Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

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Circulatory neutrophils exhibit enhanced neutrophil extracellular trap formation in early puerperium: NETs at the nexus of thrombosis and immunity?

10.1101/2021.10.18.459827 ◽

2021 ◽

Author(s):

Stavros Giaglis ◽

Chanchal Sur Chowdhury ◽

Shane Vontelin Van Breda ◽

Maria Stoikou ◽

Guenther Shaefer ◽

...

Keyword(s):

Serum Markers ◽

Late Gestation ◽

Neutrophil Extracellular Trap ◽

Coagulation Cascade ◽

Physiological Alterations ◽

Fatal Hemorrhage ◽

Circulating Microparticles ◽

Neutrophil Degranulation ◽

Extracellular Trap

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation in vitro, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal hemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs - citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activation status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

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In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

BIO-PROTOCOL ◽

10.21769/bioprotoc.2386 ◽

2017 ◽

Vol 7 (13) ◽

Author(s):

Dongsheng Jiang ◽

Mona Saffarzadeh ◽

Karin Scharffetter-Kochanek

Keyword(s):

Neutrophil Extracellular Trap ◽

Trap Formation ◽

Extracellular Trap

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In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

Journal of Visualized Experiments ◽

10.3791/58083 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ronald H.L. Li ◽

Fern Tablin

Keyword(s):

Quantitative Analysis ◽

Fluorescence Microscopy ◽

Neutrophil Extracellular Trap ◽

Trap Formation ◽

Extracellular Trap

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P063. Mesalazine and cigarette smoke inhibit neutrophil extracellular trap formation in vitro

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jju027.181 ◽

2015 ◽

Vol 9 (suppl 1) ◽

pp. S108-S108

Keyword(s):

Cigarette Smoke ◽

Neutrophil Extracellular Trap ◽

Trap Formation ◽

Extracellular Trap

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Neutrophil extracellular trap formation and nuclease activity in septic patients

10.21203/rs.2.12307/v1 ◽

2019 ◽

Author(s):

Linda Cox ◽

Kai Walstein ◽

Lena Völlger ◽

Friederike Reuner ◽

Alexandra Bick ◽

...

Keyword(s):

Healthy Volunteers ◽

Nuclease Activity ◽

Positive Control ◽

Neutrophil Extracellular Trap ◽

Average Percentage ◽

Molecular Pattern ◽

Extracellular Trap ◽

Net Release

Abstract Background: There 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) serum nuclease activities are increased as well. Methods: We included 18 septic patients and 27 volunteers in this prospective observational trial while study was registered retrospectively. Blood was withdrawn and NET formation from neutrophils in vitro was quantified (average percentage of neutrophils showing NET formation on an image) without stimulation and following incubation with mtDNA (10µg/well) or PMA (25nmol). Serum nuclease activity was assessed using gel electrophoresis. Results: In contrast to our hypothesis, compared to healthy volunteers unstimulated NET release from neutrophils in septic patients 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 hours of incubation. 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 volun-teers. As expected, PMA, as positive control, increased NET formation to 73.2% (±29.6) in septic patients and to 91.7% (±7.1) in volunteers after 4 hours of incubation (p=0.22). 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. Conclusions: Unstimulated NET formation and nuclease activity are decreased in septic patients and mtDNA can further reduce NET formation. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. Trail registration DRKS00007694, German Clinical Trials database (DRKS). Registered retrospectively 06.02.2015.

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Zearalenone Induces Estrogen-Receptor-Independent Neutrophil Extracellular Trap Release in Vitro

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.8b05948 ◽

2019 ◽

Vol 67 (16) ◽

pp. 4588-4594 ◽

Cited By ~ 6

Author(s):

Jing-Jing Wang ◽

Zheng-Kai Wei ◽

Zhen Han ◽

Zi-Yi Liu ◽

Xing-Yi Zhu ◽

...

Keyword(s):

Estrogen Receptor ◽

Neutrophil Extracellular Trap ◽

Extracellular Trap ◽

Release In Vitro

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TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4

Nature Communications ◽

10.1038/s41467-021-23881-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qian Ou ◽

Jia-qi Fang ◽

Zhe-sheng Zhang ◽

Zhe Chi ◽

Jie Fang ◽

...

Keyword(s):

Innate Immune ◽

Neutrophil Extracellular Trap ◽

Mrna Levels ◽

Peptidylarginine Deiminase ◽

E Coli ◽

Peptidylarginine Deiminase 4 ◽

Trap Formation ◽

Extracellular Trap ◽

Innate Immune Evasion

AbstractTcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.

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Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation

Scientific Reports ◽

10.1038/s41598-021-00863-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hoi Woul Lee ◽

Victor Nizet ◽

Jung Nam An ◽

Hyung Seok Lee ◽

Young Rim Song ◽

...

Keyword(s):

Inflammatory Responses ◽

Umbilical Vein ◽

Composite Endpoint ◽

Neutrophil Extracellular Trap ◽

Intracellular Adhesion Molecule ◽

Uremic Serum ◽

Control Serum ◽

Extracellular Trap

AbstractCardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

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Longitudinal characterization of circulating neutrophils uncovers distinct phenotypes associated with disease severity in hospitalized COVID-19 patients

10.1101/2021.10.04.463121 ◽

2021 ◽

Author(s):

Thomas J. LaSalle ◽

Anna L. K. Gonye ◽

Samuel S. Freeman ◽

Paulina Kaplonek ◽

Irena Gushterova ◽

...

Keyword(s):

Disease Severity ◽

Severe Disease ◽

Gene Expression Signature ◽

Suppressor Cell ◽

Gene Signature ◽

Neutrophil Extracellular Trap ◽

Effector Functions ◽

Neutrophil Degranulation ◽

Mechanistic Basis

Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19- acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.

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