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1.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570563

RESUMO

Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of Chromobacterium violaceum, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that C. violaceum produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An in silico analysis in the genome of C. violaceum revealed that genes related to synthesis and uptake of chromobactin (cba) and viobactin (vba) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of C. violaceum, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence in vivo and reduced neutrophil extracellular trap (NET) formation in in vitro assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that C. violaceum uses distinct endogenous siderophores for iron uptake and its establishment in the host.


Assuntos
Chromobacterium/genética , Chromobacterium/metabolismo , Ferro/metabolismo , Sideróforos/genética , Sideróforos/metabolismo , Animais , Transporte Biológico/fisiologia , Chromobacterium/patogenicidade , Armadilhas Extracelulares/metabolismo , Feminino , Hidroxibenzoatos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Família Multigênica/genética , Neutrófilos/metabolismo , Peptídeo Sintases/metabolismo
2.
Exp Parasitol ; 207: 107770, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31586454

RESUMO

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/parasitologia , Toxoplasma/imunologia , Animais , Gatos , Sobrevivência Celular , DNA/análise , Formazans/metabolismo , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/análise , Sais de Tetrazólio/metabolismo , Células Vero
3.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527127

RESUMO

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove's modified Dulbecco's medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.


Assuntos
Biofilmes/crescimento & desenvolvimento , Armadilhas Extracelulares/metabolismo , Nuclease do Micrococo/metabolismo , Neutrófilos/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Transferência Ressonante de Energia de Fluorescência , Humanos , Viabilidade Microbiana , Polissacarídeos Bacterianos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/metabolismo
4.
Ecotoxicol Environ Saf ; 183: 109508, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31408819

RESUMO

As a new type of antibacterial agent, nanosilver has attracted great attention in biomedical applications. However, the safety of nanosilver to humans and the environment has not been well elucidated. The objective of this study was to investigate the influence of nanosilver on novel effector mechanism of neutrophil extracellular traps (NETs), and its possible molecular mechanisms. In this study, nanosilver (10, 20 and 40 µg/mL) was incubated with neutrophils for 90 min. Then, nanosilver-induced the release of NETs was observed by laser confocal microscopy. Nanosilver-induced NETs release was also quantitatively detected by pico Green®. In addition, the role of NADPH oxidase, extracellular signal-regulated kinase (ERK) and p38 signaling pathways in nanosilver-induced NETs release were detected by the inhibitors and pico Green®. The results indicated that nanosilver significantly activated polymorphonuclear neutrophils (PMN) to release NETs, which was a DNA-based network structure modified with histones (H3) and neutrophil elastase (NE). The inhibitors of NADPH oxidase, ERK and p38 signaling pathways significantly inhibited the formation of nanosilver-induced NETs. Furthermore, nanosilver did not alter the extracellular lactate dehydrogenase (LDH) level of PMN cells. All these results showed that nanosilver significantly induced NETs release, and the potential molecular mechanisms were correlated with reactive oxygen species (ROS) production-dependent on NADPH oxidase, ERK and p38 signaling pathways, which might provide a new perspective on nanosilver-induced excess NETs release related to the host immune damage.


Assuntos
Antibacterianos/toxicidade , Armadilhas Extracelulares/metabolismo , Nanopartículas Metálicas/toxicidade , Neutrófilos/efeitos dos fármacos , Prata/toxicidade , Animais , Antibacterianos/química , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/química , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Prata/química
5.
Anesthesiology ; 131(4): 866-882, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31453815

