RESUMO
Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that neutrophils from patients with APS used glycolysis more avidly than neutrophils from people in the healthy control group, especially when the neutrophils were from patients with APS with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.
Assuntos
Síndrome Antifosfolipídica , Armadilhas Extracelulares , Glucose , Glicólise , Neutrófilos , Via de Pentose Fosfato , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Animais , Camundongos , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Síndrome Antifosfolipídica/tratamento farmacológico , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Masculino , Feminino , Glucose/metabolismo , Trombose/metabolismo , Trombose/imunologia , Trombose/patologia , Trombose/genética , Adulto , Espécies Reativas de Oxigênio/metabolismo , Pessoa de Meia-IdadeRESUMO
Our study aimed to evaluate the presence, clinical associations, and potential mechanistic roles of non-criteria antiphospholipid antibodies (aPL) and circulating calprotectin, a highly stable marker of neutrophil extracellular trap release (NETosis), in pediatric APS patients. We found that 79% of pediatric APS patients had at least one non-criteria aPL at moderate-to-high titer. Univariate logistic regression demonstrated that positive anti-beta-2 glycoprotein I domain 1 (anti-D1) IgG (p = 0.008), anti-phosphatidylserine/prothrombin (aPS/PT) IgG (p < 0.001), and aPS/PT IgM (p < 0.001) were significantly associated with venous thrombosis. Positive anti-D1 IgG (p < 0.001), aPS/PT IgG (p < 0.001), and aPS/PT IgM (p = 0.001) were also associated with non-thrombotic manifestations of APS, such as thrombocytopenia. Increased levels of calprotectin were detected in children with APS. Calprotectin correlated positively with absolute neutrophil count (r = 0.63, p = 0.008) and negatively with platelet count (r = -0.59, p = 0.015). Mechanistically, plasma from pediatric APS patients with high calprotectin levels impaired platelet viability in a dose-dependent manner.
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Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica , Humanos , Criança , Biomarcadores , beta 2-Glicoproteína I , Imunoglobulina G , Imunoglobulina M , Protrombina , Complexo Antígeno L1 LeucocitárioRESUMO
ABSTRACT: Many patients with antiphospholipid syndrome had decreased ectonucleotidase activity on neutrophils and platelets, which enabled extracellular nucleotides to trigger neutrophil-platelet aggregates. This phenotype was replicated by treating healthy neutrophils and platelets with patient-derived antiphospholipid antibodies or ectonucleotidase inhibitors.
Assuntos
Síndrome Antifosfolipídica , Humanos , Neutrófilos , Anticorpos Antifosfolipídeos , PlaquetasRESUMO
OBJECTIVE: While thrombosis and pregnancy loss are the best-known clinical features of antiphospholipid syndrome (APS), many patients also exhibit "extra-criteria" manifestations, such as thrombocytopenia. The mechanisms that drive APS thrombocytopenia are not completely understood, and no clinical biomarkers are available for predicting antiphospholipid antibody (aPL)-mediated thrombocytopenia. Calprotectin is a heterodimer of S100A8 and S100A9 that is abundant in the neutrophil cytoplasm and released upon proinflammatory neutrophil activation. Here, we sought to evaluate the presence, clinical associations, and potential mechanistic roles of circulating calprotectin in a cohort of primary APS and aPL-positive patients. METHODS: Levels of circulating calprotectin were determined in plasma by the QUANTA Flash chemiluminescent assay. A viability dye-based platelet assay was used to assess the potential impact of calprotectin on aPL-mediated thrombocytopenia. RESULTS: Circulating calprotectin was measured in 112 patients with primary APS and 30 aPL-positive (without APS criteria manifestations or lupus) patients as compared to patients with lupus (without APS), patients with unprovoked venous thrombosis (without aPL), and healthy controls. Levels of calprotectin were higher in patients with primary APS and aPL-positive patients compared to healthy controls. After adjustment for age and sex, calprotectin level correlated positively with absolute neutrophil count (r = 0.41, P < 0.001), positively with C-reactive protein level (r = 0.34, P = 0.002), and negatively with platelet count (r = -0.24, P = 0.004). Mechanistically, we found that calprotectin provoked aPL-mediated thrombocytopenia by engaging platelet surface toll-like receptor 4 and activating the NLRP3-inflammasome, thereby reducing platelet viability in a caspase-1-dependent manner. CONCLUSION: These data suggest that calprotectin has the potential to be a functional biomarker and a new therapeutic target for APS thrombocytopenia.
Assuntos
Síndrome Antifosfolipídica , Plaquetas , Complexo Antígeno L1 Leucocitário , Trombocitopenia , Humanos , Síndrome Antifosfolipídica/sangue , Feminino , Complexo Antígeno L1 Leucocitário/sangue , Masculino , Pessoa de Meia-Idade , Adulto , Trombocitopenia/sangue , Plaquetas/metabolismo , Biomarcadores/sangue , Receptor 4 Toll-Like/sangue , Anticorpos Antifosfolipídeos/sangueRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Armadilhas Extracelulares , Camundongos , Animais , Armadilhas Extracelulares/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Acetilcolina , Neutrófilos/metabolismo , Tromboxanos/metabolismoRESUMO
Risk of severe disease and death due to COVID-19 is increased in certain patient demographic groups, including those of advanced age, male sex, and obese body mass index. Investigations of the biological variations that contribute to this risk have been hampered by heterogeneous severity, with immunologic features of critical disease potentially obscuring differences between risk groups. To examine immune heterogeneity related to demographic risk factors, we enrolled 38 patients hospitalized with clinically homogeneous COVID-19 pneumonia - defined as oxygen saturation less than 94% on room air without respiratory failure, septic shock, or multiple organ dysfunction - and performed single-cell RNA-Seq of leukocytes collected at admission. Examination of individual risk factors identified strong shifts within neutrophil and monocyte/dendritic cell (Mo/DC) compartments, revealing altered immune cell type-specific responses in higher risk COVID-19 patient subgroups. Specifically, we found transcriptional evidence of altered neutrophil maturation in aged versus young patients and enhanced cytokine responses in Mo/DCs of male versus female patients. Such innate immune cell alterations may contribute to outcome differences linked to these risk factors. They also highlight the importance of diverse patient cohorts in studies of therapies targeting the immune response in COVID-19.
Assuntos
COVID-19 , Humanos , Feminino , Masculino , Idoso , Índice de Massa Corporal , Citocinas , Hipóxia , Imunidade Inata , DemografiaRESUMO
Beta-2 glycoprotein I (ß2GPI) is a phospholipid-binding plasma protein and prominent autoantigen in antiphospholipid syndrome (APS). Here, we tested the hypothesis that ß2GPI might bind to not only phospholipids, but also cell-free DNA and neutrophil extracellular traps (NETs). We report that ß2GPI interacts with cell-free DNA from different species, as well as NETs, in a dose-dependent manner, retarding their migration in an agarose-gel electrophoretic mobility shift assay. We confirm the direct binding interaction of ß2GPI with DNA and NETs by ELISA. We also demonstrate that ß2GPI colocalizes with NET strands by immunofluorescence microscopy. Finally, we provide evidence that ß2GPI-DNA complexes can be detected in the plasma of APS patients, where they positively correlate with an established biomarker of NET remnants. Taken together, our findings indicate that ß2GPI interacts with DNA and NETs, and suggest that this interaction may play a role as a perpetuator and/or instigator of autoimmunity in APS.
RESUMO
While immunologic correlates of COVID-19 have been widely reported, their associations with post-acute sequelae of COVID-19 (PASC) remain less clear. Due to the wide array of PASC presentations, understanding if specific disease features associate with discrete immune processes and therapeutic opportunities is important. Here we profile patients in the recovery phase of COVID-19 via proteomics screening and machine learning to find signatures of ongoing antiviral B cell development, immune-mediated fibrosis, and markers of cell death in PASC patients but not in controls with uncomplicated recovery. Plasma and immune cell profiling further allow the stratification of PASC into inflammatory and non-inflammatory types. Inflammatory PASC, identifiable through a refined set of 12 blood markers, displays evidence of ongoing neutrophil activity, B cell memory alterations, and building autoreactivity more than a year post COVID-19. Our work thus helps refine PASC categorization to aid in both therapeutic targeting and epidemiological investigation of PASC.
Assuntos
COVID-19 , Neutrófilos , Humanos , Síndrome de COVID-19 Pós-Aguda , Inflamação , Antivirais , Progressão da DoençaRESUMO
OBJECTIVE: This study aimed to elucidate the presence, antigen specificities, and potential clinical associations of anti-neutrophil extracellular trap (anti-NET) antibodies in a multinational cohort of antiphospholipid (aPL) antibody-positive patients who did not have lupus. METHODS: Anti-NET IgG/IgM levels were measured in serum samples from 389 aPL-positive patients; 308 patients met the classification criteria for antiphospholipid syndrome. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of the patients (n = 214), we profiled autoantibodies using an autoantigen microarray platform. RESULTS: We found elevated levels of anti-NET IgG and/or IgM in 45% of the aPL-positive patients. High anti-NET antibody levels are associated with more circulating myeloperoxidase (MPO)-DNA complexes, which are a biomarker of NETs. When considering clinical manifestations, positive anti-NET IgG was associated with lesions affecting the white matter of the brain, even after adjusting for demographic variables and aPL profiles. Anti-NET IgM tracked with complement consumption after controlling for aPL profiles; furthermore, patient serum samples containing high levels of anti-NET IgM efficiently deposited complement C3d on NETs. As determined by autoantigen microarray, positive testing for anti-NET IgG was significantly associated with several autoantibodies, including those recognizing citrullinated histones, heparan sulfate proteoglycan, laminin, MPO-DNA complexes, and nucleosomes. Anti-NET IgM positivity was associated with autoantibodies targeting single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen. CONCLUSION: These data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients, where they potentially activate the complement cascade. While anti-NET IgM may especially recognize DNA in NETs, anti-NET IgG species appear to be more likely to target NET-associated protein antigens.
Assuntos
Síndrome Antifosfolipídica , Armadilhas Extracelulares , Humanos , Anticorpos Antifosfolipídeos , Autoanticorpos , Imunoglobulina G , Imunoglobulina MRESUMO
This study analyzed the nuclease- and serum-driven degradation of millimeter-scale, circular DNA-histone mesostructures (DHMs). DHMs are bioengineered chromatin meshes of defined DNA and histone compositions designed as minimal mimetics of physiological extracellular chromatin structures, such as neutrophil extracellular traps (NETs). Taking advantage of the defined circular shape of the DHMs, an automated time-lapse imaging and image analysis method was developed and used to track DHM degradation and shape changes over time. DHMs were degraded well by 10 U/mL concentrations of deoxyribonuclease I (DNase I) but not by the same level of micrococcal nuclease (MNase), whereas NETs were degraded well by both nucleases. These comparative observations suggest that DHMs have a less accessible chromatin structure compared to NETs. DHMs were degraded by normal human serum, although at a slower rate than NETs. Interestingly, time-lapse images of DHMs revealed qualitative differences in the serum-mediated degradation process compared to that mediated by DNase I. Importantly, despite their reduced susceptibility to degradation and compositional simplicity, the DHMs mimicked NETs in being degraded to a greater extent by normal donor serum compared to serum from a lupus patient with high disease activity. These methods and insights are envisioned to guide the future development and expanded use of DHMs, beyond the previously reported antibacterial and immunostimulatory analyses, to extracellular chromatin-related pathophysiological and diagnostic studies.
Assuntos
Cromatina , Armadilhas Extracelulares , Humanos , Cromatina/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , DNA/metabolismo , Desoxirribonuclease I/metabolismoRESUMO
OBJECTIVE: Leucocyte immunoglobulin-like receptor A3 (LILRA3) belongs to a family of leucocyte receptors. Our previous study reported LILRA3 transcripts were markedly upregulated in neutrophils from patients with APS. We undertook this study to investigate clinical implications of LILRA3 in APS and its potential role in APS-associated thrombosis. METHODS: Two independent cohorts were studied. The first consisted of 294 APS patients, 48 asymptomatic aPL carriers and 150 healthy controls (HCs) from Peking University People's Hospital. The second included 99 APS patients, 25 aPL carriers and 40 HCs from United States APS centres. Serum or plasma concentrations of LILRA3 and MPO-DNA complexes were measured. Additionally, 35 patients with thrombotic APS (tAPS) were evaluated to determine potential effects of immunosuppressive therapy on serum concentrations of LILRA3 and MPO-DNA complexes. RESULTS: Both positivity and serum concentration of LILRA3 were significantly increased in APS patients, especially in those with tAPS. LILRA3-positive tAPS patients displayed more severe thrombotic manifestations. Serum LILRA3 was positively correlated with MPO-DNA complexes in LILRA3-positive tAPS. After immunosuppressive treatment, LILRA3 and MPO-DNA complexes were consistently decreased in tAPS patients. Key findings from the Peking cohort were confirmed in the United States cohort. CONCLUSION: Our study provides first evidence that LILRA3 is aberrantly expressed in APS, especially in patients with tAPS. Serum LILRA3 correlated with MPO-DNA complexes, and the two indices were consistently decreased in tAPS patients after treatment. LILRA3 may play a role in thrombosis of APS and may serve as a biomarker and/or therapeutic target in tAPS.
Assuntos
Síndrome Antifosfolipídica , Trombose , Humanos , Síndrome Antifosfolipídica/complicações , Anticorpos Antifosfolipídeos , Trombose/etiologia , Trombose/tratamento farmacológico , Biomarcadores , Estudos de Coortes , Receptores ImunológicosRESUMO
OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thromboinflammatory complications of COVID-19, the upstream mediators of endotheliopathy remain, for the most part, unknown. This study was undertaken to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID-19-related sepsis. Cell adhesion molecules (E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 [ICAM-1]) were quantified using in-cell enzyme-linked immunosorbent assay. RESULTS: Serum and plasma from COVID-19 patients increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and correlated with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly reduced the up-regulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to indicate that some COVID-19 patients have potentially diverse antibodies that drive endotheliopathy, providing important context regarding thromboinflammatory effects of autoantibodies in severe COVID-19.
