The central role of neutrophil extracellular traps in SARS-CoV-2-induced thrombogenesis and vasculitis

Objective: Severe acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) seen in SARs-CoV-2 infection has been attributed to the disruption of the immune response in COVID-19 patients. Neutrophilia and marked lymphocyte reductions are associated with disease severity and seem predictive of disease outcome in moderate and severe COVID-19 patients. Herein, we aim to decipher possible mechanisms involved in extensive tissue injury observed in COVID-19 patients, accompanied by vasculopathy, coagulopathy, and a high incidence of thrombotic complications in severe patients. Method(s): We searched PubMED for keywords including COVID-19 pathogenesis, thrombosis, and vasculities. Result(s): Neutrophils can undergo a specialized form of apoptosis to yield thread-like extracellular structures termed neutrophil extracellular traps (NETs), termed NETosis, which form web-like scaffolds of DNA, histones, and toxic protein granules and enzymes, whose primary function is to trap and eliminate microbes. However, uncontrolled NET production can lead to ALI and ARDS, coagulopathy, multiple organ failure, and autoimmune disease. Dysregulation of NETs promotes production of anti-neutrophil cytoplasmic antibodies (ANCA) which affects small vessels through ANCA-associated vasculitis (AAV). Furthermore, NETs can also induce thrombosis via formation of scaffolds that trap platelets, RBCs, fibronectin, and other proteins, which can also induce coagulation. Conclusion(s): We suggest that NET production is central during SARS-CoV-2 infection and COVID-19 pathogenesis, associated with alveolar damage accumulation of edema, endothelial injury and coagulopathy, elevated platelet activation and thrombogenesis forming a NET production feed-forward loop, causing diffuse small vessel vasculitis in the lungs and other organs. Copyright © 2020 FSG Communications Ltd. All rights reserved.

Thromboembolic Disease and Cardiac Thrombotic Complication in COVID-19: A Systematic Review.

The coronavirus 2019 pandemic has affected many healthcare systems worldwide. While acute respiratory distress syndrome (ARDS) has been well-documented in COVID-19, there are several cardiovascular complications, such as myocardial infarction, ischaemic stroke, and pulmonary embolism, leading to disability and death. The link between COVID-19 and increasing thrombogenicity potentially occurs due to numerous different metabolic mechanisms, ranging from endothelial damage for direct virus infection, associated excessive formation of neutrophil extracellular traps (NETs), pathogenic activation of the renin-angiotensin-aldosterone system (RAAS), direct myocardial injury, and ischemia induced by respiratory failure, all of which have measurable biomarkers. A search was performed by interrogating three databases (MEDLINE; MEDLINE In-Process and Other Non-Indexed Citations, and EMBASE). Evidence from randomized controlled trials (RCT), prospective series, meta-analyses, and unmatched observational studies were evaluated for the processing of the algorithm and treatment of thromboembolic disease and cardiac thrombotic complications related to COVID-19 during SARS-CoV-2 infection. Studies out with the SARS-Cov-2 infection period and case reports were excluded. A total of 58 studies were included in this analysis. The role of the acute inflammatory response in the propagation of the systemic inflammatory sequelae of the disease plays a major part in determining thromboembolic disease and cardiac thrombotic complication in COVID-19. Some of the mechanisms of activation of these pathways, alongside the involved biomarkers noted in previous studies, are highlighted. Inflammatory response led to thromboembolic disease and cardiac thrombotic complications in COVID-19. NETs play a pivotal role in the pathogenesis of the inflammatory response. Despite moving into the endemic phase of the disease in most countries, thromboembolic complications in COVID-19 remain an entity that substantially impacts the health care system, with long-term effects that remain uncertain. Continuous monitoring and research are required.

NET Formation in Systemic Lupus Erythematosus: Changes during the COVID-19 Pandemic.

