RESUMO
This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1ß production by WT neutrophils after nigericin and ATP stimulation. However, IL-1ß release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1ß release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1ß production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.
Assuntos
Anexina A1/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/metabolismo , Animais , Inflamassomos/ultraestrutura , Interleucina-1beta/metabolismo , Lipídeos/química , Masculino , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo , Neutrófilos/ultraestruturaRESUMO
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 4â¯h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3â¯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 3â¯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 , Chlorocebus aethiops , 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 VeroRESUMO
Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Hipertensão/metabolismo , Hipertensão/patologia , Neutrófilos/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Amilorida/farmacologia , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Fenômenos Biofísicos/efeitos dos fármacos , Estudos de Casos e Controles , Caveolina 1/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Canais Epiteliais de Sódio/genética , Feminino , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/genética , Masculino , Pessoa de Meia-Idade , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Necroptosis is a pro-inflammatory cell death, which happens in the context of caspase-8 inhibition, allowing activation of the receptor interacting protein kinase 1-receptor interacting protein kinase 3-mixed lineage kinase domain-like (RIPK1-RIPK3-MLKL) axis. Recently, necroptosis has emerged as a key component of resistance against pathogens including infected macrophage by Leishmania infantum, the ethiologic agent of Visceral leishmaniasis (VL). VL is the most severe form of Leishmaniasis, characterized by systemic inflammation and neutropenia. However, the role of neutrophil cell death in VL has not been characterized. Here, we showed that VL patients exhibited increased lactate dehydrogenase levels in the serum, a hallmark of cell death and tissue damage. We investigated the effect of necroptosis in neutrophil infection in vitro. Human neutrophils pretreated with zVAD-fmk (pan-caspase inhibitor) and zIETD-fmk (caspase-8 inhibitor) increased reactive oxygen species (ROS) level in response to Leishmania infection, which is associated with necroptotic cell death. MLKL, an important effector molecule downstream of necroptosis pathway, was also required for Leishmania killing. Moreover, in absence of caspases-8, murine neutrophils displayed loss of membrane integrity, higher levels of ROS, and decreased L. infantum viability. Pharmacological inhibition of RIPK1 or RIPK3 increased parasite survival when caspase-8 was blocked. Electron microscopy assays revealed morphological features associated with necroptotic death in L. infantum infected-neutrophils pretreated with caspase inhibitor, whereas infected cells pretreated with RIPK1 and RIPK3 inhibitors did not show ultra-structural alterations in membrane integrity and presented viable Leishmania within parasitophorous vacuoles. Taken together, these findings suggest that inhibition of caspase-8 contributes to elimination of L. infantum in neutrophils by triggering necroptosis. Thus, targeting necroptosis may represent a new strategy to control Leishmania replication.
Assuntos
Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Biomarcadores , Caspase 8/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Masculino , Camundongos , Necrose , Neutrófilos/parasitologia , Neutrófilos/ultraestruturaRESUMO
Cutaneous leishmaniasis is a neglected tropical disease, causing a spectrum of clinical manifestations varying from self-healing to unhealing lesions that may be very difficult to treat. Emerging evidence points to a detrimental role for neutrophils during the first hours following infection with many distinct Leishmania species (spp.) at a time when the parasite is in its nonreplicative promastigote form. Neutrophils have also been detected at later stages of infection in unhealing chronic cutaneous lesions. However, the interactions between these cells and the replicative intracellular amastigote form of the parasite have been poorly studied. Here, we show that Leishmaniamexicana amastigotes are efficiently internalized by neutrophils and that this process has only a low impact on neutrophil activation and apoptosis. In neutrophils, the amastigotes were found in acidified vesicles. Furthermore, within cutaneous unhealing lesions, heavily infected neutrophils were found with up to 6 parasites per cell. To investigate if the amastigotes could replicate within neutrophils, we generated photoconvertible fluorescent parasites. With the use of flow cytometry imaging and time-lapse microscopy, we could demonstrate that a subset of parasites replicated within neutrophils. Overall, our data reveal a novel role for neutrophils that can act as a niche for parasite replication during the chronic phase of infection, thereby contributing to disease pathology.
Assuntos
Divisão Celular , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Estágios do Ciclo de Vida/genética , Neutrófilos/parasitologia , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Animais , Feminino , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Genes Reporter , Interações Hospedeiro-Parasita/imunologia , Leishmania mexicana/patogenicidade , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/ultraestrutura , Fagocitose , Processos Fotoquímicos , Imagem com Lapso de TempoRESUMO
In spite of the numerous studies on chronic obstructive pulmonary disease (COPD), the cellular and molecular basis of the disease's development remain unclear. Neutrophils and eosinophils are known to be key players in COPD. Recently, neutrophil extracellular trap cell death (NETosis), a mechanism due to decondensation and extrusion of chromatin to form extracellular traps, has been demonstrated in COPD. However, there is limited knowledge about eosinophil extracellular trap cell death (EETosis) and its role in the pathogenesis of COPD. The aim of this study was to evaluate EETosis in stable COPD. Induced sputum obtained from healthy smokers and low exacerbation risk COPD A or B group patients or high exacerbation risk COPD C or D group patients were included. Samples were examined using electron microscopy and immunofluorescence. Healthy smokers (n=10) and COPD A (n=19) group exhibited neutrophilic or paucigranulocytic phenotypes, with NETosis being absent in these patients. In contrast, COPD B (n=29), with eosinophilic or mixed phenotypes, showed EETosis and incipient NETosis. COPD C (n=18) and COPD D groups (n=13) were differentiated from low exacerbation rate-COPD group by the abundant cellular debris, with COPD C group having an eosinophilic pattern and numerous cells undergoing EETosis. A hallmark of this group was the abundant released membranes that often appeared phagocytosed by neutrophils, which coincidentally exhibited early NETosis changes. The COPD D group included patients with a neutrophilic or mixed pattern, with abundant neutrophil extracellular trap-derived material. This study is the first to demonstrate EETosis at different stages of stable COPD. The results suggest a role for eosinophils in COPD pathophysiology, especially at the beginning and during the persistence of the disease, regardless of whether the patient quit smoking, with EETosis debris probably triggering uncontrolled NETosis. The main target of these findings should be young smokers with the potential to develop COPD.
Assuntos
Eosinófilos/ultraestrutura , Armadilhas Extracelulares/metabolismo , Pulmão/ultraestrutura , Neutrófilos/ultraestrutura , Doença Pulmonar Obstrutiva Crônica/patologia , Estudos de Casos e Controles , Morte Celular , Estudos Transversais , Eosinófilos/metabolismo , Feminino , Imunofluorescência , Volume Expiratório Forçado , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Microscopia Confocal , Microscopia Eletrônica , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/efeitos adversos , Abandono do Hábito de Fumar , Prevenção do Hábito de Fumar , Escarro/citologia , Escarro/metabolismo , Capacidade VitalRESUMO
OBJECTIVE: To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner. DESIGN: Experimental study. SETTING: University-based laboratory. PATIENT(S): Semen samples from four men and blood samples from six healthy female donors. INTERVENTION(S): Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]). MAIN OUTCOME MEASURE(S): During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 104:45 × 105], 6:18 [15 × 105:45 × 105], 9:18 [252 × 104:45 × 105]). RESULT(S): The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils. CONCLUSION(S): We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.
Assuntos
Comunicação Celular , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Adulto , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Imunofluorescência , Histonas/metabolismo , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Transdução de Sinais , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Fatores de Tempo , Adulto JovemRESUMO
Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.
Assuntos
Armadilhas Extracelulares/imunologia , Lectinas Tipo C/imunologia , Neutrófilos/imunologia , Paracoccidioides/imunologia , Receptores Mitogênicos/imunologia , DNA/imunologia , DNA/metabolismo , Desoxirribonucleases/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Histonas/imunologia , Histonas/metabolismo , Humanos , Interferon gama/farmacologia , Lectinas Tipo C/genética , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Elastase Pancreática/imunologia , Elastase Pancreática/metabolismo , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Fagocitose/efeitos dos fármacos , Receptores Mitogênicos/genética , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVES: Neutrophils play an important role in the control of pathogens through several mechanisms, including phagocytosis and the formation of neutrophil extracellular traps (NETs). The latter consists of DNA as a backbone with embedded antimicrobial peptides, histones, and proteases, providing a matrix to entrap and in some cases to kill microbes. Some metabolic requirements for NET formation have recently been described. The virus-induced formation of NETs and the role of these traps in viral infections remain scarcely reported. Here, we analyzed whether dengue virus serotype-2 (DENV-2) induces NET formation and the DENV-2 effect on phorbol myristate acetate (PMA)-induced NETs. METHODS: Peripheral blood-derived neutrophils were exposed in vitro to DENV-2 or exposed to DENV-2 and then stimulated with PMA. NET formation was assessed by fluorescence microscopy. Cell membrane Glut-1, glucose uptake, and reactive oxygen species (ROS) production were assessed. RESULTS: DENV-2 does not induce the formation of NETs. Moreover, DENV-2 inhibits PMA-induced formation of NETs by about 80%. This effect is not related to the production of ROS. The mechanism seemingly accountable for this inhibitory effect is the DENV-2-mediated inhibition of PMA-induced glucose uptake by neutrophils. CONCLUSION: Our results suggest that DENV-2 inhibits glucose uptake as a metabolism-based way to avoid the formation of NETs.
Assuntos
Vírus da Dengue/metabolismo , Armadilhas Extracelulares/virologia , Neutrófilos/virologia , Vírus da Dengue/imunologia , Armadilhas Extracelulares/imunologia , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Microscopia de Fluorescência , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Sorogrupo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Invasive aspergillosis (IA) is a severe infection that commonly occurs in immunocompromised patients after hematopoietic stem cell transplantation (HSCT). The present study explores the effect of Aspergillus fumigatus diffusates (AfDs) on phagocytic function and superoxide anion (O2(-)) burst levels in polymorphonuclear neutrophils (PMNs) from post-HSCT patients. A. fumigatus conidia with or without AfD were used to stimulate the PMN from healthy donor or HSCT patient for two hours. PMN morphology was visualized by scanning electron microscopy. The levels of respiratory burst O2(-) produced by the PMNs were determined by flow cytometry. PMN phagocytic rates and phagocytic indexes were observed and calculated using periodic acid-Schiff (PAS) staining under a light-field microscope. No difference was found between the PMN phagocytic rates, phagocytic indexes, or O2(-) respiratory burst levels in health donor PMNs following treatments of A. fumigatus conidia with or without AfD. However, significant inhibition of these indices was seen in the PMNs from HSCT patients following treatment of A. fumigatus conidia plus AfD, compared to that with conidium treatment alone (P < 0.05). Therefore, AfD significantly inhibited the phagocytic function of PMNs from HSCT patients, potentially through inhibition of intracellular respiratory burst levels during phagocytosis. This suggests that the reason underlying the greater susceptibility of HSCT patients to aspergillosis might be the existence of AfD in vivo during infection. Further research on the mechanisms by which AfD affects the phagocytic function of PMNs from HSCT patients is therefore of great significance for the prevention of IA.
Assuntos
Aspergillus fumigatus/imunologia , Transplante de Células-Tronco Hematopoéticas , Imunomodulação , Neutrófilos/microbiologia , Neutrófilos/fisiologia , Fagocitose/imunologia , Explosão Respiratória/imunologia , Adulto , Aspergilose/complicações , Aspergilose/imunologia , Aspergilose/microbiologia , Feminino , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Neutrófilos/patologia , Neutrófilos/ultraestrutura , Infecções OportunistasRESUMO
Neutrophils play a key role in the innate immune system, providing the first line of host defense. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, it has recently been shown that neutrophils can trap and kill microorganisms by the release of extracellular structures composed of DNA and antimicrobial proteins called neutrophil extracellular traps (NETs). Although physiological amounts of NETs are important as antimicrobial agents, high levels of NETs in circulation may result in severe tissue damage. Besides, the excessive generation of NETs or a disruption in their clearance mechanism might be associated with the development of certain autoimmune diseases. This review describes the structure, function and generation of NETs, and their possible implication in the initiation and/or progression of different diseases.
