scholarly journals An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular Traps

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 191
Author(s):  
Apurwa Singhal ◽  
Shubhi Yadav ◽  
Tulika Chandra ◽  
Shrikant R. Mulay ◽  
Anil Nilkanth Gaikwad ◽  
...  
Keyword(s):  
High Throughput ◽  
Lupus Erythematosus ◽  
Computational Algorithm ◽  
Single Cell Analysis ◽  
Annexin V ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps ◽  
Potential Inhibitors ◽  
Apoptosis And Necrosis

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.

scholarly journals The enrichment of neutrophil extracellular traps impair the placentas of systemic lupus erythematosus through accumulating decidual NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Jiang ◽  
Nan Shen ◽  
Haibo Zhou ◽  
You Wang ◽  
Sihan Lin ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Nk Cells ◽  
Lupus Erythematosus ◽  
Neutrophil Extracellular Traps ◽  
Clinical Samples ◽  
Systemic Lupus ◽  
Extracellular Traps ◽  
Fetal Birth Weight ◽  
Immunological Disorder ◽  
Adverse Pregnancy

AbstractDespite the advances made in the management of pregnancies in women with systemic lupus erythematosus (SLE), the rate of adverse pregnancy outcomes is still higher than that in the general population. In the last few years, neutrophil extracellular traps (NETs) were proven to be detrimental in both autoimmune diseases and placental injury. We investigated whether NETs could be detected in the placentas of pregnant individuals with SLE and explored the relationship between NETs and decidual natural killer cells (dNKs), which comprise the majority of immune cells at the maternal–fetal interface, using clinical samples and animal models. In this study, we found that the infiltration of NETs and dNKs, especially CD56+CD16+ NK cells, was significantly increased in pregnant individuals with SLE with placental insufficiency. In the murine models of SLE, the number of dNKs was significantly decreased due to the decreased formation of NETs affected by Ly6G. Moreover, the histopathological placental injury was reduced, with a remarkable increase in fetal birth weight. This study shows that NETs may contribute to immunological disorder in the placenta and the pathological changes in pregnancies with SLE, which provides a research basis for further explorations of the mechanism of SLE in placental impairment.

scholarly journals Neutrophil Extracellular Traps-DNase Balance and Autoimmunity

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2667
Author(s):  
Andrea Angeletti ◽  
Stefano Volpi ◽  
Maurizio Bruschi ◽  
Francesca Lugani ◽  
Augusto Vaglio ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Causative Factor ◽  
Neutrophil Extracellular Traps ◽  
Limiting Factor ◽  
Systemic Lupus ◽  
Extracellular Traps ◽  
Number Of Patients ◽  
Selective Depletion ◽  
Dna Antibodies ◽  
Rate Limiting

Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNASE genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.

scholarly journals MFG-E8 accelerates wound healing in diabetes by regulating “NLRP3 inflammasome-neutrophil extracellular traps” axis

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Wei Huang ◽  
Jinyu Jiao ◽  
Ju Liu ◽  
Meng Huang ◽  
Yanyan Hu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Lupus Erythematosus ◽  
Nlrp3 Inflammasome ◽  
Milk Fat ◽  
Therapeutic Potential ◽  
Neutrophil Extracellular Traps ◽  
Skin Wound ◽  
Diabetic Patients ◽  
High Dose ◽  
Extracellular Traps

