Cell death crosstalk in NET-Driven inflammation: mechanisms, disease contexts, and therapeutic perspectives

Abstract

Neutrophil extracellular traps (NETs) are chromatin-based extracellular structures consisting of DNA, histones and multiple antimicrobial proteins, which exert dual biological effects in host defense and inflammation-triggered tissue injury. This review focuses on NET-associated signaling pathways and their regulatory crosstalk with diverse forms of regulated cell death (RCD) in inflammatory disorders. First, we summarize the structural characteristics and biogenesis pathways of NETs closely linked to inflammatory amplification, including lytic and non-lytic NETosis as well as reactive oxygen species (ROS)-dependent and ROS-independent mechanisms, and elaborate the functions of NADPH oxidase, myeloperoxidase, neutrophil elastase, peptidylarginine deiminase 4 and gasdermin D during these processes. Second, we discuss how NET-derived damage-associated molecular patterns, such as DNA, histones, granular proteases, ROS and mitochondrial DNA, interact with apoptosis, necroptosis, pyroptosis, ferroptosis, autophagy and cuproptosis. Under pathological conditions, excessive NET formation or impaired NET clearance leads to autoantigen exposure, accelerated thrombosis, enhanced inflammasome activation, parenchymal cell damage and modulated tumor progression. Finally, we outline therapeutic interventions targeting NET biogenesis, NET clearance and downstream NETs-driven signaling, with an emphasis on the translational potential and safety concerns of these strategies across distinct diseases. Future investigations are required to decipher context-dependent NETs–RCD regulatory circuits, standardize NETs detection protocols, and develop precision-targeted therapeutics that restrain pathological inflammation while preserving host antimicrobial defense.

Introduction: positioning NETs at the interface of inflammation, cell death, and therapeutic translation

Neutrophils are key effector cells of the innate immune system, and phagocytosis and degranulation have long been recognized as their primary mechanisms for eliminating pathogens and clearing necrotic tissues [1]. However, since the first report of neutrophil extracellular traps (NETs) in 2004, our understanding of neutrophil functions has fundamentally changed [2]. NETs are extracellular mesh-like structures released by neutrophils, primarily composed of chromatin (nuclear DNA or mitochondrial DNA) and various granular proteins (e.g., myeloperoxidase (MPO), neutrophil elastase (NE)) [3]. This structure can physically trap pathogens such as bacteria, fungi, viruses, and parasites, and directly kill them or inhibit their proliferation through locally high concentrations of antimicrobial molecules [3]. Over the past two decades, extensive research has been conducted on the molecular mechanisms, regulatory networks, and roles of NETs in various inflammatory diseases. The process of NET formation, also known as neutrophil extracellular trap formation (NETosis), can be broadly classified into two forms: lytic pathways characterized by plasma membrane (PM) disruption and non-lytic routes involving an intact PM; these two pathways share overlapping features but differ mechanistically [4]. Extracellularly, pathogens, immune complexes (ICs), complement fragments and cytokines can all modify the process of NETosis; however, intracellular molecules such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), MPO, NE, and peptidylarginine deiminase (PAD)4 are required for NETosis regulation of NETosis [5, 6].

Inflammatory diseases are pathological conditions that result from unbalanced immune reactions in the body and are accompanied by tissue destruction or malfunction, including acute systemic inflammation represented by sepsis and chronic persistent inflammation typified by autoimmune diseases (ADs), atherosclerosis (AS), and others [7–9]. Of particular interest is that chronic inflammation has been widely recognized as a promoter of tumor initiation and progression, which represents a key link of the “inflammation-cancer” axis [10]. As NET research has advanced, it has become increasingly clear that NETs not only kill pathogens but also mediate various aforementioned inflammatory diseases. For example, in patients with systemic lupus erythematosus (SLE), the proportion of low-density granulocytes (LDGs) is higher in peripheral blood and the expression level of type I interferons (IFN-I) is high, which together promote NETs formation and release. In addition, the generated NETs can directly stimulate plasmacytoid dendritic cells (pDCs) to produce IFN-I, promoting a vicious cycle in SLE [3, 11–13]. Considering the pivotal regulatory role of NETs in inflammatory diseases, NET inhibition has emerged as a promising therapeutic approach. For example, deoxyribonuclease (DNase)I can degrade the DNA component of NETs, inhibit the toll-like receptor (TLR)4-myeloid differentiation primary response protein 88 signaling axis, reduce renal immune cell infiltration and alleviate lupus nephritis-related renal damae [14]; NE inhibitors are able to decrease chemokine and inflammatory factor levels, suppress smoke-induced NETosis in mice with chronic obstructive pulmonary disease (COPD) through improvements in lung function [15]. In contrast, over-degradation or suppression of NETs may lead to severe systemic infections and immune homeostasis disorders [16, 17]. Therefore, developing precise NETs-targeted therapeutic strategies for different types of inflammatory diseases is crucial. Furthermore, crosstalk exists between NETs and other forms of cell death [3, 18]. In-depth investigation and clarification of this intrinsic mechanism and its clinical application value will facilitate the precise regulation of inflammatory disease progression (especially tumors) through multiple pathways.

In summary, this review focuses on clinical translation and systematically covers NET research from molecular mechanisms to clinical translation. In the first place, it transcends the boundaries of one cell death model and systematically elucidates the interaction network of NETs with multiple programmed cell death types. Secondly, it systematically differentiates the role of NETs in various types of disease, such as ADs, cardiovascular diseases, and tumors. Moreover, in terms of guiding clinical translation, more emphasis is paid to the basic technologies which intersect the fields of biomedicine and materials science and thus probe into the potential application of nanoparticles/novel delivery systems for development of anti-NETs drugs. Eventually, it suggests the development of standardized methods for detection of NETs, the normalization of NETs biomarkers and personalized regimens based on NET detection that may be a reference in subsequent related research.

The biology of NETs in inflammatory diseases

Neutrophils constitute the core effector cells of innate immunity. NETs release chromatin, granular proteins, oxidants and mitochondrial DNA into the extracellular microenvironment to participate in neutrophil-mediated antibacterial responses [3]. Distinct from intracellular antimicrobial mechanisms, NETs exert biological functions by exposing DNA, histones, myeloperoxidase, NE, oxidized mitochondrial DNA and other damage-associated molecular patterns (DAMPs) to adjacent immune and parenchymal cells [19]. These mediators trigger inflammatory signaling cascades, compromise endothelial and epithelial barriers, facilitate thrombosis, and modulate apoptotic programs in neighboring cells. Figure 1 illustrates the four functions of neutrophils: phagocytosis, degranulation, and NETs formation.

Fig. 1.

A schematic diagram of the functions of neutrophils. This graph represents the four essential actions of neutrophils. These are: phagocytosis (section A), degranulation (section B), chemotaxis and migration (section C) and NETosis (section D). Neutrophils have a lobulated nucleus and various sized granules. Phagocytosis (A): neutrophils engulf the bacteria, resulting in the formation of phagolysosomes in the cell. B degranulation: stimulation causes degranulation of neutrophils, releasing various granules (primary, secondary, tertiary) and their contents (MPO, NE, defensins etc.). Chemotaxis and migration (C): cells change shape, cross the vascular endothelium, and migrate toward inflammatory cues. NETosis (D): the nuclear chromatin decondenses and is expelled to generate NETs and entrap pathogens. Among those are NETs of nuclear and mitochondrial origin. IgG: immunoglobulin G; FcR: fc receptor; CR: complement receptor; PRR: pattern recognition receptor; C3b: complement component 3b; C4b: complement component 4b; LPS: Lipopolysaccharide; PGN: Peptidoglycan; NE: neutrophil elastase; MPO: Myeloperoxidase; MMP: matrix metalloproteinase; LYN: Lyn tyrosine kinase; DAMPs: damage-associated molecular patterns; NETosis: neutrophil extracellular trap formation

Nets-relevant neutrophil biology

Neutrophils contain abundant antimicrobial granule proteins, including MPO, NE, cathepsin G, defensins, and lactoferrin. MPO and NE are core regulators of NETosis because they promote chromatin decondensation and extracellular antimicrobial activity [20]. ROS, MPO activation, NE release, and chromatin remodeling connect neutrophil activation with NET release and tissue injury. NET-associated histones can induce cytotoxicity and platelet activation [21]. NE can degrade extracellular matrix and impair tissue barrier integrity [22]. MPO can generate oxidants that intensify inflammatory damage [23]. The pathological effect of neutrophils in NET-driven diseases is determined by the balance between antimicrobial defense and excessive extracellular inflammatory injury.

Discovery, structure, and major components of NETs

NETs were formally described as extracellular chromatin structures that trap and kill microorganisms [2]. The historical evolution of NETs research, from their initial discovery to their expanding roles in inflammatory diseases, regulated cell death, and targeted therapy, is summarized in Fig. 2. Subsequent work showed that NETs can arise through lytic NETosis, non-lytic NETosis, and mitochondrial DNA-associated NET release [4]. NETs are now recognized as inflammatory platforms that expose autoantigens, activate complement and coagulation, and regulate tissue injury [3].

Fig. 2.

Key milestones timeline of NETs research. This figure depicts some of the key events in NETs research. NETs were initially identified in 2004, named as NETs at that time, the concept of NETosis was coined and mtDNA-containing NETs released from mitochondria were described in 2007 and 2009, respectively, indicating a continuous development of interest. Later studies gradually connected NETs to different mechanisms of disease and unveiled related molecular pathways. This path follows the entire course of NETs research from basic discovery to clinical application, and shows the importance of NETs in immunology, oncology and other disciplines. NETs: neutrophil extracellular traps; NETosis: neutrophil extracellular trap formation; mtNets: mitochondrial neutrophil extracellular traps; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; DNA: deoxyribonucleic acid; CCDC25: coiled-coil domain containing 25; GAPDH: glyceraldehyde-3-phosphate dehydrogenase

DNA forms the structural backbone of NETs. Nuclear DNA is the classical source of NETs DNA. Mitochondrial DNA can also be incorporated into NETs and has strong pro-inflammatory activity in interferon-associated inflammation [19]. NETs-associated proteins include nuclear components such as histones and granule-derived proteins such as NE, MPO, and cathepsin G. Histones exert direct cytotoxic and platelet-activating effects [21]. NE contributes to extracellular matrix degradation and tissue barrier disruption [22]. MPO promotes oxidative inflammatory injury through the generation of reactive oxidants [23].

NET composition varies according to stimulus type, tissue microenvironment, and disease context [24]. This heterogeneity shapes NETs-mediated inflammatory cell death. NETs-derived DNA and histones function as DAMPs. Oxidized mitochondrial DNA activates interferon-related pathways [19]. MPO-derived oxidants promote oxidative tissue damage. NET structure therefore provides a molecular basis for the interaction among innate immunity, thrombosis, tissue injury, and regulated cell death. Table 1 summarizes the major NETs components and their disease-associated inflammatory, tissue-damaging, tumor-related, degradative, and therapeutic implications.

Table 1.

Multidimensional functions of NETs core components and their associations with diseases

NETs components	molecular feature	Pro-inflammatory mechanism	Tissue damage targets	Tumor-promoting effect	degradation pathway	Targeted therapy strategy	Ref.
Citrullinated histone H3	Positively charged, antibacterial activity.	ACPA, activate neutrophils.	Vascular endothelial cells, joints, glomerulus	NA	Macrophage phagocytosis	PAD4i, PTX3, HDAC	[25–28]
MPO	Oxidase activity and antibacterial activity.	Generation of ROS, stress.	Vascular endothelial cells, glomerulus	NA	NA	ABAH	[29, 30]
NE	serine protease	Degrade ECM, activate inflammatory factors, and degrade immunoglobulins.	Lung, joints, ECM.	Promote tumor cell proliferation and enhance tumor metastasis.	α1-AT	Sivelestat	[31–34]
cfDNA	H3Cit-DNA complex	TLR9, HMGB1	Vascular endothelial cells, glomerulus	Mediate tumor metastasis and awaken dormant tumor cells.	DNase I，macrophage phagocytosis	Exogenous DNase supplementation, Inhibit TLR9 signaling.	[35, 36]
PR3	Serine protease	Autoantigen	Vascular endothelium, kidney	Promote tumor metastasis	NA	Sivelestat	[29, 37]
S100A8/A9	Form heterodimers or homodimers	RAGE, TLR4, Induce inflammatory factors	Gastrointestinal and respiratory tract epithelial cells	Inflammatory remodeling of the TME, tumor cell proliferation and survival, tumor invasion and metastasis, immune suppression.	NA	RAGE agonist, TLR4 inhibitor , Regulate the concentration of Zn2+/Mn2+, NSAID	[38]
MMP9	Zinc-dependent endopeptidase, containing catalytic domain and fibronectin domain.	Drive the activation of neutrophil function and promote the release of cytokines.	Cardiomyocytes, ECM, endothelial cells.	Awaken dormant cancer cells and promote metastasis.	NA	SB3-CT	[35, 39]

Lytic and non-lytic NETosis

NETosis is classified into lytic NETosis and non-lytic NETosis. Lytic NETosis is characterized by chromatin decondensation, nuclear envelope disruption, PM rupture, neutrophil demise, and the extracellular release of granular protein-coated chromatin [40]. Canonical lytic NETosis proceeds via NADPH oxidase-dependent ROS production, MPO activation, nuclear translocation of NE, histone modification and subsequent chromatin decondensation [20]. PAD4-catalyzed histone citrullination promotes chromatin relaxation, yet the dependence on PAD4 differs across disparate stimuli [41]. In addition, GSDMD mediates PM permeabilization to facilitate NET expulsion [42]. These distinct modes of NET release differ in membrane integrity, chromatin origin, and neutrophil viability, and their major cellular events are illustrated in Fig. 3.

Fig. 3.

