAd: writing—original draft preparation. AS, KAd, and AY: writing—review and editing. KAd and AY: supervision. All authors have read and agreed to the published version of the manuscript.