scholarly journals Convolutional Neural Networks–Based Image Analysis for the Detection and Quantification of Neutrophil Extracellular Traps

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 508
Author(s):  
Aneta Manda-Handzlik ◽  
Krzysztof Fiok ◽  
Adrianna Cieloch ◽  
Edyta Heropolitanska-Pliszka ◽  
Urszula Demkow
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Inhibitory Effect ◽  
Extracellular Dna ◽  
Neutrophil Elastase Inhibitor ◽  
Elastase Inhibitor ◽  
Extracellular Traps ◽  
Automatic Image Processing ◽  
Adopted Model ◽  
Dna Release ◽  
Detection And Quantification

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.

scholarly journals Detailed histological analysis of a thrombectomy-resistant ischemic stroke thrombus: a case report

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Senna Staessens ◽  
Olivier François ◽  
Linda Desender ◽  
Peter Vanacker ◽  
Tom Dewaele ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Von Willebrand Factor ◽  
Histological Analysis ◽  
Mechanical Thrombectomy ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Dna ◽  
Extracellular Traps ◽  
First Pass ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background Mechanical removal of a thrombus by thrombectomy can be quite challenging. For reasons that are not fully understood, some thrombi require multiple passes to achieve successful recanalization, whereas other thrombi are efficiently removed in a single pass. Since first pass success is associated with better clinical outcome, it is important to better understand the nature of thrombectomy resistant thrombi. The aim of this study was therefore to characterize the cellular and molecular composition of a thrombus that was very hard to retrieve via mechanical thrombectomy. Case presentation In a patient that was admitted with a right middle cerebral artery M1-occlusion, 11 attempts using various thrombectomy devices and techniques were required for removal of the thrombus. This peculiar case provided a rare opportunity to perform an in-depth histopathological study of a difficult to retrieve thrombus. Thrombus material was histologically analyzed using hematoxylin and eosin, Martius Scarlet Blue stain (red blood cells and fibrin), Feulgen stain (DNA), von Kossa stain (calcifications) and immunohistochemical analysis of von Willebrand factor, platelets, leukocytes and neutrophil extracellular traps. Histological analysis revealed abnormally high amounts of extracellular DNA, leukocytes, von Willebrand factor and calcifications. Extracellular DNA stained positive for markers of leukocytes and NETs, suggesting that a significant portion of DNA is derived from neutrophil extracellular traps. Conclusion In this unique case of a nearly thrombectomy-resistant stroke thrombus, our study showed an atypical composition compared to the common structural features found in ischemic stroke thrombi. The core of the retrieved thrombus consisted of extracellular DNA that colocalized with von Willebrand factor and microcalcifications. These results support the hypothesis that von Willebrand factor, neutrophil extracellular traps and microcalcifications contribute to mechanical thrombectomy resistance. Such information is important to identify novel targets in order to optimize technical treatment protocols and techniques to increase first pass success rates.

scholarly journals Neutrophil Extracellular Traps Promote the Development and Growth of Human Salivary Stones

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2139
Author(s):  
Mirco Schapher ◽  
Michael Koch ◽  
Daniela Weidner ◽  
Michael Scholz ◽  
Stefan Wirtz ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Clinical Symptoms ◽  
Neutrophil Extracellular Traps ◽  
Physicochemical Process ◽  
Extracellular Dna ◽  
Neutrophil Granulocyte ◽  
Extracellular Traps ◽  
Efferent Ducts ◽  
Small Crystals

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.

The screening of albumin as a key serum component to prevent neutrophil extracellular traps release by selectively inhibiting mitochondrial ROS generation

2020 ◽  
Author(s):  
Yue Zheng ◽  
Yuanfeng Zhu ◽  
Xin Liu ◽  
Hang Zheng ◽  
Yongjun Yang ◽  
...  
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Extracellular Dna ◽  
Organ Injury ◽  
Ros Generation ◽  
Ros Production ◽  
Serum Free ◽  
Extracellular Traps ◽  
Mitochondrial Ros ◽  
Serum Component ◽  
Microbial Defense