Molecular mechanism pathway of NETosis. This visual abstract depicts the major events of classical lytic NETosis (A) and non-lytic NETosis (B). During lytic NETosis, activated neutrophils produce high levels of intracellular ROS, which cause the release and translocation of granular cytoplasmic proteins to the nucleus. Such granule proteins are transported into the nucleus and produce physiological effects. Afterward, the nuclear envelope dissolves and the unraveled chromatin becomes mixed with cytoplasmic granule proteins and other substances. The PM is subsequently disrupted and NETs are expelled. The term non-lytic NETosis includes both nuclear-derived NETs and mtNets. The main sequence for the first is: upstream stimulation; degranulation of neutrophils, carrying chromatin (exocytosed as vesicles). Granule components associate with chromatin outside the cell to create NETs, whereas the chromatin-depleted cells produce a phagocytic vacuole mediating a different physiological response. The main mtNET-releasing mechanism is: upon stimulation, ROS generation in mitochondria results in intra-mitochondrial damage and release of mtDNA. mtDNA is then oxidized and associates with granule proteins. The mtDNA-protein complex is transported as vesicles and exocytosed to generate mtNets. Mitochondria can also directly fuse to the PM and extrude mtDNA in a localized fashion. NETosis: neutrophil extracellular trap formation; NETs: neutrophil extracellular traps; PMA: phorbol 12-myristate 13-acetate; LPS: Lipopolysaccharide; IL-8: interleukin-8; C5a: complement component 5a; DNA: deoxyribonucleic acid; mtDNA: mitochondrial DNA; PM: plasma membrane

During non-lytic NETosis, neutrophils extrude chromatin while maintaining intact PM and partial functional viability [43]. This signaling cascade can be induced by bacteria, fungi, platelets, lipopolysaccharide (LPS), and complement-derived agonists. Non-lytic NETosis permits rapid host protection alongside the retention of partial neutrophil effector capacity [44]. Once NETs generation outpaces physiological clearance capacity, both lytic and non-lytic NETosis can precipitate pathological tissue damage.

Regulation of NETosis in inflammatory diseases

NETosis is regulated by intracellular effector machineries and extracellular inflammatory triggers. Major signaling hubs linking NETosis to inflammatory regulated cell death encompass the ROS–NADPH oxidase–MPO–NE axis, PAD4-governed chromatin remodeling, GSDMD-dependent membrane permeabilization, pathogen-derived cues, immune complexes, complement fragments, cytokines, platelets, and DAMPs [6].

Regulation of NETosis by intracellular molecules

ROS-NADPH oxidase-MPO-NE axis

The ROS–NADPH oxidase–MPO–NE axis serves as the core effector module governing canonical NETosis. NADPH oxidase generates ROS that are indispensable for canonical NET biogenesis [40]. Impaired NADPH oxidase activity attenuates NET production upon stimulation with Staphylococcus aureus or phorbol 12-myristate 13-acetate (PMA). Metabolic modulation of NADPH bioavailability also alters NETs formation [45].

As downstream effectors of ROS, MPO and NE drive chromatin decondensation. NE translocates from azurophilic granules into the nucleus and proteolytically cleaves histones [20]. MPO cooperates with NE to relax chromatin and facilitate subsequent NET extrusion [46]. Defective activation of either MPO or NE compromises NETs generation in a stimulus-dependent manner [47]. ROS, MPO-derived oxidants and NE additionally trigger epithelial injury, endothelial dysfunction, inflammasome activation and oxidative regulated cell death. Collectively, the ROS–MPO–NE module mechanistically bridges NET formation and inflammatory tissue damage.

PAD4-histone remodeling and GSDMD-associated membrane rupture

PAD4 facilitates NET biogenesis by catalyzing histone citrullination and accelerating chromatin decondensation [41]. Pharmacological inhibition or genetic ablation of PAD4 curtails NETs formation across multiple inflammatory preclinical models [48]. Nevertheless, not all NETosis cascades rely on PAD4. Candida albicans triggers NETs generation via PAD4-independent pathways [49]. Notably, pharmacological inhibition of PAD4 in SARS-CoV-2 infection produces cell-type-specific effects. In neutrophils, PAD4 targeting reduces NET formation and improves clinical outcomes; however, in dendritic cells, PAD4 inhibition impairs antigen uptake and MHC-mediated antigen presentation, thereby weakening their ability to activate virus-specific T cells. In T cells, PAD4 inhibition does not affect early activation but suppresses T cell proliferation by reducing IL-2 production and downstream STAT5 signaling [50]. Therefore, when designing therapeutic strategies targeting PAD4, its pleiotropic roles across different leukocyte populations must be carefully considered.

GSDMD bridges NETosis to lytic inflammatory regulated cell death. During lytic NETosis, GSDMD oligomerizes to form PM pores, which enable the influx of bioactive mediators and calcium overload, ultimately culminating in PM breakdown [42]. GSDMD also functions as an indispensable executioner of pyroptotic cell death. Such shared molecular machinery underpins the concurrent occurrence of NETosis and pyroptosis observed in sepsis, acute lung injury and inflammasome-dependent tissue damage. Figure 4 summarizes the core ROS-dependent and ROS-independent signaling routes that converge on PAD4 activation, chromatin decondensation, and NET extrusion.

Fig. 4.

The core signaling pathways for ROS-dependent and -independent NETs formation. This diagram shows important molecules involved in ROS-dependent and -independent pathways of NETosis, mainly consisting of two pathways. The left half illustrates this ROS-dependent pathway, which is focused on the PMA/LPS → PKC → NOX2 →ROS activation → NE/MPO activation and PAD4 activation axis. The panel on the right shows the ROS-independent pathway associated with the central axis: calcium ionophore (A23187) → calcium influx → PAD4 direct activation. Both of these pathways intersect within the nucleus during chromatin depolymerization. Inhibitors are annotated as such on molecules and nodes. PMA: phorbol 12-myristate 13-acetate; LPS: Lipopolysaccharide; NE: neutrophil elastase; MPO: Myeloperoxidase; mtNets: mitochondrial neutrophil extracellular traps; GM-CSF: granulocyte-macrophage colony-stimulating factor; MEK/ERK: mitogen-activated protein kinase/extracellular signal-regulated kinase; PKC: protein kinase C; NOX2: NADPH oxidase 2; DPI: Diphenyleneiodonium; NAC: N-acetylcysteine; ABAH: 4-aminobenzoic acid hydrazide; mtROS: mitochondrial reactive oxygen species; PAD4: peptidylarginine deiminase 4

Extracellular regulatory mechanisms of NETosis

Pathogens and pathogen-derived factors

Pathogenic microorganisms represent canonical extracellular triggers for NET biogenesis. Bacteria, fungi, viruses, parasites, microbial toxins, and pathogen-associated molecular patterns (PAMPs) are all capable of stimulating NETs extrusion [3]. Although NETs constrain microbial dissemination, numerous pathogens have evolved strategies to evade host immune clearance. Certain bacterial species secrete deoxyribonucleases that degrade the DNA scaffold within NETs [51], whereas other pathogens release proteases to degrade NET-bound antimicrobial proteins [52]. In addition, bacterial outer membrane vesicles modulate NETs stability and subsequent inflammatory activation [53]. Within infection-associated inflammatory microenvironments, pathogen-initiated NETs formation triggers inflammasome assembly, drives regulated cell death, disrupts epithelial and endothelial barriers, and ultimately precipitates end-organ injury.

Immune complexes and complement fragments

Immune complexes trigger NET biogenesis via Fc receptor-dependent neutrophil activation. FcγRIIIb is tightly implicated in immune complex-initiated NETosis [54]. IgA-containing immune complexes potently activate neutrophils and promote NETs extrusion [55]. Immune complex-derived NETs induce autoantigen exposure, amplify interferon responses, activate complement cascades and sustain persistent tissue inflammation.

Complement fragments serve as another set of NETosis modulators. C5a boosts mitochondrial ROS production and subsequent NETs formation [56]. Reciprocally, NETs activate complement pathways to establish a bidirectional inflammatory amplification loop [57]. Under thromboinflammatory conditions, NETs act as a structural scaffold supporting platelet adhesion and complement deposition [58]. Platelets further augment NETosis through secretion of proinflammatory mediators. Such reciprocal crosstalk mechanistically links NET generation to thrombosis, endothelial injury and regulated cell death.

Cytokines, platelets, and DAMPs

Inflammatory cytokines modulate NET biogenesis in a context-dependent manner. Type I interferons augment ROS generation and facilitate NETs formation [59]. IL-8 drives NETs extrusion via NOX and mitogen-activated protein kinase (MAPK)-associated signaling cascades [60]. Type III interferons restrict excessive ROS-dependent NET production during viral infection [61]. Collectively, cytokines and NETs constitute an interactive inflammatory circuit.

In addition, platelets bridge vascular inflammation and NETosis. Platelet-derived P-selectin interacts with neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) to initiate PAD4-dependent NETosis [62]. Multiple NET constituents, including DNA, histones, NE and MPO, function as DAMPs. These mediators stimulate immune and parenchymal cells to amplify inflammatory responses and sustain chronic inflammatory conditions [63].

Interaction between intracellular and extracellular regulation of NETosis

The transduction of extracellular signals into intracellular execution programs is the essence of crosstalk between the intracellular and extracellular regulation of NETs. Many extracellular initiators, including pathogens, immune complexes, complement fragments, and cytokines, can trigger specialized intracellular signaling pathways through their respective receptors. These signals activate intracellular molecules, including NOX, PAD4, NE, and MPO, thereby triggering NET formation [64]. Acute pancreatitis is a prime example: P-selectin (as an extracellular initiator) liberated from platelets interacts with its ligand, PSGL-1 of neutrophils, leading to phosphorylation of Syk and elevation in intracellular calcium. This cascade in turn activates PAD4 and ultimately leads to NETosis [62]. These events demonstrate how extracellular cues are converted into intracellular NETosis programs. In addition, the soluble contents (e.g., NE and MPO) of NETs can act as DAMPs, triggering nearby immune cells to induce a positive feedback loop. This enhances the local inflammatory reaction, encourages the generation of ICs/thrombi and maintains a condition of chronic inflammation [63, 65]. In summary, extracellular substances not only serve as the end products of NETs but also function as extracellular signaling sources, thereby re-regulating the intracellular processes of NETs. Figure 5 provides further details on intracellular and extracellular regulation of NETs.

Fig. 5.

The interaction of intracellular and extracellular regulatory pathways for the formation of lytic NETs. This figure depicts the chains of events and checkpoints implicated in NE/MPO-containing nuclear-derived NETs. Part A: intracellular factors that enhance NET generation including receptor-mediated upstream activation, production of ROS, PAD4-dependent histone deamination, nuclear entry of NE and MPO as well as chromatin unpacking and their respective mechanisms are shown. Section B deals with NETs induction, with emphasis on NET components labeling and their relationship to the sources at the left. Component labels above indicate nuclear membrane lysis/NET release. Section C describes extracellular effects/regulation, representing NET clearance mechanisms, NET–immune cell interactions, NET–platelets interactions and NET-interacting anti-NET antibodies. NE: neutrophil elastase; MPO: Myeloperoxidase; PAD4: peptidylarginine deiminase 4; DNA: deoxyribonucleic acid; NETs: neutrophil extracellular traps; ROS: reactive oxygen Species; GSDMD: gasdermin D; DEK: DEK nuclear protein; DNaseI: deoxyribonuclease I; pDcs: plasmacytoid dendritic cells; TFPI: tissue factor pathway inhibitor; PAR4: protease-activated receptor 4; ANCA: anti-neutrophil cytoplasmic antibody; ACPA: anti-citrullinated protein antibody; anti-RNP: anti-ribonucleoprotein antibody

Interaction between NETs and other modes of cell death

NETosis is closely intertwined with multiple forms of regulated cell death (RCD), including apoptosis, necrosis, autophagy, pyroptosis, ferroptosis, and cuproptosis. Rather than functioning as an isolated neutrophil-specific death program, NETosis operates within a broader inflammatory cell-death network. In this network, NET-derived DNA, histones, granular enzymes, ROS, and other DAMPs can directly modulate death programs in neighboring immune and parenchymal cells. Conversely, mediators released during apoptosis, pyroptosis, ferroptosis, autophagy-related stress, and other forms of RCD can prime neutrophils, reshape the inflammatory microenvironment, and either promote or restrain NETs formation. Therefore, the pathological significance of NETs in inflammatory diseases should be understood not only from the perspective of NETs generation itself, but also through their reciprocal crosstalk with other cell-death pathways. Figure 6 illustrates the intrinsic interaction network between NETosis and these six other forms of RCD.

Fig. 6.

The cross-regulatory network of NETosis and other forms of cell death. The crosstalks of NETosis with other types of cell death are depicted in this scheme. NETosis cross-regulates apoptosis, necroptosis, pyroptosis, and autophagy that occurs in the center among them promoting or inhibiting the progression of these modules. Connecting lines indicate common or important signaling pathways and molecules between NETosis and the respective cell death types. NETosis: neutrophil extracellular trap formation; GSDMD: gasdermin D; ROS: reactive oxygen Species; MAPK: mitogen-activated protein kinase; BCL-2: B-cell lymphoma 2; GSDME: gasdermin E; PAD4: peptidylarginine deiminase 4; mTOR: mechanistic target of rapamycin; REDD1: regulated in experimentally induced diabetes and development 1; RIPK: receptor-interacting protein kinase; MLKL: mixed lineage kinase domain-like protein; HMGB1: high mobility group box 1; METTL3: methyltransferase-like 3; cGAS-STING: cyclic GMP-AMP synthase –stimulator of interferon genes

Netosis and apoptosis

Apoptosis, a representative form of RCD, is primarily executed by caspases. It is mediated mainly through intrinsic mitochondrial, extrinsic death receptor, and cytotoxic lymphocyte-associated pathways, involving MAPK, phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and nuclear factor kappa B (NF-κB) signaling [66]. Apoptotic cells are characterized by morphological features such as chromatin condensation, nuclear fragmentation, and vacuolization of the cytoplasm while maintaining PM integrity, in marked contrast to NETosis [40]. Nevertheless, in neutrophils, NETosis and apoptosis are usually seen as antagonistic death pathways. The balance between these pathways is tightly regulated by the unique signaling microenvironment and conversion between them may occur. For example, in response to ultraviolet (UV) irradiation neutrophil-apoptosis can be induced at all doses of UV through a NOX-independent NETotic process; however, at high UV doses, NOX-independent NETosis predominates in neutrophils [67]. Mesenchymal stem cell-derived apoptotic bodies can suppress the LPS- or PMA-induced formation of NETs through the fas cell surface death receptor (Fas)-FasL-ROS pathway. At the same time, they enhance expression of Fas-associated death domain protein and Caspase-3, thereby upregulating apoptosis in neutrophils [68]. Under pathological conditions, an activated apoptosis program may be re-directed to NETosis. For instance, in a fatty liver model, the transformation is driven through signaling involving sphingosine-1-phosphate receptor 2 activation by G protein alpha inhibitory/other subunits and MAPK and ROS pathways [69]. A key molecular mediator of this transition is likely to be gasdermin E (GSDME): Caspase-3 activated by apoptotic cues can cleave GSDME which in turn induces a large cellular influx of calcium. This in turn indirectly upregulates PAD4, which is one of the effectors of NETosis, thereby inducing NETotic cell death [70, 71]. This indicates an important pathogenic mechanism: In the absence of efficient cell clearance, apoptotic cells can reciprocally induce NETosis through this “molecular short circuit,” further promoting inflammation. Furthermore, the lack of cystic fibrosis transmembrane conductance regulator in cystic fibrosis patients’ neutrophils delays apoptosis and exaggerates production of NETs. This anti-apoptotic effect is not linked to inflammation and NETs production can trigger macrophage inflammation [72].

