Neutrophil extracellular traps (NETs) have become a significant area of investigation due to their crucial role in innate immunity and various pathological processes. The rapid pace of discovery in NET research necessitates a review of landmark discoveries alongside a discussion of contentious concepts, such as the role of protein arginine deiminase 4 (PAD4) in Nox-dependent NET formation. The context-dependency of NET formation has also emerged as a critical factor, highlighting differences between bloodstream and tissue environments, and under varied conditions like alkaline or hypertonic states. This review will explore how and why NETs are implicated in the pathogenesis of diseases ranging from atherosclerosis to cancer. This will inform a discussion of the mechanisms and potential efficacy of NET-based therapeutics, including gasdermin D inhibitors, transcription inhibitors, and DNase administration. The ultimate goal is to provide a comprehensive overview of the latest updates in NET formation research and contribute to a unified model of this complex process.

The review extensively cites previous research on NET formation, covering various aspects including the discovery of NETs, their role in complement activation, the mechanisms of Nox-dependent and Nox-independent NET formation, vital NET formation, the role of histone modifications, and the physiological contexts of NET formation (bloodstream vs. tissue, alkaline vs. neutral pH, hypertonic vs. isotonic conditions). It also delves into the existing literature on NET clearance mechanisms and the involvement of NETs in a wide range of pathologies, such as immunodeficiencies, autoimmunity, diabetes, cardiovascular disease, cancer, and cystic fibrosis. The authors highlight discrepancies and controversies in the literature, particularly concerning the role of PAD4 in Nox-dependent NET formation and the role of transcription in NET production, providing a critical analysis of existing knowledge and identify areas requiring further investigation.

This is a review article; therefore, it does not employ original research methodologies. The authors synthesize and analyze information from a broad range of existing studies focusing on neutrophil extracellular traps (NETs). Their approach involves: 1) A comprehensive literature search to identify relevant publications on the various aspects of NET formation, mechanisms, and pathological roles; 2) Critical evaluation of the existing literature to identify key findings, controversies, and areas requiring further investigation; 3) Organization and presentation of the synthesized information in a structured manner, starting from the discovery of NETs, moving through the detailed mechanisms of formation (Nox-dependent, Nox-independent, vital NET formation, and histone modifications), and concluding with the roles of NETs in various disease states and potential therapeutic targets; 4) The synthesis and presentation of information is structured to illustrate the current understanding of the complex biology of NET formation and its diverse roles in health and disease.

The review highlights several key findings: 1) Different agonists induce either Nox-dependent or Nox-independent NET formation via distinct pathways involving different kinases and transcription factors; 2) The role of PAD4 in Nox-dependent NET formation remains controversial; 3) Transcriptional firing is a necessary step in both Nox-dependent and independent NET formation, promoting DNA decondensation; 4) Vital NET formation, where neutrophils survive NET release, occurs in several ways, faster than suicidal NETosis, and involves nuclear or mitochondrial DNA; 5) Histone modifications (citrullination, methylation, and acetylation) play important roles in NET formation; 6) NET formation varies across physiological contexts (bloodstream vs. tissue, pH, tonicity); 7) NET clearance depends on DNase I and macrophages; 8) NETs are implicated in diverse pathologies (immunodeficiencies, autoimmunity, diabetes, cardiovascular disease, cancer, cystic fibrosis); and 9) Promising therapeutic strategies include manipulating NET formation (transcription inhibition, gasdermin D inhibition) and NET degradation (DNase administration).

The review successfully addresses the research question by providing a comprehensive overview of the current state of NET research. The significance of the results lies in its contribution towards building a unified model of NET formation, addressing the gap in understanding the complex interplay of various regulatory mechanisms and physiological contexts. The review highlights the duality of NETs—beneficial in host defense but also detrimental in various pathologies. The discussion on potential therapeutic interventions, particularly those targeting transcriptional regulation and gasdermin D, opens new avenues for future research, offering more selective and effective approaches compared to broad inhibitors like DPI. The comprehensive discussion of NET involvement in diverse diseases underscores the importance of further research to fully understand the implications of NET formation in these conditions.

This review provides a comprehensive summary of NET formation research. Key advances include the identification of vital NET formation mechanisms, the clarification of established NET pathways, and the elucidation of the role of previously overlooked neutrophil processes like transcription and mitochondrial function. The review also emphasizes the diverse roles of NETs in various physiological contexts and disease states. Future research should focus on developing more targeted therapies to selectively modulate NET formation while preserving the essential functions of neutrophils in innate immunity.

As a review article, this study is limited by its reliance on existing literature. The interpretations and conclusions are contingent on the quality and completeness of the primary research included. The authors acknowledge controversies and gaps in the literature, particularly regarding the role of PAD4 and transcription in specific NET formation pathways and the complex interplay of these factors in different physiological settings. The review’s conclusions are inherently limited by the current scope of research, and future studies may reveal new insights into the intricacies of NET formation and regulation.