Assuntos
Infecções Bacterianas/imunologia , DNA/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , alfa-Defensinas/metabolismo , Animais , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Bacteriólise , Degranulação Celular , Eosinófilos/imunologia , Eosinófilos/metabolismo , Espaço Extracelular , Humanos , Imunidade Inata , Microcirculação , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Trombose/etiologiaRESUMO
INTRODUCTION: During human immunodeficiency virus (HIV) infection a dysfunction of polymorphonuclear (PMN) cells has been described including a progressively altered superoxide production as disease progression. The NADPH oxidase has been described as a major source of superoxide. The neutrophil NADPH oxidase comprises a plasma membrane-bound cytochrome b558 (which is a heterodimer of one p22-phox and one gp91-phox subunit) and cytosolic subunits, namely p47-phox, p67-phox and p40-phox. During neutrophil activation in response to various agonists, the cytosolic subunits translocate to and associate with the cytochrome b558, a process that results in oxidase activation. Therefore, an altered superoxide production could be a consequence of abnormal distribution or translocation of NADPH oxidase components in response to HIV infection. MATERIAL AND METHODS: We used several strategies including: confocal microscopy, subcellular fractionation and sucrose gradients, to analyze the cellular distribution of two of the NADPH oxidase components (p22-phox and p47-phox). RESULTS: We observed that in resting cells, a substantial proportion of p22-phox from HIV positive patients is distributed in regions close to the cytoplasmic membrane, sediment in high density sucrose fractions and is located in the cytoplasmic insoluble fraction. Additionally, a diffuse cytosolic distribution of p47-phox was observed in neutrophils from HIV infected patients. The results demonstrate an inappropriate cell distribution of NADPH-complex in PMN from HIV positive patients.
Assuntos
Infecções por HIV/enzimologia , NADPH Oxidases/sangue , Neutrófilos/química , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Citoplasma/química , Citosol/química , Ativação Enzimática , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Microscopia Confocal , Neutrófilos/fisiologia , Neutrófilos/ultraestrutura , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Solubilidade , Frações Subcelulares/químicaRESUMO
BACKGROUND: The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. METHODS: Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. RESULTS: Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. CONCLUSIONS: This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain.
Assuntos
Micropartículas Derivadas de Células/imunologia , Ativação de Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Neutrófilos/imunologia , Tuberculose/imunologia , Comunicação Celular/imunologia , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Corantes Fluorescentes , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Microscopia Eletrônica , Neutrófilos/microbiologia , Neutrófilos/ultraestrutura , Compostos OrgânicosRESUMO
Recently it was discovered that neutrophils can generate extracellular fibers called NET (neutrophil extracellular traps), which are composed of a skeleton of DNA "decorated" with many cytoplasmic including enzymes and nuclear components.The NET are a physical barrier that prevent the spread of microorganisms and facilitate the cell death by promoting a high local concentration of antimicrobial molecules. On the other hand, the fibrous structure limits the damage to the tissue where they are generated by restricting the range of molecules that are released by the neutrophil. This paper describes this new form of cell death and the implications this may have on different diseases.
Assuntos
Autofagia/fisiologia , Neutrófilos/fisiologia , Humanos , Neutrófilos/ultraestruturaRESUMO
Oleic acid (OA) is a nonesterified fatty acid that is released into the blood during lipomobilization at the time of calving in cows, a period where increased risk of infection and acute inflammation is observed. These data suggest potential OA-mediated regulation of innate immune responses. In the present study, we assessed the effects of OA on intracellular calcium release, ERK1/2 phosphorylation, superoxide production, CD11b expression and matrix metalloproteinase-9 (MMP-9) release in bovine neutrophils. Furthermore, the presence of GPR40, an OA receptor, was assessed by RT-PCR, immunoblotting and confocal microscopy. OA induced, in a dose-dependent manner, intracellular calcium mobilization, superoxide production and CD11b expression in bovine neutrophils; these effects were reduced by the intracellular chelating agent BAPTA-AM. OA also induced ERK2 phosphorylation and MMP-9 release. RT-PCR analysis detected mRNA expression of a bovine ortholog of the GPR40 receptor. Using a polyclonal antibody against human GPR40, we detected a protein of 31kDa by immunoblotting that was localized predominately in the plasma membrane. The selective agonist of GPR40, GW9508, induced intracellular calcium mobilization and ERK2 phosphorylation. In conclusion, OA can modulate bovine neutrophil responses in an intracellular calcium-dependent manner; furthermore, these responses could be induced by GPR40 activation.
