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1.
Scand J Immunol ; 91(5): e12870, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32034957

RESUMO

Two novel enzyme-linked immunosorbent assays (ELISAs), designed to detect complexes containing DNA, leucocyte calprotectin and S100A12 proteins, were generated for improved specificity and rapid measurement of neutrophil extracellular traps (NETs). The assays were applied on plasma and serum samples from blood donors for establishment of reference values, and from patients with multiple myeloma (MM) or rheumatoid arthritis (RA) in order to examine putatively increased values in the two different inflammatory conditions. Although NETs were hardly detectable in healthy individuals, NET levels were as expected highly and statistically significantly increased in RA patients. The detection of statistically significantly increased NET levels in MM is a novel finding.


Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/metabolismo , Adulto , Idoso , Artrite Reumatoide/patologia , Doadores de Sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Projetos Piloto , Adulto Jovem
2.
Nat Commun ; 11(1): 105, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913271

RESUMO

Psoriasis is an inflammatory skin disease with strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37. LL37 in complex with DNA and RNA is thought to initiate disease exacerbation via plasmacytoid dendritic cells. However, the source of nucleic acids supposed to start this initial inflammatory event remains unknown. We show here that primary murine and human PMNs mount a fulminant and self-propagating neutrophil extracellular trap (NET) and cytokine response, but independently of the canonical NET component, DNA. Unexpectedly, RNA, which is abundant in NETs and psoriatic but not healthy skin, in complex with LL37 triggered TLR8/TLR13-mediated cytokine and NET release by PMNs in vitro and in vivo. Transfer of NETs to naive human PMNs prompts additional NET release, promoting further inflammation. Our study thus uncovers a self-propagating vicious cycle contributing to chronic inflammation in psoriasis, and NET-associated RNA (naRNA) as a physiologically relevant NET component.


Assuntos
Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Psoríase/imunologia , Adulto , Animais , Peptídeos Catiônicos Antimicrobianos , Citocinas/genética , Citocinas/imunologia , Armadilhas Extracelulares/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Psoríase/genética , RNA/genética , RNA/imunologia , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/imunologia , Adulto Jovem
3.
Nat Immunol ; 21(2): 135-144, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932813

RESUMO

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.


Assuntos
Ritmo Circadiano/imunologia , Inflamação/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Animais , Degranulação Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação/imunologia , Camundongos , Neutrófilos/imunologia , Pneumonia/complicações , Pneumonia/imunologia , Proteoma/imunologia , Proteoma/metabolismo , Síndrome do Desconforto Respiratório do Adulto/imunologia
4.
J Surg Res ; 246: 52-61, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31561178

RESUMO

BACKGROUND: Low-density neutrophils (LDN) have been shown to be increased in peripheral blood in patients with various diseases and closely related to immune-mediated pathology. However, the frequency and function of LDN in circulating blood of the patients following abdominal surgery have not been well understood. METHODS: LDN were determined by CD66b(+) cells, which were copurified with mononuclear cells by density gradient preparations of peripheral blood of surgical patients. The effects of the purified LDN on T cell proliferation and tumor cell lysis were examined in vitro. Neutrophil extracellular traps (NETs) production was examined by extracellular nuclear staining. RESULTS: The number of LDN with an immature phenotype is markedly increased in peripheral blood samples in patients after abdominal surgery. The frequency of LDN correlated positively with operative time and intraoperative blood loss. The purified LDN significantly suppressed the proliferation of autologous T cells stimulated with anti-CD3 mAb coated on plate and partially inhibited the cytotoxicity of lymphocytes activated with recombinant interleukin-2 against a human gastric cancer cell, OCUM-1. The LDN also produced NETs after short-term culture in vitro, which efficiently trap many OCUM-1. These results suggest that surgical stress recruits immunosuppressive LDN in the circulation in the early postoperative period. CONCLUSIONS: The LDN may support the lodging of circulating tumor cells via NETs formation and inhibit T cell-mediated antitumor response in target organs, which may promote postoperative cancer metastases. Functional blockade of LDN might be an effective strategy to reduce tumor recurrence after abdominal surgery.


