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During the inflammatory storm of sepsis, a significant quantity of neutrophil extracellular traps (NETs) are generated, which act as a double-edged sword and not only impede the invasion of foreign microorganisms but also exacerbate organ damage. This study provides evidence that NETs can cause damage to alveolar epithelial cells in vitro. The sepsis model developed in this study showed a significant increase in NETs in the bronchoalveolar lavage fluid (BALF). The development of NETs has been shown to increase the lung inflammatory response and aggravate injury to alveolar epithelial cells. Bay-117082, a well-known NF-κB suppressor, is used to modulate inflammation. This analysis revealed that Bay-117082 efficiently reduced total protein concentration, myeloperoxidase activity, and inflammatory cytokines in BALF. Moreover, Bay-117082 inhibited the formation of NETs, which in turn prevented the activation of the pore-forming protein gasdermin D (GSDMD). In summary, these results indicated that excessive NET production during sepsis exacerbated the onset and progression of acute lung injury (ALI). Therefore, Bay-117082 could serve as a novel therapeutic approach for ameliorating sepsis-associated ALI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Exogenous inhibition of neutrophil extracellular traps (NETs) was believed to alleviate acute pancreatitis (AP). This study aimed to comprehensively explore the key biological behavior of NETs including timing and pathogenesis in AP by integrating of single cell RNA sequencing(scRNA-seq) and bulk RNA-seq., Methods: Differentially expressed NETs-related genes and the hub genes of NETs were screened by bulk RNA-seq. ScRNA-seq was used to identify the cell types in pancreas of AP mice and to depict the transcriptomic maps in neutrophils. The mouse AP models were build to verify the timing of initiation of NETs and underlying pathogenesis of damage on pancreas acinar cells., Results: Tlr4 and Ccl3 were screened for hub genes by bulk RNA-seq. The trajectory analysis of neutrophils showed that high expression of Ccl3, Cybb and Padi4 can be observed in the middle stage during AP. Macrophages might be essential in the biological behavior of neutrophils and NETs. Through animal models, we presented that extensive NETs structures were formed at mid-stage of inflammation, accompanied by more serious pancreas and lung damage. NETs might promote necroptosis and macrophage infiltration in AP, and the damage on pancreatic injury could be regulated by Tlr4 pathway. Ccl3 was considered to recruit neutrophils and promote NETs formation., Conclusion: The findings explored the underlying timing and pathogenesis of NETs in AP for the first time, which provided gene targets for further studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metastasis to the lungs is a leading cause of death for patients with breast cancer. Therefore, effective therapies are urgently needed to prevent and treat lung metastasis. In this study, we uncovered a mechanism by which NAD(P)H:quinone oxidoreductase 1 (NQO1) orchestrates lung metastasis. NQO1 stabilized and upregulated peptidyl-prolyl cis-trans isomerase A (PPIA), a chaperone that regulates protein conformation and activity, by preventing its oxidation at a critical cysteine residue C161. PPIA subsequently activated CD147, a membrane protein that facilitates cell invasion. Moreover, NQO1-induced secretion of PPIA modulated the immune landscape of both primary and lung metastatic sites. Secreted PPIA engaged CD147 on neutrophils and triggered the release of neutrophil extracellular traps (NET) and neutrophil elastase, which enhanced tumor progression, invasiveness, and lung colonization. Pharmacological targeting of PPIA effectively inhibited NQO1-mediated breast cancer lung metastasis. These findings reveal a previously unrecognized NQO1-PPIA-CD147-NET axis that drives breast cancer lung metastasis. Inhibiting this axis is a potential therapeutic strategy to limit lung metastasis in patients with breast cancer.  Significance: NQO1 stabilizes and promotes the secretion of PPIA to activate CD147 in neutrophils and stimulate NET formation, promoting breast cancer lung metastasis and providing therapeutic targets for this fatal condition., (©2024 American Association for Cancer Research.)

The role of Neutrophil extracellular traps (NETs) in the immunopathogenesis of Diabetic Kidney Disease (DKD) remains elusive. We used a machine learning approach to identify differentially expressed genes (DEGs) associated with NETs in human DKD kidney biopsy datasets and validated the results using single-nucleus RNA sequencing datasets. The expressions of these candidate genes and related cytokines were verified in blood obtained from DKD patients. Three NETs-associated genes (ITGAM, ITGB2 and TLR7) were identified, which all showed significant upregulation in both glomerular and tubulointerstitial compartments in human DKD kidneys. DKD patients showed significantly higher number of activated neutrophils with increased ITGAM and ITGB2 expression, higher serum IL-6 but lower IL-10, compared to healthy controls (p all <0.01). This study suggests that dysregulation of NETs-associated genes ITGAM and ITGB2 are related to the pathogenesis of DKD, and may serve as novel diagnostic markers and therapeutic targets in DKD., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mechanisms underlying the development and progression of colon cancer are not fully understood. Herein, Src kinase associated phosphoprotein 1 (SKAP1), an immune cell adaptor, is identified as a novel colon cancer-related gene. SKAP1 expression is significantly increased in colon cancer cells. High SKAP1 levels are independently predictive of poor survival in patients with colon cancer. Notably, SKAP1 expression in colon cancer cells exerted a significant tumor-promoting effect in vivo rather than in vitro. Screening of tumor-infiltrating immune cells revealed the involvement of neutrophils in SKAP1-induced colon tumor promotion. Enhanced formation of neutrophil extracellular traps (NETs) is found to be a key downstream event that contributed to the pro-tumor role of SKAP1. In colon cancer cells, SKAP1 increased the expression of C-X-C motif chemokine ligand 8 (CXCL8) via nuclear factor of activated T cells c1 (NFATc1). The blockade of CXCL8 or NFATc1 largely attenuated neutrophil infiltration, NET formation, and tumor promotion induced by SKAP1. Furthermore, inhibiting SKAP1-induced NET significantly enhanced the antitumor efficiency of adoptive natural killer cell therapy in colon tumor models. In conclusion, SKAP1 significantly promotes colon cancer growth via the cancer cell/neutrophil NFATc1/CXCL8/NET axis, suggesting that SKAP1 is a potential target for colon cancer therapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Gout is a self-limited inflammatory disease caused by the deposition of monosodium urate (MSU) crystals in joints and surrounding tissues due to abnormal purine metabolism. Neutrophil extracellular traps (NETs) are formed by neutrophils in response to pathogen attack. During gout, NETs induced by MSU crystals exacerbate inflammation, and aggregated NETs (aggNETs) promote the resolution of gout-associated inflammation by encapsulating MSU crystals, degrading cytokines and chemokines, and blocking the recruitment and activation of neutrophils. With disease progression, NETs participate in the formation of tophi. Therefore, aggNETs are a possible mechanism of spontaneous gout regression. Studying the specific mechanism by which NETs affect inflammatory bursts and spontaneous regression in gout patients is important. This review summarises the role of NETs in different stages of gout and the specific pathogenesis of NETs in gout to provide new ideas for the diagnosis and treatment of gout.

Excessive formation of macrophage extracellular trap (MET) has been implicated in several autoimmune disease pathogeneses; however, its impact on type 1 diabetes (T1D) and related mechanisms remains enigmatic. We demonstrated the pivotal role of peptidyl arginine deiminase 4 (PAD4) in driving profuse MET formation and macrophage M1 polarization in intestinal inflammation in NOD mice. Genetic knockout of PAD4 or adoptive transfer of METs altered the proportion of proinflammatory T cells in the intestine, subsequently influencing their migration to the pancreas. Combining RNA sequencing and CUT&Tag analysis, we found activated PAD4 transcriptionally regulated CXCL10 expression. This study comprehensively investigated how excessive PAD4-mediated MET formation in the colon increases the aggravation of intestinal inflammation and proinflammatory T-cell migration and finally is involved in T1D progression, suggesting that inhibition of MET formation may be a potential therapeutic target in T1D., (© 2024 by the American Diabetes Association.)

Systemic sclerosis (SSc) is a challenging autoimmune disease characterized by progressive fibrosis affecting the skin and internal organs. Despite the known infiltration of macrophages and neutrophils, their precise contributions to SSc pathogenesis remain elusive. In this study, we elucidated that CD206 hi MHCII lo M2-like macrophages constitute the predominant pathogenic immune cell population in the fibrotic skin of a bleomycin-induced SSc mouse model. These cells emerged as pivotal contributors to the profibrotic response by orchestrating the production of TGF-β1 through a MerTK signaling-dependent manner. Notably, we observed that neutrophil infiltration was a prerequisite for accumulation of M2-like macrophages. Strategies such as neutrophil depletion or inhibition of CXCR1/2 were proven effective in reducing M2-like macrophages, subsequently mitigating SSc progression. Detailed investigations revealed that in fibrotic skin, neutrophil-released neutrophil extracellular traps were responsible for the differentiation of M2-like macrophages. Our findings illuminate the significant involvement of the neutrophil-macrophage-fibrosis axis in SSc pathogenesis, offering critical information for the development of potential therapeutic strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, SEZ) is an essential zoonotic bacterial pathogen that can cause various inflammation, such as meningitis, endocarditis, and pneumonia. UDP-glucose dehydrogenase (hasB) is indispensable in synthesizing SEZ virulence factor hyaluronan capsules. Our study investigated the infection of hasB on mice response to SEZ by employing a constructed capsule-deficient mutant strain designated as the ΔhasB strain. This deficiency was associated with a reduced SEZ bacterial load in the mice's blood and peritoneal lavage fluid (PLF) post-infection. Besides, the ΔhasB SEZ strain exhibited a higher propensity for neutrophil infiltration and release of cell-free DNA (cfDNA) in vivo compared to the wild-type (WT) SEZ strain. In vitro experiments further revealed that ΔhasB SEZ more effectively induced the formation of neutrophil extracellular traps (NETs) containing histone 3 (H3), neutrophil elastase (NE), and DNA, than its WT counterpart. Moreover, the release of NETs was determined to be gasdermin D (GSDMD)-dependent during the infection process. Taken together, these findings underscore that the deficiency of the hasB gene in SEZ leads to enhanced GSDMD-dependent NET release from neutrophils, thereby reducing SEZ's capacity to resist NETs-mediated eradication during infection. Our finding paves the way for the development of innovative therapeutic strategies against SEZ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sepsis is a life-threatening condition caused by an excessive inflammatory response to an infection. However, the precise regulatory mechanism of sepsis remains unclear. Using a strand-specific RNA-sequencing, we identified 115 hub differentially expressed long noncoding RNAs (lncRNAs) and 443 mRNAs in septic patients, primarily participated in crucial pathways including neutrophil extracellular trap (NET) formation and toll-like receptor signaling. Notably, NETs related gene aquaporin-9 (AQP9) and its associated lncRNAs exhibited significant upregulation in septic neutrophils. Functional experiments revealed AQP9 interacts with its lncRNAs to augment the formation of neutrophil NETs. In murine sepsis models, AQP9 inhibition with phloretin reduced proinflammatory cytokine production and lung damage. These findings provide crucial insights into the regulatory role of AQP9 in sepsis, unraveling its interaction with associated lncRNAs in transmitting downstream signals, holding promise in informing the development of novel therapeutic strategies aimed at ameliorating the debilitating effects of sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The tumor microenvironment (TME) is intricately associated with cancer progression, characterized by dynamic interactions among various cellular and molecular components that significantly impact the carcinogenic process. Notably, neutrophils play a crucial dual role in regulating this complex environment. These cells oscillate between promoting and inhibiting tumor activity, responding to a multitude of cytokines, chemokines, and tumor-derived factors. This response modulates immune reactions and affects the proliferation, metastasis, and angiogenesis of cancer cells. A significant aspect of their influence is their interaction with the endoplasmic reticulum (ER) stress responses in cancer cells, markedly altering tumor immunodynamics by modulating the phenotypic plasticity and functionality of neutrophils. Furthermore, neutrophil extracellular traps (NETs) exert a pivotal influence in the progression of malignancies by enhancing inflammation, metastasis, immune suppression, and thrombosis, thereby exacerbating the disease. In the realm of immunotherapy, checkpoint inhibitors targeting PD-L1/PD-1 and CTLA-4 among others have underscored the significant role of neutrophils in enhancing therapeutic responses. Recent research has highlighted the potential of using neutrophils for targeted drug delivery through nanoparticle systems, which precisely control drug release and significantly enhance antitumor efficacy. This review thoroughly examines the diverse functions of neutrophils in cancer treatment, emphasizing their potential in regulating immune therapy responses and as drug delivery carriers, offering innovative perspectives and profound implications for the development of targeted diagnostic and therapeutic strategies in oncology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a significant pulmonary complication of RA. This study tried to elucidate the mechanisms enhancing inflammation and causing lung injury in RA-ILD, focusing on the role of neutrophil extracellular traps (NETs). The study also investigated the potential benefits of nintedanib in advanced disease., Methods: Nine RA-ILD patients and nine healthy controls were included in the study. Inflammatory markers in patients' circulation were evaluated with immunoassays. The formation of NETs was examined using a citrullinated histone H3 (CitH3) ELISA and cell immunofluorescence. Inflammatory proteins expressed in neutrophils/NETs were studied with real-time qPCR and NET ELISA. To assess the effect of nintedanib, an intracellular tyrosine kinase inhibitor with antifibrotic properties, in RA-ILD a paired study was conducted in five patients before treatment administration and 16 weeks later., Results: The soluble terminal complement complex sC5b-9 and the levels of CitH3 were significantly elevated in patients with RA-ILD, compared to healthy controls. In addition, neutrophils isolated from RA-ILD patients released NETs enriched with tissue factor and interleukin-17A. Inflammatory NETs had a dynamic role, increasing the fibrotic potential of human pulmonary fibroblasts (HPFs). On the other hand, nintedanib treatment decreased NETs and sC5b-9 levels in RA-ILD patients., Conclusion: The findings propose an interplay between circulating NETs and HPFs, establishing the immunofibrotic aspects of RA-ILD. They also support the effectiveness of nintedanib in reducing key pathological processes of the disease. Further research is needed to fully understand these mechanisms and optimize treatment strategies for RA-ILD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Venetsanopoulou, Ntinopoulou, Papagianni, Koletsos, Voulgari and Chrysanthopoulou.)

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa ( P. aeruginosa ) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro , bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro , PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Baird, Yusuf, Forde, Pohl, Kavanagh, Fitzpatrick, Gogoi and Reeves.)