The severity of the coronavirus disease in 2019 (COVID-19) is strongly linked to a dysregulated immune response. This fuels the fear of severe disease in patients with autoimmune disorders continuously using immunosuppressive/immunomodulating medications. One complication of COVID-19 is thromboembolism caused by intravascular aggregates of neutrophil extracellular traps (NETs) occluding the affected vessels. Like COVID-19, systemic lupus erythematosus (SLE) is characterized by, amongst others, an increased risk of thromboembolism. An imbalance between NET formation and clearance is suggested to play a prominent role in exacerbating autoimmunity and disease severity. Serologic evidence of exposure to SARS-CoV-2 has a minor impact on the SLE course in a Swedish cohort reportedly. Herein, we assessed NET formation in patients from this cohort by neutrophil elastase (NE) activity and the presence of cell-free DNA, MPO-DNA, and NE-DNA complexes and correlated the findings to the clinical parameters. The presence of NE-DNA complexes and NE activity differed significantly in pre-pandemic versus pandemic serum samples. The latter correlated significantly with the hemoglobin concentration, blood cell counts, and complement protein 3 and 4 levels in the pre-pandemic but only with the leukocyte count and neutrophil levels in the pandemic serum samples. Taken together, our data suggest a change, especially in the NE activity independent of exposure to SARS-CoV-2.

Insights Into Immunothrombotic Mechanisms in Acute Stroke due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

Novel aspects of sepsis pathophysiology: NETs, plasma glycoproteins, endotheliopathy and COVID-19.

In 2016, sepsis was newly defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis remains one of the crucial medical problems to be solved worldwide. Although the world health organization has made sepsis a global health priority, there remain no specific and effective therapy for sepsis so far. Indeed, over the previous decades almost all attempts to develop novel drugs have failed. This may be partly ascribable to the multifactorial complexity of the septic cascade and the resultant difficulties of identifying drug targets. In addition, there might still be missing links among dysregulated host responses in vital organs. In this review article, recent advances in understanding of the complex pathophysiology of sepsis are summarized, with a focus on neutrophil extracellular traps (NETs), the significant role of NETs in thrombosis/embolism, and the functional roles of plasma proteins, histidine-rich glycoprotein (HRG) and inter-alpha-inhibitor proteins (IAIPs). The specific plasma proteins that are markedly decreased in the acute phase of sepsis may play important roles in the regulation of blood cells, vascular endothelial cells and coagulation. The accumulating evidence may provide us with insights into a novel aspect of the pathophysiology of sepsis and septic ARDS, including that in COVID-19.

Insights into the Role of Neutrophils and Neutrophil Extracellular Traps in Causing Cardiovascular Complications in Patients with COVID-19: A Systematic Review.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus has resulted in significant mortality and burdening of healthcare resources. While initially noted as a pulmonary pathology, subsequent studies later identified cardiovascular involvement with high mortalities reported in specific cohorts of patients. While cardiovascular comorbidities were identified early on, the exact manifestation and etiopathology of the infection remained elusive. This systematic review aims to investigate the role of inflammatory pathways, highlighting several culprits including neutrophil extracellular traps (NETs) which have since been extensively investigated. METHOD: A search was conducted using three databases (MEDLINE; MEDLINE In-Process & Other Non-Indexed Citations and EMBASE). Data from randomized controlled trials (RCT), prospective series, meta-analyses, and unmatched observational studies were considered for the processing of the algorithm and treatment of inflammatory response during SARS-CoV-2 infection. Studies without the SARS-CoV-2 Infection period and case reports were excluded. RESULTS: A total of 47 studies were included in this study. The role of the acute inflammatory response in the propagation of the systemic inflammatory sequelae of the disease plays a major part in determining outcomes. Some of the mechanisms of activation of these pathways have been highlighted in previous studies and are highlighted. CONCLUSION: NETs play a pivotal role in the pathogenesis of the inflammatory response. Despite moving into the endemic phase of the disease in most countries, COVID-19 remains an entity that has not been fully understood with long-term effects remaining uncertain and requiring ongoing monitoring and research.

Association between COVID-19 Diagnosis and Coronary Artery Thrombosis: A Narrative Review.

Coronavirus disease 2019 is characterized by its severe respiratory effects. Data early on indicated an increased risk of mortality in patients with cardiovascular comorbidities. Early reports highlighted the multisystem inflammatory syndrome, cytokine storm, and thromboembolic events as part of the disease processes. The aim of this review is to assess the association between COVID-19 and its thrombotic complications, specifically related to the cardiovascular system. The role of neutrophil extracellular traps (NETs) is explored in the pathogenesis of the disease. The structure and anatomy of the virus are pivotal to its virulence in comparison to other α and ß Coronaviridae (HCoV-229E, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1). In particular, the host interaction and response may explain the variability of severity in patients. Angio tensin-converting enzyme 2 (ACE2) activation may be implicated in the cardiovascular and throm bogenic potential of the disease. The virus may also have direct effects on the endothelial lining affecting hemostasis and resulting in thrombosis through several mechanisms. Dipyridamole may have a therapeutic benefit in NET suppression. Therapeutic avenues should be concentrated on the different pathophysiological steps involving the virus and the host.

Citrullination in the pathology of inflammatory and autoimmune disorders: recent advances and future perspectives.

Numerous post-translational modifications (PTMs) govern the collective metabolism of a cell through altering the structure and functions of proteins. The action of the most prevalent PTMs, encompassing phosphorylation, methylation, acylations, ubiquitination and glycosylation is well documented. A less explored protein PTM, conversion of peptidylarginine to citrulline, is the subject of this review. The process of citrullination is catalysed by peptidylarginine deiminases (PADs), a family of conserved enzymes expressed in a variety of human tissues. Accumulating evidence suggest that citrullination plays a significant role in regulating cellular metabolism and gene expression by affecting a multitude of pathways and modulating the chromatin status. Here, we will discuss the biochemical nature of arginine citrullination, the enzymatic machinery behind it and also provide information on the pathological consequences of citrullination in the development of inflammatory diseases (rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, periodontitis and COVID-19), cancer and thromboembolism. Finally, developments on inhibitors against protein citrullination and recent clinical trials providing a promising therapeutic approach to inflammatory disease by targeting citrullination are discussed.

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.

Lung-Centric Inflammation of COVID-19: Potential Modulation by Vitamin D.

SARS-CoV-2 infects the respiratory tract and leads to the disease entity, COVID-19. Accordingly, the lungs bear the greatest pathologic burden with the major cause of death being respiratory failure. However, organs remote from the initial site of infection (e.g., kidney, heart) are not spared, particularly in severe and fatal cases. Emerging evidence indicates that an excessive inflammatory response coupled with a diminished antiviral defense is pivotal in the initiation and development of COVID-19. A common finding in autopsy specimens is the presence of thrombi in the lungs as well as remote organs, indicative of immunothrombosis. Herein, the role of SARS-CoV-2 in lung inflammation and associated sequelae are reviewed with an emphasis on immunothrombosis. In as much as vitamin D is touted as a supplement to conventional therapies of COVID-19, the impact of this vitamin at various junctures of COVID-19 pathogenesis is also addressed.

Plasmin Cascade Mediates Thrombotic Events in SARS-CoV-2 Infection via Complement and Platelet-Activating Systems.

Objective: Recently emerged beta-coronavirus SARS-CoV-2, has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age. Recent clinical evidence suggests that the development of ARDS and subsequent pulmonary failure result from a complex interplay between cell types (endothelial, epithelial and immune) within the lung promoting inflammatory infiltration and a pro-coagulative state. How the complex molecular events mediated by SARS-CoV-2 in infected lung epithelial cells lead to thrombosis and pulmonary failure, is yet to be fully understood. Methods: We address these questions here, using publicly available transcriptomic data in the context of lung epithelia affected by SARS-CoV-2 and other respiratory infections, in vitro. We then extend our results to the understanding of in vivo lung, using a publicly available COVID-19 lung transcriptomic study. Results and Conclusions: Our analysis indicates that there exists a complex interplay between the fibrinolytic system particularly plasmin, and the complement and platelet-activating systems upon SARS-CoV-2 infection, with a potential for therapeutic intervention.