Assuntos
Anticorpos Antifosfolipídeos , COVID-19 , Células Endoteliais , Anticorpos Antifosfolipídeos/imunologia , COVID-19/imunologia , Moléculas de Adesão Celular/metabolismo , Selectina E , Células Endoteliais/metabolismo , Endotélio Vascular , Humanos , Imunoglobulina G/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVE: Defibrotide is a heterogenous mixture of polyanionic oligonucleotides currently approved for treatment of transplant-associated venoocclusive disease. While defibrotide has a known role in limiting endothelial cell activation, some studies have also demonstrated anti-leukocyte properties. In a recent study, we found that neutrophil extracellular traps (NETs) play a role in the thrombotic complications of antiphospholipid syndrome (APS). In the present study, we investigated the hypothesis that defibrotide might act to mitigate APS-relevant NET formation in vitro and in mouse models. METHODS: We used in vitro assays and a mouse model to determine the mechanisms by which defibrotide inhibits NET formation and venous thrombosis in APS. RESULTS: At doses ranging from 1 to 10 µg/ml, defibrotide significantly suppressed NET formation from control neutrophils stimulated with IgG isolated from patients with APS. Defibrotide increased levels of intracellular cyclic AMP in neutrophils, and its suppressive effects on NET formation were mitigated by blocking adenosine A2A receptor or by inhibiting the cyclic AMP-dependent kinase protein kinase A. Defibrotide at doses ranging from 15 to 150 mg/kg/day inhibited NET formation and venous thrombosis in a model of antiphospholipid antibody-accelerated thrombosis-an effect that was reduced in adenosine A2A receptor-knockout mice. CONCLUSION: This study is the first to demonstrate mechanisms by which defibrotide counteracts neutrophil-mediated thrombotic inflammation inherent to APS.
Assuntos
Síndrome Antifosfolipídica , Armadilhas Extracelulares , Trombose , Trombose Venosa , Animais , Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Humanos , Camundongos , Neutrófilos/metabolismo , Polidesoxirribonucleotídeos , Receptor A2A de Adenosina , Trombose/etiologia , Trombose/prevenção & controleRESUMO
OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thrombo-inflammatory complications of coronavirus disease-19 ( COVID-19 ), the upstream mediators of endotheliopathy remain for the most part cryptic. Our aim was to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID sepsis. Cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) were quantified by in-cell ELISA. RESULTS: Serum and plasma from patients with COVID-19 increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and tracked with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly restrained upregulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to suggest that some patients with COVID-19 have potentially diverse antibodies that drive endotheliopathy, adding important context regarding thrombo-inflammatory effects of autoantibodies in severe COVID-19.
RESUMO
Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19.
Assuntos
COVID-19/patologia , Di-Hidrotestosterona/farmacologia , Endotélio Vascular/patologia , Inflamação , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Espironolactona/farmacologia , Antagonistas de Receptores de Angiotensina/farmacologia , COVID-19/fisiopatologia , COVID-19/virologia , Moléculas de Adesão Celular/sangue , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Humanos , Masculino , Caracteres Sexuais , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/fisiologia , Valsartana/farmacologiaRESUMO
Neutrophil-mediated activation and injury of the endothelium play roles in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap-derived [NET-derived] histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the US Food and Drug Administration-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.
Assuntos
Fibrinolíticos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Trombose/tratamento farmacológico , Animais , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Fibrinolíticos/uso terapêutico , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polidesoxirribonucleotídeos/uso terapêutico , PiroptoseRESUMO
The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19, where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM. Both anti-NET IgG and anti-NET IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and, to a lesser extent, anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID-19 sera. Furthermore, purified IgG from COVID-19 sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.
Assuntos
Autoanticorpos/imunologia , COVID-19/imunologia , Armadilhas Extracelulares/imunologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Autoanticorpos/sangue , COVID-19/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Adulto JovemRESUMO
The release of neutrophil extracellular traps ( NETs ) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p<0.0001). Both anti-NET IgG and IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and to a lesser extent anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID sera. Furthermore, purified IgG from COVID sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.
RESUMO
Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro , defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo , defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.
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Patients with coronavirus disease-19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. However, bleeding complications have also been observed in some patients. Understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. 118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot-lysis assays. We found markedly elevated tPA and PAI-1 levels in patients hospitalized with COVID-19. Both factors demonstrated strong correlations with neutrophil counts and markers of neutrophil activation. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were strongly correlated with mortality and a significant enhancement in spontaneous ex vivo clot-lysis. While both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis. Further study of tPA as a biomarker is warranted.