RESUMO

BACKGROUND: In multiple-organ dysfunction, an injury affecting one organ remotely impacts others, and the injured organs synergistically worsen outcomes. Recently, several mediators, including extracellular histones and neutrophil extracellular traps, were identified as contributors to distant organ damage. This study aimed to elucidate whether these mediators play a crucial role in remote organ damage induced by intestinal ischemia-reperfusion. This study also aimed to evaluate the protective effects of recombinant thrombomodulin, which has been reported to neutralize extracellular histones, on multiple-organ dysfunction after intestinal ischemia-reperfusion. METHODS: Intestinal ischemia was induced in male C57BL/6J mice via clamping of the superior mesenteric artery. Recombinant thrombomodulin (10 mg/kg) was administered intraperitoneally with the initiation of reperfusion. The mice were subjected to a survival analysis, histologic injury scoring, quantitative polymerase chain reaction analysis of tumor necrosis factor-α and keratinocyte-derived chemokine expression, Evans blue dye vascular permeability assay, and enzyme-linked immunosorbent assay analysis of histones in the jejunum, liver, lung, and kidney after 30- or 45-min ischemia. Neutrophil extracellular trap formation was evaluated by immunofluorescence staining. RESULTS: Recombinant thrombomodulin yielded statistically significant improvements in survival after 45-min ischemia (ischemia-reperfusion without vs. with 10 mg/kg recombinant thrombomodulin: 0% vs. 33%, n = 21 per group, P = 0.001). Recombinant thrombomodulin reduced the histologic injury score, expression of tumor necrosis factor-α and keratinocyte-derived chemokine, and extravasation of Evans blue dye, which were augmented by 30-min ischemia-reperfusion, in the liver, but not in the intestine. Accumulated histones and neutrophil extracellular traps were found in the livers and intestines of 30-min ischemia-reperfusion-injured mice. Recombinant thrombomodulin reduced these accumulations only in the liver. CONCLUSIONS: Recombinant thrombomodulin improved the survival of male mice with intestinal ischemia-reperfusion injury. These findings suggest that histone and neutrophil extracellular trap accumulation exacerbate remote liver injury after intestinal ischemia-reperfusion. Recombinant thrombomodulin may suppress these accumulations and attenuate liver injury.


Assuntos
Armadilhas Extracelulares/metabolismo , Mucosa Intestinal/metabolismo , Neutrófilos/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Trombomodulina/metabolismo , Animais , Modelos Animais de Doenças , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
Thromb Res ; 180: 87-97, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31271975

RESUMO

Patients with colorectal cancer (CRC) are at increased risk of venous thrombosis, but the precise mechanisms of thrombogenesis in CRC remain largely unknown. We aimed to identify the novel role of neutrophil extracellular traps (NETs) in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). In this study, we first showed that the levels of NETs in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage II patients compared to healthy subjects. In addition, neutrophils from CRC patients were more prone to produce NETs, resulting in shortened coagulation time, significantly increased thrombin-antithrombin (TAT) complexes and fibrin fibrils compared to healthy controls. Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1. Conversely, NETs from CRC patients could also induce the exposure of PS on platelets, leading to markedly enhanced PCA. Importantly, ECs were also converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC). Our results reveal the complex interactions between neutrophils, platelets and ECs and their potential role in the hypercoagulable state in CRC. We propose that NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC.


Assuntos
Plaquetas/metabolismo , Neoplasias Colorretais/complicações , Armadilhas Extracelulares/metabolismo , Ativação Plaquetária , Trombose/etiologia , Idoso , Coagulação Sanguínea , Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombose/sangue , Trombose/metabolismo
7.
Mol Cell Biochem ; 461(1-2): 1-14, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31273604

RESUMO

Neutrophils have been thought to play a major role in inflammation and diabetic complications especially in poor glycemic control patients as demonstrated by their aberrant inflammatory markers. The aim of the present study was to compare neutrophil proteome profiles between diabetic patients with good glycemic control and those with poor glycemic control to see whether there might be any differences that could be related to the cause of complications which are found more commonly in the latter. Using 2-dimensional gel electrophoresis (2-DE) followed by quadrupole time of flight mass spectrometry (Q-TOF MS) and/or tandem mass spectrometry (MS/MS), we identified 35 differentially expressed proteins, some of which were protein components of neutrophil extracellular traps (NETs), in the poor glycemic control group compared to the good glycemic control group. The observed alterations of protein components of NETs included downregulation of myeloperoxidase, azurocidin (CAP37), and S100A9; and upregulation of the glycolytic enzymes transketolase and alpha-enolase. Manganese superoxide dismutase (MnSOD), functioning in cellular response and defense, was also found downregulated in the poor control group. Most of the glycolysis-related proteins were downregulated in the good control group but upregulated in the poor control group, including phosphoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase B chain (LDHB). The findings of this study demonstrate the dysregulation of protein components of NETs in neutrophils in patients with poorly controlled diabetes. More specifically, these findings suggest association between NETs and inflammation in diabetes and provide further insights into the role of neutrophils in the complications of poorly controlled diabetes.


