The role of platelets in central hubs of inflammation: A literature review.

Platelets are increasingly recognized for their multifaceted roles in inflammation beyond their traditional involvement in haemostasis. This review consolidates knowledge on platelets as critical players in inflammatory responses. This study did an extensive search of electronic databases and identified studies on platelets in inflammation, focusing on molecular mechanisms, cell interactions, and clinical implications, emphasizing recent publications. Platelets contribute to inflammation via surface receptors, release of mediators, and participation in neutrophil extracellular trap formation. They are implicated in diseases like atherosclerosis, rheumatoid arthritis, and sepsis, highlighting their interaction with immune cells as pivotal in the onset and resolution of inflammation. Platelets are central to regulating inflammation, offering new therapeutic targets for inflammatory diseases. Future research should explore specific molecular pathways of platelets in inflammation for therapeutic intervention.

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

The Bifunctional Effects of Lactoferrin (LFcinB11) in Inhibiting Neural Cell Adhesive Molecule (NCAM) Polysialylation and the Release of Neutrophil Extracellular Traps (NETs).

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. Thus, it is important to select a proper drug to block tumor cell migration during clinical treatment. In this study, we proposed that lactoferrin (LFcinB11) may be a better candidate for inhibiting NCAM polysialylation when compared with CMP and low-molecular-weight heparin (LMWH), which were determined based on our NMR studies. Furthermore, neutrophil extracellular traps (NETs) represent the most dramatic stage in the cell death process, and the release of NETs is related to the pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. In this study, the molecular mechanisms involved in the inhibition of NET release using LFcinB11 as an inhibitor were also determined. Based on these results, LFcinB11 is proposed as being a bifunctional inhibitor for inhibiting both NCAM polysialylation and the release of NETs.

CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1-CXCR2 induced NETs.

BACKGROUND: CD276 (B7-H3), a pivotal immune checkpoint, facilitates tumorigenicity, invasiveness, and metastasis by escaping immune surveillance in a variety of tumors; however, the underlying mechanisms facilitating immune escape in esophageal squamous cell carcinoma (ESCC) remain enigmatic. METHODS: We investigated the expression of CD276 in ESCC tissues from patients by using immunohistochemistry (IHC) assays. In vivo, we established a 4-nitroquinoline 1-oxide (4NQO)-induced CD276 knockout (CD276wKO) and K14cre; CD276 conditional knockout (CD276cKO) mouse model of ESCC to study the functional role of CD276 in ESCC. Furthermore, we used the 4NQO-induced mouse model to evaluate the effects of anti-CXCL1 antibodies, anti-Ly6G antibodies, anti-NK1.1 antibodies, and GSK484 inhibitors on tumor growth. Moreover, IHC, flow cytometry, and immunofluorescence techniques were employed to measure immune cell proportions in ESCC. In addition, we conducted single-cell RNA sequencing analysis to examine the alterations in tumor microenvironment following CD276 depletion. RESULTS: In this study, we elucidate that CD276 is markedly upregulated in ESCC, correlating with poor prognosis. In vivo, our results indicate that depletion of CD276 inhibits tumorigenesis and progression of ESCC. Furthermore, conditional knockout of CD276 in epithelial cells engenders a significant downregulation of CXCL1, consequently reducing the formation of neutrophil extracellular trap networks (NETs) via the CXCL1-CXCR2 signaling axis, while simultaneously augmenting natural killer (NK) cells. In addition, overexpression of CD276 promotes tumorigenesis via increasing NETs' formation and reducing NK cells in vivo. CONCLUSIONS: This study successfully elucidates the functional role of CD276 in ESCC. Our comprehensive analysis uncovers the significant role of CD276 in modulating immune surveillance mechanisms in ESCC, thereby suggesting that targeting CD276 might serve as a potential therapeutic approach for ESCC treatment.

NET-Related Gene as Potential Diagnostic Biomarkers for Diabetic Tubulointerstitial Injury.

Background: Tubulointerstitial injury plays a pivotal role in the progression of diabetic kidney disease (DKD), yet the link between neutrophil extracellular traps (NETs) and diabetic tubulointerstitial injury is still unclear. Methods: We analyzed microarray data (GSE30122) from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) associated with DKD's tubulointerstitial injury. Functional and pathway enrichment analyses were conducted to elucidate the involved biological processes (BP) and pathways. Weighted gene coexpression network analysis (WGCNA) identified modules associated with DKD. LASSO regression and random forest selected NET-related characteristic genes (NRGs) related to DKD tubulointerstitial injury. Results: Eight hundred ninety-eight DEGs were identified from the GSE30122 dataset. A significant module associated with diabetic tubulointerstitial injury overlapped with 15 NRGs. The hub genes, CASP1 and LYZ, were identified as potential biomarkers. Functional enrichment linked these genes with immune cell trafficking, metabolic alterations, and inflammatory responses. NRGs negatively correlated with glomerular filtration rate (GFR) in the Neph v5 database. Immunohistochemistry (IHC) validated increased NRGs in DKD tubulointerstitial injury. Conclusion: Our findings suggest that the CASP1 and LYZ genes may serve as potential diagnostic biomarkers for diabetic tubulointerstitial injury. Furthermore, NRGs involved in diabetic tubulointerstitial injury could emerge as prospective targets for the diagnosis and treatment of DKD.

Killer Function of Circulating Neutrophils in Relation to Cytokines in Uterine Myoma and Endometrial Cancer.

The study presents the killer functions of circulating neutrophils: myeloperoxidase activity, the ability to generate ROS, phagocytic activity, receptor status, NETosis, as well as the level of cytokines IL-2, IL-4, IL-6, IL-17A, and IL-18, granulocyte CSF, monocyte chemotactic protein 1, and neutrophil elastase in the serum of patients with uterine myoma and endometrial cancer (FIGO stages I-III). The phagocytic ability of neutrophils in uterine myoma was influenced by serum levels of granulocyte CSF and IL-2 in 54% of the total variance. The degranulation ability of neutrophils in endometrial cancer was determined by circulating IL-18 in 50% of the total variance. In uterine myoma, 66% of the total variance in neutrophil myeloperoxidase activity was explained by a model dependent on blood levels of IL-17A, IL-6, and IL-4. The risk of endometrial cancer increases when elevated levels of monocyte chemotactic protein 1 in circulating neutrophils are associated with reduced ability to capture particles via extracellular traps (96% probability).

Neutrophil extracellular traps formation may be involved in the association of propranolol with the development of portal vein thrombosis.

BACKGROUND & AIMS: Nonselective ß blockers (NSBBs) facilitate the development of portal vein thrombosis (PVT) in liver cirrhosis. Considering the potential effect of NSBBs on neutrophils and neutrophil extracellular traps (NETs), we speculated that NSBBs might promote the development of PVT by stimulating neutrophils to release NETs. MATERIALS AND METHODS: Serum NETs biomarkers were measured, use of NSBBs was recorded, and PVT was evaluated in cirrhotic patients. Carbon tetrachloride and ferric chloride (FeCl3) were used to induce liver fibrosis and PVT in mice, respectively. After treatment with propranolol and DNase I, neutrophils in peripheral blood, colocalization and expression of NETs in PVT specimens, and NETs biomarkers in serum were measured. Ex vivo clots lysis analysis was performed and portal vein velocity and coagulation parameters were tested. RESULTS: Serum MPO-DNA level was significantly higher in cirrhotic patients treated with NSBBs, and serum H3Cit and MPO-DNA levels were significantly higher in those with PVT. In fibrotic mice, following treatment with propranolol, DNase I significantly shortened the time of FeCl3-induced PVT formation, lowered the peripheral blood neutrophils labelled by CD11b/Ly6G, inhibited the positive staining of H3Cit and the expression of H3Cit and MPO proteins in PVT tissues, and reduced serum nucleosome level. Furthermore, the addition of DNase I to tissue plasminogen activator (tPA) significantly accelerated clots lysis as compared with tPA alone. Propranolol reduced portal vein velocity in fibrotic mice, but did not influence coagulation parameters. CONCLUSION: Our study provides a clue to the potential impact of NETs formation on the association of NSBBs with the development of PVT.

Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophil extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. METHODS: We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. RESULTS: We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. CONCLUSION: In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.

[Expression of neutrophil extracellular traps and phagocytic functions among patients with hepatic alveolar echinococcosis].

OBJECTIVE: To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions. METHODS: A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated in vitro using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients. RESULTS: The peripheral blood plasma MPOï¼»(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; t = 10.28, P < 0.05ï¼½, NEï¼»(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; t = 10.65, P < 0.05ï¼½and CitH3 levelsï¼»(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; t = 4.34, P < 0.05ï¼½were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controlsï¼»(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; t = 20.18, P < 0.05ï¼½, and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following in vitro PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level (rs = -0.515 to -0.392, all P values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 (rs = 0.333 to 0.445, all P values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients (rs = 0.290 to 0.628, all P values < 0.001). CONCLUSIONS: Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.

Neutrophil extracellular traps as a unique target in the treatment of inflammatory pain.