Assuntos
Cálcio/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/efeitos dos fármacos , Ácido Oleico/farmacologia , Superóxidos/metabolismo , Animais , Antígenos CD1/genética , Antígenos CD1/metabolismo , Bovinos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metilaminas/farmacologia , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Ácido Oleico/metabolismo , Fosforilação/efeitos dos fármacos , Propionatos/farmacologia , RNA Mensageiro/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania-neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.
Assuntos
Espaço Extracelular/imunologia , Leishmania/citologia , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Neutrófilos/imunologia , Neutrófilos/parasitologia , Animais , Morte Celular/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Glicoesfingolipídeos/farmacologia , Histonas/metabolismo , Humanos , Leishmania/imunologia , Leishmania/ultraestrutura , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The purpose of this study was twofold: to reveal cellular events associated with the protective role of endogenous annexin A1 (AnxA1) in inflammation and to highlight the potential involvement of members of the formyl peptide receptor (Fpr) family in this process. We found that wild-type, AnxA1-null, and Fpr1-null mice all displayed an intense neutrophil recruitment into the peritoneal cavity as assessed 4 hours after carrageenin injection, and that this recruitment was most pronounced in AnxA1-null mice. In addition, this cell influx could be inhibited by the AnxA1 pharmacophore peptide, Ac2-26, in wild-type, AnxA1-null, and Fpr1-null mice, but was restored when co-treated with the pan-receptor antagonist Boc2. Using the LacZ gene reporter assay, an enhancement of AnxA1 gene promoter activity in extravasated neutrophils was evident in AnxA1-null mice; again this response was reduced after peptide treatment. The lack of functional involvement of Fpr1 prompted us to monitor the structurally related receptor Fpr2. We report, for the first time, the ultrastructural immunocytochemical co-localization of Fpr2 with AnxA1 in neutrophils that migrate into the mesenteric microcirculation and extravasate into the peritoneal fluid. Collectively, these data provide in vivo support to the hypothesis that endogenous AnxA1 is an essential effector of endogenous anti-inflammation and provide an ultrastructural indication that this mediator interacts with Fpr2 in murine neutrophils. We believe that these findings could significantly affect the development of novel therapeutics, which are modeled after the anti-migratory actions of AnxA1.
Assuntos
Anexina A1/metabolismo , Quimiotaxia de Leucócito/imunologia , Inflamação/metabolismo , Neutrófilos/metabolismo , Animais , Anexina A1/ultraestrutura , Expressão Gênica , Imuno-Histoquímica , Inflamação/imunologia , Masculino , Camundongos , Camundongos Mutantes , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Receptores de Formil Peptídeo/imunologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Formil Peptídeo/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Paracoccidioidomycosis, caused by the dimorphic fungus Paracoccidioides brasiliensis, is the most prevalent systemic mycosis of Latin America, with Brazil accounting for 80% of the reported cases. The great number of neutrophils found in P. brasiliensis granulomas demonstrates the importance of polymorphonuclear neutrophils (PMN) cells during this mycotic infection. It has been found that neutrophils from healthy human donors can ingest and kill the fungus through a typical phagocytic process. The present work tests the phagocytic ability of neutrophils collected from patients that had had and were considered cured of paracoccidioidomycosis. Transmission electron microscopy and cytochemical studies indicate that patients' neutrophils eventually degenerate during phagocytosis of P. brasiliensis. Endogen peroxidase and NAD(P)H-oxidase are activated during the process showing that the respiratory burst and the neutrophil degranulation are triggered by the attachment of the yeast cells. Apparently these processes are not enough to kill P. brasiliensis. Although fungicidal activity can be determined by colony forming unit (CFU) counting, qualitative data suggest, as noted, that neutrophils from patients with treated paracoccidioidomycosis degenerate during the phagocytosis process. Hence, this work demonstrates the existence of a functional neutrophil deficiency against P. brasiliensis in susceptible individuals. The exact origin of this susceptibility is still to be determined in further studies.
Assuntos
Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Adulto , Idoso , Brasil , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Paracoccidioidomicose/microbiologia , Peroxidase/metabolismo , Fagocitose , Leveduras/imunologiaRESUMO
Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.