Assuntos
Diabetes Mellitus/metabolismo , Armadilhas Extracelulares/metabolismo , Leucócitos Mononucleares/metabolismo , Proteômica , Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Mapeamento de Interação de Proteínas
8.
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
9.
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
10.
Clin Chim Acta ; 495: 606-610, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181192

RESUMO

BACKGROUND: Previous study from this lab has discerned oxidative, nitrosative stress and their relationship with cytokines contributing to the severity of sepsis and organ dysfunction. Cytokines are known to induce neutrophil extracellular traps (NETs) formation via free radicals generation. Hyper-activation of neutrophil leads to the increased NETs formation or ineffective clearance of NETs would likely increase the risk of auto-antibody generation against NETs components and being partly responsible for the sepsis severity and organ dysfunction. The present study was undertaken to further assess the status of NETs formation and their correlation with severity of sepsis, with the cytokines and organ dysfunction. METHODS: The level of NETs formation, DNA release, elastase release, and inflammatory cytokines was determined in 80 sepsis patients and 45 healthy volunteers. Their linearity with organ parameters and associations with sepsis severity were also assessed. RESULTS: NETs formation experiment was carried out and it was significantly higher in sepsis (70%) compared to control (30%). NETs % were positively correlated with severity of sepsis and organ dysfunction. Pearson's correlation coefficient demonstrated a direct relation between NETs components and organ parameters with Sepsis severity scores. CONCLUSION: NETs formation is significantly higher due to which it is contributing to the sepsis severity and organ failure.


Assuntos
Armadilhas Extracelulares/metabolismo , Insuficiência de Múltiplos Órgãos/imunologia , Neutrófilos/citologia , Sepse/imunologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
Nutrients ; 11(6)2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31212992

RESUMO

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50-200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/106 cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.


Assuntos
Ácido Ascórbico/fisiologia , Neutrófilos/metabolismo , Transporte Biológico , Quimiotaxia , Ácido Desidroascórbico/metabolismo , Armadilhas Extracelulares/metabolismo , Voluntários Saudáveis , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo
12.
J Photochem Photobiol B ; 196: 111511, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31129510

RESUMO

Prolonged exposure of the skin to ultraviolet radiation (UV) leads to its damage and loss of protective properties. This condition called photoaging of the skin is caused by a number of destructive factors, such as reactive oxygen species (ROS) and proteolytic enzymes that cause damage to the extracellular matrix, e.g. collagen fibers. Many cells of the immune system, including neutrophils, are involved in the photoaging process. The presence of neutrophils in the skin exposed to UV irradiation is known; however, the mechanism of neutrophil activity at these conditions remains unclear. In our study, we focused on the ability of neutrophils to release neutrophil extracellular traps (NETs) and the role of these structures in the photoaging process. NET release occurs in response to various stimuli; however, we hereby showed that the UVA and UVB radiation that reaches the Earth's surface could activate the mechanism of netosis. UV-induced netosis was much faster than that activated by chemical or biological factors; however, it also occurred due to the production of ROS, known signal mediators in netosis. In this work, we also identified the probable netosis signaling pathway involved in the neutrophil response to UV. The participation of NET components may explain the ongoing process of skin photoaging, but it is also important to indicate netosis as a potential target for skin protection therapy. Antioxidants tested in this work, such as N-acetylcysteine, ethamsylate, as well as vitamin B1 (thiamine), can successfully inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/efeitos da radiação , Raios Ultravioleta , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células Cultivadas , Armadilhas Extracelulares/química , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Fosforilação/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Quinase Syk/metabolismo
13.
Biomolecules ; 9(5)2019 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083537