Neutrophil extracellular traps (NETs) are associated with rheumatoid arthritis pathogenesis and severity. Since homeostatic NET-forming neutrophils [NET+Ns] have beneficial roles in defense against pathogens, their distinction from pro-injury [NET+N] subtypes is important, especially if they are to be therapeutically targeted. Having identified circulating, pro-injury DEspR+CD11b+[NET+Ns] in patients with neutrophilic secondary tissue injury, we determined whether DEspR+[NET+Ns] are present in rheumatoid arthritis (RA) flares. Whole blood samples of patients with RA flares on maintenance therapy (n = 6) were analyzed by flow cytometry (FCM) and immunofluorescence cytology followed by semi-automated quantitative confocal microscopy (qIFC). We assessed clinical parameters, levels of neutrophils and [NET+Ns], and plasma S100A8/A9. qIFC detected circulating DEspR+CD11b+neutrophils and [NET+Ns] in RA-flare patients but not healthy controls. DEspR+[NET+Ns] were positive for citrullinated histone H3 (citH3+), extruded DNA, decondensed but recognizable polymorphic nuclei, and [NET+N] doublet interactions in mostly non-ruptured NET-forming neutrophils. Circulating DNA+/DEspR+/CD11b+/citH3+microvesicles (netMVs) were observed. FCM detected increased %DEspR+CD11b+neutrophils and DEspR+ cell-cell doublets whose levels trended with DAS28 scores, as did plasma S100A8/A9 levels. This study identifies circulating DEspR+/CD11b+neutrophils and [NET+Ns] in RA-flare patients on maintenance therapy. Detection of circulating DEspR+citH3+[NET+Ns] and netMVs indicate a systemic neutrophilic source of citH3-antigen concordant with multi-joint RA pathogenesis. Increased S100A8/A9 alarmin levels are associated with cell injury and released upon NET-formation. As a ligand for TLR4, S100A8/A9 forms a positive feedback loop for TLR4-induced DEspR+neutrophils. These data identify DEspR+neutrophils and [NET+Ns] in RA pathogenesis as a potential biomarker and/or therapeutic target., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/ S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/ S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo . Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV., Competing Interests: The authors declare no conflict of interest.

Fusobacterium nucleatum as a Gram-negative anaerobe plays a key bridging role in oral biofilms. It is involved in periodontal and extraoral diseases, the most prominent being colorectal cancer. Five subspecies are recognised: animalis, fusiforme, nucleatum, polymorphum and vincentii . Subspecies interact with neutrophils constantly patrolling tissues to remove microbial intruders. Neutrophil antimicrobial activities include generation of reactive oxygen species (ROS), formation of neutrophil extracellular traps (NETs) and release of cytokines and neutrophil enzymes. Subspecies-specific differences in immunogenicity have previously been observed in a neutrophil-like cell line but were not investigated in human neutrophils. Additionally, neutrophil responses to planktonic and biofilm-grown F. nucleatum have not been studied to date. The aims of this study were to compare the immunogenicity of planktonic and biofilm-grown F. nucleatum and to investigate potential differences in human neutrophil responses when stimulated with individual F. nucleatum subspecies. Human neutrophils isolated from peripheral blood were stimulated with planktonic and biofilm-grown F. nucleatum subspecies. Generation of ROS and NET formation were quantified by luminescence and fluorescence assays, respectively. Secretion of cytokines (IL-1β, TNF-α, IL-6, IL-8), neutrophil elastase and matrix metalloproteinase-9 was quantified by enzyme-linked immunosorbent assay (ELISA). Neutrophil responses showed biofilm-grown bacteria induced a significantly higher total and intracellular ROS response, as well as shorter time to total ROS release. Biofilm-grown F. nucleatum led to significantly lower IL-1β release. We found significant differences among individual subspecies in terms of total, intracellular ROS and extracellular superoxide. Subspecies polymorphum stimulated the highest mean amount of NET release. Amounts of cytokines released differed significantly among subspecies, while no differences were found in lysosomal enzyme release. Immunogenicity of F. nucleatum in human neutrophils is highly subspecies-specific in vitro with regard to ROS release and cytokine production. Understanding subspecies-specific immunogenicity of F. nucleatum may facilitate the discovery of novel therapeutic targets in F. nucleatum -mediated diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Muchova, Kuehne, Grant, Smith, Nagi, Chapple and Hirschfeld.)

Activated neutrophils release neutrophil extracellular traps (NETs), complex structures composed of extracellular genetic material and proteins sourced from the nucleus, granules, and cytoplasm in response to pathogenic inflammatory conditions. These NETs play a crucial role in the host's innate immune defense against invasive infections. Notably, in conditions like atherosclerosis, these extracellular formations can also be elicited by inflammatory stimuli such as lipids, prothrombotic factors, platelet aggregation, or proinflammatory cytokines. NETs have been identified on the inner arterial walls in cardiovascular disease states. By promoting inflammation through NETosis-mediated cell adhesion processes and exerting cytotoxic effects leading to cellular dysfunction and tissue damage, NETs contribute to the pathogenesis of inflammatory conditions.

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Introduction: Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils that may modulate different steps of the metastatic cascade. The aim of our study was to investigate how NETs can modulate the adhesion properties of cancer cells and whether cell exposure to NETs can activate the epithelial-to-mesenchymal transition (EMT) program thus enhancing the migratory and invasive properties of tumor cells., Materials and Methods: Different cancer cell lines were subjected to a solid-phase adhesion assay using NET-coated plates with or without the addition of antibodies against α5β1 or CCDC25 receptor. After 1-4 h of incubation, adherent cells were expressed as the percentage of total cell number. To test EMT occurrence, cells were treated with NETs for up to 48 h and then the levels of E-cadherin, vimentin, Snail, Slug, Zeb 1 and Twist 1 along with levels of Notch 1 and cleaved Notch 1 were determined by western blotting. Untreated and NET-treated cells were subjected to migration assays using 24-multiwell plates with transwell and FBS as chemoattractant., Results: Cancer cell adhesion to NET-coated plates varied between 30% and 92.7% and was significantly higher than that obtained in uncoated plates. The addition of antibodies against α5β1 or CCDC25 caused a strong reduction of cell adhesion to NETs. The prolonged exposure of EGFR-driven cancer cell lines to NETs caused the activation of the EMT program through the upregulation and cleavage of Notch 1 and was confirmed by the enhanced expression of EMT markers. The consequent loss of the epithelial phenotype induced a strong reduction of the expression of the oncogene driver. Cell migration was significantly enhanced in NET-treated cells as compared to untreated cells., Discussion: Our findings reveal the dynamic role of NETs that may provide a DNA and fibronectin rich environment for binding of many cancer cells at distant sites where the prolonged exposure to NETs triggers the EMT through the activation of Notch 1 signaling pathway with the subsequent enhancement of migratory and invasive properties of cancer cells. Furthermore, our findings provide an example of how an immune/inflammatory microenvironment may directly modulate the sensitivity of cancer cells to oncogene targeted agents., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dimitrov, Maddalena, Terlizzi, Altobelli, Pellegrino, Mehmood, De Rosa, Iommelli and Del Vecchio.)

