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Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H ions reducing the intracellular pH (pH ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH (ranging from 6.6 to 7.8; every 0.2 units) increased pH of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H ions, pH is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased lipopolysaccharide-, (Gram-negative)-, and (Gram-positive)-induced NETosis. Thus, higher pH promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H ions, whereas each bicarbonate HCO3 ion binds 1H ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from ), and bacteria ( and ). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, LPS (0111:B4 and O128:B12), and . Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H O reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter -induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Pulmonary fibrosis, a heterogeneous and fatal interstitial lung disease, lacks curative therapies and specific biomarkers, posing great clinical challenges. Neutrophil extracellular traps (NETs) are key inflammatory mediators in pulmonary fibrosis pathogenesis, yet subtype-specific regulatory mechanisms and targeted therapeutic optimization remain unclear. This review systematically elucidates the distinct NETosis pathways across various subtypes. We further elaborate the multi-layered mechanisms of NETs in mediating inflammation-fibrosis transition, fibroblast activation, and innate-adaptive immune crosstalk, revealing subtype-specific pathological effects of NETs in pulmonary fibrosis. Additionally, we conduct a critical comparison of three NET-targeted therapeutic strategies and their advantages, limitations as well as subtype adaptability. Finally, we summarize the clinical transformation challenges of NET-targeted therapies and propose optimization directions. This review provides a precise theoretical framework for understanding PF immunopathogenesis and offers actionable insights for advancing NET-targeted precision medicine in pulmonary fibrosis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with significantly accelerated atherosclerosis (AS) and increased cardiovascular risk. This review elucidates the complex immunopathological mechanisms through which SLE promotes AS, involving both innate and adaptive immune dysregulation. Aberrant activation of the type I interferon signaling pathway and dysfunctional neutrophil/NETosis reciprocally amplify each other, forming a core upstream positive feedback loop. This loop accelerates atherosclerosis progression through multiple pathways, including driving endothelial dysfunction, promoting foam cell formation, and undermining plaque stability. Dysregulation of innate immune cells is prominent. Monocytes and macrophages exhibit altered polarization and impaired efferocytosis. Complement activation further exacerbates vascular injury. Within the adaptive immune system, T-cell subsets are imbalanced, promoting inflammation and AS progression. B cells and autoantibodies play dual roles. Although certain natural IgM antibodies may be protective, class-switched IgG autoantibodies often promote atherosclerosis. The role of B-cell activating factor (BAFF) and its inhibition in AS remains complex and context dependent. Animal models have been instrumental in dissecting these pathways, revealing interactions between lupus-like autoimmunity and atherogenic processes. Despite these advances, accurately assessing cardiovascular risk in SLE patients remains challenging, underscoring the need for SLE-specific risk prediction tools. Future directions should focus on identifying specific immune mechanisms, developing targeted immunomodulatory therapies, and establishing improved risk stratification strategies to enable early intervention and improve long-term outcomes for patients with SLE.

[This corrects the article DOI: 10.3389/fimmu.2016.00557.].

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that can lead to clinical manifestations of systemic diseases. Its leading features include chronic synovial inflammation and degeneration of the bones and joints. In the past decades, multiple susceptibilities for rheumatoid arthritis have been identified along with the development of a remarkable variety of drugs for its treatment; which include analgesics, glucocorticoids, nonsteroidal anti-inflammatory medications (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and biologic response modifiers (bDMARDs). Despite the existence of many clinical treatment options, the prognosis of some patients remains poor due to complex mechanism of the disease. Programmed cell death (PCD) has been extensively studied and ascertained to be one of the essential pathological mechanisms of RA. Its dysregulation in various associated cell types contributes to the development of RA. In this review, we summarize the role of apoptosis, cell death-associated neutrophil extracellular trap formation, necroptosis, pyroptosis, and autophagy in the pathophysiology of RA to provide a theoretical reference and insightful direction to the discovery and development of novel therapeutic targets for RA.

Background: Neutrophils are one of the major early role players in antimycobacterial immunity. Upon infection, neutrophils can undergo NETosis, a cell death characterized by release of neutrophil extracellular traps (NETs). The role of NETosis in TB progression remains poorly characterized. We aim to characterize mechanisms underlying NETosis during TB pathogenesis by identifying genes that drive the cell death, and to determine their potential as markers of disease progression in high-risk individuals. Finally, we intend to evaluate neutrophil associated genes as targets for host directed therapy to reduce pathological damage caused by NETosis. Methods: Quantitative PCR will be used to quantify expression of specific genes identified in the blood of individuals with active lung disease (n=30), compared to those from healthy (n=30) and latently infected individuals (LTBI) (n=30). In addition, temporal events associated with NETosis will be measured using live microscopy in a neutrophil in vitro model of Mycobacterium tuberculosis (Mtb) infection. Candidate genes found to be associated with NETosis will be targeted with pharmaceutical inhibitors. Conclusion: Genes associated with neutrophil mediated cell death may serve as potential biomarkers of pathological damage and disease progression, as well as targets for host-directed therapy.

Evidence shows that neutrophils can protect the host against pathogens in multiple ways, including the formation and release of neutrophil extracellular traps (NETs). NETs are web‐like structures composed of fibers, DNA, histones, and various neutrophil granule proteins. NETs can capture and kill pathogens, including bacteria, viruses, fungi, and protozoa. The process of NET formation is called NETosis. According to whether they depend on nicotinamide adenine dinucleotide phosphate (NADPH), NETosis can be divided into two categories: “suicidal” NETosis and “vital” NETosis. However, NET components, including neutrophil elastase, myeloperoxidase, and cell‐free DNA, cause a proinflammatory response and potentially severe diseases. Compelling evidence indicates a link between NETs and the pathogenesis of a number of diseases, including sepsis, systemic lupus erythematosus, rheumatoid arthritis, small‐vessel vasculitis, inflammatory bowel disease, cancer, COVID‐19, and others. Therefore, targeting the process and products of NETosis is critical for treating diseases linked with NETosis. Researchers have discovered that several NET inhibitors, such as toll‐like receptor inhibitors and reactive oxygen species scavengers, can prevent uncontrolled NET development. This review summarizes the mechanism of NETosis, the receptors associated with NETosis, the pathology of NETosis‐induced diseases, and NETosis‐targeted therapy. NETs are involved in the pathogenesis and progression of various diseases, such as sepsis, SLE, RA, SVV, IBD, cancer, and COVID‐19. Components of NETs may act as autoantigens, leading to inflammation and autoimmune diseases. In addition, some diseases aggravate NETosis and cause a vicious circle.

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy.