COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

BACKGROUND: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. METHODS: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (N=24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. RESULTS: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. CONCLUSIONS: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

Suggested role for neutrophil extracellular trap formation in Ewing sarcoma immune microenvironment.

Ewing sarcoma (EWS) is a highly aggressive cancer with a survival rate of 70%-80% for patients with localized disease and under 30% for those with metastatic disease. Tumor-infiltrating neutrophils (TIN) can generate extracellular net-like DNA structures known as neutrophil extracellular traps (NETs). However, little is known about the presence and prognostic significance of tumor-infiltrating NETs in EWS. Herein, we investigated 46 patients diagnosed with EWS and treated in the Tel Aviv Medical Center between 2010 and 2021. TINs and NETs were identified in diagnostic biopsies of EWS by immunofluorescence. In addition, NETs were investigated in neutrophils isolated from peripheral blood samples of EWS patients at diagnosis and following neoadjuvant chemotherapy. The relationships between the presence of TINs and NETs, pathological and clinical features, and outcomes were analyzed. Our results demonstrate that TIN and NETs at diagnosis were higher in EWS patients with metastatic disease compared with those with local disease. High NET formation at diagnosis predicted poor response to neoadjuvant chemotherapy, relapse, and death from disease (p < 0.05). NET formation in peripheral blood samples at diagnosis was significantly elevated among patients with EWS compared with pediatric controls and decreased significantly following neoadjuvant chemotherapy. In conclusion, NET formation seems to have a role in the EWS immune microenvironment. Their presence can refine risk stratification, predict chemotherapy resistance and survival, and serve as a therapeutic target in patients with EWS.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Calpain-1 weakens the nuclear envelope and promotes the release of neutrophil extracellular traps.

The inducers of neutrophil extracellular trap (NET) formation are heterogeneous and consequently, there is no specific pathway or signature molecule indispensable for NET formation. But certain events such as histone modification, chromatin decondensation, nuclear envelope breakdown, and NET release are ubiquitous. During NET formation, neutrophils drastically rearrange their cytoplasmic, granular and nuclear content. Yet, the exact mechanism for decoding each step during NET formation still remains elusive. Here, we investigated the mechanism of nuclear envelope breakdown during NET formation. Immunofluorescence microscopic evaluation revealed a gradual disintegration of outer nuclear membrane protein nesprin-1 and alterations in nuclear morphology during NET formation. MALDI-TOF analysis of NETs that had been generated by various inducers detected the accumulation of nesprin-1 fragments. This suggests that nesprin-1 degradation occurs before NET release. In the presence of a calpain-1, inhibitor nesprin-1 degradation was decreased in calcium driven NET formation. Microscopic evaluation confirmed that the disintegration of the lamin B receptor (LBR) and the collapse of the actin cytoskeleton occurs in early and later phases of NET release, respectively. We conclude that the calpain-1 degrades nesprin-1, orchestrates the weakening of the nuclear membrane, contributes to LBR disintegration, and promoting DNA release and finally, NETs formation.

The role of neutrophil extracellular traps in ß-methylamino L-alanine-induced liver injury in mice.

The non-protein amino acid ß-N-methylamino-L-alanine (BMAA), produced by cyanobacteria, has been recognized as a neurotoxin. L-serine as an antagonist of BMAA can effectively alleviate BMAA-induced neurotoxicity. Although BMAA has long been emphasized as a neurotoxin, with the emergence of BMAA detected in a variety of algae in freshwater around the world and its clear biological enrichment effect, it is particularly important to study the non-neurotoxic adverse effects of BMAA. However, there is only limited evidence to support the ability of BMAA to cause oxidative damage in the liver. The exact molecular mechanism of BMAA-induced liver injury is still unclear. The formation of neutrophil extracellular traps (NETs) is a 'double-edged sword' for the organism, excessive formation of NETs is associated with inflammatory diseases of the liver. Our results innovatively confirmed that BMAA was able to cause the formation of NETs in the liver during the liver injury. The possible mechanism may associated with the regulation of ERK/p38 and cGAS/STING signaling pathways. The massive formation of NETs was able to exacerbate the BMAA-induced oxidative stress and release of inflammatory factors in the mice liver. And the removal of NETs could alleviate this injury. This article will bring a new laboratory evidence for BMAA-induced non-neurotoxicity and immunotoxicity.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

Mitochondrial Plasticity and Glucose Metabolic Alterations in Human Cancer under Oxidative Stress-From Viewpoints of Chronic Inflammation and Neutrophil Extracellular Traps (NETs).

Oxidative stress elicited by reactive oxygen species (ROS) and chronic inflammation are involved both in deterring and the generation/progression of human cancers. Exogenous ROS can injure mitochondria and induce them to generate more endogenous mitochondrial ROS to further perpetuate the deteriorating condition in the affected cells. Dysfunction of these cancer mitochondria may possibly be offset by the Warburg effect, which is characterized by amplified glycolysis and metabolic reprogramming. ROS from neutrophil extracellular traps (NETs) are an essential element for neutrophils to defend against invading pathogens or to kill cancer cells. A chronic inflammation typically includes consecutive NET activation and tissue damage, as well as tissue repair, and together with NETs, ROS would participate in both the destruction and progression of cancers. This review discusses human mitochondrial plasticity and the glucose metabolic reprogramming of cancer cells confronting oxidative stress by the means of chronic inflammation and neutrophil extracellular traps (NETs).

The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Neutrophil Extracellular Traps in Cardiovascular and Aortic Disease: A Narrative Review on Molecular Mechanisms and Therapeutic Targeting.

Neutrophil extracellular traps (NETs), composed of DNA, histones, and antimicrobial proteins, are released by neutrophils in response to pathogens but are also recognized for their involvement in a range of pathological processes, including autoimmune diseases, cancer, and cardiovascular diseases. This review explores the intricate roles of NETs in different cardiovascular conditions such as thrombosis, atherosclerosis, myocardial infarction, COVID-19, and particularly in the pathogenesis of abdominal aortic aneurysms. We elucidate the mechanisms underlying NET formation and function, provide a foundational understanding of their biological significance, and highlight the contribution of NETs to inflammation, thrombosis, and tissue remodeling in vascular disease. Therapeutic strategies for preventing NET release are compared with approaches targeting components of formed NETs in cardiovascular disease. Current limitations and potential avenues for clinical translation of anti-NET treatments are discussed.

IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury.

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Parvovirus B19 Infection Is Associated with the Formation of Neutrophil Extracellular Traps and Thrombosis: A Possible Linkage of the VP1 Unique Region.

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.

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.

NETosis-Inspired Cell Surface-Constrained Framework Nucleic Acids Traps (FNATs) for Cascaded Extracellular Recognition and Cellular Behavior Modulation.

Upon pathogenic stimulation, activated neutrophils release nuclear DNA into the extracellular environment, forming web-like DNA structures known as neutrophil extracellular traps (NETs), which capture and kill bacteria, fungi, and cancer cells. This phenomenon is commonly referred to as NETosis. Inspired by this, we introduce a cell surface-constrained web-like framework nucleic acids traps (FNATs) with programmable extracellular recognition capability and cellular behavior modulation. This approach facilitates dynamic key chemical signaling molecule recognition such as adenosine triphosphate (ATP), which is elevated in the extracellular microenvironment, and triggers FNA self-assembly. This, in turn, leads to in situ tightly interwoven FNAs formation on the cell surface, thereby inhibiting target cell migration. Furthermore, it activates a photosensitizer-capturing switch, chlorin e6 (Ce6), and induces cell self-destruction. This cascade platform provides new potential tools for visualizing dynamic extracellular activities and manipulating cellular behaviors using programmable in situ self-assembling DNA molecular devices.

Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis.

BACKGROUND & AIMS: In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH. METHODS: We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics. RESULTS: We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release in vitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs in vivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice. CONCLUSION: Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH. IMPACT AND IMPLICATIONS: In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

Platelet-derived extracellular vesicles promote endothelial dysfunction in sepsis by enhancing neutrophil extracellular traps.

BACKGROUND: The role of platelet-derived extracellular vesicles (PEVs) in the development of sepsis was investigated in this study. METHODS: After collection of blood samples from sepsis patients and normal volunteers, the extracellular vesicles (EVs) were separated, followed by the isolation of PEVs from the blood of rats. Next, a sepsis rat model was constructed by cecal ligation and puncture (CLP), and rats received tail vein injection of PEVs to explore the role of PEVs in sepsis. Subsequently, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were adopted to determine the diameter of EVs and observe the morphology of PEVs, respectively; flow cytometry to detect the percentage of CD41-and CD61-positive EVs in isolated EVs; and ELISA to assess neutrophil extracellular trap (NET) formation, endothelial function injury-related markers in clinical samples or rat blood and serum inflammatory factor level. RESULTS: Compared with normal volunteers, the percentage of CD41- and CD61-positive EVs and the number of EVs were significantly elevated in sepsis patients. Moreover, sepsis patients also presented notably increased histone H3, myeloperoxidase (MPO), angiopoietin-2 and endocan levels in the blood, and such increase was positively correlated with the number of EVs. Also, animal experiments demonstrated that PEVs significantly promoted NET formation, mainly manifested as up-regulation of histone H3, high mobility group protein B1 (HMGB1), and MPO; promoted endothelial dysfunction (up-regulation of angiopoietin-2, endocan, and syndecan-1); and stimulated inflammatory response (up-regulation of interleukin (IL) -1ß, IL-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP) -1) in the blood of sepsis rats. CONCLUSION: PEVs aggravate endothelial function injury and inflammatory response in sepsis by promoting NET formation.

NETosis promotes chronic inflammation and fibrosis in systemic lupus erythematosus and COVID-19.

Pulmonary fibrosis, a serious complication of systemic lupus erythematosus (SLE) and coronavirus disease 2019 (COVID-19), leads to irreversible lung damage. However, the underlying mechanism of this condition remains unclear. In this study, we revealed the landscape of transcriptional changes in lung biopsies from individuals with SLE, COVID-19-induced pulmonary fibrosis, and idiopathic pulmonary fibrosis (IPF) using histopathology and RNA sequencing, respectively. Despite the diverse etiologies of these diseases, lung expression of matrix metalloproteinase genes in these diseases showed similar patterns. Particularly, the differentially expressed genes were significantly enriched in the pathway of neutrophil extracellular trap formation, showing similar enrichment signature between SLE and COVID-19. The abundance of Neutrophil extracellular traps (NETs) was much higher in the lungs of individuals with SLE and COVID-19 compared to those with IPF. In-depth transcriptome analyses revealed that NETs formation pathway promotes epithelial-mesenchymal transition (EMT). Furthermore, stimulation with NETs significantly up-regulated α-SMA, Twist, Snail protein expression, while decreasing the expression of E-cadherin protein in vitro. This indicates that NETosis promotes EMT in lung epithelial cells. Given drugs that are efficacious in degrading damaged NETs or inhibiting NETs production, we identified a few drug targets that were aberrantly expressed in both SLE and COVID-19. Among these targets, the JAK2 inhibitor Tofacitinib could effectively disrupted the process of NETs and reversed NET-induced EMT in lung epithelial cells. These findings support that the NETs/EMT axis, activated by SLE and COVID-19, contributes to the progression of pulmonary fibrosis. Our study also highlights that JAK2 as a potential target for the treatment of fibrosis in these diseases.

Interferon alpha-2 treatment reduces circulating neutrophil extracellular trap levels in myeloproliferative neoplasms.

Neutrophil extracellular traps (NETs) may play a pathogenic role in the thrombosis associated with myeloproliferative neoplasms (MPNs). We measured serum NET levels in 128 pretreatment samples from patients with MPNs and in 85 samples taken after 12 months of treatment with interferon alpha-2 (PEG-IFNα-2) formulations or hydroxyurea (HU). No differences in NET levels were observed across subdiagnoses or phenotypic driver mutations. In PV, a JAK2V617F+ allele burden ≥50% associated with increased NET levels (p = 0.006). Baseline NET levels correlated with neutrophil count (r = 0.29, p = 0.001), neutrophil-to-lymphocyte ratio (r = 0.26, p = 0.004) and JAK2V617F allele burden (r = 0.22, p = 0.03), particularly in patients with PV and with allele burden ≥50% (r = 0.50, p = 0.01, r = 0.56, p = 0.002 and r = 0.45, p = 0.03 respectively). In PV, after 12 months of treatment, NET levels decreased on average by 60% in patients with allele burden ≥50%, compared to only 36% in patients with an allele burden <50%. Overall, treatment with PEG-IFNα-2a or PEG-IFNα-2b reduced NETs levels in 77% and 73% of patients, respectively, versus only 53% of HU-treated patients (average decrease across treatments: 48%). Normalization of blood counts did not per se account for these reductions. In conclusion, baseline NET levels correlated with neutrophil count, NLR and JAK2V617F allele burden, and IFNα was more effective at reducing prothrombotic NET levels than HU.

Prognostic analysis and validation of lncRNAs in bladder cancer on the basis of neutrophil extracellular traps.

BACKGROUND: Complex interactions in the tumor microenvironment (TME) between bladder cancer (BLCA) and immune cells are critical for cancer progression. However, studies of neutrophil extracellular trap-associated long non-coding RNAs (NET-lncRNAs) in the TME of BLCA have not been reported. This study aims to screen for NET-lncRNAs in BLCA and to preliminarily explore their effects on BLCA development. METHODS: The correlation of NET-related gene sets, which were identified from the cancer genome atlas (TCGA) BLCA datasets, with lncRNAs was analyzed and the prognosis-related genes were identified through random forest analysis. The least absolute shrinkage and selection operator (LASSO) model was utilized to obtain prognostic risk scores for NET-lncRNAs (NET-Score). We collected clinical BLCA samples, as well as SV-HUC-1 and BLCA cells, to validate the expression of NET-lncRNAs. Survival and independent prognostic analysis were performed. In J82 and UM-UC-3 cells, after NKILA expression was inhibited, cell proliferation and apoptosis levels were detected. RESULTS: NET-related gene sets mainly included CREB5, MMP9, PADI4, CRISPLD2, CD93, DYSF, MAPK3, TECPR2, MAPK1 and PIK3CA. Then, four NET-lncRNAs, MAP 3 K4-AS1, MIR100HG, NKILA and THY1-AS1, were identified. NET-Score had the highest hazard ratio for BLCA. An elevated NET-Score was linked to a significant increase in immune cell infiltration and copy number variation, as well as a notable decrease in survival rate and drug sensitivity. NET-lncRNA-related genes were mainly enriched in the pathways of angiogenesis, immune response, cell cycle and T cell activation. MAP 3 K4-AS1, MIR100HG, NKILA and THY1-AS1 expressions were significantly increased in BLCA tissues. Compared with SV-HUC-1 cells, NKILA expression was elevated in J82 and UM-UC-3 cells. Inhibition of NKILA expression inhibited the proliferation and promoted apoptosis of J82 and UM-UC-3 cells. CONCLUSIONS: Several NET-lncRNAs, including MAP 3 K4-AS1, MIR100HG, NKILA and THY1-AS1, were successfully screened in the BLCA. The NET-Score was an independent prognostic factor for BLCA. In addition, inhibition of NKILA expression suppressed BLCA cell development. The above NET-lncRNAs could serve as potential prognostic markers and targets in BLCA.

Taxifolin inhibits NETosis through activation of Nrf2 and provides protective effects in models of lupus and antiphospholipid syndrome.

OBJECTIVES: Taxifolin (dihydroquercetin) is a bioactive plant flavonoid that exhibits anti-inflammatory and anti-oxidative properties. We hypothesized that taxifolin might be an effective dietary supplement to ameliorate symptoms arising from thrombo-inflammatory diseases such as lupus and antiphospholipid syndrome (APS). METHODS: We used in vitro assays and a mouse model to determine mechanisms by which taxifolin inhibits neutrophil extracellular trap (NET) formation (i.e., NETosis) and venous thrombosis in lupus and APS. RESULTS: At doses ranging from 0.1 to 1 µg/ml, taxifolin inhibited NETosis from control neutrophils stimulated with autoantibodies isolated from lupus and APS patients, and its suppressive effects were mitigated by blocking the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Furthermore, taxifolin at a dose as low as 20 mg/kg/day reduced in vivo NETosis in thrombo-inflammatory mouse models of lupus and APS while also significantly attenuating autoantibody formation, inflammatory cytokine production, and large-vein thrombosis. CONCLUSION: Our study is the first to demonstrate the protective effects of taxifolin in the context of lupus and APS. Importantly, our study also suggests a therapeutic potential to neutralize neutrophil hyperactivity and NETosis that could have relevance to a variety of thrombo-inflammatory diseases.