Association of Neutrophil Activation, More Than Platelet Activation, With Thrombotic Complications in Coronavirus Disease 2019.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 infection is associated with hypercoagulability, which predisposes to venous thromboembolism (VTE). We analyzed platelet and neutrophil activation in patients with coronavirus disease 2019 (COVID-19) and their association with VTE. METHODS: Hospitalized patients with COVID-19 and age- and sex-matched healthy controls were studied. Platelet and leukocyte activation, neutrophil extracellular traps (NETs), and matrix metalloproteinase 9, a neutrophil-released enzyme, were measured. Four patients were restudied after recovery. The activating effect of plasma from patients with COVID-19 on control platelets and leukocytes and the inhibiting activity of common antithrombotic agents on it were studied. RESULTS: A total of 36 patients with COVID-19 and 31 healthy controls were studied; VTE developed in 8 of 36 patients with COVID-19 (22.2%). Platelets and neutrophils were activated in patients with COVID-19. NET, but not platelet activation, biomarkers correlated with disease severity and were associated with thrombosis. Plasmatic matrix metalloproteinase 9 was significantly increased in patients with COVID-19. Platelet and neutrophil activation markers, but less so NETs, normalized after recovery. In vitro, plasma from patients with COVID-19 triggered platelet and neutrophil activation and NET formation, the latter blocked by therapeutic-dose low-molecular-weight heparin, but not by aspirin or dypiridamole. CONCLUSIONS: Platelet and neutrophil activation are key features of patients with COVID-19. NET biomarkers may help to predict clinical worsening and VTE and may guide low-molecular-weight heparin treatment.

COVID-19 effects on the kidney.

Apart from pulmonary disease, acute kidney injury (AKI) is one of the most frequent and most severe organ complications in severe coronavirus disease 2019 (COVID-19). The SARS-CoV­2 virus has been detected in renal tissue. Patients with chronic kidney disease (CKD) before and on dialysis and specifically renal transplant patients represent a particularly vulnerable population. The increasing number of COVID-19 infected patients with renal involvement led to an evolving interest in the analysis of its pathophysiology, morphology and modes of virus detection in the kidney. Meanwhile, there are ample data from several autopsy and kidney biopsy studies that differ in the quantity of cases as well as in their quality. While the detection of SARS-CoV­2 RNA in the kidney leads to reproducible results, the use of electron microscopy for visualisation of the virus is difficult and currently critically discussed due to various artefacts. The exact contribution of indirect or direct effects on the kidney in COVID-19 are not yet known and are currently the focus of intensive research.

Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19.

BACKGROUND: Many arguments suggest that neutrophils could play a prominent role in COVID-19. However, the role of key components of neutrophil innate immunity in severe forms of COVID-19 has deserved insufficient attention. We aimed to evaluate the involvement of neutrophil elastase, histone-DNA, and DNases in systemic and multi-organ manifestations of COVID-19. METHODS: We performed a multicenter study of markers of neutrophil innate immunity in 155 cases consecutively recruited in a screening center, local hospitals, and two regional university hospitals. The cases were evaluated according to clinical and biological markers of severity and multi-organ manifestations and compared to 35 healthy controls. RESULTS: Blood neutrophil elastase, histone-DNA, myeloperoxidase-DNA, and free dsDNA were dramatically increased, and DNase activity was decreased by 10-fold, compared with controls. Neutrophil elastase and histone-DNA were associated with intensive care admission, body temperature, lung damage, and markers of cardiovascular outcomes, renal failure, and increased interleukin-6 (IL-6), IL-8, and CXCR2. Neutrophil elastase was an independent predictor of the computed tomography score of COVID-19 lung damage and the number of affected organs, in multivariate analyses. The increased blood concentrations of NE and neutrophil extracellular traps were related to exacerbation of neutrophil stimulation through IL-8 and CXCR2 increased concentrations and increased serum DAMPs, and to impaired degradation of NETs as a consequence of the dramatic decrease in blood DNase activity. CONCLUSION: Our results point out the key role of neutrophil innate immunity exacerbation in COVID-19. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID-19.

COVID-19, neutrophil extracellular traps and vascular complications in obstetric practice.

