Neutrophil Neutrophil Extracellular Traps Research Articles

LncRNA-mRNA co-expression analysis reveals aquaporin-9-promoted neutrophil extracellular trap formation and inflammatory activation in sepsis

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.

Astilbin inhibited neutrophil extracellular traps in gouty arthritis through suppression of purinergic P2Y6 receptor

BackgroundGouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated. PurposeThis study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo. Study designFirstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway. MethodsMSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting. ResultsUnder MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R. ConclusionThis study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.

Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.

Neutrophil Virucidal Activity Against SARS-CoV-2 Is Mediated by Neutrophil Extracellular Traps.

Inflammation in the lungs and other vital organs in COVID-19 is characterized by the presence of neutrophils and a high concentration of neutrophil extracellular traps (NETs), which seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells and what the consequence of NETs degradation would be in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. Here, by immunofluorescence microscopy, we observed that viral particles colocalize with NETs in neutrophils isolated from patients with COVID-19 or healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 hours of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice, we observed a higher viral load in animals treated with DNase I. However, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

EQUOL, A SOYBEAN METABOLITE WITH ESTROGEN-LIKE FUNCTIONS, DECREASES LIPOPOLYSACCHARIDE-INDUCED HUMAN NEUTROPHIL EXTRACELLULAR TRAPS IN VITRO.

Objective: Neutrophil extracellular traps (NETs) defend against acute infections. However, their overexpression causes organ failure during sepsis. Control of NET formation may improve the outcomes of patients with sepsis. Equol, a soybean isoflavone, is a female hormone analog, which prevents inflammation. We evaluated the effects of equol on NET formation in human neutrophils during inflammatory stimulation in vitro . Methods: Healthy volunteers provided blood samples. An enzyme-linked immunosorbent assay assessed serum equol concentrations. Neutrophil extracellular trap formation in neutrophils was induced by lipopolysaccharide treatment. Enzyme-linked immunosorbent assay quantified DNA-binding elastase, and immunostaining assessed NET formation. Reverse-transcription quantitative polymerase chain reaction and Western blotting detected G-protein-coupled receptor 30 (GPR30) or peptidyl arginine deiminase 4 (PAD4) expression. Flow cytometry assessed neutrophil phagocytic ability with inactivated Escherichia coli . Results: In neutrophils derived from males with low-serum equol levels (low-serum equol group), equol significantly decreased DNA-binding elastase levels and NET formation. Equol did not decrease NETs in neutrophils from males with high-serum equol levels. GPR30 expression of neutrophils was higher in the low-serum than in the high-serum equol group. PAD4 mRNA levels and nuclear PAD4 protein expression also decreased more than the vehicle control in the low-serum equol group. Equol did not alter the phagocytic ability of neutrophils. In neutrophils from young females, equol had no inhibitory effect on NET formation. Conclusions: Equol decreases lipopolysaccharide-induced NET formation in neutrophils from males via inhibition of PAD4 expression. Our findings provide a rationale for investigating a new therapeutic approach using equol to control neutrophil activity during sepsis.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis

IntroductionRheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. ObjectivesTo evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. MethodsThe effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. ResultsWe found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. ConclusionOur results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses.

Neutrophils display an array of biological functions including the formation of neutrophil extracellular traps (NETs), web-like structures specialized in trapping, neutralizing, killing and preventing microbial dissemination within the host. However, NETs contribute to a number of inflammatory pathologies, including severe equine asthma. Tamoxifen (TX) is a selective estrogen receptor modulator which belongs to the triphenylethyllenes group of molecules, and which is used as a treatment in all stages of estrogen-positive human breast cancer. Our previous results suggest that tamoxifen can modulate neutrophil functionality and promote resolution of inflammation; this would partly explain the clinical beneficial effect of this drug in horses with airway inflammation. Enhanced NETs production has been reported with tamoxifen use in humans, but minimal data exists regarding the drug's effect on NETs in horses. The aim of this study is to assess the in vitro effect of TX on NETs formation from peripheral blood of healthy horses. Five clinically healthy mixed-breed adult horses were enrolled in the study. For this, cellular free DNA quantification, immunofluorescence for the visualization of NETs, assessment of different types of NETs, and detection of mitochondrial superoxide. TX induced NETs formation at a concentration of 10 uM. Our results show that only two types of NETs were induced by TX: 95% spread NETs (sprNETs) and 5% aggregated NETs (aggNETs). Furthermore, induction of these NETs could be influenced by mitochondrial ROS. Future research should involve an In vivo study of horses with severe asthma and TX treatment, to evaluate BALF neutrophil NET formation. In conclusion, this in vitro study suggests that the resolution of inflammation by TX in horses with airway inflammation is due to inhibition of other neutrophilic functions but not to NET formation.

Effects and mechanisms of Celastrol on the formation of neutrophil extracellular traps (NETs).

To investigate the effect and mechanism of Celastrol on the formation of neutrophil extracellular traps (NETs), and to provide a theoretical basis for the clinical application of Tripterygium wilfordii. First, we isolated neutrophils from the peripheral blood of healthy volunteers, and then observed the effect of Celastrol on Phorbol Myristate Acetate (PMA)-induced neutrophil release of NETs. The level of NETs was detected by using the membrane-impermeable nucleic acid dye, SytoxGreens. In addition, the levels of reactive oxygen species (ROS) were also examined to determine whether Celastrol affects ROS production during PMA-induced NETs. Celastrol produced significant cytotoxicity at a concentration of 5 µM (213.2±75.07), and the effect of stimulant PMA (25 nM) treatment was not statistically different (197.3±25.15) (P=0.9167). Celastrol (1.25, 0.625, and 0.3125 µM) did not exhibit cytotoxicity when treating neutrophils. Compared with the PMA (25 nM) + Celastrol (1.25, 0.625, and 0.3125 µM) group and the PMA (25 nM) monotherapy group, SytoxGreen showed a statistically significant reduction in fluorescence at 528 µM under 485 µM light excitation. Also, under the co-localization marker of Hochest and SytoxGreen double staining, we observed that the release of NETs in the PMA-treated group was higher than that in the control group. The PMA-induced neutrophil release of NETs was markedly reduced compared to the PMA-treated group. The NET release was substantially decreased under double staining with the Hochest and SytoxGreen co-localization markers. The fluorescence intensity of the Celastrol plus PMA group was significantly lower than that of the PMA treatment group alone, indicating a decrease in the level of intracellular ROS. Interestingly, the level of ROS in the treatment group who received Celastrol alone was lower than that in the control group, indicating that Tripterygium wilfordii could inhibit the spontaneous production of ROS by neutrophils in the absence of stimulation. The molecular mechanism of Celastrol involves inhibition of PMA-stimulated neutrophil NETs formation in vitro, which is possibly related to the reduction of ROS levels. This indicates that Celastrol, the main component in Tripterygium wilfordii, can inhibit the formation of NETs, which provides a theoretical basis for the study of NETs-related diseases.

Tyrosine phosphatase SHP2 exacerbates psoriasis-like skin inflammation in mice via ERK5-dependent NETosis.

Psoriasis is a chronic inflammatory skin disease, often accompanied by increased infiltration of immune cells, especially neutrophils. However, the detailed mechanism of the neutrophil function in psoriasis progression remains unclear. Here, we found that both Src homology‐2 domain‐containing protein tyrosine phosphatase‐2 (SHP2) and neutrophils were highly correlated to developing psoriasis by single‐cell ribonucleic acid (RNA) sequencing and experiment verification. The deficiency of SHP2 in neutrophils significantly alleviated psoriasis‐like phenotype in an imiquimod‐induced murine model. Interestingly, high levels of neutrophil extracellular traps (NETs) were produced in the inflamed lesions of psoriatic patients. In addition, imiquimod‐induced psoriasis‐like symptoms were remarkably ameliorated in peptidyl arginine deiminase 4 (PAD4) knockout mice, which cannot form NETs. Mechanistically, RNA‐seq analysis revealed that SHP2 promoted the formation of NETs in neutrophils via the ERK5 pathway. Functionally, this mechanism resulted in the infiltration of pro‐inflammatory cytokines such as TNF‐α, IL‐1β, IL‐6, IL‐17A, and CXCL‐15, which enhances the inflammatory response in skin lesions and reinforces the cross‐talk between neutrophils and keratinocytes, ultimately aggravating psoriasis. Our findings uncover a role for SHP2 in NET release and subsequent cell death known as NETosis in the progression of psoriasis and suggest that SHP2 may be a promising therapeutic target for psoriasis.

Human neutrophil FcγRIIIb regulates neutrophil extracellular trap release in response to electrospun polydioxanone biomaterials

During the acute inflammatory response, the release of neutrophil extracellular traps (NETs) is a pro-inflammatory, preconditioning event on a biomaterial surface. Therefore, regulation of NET release through biomaterial design is one strategy to enhance biomaterial-guided in situ tissue regeneration. In this study, IgG adsorption on electrospun polydioxanone biomaterials with differing fiber sizes was explored as a regulator of in vitro human neutrophil NET release. The propensity to release NETs was increased and decreased by modulating adsorbed IgG, suggesting a functional link between IgG and NET formation. Fiber-size dependent NET release was reduced by blocking FcγRIIIb, but not FcγRI, FcγRIIa, or Mac-1 (CD11b/CD18), indicating a specific receptor mediated neutrophil response. Inhibition of transforming growth factor-β-activated kinase 1 (TAK1), which is activated downstream of FcγRIIIb, significantly reduced the release of NETs in a fiber size-independent manner. These results indicate that in vitro electrospun biomaterial-induced NET release is largely regulated by IgG adsorption, engagement of FcγRIIIb, and signaling through TAK1. Modulation of this pathway may have beneficial therapeutic effects for regulating neutrophil-mediated inflammation by avoiding the adverse effects of NETs and increasing the potential for in situ tissue regeneration. Statement of significanceElectrospun biomaterials have great potential for in situ tissue engineering because of their versatility and biomimetic properties. However, understanding how to design the biomaterial to regulate acute inflammation, dominated by neutrophils, remains a great challenge for successful tissue integration and regeneration. In this work, we demonstrate for the first time how protein adsorption on the biomaterial surface and engagement of a specific neutrophil receptor induces intracellular signals that regulate the pro-inflammatory release of neutrophil extracellular traps (NETs). Given the deleterious effects of NETs during the acute inflammatory response to a biomaterial, our work highlights the importance of considering biomaterial-neutrophil interactions on degradable and non-degradable biomaterials to achieve the desired biological outcome.

Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release

Preterm birth is a major contributor to neonatal mortality and morbidity. While the causes of preterm birth remain incompletely understood, infection is a major risk factor, and chorioamnionitis is commonly observed. Chorioamnionitis is characterized by inflammation and neutrophil infiltration of the fetal membranes (FM). We recently reported that human FMs which had been exposed to low levels of bacterial lipopolysaccharide (LPS) recruit neutrophils and activate them, increasing their secretion of pro-inflammatory cytokines, degranulation of myeloperoxidase (MPO), and release of neutrophil extracellular traps (NETs). Herein, we demonstrate that conditioned media (CM) from viral dsRNA (Poly(I:C))-stimulated FMs also increased neutrophil migration, and induced the secretion of inflammatory IL-8 and the release of NETs. Furthermore, CM from FMs stimulated by a combination of bacterial LPS and Poly(I:C) augmented neutrophil NET release, compared to CM from FMs stimulated with either Poly(I:C) or LPS alone. NETs induced by FMs exposed to Poly(I:C), with or without LPS, were released and degraded quicker than those induced by resting or LPS-stimulated FM-CM. These findings indicate that FMs exposed to viral dsRNA promote neutrophil recruitment, activation and NET formation, similar to FMs exposed to bacterial LPS alone. However, in response to FM polymicrobial stimulation the levels and kinetics of NET release are augmented. This work builds upon our understanding of how infections at the maternal-fetal interface may affect neutrophil function.

Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues.

One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry-based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC. Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow Alternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytes Alternate Protocol 2: Detection of nuclear decondensation in neutrophils from blood Support Protocol 1: Cryopreservation and defrosting of samples Support Protocol 2: Paraformaldehyde fixation of samples.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ Tcells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism.

BackgroundSmall vessel vasculitis (SVV) is a group of systemic autoimmune diseases that are mediated by neutrophil extracellular traps (NETs) in response to cathelicidin LL37, an aging molecular marker, which could be induced by telomere dysfunction. Therefore, in this study, we evaluated the hypothesis that telomere dysfunction in neutrophils may promote SVV via an LL37-NETs-dependent mechanism.MethodsWe contrasted the release of neutrophil NETs from mice with telomere dysfunction, mice with DNA damage and wide-type mice. Neutrophil telomere length, the expression of LL37, and the formation of NETs were measured in SVV patients and healthy controls (HCs). The co-expression of γH2AX, LL37, and NETs were detected in SVV patients to evaluate the association of the immune aging of neutrophils and pro-inflammatory conditions. LL37 inhibitor was used to verify its key role in NETs release in SVV patients and DNA damage mice.ResultsWe found that NETs were over-induced by telomere dysfunction and DNA damage in mice, which may be associated with a marked increase in LL37. For patients with SVV, telomeres in neutrophils were significantly shortened, which was also associated with higher levels of LL37 and NETs. Inhibition of LL37 reduced the NETs released from neutrophils.ConclusionsTaken together, the results of these studies suggest that dysfunction of telomeres may promote SVV through the mechanism of LL37-dependent NETs. Thus, targeting the LL37-NETs may be a novel therapy for SVV.

Neutrophil extracellular traps enhance procoagulant activity in patients with oral squamous cell carcinoma.

Hypercoagulability is a major cancer-associated complication linked to poor patient prognosis. The production of neutrophil extracellular traps (NETs) is increasingly found to be linked with the development and metastasis of cancer, as well as with thrombi formation in cancer patients. We hypothesized that the neutrophil NET release may be triggered by specific cytokines in oral squamous cell carcinoma (OSCC) patients, thereby predisposing them to a hypercoagulable state. Moreover, we have evaluated the interaction between NETs and endothelial cells (ECs). NET procoagulant activity was assessed based on fibrin and purified coagulation complex production assays, as well as by measuring coagulation time (CT). We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands, and cell FVa/Xa binding. OSCC patients with stage III/IV exhibited elevated plasma NET levels compared to stage I/II or CTR (all P < 0.05). Neutrophils from OSCC patients are predisposed to amplified NET release compared to those from CTR. Furthermore, depleting IL-8, IL-6, and TNF-α led to a reduction in NET release in the plasma. OSCC NETs increased thrombin and fibrin generation and decreased CT significantly (P < 0.05). When NETs were isolated and used to treat ECs, these cells exhibited disrupted morphology by retracting from their cell-cell junctions and convert to a procoagulant phenotype. These effects could be attenuated by approximately 70% using DNase I. Our findings are consistent with a model wherein OSCC drives a systemic inflammatory state, which, in turn, drives neutrophils to prime and release NETs, which drive the development of a hypercoagulable state. Intervening in this process may be a viable means of disrupting these undesirable coagulation dynamics in stage III/IV OSCC patients.

Therapeutic Targeting of Neutrophil Extracellular Traps in Atherogenic Inflammation.

Neutrophils and neutrophil extracellular traps (NETs) have a robust relationship with atherothrombotic disease risk, which led to the idea that interfering with the release of NETs therapeutically would ameliorate atherosclerosis. In human studies, acute coronary events and the pro-thrombotic state cause markedly elevated levels of circulating deoxyribonucleic acid (DNA) and chromatin, suggesting that DNase I might produce cardiovascular benefit. DNase I reproduced the phenotype of peptidylarginine deiminase 4 (PAD4) deficiency and showed a significant benefit for atherothrombotic disease in experimental mouse models. However, the mechanisms of benefit remain unclear. Insights into the mechanisms underlying NET release and atherogenic inflammation have come from transgenic mouse studies. In particular, the importance of neutrophil NET formation in promoting atherothrombotic disease has been shown and linked to profound pro-inflammatory and pro-thrombotic effects, complement activation and endothelial dysfunction. Recent studies have shown that myeloid deficiency of PAD4 leads to diminished NET formation, which in turn protects against atherosclerosis burden, propagation of its thrombotic complications and notably macrophage inflammation in plaques. In addition, oxidative stress and neutrophil cholesterol accumulation have emerged as important factors driving NET release, likely involving mitochondrial reactive oxidants and neutrophil inflammasome activation. Further elucidation of the mechanisms linking hyperlipidaemia to the release of NETs may lead to the development of new therapeutics specifically targeting atherogenic inflammation, with likely benefit for cardiovascular diseases.

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Impaired neutrophil extracellular traps and inflammatory responses in the peritoneal fluid of patients with liver cirrhosis.

