Neutrophil Extracellular Traps Formation In Neutrophils Research Articles

#944 Soluble uric acid inhibits neutrophil functions during infections in patients with kidney disease

Abstract Background and Aims Chronic kidney disease (CKD) is one of the most common diseases affecting 8-16% of the world population. Beside cardiovascular complications, infections including sepsis are the second most common cause of death in patients with acute kidney injury (AKI) and CKD. However, the underlying pathomechanisms that contribute to the secondary immunodeficiency related to kidney disease [1] are not well understood. Recent evidence suggests that CKD-related hyperuricemia characterised by elevated serum uric acid (UA) levels suppresses immune cell functions during sterile inflammation [2]. Whether this is also the case during host defense is subject of our current investigation. In this study, we hypothesized that soluble UA inhibits neutrophil effector functions and therefore contributes to the increased infection risk in patients with kidney disease. Method Blood neutrophils were isolated from healthy individuals as well as from patients with kidney diseases, and incubated ex vivo in the presence or absence of 10 mg/dl soluble UA prior to stimulation with bacterial peptides including LPS. Immune cell functions including cytoskeletal changes, immune cell activation, maturation, endo- and phagocytosis (pHrodo Dextran particles, IgG-FITC beads, pHrodo E. coli bioparticles), ROS production, and neutrophil extracellular trap (NET) formation were analysed and quantified using flow cytometry, fluorescence and colorimetric assays, and fluorescence microscopy. In addition, to verify our results we stimulated neutrophils from healthy individuals with sera from hyperuricemic CKD patients. Results Our results show for the first time that soluble UA significantly inhibits the ability of neutrophils to endocytose small particles and phagocytose beads in neutrophils from healthy individuals comparable to the inhibitory effect of Cytochalasin D, an inhibitor of endo- and phagocytosis. Interestingly, unlike neutrophils from AKI patients, neutrophils from CKD patients were significantly less able to phagocytose beads compared to healthy controls with and without stimulation. To mimic the uptake of pathogens more physiologically, pHrodo E. coli bioparticles were used. We found that soluble UA inhibited the phagocytosis of such bioparticles in neutrophils. In addition, our investigation into the formation of ROS showed that soluble UA inhibited specifically nitric oxide, peroxynitrite, hydroxyl radicals and hydrogen peroxide, but to a lower extent the formation of superoxides in neutrophils from healthy individuals, similar to the effectiveness of the radical scavenger N-acetylcysteine. These data were comparable with our cohort study of neutrophils from CKD patients. To examine the mechanism of reduced ROS production, we inhibited NADPH oxidase (DPI) and MPO (4-ABAH) in neutrophils and found that soluble UA has similar inhibitory effects on ROS production like DPI whereas 4-ABAH was more potent. This indicated that soluble UA modulates NADPH oxidase and ROS production, which subsequently resulted in reduced NET formation in neutrophils. Conclusion Our data identify soluble UA as potential immune regulator of the secondary immunodeficiency in patients with kidney disease by inhibiting the endo- and phagocytosis of particles, NADPH-mediated ROS production and NET formation in neutrophils, processes that are important to kill pathogens and fight an infection. Thus, specifically targeting UA with urate-lowering therapy might overcome the suppressed host defence against infection in patients with kidney disease.

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.

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.

High expression of P-selectin induces neutrophil extracellular traps via the PSGL-1/Syk/Ca2+/PAD4 pathway to exacerbate acute pancreatitis.

Excessive neutrophil extracellular traps (NETs) is involved in the progression of acute pancreatitis (AP) but the mechanisms controlling NETs formation in AP are not fully understood. Therefore, our study sought to investigate the mechanism of the highly expressed P-selectin stimulating the formation of NETs in AP. NETs formation was detected by flow cytometry, immunofluorescence staining, and cf-DNA and MPO-DNA complexes were measured as biomarkers of NETs formation. Neutrophils treated with P-selectin and pharmacological inhibitors were examined by western blot, immunofluorescence staining and flow cytometry. Mouse model of AP was established by caerulein and the effect of inhibiting P-selectin by PSI-697 on the level of NETs and PAD4 in pancreatic tissue was observed. The severity of AP was evaluated by histopathological score and the detection of serum amylase and lipase. Patients with AP had elevated levels of NETs and P-selectin compared with healthy volunteers. Stimulation of P-selectin up-regulated the expression of PSGL-1, increased the phosphorylation of Syk, mediated intracellular calcium signal and led to the activation and expression of PAD4, which modulated NETs formation in neutrophils. Pretreament with PSI-697 blunted NETs formation and PAD4 expression in the pancreatic tissue, and ameliorated the severity of AP in mice. Taken together, these results suggest that P-selectin induces NETs through PSGL-1 and its downstream Syk/Ca2+/PAD4 signaling pathway, and that targeting this pathway might be a promising strategy for the treatment of AP.

