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1.
mBio ; 12(1)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33593982

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to public health crises worldwide. An understanding of the pathogenesis and the development of treatment strategies is of high interest. Recently, neutrophil extracellular traps (NETs) have been identified as a potential driver of severe SARS-CoV-2 infections in humans. NETs are extracellular DNA fibers released by neutrophils after contact with various stimuli and accumulate antimicrobial substances or host defense peptides. When massively released, NETs are described to contribute to immunothrombosis in acute respiratory distress syndrome and in vascular occlusions. Based on the increasing evidence that NETs contribute to severe COVID-19 cases, DNase treatment of COVID-19 patients to degrade NETs is widely discussed as a potential therapeutic strategy. Here, we discuss potential detrimental effects of NETs and their nuclease degradation, since NET fragments can boost certain bacterial coinfections and thereby increase the severity of the disease.


Assuntos
/metabolismo , Coinfecção/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Infecções Bacterianas/metabolismo , Coinfecção/microbiologia , Coinfecção/virologia , Humanos
2.
Int J Med Sci ; 18(3): 846-851, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33437221

RESUMO

In the last 50 years we have experienced two big pandemics, the HIV pandemic and the pandemic caused by SARS-CoV-2. Both pandemics are caused by RNA viruses and have reached us from animals. These two viruses are different in the transmission mode and in the symptoms they generate. However, they have important similarities: the fear in the population, increase in proinflammatory cytokines that generate intestinal microbiota modifications or NETosis production by polymorphonuclear neutrophils, among others. They have been implicated in the clinical, prognostic and therapeutic attitudes.


Assuntos
/epidemiologia , Infecções por HIV/epidemiologia , HIV-1/patogenicidade , Pandemias/história , /patogenicidade , /imunologia , /transmissão , Citocinas/sangue , Citocinas/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Medo , Carga Global da Doença/estatística & dados numéricos , Infecções por HIV/imunologia , Infecções por HIV/psicologia , Infecções por HIV/transmissão , HIV-1/imunologia , HIV-1/isolamento & purificação , História do Século XX , História do Século XXI , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Mortalidade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pandemias/estatística & dados numéricos , Prognóstico , /isolamento & purificação
3.
Methods Mol Biol ; 2241: 193-198, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486738

RESUMO

The process of extracellular DNA trap release by leukocytes, including eosinophils, has been considered as an important cell-mediated immune response to different inflammatory stimuli helping to understand the physiopathology of many diseases. Here we describe in detail two useful and simple protocols for a semiquantitative and a qualitative analysis of extracellular DNA traps released by human eosinophils, based on fluorimetry and fluorescence microscopy, respectively. These methods can also be applied to detect the DNA trap release by other leukocytes such as neutrophils and even other cell types.


Assuntos
Eosinófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Microscopia de Fluorescência/métodos , Eosinófilos/fisiologia , Armadilhas Extracelulares/imunologia , Humanos , Leucócitos , Neutrófilos/imunologia
4.
Nat Commun ; 12(1): 683, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514748

RESUMO

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid ß, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and ß-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid ß as the protagonist and potential therapeutic target.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Armadilhas Extracelulares/metabolismo , Neoplasias/patologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Medula Óssea/patologia , Antígeno CD11b/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Carcinogênese/patologia , Comunicação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias/sangue , Neoplasias/genética , Neoplasias/mortalidade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estudos Observacionais como Assunto , Cultura Primária de Células , Prognóstico , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos
5.
Cells ; 10(1)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466589

RESUMO

Severe contagious respiratory disease-COVID-19-caused by the SARS-CoV-2 coronavirus, can lead to fatal respiratory failure associated with an excessive inflammatory response. Infiltration and spread of SARS-CoV-2 are based on the interaction between the virus' structural protein S and the cell's receptor-angiotensin-converting enzyme 2 (ACE2), with the simultaneous involvement of human trans-membrane protease, serine 2 (TMPRSS2). Many scientific reports stress the importance of elevated recruitment and activity of neutrophils, which can form extracellular neutrophil traps (NETs) playing a significant role in the mechanism of combating pathogens, in the pathogenesis of COVID-19. Excessive generation of NETs during prolonged periods of inflammation predisposes for the occurrence of undesirable reactions including thromboembolic complications and damage to surrounding tissues and organs. Within the present manuscript, we draw attention to the impact of NET generation on the severe course of COVID-19 in patients with concurrent cardiovascular and metabolic diseases. Additionally, we indicate the necessity to explore not only the cellular but also the molecular bases of COVID-19 pathogenesis, which may aid the development of dedicated therapies meant to improve chances for the successful treatment of patients. We also present new directions of research into medications that display NETs formation regulatory properties as potential significant therapeutic strategies in the progress of COVID-19.


