Neutrophil extracellular traps (NETs) have become a subject of intense investigation due to their significant roles in innate immunity and various pathological processes. The mechanisms of NET formation, including both NADPH oxidase (Nox)-dependent and independent pathways, are being rapidly elucidated, along with the discovery of various forms of vital NET formation. This review aims to provide a comprehensive overview of the latest advancements in NET research, addressing both established and contentious concepts, such as the role of protein arginine deiminase 4 (PAD4) in Nox-dependent NET formation. The review will explore the context-dependency of NET formation, highlighting differences between bloodstream and tissue environments and the influence of factors like alkaline pH and hypertonic conditions. Finally, it will discuss the implications of NETs in the pathogenesis of various diseases (atherosclerosis to cancer) and analyze the potential efficacy of emerging NET-based therapeutics, including gasdermin D inhibitors, transcription inhibitors, and DNase administration. The goal is to offer a concise yet thorough summary of current knowledge, identify key challenges, and stimulate further investigation toward a unified model of NET formation.

Early studies focused on identifying NET formation as a distinct process from apoptosis or necrosis, initially characterized by chromatin decondensation, nuclear membrane dissolution, and release of chromatin coated with granular proteins. Further research revealed that NETs, initially believed to be formed by viable neutrophils within minutes of stimulation, are primarily released by dying neutrophils 2–4 hours post-activation. While initially thought to be solely NADPH oxidase (Nox)-dependent, Nox-independent NET formation has been demonstrated, involving calcium influx and mitochondrial ROS production. The discovery of vital NET formation—where neutrophils survive and maintain function after releasing NETs—marked a significant shift in understanding. Three types have been described: mitochondrial DNA-based vital NETs released in response to C5a and GM-CSF or LPS; nuclear bleb-based vital NETs induced by Panton-Valentine leukocidin; and platelet-surface TLR4-dependent vital NETs responding to lipopolysaccharide. The role of histone modifications, particularly citrullination by PAD4 and acetylation, has been explored. While there is some controversy regarding the necessity of PAD4 in Nox-dependent NET formation, its importance in Nox-independent formation is well-established. The role of autophagy and transcription in NET formation also emerged as critical factors in the process. The nomenclature surrounding the process, specifically whether to utilize “NETosis,” remains a subject of ongoing debate within the field.

This review is a comprehensive literature review. The authors systematically synthesized existing research findings on NET formation from various published studies and research articles. The methods involved reviewing published literature using key search terms, including "neutrophil extracellular traps," "NETosis," "Nox-dependent NETs," "Nox-independent NETs," "vital NETs," "cystic fibrosis," "autoimmunity," "diabetes," and "cancer metastasis." The authors then extracted relevant information, analyzing diverse aspects of NET formation, including the mechanisms of NET formation (Nox-dependent and Nox-independent, vital NETs), the roles of transcription and histone modifications (citrullination, methylation, acetylation), the various physiological contexts of NET formation (bloodstream vs. tissue, alkaline pH, hypertonic conditions), the process of NET clearance, and the involvement of NETs in various pathologies (immunodeficiencies, autoimmunity, diabetes, cardiovascular disease, cancer, and cystic fibrosis). The collected information has been integrated to present a current, detailed, and critical appraisal of the existing knowledge on NET formation. The goal was to achieve a comprehensive and updated understanding of the different mechanistic aspects of NET formation, bridging various areas of research to build a more unified model. The authors included the latest published studies to assure their review is the most up-to-date available.