RESUMO

Acetylation is an important post translational modification of histone that plays a role in regulation of physiological and pathological process in the body. We have recently shown that the inhibition of histone deacetylases (HDAC) by low concentrations of HDAC inhibitors (HDACis), belinostat (up to 0.25 µM) and panobinostat (up to 0.04 µM) promote histone acetylation (e.g., AcH4) and neutrophil extracellular trap formation (NETosis). Clinical use of belinostat and panobinostat often leads to neutropenia and the in vivo concentrations vary with time and tissue locations. However, the effects of different concentrations of these HDACis on neutrophil death are not fully understood. We considered that increasing concentrations of belinostat and panobinostat could alter the type of neutrophil death. To test this hypothesis, we treated human neutrophils with belinostat and panobinostat in the presence or absence of agonists that promote NOX-dependent NETosis (phorbol myristate acetate or lipopolysaccharide from Escherichia coli 0128) and NOX-independent NETosis (calcium ionophores A23187 or ionomycin from Streptomyces conglobatus). Increasing concentrations of HDACis induced histone acetylation in a dose-dependent manner. ROS analyses showed that increasing concentrations of HDACis, increased the degree of NOX-derived ROS production. Higher levels (>1 µM belinostat and >0.2 µM panobinostat) of AcH4 resulted in a significant inhibition of spontaneous as well as the NOX-dependent and -independent NETosis. By contrast, the degree of neutrophil apoptosis significantly increased, particularly in non-activated cells. Collectively, this study establishes that increasing concentrations of belinostat and panobinostat initially increases NETosis but subsequently reduces NETosis or switches the form of cell death to apoptosis. This new information indicates that belinostat and panobinostat can induce different types of neutrophil death and may induce neutropenia and regulate inflammation at different concentrations.


Assuntos
Apoptose , Armadilhas Extracelulares/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Neutrófilos/efeitos dos fármacos , Acetilação , Células Cultivadas , Armadilhas Extracelulares/efeitos dos fármacos , Código das Histonas , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Lipopolissacarídeos/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Panobinostat/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
14.
Stroke ; 50(6): 1548-1557, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31084324

RESUMO

Background and Purpose- Ischemia attracts neutrophils to the injured brain. However, neutrophil location and access to the damaged brain tissue is not yet entirely understood. We aimed to investigate neutrophil location in a mouse model of cerebral ischemia/reperfusion. Methods- Adult male C57BL/6 mice (n=52) received 45-minute intraluminal middle cerebral artery occlusion followed by 14, 24, 48, or 96 hours of reperfusion. Sham-operated mice (n=9) were subjected to the entire surgical procedure. We used wild-type mice and CatchupIVM mice expressing a red fluorescent protein in neutrophils. In addition, fluorescent neutrophils obtained from reporter DsRed (discosoma red fluorescent protein) mice were transferred intravenously to wild-type mice after ischemia. Mice received transcardial paraformaldehyde perfusion, the brain was cryoprotected, frozen, and cryostat sections were studied by immunofluorescence and confocal microscopy. Results- Ischemia induced a time-dependent increase in brain neutrophil numbers versus sham operation. We detected neutrophils in the leptomeninges, ventricles, capillary lumen, perivascular spaces, and parenchyma within the infarcted core. Most ischemic mice showed neutrophils in the leptomeninges and perivascular spaces, whereas the presence and number of neutrophils in the parenchyma was variable among ischemic mice. During the first 24 hours, only a few mice showed parenchymal neutrophils, but the frequency of mice showing neutrophils in the parenchyma and neutrophil numbers increased at 48 and 96 hours. We also detected signs of basement membrane disruption and hints of occasional neutrophil degranulation and formation of neutrophil extracellular traps. Conclusions- After ischemia/reperfusion, neutrophils accumulate in the leptomeninges and perivascular spaces, and eventually can reach the infarcted brain parenchyma.


Assuntos
Lesões Encefálicas , Encéfalo , Degranulação Celular , Armadilhas Extracelulares/metabolismo , Neutrófilos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Tempo
15.
Cardiovasc Diabetol ; 18(1): 49, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992036