Assuntos
/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , /metabolismo , Animais , Humanos
6.
Sci Rep ; 10(1): 19630, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184506

RESUMO

The novel coronavirus SARS-CoV-2 causes COVID-19, a highly pathogenic viral infection threatening millions. The majority of the individuals infected are asymptomatic or mildly symptomatic showing typical clinical signs of common cold. However, approximately 20% of the patients can progress to acute respiratory distress syndrome (ARDS), evolving to death in about 5% of cases. Recently, angiotensin-converting enzyme 2 (ACE2) has been shown to be a functional receptor for virus entry into host target cells. The upregulation of ACE2 in patients with comorbidities may represent a propensity for increased viral load and spreading of infection to extrapulmonary tissues. This systemic infection is associated with higher neutrophil to lymphocyte ratio in infected tissues and high levels of pro-inflammatory cytokines leading to an extensive microthrombus formation with multiorgan failure. Herein we investigated whether SARS-CoV-2 can stimulate extracellular neutrophils traps (NETs) in a process called NETosis. We demonstrated for the first time that SARS-CoV-2 in fact is able to activate NETosis in human neutrophils. Our findings indicated that this process is associated with increased levels of intracellular Reactive Oxygen Species (ROS) in neutrophils. The ROS-NET pathway plays a role in thrombosis formation and our study suggest the importance of this target for therapy approaches against disease.


Assuntos
Infecções por Coronavirus/imunologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/citologia , Pneumonia Viral/imunologia , Adolescente , Idoso , Infecções por Coronavirus/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Pandemias , Fagocitose , Pneumonia Viral/patologia , Espécies Reativas de Oxigênio/metabolismo
7.
Am J Physiol Lung Cell Mol Physiol ; 319(4): L661-L669, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783617

RESUMO

The past two decades have witnessed a resurgence in neutrophil research, inspired in part by the discovery of neutrophil extracellular traps (NETs) and their myriad roles in health and disease. Within the lung, dysregulation of neutrophils and NETosis have been linked to an array of diseases including pneumonia, cystic fibrosis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and severe asthma. However, our understanding of pathologic neutrophil responses in the lung remains incomplete. Two methodologic issues have contributed to this gap: first, an emphasis on studying neutrophils from blood rather than the lung and second, the technical difficulties of interrogating neutrophil responses in mice, which has largely restricted basic murine research to specialized laboratories. To address these limitations, we have developed a suite of techniques for studying neutrophil effector functions specifically in the mouse lung. These include ex vivo assays for phagocytosis and NETosis using bronchoalveolar neutrophils and in situ evaluation of NETosis in a murine model of pneumonia. Throughout, we have prioritized technical ease and robust, quantitative readouts. We hope these assays will help to standardize research on lung neutrophils and improve accessibility to this burgeoning field.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/patologia , Fagocitose/fisiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Animais , Modelos Animais de Doenças , Pulmão/patologia , Camundongos , Pneumonia/diagnóstico , Pneumonia/patologia , /patologia
8.
Adv Biol Regul ; 77: 100741, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32773102

RESUMO

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.


Assuntos
Antioxidantes/uso terapêutico , Infecções por Coronavirus/epidemiologia , Armadilhas Extracelulares/imunologia , Linfopenia/epidemiologia , Neutrófilos/imunologia , Pandemias , Pneumonia Viral/epidemiologia , Acetilcisteína/uso terapêutico , Ácido Ascórbico/uso terapêutico , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/genética , Citocinas/imunologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Linfopenia/tratamento farmacológico , Linfopenia/imunologia , Linfopenia/virologia , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/virologia , Estresse Oxidativo/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia
9.
EBioMedicine ; 59: 102951, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32818801

RESUMO

BACKGROUND: . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. METHODS: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. FINDINGS: SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. INTERPRETATION: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.


Assuntos
Infecções por Coronavirus/transmissão , Placenta/virologia , Pneumonia Viral/transmissão , Adulto , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Recém-Nascido , Macrófagos/virologia , Microscopia Eletrônica , Nasofaringe/virologia , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Placenta/citologia , Placenta/patologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Gravidez , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
PLoS Pathog ; 16(8): e1008230, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797076

RESUMO

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.


Assuntos
Eritrócitos/imunologia , Armadilhas Extracelulares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária/imunologia , Neutrófilos/imunologia , Plasmodium/imunologia , Receptores CXCR4/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Malária/metabolismo , Malária/parasitologia , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/parasitologia , Parasitemia/patologia
11.
Circulation ; 142(12): 1176-1189, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32755393

RESUMO

BACKGROUND: Severe acute respiratory syndrome corona virus 2 infection causes severe pneumonia (coronavirus disease 2019 [COVID-19]), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in patients with COVID-19. METHODS: A total of 62 subjects were included in our study (n=38 patients with reverse transcriptase polymerase chain reaction-confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathologic assessment of autopsy cases, surface marker-based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions, and coagulation tests, as well. RESULTS: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that, in COVID-19, inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. Patients with COVID-19 also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, patients severely affected with COVID-19 are characterized by excessive platelet and neutrophil activation in comparison with healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in severe acute respiratory syndrome corona virus 2 pneumonia is linked to both acute respiratory distress syndrome and systemic hypercoagulability. CONCLUSIONS: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19.


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Insuficiência Respiratória/etiologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Transtornos da Coagulação Sanguínea/diagnóstico , Transtornos da Coagulação Sanguínea/etiologia , Plaquetas/citologia , Plaquetas/metabolismo , Plaquetas/patologia , Estudos de Casos e Controles , Infecções por Coronavirus/complicações , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/metabolismo , Humanos , Rim/patologia , Pulmão/patologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Pandemias , Fenótipo , Ativação Plaquetária , Pneumonia Viral/complicações , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Insuficiência Respiratória/diagnóstico , Índice de Gravidade de Doença , Trombose/complicações , Trombose/diagnóstico
12.
EBioMedicine ; 58: 102925, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32745993

RESUMO

BACKGROUND: Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood. METHODS: Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry. PATIENT FINDINGS: Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage. INTERPRETATION: These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage. FUNDING: Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung.


Assuntos
Infecções por Coronavirus/patologia , Armadilhas Extracelulares/metabolismo , Microvasos/patologia , Neutrófilos/metabolismo , Pneumonia Viral/patologia , Trombose/metabolismo , Células Cultivadas , Infecções por Coronavirus/complicações , Infecções por Coronavirus/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Microvasos/metabolismo , Neutrófilos/patologia , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/metabolismo , Trombose/etiologia , Trombose/patologia
13.
J Clin Invest ; 130(11): 6151-6157, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32759504

RESUMO

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.


Assuntos
Betacoronavirus , Complexo de Ataque à Membrana do Sistema Complemento , Infecções por Coronavirus , Armadilhas Extracelulares , Neutrófilos , Pandemias , Pneumonia Viral , Tromboplastina , Trombose , Idoso , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Ativação do Complemento/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos Cíclicos/farmacologia , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/sangue , Receptor da Anafilatoxina C5a/imunologia , /imunologia , Trombina/imunologia , Trombina/metabolismo , Tromboplastina/imunologia , Tromboplastina/metabolismo , Trombose/sangue , Trombose/imunologia , Trombose/virologia
15.
Nat Rev Immunol ; 20(9): 515-516, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32728221

Assuntos
Antioxidantes/uso terapêutico , Betacoronavirus/patogenicidade , Infecções por Coronavirus/tratamento farmacológico , Pulmão/imunologia , Neutrófilos/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Acetilcisteína/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/genética , Citocinas/imunologia , Quimioterapia Combinada , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Regulação da Expressão Gênica , Glicina/análogos & derivados , Glicina/uso terapêutico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/virologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/imunologia , Neutrófilos/virologia , Estresse Oxidativo/efeitos dos fármacos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas Secretadas Inibidoras de Proteinases/uso terapêutico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Sulfonamidas/uso terapêutico , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia
16.
PLoS One ; 15(7): e0236744, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730309

RESUMO

Repeated exposures to environmental allergens in susceptible individuals drive the development of type 2 inflammatory conditions such as asthma, which have been traditionally considered to be mainly mediated by Th2 cells. However, emerging evidence suggest that a new innate cell type, group 2 innate lymphoid cells (ILC2), plays a central role in initiating and amplifying a type 2 response, even in the absence of adaptive immunity. At present, the regulatory mechanisms for controlling ILC2 activation remain poorly understood. Here we report that respiratory delivery of immunogenic extracellular RNA (exRNAs) derived from RNA- and DNA-virus infected cells, was able to activate a protective response against acute type 2 lung immunopathology and airway hyperresponsiveness (AHR) induced by IL-33 and a fungal allergen, A. flavus, in mice. Mechanistically, we found that the innate immune responses triggered by exRNAs had a potent suppressive effect in vivo on the proliferation and function of ILC2 without the involvement of adaptive immunity. We further provided the loss-of-function genetic evidence that the TLR3- and MAVS-mediated signaling axis is essential for the inhibitory effects of exRNAs in mouse lungs. Thus, our results indicate that the host detection of extracellular immunostimulatory RNAs generated during respiratory viral infections have an important function in the regulation of ILC2-driven acute lung inflammation.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Pneumonia/imunologia , RNA/imunologia , Hipersensibilidade Respiratória/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Citocinas/metabolismo , Armadilhas Extracelulares/metabolismo , Interleucina-33/imunologia , Interleucina-33/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/metabolismo , Pneumonia/patologia , RNA/metabolismo , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Receptor 3 Toll-Like/fisiologia
17.
J Vis Exp ; (160)2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32628162

RESUMO

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.