This review highlights several key findings regarding NET formation and its implications: 1. **Multiple Mechanisms of NET Formation:** The authors detail both Nox-dependent and Nox-independent pathways, and the newly identified vital NET formation, where neutrophils release NETs without undergoing cell death. Nox-dependent NET formation, typically stimulated by PMA or LPS, involves ROS production and the release of granular proteins (NE and MPO) that contribute to chromatin decondensation. Nox-independent NET formation, induced by calcium ionophores (A23187, ionomycin), relies on calcium influx, mitochondrial ROS production, and PAD4-mediated histone citrullination. 2. **Role of Transcription and Histone Modifications:** The study emphasizes the previously underappreciated roles of transcription and various histone modifications in NET formation. Transcriptional firing at promoter regions promotes DNA decondensation, necessary for both Nox-dependent and -independent NET formation. Histone modifications, including citrullination, methylation, and acetylation, have been shown to influence NET formation. Controversies surrounding the role of PAD4 in Nox-dependent NET formation are also addressed. 3. **Context-Dependent NET Formation:** NET formation differs significantly depending on the physiological context. Bloodstream NET formation is often rapid, vital, platelet-dependent, and Nox-independent, in contrast to tissue NET formation, which is usually slower, suicidal, Nox-dependent, and often elicited by specific LPS serotypes. Alkaline pH and hypertonic conditions have been shown to promote Nox-independent NET formation. 4. **NET Clearance and Pathologies:** The mechanisms of NET clearance involving DNase I and macrophages are discussed. Impaired NET clearance is implicated in pathologies like lupus nephritis and ARDS. NETs play a dual role in immunology, exacerbating immunodeficiencies (like CGD) and driving autoimmune conditions (SLE, RA). They are also implicated in diabetes and cardiovascular diseases (atherosclerosis), cancer (promoting tumor growth and metastasis), and cystic fibrosis (exacerbating mucus viscosity and tissue damage). 5. **Pharmacological Manipulation of NET Formation:** The review examines various approaches to manipulate NET formation therapeutically, including gene therapy to restore Nox function in CGD, Nox inhibitors (like DPI, with limitations), transcription inhibitors (as a potential alternative), and GSDMD inhibitors (selectively targeting NET formation without impairing phagocytosis). DNase administration, while having potential, presents certain challenges as it may not completely clear harmful NET components, such as histones and proteases.

The findings of this review significantly advance our understanding of NET formation, moving beyond a simplistic “suicidal” model to a more nuanced view that encompasses vital NET formation and the complexities of context-dependent regulation. The identification of the roles of transcription and mitochondrial ROS, as well as the various histone modifications, provides new therapeutic targets. The context-dependent differences in NET formation mechanisms suggest that therapeutic interventions should be tailored to the specific disease context. For instance, targeting Nox may be suitable for tissue-based pathologies, while approaches focused on transcription or GSDMD might be more suitable for bloodstream infections. The challenges related to indiscriminate DNase use highlight the need for more sophisticated strategies that precisely target pathological NET formation while preserving beneficial immune responses. Further research is needed to fully elucidate the interplay between NET formation, clearance mechanisms, and disease progression. The ultimate goal is to develop precise therapies that modulate NET formation in a way that balances the benefits of these structures in host defense against their damaging effects in various disease states.

This review has comprehensively summarized the current state of knowledge regarding NET formation, emphasizing the multifaceted nature of this process. The identification of novel mechanisms, the roles of various regulatory factors, and the context-dependent nature of NET formation have paved the way for developing targeted therapeutic strategies. Future research should focus on further characterizing the diverse types of NET formation, understanding the precise interplay between different signaling pathways, and developing more specific therapeutic agents that modulate NET formation without compromising essential neutrophil functions. This includes the further development and testing of transcription inhibitors, GSDMD inhibitors, and improved DNase strategies. This integrated approach will allow for the development of more effective treatments for various NET-associated diseases.

As a literature review, this paper's findings are limited by the available published literature. Although the authors included current research, the field of NET research is rapidly evolving, and newer findings could potentially alter some of the conclusions presented. Moreover, the interpretations of some findings remain controversial, particularly regarding the role of PAD4 in Nox-dependent NET formation and the precise impact of various histone modifications. Further research is needed to address these controversies and refine our understanding of NET formation and its regulation.