RESUMO

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with a hypercoagulable state and increased neutrophil extracellular traps formation (NETosis). We investigated predictors of NETosis and cell death markers in circulating blood and their association with a prothrombotic state in T2DM. METHODS: In a cross-sectional study involving 113 T2DM patients aged 63.7 ± 8.2 years, we investigated citrullinated histone H3 (H3Cit), cell-free deoxyribonucleic acid (cfDNA), myeloperoxidase, neutrophil elastase, and inflammation markers, along with thrombin generation (TG), plasma clot lysis time (CLT), clot permeability (Ks) and fibrinolysis inhibitors. RESULTS: On multivariate logistic regression analysis adjusted for age and gender, predictors of high H3Cit (≥ 7.36 ng/mL, upper quartile) were: glycated hemoglobin (HbA1c) ≥ 7.0% and interleukin-6. Interleukin-6 was also found to be a predictor of high cfDNA (≥ 2.84 µg/mL, upper quartile) along with glucose. Citrullinated histone H3 and cfDNA correlated positively with CLT and inversely with Ks, while TG associated solely with cfDNA. These associations were not seen with myeloperoxidase and neutrophil elastase. Patients with previous myocardial infarction (n = 21, 18.6%) had higher H3Cit (+108%, p < 0.001) and cfDNA (+45%, p = 0.022). On multivariable analysis adjusted for potential confounders, H3Cit and cfDNA, along with plasminogen activator inhibitor-1 and concomitant cardiovascular disease, were predictors of CLT. Citrullinated histone H3 alone was a predictor of Ks and only cfDNA was a predictor of peak thrombin generated. CONCLUSIONS: In T2DM, NETosis detectable in circulating blood is associated with inflammatory state and a prothrombotic state, especially hypofibrinolysis.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Armadilhas Extracelulares/metabolismo , Fibrinólise , Trombose/sangue , Idoso , Biomarcadores/sangue , Glicemia/metabolismo , Ácidos Nucleicos Livres/sangue , Citrulinação , Estudos Transversais , DNA/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Hemoglobina A Glicada/metabolismo , Histonas/sangue , Humanos , Mediadores da Inflamação/sangue , Elastase de Leucócito/sangue , Masculino , Pessoa de Meia-Idade , Peroxidase/sangue , Fatores de Risco , Trombina/metabolismo , Trombose/diagnóstico , Trombose/etiologia
16.
Nat Commun ; 10(1): 1780, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992428

RESUMO

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.


Assuntos
Plaquetas/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Ativação Plaquetária/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Complemento C3/imunologia , Complemento C3/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/sangue , Influenza Humana/virologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo
17.
Nutrients ; 11(4)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013737

RESUMO

The innate immune response plays an important role in the pathophysiology of acute respiratory distress syndrome (ARDS). Glutamine (Gln) decreases lung inflammation in experimental ARDS, but its impact on the formation of extracellular traps (ETs) in the lung is unknown. In a mouse model of endotoxin-induced pulmonary ARDS, the effects of Gln treatment on leukocyte counts and ET content in bronchoalveolar lavage fluid (BALF), inflammatory profile in lung tissue, and lung morphofunction were evaluated in vivo. Furthermore, ET formation, reactive oxygen species (ROS) production, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were tested in vitro. Our in vivo results demonstrated that Gln treatment reduced ET release (as indicated by cell-free-DNA content and myeloperoxidase activity), decreased lung inflammation (reductions in interferon-γ and increases in interleukin-10 levels), and improved lung morpho-function (decreased static lung elastance and alveolar collapse) in comparison with ARDS animals treated with saline. Moreover, Gln reduced ET and ROS formation in BALF cells stimulated with lipopolysaccharide in vitro, but it did not alter GPx or GR activity. In this model of endotoxin-induced pulmonary ARDS, treatment with Gln reduced pulmonary functional and morphological impairment, inflammation, and ET release in the lung.


Assuntos
Armadilhas Extracelulares/metabolismo , Glutamina/uso terapêutico , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Síndrome do Desconforto Respiratório do Adulto/tratamento farmacológico , Animais , DNA , Modelos Animais de Doenças , Endotoxinas , Feminino , Glutamina/farmacologia , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Inflamação/etiologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Contagem de Leucócitos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Peroxidase/metabolismo , Pneumonia/etiologia , Alvéolos Pulmonares , Espécies Reativas de Oxigênio/metabolismo , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Síndrome do Desconforto Respiratório do Adulto/patologia
18.
Lab Chip ; 19(10): 1736-1746, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31020286

RESUMO

Circulating leukocytes are indispensable components of the immune system, and rapid analysis of their native state or functionalities can help to unravel their pathophysiological roles and identify novel prognostic biomarkers in health and diseases. Herein we report a novel high throughput "sample-in-answer-out" integrated platform for continuous leukocyte sorting and single-cell electrical profiling in a label-free manner. The multi-staged platform enables isolation of neutrophils and monocytes from diluted or lysed blood samples directly within minutes based on Dean flow fractionation (DFF) (stage 1). Next DFF-purified leukocytes are inertially focused in serpentine channels into a single stream (stage 2) prior to impedance detection (stage 3). As a proof-of-concept for neutrophil functional characterization towards diabetes testing, we characterized the formation of neutrophil extracellular traps (NETosis) of healthy and glucose-treated neutrophils and observed significant changes in dielectric properties (size and opacity) between both groups. Interestingly, the NETosis profiles induced by calcium ionophore (CaI) and phorbol 12-myristate 13-acetate (PMA) were also electrically different, which could be attributed to the differential rates of cell enlargement and attenuated membrane permeability. Taken together, these results clearly demonstrated the potential of the developed platform for rapid (∼mins) and label-free leukocyte profiling and the use of impedance signatures as novel functional biomarkers for point-of-care testing in diabetes.


Assuntos
Armadilhas Extracelulares/metabolismo , Citometria de Fluxo , Leucócitos/citologia , Neutrófilos/metabolismo , Ionóforos de Cálcio/farmacologia , Impedância Elétrica , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
19.
J Cancer Res Clin Oncol ; 145(7): 1695-1707, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31020419

RESUMO

BACKGROUND: Hypercoagulability is a major cancer-associated complication linked to poor patient prognosis. The production of neutrophil extracellular traps (NETs) is increasingly found to be linked with the development and metastasis of cancer, as well as with thrombi formation in cancer patients. We hypothesized that the neutrophil NET release may be triggered by specific cytokines in oral squamous cell carcinoma (OSCC) patients, thereby predisposing them to a hypercoagulable state. Moreover, we have evaluated the interaction between NETs and endothelial cells (ECs). METHODS: NET procoagulant activity was assessed based on fibrin and purified coagulation complex production assays, as well as by measuring coagulation time (CT). We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands, and cell FVa/Xa binding. RESULTS: OSCC patients with stage III/IV exhibited elevated plasma NET levels compared to stage I/II or CTR (all P < 0.05). Neutrophils from OSCC patients are predisposed to amplified NET release compared to those from CTR. Furthermore, depleting IL-8, IL-6, and TNF-α led to a reduction in NET release in the plasma. OSCC NETs increased thrombin and fibrin generation and decreased CT significantly (P < 0.05). When NETs were isolated and used to treat ECs, these cells exhibited disrupted morphology by retracting from their cell-cell junctions and convert to a procoagulant phenotype. These effects could be attenuated by approximately 70% using DNase I. CONCLUSIONS: Our findings are consistent with a model wherein OSCC drives a systemic inflammatory state, which, in turn, drives neutrophils to prime and release NETs, which drive the development of a hypercoagulable state. Intervening in this process may be a viable means of disrupting these undesirable coagulation dynamics in stage III/IV OSCC patients.


Assuntos
Armadilhas Extracelulares/metabolismo , Neoplasias Bucais/sangue , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangue , Trombofilia/sangue , Citocinas/sangue , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Neutrófilos/metabolismo , Neutrófilos/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Trombofilia/patologia
20.
Nat Commun ; 10(1): 1916, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015489

RESUMO

Potentiation of neutrophil extracellular trap (NET) release is one mechanism by which antiphospholipid antibodies (aPL Abs) effect thrombotic events in patients with antiphospholipid syndrome (APS). Surface adenosine receptors trigger cyclic AMP (cAMP) formation in neutrophils, and this mechanism has been proposed to regulate NETosis in some contexts. Here we report that selective agonism of the adenosine A2A receptor (CGS21680) suppresses aPL Ab-mediated NETosis in protein kinase A-dependent fashion. CGS21680 also reduces thrombosis in the inferior vena cavae of both control mice and mice administered aPL Abs. The antithrombotic medication dipyridamole is known to potentiate adenosine signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP. Like CGS21680, dipyridamole suppresses aPL Ab-mediated NETosis via the adenosine A2A receptor and mitigates venous thrombosis in mice. In summary, these data suggest an anti-inflammatory therapeutic paradigm in APS, which may extend to thrombotic disease in the general population.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina/análogos & derivados , Síndrome Antifosfolipídica/tratamento farmacológico , Armadilhas Extracelulares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fenetilaminas/farmacologia , Trombose Venosa/tratamento farmacológico , Adenosina/imunologia , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/genética , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/patologia , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Dipiridamol/farmacologia , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Fibrinolíticos/farmacologia , Regulação da Expressão Gênica , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/imunologia , Transdução de Sinais , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/imunologia , Veia Cava Inferior/metabolismo , Trombose Venosa/genética , Trombose Venosa/imunologia , Trombose Venosa/patologia
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