Assuntos
DNA/análise , Espaço Extracelular/metabolismo , Glomerulonefrite/genética , Aprendizado de Máquina Supervisionado , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Biópsia , Armadilhas Extracelulares/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peroxidase/metabolismo
18.
Mol Immunol ; 124: 211-217, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32603960

RESUMO

The primary immune response against Staphylococcus aureus is mediated by neutrophils. In response to S. aureus and its proteins, neutrophil shows two different kinds of NETosis, viz. suicidal and vesicular NETosis. Glucose is the major energy source of neutrophils for performing NETosis. However, NETosis was found altered in response to high glucose levels. Growth of S. aureus was also found modulated in response to high glucose and they behave differently at different glucose levels. This work was attempted to study NET release in response to S. aureus cell-free culture supernatant at different glucose concentrations. Freshly isolated neutrophils were treated with different concentrations of glucose along with S. aureus cell-free culture supernatant and were analyzed for neutrophil extracellular trap formation, ROS production, and peptidylarginine deiminase 4 activities. Influence of calcium on NETosis was analyzed using calcium chelator (EDTA) and calcium inhibitor (TMB-8). With increasing glucose levels, NET release in response to S. aureus cell-free culture supernatant was increased. Oxidant level was also increased dose-dependently with increasing concentrations of glucose. At very high glucose concentrations (> 15 mM), vesicular NETosis was predominantly observed. At these glucose concentrations, peptidylarginine deiminase activity was found to be decreased. Furthermore, calcium quenching in the medium facilitated vesicular mode of NET release. In conclusion, calcium depletion occurring at high glucose concentrations can reduce peptidylarginine deiminase 4 activity and can thereby promote the vesicular NET release.


Assuntos
Cálcio/metabolismo , Armadilhas Extracelulares/metabolismo , Glucose/metabolismo , Neutrófilos/imunologia , Staphylococcus aureus/imunologia , Células Cultivadas , Armadilhas Extracelulares/imunologia , Humanos , Vesículas Secretórias/metabolismo , Infecções Estafilocócicas/imunologia
19.
Arterioscler Thromb Vasc Biol ; 40(9): 2279-2292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32611241

RESUMO

OBJECTIVE: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-ß) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. CONCLUSIONS: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.


Assuntos
Armadilhas Extracelulares/metabolismo , Microbioma Gastrointestinal , Isquemia Mesentérica/metabolismo , Mesentério/irrigação sanguínea , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Traumatismo por Reperfusão/metabolismo , Vênulas/metabolismo , Animais , Bacillus subtilis/patogenicidade , Adesão Celular , Células Cultivadas , Modelos Animais de Doenças , Escherichia coli/patogenicidade , Armadilhas Extracelulares/microbiologia , Feminino , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/microbiologia , Masculino , Isquemia Mesentérica/microbiologia , Isquemia Mesentérica/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/microbiologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Vênulas/microbiologia , Vênulas/patologia
20.
Am J Pathol ; 190(11): 2282-2289, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32702358

RESUMO

Idiopathic osteonecrosis of the femoral head (ONFH) is defined as necrosis of osteocytes due to a non-traumatic ischemia of the femoral head. Iatrogenic glucocorticoid administration and habitual alcohol intake are regarded as risk factors. It has been suggested that glucocorticoid-induced activation of platelets contributes to the local blood flow disturbance of the femoral head. Both activated platelets and alcohol can induce neutrophil extracellular traps (NETs). To determine the association of NETs with the development of idiopathic ONFH, surgically resected femoral heads of patients with idiopathic ONFH and osteoarthritis were assessed for existence of NET-forming neutrophils by immunofluorescence staining. NET-forming neutrophils were present in small vessels surrounding the femoral head of patients with idiopathic ONFH but not osteoarthritis. Moreover, Wistar-Kyoto rats were intravenously injected with NET-forming neutrophils or neutrophils without NET induction, and then the ischemic state of the tissue around the femoral head was evaluated by immunohistochemistry for hypoxia-inducible factor-1α. NET-forming neutrophils circulated into the tissue around the femoral head, and hypoxia-inducible factor-1α expression in the tissue was higher compared with that of rats intravenously administered with neutrophils without NET induction. Furthermore, ischemic change of osteocytes was observed in the femoral head of rats given an i.v. injection of NET-forming neutrophils. The collective findings suggest that NETs are possibly associated with the development of idiopathic ONFH.


Assuntos
Armadilhas Extracelulares/metabolismo , Necrose da Cabeça do Fêmur/metabolismo , Cabeça do Fêmur/metabolismo , Neutrófilos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Cabeça do Fêmur/irrigação sanguínea , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Ratos , Ratos Endogâmicos WKY
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