Pain is a widespread motivation for seeking healthcare and stands as a substantial global public health concern. Despite comprehensive investigations into the mechanisms of pain sensitization induced by inflammation, efficacious treatments options remain scarce. Neutrophil extracellular traps (NETs) have been associated with the progression and tissue damage of diverse inflammatory diseases. This study aims to explore the impact of NETs on the progression of inflammatory pain and explore potential therapeutic approaches. Initially, we observed neutrophil infiltration and the formation of NETs in the left hind paw of mice with inflammatory pain induced by complete Freund's adjuvant (CFA). Furthermore, we employed the peptidyl arginine deiminase 4 (PAD4) inhibitor Cl-amidine (diluted at 50 mg/kg in saline, administered via tail vein injection once daily for three days) to impede NETs formation and administered DNase1 (diluted at 10 mg/kg in saline, once daily for three days) to break down NETs. We investigated the pathological importance of peripheral NETs formation in inflammatory pain and its influence on the activation of spinal dorsal horn microglia. The findings indicate that neutrophils infiltrating locally generate NETs, leading to an increased release of inflammatory mediators that worsen peripheral inflammatory reactions. Consequently, this results in the transmission of more harmful peripheral stimuli to the spinal cord, triggering microglial activation and NF-κB phosphorylation, thereby escalating neuroinflammation and fostering pain sensitization. Suppression of peripheral NETs can mitigate peripheral inflammation in mice with inflammatory pain, reverse mechanical and thermal hypersensitivity by suppressing microglial activation in the spinal cord, ultimately diminishing inflammatory pain. In conclusion, these discoveries propose that obstructing or intervening with NETs introduces a novel therapeutic avenue for addressing inflammatory pain.

Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

NETosis in tumour microenvironment of liver: From primary to metastatic hepatic carcinoma.

BACKGROUND: Hepatocellular carcinoma is a common and highly lethal tumour. The tumour microenvironment (TME) plays an important role in the progression and metastasis of hepatocellular carcinoma (HCC). A cell death mechanism, termed NETosis, has been found to play an important role in the TME of HCC. SUMMARY: This review article focuses on the role of NETosis in the TME of HCC, a novel form of cell death in which neutrophils capture and kill microorganisms by releasing a type of DNA meshwork fibres called "NETs". This process is associated with neutrophil activation, local inflammation and cytokines. The study suggests that NETs play a multifaceted role in the development and metastasis of HCC. The article also discusses the role of NETs in tumour proliferation and metastasis, epithelial-mesenchymal transition (EMT), and surgical stress. In addition, the article discusses the interaction of NETosis with other immune cells in the TME and related therapeutic strategies. A deeper understanding of NETosis can help us better understand the complexity of the immune system and provide a new therapeutic basis for the treatment and prevention of HCC. KEY INFORMATION: In conclusion, NETosis is important in the TME of liver. NETs have been shown to contribute to the progression and metastasis of liver cancer. The interaction between NETosis and immune cells in the TME, as well as related therapies, are important areas of research.

A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis.

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

Modelling the Impact of NETosis During the Initial Stage of Systemic Lupus Erythematosus.

The development of autoimmune diseases often takes years before clinical symptoms become detectable. We propose a mathematical model for the immune response during the initial stage of Systemic Lupus Erythematosus which models the process of aberrant apoptosis and activation of macrophages and neutrophils. NETosis is a type of cell death characterised by the release of neutrophil extracellular traps, or NETs, containing material from the neutrophil's nucleus, in response to a pathogenic stimulus. This process is hypothesised to contribute to the development of autoimmunogenicity in SLE. The aim of this work is to study how NETosis contributes to the establishment of persistent autoantigen production by analysing the steady states and the asymptotic dynamics of the model by numerical experiment.

Extracellular traps development in canine neutrophils induced by infective stage Toxocara canis larvae.

Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae.

Neutrophil extracellular traps (NETs) and fibrotic diseases.

Fibrosis, a common cause and serious outcome of organ failure that can affect any organ, is responsible for up to 45% of all deaths in various clinical settings. Both preclinical models and clinical trials investigating various organ systems have shown that fibrosis is a highly dynamic process. Although many studies have sought to gain understanding of the mechanism of fibrosis progression, their findings have been mixed. In recent years, increasing evidence indicates that neutrophil extracellular traps (NETs) are involved in many inflammatory and autoimmune disorders and participate in the regulation of fibrotic processes in various organs and systems. In this review, we summarize the current understanding of the role of NETs in fibrosis development and progression and their possibility as therapeutic targets.

Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration.

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.