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.     

Neutrophil Extracellular Traps and Their Implications in Cardiovascular and Inflammatory Disease

Neutrophils are primary effector cells of innate immunity and fight infection by phagocytosis and degranulation. Activated neutrophils also release neutrophil extracellular traps (NETs) in response to a variety of stimuli. These NETs are net-like complexes composed of cell-free DNA, histones and neutrophil granule proteins. Besides the evolutionarily conserved mechanism to capture and eliminate pathogens, NETs are also associated with pathophysiological processes of various diseases. Here, we elucidate the mechanisms of NET formation and their different implications in disease. We focused on autoinflammatory and cardiovascular disorders as the leading cause of death. Neutrophil extracellular traps are not only present in various cardiovascular diseases but play an essential role in atherosclerotic plaque formation, arterial and venous thrombosis, as well as in the development and progression of abdominal aortic aneurysms. Furthermore, NETosis can be considered as a source of autoantigens and maintains an inflammatory milieu promoting autoimmune diseases. Indeed, there is further need for research into the balance between NET induction, inhibition, and degradation in order to pharmacologically target NETs and their compounds without impairing the patient’s immune defense. This review may be of interest to both basic scientists and clinicians to stimulate translational research and innovative clinical approaches.     

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

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.     

Tetrahydroisoquinolines: New Inhibitors of Neutrophil Extracellular Trap (NET) Formation

Neutrophils are short‐lived leukocytes that migrate to sites of infection as part of the acute immune response, where they phagocytose, degranulate, and form neutrophil extracellular traps (NETs). During NET formation, the nuclear lobules of neutrophils disappear and the chromatin expands and, accessorized with neutrophilic granule proteins, is expelled. NETs can be pathogenic in, for example, sepsis, cancer, and autoimmune and cardiovascular diseases. Therefore, the identification of inhibitors of NET formation is of great interest. Screening of a focused library of natural‐product‐inspired compounds by using a previously validated phenotypic NET assay identified a group of tetrahydroisoquinolines as new NET formation inhibitors. This compound class opens up new avenues for the study of cellular death through NET formation (NETosis) at different stages, and might inspire new medicinal chemistry programs aimed at NET‐dependent diseases.     

CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet–Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet–neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b−/− mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b−/− animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.     

Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α

Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by Methyl-β-cyclodextrin (MβCD) is known as one of the processes initiating NET formation. Since neutrophils mainly act in an inflammatory environment with decreased, so-called hypoxic, oxygen conditions, we aimed to study the effect of oxygen and the oxygen stress regulator hypoxia-inducible factor (HIF)-1α on cholesterol-dependent NET formation. Thus, murine bone marrow-derived neutrophils from wild-type and HIF-knockout mice or human neutrophils were stimulated with MβCD under normoxic (21% O₂) compared to hypoxic (1% O₂) conditions, and the formation of NETs were studied by immunofluorescence microscopy. We found significantly induced NET formation after treatment with MβCD in murine neutrophils derived from wild-type as well as HIF-1α KO mice at both hypoxic (1% O₂) as well as normoxic (21% O₂) conditions. Similar observations were made in freshly isolated human neutrophils after stimulation with MβCD or statins, which block the HMG-CoA reductase as the key enzyme in the cholesterol metabolism. HPLC was used to confirm the reduction of cholesterol in treated neutrophils. In summary, we were able to show that NET formation via MβCD or statin-treatment is oxygen and HIF-1α independent.     

Role of Neutrophils and NETs in Animal Models of Thrombosis

Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as “neutrophil extracellular traps” (NETs). Initially, described as “dead sacrifices for a good cause” that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research.     

The Role of the Neutrophilic Network in the Pathogenesis of Psoriasis

One role of neutrophils, the most abundant innate immune sentinels, is neutrophil extracellular trap (NET) formation, which plays a significant role in immune surveillance. However, NET operation is bidirectional. Recent studies report that NETs may contribute to the development of autoimmune diseases such as psoriasis. The participation of neutrophils in the pathogenesis of that disease is dependent on an autoinflammatory feedback loop between neutrophils, lymphocytes, dendritic cells and keratinocytes. Our aim was to clarify the field of NET research in psoriasis and highlight the main factors required for NET generation, which may be a target of new therapies. This article presents a comphrehensive review concerning studies addressing the participation of neutrophils in the pathogenesis of psoriasis. Based on the available English-language literature, we discuss original papers presenting significant research findings which may help to understand and interpret the NET formation process in psoriasis, as well as the newest systematic reviews on PubMed. Next, the comparison, synthesis and summary of reported results were performed to clearly indicate the specific component of the NET which participates in the development of psoriasis.     

Anti-Inflammatory Effects and Decreased Formation of Neutrophil Extracellular Traps by Enoxaparin in COVID-19 Patients

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO–DNA and Histone–DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.     

High Mobility Group Box 1 Protein in Cerebral Thromboemboli

High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6–6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke.     

Neutrophil Extracellular Traps in Colorectal Cancer Progression and Metastasis

Neutrophils form sticky web-like structures known as neutrophil extracellular traps (NETs) as part of innate immune response. NETs are decondensed extracellular chromatin filaments comprising nuclear and cytoplasmic proteins. NETs have been implicated in many gastrointestinal diseases including colorectal cancer (CRC). However, the regulatory mechanisms of NET formation and potential pharmacological inhibitors in the context of CRC have not been thoroughly discussed. In this review, we intend to highlight roles of NETs in CRC progression and metastasis as well as the potential of targeting NETs during colon cancer therapy.     

High-Fat Diet Aggravates Cerebral Infarct,Hemorrhagic Transformation and Neuroinflammation in a Mouse Stroke Model

Background: Stroke in context of type 2 diabetes (T2D) is associated with a poorer outcome than in non-diabetic conditions. We aimed at creating a new reproducible mouse model of stroke in impaired glucose tolerance conditions induced by high-fat diet. Methods: Adult C57BL6 mice were fed for 2 months with either normal diet (ND) or high-fat diet (HFD). We used a model of Middle Cerebral Artery Occlusion (MCAO) for 90 min. Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT) were used to assess pre-diabetic status. Brain infarct volume, hemorrhagic transformation (HT) as well as systemic and cerebral inflammatory markers were evaluated. Results: HFD was associated with an increased body weight and glycemia following OGTT. The HFD group presented a significant increase in brain infarct volume (38.7 (IQR 30–46.7%) vs. 28.45 (IQR 21–30%); p = 0.016) and HT (HFD: 2 (IQR 1–5) vs. ND: 0 (IQR 0–1); p = 0.012) and higher levels of IL-6 and MCP-1 in infarcted hemisphere compared to the ND group. Conclusion: Two months of HFD in adult mice were sufficient to alter the lipid profile and the control of hyperglycemia. These metabolic perturbations were significantly associated with increased infarct volume and hemorrhagic complications.     

Short-Term Effect of SARS-CoV-2 Spike Protein Receptor-Binding Domain-Specific Antibody Induction on Neutrophil-Mediated Immune Response in Mice

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2⁺CD101⁺ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.     

Redefining the Role of ADAM17 in Renal Proximal Tubular Cells and Its Implications in an Obese Mouse Model of Pre-Diabetes

Acute and chronic kidney lesions induce an increase in A Disintegrin And Metalloproteinase domain 17 (ADAM17) that cleaves several transmembrane proteins related to inflammatory and fibrotic pathways. Our group has demonstrated that renal ADAM17 is upregulated in diabetic mice and its inhibition decreases renal inflammation and fibrosis. The purpose of the present study was to analyze how Adam17 deletion in proximal tubules affects different renal structures in an obese mice model. Tubular Adam17 knockout male mice and their controls were fed a high-fat diet (HFD) for 22 weeks. Glucose tolerance, urinary albumin-to-creatinine ratio, renal histology, and pro-inflammatory and pro-fibrotic markers were evaluated. Results showed that wild-type mice fed an HFD became obese with glucose intolerance and renal histological alterations mimicking a pre-diabetic condition; consequently, greater glomerular size and mesangial expansion were observed. Adam17 tubular deletion improved glucose tolerance and protected animals against glomerular injury and prevented podocyte loss in HFD mice. In addition, HFD mice showed more glomerular macrophages and collagen accumulation, which was prevented by Adam17 deletion. Galectin-3 expression increased in the proximal tubules and glomeruli of HFD mice and ameliorated with Adam17 deletion. In conclusion, Adam17 in proximal tubules influences glucose tolerance and participates in the kidney injury in an obese pre-diabetic murine model. The role of ADAM17 in the tubule impacts on glomerular inflammation and fibrosis.     

