Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation
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ABSTRACT:
SUBMITTER: Thazhathveettil J
PROVIDER: S-EPMC10960408 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.
| S-EPMC6138659 | biostudies-literature
Project description:Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.
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Project description:Novel metal oxide nanoparticle (NP) contrast agents may offer safety and functionality advantages over conventional gadolinium-based contrast agents (GBCAs) for cancer diagnosis by magnetic resonance imaging. However, little is known about the behavior of metal oxide NPs, or of their effect, upon coming into contact with the innate immune system. As neutrophils are the body's first line of defense, we sought to understand how manganese oxide and iron oxide NPs impact leukocyte functionality. Specifically, we evaluated whether contrast agents caused neutrophils to release web-like fibers of DNA known as neutrophil extracellular traps (NETs), which are known to enhance metastasis and thrombosis in cancer patients. Murine neutrophils were treated with GBCA, bare manganese oxide or iron oxide NPs, or poly(lactic-co-glycolic acid) (PLGA)-coated metal oxide NPs with different incorporated levels of poly(ethylene glycol) (PEG). Manganese oxide NPs elicited the highest NETosis rates and had enhanced neutrophil uptake properties compared to iron oxide NPs. Interestingly, NPs with low levels of PEGylation produced more NETs than those with higher PEGylation. Despite generating a low rate of NETosis, GBCA altered neutrophil cytokine expression more than NP treatments. This study is the first to investigate whether manganese oxide NPs and GBCAs modulate NETosis and reveals that contrast agents may have unintended off-target effects which warrant further investigation.
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Project description:During the acute inflammatory response, the release of neutrophil extracellular traps (NETs) is a pro-inflammatory, preconditioning event on a biomaterial surface. Therefore, regulation of NET release through biomaterial design is one strategy to enhance biomaterial-guided in situ tissue regeneration. In this study, IgG adsorption on electrospun polydioxanone biomaterials with differing fiber sizes was explored as a regulator of in vitro human neutrophil NET release. The propensity to release NETs was increased and decreased by modulating adsorbed IgG, suggesting a functional link between IgG and NET formation. Fiber-size dependent NET release was reduced by blocking FcγRIIIb, but not FcγRI, FcγRIIa, or Mac-1 (CD11b/CD18), indicating a specific receptor mediated neutrophil response. Inhibition of transforming growth factor-β-activated kinase 1 (TAK1), which is activated downstream of FcγRIIIb, significantly reduced the release of NETs in a fiber size-independent manner. These results indicate that in vitro electrospun biomaterial-induced NET release is largely regulated by IgG adsorption, engagement of FcγRIIIb, and signaling through TAK1. Modulation of this pathway may have beneficial therapeutic effects for regulating neutrophil-mediated inflammation by avoiding the adverse effects of NETs and increasing the potential for in situ tissue regeneration. STATEMENT OF SIGNIFICANCE: Electrospun biomaterials have great potential for in situ tissue engineering because of their versatility and biomimetic properties. However, understanding how to design the biomaterial to regulate acute inflammation, dominated by neutrophils, remains a great challenge for successful tissue integration and regeneration. In this work, we demonstrate for the first time how protein adsorption on the biomaterial surface and engagement of a specific neutrophil receptor induces intracellular signals that regulate the pro-inflammatory release of neutrophil extracellular traps (NETs). Given the deleterious effects of NETs during the acute inflammatory response to a biomaterial, our work highlights the importance of considering biomaterial-neutrophil interactions on degradable and non-degradable biomaterials to achieve the desired biological outcome.
| S-EPMC8316391 | biostudies-literature
Project description:Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease characterized by collagen deposition within the interstitium of the lung. This impairs gas exchange and results in eventual respiratory failure. Clinical studies show a correlation between elevated neutrophil numbers and IPF disease progression; however, the mechanistic roles neutrophils play in this disease are not well described. In the present study, we describe alterations to the trafficking and function of neutrophils after the development of fibrosis. We observed increased numbers of total and aged neutrophils in peripheral tissues of fibrotic mice. This appeared to be driven by an upregulation of neutrophil chemokine Cxcl2 by lung cells. In addition, neutrophil recruitment back to the bone marrow for clearance appeared to be impaired, because we saw decreased aged neutrophils in the bone marrow of fibrotic mice. Neutrophils in fibrosis were activated, because ex vivo assays showed increased elastase and extracellular trap release by neutrophils from fibrotic mice. This likely mediated disease exacerbation, because mice exhibiting a progressive disease phenotype with greater weight loss and mortality had more activated neutrophils and increased levels of extracellular DNA present in their lungs than did mice with a nonprogressive disease phenotype. These findings further our understanding of the dynamics of neutrophil populations and their trafficking in progressive fibrotic lung disease and may help inform treatments targeting neutrophil function for patients with IPF experiencing disease exacerbation in the future.
