The effect and possible clinical efficacy of in vivo inhibition of neutrophil extracellular traps by blockade of PI3K-gamma on the pathogenesis of microscopic polyangiitis

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

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The Emerging Role of Neutrophil Extracellular Traps in Arterial, Venous and Cancer-Associated Thrombosis

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.786387 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yilu Zhou ◽

Weimin Tao ◽

Fuyi Shen ◽

Weijia Du ◽

Zhendong Xu ◽

...

Keyword(s):

Current Knowledge ◽

Neutrophil Extracellular Traps ◽

Vital Role ◽

Extracellular Traps ◽

Protein Components ◽

Cancer Associated Thrombosis ◽

Coagulation Pathway

Neutrophils play a vital role in the formation of arterial, venous and cancer-related thrombosis. Recent studies have shown that in a process known as NETosis, neutrophils release proteins and enzymes complexed to DNA fibers, collectively called neutrophil extracellular traps (NETs). Although NETs were originally described as a way for the host to capture and kill bacteria, current knowledge indicates that NETs also play an important role in thrombosis. According to recent studies, the destruction of vascular microenvironmental homeostasis and excessive NET formation lead to pathological thrombosis. In vitro experiments have found that NETs provide skeletal support for platelets, red blood cells and procoagulant molecules to promote thrombosis. The protein components contained in NETs activate the endogenous coagulation pathway to promote thrombosis. Therefore, NETs play an important role in the formation of arterial thrombosis, venous thrombosis and cancer-related thrombosis. This review will systematically summarize and explain the study of NETs in thrombosis in animal models and in vivo experiments to provide new targets for thrombosis prevention and treatment.

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Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

Cardiovascular Research ◽

10.1093/cvr/cvy036 ◽

2018 ◽

Vol 114 (8) ◽

pp. 1178-1188 ◽

Cited By ~ 14

Author(s):

Daniel S Gaul ◽

Julien Weber ◽

Lambertus J van Tits ◽

Susanna Sluka ◽

Lisa Pasterk ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Infarction ◽

Bone Marrow ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Wild Type ◽

Rotational Thromboelastometry ◽

Extracellular Traps

AbstractAimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).ConclusionsSirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

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Neutrophil Extracellular Traps May Contribute to the Pathogenesis in Adult-onset Still Disease

The Journal of Rheumatology ◽

10.3899/jrheum.181058 ◽

2019 ◽

Vol 46 (12) ◽

pp. 1560-1569 ◽

Cited By ~ 4

Author(s):

Mi-Hyun Ahn ◽

Jae Ho Han ◽

Young-Jun Chwae ◽

Ju-Yang Jung ◽

Chang-Hee Suh ◽

...

Keyword(s):

Immunohistochemical Analysis ◽

Serum Levels ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Adult Onset ◽

Cell Free Dna ◽

Extracellular Traps ◽

Free Dna ◽

Still Disease

Objective.Release of neutrophil extracellular traps (NET) has been described as an effector mechanism of polymorphonuclear neutrophils in several inflammatory diseases. Thus, this study was performed to evaluate the role of NET in the pathogenesis of adult-onset Still disease (AOSD).Methods.We determined the serum levels of NET molecules and investigated their associations with clinical disease activities in patients with AOSD. Further, we analyzed the differences in the NETosis response in AOSD patients compared to healthy controls (HC). To explore the in vivo involvement of NET in AOSD, we performed immunohistochemical analysis of skin and lymph node (LN) biopsies for proteins related to NET in patients with active AOSD.Results.Serum levels of cell-free DNA, myeloperoxidase (MPO)-DNA complex, and α-defensin were significantly increased in patients with AOSD compared to HC. Serum levels of the NET molecules, cell-free DNA, MPO-DNA, and α-defensin were correlated with several disease activity markers for AOSD. In followup of patients with AOSD after treatment with corticosteroid, the levels of cell-free DNA and α-defensin decreased significantly. On immunohistochemistry, neutrophil elastase–positive and MPO-positive inflammatory cells were detected in skin and LN of patients with AOSD, and were expressed in fiber form in the lesions. The serum from patients with active AOSD induced NETosis in neutrophils from HC. NET molecules induced interleukin 1β production in monocytes, representing a novel mechanism in the pathogenesis of AOSD.Conclusion.The findings presented here suggest that NET may contribute to the inflammatory response and pathogenesis in AOSD.

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Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00144.2019 ◽

2020 ◽

Vol 319 (1) ◽

pp. L137-L147 ◽

Cited By ~ 1

Author(s):

Kristin M. Hudock ◽

Margaret S. Collins ◽

Michelle Imbrogno ◽

John Snowball ◽

Elizabeth L. Kramer ◽

...

Keyword(s):

Neutrophil Extracellular Traps ◽

Gm Csf ◽

Extracellular Traps ◽

Tnf Α ◽

Multiple Donors ◽

Novel Target ◽

Induced Changes ◽

Air Liquid Interface

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

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Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo

Blood ◽

10.1182/blood-2011-12-401133 ◽

2012 ◽

Vol 120 (22) ◽

pp. 4421-4431 ◽

Cited By ~ 118

Author(s):

Kan Chen ◽

Hiroshi Nishi ◽

Richard Travers ◽

Naotake Tsuboi ◽

Kimberly Martinod ◽

...

Keyword(s):

Immune Complexes ◽

Fluid Phase ◽

Neutrophil Extracellular Traps ◽

Humoral Factors ◽

Extracellular Traps ◽

Fluid Phase Endocytosis ◽

Soluble Immune Complexes ◽

The Individual

Abstract Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)–linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.

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How Neutrophil Extracellular Traps Become Visible

Journal of Immunology Research ◽

10.1155/2016/4604713 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13 ◽

Cited By ~ 47

Author(s):

Nicole de Buhr ◽

Maren von Köckritz-Blickwede

Keyword(s):

Electron Microscopy ◽

Nuclear Dna ◽

Defense Mechanism ◽

Neutrophil Extracellular Traps ◽

Immune Defense ◽

Extracellular Traps ◽

Activated Neutrophils ◽

Microscopy Techniques

Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one handin vitrolive-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital,in vivo, andin situmicroscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.

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