The IL-23-Th17 axis is responsible for neutrophilic inflammation in various inflammatory diseases. Here, we discover a potential pathway to inhibit neutrophilic asthma. In our neutrophil-dominant asthma (NDA) model, single-cell RNA-seq analysis identifies a subpopulation of CD39 + CD9 + interstitial macrophages (IMs) suppressed by IL-23 in NDA conditions but increased by an IL-23 inhibitor αIL-23p19. Adoptively transferred CD39 + CD9 + IMs suppress neutrophil extracellular trap formation (NETosis), a representative phenotype of NDA, and also Th17 cell activation and neutrophilic inflammation. CD39 + CD9 + IMs first attach to neutrophils in a CD9-dependent manner, and then remove ATP near neutrophils that contribute to NETosis in a CD39-dependent manner. Transcriptomic data from asthmatic patients finally show decreased CD39 + CD9 + IMs in severe asthma than mild/moderate asthma. Our results suggest that CD39 + CD9 + IMs function as a potent negative regulator of neutrophilic inflammation by suppressing NETosis in the IL-23-Th17 axis and can thus serve as a potential therapeutic target for IL-23-Th17-mediated neutrophilic asthma., (© 2024. The Author(s).)

Non-melanoma skin cancers (NMSC), including basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and Merkel cell carcinoma (MCC), are increasingly common and present significant healthcare challenges. Neutrophil extracellular traps (NETs), chromatin fibers expulsed by neutrophil granulocytes, can promote immunotherapy resistance via an impairment of CD8 + T cell-mediated cytotoxicity. Here, to identify a potential therapeutic target, we investigate the expulsion of NETs and their relation to CD8 + T cell infiltration in NMSC. Immunofluorescence staining for neutrophils (CD15) and NETs (H3cit), as well as immunohistochemistry for cytotoxic T cells (CD8 + ) on human cSCCs ( n = 24), BCCs ( n = 17) and MCCs ( n = 12), revealed a correlation between neutrophil infiltration and ulceration diameter in BCC and MCC, but not in cSCC. In BCC and cSCC, neutrophil infiltration also correlated with the cross-sectional area (CSA). NETs were not associated with established risk factors but with the presence of an ulceration, and, in cSCC, with abscess-like structures. CD8 + T cell infiltration was not reduced in tumors that were NET-positive nor in those with a denser neutrophil infiltration. This study is the first to report and characterize NETs in NMSC. Thus, it gives an incentive for further research in this relevant yet understudied topic.

Objective To investigate the phenotypic and functional characteristics of normal human peripheral blood neutrophils. Methods Normal human peripheral blood neutrophils were isolated through density gradient centrifugation followed by red blood cell lysis, and their surface markers and distribution characteristics in different neutrophil subsets were detected using multi-color immunofluorescence staining and flow cytometry. The phagocytic activity of neutrophils was evaluated with pHrodo TM Green E.coli BioParticles TM . The morphological characteristics of neutrophils undergoing the formation of neutrophil extracellular traps (NET) in vitro were examined via Wright-Giemsa staining and immunofluorescence staining, and were observed using standard light microscopy and confocal microscopy, respectively. Results CD66b, CD11b and CD16 were all highly expressed on normal human peripheral blood neutrophils and the percentage of CD16 positive neutrophils was higher compared to CD11b positive neutrophils. Neutrophils could be classified into three subsets based on the density of CD16 expression: namely, CD16 hi neutrophil, CD16 int neutrophil and CD16 lo neutrophil. The proportion of the CD16 hi neutrophil was the highest among the three subsets. The inhibitory molecule programmed cell death ligand 1 (PD-L1) was expressed on all neutrophil subsets without significant differences; however, the expression level of another inhibitory molecule, CD300LD, was markedly higher in the CD16 hi neutrophil compared to CD16 int or CD16 lo neutrophil. Each neutrophil subset demonstrated high levels of adhesion molecule CD62L without notable differences. Notably, the mean fluorescence intensity (MFI) value for pHrodo TM Green E.coli BioParticles TM in the CD16 int neutrophil was the highest among three subsets; and that in CD16 hi neutrophil was higher compared to CD16 lo neutrophil. Results of Wright-Giemsa staining showed that normal peripheral blood neutrophils typically possessed lobulated nuclei; upon stimulation with phorbol 12-myristate 13-acetate (PMA), the nuclei of these neutrophils formed a network structure while losing their lobulated nuclear morphology. Laser confocal microscopy results showed an increased release of neutrophil elastase (NE) with network formation in neutrophils after PMA stimulation. Conclusions Normal human peripheral blood neutrophils are heterogenous population with different expression of surface marker molecules, which might exert different immune functions. NET formation can be induced by PMA stimulation, and the differences of NET formation in different neutrophil subsets need to be further studied.

