Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

Manda-Handzlik, Aneta; Bystrzycka, Weronika; Cieloch, Adrianna; Głodkowska-Mrówka, Eliza; Jankowska‐Steifer, Ewa; Heropolitańska-Pliszka, Edyta; Skrobot, Agnieszka; Muchowicz, Angelika; Ciepiela, Olga; Wachowska, Malgorzata; Demkow, Urszula

doi:10.1007/s00018-019-03331-x

Cited by 58 publications

(59 citation statements)

References 54 publications

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“…At the indicated timepoint, cells were simultaneously stained with Hoechst 33342 and SYTOX Green and visualized. As we and others reported previously [44][45][46], all three stimuli efficiently induced NETs release and preincubation with NEi diminished NETs formation (Supplementary Figure S1). NETs formation could be consecutively observed as the loss of lobulated nuclear shape, rounding of the nucleus and loosening of the chromatin structure followed by the rupture of cell membrane and release of cloud-like NETs structures.…”

Section: The Adopted Model Detects Objects With Quality Comparable Tosupporting

confidence: 85%

“…To enlarge NETs dataset, we included similarly processed images of neutrophils isolated from CGD patients and stimulated with SNAP or peroxynitrite. As we reported previously, peroxynitrite efficiently stimulated NETs release, whilst no or very weak NETs release upon incubation with SNAP was observed [46] (Supplementary Figure S2).…”

Section: The Adopted Model Detects Objects With Quality Comparable Tosupporting

confidence: 83%

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

Manda-Handzlik

Fiok

Cieloch

et al. 2020

Cells

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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.

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Section: The Adopted Model Detects Objects With Quality Comparable Tosupporting

confidence: 85%

Section: The Adopted Model Detects Objects With Quality Comparable Tosupporting

confidence: 83%

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

Manda-Handzlik

Fiok

Cieloch

et al. 2020

Cells

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“…Neutropenia observed in a large portion of our patients further limited the availability of the clinical material for mechanistic evaluations. In addition to that, although the quantitative NETs assay using SYTOX green is commonly used in NET-release studies [ 8 , 33 ], its main limitation is inability to differentiate between DNA released during NETosis and DNA from dead cells. To limit the risk of bias, viability of all neutrophils was tested before the experiments and only samples with viability >85% were used.…”

Section: Discussionmentioning

confidence: 99%

Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias

Ostafin

Ciepiela

Pruchniak

et al. 2021

IJMS

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Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications—a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

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“…Notwithstanding the great interest and relevant roles of NETs, their release mechanisms are not fully understood and some aspects of the process still remain unclear (Manda-Handzlik et al, 2019). In addition, the structure and possible functional roles of exDNA in NETs organization is still matter of investigation.…”

Section: Netsmentioning

confidence: 99%

The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms

et al. 2020

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The capacity to actively release genetic material into the extracellular environment has been reported for bacteria, archaea, fungi, and in general, for microbial communities, but it is also described in the context of multicellular organisms, animals and plants. This material is often present in matrices that locate outside the cells. Extracellular matrices have important roles in defense response and disease in microbes, animal and plants cells, appearing as barrier against pathogen invasion or for their recognition. Specifically, neutrophils extracellular traps (NETs) in animals and root extracellular traps (RETs) in plants, are recognized to be important players in immunity. A growing amount of evidence revealed that the extracellular DNA, in these contexts, plays an active role in the defense action. Moreover, the protective role of extracellular DNA against antimicrobials and mechanical stress also appears to be confirmed in bacterial biofilms. In parallel, recent efforts highlighted different roles of self (homologous) and non-self (heterologous) extracellular DNA, paving the way to discussions on its role as a “Damage-associated molecular pattern” (DAMP). We here provide an evolutionary overview on extracellular DNA in extracellular matrices like RETs, NETs, and microbial biofilms, discussing on its roles and inferring on possible novel functionalities.

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Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

Cited by 58 publications

References 54 publications

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

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

Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias

The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms

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