Assuntos
Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Neoplasias Gastrointestinais/cirurgia , Recidiva Local de Neoplasia/imunologia , Neutrófilos/imunologia , Estresse Fisiológico/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Perda Sanguínea Cirúrgica/estatística & dados numéricos , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/patologia , Humanos , Contagem de Leucócitos , Recidiva Local de Neoplasia/epidemiologia , Células Neoplásicas Circulantes/imunologia , Neutrófilos/metabolismo , Duração da Cirurgia , Linfócitos T/imunologia
6.
Breast Cancer Res ; 21(1): 145, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852512

RESUMO

The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Microambiente Tumoral , Biomarcadores , Neoplasias da Mama/etiologia , Neoplasias da Mama/terapia , Gerenciamento Clínico , Armadilhas Extracelulares/imunologia , Feminino , Humanos , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Neutrófilos/imunologia , Neutrófilos/patologia , Trombose
7.
PLoS Negl Trop Dis ; 13(9): e0007368, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31504035

RESUMO

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata , Hanseníase/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/imunologia , Doenças Autoimunes/microbiologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Hanseníase/tratamento farmacológico , Hanseníase/patologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Mycobacterium leprae/patogenicidade , Neutrófilos/patologia , Talidomida/administração & dosagem , Talidomida/uso terapêutico
8.
J Agric Food Chem ; 67(40): 11230-11235, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31523955

RESUMO

Ochratoxin A (OTA) is a mycotoxin which could cause strong immunosuppressive toxicological effects in animals and humans. Heterophil extracellular traps (HETs) as a novel defense of chicken heterophils play an important role against pathogen infection. It has been reported that OTA can weaken the phagocytosis function of neutrophils. However, whether or not OTA shows immunosuppressive effects on HET release remains unclear. In the present study, we aim to first investigate the effects of OTA on HET release and then try to clarify the mechanisms in this process. OTA-induced HET structures were observed and analyzed by fluorescence confocal microscopy. The quantitative determination of OTA-induced HETs was measured by PicoGreen and a fluorescence microplate. The results clearly showed that OTA obviously induced the release of HET-like structures in heterophils, and these extracellular networks were composed by chromatin decorated with histones and neutrophil elastase. Reactive oxygen species (ROS) production was also increased in the process of OTA-induced HET formation. Furthermore, the inhibitors of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways significantly decreased OTA-induced HET formation. The abovementioned results suggest that OTA-induced HET formation is related to ROS production dependent on the activation of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways. Taken together, this study first shows that OTA possesses the ability to trigger HET formation, which provides our understanding of the host that continuously suffered OTA exposure leading to the hyporeactivity of the immune system against infection.


Assuntos
Galinhas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Armadilhas Extracelulares/imunologia , NADPH Oxidases/imunologia , Neutrófilos/efeitos dos fármacos , Ocratoxinas/toxicidade , Espécies Reativas de Oxigênio/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Galinhas/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Armadilhas Extracelulares/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , NADPH Oxidases/genética , Neutrófilos/enzimologia , Neutrófilos/imunologia , Fagocitose , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Nat Commun ; 10(1): 3422, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366921

RESUMO

Severe influenza infection has no effective treatment available. One of the key barriers to developing host-directed therapy is a lack of reliable prognostic factors needed to guide such therapy. Here, we use a network analysis approach to identify host factors associated with severe influenza and fatal outcome. In influenza patients with moderate-to-severe diseases, we uncover a complex landscape of immunological pathways, with the main changes occurring in pathways related to circulating neutrophils. Patients with severe disease display excessive neutrophil extracellular traps formation, neutrophil-inflammation and delayed apoptosis, all of which have been associated with fatal outcome in animal models. Excessive neutrophil activation correlates with worsening oxygenation impairment and predicted fatal outcome (AUROC 0.817-0.898). These findings provide new evidence that neutrophil-dominated host response is associated with poor outcomes. Measuring neutrophil-related changes may improve risk stratification and patient selection, a critical first step in developing host-directed immune therapy.


Assuntos
Armadilhas Extracelulares/imunologia , Influenza Humana/imunologia , Influenza Humana/patologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Ciclo Celular/imunologia , Feminino , Expressão Gênica/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/imunologia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/mortalidade , Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Respiração Artificial , Insuficiência Respiratória/mortalidade , Insuficiência Respiratória/patologia , Insuficiência Respiratória/virologia
10.
Immunity ; 51(3): 443-450.e4, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31422870

RESUMO

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.