Neutrophil extracellular traps (NETs) are extracellular DNA webs released from neutrophils to mediate host anti-microbial defense. As NETs could also induce thrombosis and cause organ injury, their release should be strictly controlled. However, it is not well understood about the intrinsic mechanisms that prevent unfavorable NETs. Herein, an accidental finding of NETs release from human peripheral neutrophils was firstly described in serum free culture, and it was also determined as a conserved effect for serum to prevent NETs. In contrast to canonical NETs induced by phorbol-12-myristate-13-acetate (PMA), NETs formation by serum free culture was rapid and without prevalent NETosis. Next, albumin was screened out as a key serum component that mediated the suppression of NETs. Moreover, NETs induced upon serum or albumin deficiency were independent of the canonical pathway that involves NOX2 activation and cytosol ROS production. Instead, the generation of mitochondrial ROS (mtROS) was upregulated to promote NETs release. Albumin exhibited mtROS scavenging activity and thus inhibited NETs. Serum free culture also induces the release of NET-bound oxidized mtDNA which stimulated IFN-β production. Overall, our research provides new evidences that characterize the NETs production in serum free culture and determine the mechanisms of serum albumin to inhibit NETs.

scholarly journals The inhibitory effect of secretory leukocyte protease inhibitor (SLPI) on formation of neutrophil extracellular traps

2015 ◽  
Vol 98 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Katarzyna Zabieglo ◽  
Pawel Majewski ◽  
Monika Majchrzak-Gorecka ◽  
Agnieszka Wlodarczyk ◽  
Beata Grygier ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Neutrophil Extracellular Traps ◽  
Inhibitory Effect ◽  
Secretory Leukocyte Protease Inhibitor ◽  
Extracellular Traps

Proinflammatory role of neutrophil extracellular traps in abdominal sepsis

2014 ◽  
Vol 307 (7) ◽  
pp. L586-L596 ◽  
Author(s):  
Lingtao Luo ◽  
Su Zhang ◽  
Yongzhi Wang ◽  
Milladur Rahman ◽  
Ingvar Syk ◽  
...  
Keyword(s):  
Lung Injury ◽  
Peritoneal Cavity ◽  
Tissue Injury ◽  
Cecal Ligation ◽  
Neutrophil Extracellular Traps ◽  
Abdominal Sepsis ◽  
Extracellular Dna ◽  
Extracellular Traps ◽  
Proinflammatory Role

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-α, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury.

scholarly journals Bacillus licheniformis and Bacillus subtilis, Probiotics That Induce the Formation of Macrophage Extracellular Traps

2021 ◽  
Vol 9 (10) ◽  
pp. 2027
Author(s):  
Carol M. Romo-Barrera ◽  
Laura E. Castrillón-Rivera ◽  
Alejandro Palma-Ramos ◽  
Jorge I. Castañeda-Sánchez ◽  
Julieta Luna-Herrera
Keyword(s):  
Pathogenic Bacteria ◽  
Mouse Cell ◽  
Extracellular Dna ◽  
Microbial Pathogens ◽  
Bacillus Species ◽  
Human Consumption ◽  
Genus Bacillus ◽  
Beneficial Effects ◽  
Extracellular Traps ◽  
Dna Release

Probiotics are considered living microorganisms that help preserve the health of the host who uses them. Bacillus are a genus of Gram-positive bacteria used as probiotics for animal and human consumption. They are currently distributed in various commercial forms. Two of the species used as probiotics are B. licheniformis and B. subtilis. Macrophages are central cells in the immune response, being fundamental in the elimination of microbial pathogens, for which they use various mechanisms, including the formation of extracellular traps (METs). There have been very few studies carried out on the participation of macrophages in response to the interaction of probiotics of the genus Bacillus with the host. In this work, we used macrophages from the J774A mouse cell line.1, and we found that they are susceptible to infection by the two Bacillus species. However, both species were eliminated as the infection progressed. Using confocal microscopy, we identified the formation of METs from the first hours of infection, which were characterized by the presence of myeloperoxidase (MPO) and citrullinated histone (Hit3Cit). Quantitative data on extracellular DNA release were also obtained; release was observed starting in the first hour of infection. The induction of METs by B. licheniformis caused a significant decrease in the colony-forming units (CFU) of Staphylococcus aureus. The induction of METS by bacteria of the Bacillus genus is a mechanism that participates in controlling the probiotic and potentially pathogenic bacteria such as S. aureus. The induction of METs to control pathogens may be a novel mechanism that could explain the beneficial effects of probiotics of the genus Bacillus.

scholarly journals Tissue Accumulation of Neutrophil Extracellular Traps Mediates Muscle Hyperalgesia in a Mouse Model