Abstract Sustained activation of NLRP3 inflammasome and release of neutrophil extracellular traps (NETs) impair wound healing of diabetic foot ulcers (DFUs). Our previous study reported that milk fat globule epidermal growth factor VIII (MFG-E8) attenuates tissue damage in systemic lupus erythematosus. However, the functional effect of MFG-E8 on “NLRP3 inflammasome-NETs” inflammatory loop in wound healing of diabetes is not completely elucidated. In this study, neutrophils from DFU patients are susceptible to undergo NETosis, releasing more NETs. The circulating levels of NET components neutrophil elastase and proteinase 3 and inflammatory cytokines IL-1β and IL-18 were significantly elevated in DFU patients compared with healthy controls or diabetic patients, in spite of higher levels of MFG-E8 in DFU patients. In Mfge8−/− diabetic mice, skin wound displayed exaggerated inflammatory response, including leukocyte infiltration, excessive activation of NLRP3 inflammasome (release of higher IL-1β, IL-18, and TNF-α), largely lodged NETs, resulting in poor angiogenesis and wound closure. When stimulated with high-dose glucose or IL-18, MFG-E8-deficient neutrophils release more NETs than WT neutrophils. After administration of recombinant MFG-E8, IL-18-primed NETosis of WT or Mfge8−/− neutrophils was significantly inhibited. Furthermore, NET and mCRAMP (component of NETs, the murine equivalent of cathelicidin LL-37 in human)-mediated activation of NLRP3 inflammasome and production of IL-1β/IL-18 were significantly elevated in Mfge8−/− macrophages compared with WT macrophages, which were also significantly dampened by the administration of rmMFG-E8. Therefore, our study demonstrated that as inhibitor of the “NLRP3 inflammasome-NETs” inflammatory loop, exogenous rMFG-E8 improves angiogenesis and accelerates wound healing, highlighting possible therapeutic potential for DFUs.

Neutrophil extracellular traps-associated markers are elevated in patients with systemic lupus erythematosus

2019 ◽  
Vol 39 (11) ◽  
pp. 1849-1857 ◽  
Author(s):  
Ivica Jeremic ◽  
Olivera Djuric ◽  
Milos Nikolic ◽  
Marina Vlajnic ◽  
Aleksandra Nikolic ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Neutrophil Extracellular Traps ◽  
Systemic Lupus ◽  
Extracellular Traps

scholarly journals Neutrophil Extracellular Traps in the Autoimmunity Context

2021 ◽  
Vol 8 ◽  
Author(s):  
Maurizio Bruschi ◽  
Gabriella Moroni ◽  
Renato Alberto Sinico ◽  
Franco Franceschini ◽  
Micaela Fredi ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Critical Role ◽  
Basic Research ◽  
Neutrophil Extracellular Traps ◽  
New Drugs ◽  
Post Translational Modification ◽  
Clinical Trial Registration ◽  
Membrane Breakdown ◽  
Extracellular Traps ◽  
Registration Number

The formation of neutrophil extracellular traps (NETs) is a strategy utilized by neutrophils for capturing infective agents. Extracellular traps consist in a physical net made of DNA and intracellular proteins externalized from neutrophils, where bacteria and viruses are entrapped and killed by proteolysis. A complex series of events contributes to achieving NET formation: signaling from infectious triggers comes first, followed by decondensation of chromatin and extrusion of the nucleosome components (DNA, histones) from the nucleus and, after cell membrane breakdown, outside the cell. NETs are composed of either DNA or nucleosome proteins and hundreds of cytoplasm proteins, a part of which undergo post-translational modification during the steps leading to NETs. There is a thin balance between the production and the removal of circulating NETs from blood where digestion of DNA by circulating DNases 1 and IL3 has a critical role. A delay in NET removal may have consequences for autoimmunity. Recent studies have shown that circulating NET levels are increased in systemic lupus erythematosus (SLE) for a functional block of NET removal mediated by anti-DNase antibodies or, in rare cases, by DNase IL3 mutations. In SLE, the persistence in circulation of NETs signifies elevated concentrations of either free DNA/nucleosome components and oxidized proteins that, in some cases, are recognized as non-self and presented to B-cells by Toll-like receptor 9 (TLR9). In this way, it is activated as an immunologic response, leading to the formation of IgG2 auto-antibody. Monitoring serum NET levels represents a potential new way to herald the development of renal lesions and has clinical implications. Modulating the balance between NET formation and removal is one of the objectives of basic research that are aimed to design new drugs for SLE.Clinical Trial Registration Number: The Zeus study was registered at https://clinicaltrials.gov (study number: NCT02403115).

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