| S-EPMC10542701 | biostudies-literature
Project description:ObjectiveThe release of neutrophil extracellular traps (NETs) by hyperactive neutrophils has recently been recognized to play an important role in antiphospholipid syndrome (APS). This study was undertaken to evaluate autoantibodies targeting NETs in patients with primary APS, and to determine their potential functions and clinical associations.MethodsWe measured global anti-NET activity in 76 patients with primary APS, 23 patients with systemic lupus erythematosus without antiphospholipid antibodies (aPL), 11 patients with a history of unprovoked venous thrombosis without aPL, and 44 healthy controls. The ability of APS sera to degrade NETs was also assessed.ResultsWe found markedly elevated levels of anti-NET IgG and IgM in patients with primary APS compared with healthy controls (for IgG, mean ± SD optical density 0.55 ± 0.34 versus 0.33 ± 0.17; for IgM, mean ± SD optical density 0.76 ± 0.51 versus 0.26 ± 0.23). This anti-NET activity did not correlate with levels of traditional aPL and was relatively stable over time. Mechanistically, anti-NET antibodies (especially of the IgG isotype) impaired the ability of patient sera to degrade NETs (r = 0.4, P = 0.003). Levels of anti-NET IgM inversely correlated with complement C4 (r = 0.4, P = 0.019). Clinically, anti-NET antibodies associated with certain APS clinical manifestations, and in particular recurrent venous thrombosis (odds ratio 4.3; P = 0.002). Interestingly, anti-NET antibody levels also appeared to be associated with unprovoked venous thrombosis in the general population (for IgM, mean ± SD optical density 0.67 ± 0.34 versus 0.26 ± 0.23).ConclusionOur data indicate high levels of anti-NET antibodies in patients with primary APS, which may impair NET clearance and activate the complement cascade. These findings may ultimately enable more effective risk stratification.
| S-EPMC7722115 | biostudies-literature
Project description:Excessive release of neutrophil extracellular traps (NETs) has been reported in various human pathologies, including COVID-19 patients. Elevated NET levels serve as a biomarker, indicating increased coagulopathy and immunothrombosis risks in these patients. Traditional immunoassays employed to quantify NET release focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NET release from primary human neutrophils with single-cell resolution in the presence of the motile bacteria Pseudomonas aeruginosa PAO1 and inflammatory mediators. The device was engineered to have wide chambers and constricted loops to measure NET release in variably confined spaces. Our open NET-array device enabled immunofluorescent labeling of citrullinated histone H3, a NET release marker. We took time-lapse images of primary healthy human neutrophils releasing NETs in clinically relevant infection and inflammation-rich microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α [20 ng mL-1] and interleukin-6 [50 ng mL-1], but not leukotriene B4 [20 nM], compared to the infection alone. We also quantified the temporal dynamics of NET release and differences in the relative areas of NETs, showing a high percentage of variable size NET release with combined PAO1 - inflammatory mediator treatment, in the device chambers. Importantly, we demonstrated reduced NET release in the confined loops of our combined infection-inflammation microsystem. Ultimately, our NET-array device stands as a valuable tool, facilitating experiments that enhance our comprehension of the spatiotemporal dynamics of NET release in response to infection within a defined microenvironment. In the future, our system can be used for high throughput and cost-effective screening of novel immunotherapies on human neutrophils in view of the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.
| S-EPMC10826461 | biostudies-literature
Project description:A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.
| S-EPMC5529471 | biostudies-literature
Project description:While neutrophil extracellular traps (NETs) have previously been linked to some diabetes-associated complications, such as dysfunctional wound healing, their potential role in diabetic vascular dysfunction has not been studied. Diabetic Akita mice were crossed with either Elane-/- or Pad4-/- mice to generate NET-deficient diabetic mice. By 24 weeks of age, Akita aortae showed markedly impaired relaxation in response to acetylcholine, indicative of vascular dysfunction. Both Akita-Elane-/- mice and Akita-Pad4-/- mice had reduced levels of circulating NETs and improved acetylcholine-mediated aortic relaxation. Compared with wild-type aortae, the thromboxane metabolite TXB2 was roughly 10-fold higher in both intact and endothelium-denuded aortae of Akita mice. In contrast, Akita-Elane-/- and Akita-Pad4-/- aortae had TXB2 levels similar to wild type. In summary, inhibition of NETosis by two independent strategies prevented the development of vascular dysfunction in diabetic Akita mice. Thromboxane was up-regulated in the vessel walls of NETosis-competent diabetic mice, suggesting a role for neutrophils in driving the production of this vasoconstrictive and atherogenic prostanoid.
| S-EPMC10599623 | biostudies-literature
Project description:BackgroundTriptolide (PG490), as a triterpene dicyclic oxide has been reported to increase the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induce apoptosis of RAW 264.7 cells in a dose-dependent manner. The activity of death NETs plays an important role in anti-bacterial processes in the human body. This study aimed to investigate the effect of triptolide (PG490) on neutrophil extracellular traps (NETs) formation.MethodsAfter isolating peripheral blood neutrophils from healthy volunteers, cells were incubated with PG490 to observe and detect the level of NETs and detect the level of reactive oxygen species (ROS). The cells were cultured, stained and analyzed by fluorescence microscopy.ResultsCompared with the 12-myristate-13-acetate (PMA) group, the average fluorescence intensity of SYTOX Green in the PG490 + PMA group, as detected by a multifunctional microplate reader, was significantly decreased. Intracellular ROS were labeled by fluorescence, with fluorescence intensity then measured by multifunctional microplate reader and flow cytometry. The results showed that compared with the control group, the fluorescence intensity of the PMA group was significantly increased, while there was no significant difference between PMA group and PG490 + PMA group.ConclusionsThe production of NETs is inhibited by PG490 in vitro, which is not associated with the level of cellular ROS. This suggests that PG490in Tripterygium wilfordii Hook F can suppress related diseases.
| S-EPMC8506553 | biostudies-literature