Investigation of Neutrophil Extracellular Trap (NET) Induction in Patients with Chronic Idiopathic Neutropenia Bearing Mutations in the Mediterranean Fever (MEFV) Gene

Introduction: Chronic idiopathic neutropenia (CIN) is a disorder characterized by the prolonged and unexplained reduction in the number of circulating neutrophils. The pathophysiology of CIN has been associated with an inflammatory bone marrow microenvironment consisting of activated T-cells with an oligoclonal/monoclonal profile and intermediate CD14bright/CD16+ monocytes that induce the apoptotic death of the granulocytic progenitor cells. To probe further the basis of immune cell activation in CIN we have recently investigated by next generation sequencing (NGS) the frequency of mutations in the MEFV gene encoding pyrin, in a cohort of 50 CIN patients without any signs/symptoms of Familial Mediterranean Fever (FMF). We have found that 9/50 patients displayed variants associated with typical or atypical FMF phenotype in the Greek population implicating exon 10 and/or exon 2 (Skendros P et al. Blood, 138, Sup.1, p3124;2021). Aim:MEFV mutations have been associated with increased neutrophil activation in FMF and other inflammatory diseases, neutrophil entry in autophagy and extrusion of Neutrophil Extracellular Traps (NETs) decorated with the pro-inflammatory cytokine IL-1β. The aim of the current study is to investigate the association between MEFV mutations and NET formation in neutrophils of CIN patients. Patients-Methods: We studied 20 CIN patients (17 females and 3 males) aged 27 to 78 years. Six CIN patients harbored at least one MEFV mutation with potential clinical significance (A744S homo/R202Q homo, I720M hetero, R202Q homo, K965R hetero, A744S hetero, M694V hetero) and 14 were negative for MEFV pathogenic mutations. The MEFV R202Q mutation is located in exon 2 whereas all the rest mutations are located in exon 10. We isolated neutrophils from healthy subjects (n=5), age- and sex- matched with the patients, and co-cultured them with serum from CIN patients. We used the supernatants of the cultures to quantify NETs with Sytox green staining for extracellular DNA and/or ELISA for dsDNA/MPO complexes. We also used the serum-treated neutrophils for the evaluation of NETosis by immunostaining using confocal microscopy. In addition, neutrophils isolated from CIN patients were used to detect their direct response to spontaneous NETosis induction. Statistical analysis was performed with one-way ANOVA adjusting for multiple comparisons. Results: By co-culturing patients' sera with healthy neutrophils obtained from five different healthy donors, we did not identify statistically significant differences in NETosis evaluation between the three study groups i.e. CIN patients with MEFV mutations, CIN patients negative for pathogenic MEFV mutations, and healthy individuals. On an individual basis, the patient harboring the double homozygous MEFV mutations (A744S/R202Q) displayed increased NETosis in immunostaining, Sytox and ELISA evaluation, as compared to MEFV unmutated CIN patients and healthy individuals. In addition, ex vivo culture of patients' neutrophils and microscopic observation gave us the indication that CIN patients, regardless of their MEFV gene variants, present increased NETosis compared to healthy individuals, suggesting an overactivation status of neutrophils in CIN. Conclusions: The neutrophils of CIN patients display increased NETosis irrespectively of the MEFV mutation status and may thus contribute to the inflammatory processes associated with the disease, a field that has not been investigated so far. Although in our study MEFV mutations did not appear to contribute significantly to inflammatory processes through the NETosis mechanism in CIN patients, their involvement in other aspects of immune deregulation such as through the mTOR/autophagy pathway remains to be further explored.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser’s disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5′-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

Role of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil extracellular trap formation in dairy cows with subclinical hypocalcemia

Hypocalcemia in dairy cows is associated with decreased neutrophil phagocytosis, adhesion capacity, migration, and reactive oxygen species (ROS) production through alterations in ORAI calcium release-activated calcium modulator 1 (ORAI1). Neutrophils can resist the invasion of pathogenic microorganisms by releasing neutrophil extracellular traps (NET). However, the mechanisms controlling NET formation during hypocalcemia are unknown. To address the role of ORAI1 in NET formation, neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (n = 10 per group) diagnosed as clinically healthy (control) or with plasma concentrations of Ca2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (SCH). A series of ex vivo experiments were conducted as follows: first, neutrophils isolated from both groups of cows were treated with phorbol 12-myristate 13-acetate (PMA) to stimulate NET formation; second, neutrophils from control and SCH were pretreated with or without the ROS scavenger N-acetylcysteine (NAC), the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, or ORAI1 blocker 2APB and then treated with PMA to stimulate NET formation; and third, neutrophils were transfected with small interfering (si)ORAI1 or nontarget siRNA (siNEG) and then stimulated with PMA to induce formation of NET. A one-way ANOVA was used for statistical analysis of individual experiments. In the first experiment, neutrophils from SCH cows formed NET with fewer DNA filaments, more diffused nuclei, and reduced translocation of myeloperoxidase (MPO) and neutrophil elastase (NE) to the nucleus. Neutrophils from SCH cows stimulated with PMA had a lower mitochondrial permeability, the state of mitochondrial permeability transition pore was open, ROS production was lower and there was increased mitochondrial damage. In the second experiment, in both control and SCH-PMA stimulated neutrophils, exogenous NAC decreased NET formation (assessed via Hoechst 33342 dye; Beyotime). Furthermore, following the challenge with PMA, thapsigargin increased NET formation and ROS production, but blocking ORAI1 with 2APB decreased NADPH oxidase activation, ROS production, and NET formation. In the third experiment, neutrophils transfected with siORAI1 before stimulation with PMA had lower intracellular concentrations of Ca2+, NET formation, and ROS production. Overall, the data indicated that SCH reduces NET formation in neutrophils partly due to damaged mitochondria. The reduction in ORAI1 abundance in neutrophils of dairy cows with hypocalcemia also decreases ROS production.

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Context: Cardiovascular (CVS) complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). Neutrophil extracellular traps (NETs) have previously been implicated in the pathogenesis of cancer-associated thrombosis. Objective: To assess NET formation in neutrophils from patients with CML, to characterize the role of reactive oxygen species (ROS) and peptidyl arginine deiminase 4 (PAD4) in NET formation in CML, and to assess the effects of TKIs on NET formation in CML. Design: Blood samples were obtained from patients with newly diagnosed CML and from age/gender-matched controls. NETs were assessed after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin (IO), with or without pre-treatment with imatinib, ponatinib, nilotinib, and dasatinib at clinically relevant concentrations. NET formation was assessed by morphology, immunofluorescence (IF), and the expression of proteins known to be associated with NET formation (NET-associated elastase, citrullinated histone H3 [H3cit], etc.). ER-HoxB8 conditionally immortalized murine myeloid progenitor cell line was differentiated into functional neutrophils and transduced with a BCR-ABL1 oncogene (or empty vector control). Results: Neutrophils isolated from treatment-naive patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with IO as assessed by IF and after stimulation with PMA as assessed by NET-associated elastase expression. Expression of H3cit, PAD4, and ROS was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation. Exposure to ponatinib significantly augmented NET-associated elastase and ROS levels as compared to control and other TKIs in neutrophils derived from patients with CML. BCR-ABL1 retrovirally transduced HoxB8-neutrophils demonstrated increased H3cit and myeloperoxidase expression, consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib and nilotinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. Conclusions: CML is associated with increased NET formation, which is further augmented by ponatinib and nilotinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the ER-HoxB8-BCR-ABL1 model, NET formation is PAD4- but not ROS-dependent.

Effects of Fluorine on Neutrophil Extracellular Trap Formation through Regulating AMPK/p38 Signaling Pathway.

Fluorine is an important trace element that is widely dispersed, and studies showed that fluorine could cause severe toxicity to fish. The aim of this study was to investigate the effects of fluorine on neutrophil extracellular trap (NET) formation in common carp and clarify the possible mechanism. The neutrophils were isolated and exposed to 0.25, 0.5, or 1 mM sodium fluoride (NaF). The results showed that NaF could induce the formation of NETs which exhibited a DNA-based network structure modified with histones and myeloperoxidase (MPO). Furthermore, NaF led to the production of reactive oxygen species (ROS) in neutrophils. Western blot results showed that NaF significantly increased the phosphorylation of AMPK and p38. In addition, our results showed that NaF-induced NET formation could be inhibited by an AMPK or p38 inhibitor. In conclusion, our results showed that NaF induced NET formation in neutrophils through regulation of the AMPK/p38 signaling pathway.

Recent progress in the mechanistic understanding of NET formation in neutrophils.

