Search Results (7)

Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte^® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol–myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte^® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease. Full article

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation. Full article

Neutrophil extracellular traps (NETs) are associated with multiple disease pathologies including sepsis, asthma, rheumatoid arthritis, cancer, systemic lupus erythematosus, acute respiratory distress syndrome, and COVID-19. NETs, being a disintegrated death form, suffered inconsistency in their identification, nomenclature, and quantifications that hindered therapeutic approaches using NETs as a target. Multiple strategies including microscopy, ELISA, immunoblotting, flow cytometry, and image-stream-based methods have exhibited drawbacks such as being subjective, non-specific, error-prone, and not being high throughput, and thus demand the development of innovative and efficient approaches for their analyses. Here, we established an imaging and computational algorithm using high content screening (HCS)—cellomics platform that aid in easy, rapid, and specific detection as well as analyses of NETs. This method employed membrane-permeable and impermeable DNA dyes in situ to identify NET-forming cells. Automated algorithm-driven single-cell analysis of change in nuclear morphology, increase in nuclear area, and change in intensities provided precise detection of NET-forming cells and eliminated user bias with other cell death modalities. Further combination with Annexin V staining in situ detected specific death pathway, e.g., apoptosis, and thus, discriminated between NETs, apoptosis, and necrosis. Our approach does not utilize fixation and permeabilization steps that disturb NETs, and thus, allows the time-dependent monitoring of NETs. Together, this specific imaging-based high throughput method for NETs analyses may provide a good platform for the discovery of potential inhibitors of NET formation and/or agents to modulate neutrophil death, e.g., NETosis-apoptosis switch, as an alternative strategy to enhance the resolution of inflammation. Full article

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. Full article

Over a decade ago, the formation of neutrophil extracellular traps (NETs) was described as a novel mechanism employed by neutrophils to tackle infections. Currently applied methods for NETs release quantification are often limited by the use of unspecific dyes and technical difficulties. Therefore, we aimed to develop a fully automatic image processing method for the detection and quantification of NETs based on live imaging with the use of DNA-staining dyes. For this purpose, we adopted a recently proposed Convolutional Neural Network (CNN) model called Mask R-CNN. The adopted model detected objects with quality comparable to manual counting—Over 90% of detected cells were classified in the same manner as in manual labelling. Furthermore, the inhibitory effect of GW 311616A (neutrophil elastase inhibitor) on NETs release, observed microscopically, was confirmed with the use of the CNN model but not by extracellular DNA release measurement. We have demonstrated that a modern CNN model outperforms a widely used quantification method based on the measurement of DNA release and can be a valuable tool to quantitate the formation process of NETs. Full article

This proof-of-concept study aimed to evaluate a novel method of flow cytometry-based quantification of neutrophil extracellular traps (NETs) in septic shock patients and to identify possible interactions between the number of free-circulating NETs and alterations of the coagulatory system. Patients suffering from septic shock, a matched control group (CTRL), and patients suffering from systemic inflammation after cardiac (CABG) or major abdominal surgery (MAS) were enrolled in this prospective proof-of-concept study. Compared to the matched controls, free-circulating NETs were significantly elevated in septic shock and postsurgical patients (data are presented in median (IQR)); septic shock: (2.7 (1.9–3.9); CABG: 2.7 (2.1–3.7); MAS: 2.7 (2.1–3.9); CTRL: 1.6 (1–2); CTRL vs. septic shock: p = 0.001; CTRL vs. CABG: p < 0.001; CTRL vs. MAS: p < 0.001). NETs correlated positively with FIBTEM mean clot firmness (MCF) in septic shock patients (r = 0.37, p < 0.01) while they correlated negatively in surgical patients (CABG: r = −0.28, p < 0.01; MAS: r = −0.25, p = 0.03). Flow-cytometric quantification of NETs showed a significant increase in free-circulating NETs under inflammatory conditions. Furthermore, this study hints to an association of the number of NETs with hypercoagulation in septic shock patients and hypocoagulation in surgery-induced inflammation. Full article

Introduction: Neutrophil extracellular trap (NET) formation was recently identified as a novel mechanism to kill pathogens. However, excessive NET formation in sepsis can injure host tissues. We have recently shown that parenteral vitamin C (VitC) is protective in sepsis. Whether VitC alters NETosis is unknown. Methods: We used Gulo^−/− mice as they lack the ability to synthesize VitC. Sepsis was induced by intraperitoneal infusion of a fecal stem solution (abdominal peritonitis, FIP). Some VitC deficient Gulo^−/− mice received an infusion of ascorbic acid (AscA, 200 mg/kg) 30 min after induction of FIP. NETosis was assessed histologically and by quantification for circulating free DNA (cf-DNA) in serum. Autophagy, histone citrullination, endoplasmic reticulum (ER) stress, NFκB activation and apoptosis were investigated in peritoneal PMNs. Results: Sepsis produced significant NETs in the lungs of VitC deficient Gulo^−/− mice and increased circulating cf-DNA. This was attenuated in the VitC sufficient Gulo^−/− mice and in VitC deficient Gulo^−/− mice infused with AscA. Polymorphonuclear neutrophils (PMNs) from VitC deficient Gulo^−/− mice demonstrated increased activation of ER stress, autophagy, histone citrullination, and NFκB activation, while apoptosis was inhibited. VitC also significantly attenuated PMA induced NETosis in PMNs from healthy human volunteers. Full article