Despite their relatively short lifespan, neutrophils are tasked with counteracting pathogens through various functions, including phagocytosis, production of reactive oxygen species, neutrophil extracellular traps (NETs), and host defense peptides. Regarding the latter, small cationic cathelicidins present a conundrum in neutrophil function. Although primarily recognized as microbicides with an ability to provoke pores in microbial cell walls, the ability of cathelicidin to modulate key neutrophil functions is also of great importance, including the release of chemoattractants, cytokines, and reactive oxygen species, plus prolonging neutrophil lifespan. Cumulative evidence indicates a less recognized role of cathelicidin as an "immunomodulator"; however, this term is not always explicit, and its relevance in neutrophil responses during infection and inflammation is seldom discussed. This review compiles and discusses studies of how neutrophils use cathelicidin to respond to infections, while also acknowledging immunomodulatory aspects of cathelicidin through potential crosstalk between sources of the peptide., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Sepsis is a severe disease that occurs when the body's immune system reacts excessively to infection. The body's response, which includes an intense antibacterial reaction, can damage its tissues and organs. Neutrophils are the major components of white blood cells in circulation, play a vital role in innate immunity while fighting against infections, and are considered a feature determining sepsis classification. There is a plethora of basic research detailing neutrophil functioning, among which, the study of neutrophil extracellular traps is providing novel insights into mechanisms and treatments of sepsis. This review explores their functions, dysfunctions, and influences in the context of sepsis. The interplay between neutrophils and the human microbiome and the impact of DNA methylation on neutrophil function in sepsis are crucial areas of study. The interaction between neutrophils and the human microbiome is complex, particularly in the context of sepsis, where dysbiosis may occur. We highlight the importance of deciphering neutrophils' functional alterations and their epigenetic features in sepsis because it is critical for defining sepsis endotypes and opening up the possibility for novel diagnostic methods and therapy. Specifically, epigenetic signatures are pivotal since they will provide a novel implication for a sepsis diagnostic method when used in combination with the cell-free DNA. Research is exploring how specific patterns of DNA methylation in neutrophils, detectable in cell-free DNA, could serve as biomarkers for the early detection of sepsis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose of Review: The process of neutrophil extracellular traps (NETs) formation, called NETosis, is a peculiar death modality of neutrophils, which was first observed as an immune response against bacterial infection. However, an ongoing and exaggerated NETs formation may have adverse clinical consequences and even promote cancer progression. This review will discuss the complex relationship between NETosis and cancer progression., Recent Findings: NETs exhibits cancer-promoting effects by causing cancer metastaisis and tumor-associated thrombosis. Many studies have found that many mechanisms are involved in the process, and the corresponding targets could be applied for cancer therapy. Although NETs may have anti-bacteria effects, it is necessary to inhibit an excessive NETs formation, mostly showing cancer-promoting effects. The contribution of NETs to cancer progression has gained a growing appreciation and the approaches to targeting NETs deposition exhibited beneficial effects both in primary and metastatic tumors, which, however, has been challenged by a recent finding demonstrating an opposite effect of NETs to suppress tumor growth via the activation of immune response against tumor. This seeming discrepancy reflects we are in the early stage of NETs study facing fundamental questions and a better understanding of the underlying mechanism is urgently needed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Inflammation contributes to the development of thrombosis, but the mechanistic basis for this association remains poorly understood. Innate immune responses and coagulation pathways are activated in parallel following infection or injury, and represent an important host defense mechanism to limit pathogen spread in the bloodstream. However, dysregulated proinflammatory activity is implicated in the progression of venous thromboembolism and arterial thrombosis. In this review, we focus on the role of myeloid cells in propagating thromboinflammation in acute inflammatory conditions, such as sepsis and coronavirus disease 2019 (COVID-19), and chronic inflammatory conditions, such as obesity, atherosclerosis, and inflammatory bowel disease. Myeloid cells are considered key drivers of thromboinflammation via upregulated tissue factor activity, formation of neutrophil extracellular traps (NETs), contact pathway activation, and aberrant coagulation factor-mediated protease-activated receptor (PAR) signaling. We discuss how strategies to target the intersection between myeloid cell-mediated inflammation and activation of blood coagulation represent an exciting new approach to combat immunothrombosis. Specifically, repurposed anti-inflammatory drugs, immunometabolic regulators, and NETosis inhibitors present opportunities that have the potential to dampen immunothrombotic activity without interfering with hemostasis. Such therapies could have far-reaching benefits for patient care across many thromboinflammatory conditions., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Neutrophil extracellular traps (NETs) have long been consistently considered an innate immune defense against foreign pathogens, but this oversimplified view has decelerated the progression of perceiving NET biology in chronic diseases. It is now increasingly accepted that NETs are not exclusive to anti-infection responses, but are also central players with a double-edged sword role in cancer progression. NETs have gradually emerged as tumor diagnostic, predictive, and prognostic biomarkers, and strenuous endeavors have been devoted to tapping their potential as new therapeutic targets. Correspondingly, the boom in studies on NETs and tumors in recent years has achieved a series of scientific outputs, which opens up a new perspective for perceiving the sophisticated landscapes of the tumor immune microenvironment. However, there is still much room to translate NET-targeted immunotherapies into clinical practice. Therefore, it is necessary to explore the knowledge structure and latent hotspots of the links between NETs and tumors using bibliometric analysis., Methods: NETs and tumor publications from 2006 to 2024 were extracted from the Web of Science Core Collection. Bibliometric analysis and visualization were conducted using Microsoft Excel, VOSviewer, CiteSpace, and R-bibliometrix., Results: The analysis included 1,339 publications authored by 7,747 scholars affiliated with 1,926 institutions across 70 countries/regions with relevant articles published in 538 journals. Despite China's maximum number of publications, the United States has continued to dominate the field as a global cooperation center with overwhelming citation counts. Frontiers in Immunology published the most number of publications, whereas Blood was the most cited journal. Wagner, Denisa D. and Kaplan, Mariana J. are concurrently in both the top 10 most prolific authors and cited author lists. Tumor microenvironment and immunotherapy will likely be the focus of future research., Conclusions: A comprehensive bibliometric analysis was first conducted to map the current landscape and knowledge structure of the link between NETs and tumors in the hope of providing guidance and fresh perspectives for further research in this field. NETs are promising antitumor targets, and perhaps the eventual destination in the realm is to translate NET-targeted immunotherapies into clinical practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xiao, Feng, Zhao, Ding and Gao.)

Neutrophil extracellular traps (NETs), crucial in immune defense mechanisms, are renowned for their propensity to expel decondensed chromatin embedded with inflammatory proteins. Our comprehension of NETs in pathogen clearance, immune regulation and disease pathogenesis, has grown significantly in recent years. NETs are not only pivotal in the context of infections but also exhibit significant involvement in sterile inflammation. Evidence suggests that excessive accumulation of NETs can result in vessel occlusion, tissue damage, and prolonged inflammatory responses, thereby contributing to the progression and exacerbation of various pathological states. Nevertheless, NETs exhibit dual functionalities in certain pathological contexts. While NETs may act as autoantigens, aggregated NET complexes can function as inflammatory mediators by degrading proinflammatory cytokines and chemokines. The delineation of molecules and signaling pathways governing NET formation aids in refining our appreciation of NETs' role in immune homeostasis, inflammation, autoimmune diseases, metabolic dysregulation, and cancer. In this comprehensive review, we delve into the multifaceted roles of NETs in both homeostasis and disease, whilst discussing their potential as therapeutic targets. Our aim is to enhance the understanding of the intricate functions of NETs across the spectrum from physiology to pathology., (© 2024. The Author(s).)

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) ( n = 24) or vehicle ( n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19.

Introduction: Crohn's disease (CD) is characterized by chronic inflammation and intestinal fibrosis leading to lifelong complications. However, the disease pathogenesis remains elusive, and the therapeutic options are limited. Here, we investigated the interaction between neutrophils and intestinal fibroblasts in the development of CD immunofibrosis, a disease mechanism predisposing to inflammatory and fibrotic complications., Methods: Peripheral neutrophils, enriched neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies from CD, ulcerative colitis (UC) patients, and healthy individuals (HI), were studied. Transcriptome analysis of neutrophils, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed., Results: Compared to UC, PIFs from CD patients, independently to the presence of strictures, displayed a distinct pro-fibrotic phenotype characterized by negative Krüppellike Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. In both UC and CD, PIFs-derived IL-8 acted as a culprit chemoattractant for neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs induced a CD-like phenotype in HI PIFs, suggesting their fibrotic plasticity. High IFNa in serum and IFΝ-responsive signature in peripheral neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum stimulated the release of fibrogenic eNETs from neutrophils in an IFNa-dependent manner, suggesting the priming role of IFNa in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, both serum IFNa levels and mRNA levels of key IFN signaling components in neutrophils were wellcorrelated with CD severity., Conclusions: This study reveals the important role of the IFNa/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and putative therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gavriilidis, Divolis, Natsi, Kafalis, Kogias, Antoniadou, Synolaki, Pavlos, Koutsi, Didaskalou, Papadimitriou, Tsironidou, Gavriil, Papadopoulos, Agelopoulos, Tsilingiris, Koffa, Giatromanolaki, Kouklakis, Ritis and Skendros.)

Extracorporeal membrane oxygenation (ECMO), as an extracorporeal life support technique, can save the lives of reversible critically ill patients when conventional treatments fail. However, ECMO-related acute organ injury is a common complication that increases the risk of death in critically ill patients, including acute kidney injury, acute brain injury, acute lung injury, and so on. In ECMO supported patients, an increasing number of studies have shown that activation of the inflammatory response plays an important role in the development of acute organ injury. Cross-cascade activation of the complement system, the contact system, and the coagulation system, as well as the mechanical forces of the circuitry are very important pathophysiological mechanisms, likely leading to neutrophil activation and the production of neutrophil extracellular traps (NETs). NETs may have the potential to cause organ damage, generating interest in their study as potential therapeutic targets for ECMO-related acute organ injury. Therefore, this article comprehensively summarized the mechanism of neutrophils activation and NETs formation following ECMO treatment and their actions on acute organ injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Li, Ying and Qu.)

