Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology

NETs have become central to understanding innate immunity and multiple diseases. The review aims to synthesize landmark discoveries and rapidly evolving mechanistic details of Nox-dependent and Nox-independent NET formation, and emerging forms of vital NET release. It addresses contentious issues such as the role of PAD4 in Nox-dependent NET formation and the contribution of transcriptional firing, and emphasizes context-dependency (bloodstream vs tissue; alkaline or hypertonic environments). The purpose is to provide a succinct, comprehensive update, relate NETs to major diseases, and inform discussion of NET-targeted therapeutics, culminating in an updated unified model of NET formation.

The review traces the discovery and evolution of NET concepts: from PMA-induced chromatin decondensation and release (1996; 2004) to recognition that most NETs are released during suicidal death over 2–4 h and can also occur vitally within minutes. It summarizes evidence for both Nox-dependent mechanisms (e.g., PMA, LPS) and Nox-independent mechanisms (e.g., calcium ionophores A23187/ionomycin), including roles for ROS, kinases (Akt, ERK, p38, JNK, Src/PyK2), MPO/NE nuclear translocation, and PAD4-mediated histone citrullination in Nox-independent NET formation. It details vital NET formation variants: platelet TLR4–dependent nuclear DNA release in bloodstream infections; mitochondrial DNA release requiring GM-CSF with LPS or C5a; and toxin-induced rapid nuclear blebbing. The paper reviews autophagy’s involvement, ApoNETosis with UV, and nomenclature issues, recommending use of “NET formation” rather than “NETosis” without evidence of death. It compiles evidence that NETs activate complement (alternative pathway components on NETs and MAC deposition) and surveys histone modifications (citrullination, acetylation promoting NETs; potential interplay with methylation). Physiological modulators are summarized: context-specific LPS serotype responses in blood vs tissue; alkaline pH enhancing both Nox-dependent and -independent NETs; extracellular/intracellular acidification suppressing NETs; and hypertonic saline/osmolytes suppressing Nox-dependent NETs via ROS reduction. Clearance mechanisms by DNase I and macrophage efferocytosis are reviewed, including impaired clearance in SLE and ARDS. Disease associations include CGD, RA, diabetes/diabetic foot ulcers, atherosclerosis/coronary disease, cancer growth/metastasis, and cystic fibrosis. Therapeutic avenues surveyed include gene therapy in CGD, Nox inhibitors (DPI; limitations), transcription inhibitors, gasdermin D inhibitors (e.g., LDC7559), and DNase to degrade extracellular DNA, with cautions regarding unintended consequences.

This is a narrative review synthesizing findings from previously published studies across mechanistic, physiological, and pathological aspects of NET biology. No primary experimental methodology or systematic review protocol is described. The authors integrate data from cellular, animal, and clinical studies and compile an updated unified model (Figure 1).