NETs released into the extracellular space can act as DAMPs through their components (eg, DNA and histones), directly or indirectly leading to apoptosis of local cells and exacerbating tissue damage. In nerve injury models, NETs induce neuronal apoptosis through activation of the neuronal stimulator of interferon genes (STING)-inositol-requiring enzyme 1α (IRE1α)/apoptosis signal-regulating kinase 1/stress-activated protein kinase pathway or via TLR9-mediated endoplasmic reticulum stress. Inhibition of NETs formation (e.g., PAD4 inhibition) or blockade of their recognition (e.g., TLR9 antagonists) is also known to effectively protect neurons [73, 74]. In lung injury models, NETs deposition is associated with enhanced apoptosis of alveolar epithelial cells, which coincides with increased expression of proinflammatory mediators (IL-8 and HMGB1) as well as downregulation of anti-apoptotic miR-146a. Ectopic expression of miR-146a consistently inhibits NETosis and cell apoptosis indicating a potential common node of regulation [75]. In gestational diabetes mellitus, low maternal adiponectin levels promote NETs formation. These NETs subsequently induce trophoblast apoptosis by triggering mitochondrial ROS generation and inhibiting the ERK1/2 survival signaling pathway, which may be closely related to adverse pregnancy outcomes [76].

Collectively, NETosis and apoptosis form a context-dependent regulatory axis in inflammatory diseases. Apoptotic signaling can antagonize NETosis to promote resolution, or shift toward NETs formation under excessive ROS, calcium influx, GSDME activation, or defective efferocytosis. Conversely, NETs can induce apoptosis in neighboring parenchymal cells via DAMPs sensing, mitochondrial injury, oxidative stress, and ER stress. Thus, NETs are not merely terminal neutrophil death products but apoptosis-amplifying inflammatory platforms that propagate secondary tissue injury. Elucidating the molecular switches governing neutrophil apoptosis versus NETosis could provide therapeutic opportunities to limit NETs-driven inflammation while preserving physiological immune clearance.

Nets and necrosis

Necrosis is another type of cell death with loss of PM integrity and pro-inflammatory effects. Some forms of necrosis are genetically regulated, including necroptosis, such as necroptosis [66]. The central executioner molecules of necroptosis are receptor-interacting protein kinase (RIPK)1, its paralogue RIPK3 and the substrate mixed lineage kinase domain-like protein (MLKL), a pseudokinase known as MLKL. It has also been confirmed that these molecules are the crucial nodes that control NETosis, which provides a molecular basis for the crosstalk between these two cell death pathways. Under autoimmune stimulation, antineutrophil cytoplasmic antibody (ANCA) can trigger NETosis through the activation of the upstream signal of RIPK3-dependent necroptosis [77]. Subsequent studies reported that MLKL is able to directly induce NETs formation through PAD4 [78]. Indeed, RIPK1 can function as a “molecular switch” to guide how the cell will enter into either necroptosis or NETosis by using distinct downstream substrates [78]. In thrombosis and infection, activated platelets can induce MLKL-dependent necroptosis leading to NET release; conversely, blocking necroptosis prevents NETs release [79]. The Mycobacterium tuberculosis virulence factor ESAT-6 can cause calpain-dependent necrosis and a NETosis-like pathway, raising the possibility that different necrotic stimuli could perhaps converge on a similar terminal pathway of NET formation [80].

NETosis also has the capacity to trigger and foster necrosis in other cells. Renal ischemic-reperfusion leads to necrosis of renal tubular epithelial cells, producing DAMPs that promote the formation of NETs; in turn, histones and NETs exacerbate tubular epithelial cell necrosis. Both NETs formation and tubular necrosis may be inhibited by anti-histone antibodies [81]. Similarly in intestinal I/R injury, NETs suppress mitophagy through TLR4–RIPK3–FUN14 domain containing 1 (FUNDC1), resulting in necroptosis of intestinal epithelial cells and leading to the loss of intestinal barrier function. In mice with a defect in NETs formation, the production of NETs and the necroptosis of intestinal epithelial cells were decreased after intestinal I/R [82]. In addition, NETs can activate the cyclic GMP-AMP synthase (cGAS)-STING pathway through endocytosis, thereby driving necroptosis in alveolar epithelial cells and triggers a local/cascade inflammatory response, contributing to acute lung injury [83].

Taken together, NETosis and necroptosis share lytic features and overlapping molecular regulators. RIPK1, RIPK3, MLKL, and PAD4 can function as common signaling nodes that determine whether inflammatory stimuli induces necroptosis, NETosis, or a mixed lytic phenotype. Conversely, extracellular NETs can promote necrotic or necroptotic injury in parenchymal cells through histone-mediated cytotoxicity, DAMP sensing, TLR4–RIPK3 signaling, impaired mitophagy, and cGAS-STING activation. Thus, NETosis-necroptosis crosstalk may form a feedforward inflammatory circuit in which membrane rupture, extracellular DNA, histones, and cytokine amplification jointly drive tissue injury. Targeting shared nodes may help interrupt this circuit while preserving neutrophil host defense.

Netosis and pyroptosis

Pyroptosis is a form of RCD induced by pore-forming activity of GSDM family proteins. As a crucial molecule in the canonical pyroptosis signal pathway, caspase-1 also contributes to the release of inflammatory cytokines, giving it bidirectional regulatory roles in the progression of disease [66]. In recent years, studies on the association between NETosis and pyroptosis have increasingly shown that NETs are closely related to pyroptosis of different cells. Under certain pathological circumstances, however, the two could functionally exert mutual support to exacerbate the progression of related diseases including sepsis, diabetes and acute respiratory distress syndrome (ARDS). NETs acting as DAMPs to promote macrophage pyroptosis is one of the most well-understood mechanisms. In models of sepsis, NETs generated prior to macrophage pyroptosis can exacerbate this program. Mechanistically, NET-released HMGB1 activates Cathepsin B through RAGE, leading to the formation of ASC specks and activation of Caspase-1, hence pyroptosis [84]. A few later studies have also demonstrated a variety of molecular pathways that NETs engage to induce pyroptosis through inflammasome activation: 1) AIM2 inflammasome signaling: DNA within NETs can be recognized by alveolar macrophages, thereby activating the AIM2 inflammasome and triggering caspase-1-dependent pyroptosis during ARDS. In those studies, pyroptosis derived from macrophages in such a context might produce more pro-inflammatory stimuli and thereby enhance NETosis; thus mimicking an auto-amplified inflammation loop [85]. 2) NLRP3 inflammasome pathway: In sepsis-associated lung injury and Kawasaki disease models, NETs induce the assembly and activation of the NLRP3 inflammasome via an ROS burst, resulting in pyroptosis in macrophages or peripheral blood mononuclear cells [86, 87]. 3) The non-receptor-dependent physical interaction: the positively charged protein components (such as histones) of NETs generated under high-glucose conditions might physically interact directly with glomerular endothelial cells, which have a negative charge, leading to pyroptosis. This indicates the presence of pyroptosis-regulatory machinery that is upstream of DAMPs receptors [88].

It is notable that NETs play specific roles in regulating tumor cell pyroptosis. NETs suppress oral squamous cell carcinoma (OSCC) cell pyroptosis and enhance cancer cell invasion in the setting of OSCC by reducing NLRP3 expression and GSDMD cleavage. In addition, tryptase-producing NETs within TME can significantly suppress epithelial-mesenchymal transition (EMT) of OSCC cells by inhibiting NE expression and enhancing NLRP3 inflammasome activation [89]. It suggests that NETs are also involved in the regulation of tumor cell pyroptosis.

Overall, NETosis and pyroptosis are connected through mechanisms centered on the inflammasome. NET-derived HMGB1, DNA, histones, ROS, and granular proteins activate RAGE-Cathepsin B-ASC-Caspase-1, AIM2, NLRP3, or receptor-independent membrane injury pathways, inducing pyroptosis in macrophages, endothelial, epithelial, and tumor cells. Conversely, pyroptotic cells release IL-1β, IL-18, HMGB1, ATP, and other DAMPs that can further recruit and prime neutrophils, creating a self-amplifying NETosis-pyroptosis inflammatory loop. Therefore, therapeutic strategies aimed at this crosstalk should not simply block NETs formation or pyroptosis globally, but rather target disease-specific nodes.

Netosis and ferroptosis

Ferroptosis is classified as a type of RCD that depends on the iron-mediated peroxidation of membrane lipids. Its primary characteristics are disordered iron metabolism, imbalance of the antioxidant defense system with subsequent irreversible peroxidation of phospholipids and rupture of the cell membrane [90]. The first evidence of NETs inducing ferroptosis came from alveolar epithelial cells. Early studies in sepsis-induced ALI models showed that NETs induce ferroptosis of alveolar epithelial cells through a methyltransferase-like 3 (METTL3)-mediated m6A methylation [91]. This regulatory pathway was further elucidated: NETs induced p300 activation and histone H3K27 acetylation, which mediated the transcriptional upregulation of METTL3 by p21. Activated METTL3, in turn, enhanced the expression of the EMT-related gene hypoxia-inducible factor-1α (HIF-1α) through m6A-driven IGF2BPs. This process promotes ferroptosis, promotes glycolysis but suppresses oxidative phosphorylation in alveolar epithelial cells [92]. More importantly, NETs also have a role in the regulation of ferroptosis in various cell types. Excessive NETs significantly induce ferroptosis in intestinal epithelial cells by inhibiting mitophagy in intestinal I/R injury, and this process is regulated by FUNDC1; PAD4 deficiency counters this trend of injury [93]. NETs may also inhibit expression of SLC25A11 in vascular smooth muscle cells (VSMCs), resulting in mitochondrial glutathione depletion and induction of VSMCs ferroptosis [94]. A subsequent study has demonstrated that NETs-mediated VSMCs ferroptosis is caused by suppression of PI3K/Akt signaling, and administration of extracellular vesicles derived from mesenchymal stem cells reversed a mode of neutrophil death from NETosis to apoptosis, alleviating the formation of NETs as well as the occurrence of VSMCs ferroptosis [95].

Conversely, ferroptosis can also induce NETosis. In non-alcoholic steatohepatitis (NASH), hepatocytes highly expressing amino acid synthesis control factor 5-like 1 can promote mitochondrial permeability transition pore (mPTP) opening by acetylating cyclophilin D, inducing mitochondrial ROS release and hepatocyte ferroptosis. Ferroptotic hepatocytes release large amounts of HMGB1, which recruits and activates neutrophils to form NETs, constituting a “ferroptosis-NETs” inflammation amplification loop that drives hepatitis progression [96].

NETosis and ferroptosis appear to form a bidirectional inflammatory amplification loop. NETs promote ferroptosis by inducing mitochondrial dysfunction, mitophagy impairment, glutathione depletion, lipid peroxidation, and METTL3/m6A- or PI3K/Akt-related metabolic reprogramming. Conversely, ferroptotic cells release DAMPs which recruit and activate neutrophils and further enhance NETs formation. Therefore, NETosis-ferroptosis crosstalk may be a key mechanism connecting oxidative tissue injury with sustained inflammation in multiple inflammatory diseases.

Netosis and autophagy

Autophagy, as a core biological process maintaining intracellular homeostasis, plays a key supporting and regulatory role in NETosis [66]. Early studies confirmed that in PMA-induced NETosis, autophagy and NOX2-derived ROS signals are two indispensable and synergistic core elements; blocking either pathway inhibits NETosis, shifting the mode of cell death toward apoptosis [97]. Mincle, a C-type lectin receptor capable of recognizing host- and pathogen-associated molecular patterns, is a key regulator of NETs formation, acting downstream of the mechanistic target of rapamycin (mTOR). Mincle-knockout (Mincle^-/^-) neutrophils exhibit impaired autophagy activation and reduced NETs formation, whereas tamoxifen treatment can restore NETs formation by activating autophagy [98]. Furthermore, treatment with low molecular weight heparin inhibits neutrophil autophagy activation, thereby blocking NETs generation [99]. Similarly, platelet-derived microparticles from patients with systemic sclerosis activate neutrophil autophagy, which is closely related to NETs formation [100], although the direct regulatory relationship between autophagy and NETosis was not explicitly clarified in that study. Other research indicates that in familial Mediterranean fever and systemic lupus erythematosus, autophagy pathway mediated by DNA damage-inducible transcript 1 regulates NETosis and participates in disease pathology [101, 102]. Notably, in aging-related spontaneous NETs formation, autophagy regulates NETosis via an mTOR-independent pathway [103].

In conclusion, current data strongly support autophagy as an important upstream node to control NETosis. It may play a hub role in intracellular signaling networks upon receiving different stress signals, including ROS, metabolic changes and receptor activation, to decide whether neutrophils die via NETosis or other types of cell death. It remains to be determined in future studies the molecular interplay between autophagosomes and downstream NETosis execution molecules (e.g., PAD4 and NE) as well as how exactly autophagy drives NET formation in different pathophysiological settings.