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis.

Objectives: There is already substantial evidence indicating that neutrophil extracellular trap (NET) formation contributes to the inflammatory cascade in ulcerative colitis (UC). However, the precise regulatory mechanisms governing this process remain elusive. This study aimed to determine the role of NET-related genes in UC and reveal possible mechanisms., Methods: Employing a two-sample MR methodology, we investigated the correlations between NET-associated genes (NRGs) and UC with summary data derived from a genome-wide association study (12,366 cases vs. 33,609 controls) and FinnGen (8,279 cases vs. 261,098 controls). The main analysis employed the inverse variance weighted method, supplemented by the MR-Egger method and weighted median method. Sensitivity analysis was conducted to rule out the interference of heterogeneity and pleiotropy among utilized instrument variables. The colocalization analysis was used to determine whether the identified NRGs and UC shared casual variants. Cross-tissue expression analysis was performed to characterize the expression patterns of target NRGs, while multi-gene correlation analysis and GSEA analysis were conducted to explore the mechanisms by which target NRGs promote UC and NET formation. Immunohistochemistry was used to validate the protein expression of target NRGs in the colon tissue of UC patients., Results: After the validation of two datasets, seven NRGs were associated with the risk of UC. The higher expression of ITGB2 was associated with increased UC risk, while the expression of CXCR1, CXCR2, IRAK4, MAPK3, SIGLEC14, and SLC22A4 were inversely associated with UC risk. Colocalization analysis supported the correlation between CXCR1/2 and UC risk. Expression analysis indicated that CXCR1/2 were down-regulated in peripheral blood, but up-regulated in colon tissue. GSEA analysis and correlation analysis indicated that CXCR1/2 promoted UC and NET formation through neutrophil chemotaxis and PAD4-mediated pathways, separately. Immunohistochemical results confirmed the high expression of CXCR1/2 in colon tissues of UC patients., Conclusions: Our study identified CXCR1/2 as candidate targets in UC among all NRGs through multi-method argumentation, providing new insights of the regulation mechanisms of NET formation in the pathogenesis of UC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xv, Feng and Lin.)

Neutrophilia occurs in patients infected with SARS-CoV-2 (COVID-19) and is predictive of poor outcomes. Here, we link heterogenous neutrophil populations to disease severity in COVID-19. We identified neutrophils with features of cellular aging and immunosuppressive capacity in mild COVID-19 and features of neutrophil immaturity and activation in severe disease. The low-density neutrophil (LDN) number in circulating blood correlated with COVID-19 severity. Many of the divergent neutrophil phenotypes in COVID-19 were overrepresented in the LDN fraction and were less detectable in normal-density neutrophils. Functionally, neutrophils from patients with severe COVID-19 displayed defects in neutrophil extracellular trap formation and reactive oxygen species production. Soluble factors secreted by neutrophils from these patients inhibited T cell proliferation. Neutrophils from patients with severe COVID-19 had increased expression of arginase-1 protein, a feature that was retained in convalescent patients. Despite this increase in intracellular expression, there was a reduction in arginase-1 release by neutrophils into serum and culture supernatants. Furthermore, neutrophil-mediated T cell suppression was independent of arginase-1. Our results indicate the presence of dysfunctional, activated, and immature neutrophils in severe COVID-19.

Background: Sepsis is considered a high risk factor for new-onset atrial fibrillation (NOAF), with neutrophil extracellular traps (NETs) being implicated in the pathogenesis of numerous diseases. However, the precise role of NETs and NETs-related genes (NRGs) in the occurrence of NOAF in sepsis remains inadequately elucidated. The objective of this study was to identify hub NRGs connecting sepsis and AF, and to investigate the potential association between NETs and NOAF in sepsis., Methods: The AF and sepsis microarray datasets were retrieved from the Gene Expression Omnibus (GEO) database for analysis of shared pathophysiological mechanisms and NRGs implicated in both sepsis and AF using bioinformatics techniques. The CIBERSORT algorithm was employed to assess immune cell infiltration and identify common immune characteristics in these diseases. Additionally, a rat model of lipopolysaccharide (LPS)-induced sepsis was utilized to investigate the association between NETs, NRGs, and sepsis-induced AF. Western blotting, enzyme-linked immunosorbent assay, hematoxylin-eosin staining, immunohistochemistry, and immunofluorescence were employed to assess the expression of NRGs, the formation of NETs, and the infiltration of neutrophils. Electrophysiological analysis and multi-electrode array techniques were utilized to examine the vulnerability and conduction heterogeneity of AF in septic rats. Furthermore, intervention was conducted in LPS-induced sepsis rats using DNase I, a pharmacological agent that specifically targets NETs, in order to assess its impact on neutrophil infiltration, NETs formation, hub NRGs protein expression, and AF vulnerability., Results: A total of 61 commonly differentially expressed genes (DEGs) and four hub DE-NRGs were identified in the context of sepsis and AF. Functional enrichment analysis revealed that these DEGs were predominantly associated with processes related to inflammation and immunity. Immune infiltration analysis further demonstrated the presence of immune infiltrating cells, specifically neutrophil infiltration, in both sepsis and AF. Additionally, a positive correlation was observed between the relative expression of the four hub DE-NRGs and neutrophil infiltration. In rats with LPS-induced sepsis, we observed a notable upregulation in the expression of four DE-NRGs, the formation of NETs, and infiltration of neutrophils in atrial tissue. Through electrophysiological assessments, we identified heightened vulnerability to AF, reduced atrial surface conduction velocity, and increased conduction heterogeneity in LPS-induced sepsis rats. Notably, these detrimental effects can be partially ameliorated by treatment with DNase I., Conclusions: Through bioinformatics analysis and experimental validation, we identified four hub NRGs in sepsis and AF. Subsequent experiments indicated that the formation of NETs in the atria may contribute to the pathogenesis of NOAF in sepsis. These discoveries offer potential novel targets and insights for the prevention and treatment of NOAF in sepsis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Ischemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI., Methods: We conducted our study on amputated pig forelimbs exposed to 1 h or 9 h of ischemia and then reperfused in vivo for 12 h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot., Results: Our data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9 h ischemia compared to the 1 h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9 h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs' and NETs' intracellular regulatory functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zollet, Arenas Hoyos, Hirsiger, Brahim, Petrucci, Casoni, Wang, Spirig, Nettelbeck, Garcia, Fuest, Vögelin, Constantinescu and Rieben.)