- NET formation encompasses suicidal (typically 2–4 h) and vital (5–60 min) processes, with both Nox-dependent and Nox-independent pathways. - Nox-dependent NET formation: PMA and LPS trigger ROS via Nox2; PMA acts via PKC and Nox assembly; LPS engages TLR4 and JNK signaling. NE and MPO translocate to the nucleus aiding chromatin decondensation. PAD4’s role here remains controversial. - Nox-independent NET formation: Calcium influx activates PAD4 leading to histone hypercitrullination and chromatin decondensation; SK3 channels and mitochondrial ROS are necessary; mitochondrial function serves as a ROS source in neutrophils. - Transcriptional firing at promoter regions promotes chromatin decondensation and is required for both Nox-dependent and -independent NET formation in serum-free conditions; inhibitors of transcription (but not translation) suppress NETs without impairing antimicrobial ROS. Conflicting data under serum conditions highlight context dependence. - Vital NET formation variants include: platelet TLR4–dependent nuclear DNA release in bloodstream (Nox-independent), mitochondrial DNA release requiring GM-CSF with LPS or C5a (Nox-dependent), and toxin-induced rapid NETs. - Histone modifications modulate NETs: PAD4-mediated citrullination is critical for Nox-independent NETs; histone acetylation promotes NET formation; HDAC inhibitors show biphasic effects (e.g., belinostat promotes NETs at ≤0.25 µM, inhibits at >1 µM). - Physiological context: Intravascular LPS induces rapid, vital, platelet-dependent NETs; tissue LPS induces suicidal, Nox-dependent NETs over 2–4 h. Specific LPS serotypes trigger NETs differentially in blood vs tissue. Alkaline pH enhances both pathways (increased intracellular pH, Ca2+, mROS, PAD4 activity; increased ROS and histone cleavage); acidification suppresses NETs. Hypertonic saline/osmolytes suppress Nox-dependent NETs via ROS reduction. - Complement: NETs bind and activate alternative pathway components leading to MAC (C5b-9) deposition; DNase abrogates MAC deposition on NETs. - Clearance: DNase I and macrophages collaborate; physiological DNase I alone is insufficient for full degradation; macrophage endocytosis is active and enhanced by DNase; clearance is impaired in SLE and ARDS. - Disease links: CGD lacks Nox-dependent NETs and is susceptible to infections; RA shows enhanced NET markers (MPO-DNA, PAD4 translocation); diabetes/DFU have elevated NET components and delayed healing improved by DNase; atherosclerosis involves cholesterol crystal–induced, PAD4-independent, Nox-dependent NETs that enhance IL-1β and TH17 responses; NET biomarkers associate with severe coronary disease; NETs foster tumor growth/metastasis (NE, MMP-9, tumor cell adhesion); in CF, NETs increase mucus viscosity and tissue damage. - Therapeutics: Gene therapy restores NETs in CGD; DPI inhibits Nox-dependent NETs but affects neutrophil function; transcription and gasdermin D inhibitors (e.g., LDC7559) can block NETs while sparing phagocytosis; DNase degrades extracellular DNA (beneficial in CF, SLE contexts) but may release harmful components or disrupt protective NET aggregates in some conditions.

The review integrates disparate findings to propose a unified, context-dependent model of NET formation. It clarifies that distinct stimuli engage different ROS sources, kinase cascades, transcriptional programs, and histone modifications to drive chromatin decondensation and NET release, and that environmental factors (pH, tonicity) and anatomical context (blood vs tissue) bias toward vital or suicidal pathways. These insights address the central aim of reconciling mechanisms with physiological and pathological outcomes, informing therapeutic strategies that inhibit pathological NETs while preserving essential neutrophil functions like phagocytosis and degranulation. The discussion underscores controversies (PAD4 in Nox-dependent NETs; necessity of transcription) as opportunities for targeted experimentation, highlights complement activation on NETs as a mechanistic bridge to inflammation and thrombosis, and emphasizes that clearance efficiency (DNase, macrophages) modulates disease severity. Therapeutic implications include targeting upstream regulators (transcription, GSDMD) to prevent NET formation selectively versus downstream degradation (DNase) that may have context-specific risks.

Key advances support a unified model encompassing Nox-dependent, Nox-independent, and vital NET formation, with newly appreciated roles for transcriptional firing and mitochondrial ROS. NET generation is strongly context dependent (site, pH, tonicity, stimulus). NETs contribute to major diseases (autoimmunity, diabetes, atherosclerosis, cancer, cystic fibrosis) and interact with complement and coagulation. Therapeutic avenues include selective inhibition of NET formation (transcriptional and gasdermin D inhibitors) that preserve neutrophil antimicrobial functions, and judicious use of DNase to degrade extracellular DNA where beneficial. Future research should resolve mechanistic controversies (PAD4 in Nox-dependent NETs; transcriptional requirements under physiological conditions), standardize experimental conditions (e.g., pH, serum), dissect environmental/contextual modulators, and evaluate NET-targeted therapies in disease-specific models while ensuring preservation of host defense.

As a narrative review, no systematic methodology is provided, and publication bias cannot be excluded. Several mechanistic areas remain contentious (e.g., PAD4’s role in Nox-dependent NETs; requirement for transcription under serum-containing conditions). Many insights rely on non-physiological agonists (PMA, ionophores), and findings are context sensitive (blood vs tissue, pH, tonicity), limiting generalizability. Some observations (e.g., biphasic effects of HDAC inhibitors; ApoNETosis; hypertonic effects on Nox-independent NETs) require independent corroboration. Differential responses to LPS serotypes and species/model differences may complicate translation. DNase-based strategies may incompletely remove harmful NET components or disrupt protective NET aggregates, necessitating careful clinical evaluation.