Liver cirrhosis (LC) is an inflammatory process associated with impaired functions in adaptive and innate immune responses at both systemic and local levels, also referred as Cirrhosis-Associated Immune Dysfunction. In this study, we evaluated the functionality of neutrophils from ascitic fluid (AF) of patients with hepatic cirrhosis by testing their ability to generate neutrophil extracellular traps (NETs) invitro. To further determine the activation state of neutrophils, expression of the activation markers CD66b, CD69, and CD80 on these cells was analysed by flow cytometry. The inflammatory environment in AF was assessed by measured concentration of pro- and anti-inflammatory cytokines. Samples were collected from 40 patients with LC, 20 of them with uncomplicated ascites (ASC) and 20 with spontaneous bacterial peritonitis (SBP). Peripheral blood (PB) neutrophils from healthy individuals were used as control (HC). Our results revealed a significant decrease in the release of NETs in neutrophils from the SBP group compared with HC. Low expression of CD69 and CD80 on neutrophils from AF of SBP patients was also observed. Comparisons of inflammatory cytokine levels in AF from the different study groups (SBP and ASC) revealed significant differences. In conclusion, we demonstrate that the development of complications, such as SBP, increases initially the inflammatory status, but chronically results in impaired neutrophil function as demonstrated by the decreased capability of NETs formation. There is also an increase in both pro-inflammatory and anti-inflammatory cytokines, thus predisposing for new episodes of SPB and increasing morbidity and mortality in cirrhotic patients.

1146 Nuclear translocation of PKCα is important in neutrophil NETosis and UVB induced-skin inflammation

Neutrophils are the most abundant leukocytes and the first to be recruited to the site of photodamage after ultraviolet B (UVB) exposure. However, little is known about the role of neutrophils in UVB-induced skin inflammation. Neutrophils release neutrophil extracellular traps (NETs) during NETosis, which are important in autoimmune inflammation, including lupus. Studies have shown the importance of actin cytoskeleton regulator Rho kinase (ROCK) in PMA-induced neutrophil NETosis, and we have found the NETotic neutrophils in UVB-induced skin inflammation. However it not clear how ROCK is involved in neutrophil NETosis and UVB-induced skin inflammation. To explore the effects of ROCK, we studied the effects of ROCK inhibition on neutrophil NETosis in vitro and on neutrophil NETosis in the UVB-induced skin inflammation in mice. Following PMA stimulation, cytosolic PKCα is gradually translocated to the nuclear membrane. Particularly the phosphorylated PKCα (phosphor-PKCα at ser657) is accumulated at the site of nuclear membrane rupture in the NETotic neutrophils in our confocal analysis. Inhibition of actin, myosin, MLCK (myosin light chain kinase) of actomyosin networks with cytochalasin D, Blebbistatin, or ML7 can attenuate both nuclear translocation of PKCα and neutrophil NETosis. Similarly, dual ROCK1/2 inhibitor HA1077 attenuated nuclear translocation of PKCα and neutrophil NETosis. Most importantly, application of HA1077 intraperitoneally (i.p.) can also attenuate neutrophil NETosis and skin inflammation in UVB-induced skin inflammation. Our preliminary studies therefore elucidates a novel mechanism that ROCK-mediated nuclear translocation and phosphorylation of PKCα regulates neutrophil NET formation, and demonstrates the protective role of ROCK inhibition in neutrophil NETosis in vitro and in vivo. These findings may provide insights into a novel therapeutic target for treatment of UVB-induced skin inflammation.

Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis.

Neutrophil extracellular traps (NETs) are networks of extracellular fibres produced from neutrophil DNA with a pathogenic role in infection, thrombosis and other conditions. Reliable assays for measuring NETs are desirable as novel treatments targeting NETs are being explored for the treatment of these conditions. We compare a whole blood flow cytometry method with serum assays to measure NETs-associated markers in patients with sepsis and thrombosis. Patients with deep venous thrombosis (n=25), sepsis (n=21) and healthy controls (n=23) were included in the study. Neutrophil surface NETs markers were determined by flow cytometry on whole blood samples by gating of neutrophils stained for surface citrullinated histone (H3cit) and myeloperoxidase (MPO). Serum double-stranded (ds) DNA, MPO, myeloid-related protein, nucleosomes, DNAse, elastase, human high-mobility group box 1 and MPO-DNA complexes were quantified as circulating markers of NETs. Neutrophil NETs markers by flow cytometry and serum NETs markers were significantly higher in patients with thrombosis and sepsis compared with healthy controls. Neutrophil NETs markers significantly correlated with the serum marker dsDNA. Flow cytometry detection of neutrophil NETs markers is feasible in whole blood and correlates with serum markers of NETs. We propose the flow cytometry detection of MPO/H3cit positive neutrophils and serum dsDNA as simple methods to quantify cellular and extracellular NET markers in patients with thrombosis and sepsis.