Background: Neutrophils (polymorphonuclear leukocytes, PMNs) are the most abundant subtype of white blood cells and are among the main actors in the inflammatory response. Psoriatic arthritis (PsA) is a chronic inflammatory disease affecting both the axial and peripheral joints. Typically associated with psoriasis, PsA can also affect multiple systems and organs, including the nails and entheses. Despite the involvement of PMNs in PsA, their specific role in the disease remains poorly understood. This study aimed to characterize the biological functions of PMNs and neutrophil-related mediators in PsA patients., Materials and Methods: 31 PsA patients and 22 healthy controls (HCs) were prospectively recruited. PMNs were isolated from peripheral blood and subjected to in vitro stimulation with lipopolysaccharide (LPS), N-Formylmethionyl-leucyl-phenylalanine (fMLP), tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate (PMA), or control medium. Highly purified peripheral blood PMNs (>99%) were evaluated for activation status, reactive oxygen species (ROS) production, phagocytic activity, granular enzyme and neutrophil extracellular traps (NETs) release. Serum levels of matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), TNF, interleukin 23 (IL-23), and interleukin 17 (IL-17) were measured by ELISA. Serum Citrullinated histone H3 (CitH3) was measured as a NET biomarker., Results: Activated PMNs from PsA patients displayed reduced activation, decreased ROS production, and impaired phagocytic activity upon stimulation with TNF, compared to HCs. PMNs from PsA patients also displayed reduced granular enzyme (MPO) and NET release. Serum analyses revealed elevated levels of MMP-9, MPO, TNF, IL-23, IL-17, and CitH3 in PsA patients compared to HCs. Serum CitH3 levels positively correlated with MPO and TNF concentrations, and IL-17 concentrations were positively correlated with IL-23 levels in PsA patients. These findings indicate that PMNs from PsA patients show reduced in vitro activation and function, and an increased presence of neutrophil-derived mediators (MMP-9, MPO, TNF, IL-23, IL-17, and CitH3) in their serum., Conclusions: Taken together, our findings suggest that PMNs from PsA patients exhibit an "exhausted" phenotype, highlighting their plasticity and multifaceted roles in PsA pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Modestino, Tumminelli, Mormile, Cristinziano, Ventrici, Trocchia, Ferrara, Palestra, Loffredo, Marone, Rossi, de Paulis and Galdiero.)

B-1a cells, a regulatory subset of B lymphocytes, produce natural IgM and interleukin-10. Neutrophil extracellular traps (NETs) play a crucial role in pathogen defense, but their excessive formation during sepsis can cause further inflammation and tissue damage. In sepsis, extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released to induce NET formation. We hypothesize that B-1a cells clear NETs to prevent sepsis-induced injury. Sepsis in mice was induced by injecting 1 × 107 and 5 × 107 colony-forming units of Escherichia coli intraperitoneally. After 4 and 20 h, we assessed the number of B-1a cells in the peritoneal cavity using flow cytometry. Our results showed that the number of peritoneal B-1a cells was significantly decreased in E. coli sepsis mice. Importantly, replenishing B-1a cells via intraperitoneal injection in sepsis mice significantly decreased NETs in peritoneal neutrophils. We also observed a decrease in serum inflammation and injury markers and a significant increase in the overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we cocultured bone marrow-derived neutrophils with peritoneal B-1a cells in a contact or noncontact condition using an insert and stimulated them with eCIRP. After 4 h, we found that eCIRP significantly increased NET formation in bone marrow-derived neutrophils. Interestingly, we observed that B-1a cells inhibited NETs by 67% in a contact-dependent manner. Surprisingly, when B-1a cells were cultured in inserts, there was no significant decrease in NET formation, suggesting that direct cell-to-cell contact is crucial for this inhibitory effect. We further determined that B-1a cells promoted NET phagocytosis, and this was mediated through natural IgM, as blocking the IgM receptor attenuated the engulfment of NETs by B-1a cells. Finally, we identified that following their engulfment, NETs were localized into the lysosomal compartment for lysis. Thus, our study suggests that B-1a cells decrease NET content in eCIRP-treated neutrophils and E. coli sepsis mice., Competing Interests: Conflict of interest statement. The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objectives: To investigate whether the interplay of anti-galectin-3 antibodies (anti-Gal3 Abs) with neutrophils contributes to the development of lupus cutaneous vasculitis., Methods: Enzyme-linked immunosorbent assay was used to determine the serum level of anti-Gal3 Abs in lupus patients. Flow cytometry, quantitative PCR and western blot were performed to investigate the expression of cell surface receptors, proinflammatory cytokines and signalling molecules in neutrophils stimulated by serum from lupus patients or healthy controls (HCs) or anti-Gal3 Ab, respectively. Immunofluorescence was performed to visualise the formation of neutrophil extracellular traps (NETs). Human umbilical vein endothelial cells were co-cultured with the supernatants from neutrophils stimulated by anti-Gal3 Ab, and cytokine production was measured at mRNA and protein levels. Immunohistochemistry was adopted to reveal the distribution of Gal3, cytokines and myeloperoxidase within lupus skin lesions., Results: Serum levels of anti-Gal3 Abs were negatively correlated with peripheral counts of neutrophils. Anti-Gal3 Abs positive sera from SLE patients accelerated neutrophil death, altered cell phenotype and promoted formation of NETs with the involvement of p38 MAPK pathway. Supernatants collected from neutrophils co-cultured with anti-Gal3 Ab provoked endothelial cells to produce cytokines such as IL-1, ICAM-1, SELE and particularly IL-6. Consistently, IL-6 was higher in SLE patients with anti-Gal3 Ab positive sera and enriched in the area of vascular inflammation together with enhanced expression of Gal3 protein and infiltration of neutrophils., Conclusions: Overall, these findings suggested that neutrophils were crucial mediators in anti-Gal3 Ab induced lupus cutaneous vasculitis.

Triple-negative breast cancer (TNBC) is labeled as an aggressive type of breast cancer and still has limited therapeutic targets despite the advanced development of cancer therapy. Neutrophils, representing the conventional inflammatory response, significantly influence the malignant phenotype of tumors, supported by abundant evidence. As a vital function of neutrophils, NETs are the extracellular fibrous networks including the depolymerized chromatin DNA frames with several antimicrobial proteins. They are produced by activated neutrophils and are involved in host defence or immunological reactions. This review focuses more on the interactions between neutrophils and TNBC, focusing on how neutrophils modulate the immune response within the tumor milieu. Specifically, we delve into the role of NETs, which are involved in promoting tumor growth and metastasis, inhibiting anti-tumor immunity, and promoting tumor-associated thrombosis. Furthermore, we discuss recent advancements in therapeutic strategies aimed at targeting NETs to enhance the efficacy of TNBC treatment. The advances in the knowledge of the dynamics between neutrophils and TNBC may lead to the opportunity to devise new immunotherapeutic strategies targeted to fight this hostile type of breast cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Psoriasis is a chronic inflammatory skin disease connected with immune dysregulation. Macrophages are key inflammatory cells in psoriasis but the specific mechanism of their activation is not fully understood. Neutrophil extracellular traps (NETs) have been shown to regulate macrophage function. Here, we found that NET deposition was increased in psoriasis lesions. Peptidylarginine deaminase 4 (PAD4, a key enzyme for NET formation) deficiency attenuated skin lesions and inflammation in an imiquimod-induced psoriatic mouse model. Furthermore, the STING signaling pathway was markedly activated in psoriasis and abolished by PAD4 deficiency. PAD4-deficient mice treated with the STING agonist DMXAA exhibited more severe symptoms and inflammation than control mice. Mechanistically, the STING inhibitor C-176 inhibited NET-induced macrophage inflammation and further inhibited the proliferation of HaCaT cells. Our findings suggest an important role of NETs in the pathogenesis of psoriasis, and activation of macrophage STING/NF-κB signaling pathway might involve in NETs related psoriasis., (Copyright © 2024. Published by Elsevier Inc.)