Assuntos
Armadilhas Extracelulares/imunologia , Cálculos Biliares/imunologia , Neutrófilos/imunologia , Animais , Feminino , Humanos , Imunidade Inata/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/imunologia
11.
Andrologia ; 51(9): e13356, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31287181

RESUMO

Leucocytospermia has been associated with loss of sperm function. Extracellular traps (ETs) of leucocytes are produced during innate immune response. ETs can be activated by spermatozoa in contact with polymorphonuclear (in vitro), inducing sperm entrapment and decrease motility. In this pilot study, we describe the results of ETosis ex vivo, in seminal fluid (SF) smear of infertile patients, associating ETs with leucocytospermia and bacteriospermia. In 21 infertile patients, semen parameters (WHO, 2010), microbiological study, leucocytospermia and presence of ETs in SF were determined. Leucocytes (CD45, CD15 and CD68) were evaluated by immunostaining in SF smears. Indirect immunofluorescence (global histone and H4-citrullinated 3) and scanning electron microscopy (SEM) were used to determine ETs morphology. In 28.6% of patients presented leucocytospermia without bacteriospermia, all of them presented a large number of ETs in the SF smears examined. About 76.6% of the patients without leucocytospermia were positive for ETs. Samples with leucocytospermia have a higher number of ETs and would be related to the amount of leucocytes in the SF. The morphological predominant ETs were diffuse (diffETs) and spread (sprETs). The formation of ETs indicates leucocyte activation in semen, and it was observed that ETosis does not depend exclusively on the presence of bacterial contamination.


Assuntos
Armadilhas Extracelulares/imunologia , Infertilidade Masculina/imunologia , Leucócitos/imunologia , Sêmen/citologia , Adulto , Bactérias/isolamento & purificação , Humanos , Infertilidade Masculina/microbiologia , Leucócitos/citologia , Leucócitos/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Oligopeptídeos , Projetos Piloto , Sêmen/imunologia , Sêmen/microbiologia , Análise do Sêmen/métodos
12.
Autoimmun Rev ; 18(8): 751-760, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181324

RESUMO

Neutrophils derive from hematopoietic stem cells (HSCs) with systemic inflammation driving their activation and differentiation to myeloid progenitors to ensure enhanced myelopoiesis. Epigenetic reprograming and re-education of these HSCs produces neutrophils primed towards elimination of pathogens and increased inflammatory response. Neutrophils -an important component of acute inflammation- are not present in chronic inflammatory tissues leading to the false assumption that they may not be as important for the latter. Activated neutrophils may release Neutrophil Extracellular Traps (NETs) during a distinct form of cell death, named NETosis; NETs are rich in bioactive molecules that promote thrombosis (including atherothrombosis), inflammation and fibrosis. Thus, although neutrophils may not be present in chronic inflammatory lesions, their remnants may amplify the inflammatory response beyond their short life-span in the tissues. Herein, we review current evidence supporting a role of neutrophils and NETosis in tissue injury and dysfunction in systemic autoimmunity using as disease paradigms Systemic Lupus Erythematosus (SLE) and the ANCA-associated vasculitides (AAV). We also discuss the mechanisms involved and their potential as targets for novel therapy and drug repositioning.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/imunologia , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/terapia , Morte Celular , Diferenciação Celular , Armadilhas Extracelulares/imunologia , Fibrose , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Mielopoese , Neutrófilos/patologia
13.
Mol Immunol ; 112: 188-197, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176198

RESUMO

Rheumatoid arthritis (RA) is a chronic, systemic, synovitis-based inflammatory disease with unknown etiology. Neutrophils play important roles in the pathogenesis of RA. Apoptosis and NETosis of neutrophils are two major mechanisms of programmed cell death that differ in their morphological characteristics and effects on the immune system. In rheumatoid arthritis, delayed neutrophil apoptosis amplifies the inflammatory response; and massive release of NETs and their components may cause tissue damage and provide self-antigens. Emodin is a natural anthraquinone derivative that occurs in many widely used Chinese medicinal herbs. In this study, we evaluated the effect of emodin on a murine adjuvant-induced arthritis (AA) model of RA in vivo and on neutrophil apoptosis and NETosis in vitro. Our results show that emodin alleviated AA by reducing neutrophil infiltration and proinflammatory cytokine (interleukin-6, interferon-gamma and tumor necrosis factor-α) release. Emodin promoted apoptosis and inhibited autophagy and NETosis in neutrophils. These findings indicate that emodin represents a potential therapeutic agent for RA.


Assuntos
Apoptose/imunologia , Artrite Reumatoide/imunologia , Emodina/imunologia , Armadilhas Extracelulares/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Animais , Artrite Experimental/imunologia , Autoantígenos/imunologia , Autofagia/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/imunologia
14.
Lakartidningen ; 1162019 May 21.
Artigo em Sueco | MEDLINE | ID: mdl-31192424

RESUMO

Neutrophil extracellular traps (NETs) were first described over a decade ago as part of our innate immune system. Through the extracellular release of web-like structures composed of DNA and histones coated with antimicrobial peptides, the neutrophil was shown to entrap and disarm invading microorganisms. Recent data now propose a central role of NETs in a variety of non-infectious conditions - such as autoimmunity, thrombosis, and cancer - revealing that NETs may not only be beneficial, but also harmful if uncontrolled. Continued investigations into the clinical relevance of NETs will shed further light on the utility of biomarkers associated with NETs, and may open for new therapeutic options in several disease settings.


Assuntos
Armadilhas Extracelulares , Doenças Autoimunes/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/fisiologia , Humanos , Imunidade Inata , Neoplasias/imunologia , Trombose/imunologia
15.
Nat Commun ; 10(1): 2402, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160588

RESUMO

Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induce neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared to  stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30 to 90%. The identification of critical roles of CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.


Assuntos
Plaquetas/metabolismo , Vírus da Dengue/imunologia , Dengue/imunologia , Vesículas Extracelulares/imunologia , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Citocinas/imunologia , Dengue/virologia , Exossomos/imunologia , Exossomos/metabolismo , Armadilhas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Inflamação , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Ativação Plaquetária , Receptores de Superfície Celular/genética , Fator de Transcrição STAT1/genética , Taxa de Sobrevida
16.
J Immunol ; 203(2): 500-510, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31167775

RESUMO

Preterm birth is a major contributor to neonatal mortality and morbidity, and infection is a major risk factor. Chorioamnionitis, inflammation of the placenta, and fetal membranes (FMs) are commonly observed in preterm birth and are characterized by neutrophil infiltration. However, interactions between FMs and neutrophils remain incompletely understood. The objectives of this study were to determine how FMs, with or without bacterial LPS stimulation, affect neutrophil recruitment, activation, and the formation of neutrophil extracellular traps (NETs) and to elucidate the signaling mechanisms involved. Using a combination of in vitro, ex vivo, and in vivo approaches, we show that human resting FMs can directly recruit neutrophils and induce them to produce proinflammatory factors. Furthermore, neutrophils release vital NETs in response to FM-derived factors. LPS-stimulated FMs further augmented neutrophil recruitment, inflammatory cytokine/chemokine secretion, and vital NET release and also induced reactive oxygen species production and degranulation. We demonstrate a role for FM-derived TNF-α in mediating these effects through activation of neutrophil p38 MAPK. We propose that, during infection, neutrophil recruitment and activation may neutralize pathogens, vital NET formation, and prolonged neutrophil viability, and in combination with degranulation, reactive oxygen species production and inflammatory chemokine/cytokine production may contribute to tissue injury at the maternal/fetal interface.


Assuntos
Armadilhas Extracelulares/imunologia , Membranas Extraembrionárias/imunologia , Lipopolissacarídeos/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Placenta/imunologia , Animais , Corioamnionite/imunologia , Citocinas/imunologia , Feminino , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/imunologia , Gravidez , Espécies Reativas de Oxigênio/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
17.
Methods Mol Biol ; 1982: 517-528, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172493

RESUMO

Neutrophil extracellular traps (NETs) are made of a network of extracellular strings of DNA that bind pathogenic microbes. Histones and several neutrophil granule proteins associated with the DNA framework damage entrapped microorganisms. Reactive oxygen species generated by the neutrophil NADPH oxidase have been shown to be essential to mediate NET release by several stimuli including numerous pathogenic bacteria. Although several methods have been used in the literature to detect NETs in vitro and in vivo, a consensus is urgently needed on the field to standardize the best NET-specific assays. In this chapter, two methods are described in details that can be used to detect NETs and to distinguish them from other mechanisms of neutrophil cell death. While NET-specific, these assays are also relatively simple and straightforward enabling their potential use by a wide audience.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/fisiologia , Biomarcadores , Ensaio de Imunoadsorção Enzimática , Armadilhas Extracelulares/imunologia , Imunofluorescência , Humanos
18.
Int J Mol Sci ; 20(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242568

RESUMO

Autoantibodies against citrullinated proteins are a hallmark of rheumatoid arthritis, a destructive inflammatory arthritis. Peptidylarginine deiminase 4 (PAD4) has been hypothesized to contribute to rheumatoid arthritis by citrullinating histones to induce neutrophil extracellular traps (NETs), which display citrullinated proteins that are targeted by autoantibodies to drive inflammation and arthritis. Consistent with this theory, PAD4-deficient mice have reduced NETs, autoantibodies, and arthritis. However, PAD4's role in human rheumatoid arthritis is less clear. Here, we determine if single nucleotide polymorphism rs2240335 in PADI4, whose G allele is associated with reduced PAD4 in neutrophils, correlates with NETs, anti-histone antibodies, and rheumatoid arthritis susceptibility in North Americans. Control and rheumatoid arthritis subjects, divided into anti-cyclic citrullinated peptide (CCP) antibody positive and negative groups, were genotyped at rs2240335. In homozygotes, in vitro NETosis was quantified in immunofluorescent images and circulating NET and anti-histone antibody levels by enzyme linked immunosorbent assay (ELISA). Results were compared by t-test and correlation of rheumatoid arthritis diagnosis with rs2240335 by Armitage trend test. NET levels did not significantly correlate with genotype. G allele homozygotes in the CCP- rheumatoid arthritis group had reduced anti-native and anti-citrullinated histone antibodies. However, the G allele conferred increased risk for rheumatoid arthritis diagnosis, suggesting a complex role for PAD4 in human rheumatoid arthritis.


Assuntos
Artrite Reumatoide/etiologia , Autoanticorpos/imunologia , Suscetibilidade a Doenças , Histonas/imunologia , Polimorfismo de Nucleotídeo Único , /genética , Alelos , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/metabolismo , Autoanticorpos/sangue , Autoantígenos/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Genótipo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo
19.
PLoS Pathog ; 15(5): e1007773, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31107907

RESUMO

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.


Assuntos
Adesinas Bacterianas/imunologia , Infecções por Bacteroidaceae/imunologia , Cisteína Endopeptidases/imunologia , Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Peritonite/imunologia , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Receptor PAR-2/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/microbiologia , Infecções por Bacteroidaceae/patologia , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Armadilhas Extracelulares/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/microbiologia , Neutrófilos/patologia , Peritonite/metabolismo , Peritonite/microbiologia , Receptor PAR-2/imunologia , Transdução de Sinais
20.
J Immunol Res ; 2019: 6252138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31093511

RESUMO

In this review, we will highlight several studies that revolve around interleukin-8 (IL-8) and show the multiple facets that could take in the tumor microenvironment. Chemokines that attract neutrophils (to a large extent, IL-8) can have a bimodal behavior inducing the migration of them in the first place and later favoring the formation of NETs in the place of emission focus of the chemokine. Also, this mechanism occurs when neutrophils migrate to tumor cells and where the extrusion of NETs in the tumor is observed. A possible participation of NETs in cancer progression was considered; however, until now, it is difficult to decide if NETosis plays a pro- or antitumor role, although it is necessary to emphasize that there is more experimentation focused on the protumorigenic aspect of the NETs. The formation of NETs has a relevant role in the inhibition of the immune response against the tumor generated by neutrophils and in turn favoring the processes involved in the development of tumor metastasis. It is striking that we do not have more complete information about the effects of circulating chemokines on neutrophils in cancer patients and hence the suitability of this review. No one has observed to date the impact that it could have on other cell populations to inhibit the arrival of neutrophils and the formation/elimination of NETs. However, the extent to which NETs affect the function of other cells of the immune system in the tumor context has not been directly demonstrated. It is necessary to identify possible combinations of immunotherapy that involve the modulation of neutrophil activity with other strategies (immunomodulatory antibodies or adoptive cell therapy). Therefore, knowing the mechanisms by which tumors take advantage of this ability of neutrophils to form NETs is very important in the search for antitumor therapies and thus be able to take advantage of the possible immunotherapeutic combinations that we currently have in clinical practice.


Assuntos
Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neoplasias/patologia , Neutrófilos/imunologia , Microambiente Tumoral/imunologia , Animais , Humanos , Camundongos , Neoplasias/imunologia , Ativação de Neutrófilo
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