Beyond Hemostasis: Platelet Innate Immune Interactions and Thromboinflammation

There is accumulating evidence that platelets play roles beyond their traditional functions in thrombosis and hemostasis, e.g., in inflammatory processes, infection and cancer, and that they interact, stimulate and regulate cells of the innate immune system such as neutrophils, monocytes and macrophages. In this review, we will focus on platelet activation in hemostatic and inflammatory processes, as well as platelet interactions with neutrophils and monocytes/macrophages. We take a closer look at the contributions of major platelet receptors GPIb, α_IIbβ₃, TLT-1, CLEC-2 and Toll-like receptors (TLRs) as well as secretions from platelet granules on platelet–neutrophil aggregate and neutrophil extracellular trap (NET) formation in atherosclerosis, transfusion-related acute lung injury (TRALI) and COVID-19. Further, we will address platelet–monocyte and macrophage interactions during cancer metastasis, infection, sepsis and platelet clearance.     

Endothelial Dysfunction Induced by Extracellular Neutrophil Traps Plays Important Role in the Occurrence and Treatment of Extracellular Neutrophil Traps-Related Disease

Many articles have demonstrated that extracellular neutrophil traps (NETs) are often described as part of the antibacterial function. However, since the components of NETs are non-specific, excessive NETs usually cause inflammation and tissue damage. Endothelial dysfunction (ED) caused by NETs is the major focus of tissue damage, which is highly related to many inflammatory diseases. Therefore, this review summarizes the latest advances in the primary and secondary mechanisms between NETs and ED regarding inflammation as a mediator. Moreover, the detailed molecular mechanisms with emphasis on the disadvantages from NETs are elaborated: NETs can use its own enzymes, release particles as damage-associated molecular patterns (DAMPs) and activate the complement system to interact with endothelial cells (ECs), drive ECs damage and eventually aggravate inflammation. In view of the role of NETs-induced ED in different diseases, we also discussed possible molecular mechanisms and the treatments of NETs-related diseases.     

Methylprednisolone Induces Extracellular Trap Formation and Enhances Bactericidal Effect of Canine Neutrophils

Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.     

The Emerging Role of Neutrophils in the Pathogenesis of Thrombosis in COVID-19

Previous studies have shown that COVID-19 leads to thrombotic complications, which have been associated with high morbidity and mortality rates. Neutrophils are the largest population of white blood cells and play a pivotal role in innate immunity. During an infection, neutrophils migrate from circulation to the infection site, contributing to killing pathogens. This mechanism is regulated by chemokines such as IL-8. Moreover, it was shown that neutrophils play an important role in thromboinflammation. Through a diverse repertoire of mechanisms, neutrophils, apart from directly killing pathogens, are able to activate the formation of thrombi. In COVID-19 patients, neutrophil activation promotes neutrophil extracellular trap (NET) formation, platelet aggregation, and cell damage. Furthermore, neutrophils participate in the pathogenesis of endothelitis. Overall, this review summarizes recent progress in research on the pathogenesis of COVID-19, highlighting the role of the prothrombotic action of neutrophils in NET formation.     

The Impact of Cytokines on Neutrophils’ Phagocytosis and NET Formation during Sepsis—A Review

Sepsis is an overwhelming inflammatory response to infection, resulting in multiple-organ injury. Neutrophils are crucial immune cells involved in innate response to pathogens and their migration and effector functions, such as phagocytosis and neutrophil extracellular trap (NET) formation, are dependent on cytokine presence and their concentration. In the course of sepsis, recruitment and migration of neutrophils to infectious foci gradually becomes impaired, thus leading to loss of a crucial arm of the innate immune response to infection. Our review briefly describes the sepsis course, the importance of neutrophils during sepsis, and explains dependence between cytokines and their activation. Moreover, we, for the first time, summarize the impact of cytokines on phagocytosis and NET formation. We highlight and discuss the importance of cytokines in modulation of both processes and emphasize the direction of further investigations.