Modulating SYK has been demonstrated to have impacts on pathogenic neutrophil responses in COVID-19. During sepsis, neutrophils are vital in early bacterial clearance but also contribute to the dysregulated immune response and organ injury when hyperactivated. Here, we evaluated the impact of R406, the active metabolite of fostamatinib, on neutrophils stimulated by LPS. We demonstrate that R406 was able to effectively inhibit NETosis, degranulation, ROS generation, neutrophil adhesion, and the formation of CD16 low neutrophils that have been linked to detrimental outcomes in severe sepsis. Further, the neutrophils remain metabolically active, capable of releasing cytokines, perform phagocytosis, and migrate in response to IL-8. Taken together, this data provides evidence of the potential efficacy of utilizing fostamatinib in bacterial sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Historically, neutrophils have been primarily regarded as short-lived immune cells that act as initial responders to antibacterial immunity by swiftly neutralizing pathogens and facilitating the activation of adaptive immunity. However, recent evidence indicates that their roles are considerably more complex than previously recognized. Neutrophils comprise distinct subpopulations and can interact with various immune cells, release granular proteins, and form neutrophil extracellular traps. These functions are increasingly recognized as contributing factors to tissue damage in autoimmune diseases. This review comprehensively examines the physiological functions and heterogeneity of neutrophils, their interactions with other immune cells, and their significance in autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, antineutrophil cytoplasmic antibody-associated vasculitis, multiple sclerosis, and others. This review aims to provide a deeper understanding of the function of neutrophils in the development and progression of autoimmune disorders., Competing Interests: Declaration of competing interest We declare no Conflict of Interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Crystalline material can cause a multitude of acute and chronic inflammatory diseases, such as gouty arthritis, silicosis, kidney disease, and atherosclerosis. Crystals of various types are thought to cause similar inflammatory responses, including the release of proinflammatory mediators and formation of neutrophil extracellular traps (NETs), processes that further promote necroinflammation and tissue damage. It has become apparent that the intensity of inflammation and the related mechanisms of NET formation and neutrophil death in crystal-associated diseases can vary depending on the crystal type, amount, and site of deposition. This review details new mechanistic insights into crystal biology, highlights the differential effects of various crystals on neutrophils and extracellular trap (ET) formation, and discusses treatment strategies and potential future approaches for crystal-associated disorders., Competing Interests: Declaration of interests The authors declare that there are no conflicts of interest related to this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage 1,2 . Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS-PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies to MICL inhibit key functions of this receptor. Of note, we also detect similarly inhibitory anti-MICL autoantibodies in patients with other diseases linked to aberrant NET formation, including lupus and severe COVID-19. By contrast, dysregulation of NET release is protective during systemic infection with the fungal pathogen Aspergillus fumigatus. Together, we show that the recognition of NETs by MICL represents a fundamental autoregulatory pathway that controls neutrophil activity and NET formation., (© 2024. The Author(s).)

The hallmark of heart failure (HF) is structural myocardial remodeling including cardiomyocyte hypertrophy, fibrosis, cardiomyocyte cell death, and a low-grade aseptic inflammation. The initiation and maintenance of persistent chronic low-grade inflammation in HF are not fully understood. Oxidative stress-mediated neutrophil extracellular traps (NETs) are the main immune defense mechanism against external bacterial infections. Furthermore, NETs play important roles in noninfectious diseases. In the settings of myocardial infarction, myocarditis, or cardiomyopathies, neutrophils infiltrate the cardiac tissue and undergo NETosis that further aggravate the inflammation. A number of stimuli may cause NETosis that is a form of programmed cell death of neutrophils that is different from apoptosis of these cells. Whether NETosis is directly involved in the pathogenesis and development of HF is still unclear. In this review, we analyzed the mechanisms and markers of NETosis, especially placing the accent on the activation of the neutrophil-specific myeloperoxidase (MPO), elastase (NE), and peptidylarginine deiminase 4 (PAD4). These conclusions are supported by the recent genetic and pharmacological studies which demonstrated that MPO, NE, and PAD4 inhibitors are effective at least in the settings of post-myocardial infarction adverse remodeling, cardiac valve diseases, cardiomyopathies, and decompensated left ventricular hypertrophy whose deterioration can lead to HF. This is essential for understanding NETosis as a contributor to pathophysiology of HF and developments of new therapies of HF., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that primarily affects young women and causes a wide range of inflammatory manifestations. The hallmark of SLE is the production of antibodies to components of the cell nucleus (antinuclear antibodies [ANAs]). These antibodies can bind to DNA, RNA, and protein complexes with nucleic acids. Among ANAs, antibodies to DNA (anti-DNA) are markers for classification and disease activity, waxing and waning disease activity in many patients. In the blood, anti-DNA antibodies can bind to DNA to form immune complexes with two distinct roles in pathogenesis: (1) renal deposition to provoke nephritis and (2) stimulation of cytokine production following uptake into innate immune cells and interaction with internal nucleic acid sensors. These sensors are part of an internal host defense system in the cell cytoplasm that can respond to DNA from infecting organisms; during cell stress, DNA from nuclear and mitochondrial sources can also trigger these sensors. The formation of immune complexes requires a source of extracellular DNA in an immunologically accessible form. As shown in in vivo and in vitro systems, extracellular DNA can emerge from dead and dying cells in both a free and a particulate form. Neutrophils undergoing the process of NETosis can release DNA in mesh-like structures called neutrophil extracellular traps. In SLE, therefore, the combination of ANAs and immunologically active DNA can create new structures that can promote inflammation throughout the body as well as drive organ inflammation and damage., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Rationale: Nitric oxide (NO) is increased in the airways and serum of patients with allergic asthma, suggesting an important role in asthma. NO production has been widely attributed to the canonical inducible NO synthase. Much effort has been made to inhibit this enzyme, with two outcomes: no asthma improvement and partial NO reduction, suggesting the involvement of an inducible NO synthase-independent source. Objectives: Neutrophils produce NO under inflammatory conditions, and their role in asthma has been overlooked. The present study analyzes their possible role as sources of NO. Methods: Our hypothesis was tested in 99 allergic patients with intermittent bronchial asthma and 26 healthy donors. NO production by blood and sputum neutrophils in response to allergens, anti-IgE, and anti-IgE receptor antibodies was assessed by Griess reagent, flow cytometry, and confocal microscopy. The formation of extracellular traps (ETs) as a possible consequence of NO production was quantified by Western blot and confocal microscopy, and reactive oxygen species were assessed with luminol-enhanced chemiluminescence. Measurements and Main Results: Among blood and sputum granulocytes from patients with allergic asthma, only neutrophils produce NO by an IgE-dependent mechanism. This production is independent of NO synthase, but dependent on a reaction between L-arginine and reactive oxygen species from NOX2 (NADPH oxidase). NO and ETosis are induced in parallel, and NO amplifies ET formation, which is a key mediator in asthma. Conclusions: Our findings reveal a novel role of neutrophils as the unique allergen/IgE-dependent NO source in allergic asthma, enhancing ET formation. These results suggest that NO produced by neutrophils needs further consideration in the treatment of allergic asthma.