Netosis and cuproptosis

Cuproptosis is a novel RCD originating from disruption of copper ion homeostasis [104]. Primary findings indicate a putative reciprocal regulatory mechanism between cuproptosis and NETosis. On the one hand, Cu²⁺ can be a danger signal by inducing IFN-I production which involves cGAS-STING pathway activation and is beneficial for enhancement of neutrophil function and NETs formation [105]. In contrast, tissue damage and inflammatory microenvironment mediated by extensive NETs formation likely have indirect influence on cellular copper homeostasis through accentuating oxidative stress, which may in turn underlie cuproptosis [105], demonstrating a potential feedback loop between the two. In the context of tumors, the interaction between NETs and cuproptosis exhibits negative regulatory characteristics. Lenvatinib, a first-line drug for primary hepatocellular carcinoma (HCC), increases the number of neutrophils in the HCC TME and promotes IL-33 secretion by HCC cells through the upregulation of NADH dehydrogenase 1 alpha subcomplex 4-like 2 expression. IL-33 upregulates PAD4 expression via the Akt/mTOR pathway, ultimately inducing NETs formation. The NETs induced by Lenvatinib can inhibit HCC cell cuproptosis, leading to Lenvatinib resistance in HCC [106]. In colorectal cancer (CRC), single-cell and spatial transcriptomics analyses show that both NETs-related markers and cuproptosis-related markers are key indicators for predicting CRC prognosis, though the specific interactive regulatory mechanisms in CRC remain to be further elucidated [107]. It must be clarified that as cuproptosis is an emerging RCD subtype, current research on the interaction mechanism between NETosis and cuproptosis is not yet systematic and is largely concentrated in the oncology field. Cu²⁺ may act as a danger signal to promote NETs formation through the cGAS-STING-IFN-I axis, whereas excessive NETs may indirectly perturb copper homeostasis by amplifying oxidative stress and inflammatory injury. Therefore, deeper study of NETosis-cuproptosis crosstalk may inform therapies targeting multiple cell death programs in inflammatory diseases and inflammation-associated tumors. Future work should expand into inflammatory diseases and focus on single-cell and multi-omics technologies for mechanistic and clinical translation.

In summary, there is a tight and complex interactive regulatory relationship between NETosis and other RCD subtypes. Deeply exploring the intrinsic molecular mechanisms of these interactions is expected to provide a theoretical basis for developing therapeutic strategies based on dual or multiple cell death regulation, which holds significant importance for the treatment of inflammatory diseases, especially inflammation-associated tumors.

Nets in inflammatory diseases and related targeted therapies

The primary purpose of NETosis is to enhance host defense and pathogen elimination. However, in the context of the immune response, NETs have a Janus-like character, involved, on one hand, with antimicrobial activity and, on the other, with promotion of inflammation. This dual effect on inflammatory control allows NETs to contribute to and mediate the pathogenesis of a variety of diseases [3, 24]. The pathogenesis of NETs in various multisystem inflammations is summarized, including immune, pulmonary, endocrine, and cardiovascular diseases, as well as neoplasms. Potential targeted drugs and specific treatment strategies are also described. As represented by SLE as an ADs, Fig. 7 illustrates a vicious cycle that exacerbated or prolonged the course of the disease due to excessive NETs.

Fig. 7.

Nets drive the vicious cycle of autoimmunity in SLE. This figure presented the pathological function of NETs in autoimmunity of SLE. The primary processes include: 1. NETs liberation: neutrophils liberate NETs rich in autoantigens (DNA/LL37, etc.); 2. Autoantibody production: PDCs phagocytose NETs, origin of type I α-interferon (left side) and generation of autoantibodies by B cells; 3. Immune complex formation between NETs antigens and autoantibodies; 4. Organ damage: immune complexes are deposited in visceral organs such as kidney (lupus nephritis) leading to activation of complement and further inflammatory cell infiltration; 5. Exaggerating and magnifying inflammation and NETosis: recruited neutrophils are activated to release more NETs. IFN-I: type I Interferon; DAMPs: damage-associated molecular patterns; DNA: deoxyribonucleic acid; NETs: neutrophil extracellular traps; pDC: plasmacytoid dendritic cell

Nets and autoimmune diseases

ADs are disorders in which the immune system erroneously recognizes self-tissues as foreign antigens. Their pathogenesis involves a complex interplay of genetic, environmental, and immune system hyperactivation factors [108]. Emerging evidence confirms a close association between NETs and various ADs, including SLE, rheumatoid arthritis (RA), and AAV [3].

Nets and SLE

SLE is a systemic autoimmune disorder that involves the immune system attacking healthy self-cells and tissues, which can involve several organ systems. Patients commonly have multiple autoantibodies such as ANA, anti-double-stranded DNA (dsDNA) and anti-ribonucleoprotein (RNP) antibodies. Furthermore, IFN-I is highly detected and correlates with disease activity. Clinically, SLE is a prototypical fluctuating multi-organ autoimmune disease with substantial heterogeneity [109]. NET formation is significantly increased in SLE patients, with changes in neutrophil status and the microenvironment being considered as one major factor. On the one hand, the percentage of LDGs is elevated in peripheral blood and those cells have a higher ability to spontaneously generate NETs [3, 13]. Moreover, as a downstream effect, the typical high IFN-I microenvironment in SLE also promotes neutrophil activity and markedly increases their sensitivity to NETosis [12]. Particularly, multiple autoimmune antibodies among SLE patients can also contribute to NETosis: anti-RNP antibody induces NETosis of neutrophils under the pathogenesis of SLE in an NADPH-dependent manner followed by IFN-I secretion from pDCs. Interestingly, this inhibition does not occur if anti-RNP antibodies are used to activate normal neutrophils [11]. Furthermore, the extracellular DNA of NETs is bound and shielded against nuclease degradation by anti-dsDNA antibodies which leads to an increase in the immunostimulatory capacity [110]. Thus, NETs serve as central mediators of the pathogenic process in SLE. NETs-related immune disarrangement in SLE is multi-lateral. Upstream excessive release of mitochondrial ROS is also a required element in the LDGs for driving spontaneous NETosis, and inhibition of this pathway has been demonstrated to reduce pathological manifestations in disease models [19]. Further downstream, it should be kept in mind that NETs initiate innate immunity as well as directly crosstalk with adaptive immune systems. Thus, they may contribute to the process of immune tolerance loss, e.g. by suppressing CD4⁺ T cell function or stimulating regulatory T cells activity [111, 112]. These data shed new light on the mechanism underlying the relationship between NETs and SLE from a network-immunology viewpoint. By these associations, NETs represent interesting biomarkers of SLE [113]. Importantly, NETs remnants (e.g., NE, HMGB1 and H3Cit) may present as prognostic markers for lupus nephritis [114, 115]; however, their diagnostic sensitivity and specificity need to be verified by large scale clinical studies.

Nets and RA

RA is a chronic inflammatory disease, which is clinically characterized primarily by the gradual destruction of articular cartilage and bone, causing joint deformity, and even disability. It is often associated with extra-articular features like rheumatoid nodules, pulmonary disease and vasculitis. In addition, marked interindividual heterogeneity as to pathological characteristics and therapy susceptibility is present [108, 116]. Studies have identified NETs as a major contributor to joint inflammation and bone erosion in RA. Del-Moral et al. showed that the neutrophils in peripheral blood and involved joints of RA patients have a significantly increased capacity for NETosis. Released components, e.g. citrullinated antigens have been strongly connected with the generation of anti-citrullinated protein antibodies (ACPA), the serological hallmark specific for RA [3, 117]. Concurrently, ACPA, rheumatoid factor (RF), and inflammatory cytokines (such as IL-17A and tumor necrosis factor [TNF]-α) in RA patients can all induce NETosis, with ROS and PAD4 participating in the regulation of this process. Autoantigens further released in NETosis can also stimulate fibroblast-like synoviocytes (FLS) to secrete more inflammatory cytokines, thus making a vicious cycle of RA development [117]. NETs and RA mutually promote each other’s development in various ways: 1) Regulation of FLS function: It has been confirmed that NETs can be engulfed by FLS through RAGE–TLR9 signaling. This internalization not only drives transition of FLS to a pro-inflammatory state but also upregulated MHC-II expression which allows the presentation of NET-derived peptides, thereby directly contributing to ACPA generation [118]. 2) Direct induction of joint destruction: NE in NETs could directly lead to cartilage degradation and promote FLS to release PAD2 to produce citrullination of cartilage proteins, which gives rise to neoantigens [119]. 3) Bone erosion promotion: NETs massively stimulate osteoclast differentiation and activation through signaling molecules, such as TLR4/TLR9, resulting in articular bone tissue destruction [120, 121].

Nets and AAV

AAV comprises a group of ADs and mainly involves small to medium vessels. Its key features are inflammation and necrosis of blood vessels, together with circulating ANCA. The kidney is the most common target organ [108, 122]. Autophagy is also involved in the AAV-NET interaction, surprisingly. In the initial studies, it was shown that ANCA is capable of inducing neutrophil autophagy (evidenced by increased expression of autophagic marker protein LC3B), which contributes to the formation of ANCA-mediated NETs [123]. Moreover, a subset of ANCA, anti-lysosome-associated membrane protein-2 antibodies have been shown to directly initiate autophagy pathway (up-regulating LC3B expression) which inhibits neutrophil apoptosis and generates NETs [123]. Importantly, the process of NETs formation in AAV may progress along two concurrent pathways: ANCA-mediated and ANCA-independent. In addition to ANCA-mediated stimulation, there is evidence that endogenous neutrophil granule components like MPO and proteinase 3 (PR3) themselves might directly be involved in or potentiate NETosis [124, 125]. Furthermore, NETs can promote DCs activation and expansion of tissue-invasive monocytes, which amplify AAV pathogenesis by enhanced immune system stimulation [126, 127].

Besides the three diseases mentioned above, NETs also participate in the pathological development of other autoimmune diseases which include antiphospholipid syndrome [128–130], Sjögren’s syndrome [131] and multiple sclerosis [132]. Thus, blocking NETs production or inhibiting their function could attenuate disease severity. Table 2 Mechanism of cross-organ injury by NETs in non-neoplastic diseases and potential intervention targets.

Table 2.

Nets-mediated trans-organ injury mechanisms in non-tumor diseases and intervention windows

Disease	Core Trigger	NETs Characteristic Components	Organ Injury Target	Key Mediators	Diagnostic Biomarker	Preventive Intervention Window	Therapeutic Bottleneck	Ref.
Psoriasis	IL-17A, IL-23, IL-6	LL37, granulysin	Skin (keratinocytes) and blood vessels	IL-1β, LL37, human β-defensin 2, NE	MPO-DNA complexes, miRNAs, CitH3, IL-17, PAD4	Inhibit LDG proliferation and activation, and suppress PAD4 activity.	Insufficient drug specificity, limitations of NETs detection technology, balance between drug safety and efficacy, and complexity of NETs’ mechanism of action in psoriasis.	[133, 134]
RA	Immune complexes, ACPA, HIF-1α, and inflammatory microenvironment.	Citrullinated histones, antimicrobial protein complexes.	Articular cartilage, bone, and synovium.	Citrullinated histones, NE, MMP, ROS, IL-8, ACPA.	MPO-DNA complexes, NE, anti-NETs autoantibodies (ANETA), citrullinated histones.	Inhibit HIF-1α activity or glycolytic enzymes, regulate neutrophil function and subsets, early use of immunosuppressants, and inhibit neutrophil migration.	Complex mechanism of NETs formation in RA, increased infection risk from NETs intervention, patient heterogeneity, and efficacy variability.	[135, 136]
SLE	Infection, UV exposure, autoantibodies, and high-level oxidative stress	Oxidized DNA, acetylated histones, granzymes.	Kidneys, skin, blood vessels, placenta, and CNS.	IFN, IL-1β, IL-6, ROS, MMPs, complement.	MPO-DNA complexes, cfDNA, LDGs, anti-NETs antibodies.	Post-infection, post-UV exposure, early vascular injury, early BBB protection, and LDG regulation.	Lack of specific NETs-targeted drugs and dual role of NETs.	[136–138]
Atherosclerosis	Cholesterol crystals, IL-1β, and low shear stress.	DNA, citrullinated histones, granzymes.	Vascular endothelial cells, VSMCs, macrophages, and platelets.	IL-1α, IL-8, ROS, TF, histones, MPO.	DNase activity，cfDNA，citrullinated histones, MPO.	Target NLRP3 inflammasome, PAD4, and MPO at the early stage of plaque formation.	Diversity of NETosis initiation pathways, dual functionality of NETs components, drug specificity, and stability.	[139]
Sepsis	DAMPs, complement, IL-8, TNF-α, and chemokines.	Nuclear DNA, histones, granzymes, LL-37.	Vascular endothelial cells, platelets, lungs, liver, and kidneys.	MPO, TF, MMPs, H4, NE, ROS.	Histone-complexed DNA (hcDNA), cfDNA，MPO, NE, circulating nucleosomes.	Inhibit NETs formation in early sepsis to block subsequent vascular injury; inhibit platelet-NETs positive feedback loop during coagulation disorders.	Insufficient specificity of NETs inhibitors, difficulty in determining intervention timing, synergistic pathogenicity of NETs via multiple targets, and patient heterogeneity.	[140, 141]
ARDS	PAMPs, ACE2, platelets, IL-8, miR-144, and miR-155.	DNA, histones, granzymes.	Alveolar epithelial cells, pulmonary microvascular endothelial cells, and pulmonary macrophages.	Histone, MPO, NE, DNA, HMGB1, IL-1β, PAD4.	Citrullinated histone H3, MPO-DNA complexes, BALF NETs levels.	Inhibit NETs formation in the early stage of ARDS; use NETs inhibitors before and after high-risk procedures (blood transfusion, mechanical ventilation, surgery).	Dual role of NETs, insufficient drug specificity, diversity of NETosis pathways, and complexity of intervention targets.	[142, 143]

Nets and infectious, respiratory, metabolic, and cardiovascular diseases

Beyond ADs, substantial evidence indicates that NETs are closely associated with infectious, respiratory, metabolic, and cardiovascular diseases [4, 139, 144]. This section will discuss the pathogenic mechanisms of NETs and targeted therapeutic strategies in these four categories of diseases, using sepsis, COPD, diabetes, and AS as examples.

Nets and sepsis

Sepsis is a life-threatening organ dysfunction triggered by a dysregulated host response to infection, and early recognition and intervention can significantly improve patient outcomes [9]. Studies have shown that neutrophils are stimulated by microbial or inflammatory signals to undergo NETosis in sepsis, and higher NET levels are associated with greater sepsis severity [145–147]. In addition, microparticles formed from activated platelets by P-selectin and LPS may also stimulate NETs release [148]. Recently, a new mechanism of NETosis and thrombotic process in sepsis was identified: cyclic adenosine monophosphate in the contextual milieu of septic conditions induced binding between autophagy-related TBC1 domain-containing protein and Syntaxin-Binding Protein 2, which assembled the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, thereby inducing NETs release to drive septic thrombosis. The key evidence that supports this pathway is the inhibitory effect of platelet-specific knockout of STING on this process, accompanied by a notably decreased level of thrombosis and NETosis in septic mice [149]. Besides, DAMPs are evidently involved in a positive feedback loop between sepsis and NETs. As another member of DAMPs, exogenous cold-inducible RNA-binding protein increases the intercellular adhesion molecule-1 (ICAM-1) expression in neutrophils through triggering receptor expressed on myeloid cells-1, triggering Rho proteins and promoting NETs formation, and eventually promoting the inflammatory response during sepsis [150]. While NETs can limit sepsis progression by entrapping microorganisms and providing antimicrobial activity, they also escalate coagulation dysfunction in sepsis contributing to thrombosis and disseminated intravascular coagulation [151]. Such thrombi act synergistically with direct tissue damage caused by NETs to aggravate organ injury and promote organ failure [151, 152]. Therefore, targeted intervention and regulation of NETs are of great significance for the treatment of sepsis patients.

Nets and COPD

According to the 2019 Global Burden of Disease data, COPD is the third leading cause of death in the world by single etiology. The disease presents with a progressive and not fully reversible airflow obstruction. Its pathogenesis is highly correlated with risk factors such as smoking and air pollution. Its clinical manifestations include respiratory distress and chronic cough, and it is characterized by recurrent acute exacerbations [153, 154]. Analysis of sputum samples from clinical COPD patients using confocal laser microscopy, electron microscopy, immunolabelling and fluorescent DNA staining techniques revealed substantial quantities of NETs and neutrophils capable of NET formation within the sputum. Furthermore, NET levels were found to correlate closely with disease severity (acute exacerbation phase > stable phase; smoking group > non-smoking group) [155, 156]. Mechanistic studies have also verified that several NET-related mechanisms are important for COPD pathogenesis. NETs-produced DNA, through the cGAS-TLR9 pathway, can trigger NF-κB-dependent autoimmune responses, aggravating smoke-caused COPD development [157]. Moreover, multi-omics studies have also shown that Haemophilus influenzae is capable of inducing robust neutrophil NETosis to elevate transduction levels of IL-6 and hence exacerbating inflammatory response in COPD [158]. Furthermore, in the context of rhinovirus infection NETs can be triggered independently of bacteria by releasing dsDNA and NETs support inflammatory responses during COPD exacerbations [159]. Other work also shows that in the diseased state of COPD, C-X-C motif chemokine receptor (CXCR) 2 facilitates neutrophil activation and migration and is critically involved in generating COPD-associated NETs [160]. Therefore, inhibitors of NETosis via CXCR2 antagonists might have potential in COPD therapy [161].

Nets and diabetes

Diabetes is a metabolic disorder with chronic hyperglycemia as its typical feature, which can be categorized into type 1 diabetes (T1D), type 2 diabetes (T2D) and other special types. Sustained hyperglycemia induces irreversible damage to the cardiovascular system, kidneys, retina and peripheral nerves with increased severity of chronic inflammatory responses and immune-complex dysfunction [162]. Hyperglycemia can trigger NETosis through NOX-dependent mechanisms, and this seems particularly relevant in diabetic retinopathy [163]. The concrete model includes the human cathelicidin antimicrobial peptide leucine-leucine-37 (LL-37) as a ligand of G protein-coupled formyl peptide receptor 2 (FPR2). Hyperglycemia works synergistically with LL-37 to increase FPR2 expression in neutrophils, promote mPTP opening and mitochondrial ROS production and finally NETs release [164]. NETs are centrally involved in regulation of the development and progression of chronic non-healing diabetic wounds, by interfering with the normal wound healing process through a variety of mechanisms [165–168], including: 1) Antiangiogenesis: NETs suppress angiogenesis in endothelial cells via the TLR9–PAK2 signaling axis by inhibiting the Yes-associated protein signaling pathway and promoting endothelial-to-mesenchymal transformation, which contributes to prevent neovascularization [169]. 2) Inflammation resolution delay: NETs reduction of macrophage phagocytosis through the PI3K-Rac1 axis reduces apoptotic cell clearance in the wound, which extends inflammation [170]. 3) Tissue repair impairment: NETs can cause fibroblast endoplasmic reticulum stress and ferroptosis by activating the IRE1α-XBP1 pathway to diminish collagen synthesis and secretion in fibroblasts, which results in impaired granulation tissue formation [171]. Recent studies have found that, in addition to delaying wound healing, NETs are closely associated with other diabetic complications. NETs can cause bone loss in T1D patients by downregulating Fn1 expression and disrupting the PI3K/Akt signaling pathway and the ECM-receptor interaction pathway [172]. In diabetic nephropathy, NETs can promote NLRP3 inflammasome activation and exacerbate glomerular endothelial dysfunction; conversely, blocking NETs formation (e.g., using PAD4 inhibitors) can significantly attenuate renal injury [173, 174].

Nets and AS

AS is a chronic granulomatous inflammatory disorder of large- and medium-sized arteries. Its underlying pathological process is a series of progressing steps in the deposition and infiltration of lipids, endothelial dysfunction, hyperplasia of vascular smooth muscles, and then formation of the atherosclerotic plaque in the arterial intima; finally, rupture in the later stage will cause a high risk of cardiovascular and cerebrovascular events. The pathogenesis of AS is so closely associated with many risk factors such as hyperlipidemia, hypertension, diabetes and immune system dysfunction [139, 175]. Current knowledge suggests the crosstalk between NETs and AS is complex [139, 176]. Cholesterol crystals were reported to be able to trigger NETs formation in early work. NETs also induced release of pro-inflammatory cytokine (i.e., IL-1β) from macrophages, the activation of T helper 17 cells, leading to further progression of AS plaques and worsening plaque stability [177]. This study offered direct evidence that NETs were implicated in the pathogenesis of AS. Clinical research has reported that the plasma level of NETs-related markers (like MPO, H3Cit and so on) is dramatically increased in patients with coronary heart diseases and acute coronary syndrome as well as being closely related to lesion degree and poor prognosis [178]. And NETs may be all around to promote the pathogenic mechanism of AS via several ways like impairing functions of vascular endothelium, worsening inflammatory reactions, and inducing thrombosis [139]. First, NETs are potentially strong damaging factors to vascular endothelium: their constituents may directly compromise the endothelial mechanical barrier and the vasomotor regulating apparatus. NE and histones (in part through TLR4 signaling) can degrade or down-regulate a variety of endothelial junction proteins (e.g. VE-cadherin), leading to the loss of endothelial barrier integrity, resulting in vascular permeabilization [179, 180]. Meanwhile MPO and its catalytic product, hypochlorous acid can directly scavenge nitric oxide and inactivate endothelial nitric oxide synthase, thus compromising vasodilatory effect and exacerbating inflammatory reactions [181–183]. Second, NETs may be involved in the progression of AS through the regulation of vascular inflammation; for example, histones from NETs can lead to adhesion molecules such as VCAM-1 and ICAM-1 expression in endothelial cells, and neutrophil MPO might induce chemokines release (CCL5, IL-8, endothelin-1). These molecules and chemokines synergically contribute to leukocytes (e.g., monocytes, T cells) infiltration in endothelial cells further activating the endothelium pro-inflammatory activation [139, 180, 184, 185]; simultaneously, NETs may stimulate endothelial cells to produce chemoattractants for monocyte recruitment and differentiate macrophages being part of a positive feedback loop that sustains NETosis [186]. Third, NETs can directly cause plaque instability and thrombosis: on the one hand, ROS derived from NETs, as well as MPO and histones from NETs facilitate apoptosis or necrosis of endothelial cells or smooth muscle cells, leading to a fragile fibrous cap strength and favoring integrity breaking risk [187–189]; on the other hand, NETs provide a matrix and procoagulant signals for thrombus formation. Their DNA backbone functions as a scaffold bridging erythrocyte and platelet adhesion, and could form complex with tissue factor (TF) so as to trigger potent coagulation cascade activation while that histones in NETs are also able to directly promote fibrin deposition [190, 191]. These mechanisms serve to turn NETs into an integral component of occlusive thrombi in ACS. Taken together, NETs and their elements are involved in AS pathogenesis through multiple pathways, and have been shown to have potential diagnostic significance for AS [192].

Nets and tumors

Cancer and inflammation are intimately associated: not only do inflammatory reactions contribute to the generation of spontaneous tumors, but also many tumors arise in organs affected by chronic inflammation induced by infections or irritations. The TME, constructed partly by innate immune cells, is capable of facilitating proliferation, survival, and migration of tumor cells and subsequently hijack signaling molecules of innate immunity in order to invasion, metastasis, and other malignant features [193]. Early studies demonstrated that tumor-associated neutrophils are activated to produce NETs in Ewing sarcoma. NETs may play a dual role in cancer progression: they possess anti-tumor effects by killing tumor cells and activating the immune system, but may also exhibit pro-tumorigenic effects by promoting metastasis [194]. It has been reported that NE on NETs can promote the nuclear translocation of β-catenin, leading to downstream signaling and EMT activation. EMT is one of the most important regulating mechanisms for tumorigenesis, development, metastasis and relevant pathologies [195, 196]. Therefore, the role of NETs in cancer should not be oversimplified as a singular pro-tumorigenic factor; rather, it should be delineated based on specific pathological contexts. Based on current evidence, NETs primarily influence tumors through five interconnected yet mechanistically distinct pathways: promoting local tumor progression, facilitating metastasis and the formation of the pre-metastatic niche, mediating tumor immunosuppression, driving tumor-associated thrombosis, and modulating tumor responses to chemotherapy, radiotherapy, and immunotherapy.

Nets as drivers of local tumor progression

Within the local TME, NETs promote local tumor progression by enhancing proliferation, stromal remodeling, angiogenesis, and EMT. Lewis lung carcinoma (LLC)-induced NETs are highly efficient inducers of tumor growth in mouse models of LLC, in which the G-CSF produced by LLC cells is necessary to induce NETosis and neutrophil activation [197]. Similarly, the inhibition of NETs could reduce macrophage infiltration and inflammation which may contribute to reducing risk for progression from NASH to HCC [198]. Moreover, NETs promote the proliferation of pancreatic cancer cells: DNA in NETs induces activation of PSCs through RAGE, and enhances cell proliferation; this is a process that can be highly related to the development and metastasis of pancreatic cancer [199]. Reportedly, NETs have been shown to trigger the NF-κB pathway through ROS and TLR4 leading to pro-angiogenic effects such as promoting proliferation, migration and permeability of endothelial cells; angiogenesis is critical for tumorigenesis and metastasis [200, 201]. Therefore, at the level of localized tumor progression, NETs predominantly exert pro-proliferative, pro-stromal activation, pro-angiogenic and pro-invasive effects.

Notably, the regulatory effect of NETs on tumor growth is not unidirectional. Several studies have demonstrated that NETs are capable of suppressing tumor growth under specific conditions. In vitro studies show that NETs can block the colon cancer cell growth and promote their apoptosis, similar effects were seen in acute myeloid leukemia cells [202]. In addition, NETs are identified in ulcerated melanoma tissues and can attach to tumor cells substantially, resulting in restraint of their migration potential or viability with accompanying induction of cell death [203]. Nevertheless, in contrast to pro-proliferative effects of NETs, the intrinsic molecular mechanisms of how NETs suppress tumor cell proliferation still need to be validated and explored.

Nets serve as driving factors for metastasis and the formation of pre-metastatic niche

In contrast to localized primary tumor progression, NET-driven tumor metastasis encompasses multiple key steps including circulating tumor cells (CTCs) trapping, endothelial adhesion, extravasation, distal organ colonization, and reactivation of dormant tumor cells. NETs have been identified as the primary inducers of tumor metastasis, acting as a powerful driver that controls essential processes including the seeding and colonization of CTCs, endothelium transmigration and remodeling of distant microenvironment [204]. NETs can seize CTCs and mediate its adhesion: sepsis-induced NETs exhibited extensive deposition on the tumor cells and their ability to form micrometastases, in addition to generating a microenvironment that promotes cancer cell migration and invasion [205]. Another work with NET-targeting nanoparticles also confirmed this adhesive effect—they degraded NETs and decreased adhesion efficacy of the tumor cell in the liver, then inhibited systemic metastasis [206]. Systemic inflammation specifically may increase adhesion of NETs to tumor cells; indeed, LPS-induced systemic inflammation increases hepatic sinusoidal adhesive interactions between cancer cells by 40%, and this phenomenon is thought to occur via selectin-selectin ligand interactions [207]. The DNA composition of NETs also has chemotactic effect on cancer cells mediated by coiled-coil domain-containing protein 25, a transmembrane protein expressed by cancer cells, and favors adhesion, migration and establishment of metastasis foci [204].

Furthermore, NET could potentiate metastasis by the injury to endothelial cells because the protein in NETs (e.g., NE and MPO) directly cause damage on endothelial cells, which can increase adherence of cancer cell to endothelial surface and result in extravasation for distant metastases [208]. At the same time, NETs might be involved in the pre-metastatic niche formation: tumor derived G-CSF and lung mesenchymal stroma cells-derived complement component 3 promote neutrophils recruitment to lung and NETosis which both contribute to breast cancer (BC) metastasis [209]. In addition, NETs may promote cancer recurrence and metastasis through their ability to wake up dormant tumor cells: constant inflammatory stimulus leads to activation of NE and MMP9 from the NET-associated proteins. It is involved in activation of the integrin α3β1 signaling pathway, which has a pivotal role in awakening cancer cells [35]. Collectively, the core function of NETs during metastasis is not merely to boost primary tumor proliferation, but to improve the survival, adhesion and colonization of CTCs as well as their adaption to the distant microenvironment.

Nets as mediators of tumor immunosuppression

In addition to directly facilitating metastasis, NETs mediate immunosuppression within the tumor microenvironment by dampening anti-tumor immune responses, thereby constructing an immune-permissive niche that favors tumor progression [210]. NETs expression is inversely correlated with the number of CD8⁺ T cells, the main effector cells in anti-tumor immunity as well as infiltration by CD8⁺ T cells in different cancers [211]. NETs transporting programmed death-ligand 1 (PD-L1) have been reported to induce an effect on CD4⁺ T cells and CD8⁺ T cells. Further studies have confirmed that NETs play a crucial role in tumor progression through the immune system [212]. Furthermore, NETs may physically prevent contact between effector cells and tumor cells: Tumor-associated CXCR1 and CXCR2 agonists can trigger NETosis; in co-culture of inflammatory spheroid-based tumors cells with neutrophils, NETs alone did not impact the survival of cancer cells, but cancer cell enwrapped by NETs dramatically lost contact with CD8⁺ T lymphocytes and had significantly improved survival rates [213]. These findings imply that NETs facilitate tumorigenesis through inhibition of immune system function. However, the mechanism of interaction between NETs and tumor immunity is still in its infancy, and further studies are required to clarify the specific regulatory relationship.

Nets as promoters of tumor-associated thrombosis

Tumor-associated thrombosis represents an important yet relatively independent mechanism whereby NETs facilitate tumor progression. The DNA backbone of NETs provides a structural scaffold for the adhesion of platelets, erythrocytes and coagulation factors, and forms complexes with tissue factor to initiate robust coagulation cascades. NETs-derived histones can also directly accelerate fibrin deposition [190, 191]. In tumor settings, DNase I, which degrades preformed NETs, alleviates tumor-associated thrombosis, confirming a tight correlation between NETs and cancer-associated coagulopathy [214]. Distinct from tumor cell proliferation and immune evasion, NET-driven thrombosis is characterized by intravascular coagulation activation, microvascular occlusion and elevated venous thrombotic risk.

Regulatory roles of NETs in therapeutic response and treatment resistance

NETs can also modulate tumor responsiveness to radiotherapy, chemotherapy and immunotherapy, while these effects display marked context dependency. Direct inhibition of NETosis has become a prevalent therapeutic intervention strategy. For example, the PAD4 inhibitor BMS-P5 delays disease progression of multiple myeloma [215], and another PAD4 inhibitor GSK484 improves radiosensitivity in colorectal cancer [216]. In addition, small-molecule agents and natural products, including epigallocatechin gallate, dihydrotanshinone and glycyrrhizic acid, suppress NET formation via downregulating the STAT3/CXCL8 and PAD4 signaling axes, thereby restraining metastasis of colon and breast cancers and potentiating the efficacy of chemo, radio and immunotherapy [217–219]. Delivery of anti-NET therapeutics via nanoparticles or biological carriers is capable of reprogramming NETosis toward apoptosis and enabling targeted drug accumulation within the tumor core, which ultimately improves therapeutic efficiency [220, 221].

Nevertheless, the correlation between NETs and therapeutic responses is not unidirectional. Several studies have demonstrated that boosting NETs formation under certain circumstances may suppress tumor progression. IFN-γ markedly facilitates NETs generation and apoptosis in microsatellite-stable colorectal cancer cell lines, leading to reduced tumor volume in mouse models and potentiation of anti-tumor activity triggered by anti-PD-1 antibodies [222]. Consistently, accumulating evidence verifies that NETs can sensitize colorectal cancer to chemotherapy [223]. Accordingly, NETs cannot be simply categorized as universal drivers of therapeutic resistance; instead, their functional roles should be analyzed individually based on cancer subtype, treatment modality, host immune status, and compositional heterogeneity of NETs.

In summary, NETs participate in multiple interconnected yet mechanistically distinct biological processes of malignancies. During local tumor progression, NETs predominantly drive tumor cell proliferation, stromal activation, angiogenesis and invasion. For metastatic cascade, NETs facilitate the capture of CTCs, distal organ colonization, pre-metastatic niche establishment, as well as reactivation of dormant tumor cells. From the perspective of immune regulation, NETs promote tumor immune escape by restraining T cell infiltration, carrying immune checkpoint-associated signals, and constructing physical barriers. At the coagulation level, NETs serve as procoagulant scaffolds to trigger tumor-associated thrombosis. With regard to therapeutic outcomes, NETs modulate responses to radiotherapy, chemotherapy and immunotherapy in a tumor type- and treatment context-dependent manner. Collectively, the roles of NETs in cancer constitute a context-dependent multi-mechanistic regulatory network rather than a uniform pro-tumor pathway. The spatial distribution of NETs in the solid tumor microenvironment and its associations with clinical prognosis are summarized in Table 3.

Table 3.

Spatial distribution of NETs in the solid tumor microenvironment and its associations with clinical prognosis

Tumor Types	NETs Density Localization	Major Inducing Cells	Key Receptors	Effector cells	Pro-/anti-tumorigenic phenotype	Clinical Prognostic Association	Targeted intervention strategy	Ref.
Pancreatic Cancer	Around tumor cells, stroma, tumor-associated vessels	Pancreatic tumor cells, platelets	RAGE, DDR1, CXCR2	Pancreatic stellate cells, tumor-associated neutrophils, pancreatic tumor cells.	Promote proliferation, primary tumor growth, and metastasis.	High NETs expression may be associated with increased metastasis risk and poor prognosis.	PAD4i, block RAGE, DNase	[199, 224–226]
Liver Cancer	Liver sinusoids, tumor tissue, surrounding metastatic lesions.	Liver tumor cells, liver sinusoidal endothelial cells.	TLR4/9	HCC cells, neutrophils	Promote metastasis, migration, and invasion.	High NETs expression is associated with metastasis/recurrence risk in HCC.	DNase, Nei, COX2 inhibitor	[205, 227, 228]
BC	Local pulmonary metastatic foci, TME stroma.	BC cells, tumor-associated senescent neutrophils.	CXCR4，CXCR2	BC cells, neutrophils	Promote proliferation and metastasis	NETs levels are highly correlated with metastasis.	Inhibit CTSC, inhibit PR3, DNase, inhibit SIRT1.	[37, 229]
CRC	Primary tumor tissue, surrounding metastatic tumor cells	CRC cells, postoperative pathogens	CEACAM1, TLR4/9, CCDC25, CD16	Colon cancer tumor cells, NK cells, neutrophils	Promote metastasis	NETs levels are associated with poor prognosis in CRC.	Anti-CEACAM1 antibody, DNase，NEi, PAD4i, inhibit CD16.	[230–232]
Glioblastoma	Within the TME	Glioma cells	RAGE, CXCR2	Glioma cells, neutrophils	Promote proliferation, migration, and invasion.	Glioma patients with high NETs expression have a poorer prognosis.	DNase, inhibit RAGE, inhibit CXCR2.	[233]
Ovarian Cancer (OC)	Omental tissue	OC cells	CXCR2	Omental stromal cells, ovarian cancer cells, neutrophils.	Promote omental metastasis of OC.	NETs density is associated with tumor malignancy.	Specific PAD4i, DNase	[234]
Prostate Cancer	Liver metastases of NEPC and surrounding areas.	Neuroendocrine neoplasm cells	NA	Liver-infiltrating neutrophils	Promote metastasis	NETs levels are highly correlated with metastasis.	DNase, inhibit 5-HT signaling	[235]

Application of anti-NETs strategies in various disease models

In the past few years, NETs are increasingly recognized to play broad roles, ranging from antimicrobial defense to the regulation of autoimmunity, inflammation, tumorigenesis, and tumor progression. The underlying mechanisms of NETs in the pathogenesis of disease are being revealed, and targeting NETs may be a promising therapeutic approach [2–4]. In this chapter, the major targets and mechanisms of anti-NETs intervention will be systematically summarized, followed by a discussion of their research advancements in various disease models and the potential for clinical translation, along with prospects for further development.

Strategies and mechanisms of anti-NETs interventions

Current research indicates that anti-NETs interventions can be categorized into three classes: inhibition of NETs formation, enhancement of the clearance of formed NETs, and blockade of NETs downstream signaling.

Inhibiting the formation of NETs

NETs are meshworks of DNA, MPO, and NE. NETosis presents multiple important steps, including histone citrullination, chromatin decondensation, nuclear envelope rupture and finally cell lysis during NETosis [2, 236]. Thus, suppressing the formation of these core factors or blocking the important pathways in NETosis would directly inhibit NETs production. PAD4 is a critical factor in histone citrullination. In multiple diseases including RA, ulcerative colitis, and multiple myeloma (MM), PAD4 inhibitors have been shown to produce antiinflammatory effects via inhibiting NET generation [215, 237–239]. NE and MPO are not only constituents of the NETs, but are also involved in the regulation of NETosis. Inhibition of NE (such as with its endogenous inhibitor α-1 antitrypsin or the synthetic inhibitor AZD9668) effectively suppressed NETs formation, and improved coagulation disorder in septic models or wound healing of the skin [240, 241]. In contrast to NE, more exploration is underway in studies have increasingly focused on how MPO induces NET formation and diseases associated with this process; however, how to alleviate disease progression by blocking NETs via intervening in MPO appears to be more feasible. In particular, conventional drugs including high-dose atorvastatin and rosuvastatin are able to concurrently reduce the expression of NETs-related proteins such as MPO, NE and H3Cit [242], also pointing to a promising path to study the therapeutic potential of available therapeutic agents. The ROS-mediated pathway is a key path in NETosis. Years of research have shown that natural products such as Polydatin [243], Kaempferol [238], Ethyl pyruvate [244], Zingerone [245], and Warifteine [246] could abrogate the NETosis process by suppressing the production of ROS, which alleviates pathological progression in diseases including SLE, sepsis and ALI. It is noteworthy to mention that, as opposed to the above-mentioned compounds, Warifteine acts on NETosis by modulating ROS signaling to prevent GSDMD-mediated neutrophil lysis [246], indicating the possibility for modulation of NETs by manipulation of cell membrane lysis. Mechanistically, the upstream signaling pathways of pattern recognition receptor TLR4 could inhibit ROS-induced NETosis and contribute to the regulation of acute otitis media [247].

Enhancing the clearance of NETs

Approaches to encourage NETs clearance are therefore centred on breaking down and clearing released NET structures. The disposal of NETs under normal physiological conditions is dependent on a two-stage cascade manner: nucleases (such as DNase I) digest the DNA backbone in NETs, and then these fragmented NETs are recognized and removed by phagocytes including macrophages as well as DCs [248]. Thus, rather than preventing NETs formation, clearance after they are formed is a more direct therapeutic strategy. Some endogenous molecules such as neonatal NET-inhibitory factor-related peptides and lactoferrin are able to inhibit the formation of NETs, yet either cannot degrade formed NETs or have low inhibitory efficiency [249, 250]. As a result, direct removal of circulating and/or tissue-deposited NETs seems to be a more attractive therapeutic approach. Studies demonstrate that the DNase I can degrade DNA contained in NETs, and disrupt to the NETs structure, while the DNase 1L3 is effective at degrading protein bound DNA; however its ability to degrade NETs needs validation through basic experiments and clinical trials [248, 251].

It should be noted that in light of the rapid development of nanotechnology, the synergistic achievement of NETs inhibition as well as clearance can now be achieved. Cerium oxide nanoparticles are able to be grafted with DNase I, which can lower intracellular ROS levels for inhibiting NETs generation and degrade produced NETs [252]. However, it is largely impeded by the inevitable defects of nanotechnology (e.g., high preparation cost, difficulty for clinical transformation and mandatory biosafety evaluation) [253].

Blocking downstream signals of NETs

NETs can not only influence (or induce) but also trigger immune responses and promote coagulation by pattern recognition receptors (eg, TLRs, RAGE) or intracellular signaling cascades (ie, cGAS-STING, the complement system and TF), modulators of both homeostasis systems [254–260]. Although these responses are protective against pathogen invasion, they may also be the fulcrum that mediates autoimmune tissue injury and lead to inflammatory or thrombotic diseases. For instance, hydroxychloroquine suppressed NETs-induced pathological responses via the inhibition of TLR9 signaling and hence ameliorated liver ischemia-reperfusion injury [261]; exogenous H₂S inhibited NETs generation through the HMGB1–TLR4-p38 MAPK-ROS axis [262]; and hydroxysafflor yellow A abrogated pathological damage in venous thromboembolism models by impairing TLR4-NF-κB-dependent NETs formation [263]. Thus, targeting downstream signaling pathways of NETs can effectively mitigate disease progression, and downstream targets related to NETs hold promise as potential therapeutic targets for various inflammatory and thrombosis-related diseases.

In conclusion, there are the following three levels of current anti-NETs intervention methods: source inhibition which intervenes in NETosis processes, terminal clearance responsible for degradation of formed NETs and signal interception which involves hindering the pathologically induced signal transduction caused by NETs. In terms of the mechanisms of action, approaches to inhibit NETs generation and facilitate their elimination are more direct with potential synergy for combination (such as a combined intervention based on nanotechnology), while strategies of blocking its downstream pathways may be used in a more flexible way especially under pathological conditions where it is hard to entirely block NETs production. However, there are substantial differences in the clinical translational maturity of distinct anti-NETs strategies. PAD4 inhibitors, ROS-targeted interventions, blockade of NETs downstream signaling pathways, and nanodelivery systems have only demonstrated efficacy in cellular assays and animal models to date, and remain at the preclinical stage. While these strategies can furnish critical mechanistic evidence, their target specificity, pharmacokinetic profiles, long-term safety, and potential impacts on the host’s anti-infective defense function still warrant further systematic validation. In contrast, DNase I-based NETs clearance strategies and NE inhibition strategies possess relatively higher translational feasibility, with relevant drugs and intervention regimens already undergoing clinical studies in inflammatory diseases, respiratory diseases, and critical care settings. Nevertheless, the optimal administration timing, eligible patient populations, dosage windows, safety margins, and adverse events associated with infection and coagulation risks of these strategies remain to be defined via large-scale clinical trials. Therefore, upcoming tasks specifically involve identifying individual biomarkers for NETs-related diseases to guide patient stratification, as well as refining pharmacokinetics and targeted delivery efficiency of currently available inhibitors (PAD4i and DNase), and rationally designing future related clinical trials based on the core regulatory mechanism of NETs in each disease.

Advances in anti-NETs strategies in inflammatory disease models

NETs are a critical player in the pathogenesis of different inflammatory diseases, and targeting NETs is likely to be a promising therapeutic strategy. Based on the above intervention mechanism, this section discusses the progress of research and the therapeutic potential of anti-NETs strategies in several common ADs models. Figure 8 depicts the use of intervention strategies against NETs applied in different disease models.

Fig. 8.

A panoramic view of the application of NETs-targeted intervention strategies in disease models. This diagram presents a summary flowchart of intervention strategies targeting NETs. The three major strategies are: formation inhibition, clearance enhancement, and signaling blockade. Under each strategy, representative intervention targets and drugs are listed, along with specific disease models where efficacy has been demonstrated. NETs: neutrophil extracellular traps; DNaseI: deoxyribonuclease I; LPS: Lipopolysaccharide; PAD4i: peptidylarginine deiminase 4 inhibitor; MiR-155: MicroRNA-155; NEi: neutrophil elastase inhibitor; GSDMDi: gasdermin D inhibitor; CXCR: C-X-C motif chemokine receptor; PAN02: pancreatic cancer 02; IR: ischemia-reperfusion; CLP: cecal ligation and puncture; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus

Nets and autoimmune diseases

In the context of ADs, the therapeutic benefit of NETs targeting has been demonstrated in a number of models that are representative for various disorders such as SLE, RA or AAV. NETs are crucial in the pathogenesis of SLE as they generate autoantigen and stimulate pDCs. Thus, the degradation of NETs has become a potential therapeutic approach. Exogenous DNase I attenuated the pathology of induced glomerulonephritis in lupus-proned mice by cleaving the DNA backbone structure on NETs, thereby inhibiting the NETs-TLR4-MyD88 signaling axis and decreasing immune cell infiltration into kidney [14]. These findings validate the therapeutic potential for NETs targeting in SLE, of which there is limited translation to date, demanding clinical trials based on eradication or blockade of NETs. NETs play a significant role in synovial inflammation and joint destruction in RA, and multi-pathway interventions aimed at NET formation and clearance have been broadly investigated. Inhibition of the fundamental pathways of NETosis would seem to be a major approach for intervention: PAD4 inhibitors have been shown to inhibit efficiently the generation of NETs and arthritis in RA models [239]. Moreover, the NLRP3 inflammasome/GSDMD pyroptosis pathway has been identified as a major upstream factor in the induction of NETosis in RA and its blockade significantly attenuates the pathogenic joint damage [264]. Interventions at other signaling nodes have also proven effective; for instance, Sinomenine and Rhaponticin reduce NETs generation by inhibiting the PDGF-PDGFR and NLRP3-GSDMD pathways, respectively [265, 266]. Furthermore, novel intervention strategies are continuously emerging, including the use of mesenchymal stem cells to regulate the PGE2-PKA-ERK axis [267], and even systemic inhibition of neutrophil activity and NETs formation by modulating the gut microbiota (e.g., Magnolia flora can convert ethanol to acetate, thereby activating GPCR43) [268]. On a technical level, to enhance the sustainability of therapeutic effects, novel nanogel delivery systems have been developed to achieve long-acting release of NETs inhibitors, demonstrating the potential for sustained disease remission in RA models [269, 270]. In AAV, NETs are a critical bridge in ANCA-mediated neutrophil stimulation and vascular damage. Experiments in MPA models have demonstrated that the NETs formation process can indeed be suppressed (for example, by inhibiting the PI3K-γ signaling pathway or by application of anti-myosin light chain 6 antibodies to prevent cytoskeletal remodeling), resulting in a reduction of NETs release and amelioration of vasculitis symptoms [271–273]. Endovascular immunoglobulin has also been shown to inhibit ANCA-induced NETs formation, but the exact mechanism remains unknown [274]. Interestingly, in relation to the oxidative stress-mediated pathway triggering NETosis, nuclear factor erythroid 2-related factor 2(Nrf2) is a pleiotropic protective transcriptional factor. It was demonstrated that Nrf2 activation can suppress ANCA-induced NETosis not only directly by opposing ROS, but also indirectly through promoting endothelial resistance to NETs’ mediated injury and systemically by controlling T-cell responses in the spleen (e.g., suppression of Th17 cells), thus preventing MPO-AAV in models [275]. This study suggests that targeting key transcriptional nodes regulating neutrophils and microenvironmental stress responses may provide a novel and more fundamental strategy for the treatment of ADs such as AAV.

Nets and infectious diseases

In the infectious disease area, it has been demonstrated that inhibition of NETosis helps to prevent the further evolution of sepsis. CXCR1/2 signalling blockade may provide reduced NETs formation and thereby sepsis-related thrombosis, pulmonary injury and mortality [276]. Suppression of NETs formation alleviates sepsis by preventing Nrf2-mediated ROS generation [245]. Actin plays important roles in NETosis, and intervention of Arp 2/3-dependent F-actin polymerization reduces NET formation and sepsis-induced lung injury [277]. Therefore, NETosis-related biomarkers may be used as sepsis diagnostic indicators and prognostic markers [278]. This gives a new perspective for the clinical translation of sepsis therapies by targeting NETs-related molecules (Table 4).

Table 4.

Characteristics of NETs-driven infectious immune dysregulation and intervention strategies

Pathogen Type	NETs Induction Intensity	Immune Escape Mechanism	Key Inflammatory Cytokine Storm	Immune Exhaustion Biomarker	NETs-Targeted Therapy	Clinical Efficacy	Drug Resistance Risk	Ref.
Staphylococcus aureus	Strong	Nuclease, adenylate synthase A, DNA binding inhibits antimicrobial peptide activity.	IL-1β, TNF-α, IL-6, IFN-γ	PD-1/PD-L1 upregulation (advanced sepsis)	BB-Cl-amidine, DNase I, ADAMTS13	Reduce bacteremia mortality and alleviate organ damage.	Bacterial nuclease secretion degrades NETs, leading to bacterial release, dissemination, and risk of secondary infection.	[279–283]
Influenza Virus (H1N1)	Weak	Viral NS1 protein inhibits NETs production; excessive NETs induce lung injury.	CXCL1, CCL2, CXCL5, IL-6	T cell functional exhaustion (TIM-3, LAG-3)	DNase I, Ly6G monoclonal antibody, DETC	Alleviate acute lung injury (ALI/ARDS) and improve oxygenation index.	Complete NETs inhibition may reduce viral clearance or induce secondary bacterial infection.	[284–288]
Candida albicans	Strong	Metabolites, cell wall remodeling, blocks ROS production	IL-1β, TNF-α, IL-6	CTLA-4, PD-1	DNase I, NAC	Reduce fungal burden and prevent biofilm-induced damage to target organs (e.g., kidneys).	Long-term use impairs antifungal defense.	[289–291]
Mycobacterium tuberculosis	Strong	Induced NETs protect mycobacterium tuberculosis; NETs damage macrophages	TNF-α, IFN-γ, IL-1α, IL-1β, IL-6, CXCL1/CXCL2	PD-1, TIM-3	SB-3CT, doxycycline, Cl-amidine, type I interferon blockers.	Reduce pulmonary cavity formation and histopathological damage, and control inflammation.	May destabilize granuloma structure and reactivate latent tuberculosis.	[292–295]
Drug-Resistant Acinetobacter baumannii	Extremely Low	Inhibits CD11a and NETs formation	TNF-α, IL-6, IL-10	HLA-DR low expression (monocytes)	NETs formation-enhancing immunomodulators (e.g., LL-37).	Restore bacterial clearance capacity and enhance infection survival rate.	Drug resistance	[5, 296, 297]
Klebsiella pneumoniae (Hypervirulent)	Weak	Resists NETs trapping and killing	IL-6, IL-8	-	Restoring the function of NETs antimicrobial proteins.	Restrict bacterial dissemination in the liver/lungs.	Hypervirulent strains are often associated with multidrug resistance; single NETs-targeted therapy has limited efficacy.	[298–300]
EB Virus (Active Phase)	Moderate	NETs interfere with T cells	CXCL10	CD8+ T cell decrease	DNase I, AMG487, GSK484	-	Long-term PAD4 inhibition may impair the formation of antiviral immune memory.	[301, 302]

Nets and respiratory diseases

In the field of respiratory diseases, multiple studies have confirmed that regulating NETs formation contributes to the treatment of COPD [303]. Mouse experiments have verified that inhibiting NE reduces the levels of neutrophil chemokines and pro-inflammatory cytokines, suppresses cigarette smoke-induced NETs formation, and improves lung function in COPD mice [15]. Additionally, erythromycin decreases airway NETs levels in mice with chronic tobacco exposure and alleviates emphysema symptoms by downregulating Th1/Th17 cell activity and inhibiting the function of CD40⁺ and CD86⁺ monocyte-derived DCs [304]. This suggests that the interaction mechanisms between specific types of antibiotics and NETs require further investigation. Recent mouse experiments confirmed that levels of 5-hydroxyindoleacetic acid (5-HIAA) are elevated in COPD models; inhibiting 5-HIAA downregulates aryl hydrocarbon receptor expression, reduces ROS generation and NETs formation, and improves lung function-related indices [305]. The latest research has revealed the regulatory relationship between traditional Chinese medicine preparations, NETs, and COPD. Ultra-high performance liquid chromatography-charge selection mass spectrometry analysis showed that Qingke Pingchuan granules can inhibit pulmonary NETs formation via the NOX2/p47phox-ROS pathway, thereby alleviating airway inflammation during the acute exacerbation stage of COPD [306].

Nets and metabolic diseases

A similar pathological process has been found in metabolic diseases (MDs), and the excessive production of NETs can promote the development of multiple diabetic complications. As to diabetic angiopathy and delayed wound healing, blocking NETs production can improve endothelial function and enhance tissue repair [307]. Particularly, the traditional anti-diabetic drug metformin has been shown to suppress NETs formation in a glucose-independent pathway by suppression of PKC-βII/NOX axis [308], suggesting another therapeutic potential for “drug-repurposing.” The antibiotic clarithromycin can promote wound healing and increase the host defense in T2DM patients by upregulating NETs‑related antimicrobial peptide LL‑37 [309]. New intervention targets have been investigated for diabetic neuropathy; such as the 5-hydroxytryptamine receptor 2A antagonist desloratadine, which suppresses NETs generation by blocking PKC/NADPH/ROS signaling pathway, thereby alleviating neuropathological damage [310], indicating that this target may be well worth studying in other microvascular complications. Taken together, the inhibition of NETosis may offer multipronged strategies for treatment of diabetic complications, although current evidence is mostly obtained from preclinical studies and systematic evaluation in terms of efficacy and safety still needs to be established.

Nets and cardiovascular diseases

In the cardiovascular field, such as AS, for example, it has been recently reported that anti-NETs therapy has been preliminarily proved to be beneficial in interference with the pathological process of AS. For example, paeonol obviously suppresses NETs formation and reduces the concentration of intra-plaque inflammatory factors, as well as inhibits foam cell inflammation in response to NETs by modulating NE through the CitH3–NLRP3-caspase-1 signal axis [311]. Furthermore, the TCM formula MTHSWD has anti-AS effect through the regulation of NETs to inhibit EC injury and apoptosis as well as reduce blood lipid levels [312]. With respect to mechanistic and translational research, the pathogenic significance of PAD4-mediated NETosis has been validated in superficial plaque erosion, and collagen IV-targeted nanocarriers have been engineered for site-specific delivery of a PAD4 inhibitor (GSK484) to endothelial denuded areas. This significantly reduced local NETs accumulation and maintained endothelial integrity [313]. This research confirms the feasibility of precise NETs intervention at the mechanistic level.

Nets and tumors

In the field of oncology, recent studies have explored NETs-targeted strategies mainly from the perspectives of inhibiting NETosis, degrading established NETs, regulating NETs-associated signaling pathways, and combining NETs modulation with conventional anticancer therapies. First, directly blocking NETosis: using PAD4 inhibitors (such as BMS-P5, GSK484) can delay MM progression and increase the radiosensitivity of CRC [215, 216]. Second, degrading formed NETs: using enzymes such as DNase I to degrade established NETs helps reduce tumor-associated thrombosis, though attention must be paid to its long-term safety [214]. Third, regulating related signaling pathways: small molecule compounds or natural products (such as EGCG, DHT, glycyrrhizic acid, etc.) can inhibit NETs formation by downregulating pathways including STAT3/C-X-C motif chemokine ligand (CXCL) 8 and PAD4, thereby inhibiting colon cancer and BC metastasis and enhancing the efficacy of chemoradiotherapy and immunotherapy [217–219]. Furthermore, delivering anti-NETs drugs via nanoparticles or biological carriers can both induce the reprogramming of NETosis toward apoptosis and achieve targeted drug delivery to the tumor core, significantly improving treatment efficiency [220, 221]. However, NET modulation in cancer is not necessarily equivalent to uniform NET inhibition, because its therapeutic consequences may be context-dependent. Research has found that IFN-γ significantly enhances NETs formation and apoptosis in microsatellite stable CRC cell lines, contributing to reduced tumor volume in mouse models and enhancing the antitumor activity induced by programmed cell death protein-1 (PD-1) antibodies [222]. Similarly, other studies have confirmed that NETs can enhance the chemosensitivity of CRC [223]. These findings indicate that NET-targeted therapy should be interpreted according to tumor type, immune context, NET burden and treatment modality. Therefore, how to precisely regulate NETs to maximize antitumor efficacy while avoiding excessive impairment of host defense remains one of the core directions for future research.

Safety and translational potential of anti-NETs therapies in inflammatory diseases

NETs have proinflammatory roles in diverse inflammatory diseases (autoimmune disorders, respiratory diseases, MDs, cardiovascular diseases and tumor) and can drive tumor initiation, progression as well as metastasis. As a result, the interventions targeting NETs or NETosis (inhibition of NETosis, degradation of NETs and blockade of downstream signaling pathway) have been applied to modulate disease progression in inflammatory diseases, showing protective effects in many animal models. Yet, there continue to exist a number of unaddressed issues in the field. According to available findings, this section reviews the safety and clinical translational value of anti-NETs strategies. Figure 9 The bidirectional role of NETs and clinico-translational objectives for intervention strategies associated with the biology of NETs.

Fig. 9.

The dual effects of NETs and the clinical translation decision-making map. This graph shows three elements. Part a depicts the bidirectional NET effects (including both physiological and pathological pathways) as well as the several functions played by them. Section B summarises the decision process of the NETs targeting, including patient disease context assessment for infectious activity and targeted NETs strategies. Section C outlines the barriers and priorities in translating NETs therapies into clinical practice, advocating for short-, medium-, and long-term goals. The goals and challenges in each time frame are specified, alongside the associated risks and challenges. NETs: neutrophil extracellular traps; DNaseI: deoxyribonuclease I; DNA: deoxyribonucleic acid; MPO: Myeloperoxidase; PAD4i: peptidylarginine deiminase 4 inhibitor; CitH3: citrullinated histone H3

As for safety, the main difficulties arise from the physiological role of NETs and the limitations of current interventions, which could be summarized as follows: 1. Innate Measurable Risks: 1) Immunosuppression Risk: NETs are part of the host defense. Systemic or long-term inhibition of their formation (e.g., PAD4 inhibition) could potentially impair the host’s capacity to eliminate pathogens, such as bacteria and viruses. This conclusion has now been substantiated in PAD4-deficient animals [48, 314]. 2) Risk of Coagulation Disequilibrium: DNase intervention to remove NETs may cause unexpected adverse effects. It has been shown that in trauma patients, soluble histones and released granular components liberated after degradation of NETs by DNase can have counterintuitive pro-thrombotic effects in serum [315, 316]. This indicates a potential need for monitoring the risk of “secondary injury” after clearance of NETs. 2. Limited Drug Specificity: Considering PAD4 inhibitors as the most clinically advanced strategy, their core issues include low selectivity and associated cytotoxicity [317]. Even though nanocarriers or biologics-based targeted delivery are promising strategies to improve selectivity, the overly complex manufacturing procedures, high R&D costs and long-term biosafety concerns of these advanced formulations currently greatly hamper them in the clinical translational process [253, 313].

For clinical translation, the development of an anti-NETs therapy seems to have many bottlenecks: 1. Preliminary Clinical Evidence: In fact, clinical trials focusing on NETs are now beginning to emerge and they are very much in the exploration phase. The most typical study concerns the strategy of nebulized inhalation of recombinant human rhDNase I (Dornase α) in severe cases. One small observational cohort study indicated a potential benefit in pulmonary inflammation [318]; however the applicability and true efficacy need to be confirmed by large RCTs. This existing situation reflects the significant discrepancy between animal models of NETs and clinical use for approved therapies. 2. Significant Translational Bottleneck -Biomarkers For the precision medicine to be achieved, reliable biomarkers are needed and need to become available. Circulating NETs components (including cfDNA and MPO-DNA complexes) are regarded as very promising biomarkers because of their rapid responsiveness to clinical status and close correlation with the disease activity [319]. However, clinical use is confronted with two central problems: On the one hand, disease specificity is only modest, hindering the identification of diseases based on NETs release caused by different etiologies. Second, there is no unified standard of detection techniques. From more complex morphological detecting techniques (including electron microscopy and immunofluorescence) to simple but low-information enzyme-linked immunosorbent assay, a “gold standard” for the standardized quantitative characterization of intact NETs has not emerged as of now [319]. The delay of biomarker studies is seriously hindering the accuracy of patient stratification and efficacy evaluation.

In summary, despite the broad therapeutic prospects of targeting NETs theoretically, its clinical translation remains mired in multiple dilemmas, including safety concerns, a paucity of effective clinical evidence, and the absence of companion diagnostic tools. Table 5 summarizes existing strategies targeting NETs and their potential for clinical translation.

Table 5.

Nets-targeted clinical intervention strategies and their translational potential

Intervention Category	Representative Drugs/Measures	Target of Action	Preclinical Model	Clinical Trial Phase	Indication	Drug Resistance Risk	Combination Therapy Potential	Ref.
DNase Enzymes	rhDNase, Pulmozyme	NETs DNA	ARDS, sepsis, ischemia-reperfusion, thrombosis.	Preclinical studies, phase 2 (pulmozyme).	ARDS, sepsis, thrombosis, SARS-CoV-2.	Low	Antibiotics, anti-inflammatory drugs, and antithrombotic drugs.	[314, 320, 321]
PAD4 Inhibitors	GSK484，BB-Cl-amidine	PAD4	Heparin-Induced Thrombocytopenia (HIT), Sepsis, SLE.	Preclinical Studies	HIT, Sepsis-Associated Encephalopathy, SLE.	Moderate	Non-heparin anticoagulants and DNase I.	[322–324]
Neutrophil-Depleting Agents	Anti-Ly6G	Ly6G (mouse neutrophils)	MASH, sepsis, viral myocarditis	Preclinical studies	Alcohol-associated fatty hepatitis, sepsis, viral myocarditis.	NA	NA	[325–327]
ROS Scavengers	Metformin DPI	PKC、NOX	SLE, obesity, HIT	Conceptual trial (metformin), preclinical studies	Diabetes, SLE, Thrombosis	NA	Glucocorticoids, immunosuppressants, and DNase I.	[308, 328–330]
TLR Pathway Antagonists	Anti-CD282，TAK-242，Cu-CPT9a	TLR2/4，TLR8	Psoriasis, SLE	Preclinical studies.	AAV, psoriasis, SLE	NA	DNase I and CI-amidine.	[331–333]
Epigenetic Modulators	Padi4−/−, Gsdme−/−, Cl-amidine.	PAD4, GSDME	SLE, acute myocardial infarction (AMI).	Preclinical studies	SLE, AMI	NA	NA	[71, 334]
NETs-Clearing Antibodies	CIT-013	Citrullinated histones H2A, H4	Neutrophilic airway inflammation model, RA, hidradenitis suppurativa (HS).	Phase 1	RA, HS	NA	NA	[335]

Discussion

NETs are major effector molecules for neutrophils and have dual roles in the process of inflammation-related diseases. During acute infection, NETs serve as scaffolds that localize pathogens at inflammatory sites in focal lesions and the release of effector factors like MPO, NE; hence the pathogens are cleared. This highly effective and fast NET formation enhances the host’s ability to limit microbial spread and accelerates lesion resolution. However, if NETs generation or clearance is redundant-especially in combination with chronic inflammatory disorders like protracted ADs, chronic infections and tumors-NETs will maintain the development phase of chronic inflammation by enhancing the autoimmune response and vascular thrombosis even to some excessive degree, thus turning into a major determinant that promotes the progression of chronic inflammatory diseases. Adding to this complexity, NETs composition and modifications are heterogeneous across different stimulation conditions (intracellular vs. extracellular). Additionally, intricate crosstalk is demonstrated between NETosis and other types of RCD such as apoptosis, necrosis, pyroptosis, autophagy, ferroptosis and cuproptosis. On the one hand, this crosstalk amplifies persistent host inflammatory responses to aggravate pathological progression; on the other hand, it confers anti-inflammatory activity and contributes to improved infection outcomes and alleviation of clinical manifestations. This complexity does not support the idea of NETs as a single pathogenic entity in disease but rather demonstrates that they form part of a complex and dynamic “NET-omics” regulatory network.

In preclinical research, there has been rapid progress in developing anti-NETs strategies in disease models over recent years. These approaches mainly focus on three major levels including suppression at the source (e.g., PAD4 inhibitors, NE inhibitors, ROS inhibitors), enhancement of clearance of formed NETs (e.g., DNase I) and interception of signaling pathways (e.g., targeting TLR9 and cGAS-STING). Detailed preclinical research has supported the significant therapeutic efficacy of these interventions in different ADs, MDs, thrombosis and tumor metastasis models, indicating that targeting NETs may confer broad-spectrum benefits. Clinical trials are, nevertheless, few in number and the majority of the studies have not moved beyond basic and preclinical exploration. Furthermore, translating evidence from experimental studies to clinical use is fraught with multiple difficulties. First, the lack of agreed upon standards for NET biomarker measurements and detection—such as circulating DNA-protein complexes or specific proteomic profiles that are known to be unique to NETs—is a major limitation in patient stratification and targeting treatment. Secondly, the precise timing and delivery modes of anti-NET therapies are still being explored. Short-term local delivery (such as inhaled DNase) has the potential of locally ameliorating tissue destruction without causing systemic immune depression, but its therapeutic window, dosage, and optimal regimen remain to be defined by clinical trials. Likewise, while nanoparticle-based delivery systems provide optimal targeting of anti-NETs agents, their safety and scalability for clinical use require further validation. Third, anti-NETs agents and their safety are yet to be established. The employment of those agents would inevitably inhibit immune system function, and thus the particular intensity of immunosuppression resulting from protracted pharmaceutical administration, together with associated risks (e.g., risk of developing coagulation disorders) need to be determined by long-term animal studies or dedicated clinical trials. Fourth, current animal models have their inherent shortfalls. In comparison to humans, animal models display inherent differences in neutrophil biology and NET-related functions that necessitate the development of humanized models and organoids more closely resembling human physiology to improve both validity and reliability of preclinical data. Meanwhile, considering that the interaction between NETs and inflammatory diseases is multi-target, multi-pathway and heterogeneous in an integrated network, it may be difficult for single-target intervention to effectively treat various sorts of inflammation. Therefore, the combinatorial use of anti-NETs strategies and other modulation of different cell death (e.g., induction of apoptosis or regulation of necrosis), anti-infection therapy and immunomodulation is highly promising for clinical translation.

Overall, the clinical translation of anti-NET therapies against inflammatory diseases is still facing many issues to be resolved. Future research directions: 1) Clarifying the specific molecular pathways by which NETs contribute to exacerbation of various inflammatory diseases at the basic research level, precisely connecting known regulatory machinery of NETs and pathophysiology of the disease, and in-depth characterization of heterogeneity present in different forms of NETs. 2) Establishing consensus standards and QC procedures for the detection assays of NETs biomarkers, and cross-validating data in a multicenter comparative studies to provide standardized support for clinical application of NETs detection technologies. 3) Systematically analyzing the safety of anti-NETs drugs, with increased intensity in preclinical safety assessment and the initiation of efficient clinical trials with an appropriate sample size as soon as possible. 4) The creation of new formulations and combination therapies targeting NETs (e.g., nanocarriers of anti-PAD4 compounds or biologics) while improving product manufacturing to make them safer and enable scaling-up. Looking ahead, as these major challenges are increasingly addressed, NET-targeting therapeutic approaches will hopefully transcend existing clinical barriers and bring new therapies to patients suffering from inflammatory diseases.

Abbrevaiation

5-HIAA

5-hydroxyindoleacetic acid

AAV

ANCA-associated vasculitis

ACPA

Anti-citrullinated protein antibody

Ads

Autoimmune diseases

Akt

Protein kinase B

ANCA

Antineutrophil cytoplasmic antibody

ARDS

Acute respiratory distress syndrome

AS

Atherosclerosis

BC

Breast cancer

BER

Base excision repair

C5a

Complement component 5a

Caspase

Cysteine-aspartic protease

cGAS

Cyclic GMP-AMP synthase

COPD

Chronic obstructive pulmonary disease

CR3

Complement receptor 3

CRC

Colorectal cancer

CTCs

Circulating tumor cells

CTSC

Cathepsin C

CXCL

C-X-C motif chemokine ligand

CXCR

C-X-C motif chemokine receptor

DAMPs

Damage-associated molecular patterns

DCs

Dendritic cells

DNase

Deoxyribonuclease

dsDNA

Double-stranded DNA

ECM

Extracellular matrix

EMT

Epithelial-mesenchymal transition

ERK

Extracellular signal-regulated kinase

Fas

Fas cell surface death receptor

FcR

Fc receptor

FcγR

Fcγ receptor

FLS

Fibroblast-like synoviocytes

Fn

Fibronectin

FPR2

Formyl peptide receptor 2

FUNDC1

FUN14 domain containing 1

G6PD

Glucose-6-phosphate dehydrogenase

G-CSF

Granulocyte colony-stimulating factor

GM-CSF

Granulocyte-macrophage colony-stimulating factor

GPCR

G-protein-coupled receptor

GSDMD

Gasdermin D

GSDME

Gasdermin E

H₂O₂

Hydrogen peroxide

H3Cit

Citrullinated histone H3

HCC

Hepatocellular carcinoma

HDACs

Histone deacetylases

HIF-1α

Hypoxia-inducible factor-1α

HMGB1

High mobility group box 1

ICAM-1

Intercellular adhesion molecule-1

ICs

Immune complexes

IFN-I

Type I interferon

IFN-III

Type III interferon

IgG

Immunoglobulin G

iICs

Immobilized immune complexes

IL

Interleukin

IRE1α

Inositol-requiring enzyme 1α

LAP

LC3-associated phagocytosis

LDGs

Low-density granulocytes

LL-37

Leucine-leucine-37

LLC

Lewis lung carcinoma

LPS

Lipopolysaccharide

MAPK

Mitogen-activated protein kinase

MDs

Metabolic diseases

METTL3

Methyltransferase-like 3

MHC

Major histocompatibility complex

MLKL

Mixed lineage kinase domain-like protein

MM

Multiple myeloma

MMP

Matrix metalloproteinase

MPO

Myeloperoxidase

mPTP

Mitochondrial permeability transition pore

mTOR

Mechanistic target of rapamycin

NADPH

Nicotinamide adenine dinucleotide phosphate

NASH

Non-alcoholic steatohepatitis

NE

Neutrophil elastase

NETosis

Neutrophil extracellular trap formation

NETs

Neutrophil extracellular traps

NF-κB

Nuclear factor kappa B

NOX

Nicotinamide adenine dinucleotide phosphate oxidase

Nrf2

Nuclear factor erythroid 2-related factor 2

OMVs

Outer membrane vesicles

OSCC

Oral squamous cell carcinoma

p38 MAPK

p38 mitogen-activated protein kinase

PAD

Peptidylarginine deiminase

PD-1

Programmed cell death protein-1

pDCs

Plasmacytoid dendritic cells

PD-L1

Programmed death-ligand 1

PI3K

Phosphoinositide 3-kinase

PIP3

Phosphatidylinositol-3,4,5-trisphosphate

PKC

Protein kinase C

PLC

Phospholipase C

PLCγ

Phosphoinositide-specific phospholipase Cγ

PM

Plasma membrane

PMA

Phorbol 12-myristate 13-acetate

PPP

Pentose phosphate pathway

PR3

Proteinase 3

PSGL-1

P-selectin glycoprotein ligand-1

RA

Rheumatoid arthritis

RAGE

Receptor for advanced glycation end products

RCD

Regulated cell death

Rho GTPases

Rho family GTPases

RIPK

Receptor-interacting protein kinase

RNP

Ribonucleoprotein

ROCK

Rho-associated coiled-coil-containing protein kinase

ROS

Reactive oxygen species

STAT3

Signal transducer and activator of transcription 3

STING

Stimulator of interferon genes

Syk

Spleen tyrosine kinase

T1D

Type 1 diabetes

T2D

Type 2 diabetes

TF

Tissue factor

TLR

Toll-like receptors

TME

Tumor microenvironment

TNF

Tumor necrosis factor

UV

Ultraviolet

VSMCs

Vascular smooth muscle cells

Author contributions

L.Z. Song, T. Zhou, Y. Shu, and J.B. Zhao contributed to the manuscript writing and figure preparation; Z. Zou, W.Z.Q. Zhang, and H.L. Zhang designed the work; Y. Liao and C.L. Zhu supervised the work.

Funding

This study was funded by Changhai Hospital Anesthesia Specialty Platform Construction Project and The National Natural Science Foundation of China (82572494).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable. This manuscript does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable. This manuscript does not include details, images, or videos relating to an individual person.

Competing interests

The authors declare no competing interests.