An issue of the novel coronavirus infection spreading is currently in the first place among others in the list of the international medical community. Due to lack of information, conflicting research findings, multicomponent effect of the virus on the body host, as well as various consequences that the virus triggers in the body, now every medical specialty does study the viral attack pathogenesis. Recent months showed that vascular complications are the most severe in the Coronavirus Disease 2019 (COVID-19) and are the main cause of death in the patients. The mechanisms of vascular complications are complex and affect both the hemostatic system and immune responses, "inflammatory storm", disorders of the renin-angiotensin-aldosterone system, endotheliopathy, etc. Due to the leading role of vascular complications in the viral infection pathogenesis, several groups of patients are at extra risk, including pregnant women, patients with a burdened obstetric history, with hereditary thrombophilia and antiphospholipid syndrome, and patients after in vitro fertilization (IVF). In this category of pregnant women, use of low-molecular-weight heparins (LMWH) is particularly important for both prevention of vascular and obstetric complications, and for pathogenetic therapy of COVID-19.

Devilishly radical NETwork in COVID-19: Oxidative stress, neutrophil extracellular traps (NETs), and T cell suppression.

Pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and poses an unprecedented challenge to healthcare systems due to the lack of a vaccine and specific treatment options. Accordingly, there is an urgent need to understand precisely the pathogenic mechanisms underlying this multifaceted disease. There is increasing evidence that the immune system reacts insufficiently to SARS-CoV-2 and thus contributes to organ damage and to lethality. In this review, we suggest that the overwhelming production of reactive oxygen species (ROS) resulting in oxidative stress is a major cause of local or systemic tissue damage that leads to severe COVID-19. It increases the formation of neutrophil extracellular traps (NETs) and suppresses the adaptive arm of the immune system, i.e. T cells that are necessary to kill virus-infected cells. This creates a vicious cycle that prevents a specific immune response against SARS-CoV-2. The key role of oxidative stress in the pathogenesis of severe COVID-19 implies that therapeutic counterbalancing of ROS by antioxidants such as vitamin C or NAC and/or by antagonizing ROS production by cells of the mononuclear phagocyte system (MPS) and neutrophil granulocytes and/or by blocking of TNF-α can prevent COVID-19 from becoming severe. Controlled clinical trials and preclinical models of COVID-19 are needed to evaluate this hypothesis.

Novel therapeutic targets for SARS-CoV-2-induced acute lung injury: Targeting a potential IL-1ß/neutrophil extracellular traps feedback loop.

Most COVID-19 infected individuals present with mild flu-like symptoms; however, 5-10% of cases suffer from life-threatening pneumonia and respiratory failure. The pathogenesis of SARS-CoV-2 and its pathology of associated acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sepsis, coagulopathy and multiorgan failure is not known. SARS-CoV-2 is an envelope virus with S (spike), M (membrane), N (nucleocapsid) and E (envelop) proteins. In a closely related coronavirus (SARS-CoV), the transmembrane E protein exerts an important role in membrane-ionic transport through viroporins, deletion of which reduced levels of IL-1ß and a remarkably reduced lung edema compared to wild type. IL-1ß is generated by macrophages upon activation of intracellular NLRP3 (NOD-like, leucine rich repeat domains, and pyrin domain-containing protein 3), part of the functional NLRP3 inflammasome complex that detects pathogenic microorganisms and stressors, while neutrophils are enhanced by increasing levels of IL-1ß. Expiring neutrophils undergo "NETosis", producing thread-like extracellular structures termed neutrophil extracellular traps (NETs), which protect against mild infections and microbes. However, uncontrolled NET production can cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), coagulopathy, multiple organ failure, and autoimmune disease. Herein, we present arguments underlying our hypothesis that IL-1ß and NETs, mediated via NLRP3 inflammasomes, form a feed-forward loop leading to the excessive alveolar and endothelial damage observed in severe cases of COVID-19. Considering such assertions, we propose potential drug candidates that could be used to alleviate such pathologies. Considering that recent efforts to ascertain effective treatments of COVID-19 in severe patients has been less than successful, investigating novel avenues of treating this virus are essential.