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

Colchicine Inhibits Neutrophil Extracellular Trap Formation in Patients With Acute Coronary Syndrome After Percutaneous Coronary Intervention.

BackgroundRelease of neutrophil extracellular traps (NETs) after percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS) is associated with periprocedural myocardial infarction, as a result of microvascular obstruction via pro‐inflammatory and prothrombotic pathways. Colchicine is a well‐established anti‐inflammatory agent with growing evidence to support use in patients with coronary disease. However, its effects on post‐PCI NET formation in ACS have not been explored.Methods and ResultsSixty patients (40 ACS; 20 stable angina pectoris) were prospectively recruited and allocated to colchicine or no treatment. Within 24 hours of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 patients with ACS post‐PCI and 4 healthy controls were treated in vitro with colchicine (25 nmol/L) and stimulated with either ionomycin (5 μmol/L) or phorbol 12‐myristate 13‐acetate (50 nmol/L). Extracellular DNA was quantified using Sytox Green and fixed cells were stained with Hoechst 3342 and anti‐alpha tubulin. Baseline characteristics were similar across both treatment and control arms. Patients with ACS had higher NET release versus patients with stable angina pectoris (P<0.001), which was reduced with colchicine treatment (area under the curve: 0.58 versus 4.29; P<0.001). In vitro, colchicine suppressed unstimulated (P<0.001), phorbol 12‐myristate 13‐acetate–induced (P=0.009) and ionomycin‐induced (P=0.002) NET formation in neutrophils isolated from patients with ACS post‐PCI, but not healthy controls. Tubulin organization was impaired in neutrophils from patients with ACS but was restored by colchicine treatment.ConclusionsColchicine suppresses NET formation in patients with ACS post‐PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomized trials powered for clinical end points.RegistrationURL: https://anzctr.org.au; Unique identifier: ACTRN12619001231134.

Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps.

Simple SummaryNeutrophil extracellular traps (NETs) are extracellular web-like structures comprised of proteins and DNA that have been associated with the development of cancer. Citrullination of histone H3 has been implicated in the formation of NETs. The aim of our study was to develop a quick and reliable method for NET quantitation. Here, we describe a novel protocol for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). This assay was validated by comparing the ability of neutrophils from control tumor-free and myeloma-bearing mice to form NETs in response to stimuli. We demonstrated that neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production as detected by microscopy correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA in our novel binding assay.Formation of neutrophil extracellular traps (NETs) has been associated with multiple pathologies including cancer. While the visualization of NETs by microscopy is a routine technique, their quantification presents a number of challenges. Commonly, as citrullination of histone H3 is required for NET formation, the presence of this modified histone along with DNA is considered to be a hallmark of NETs. Here, we describe and validate a novel assay for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). Using this assay, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on NET formation by neutrophils isolated from the bone marrow of control and myeloma-bearing mice. We found that PMA induced citrullination of histone H3, an increase in the level of CitH3DNA, and NET formation in neutrophils from both tumor-free and myeloma-bearing mice. The levels of CitH3DNA in the NET fractions, as measured by our assay directly correlated with the citrullination of histone H3 in neutrophils, as detected by Western blotting, and were significantly higher in PMA-stimulated compared to unstimulated neutrophils. Neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA. Thus, our data indicate that bone marrow neutrophils from myeloma-bearing hosts are prone to NET formation.

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.

In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals.

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

Neutrophil extracellular trap formation and nuclease activity in septic patients

BackgroundThere is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well.MethodsFollowing ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 μg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis.ResultsIn contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0–6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers.ConclusionsUnstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo.Trial registrationDRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.

461 Colchicine Inhibits Neutrophil Extracellular Trap Formation in Acute Coronary Syndrome Patients Post Percutaneous Coronary Intervention

Release of neutrophil extracellular traps (NETs) after PCI in ACS patients is associated with peri-procedural myocardial infarction, as a result of microvascular obstruction via pro-inflammatory and pro-thrombotic pathways. Colchicine is a potent, well-established anti-inflammatory agent with growing evidence to support its use in patients with coronary disease. However, its effects on post-PCI NET formation in ACS patients has not been explored. 60 patients (40 ACS; 20 stable angina pectoris[SAP]) were prospectively recruited and allocated to colchicine (1.5 mg) or no treatment. Within 24 h of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 ACS patients post-PCI and 4 healthy controls were treated with colchicine (25 nM) and stimulated with either ionomycin (5 μM) or phorbol 12-myristate 13-acetate (PMA, 50 nM) in vitro. Extracellular DNA was quantified using Sytox Green and fixed cells were stained with hoechst and anti-alpha tubulin. Baseline characteristics were similar across both treatment and control arms. ACS patients had higher NET release versus SAP patients (p<0.001), which was reduced with colchicine treatment (AUC: 0.58 vs 4.29; p<0.001). In vitro, colchicine suppressed spontaneous (p=0.004), PMA-induced (p=0.03) and ionomycin-induced (p=0.02) NET formation in neutrophils isolated from ACS patients post-PCI, but not healthy controls. Tubulin organisation was impaired in neutrophils from patients with ACS but was restored by colchicine treatment. Colchicine suppresses NET formation in ACS patients post-PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomised trials powered for clinical endpoints.

