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.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2.

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Circulating Nucleosomes as Potential Markers to Monitor COVID-19 Disease Progression.

The severity of coronavirus disease 2019 (COVID-19) varies significantly with cases spanning from asymptomatic to lethal with a subset of individuals developing Severe Acute Respiratory Syndrome (SARS) and death from respiratory failure. To determine whether global nucleosome and citrullinated nucleosome levels were elevated in COVID-19 patients, we tested two independent cohorts of COVID-19 positive patients with quantitative nucleosome immunoassays and found that nucleosomes were highly elevated in plasma of COVID-19 patients with a severe course of the disease relative to healthy controls and that both histone 3.1 variant and citrullinated nucleosomes increase with disease severity. Elevated citrullination of circulating nucleosomes is indicative of neutrophil extracellular trap formation, neutrophil activation and NETosis in severely affected individuals. Importantly, using hospital setting (outpatient, inpatient or ICU) as a proxy for disease severity, nucleosome levels increased with disease severity and may serve as a guiding biomarker for treatment. Owing to the limited availability of mechanical ventilators and extracorporal membrane oxygenation (ECMO) equipment, there is an urgent need for effective tools to rapidly assess disease severity and guide treatment selection. Based on our studies of two independent cohorts of COVID-19 patients from Belgium and Germany, we suggest further investigation of circulating nucleosomes and citrullination as biomarkers for clinical triage, treatment allocation and clinical drug discovery.

Peptidylarginine deiminases and extracellular vesicles: prospective drug targets and biomarkers in central nervous system diseases and repair.

Peptidylarginine deiminases are a family of calcium-activated enzymes with multifaceted roles in physiological and pathological processes, including in the central nervous system. Peptidylarginine deiminases cause post-translational deimination/citrullination, leading to changes in structure and function of a wide range of target proteins. Deimination can facilitate protein moonlighting, modify protein-protein interaction, cause protein dysfunction and induce inflammatory responses. Peptidylarginine deiminases also regulate the biogenesis of extracellular vesicles, which play important roles in cellular communication through transfer of extracellular vesicle-cargo, e.g., proteins and genetic material. Both peptidylarginine deiminases and extracellular vesicles are linked to a number of pathologies, including in the central nervous system, and their modulation with pharmacological peptidylarginine deiminase inhibitors have shown great promise in several in vitro and in vivo central nervous system disease models. Furthermore, extracellular vesicles derived from mesenchymal stem cells have been assessed for their therapeutic application in central nervous system injury. As circulating extracellular vesicles can be used as non-invasive liquid biopsies, their specific cargo-signatures (including deiminated proteins and microRNAs) may allow for disease "fingerprinting" and aid early central nervous system disease diagnosis, inform disease progression and response to therapy. This mini-review discusses recent advances in the field of peptidylarginine deiminase and extracellular vesicle research in the central nervous system, focusing on several central nervous system acute injury, degeneration and cancer models.

Citrullinated histone H3, a marker of extracellular trap formation, is increased in blood of stable asthma patients.

BACKGROUND: Emerging data indicates that extracellular traps (ETs), structures formed by various immune cell types, may contribute to the pathology of noninfectious inflammatory diseases. Histone hypercitrullination is an important step in ETs formation and citrullinated histone H3 (H3cit) is considered a novel and specific biomarker of that process. In the present study we have evaluated circulating H3cit in stable asthmatics and investigated its relationship with asthma severity, pulmonary function and selected blood and bronchoalveolar lavage (BAL) biomarkers. METHODS: In 60 white adult stable asthmatics and 50 well-matched controls we measured serum levels of H3cit. In asthmatics we also performed bronchoscopy with BAL. We analyzed blood and BAL biomarkers, including interleukin (IL)-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A and interferon γ. For statistical analysis, Mann-Whitney U-test, χ2 test, one-way ANCOVA, ROC curve analysis and univariate linear regression were applied. Independent determinants of H3cit were established in a multiple linear regression model. RESULTS: Asthma was characterized by elevated circulating H3cit (17.49 [11.25-22.58] vs. 13.66 [8.66-18.87] ng/ml, p = 0.03). In asthmatics positive associations were demonstrated between serum H3cit and lung function variables, including total lung capacity (TLC) (ß = 0.37 [95% CI 0.24-0.50]) and residual volume (ß = 0.38 [95% CI 0.25-0.51]). H3cit was increased in asthma patients receiving systemic steroids (p = 0.02), as well as in subjects with BAL eosinophilia above 144 cells/ml (p = 0.02). In asthmatics, but not in controls, circulating H3cit correlated well with number of neutrophils (ß = 0.31 [95% CI 0.19-0.44]) and monocytes (ß = 0.42 [95% CI 0.29-0.55]) in peripheral blood. Furthermore, BAL macrophages, BAL neutrophils, TLC, high-sensitivity C-reactive protein, Il-12p70 and bronchial obstruction degree were independent determinants of H3cit in a multivariate linear regression model. CONCLUSIONS: Asthma is characterized by increased circulating H3cit likely related to the enhanced lung ETs formation. Inhibition of ETs might be a therapeutic option in selected asthma phenotypes, such as neutrophilic asthma.

Vitamin D Deficiency and Air Pollution Exacerbate COVID-19 Through Suppression of Antiviral Peptide LL37.

Vitamin D deficiency and insufficiency (VDD) are widely recognized as risk factors for respiratory tract infections. Vitamin D influences expression of many genes with well-established relevance to airway infections and relevant to immune system function. Recently, VDD has been shown to be a risk factor for acquisition and severity of COVID-19. Thus, treating VDD presents a safe and inexpensive opportunity for modulating the severity of the disease. VDD is common in those over 60 years of age, many with co-morbid conditions and in people with skin pigmentation sufficient to reduce synthesis of vitamin D. Exposure to fine particulate air pollution is also associated with worse outcomes from COVID19. Vitamin D stimulates transcription of cathelicidin which is cleaved to generate LL37. LL37 is an innate antimicrobial with demonstrated activity against a wide range of microbes including envelope viruses. LL37 also modulates cytokine signaling at the site of infections. Fine particles in air pollution can interfere with LL37 destruction of viruses and may reduce effective immune signaling modulation by LL37. While vitamin D influences transcription of many immune related genes, the weakened antimicrobial response of those with VDD against SARS-CoV-2 may be in part due to reduced LL37. Conclusion: Vitamin D plays an important role reducing the impact of viral lung disease processes. VDD is an acknowledged public health threat that warrants population-wide action to reduce COVID-19 morbidity and mortality. While vitamin D influences transcription of many immune related genes, the weakened antimicrobial response of those with VDD against SARS-CoV-2 may be in part due to reduced LL37. Action is needed to address COVID-19 associated risks of air pollution from industry, transportation, domestic sources and from primary and second hand tobacco smoke.