2021 ◽  
Author(s):  
Kazuaki Suzuki ◽  
Masahiro Tsuchiya ◽  
Shinichiro Yoshida ◽  
Kazumi Ogawa ◽  
Weijian Chen ◽  
...  
Keyword(s):  
Uric Acid ◽  
Muscle Contraction ◽  
Dna Cleavage ◽  
Neutrophil Extracellular Traps ◽  
Neutrophil Recruitment ◽  
Triceps Surae ◽  
Extracellular Dna ◽  
Xanthine Oxidase Inhibitor ◽  
Extracellular Traps ◽  
Neutrophil Depletion

Abstract Background: Accumulation of uric acid during muscular trauma is potentially a causative factor of damage-associated molecular patterns (DAMPs) involved in the development of muscle hyperalgesia. Neutrophil extracellular traps (NETs), DNA-based reticular structures to capture DAMPs, play a central role in the onset of pain in gout attacks associated with hyperuricemia; however, their association with muscle hyperalgesia due to overuse injuries remains unknown. Therefore, the aim of this study was to investigate the involvement of NETs via the elevation of local uric acid level in muscle nociception.Methods: The triceps surae muscles (TSMs) in the unilateral hindlimb of mice were repeatedly stimulated with electrical pulses to induce excessive muscle contraction, and the contralateral TSM was used as a control. In addition to mechanical nociceptive thresholds, tissue uric acid levels, neutrophil recruitment, protein amount, and histological distribution of citrullinated histone 3 (citH3), a major marker of NETs, were investigated. Furthermore, whether neutrophil depletion, extracellular DNA cleavage (deoxyribonuclease I), and administration of the urate-lowering agent febuxostat, a xanthine oxidase inhibitor, improved muscle hyperalgesia due to NET accumulation was examined. Using a combination of multiphoton imaging analysis and intravital fluorescence staining, we also evaluated the intramuscular distribution of NET accumulation in stimulated TSMs.Results: CitH3 expression upon neutrophil recruitment significantly increased in the stimulated TSMs tissues with an increase in tissue uric acid levels. However, neutrophil depletion and extracellular DNA cleavage prevented the increase in uric acid levels in damaged muscle tissues. Furthermore, febuxostat administration significantly improved muscle hyperalgesia, with decreases not only in citH3 and tissue uric acid levels, but also in neutrophil recruitment. Interestingly, the intramuscular distribution of NETs in the stimulated TSM was predominantly observed in the myofascial region.Conclusions: Our findings suggest that NET accumulation caused by excessive muscle contraction was strongly associated with the pathogenesis of muscle hyperalgesia. Further, the mechanism underlying induction of locally recruited neutrophils forming NETs was increased tissue uric acid levels, which potentially plays a significant role in creating a vicious circle of muscle pain.

scholarly journals Netome: The Molecular Characterization of Neutrophil Extracellular Traps (NETs)

2020 ◽  
Author(s):  
David Scieszka ◽  
Yi-Han Lin ◽  
Weizhong Li ◽  
Saibyasachi Choudhury ◽  
Yanbao Yu ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Genomic Sequence ◽  
White Blood Cells ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Dna ◽  
Sterile Inflammation ◽  
Extracellular Milieu ◽  
Extracellular Traps ◽  
Genomics And Proteomics

AbstractNeutrophils are the most abundant type of white blood cells in humans with biological roles relevant to inflammation and fighting infections. The release of neutrophil extracellular DNA aims to control invasion by bacteria, viruses, fungi, and tissue damage. Neutrophil Extracellular Traps (NETs) act as antimicrobial agents triggering immune signaling through the release of the nuclear content into the extracellular space. Although intense investigations have elucidated the pathways preceding NET formation, the exact molecular composition of released NETs has not been mapped. We aimed to decode the sequences of DNA and proteins from NETs. With emerging needs to understand neutrophil functions precisely, we open the field of NETOMIC studies through isolation of NETs in combination with omics approaches including shotgun genomics and proteomics. Our in vitro NET isolation methodology allowed for unprecedented replicability with induction in a sterile inflammation model system. Enrichment of mitochondrial DNA and telomere sequences are significantly expressed in NET genomes. This study revealed that the genomic sequence released in the extracellular milieu is not stochastically serving as a scaffold for a repertoire of proteins involved in neutrophil protective functions. Collectively, we established the gene and protein signatures exclusive to the extracellular NETs in comparison to undifferentiated and differentiated neutrophil states, further guiding future detection of specific regions needed for diagnostics and targeted therapies of NET related conditions.

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