Chemokine CXCb1 stimulates formation of NETs in trunk kidney neutrophils of common carp

Both in mammals and in fish, CXC chemokines activate leukocytes and regulate their migration both under normal physiological and inflammatory conditions. Moreover, in mammalian neutrophils CXC chemokines also stimulate the formation of neutrophil extracellular traps (NETs). Here, we investigated the effects of recombinant carp CXCL8s and CXCb1 on NET formation in neutrophils from the head (HK) and trunk (TK) kidney of carp.We found that neither recombinant CXCL8s nor CXCb1 stimulated DNA release in HK-derived neutrophils, while in TK-derived cells rcCXCb1 stimulated the release of NETs, composed of extracellular DNA co-localized with citrulline H3 histone and neutrophil elastase. Furthermore, CXCb1-induced NET release required NADPH oxidase activity, while it did not change upon treatment with CXCR inhibitors.In conclusion, we demonstrated, for the first time in fish, that CXCb1 chemokine induces formation of NETs in TK-derived neutrophils and this process is ROS-dependent. The difference between HK and TK-derived neutrophils is probably related to differences in the maturation state of these cells.

A Role for Receptor-Interacting Protein Kinase-1 in Neutrophil Extracellular Trap Formation in Patients with Systemic Lupus Erythematosus: a Preliminary Study

Background/Aims: Neutrophil extracellular traps (NETs) are known to play an important role in systemic lupus erythematosus (SLE) by triggering innate and adaptive immune responses. The molecular mechanisms responsible for their formation in SLE are still unclear. In this study, we aim to characterize the role of the receptor-interacting protein kinase-1 (RIPK1), a homologous serine/threonine kinase previously implicated in the regulation of necroptosis and tissue injury, in decreasing neutrophil death and formation of NETs, and to investigate the clinical implications of RIPK1 in SLE. Methods: Patients with SLE (n = 50) and healthy donors (n = 35) were enrolled in in vitro studies. Management of SLE patients was evaluated using the SLE disease activity index 2000 (SLEDAI-2K) score and laboratory variables. The mRNA level of RIPKs was measured by quantitative polymerase chain reaction (qPCR). Intracellular RIPK1 and RIPK3 production by peripheral blood leukocytes was detected by four-color flow cytometry and confirmed by automatic western blotting. TNF-α, IFN-γ, IL-1β, IL-2, IL-8, IL-18, and RIPK1 were measured by enzyme-linked immunosorbent assay. Cell death was assayed by Sytox green dye from peripheral neutrophils stimulated by RIPK-1-stabilizer necrostatin-1 (nec-1) and phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining and confocal microscopy were used to detect NET formation ex vivo. Quantification of NETs was determined by fluorescence spectrometry. Results: IFN-γ, IL-1β, IL-8, and IL-18 levels in serum were increased in SLE patients compared to controls. However, the expression of TNF-α, IL-2, and RIPK1 were decreased. In addition, we observed significant differences in the expression of RIPK1 in peripheral blood leukocytes. Of all the leukocytes, RIPK1 expression was significantly lower in neutrophils. Furthermore, we studied NETs formation in neutrophils of SLE with decreased RIPK1 expression, and these show increased susceptibility to NETosis, when stimulated with PMA and/or nec-1. Importantly, RIPK1 expression in neutrophils negatively correlated with ESR, CRP, 24-hour urine total protein, and the disease activity index in SLE. Conclusion: These data represent the first report of decreased RIPK1 expression in neutrophils of SLE patients and imply that RIPK1 may be involved in neutrophil death and NET formation. We suggest that RIPK1 is a potential biomarker to predict disease activity.

Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

The interrelationship between phagocytosis, autophagy and formation of neutrophil extracellular traps following infection of human neutrophils by Streptococcus pneumoniae.

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen.