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1.
Int J Mol Sci ; 22(23)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34884704

RESUMO

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.


Assuntos
Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismo , Adulto , Carcinógenos/farmacologia , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Lágrimas/citologia , Acetato de Tetradecanoilforbol/farmacologia , Adulto Jovem
2.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34675073

RESUMO

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.


Assuntos
Quimiotaxia de Leucócito/imunologia , Neutrófilos/imunologia , Proteínas Ativadoras de ras GTPase/imunologia , Proteínas ras/antagonistas & inibidores , Actinas/imunologia , Movimento Celular , Polaridade Celular , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/genética , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Receptores Acoplados a Proteínas G/imunologia , Transdução de Sinais , Proteínas de Ligação a Telômeros/imunologia , Proteínas Ativadoras de ras GTPase/deficiência , Proteínas Ativadoras de ras GTPase/genética , Proteínas ras/imunologia
3.
Mol Biol (Mosk) ; 55(5): 858-869, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34671008

RESUMO

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.


Assuntos
Mitoxantrona , Neutrófilos , Plaquetas , Humanos , Mitoxantrona/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Peptídeos
4.
Mar Drugs ; 19(5)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062855

RESUMO

Two new isosarcophine derivatives, cherbonolides M (1) and N (2), were further isolated from a Formosan soft coral Sarcophyton cherbonnieri. The planar structure and relative configuration of both compounds were established by the detailed analysis of the IR, MS, and 1D and 2D NMR data. Further, the absolute configuration of both compounds was determined by the comparison of CD spectra with that of isosarcophine (3). Notably, cherbonolide N (2) possesses the unique cembranoidal scaffold of tetrahydrooxepane with the 12,17-ether linkage fusing with a γ-lactone. In addition, the assay for cytotoxicity of both new compounds revealed that they showed to be noncytotoxic toward the proliferation of A549, DLD-1, and HuCCT-1 cell lines. Moreover, the anti-inflammatory activities of both metabolites were carried out by measuring the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion and elastase release in the primary human neutrophils. Cherbonolide N (2) was found to reduce the generation of superoxide anion (20.6 ± 6.8%) and the elastase release (30.1 ± 3.3%) in the fMLF/CB-induced human neutrophils at a concentration of 30 µM.


Assuntos
Antozoários/química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Citocalasina B/farmacologia , Diterpenos/isolamento & purificação , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Elastase Pancreática/metabolismo , Superóxidos/metabolismo , Taiwan
5.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467555

RESUMO

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata/imunologia , Leucemia/imunologia , Neutrófilos/imunologia , Doença Aguda , Adolescente , Ionóforos de Cálcio/farmacologia , Células Cultivadas , Criança , Pré-Escolar , Humanos , Imunidade Inata/efeitos dos fármacos , Lactente , Leucemia/sangue , Leucemia/tratamento farmacológico , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
6.
Nat Prod Res ; 35(1): 17-24, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31135226

RESUMO

A new dimeric quaternary protoberberine alkaloid, bispalmatrubine (1), and thirteen known compounds (2-14) were purified from the tubers of Tinospora dentata. Their structures were determined by spectroscopic and spectrometric analytical methods. Among the isolates, eight compounds were examined for their in vitro anti-inflammatory potential and several tested alkaloids displayed moderate inhibitory effects of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.


Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Tinospora/química , Alcaloides/química , Alcaloides/farmacologia , Alcaloides de Berberina/química , Citocalasina B/farmacologia , Humanos , Estrutura Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Tubérculos/química , Plantas Medicinais/química , Superóxidos/metabolismo
7.
Cell Biol Toxicol ; 37(2): 177-191, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32367270

RESUMO

Owing to the excellent antibacterial and antiviral activity, silver nanoparticles have a widespread use in the food and pharmaceutical industries. With the increase in the production and use of the related products, the potential hazard of silver nanoparticles has aroused public attention. The main purpose of this study is to explore the toxicity of silver nanoparticles and induction of lung inflammation in vitro and in vivo. Here, we validated that small amounts of silver ions dissolved from silver nanoparticles caused the depolarization of plasma membrane, resulting in an overload of intracellular sodium and calcium, and eventually led to the cell necrosis. The blockers of calcium or sodium channels inversed the toxicity of silver ions. Then, we instilled silver nanoparticles or silver nitrate (50 µg per mouse) into the lungs of mice, and this induced pulmonary injury and mitochondrial content release, led to the recruitment of neutrophils to the lung tissue via p38 MAPK pathway. Altogether, these data show that released silver ions from nanoparticles induced cell necrosis through Na+ and Ca2+ influx and triggered pulmonary inflammation through elevating mitochondrial-related contents released from these necrotic cells.


Assuntos
Nanopartículas Metálicas/efeitos adversos , Mitocôndrias/metabolismo , Pneumonia/patologia , Prata/efeitos adversos , Células A549 , Animais , Cálcio/metabolismo , DNA Mitocondrial/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Íons , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Necroptose/efeitos dos fármacos , Necrose , Infiltração de Neutrófilos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Sódio/metabolismo
8.
Front Immunol ; 12: 785275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35069556

RESUMO

Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc.


Assuntos
N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Armadilhas Extracelulares/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
9.
Molecules ; 25(24)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327368

RESUMO

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/farmacologia , Fraxinus/química , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Casca de Planta/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/classificação , Anti-Inflamatórios/isolamento & purificação , Citocalasina B/antagonistas & inibidores , Citocalasina B/farmacologia , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Glucosídeos Iridoides/química , Glucosídeos Iridoides/classificação , Glucosídeos Iridoides/isolamento & purificação , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Elastase de Leucócito/imunologia , Elastase de Leucócito/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Camundongos , Estrutura Molecular , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Cultura Primária de Células , Células RAW 264.7 , Relação Estrutura-Atividade , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
10.
Cells ; 9(12)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322305

RESUMO

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCß. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.


Assuntos
N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptor Toll-Like 9/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Cloroquina/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Neurilemoma/metabolismo , Neurilemoma/patologia , Ratos , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/genética , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/genética
11.
J Phys Chem Lett ; 11(21): 8952-8957, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33030905

RESUMO

The formyl peptide receptor 2 (ALX/FPR2), a G-protein-coupled receptor (GPCR), plays an important role in host defense and inflammation. This receptor can be driven as pro- or anti-inflammatory depending on its agonist, such as N-formyl-Met-Leu-Phe-Lys (fMLFK) and resolvin D1 (RvD1) or its aspirin-triggered 17 (R)-epimer, AT-RvD1, respectively. However, the activation mechanism of ALX/FPR2 by pro- and anti-inflammatory agonists remains unclear. In this work, on the basis of molecular dynamics simulations, we evaluated a model of the ALX/FPR2 receptor activation process using two agonists, fMLFK and AT-RvD1, with opposite effects. The simulations by both fMLFK and AT-RvD1 induced the ALX/FPR2 activation through a set of receptor-core residues, in particular, R205, Q258, and W254. In addition, the activation was dependent on the disruption of electrostatic interactions in the cytoplasmic region of the receptor. We also found that in the AT-RvD1 simulations, the position of the H8 helix was similar to that of the same helix in other class-A GPCRs coupled to arrestin. Thus our results shed light on the mechanism of activation of the ALX/FPR2 receptor by pro-inflammatory and pro-resolution agonists.


Assuntos
Anti-Inflamatórios/química , Ácidos Docosa-Hexaenoicos/química , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , Receptores de Formil Peptídeo/agonistas , Receptores de Lipoxinas/agonistas , Sequência de Aminoácidos , Anti-Inflamatórios/farmacologia , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Ácidos Docosa-Hexaenoicos/farmacologia , Glucocorticoides/química , Humanos , Simulação de Dinâmica Molecular , N-Formilmetionina Leucil-Fenilalanina/química , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Conformação Proteica , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Transdução de Sinais , Eletricidade Estática
12.
Front Immunol ; 11: 2124, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013896

RESUMO

The importance of the intracellular Ca2+ concentration ([Ca2+]i) in neutrophil function has been intensely studied. However, the role of the intracellular Na+ concentration ([Na+]i) which is closely linked to the intracellular Ca2+ regulation has been largely overlooked. The [Na+]i is regulated by Na+ transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+]i. fMLF induces a sustained increase of [Na+]i, surprisingly, reaching higher values in TRPM2-/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+]i. The difference in [Na+]i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na+ concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2-/- neutrophils. Increasing the [Na+]i by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na+ homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na+ concentration gradients. Increasing the [Na+]i by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.


Assuntos
Quimiotaxia de Leucócito/imunologia , Homeostase/imunologia , Sódio/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Cálcio/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C5a/imunologia , Complemento C5a/farmacologia , Líquido Intracelular/imunologia , Leucemia Mieloide , Camundongos , Camundongos Endogâmicos C57BL , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Trocador de Sódio e Cálcio/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Canais de Cátion TRPM/deficiência
13.
Eur J Pharmacol ; 884: 173353, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32707189

RESUMO

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A2 (cPLA2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) secretion. However, at concentrations that blocked both AA release and LTC4 secretion, D609 had no significant inhibitory effect on stimulated cPLA2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC4 secretion by D609 is due to inhibition of calcium-mediated cPLA2 translocation to intracellular membranes, not inhibition of cPLA2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+]i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC4 synthesis through calcium-mediated cPLA2 activation.


Assuntos
Degranulação Celular , Eosinófilos/enzimologia , Leucotrienos/metabolismo , Fosfolipases Tipo C/metabolismo , Ácido Araquidônico/metabolismo , Sinalização do Cálcio , Degranulação Celular/efeitos dos fármacos , Citocalasina B/farmacologia , Ativação Enzimática , Eosinófilos/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Leucotrieno C4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Norbornanos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Transdução de Sinais , Tiocarbamatos/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
14.
ESC Heart Fail ; 7(5): 2250-2257, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32597024

RESUMO

AIMS: Takotsubo syndrome (TTS) episodes are primarily initiated by 'pulse' release of catecholamines inducing neutrophil infiltration and myocardial inflammation in susceptible individuals (largely ageing women). Evidence of myocardial inflammation and associated energetic impairment persists for ≥ 3 months post-acute TTS episodes, suggesting the existence of additional 'perpetuating' mechanisms. The effects of B-type natriuretic peptide (BNP) in suppressing superoxide (O2 - ) release from neutrophils are transiently impaired in acute heart failure. We also evaluated the extent and duration of BNP-induced suppression of O2 - release post-TTS. METHODS AND RESULTS: TTS patients were studied acutely (n = 34) and 3 months thereafter (n = 13) and compared with control subjects (n = 25). O2 - generation from neutrophils, triggered by N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate, and its suppression by BNP, were measured in vitro. Determinants of variability in BNP effect were sought via univariate and multivariate analyses. Relative to control subjects, in TTS patients, BNP suppression of both phorbol myristate acetate and N-formyl-methionyl-leucyl-phenylalanine-induced O2 - release was impaired acutely (P < 0.05 for both); this did not improve over the 3-month recovery period, despite treatment with conventional anti-failure medication in 85% of patients. No significant correlates of BNP effect (other than TTS) were identified. CONCLUSIONS: (1) While TTS is associated with marked and prolonged release of BNP, there is virtually total loss of the ability of BNP to suppress neutrophil O2 - release and its impact on tissue inflammation. (2) BNP responses do not recover for at least 3 months post-attacks, suggesting that this might contribute to perpetuation of myocardial inflammation in TTS patients.


Assuntos
Peptídeo Natriurético Encefálico , Cardiomiopatia de Takotsubo , Anti-Inflamatórios , Antioxidantes , Feminino , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Cardiomiopatia de Takotsubo/diagnóstico , Cardiomiopatia de Takotsubo/etiologia
15.
Dermatol Ther ; 33(3): e13388, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32277559

RESUMO

Neauvia hydrogel (N-Gel) is a hyaluronic acid-based dermal filler, cross-linked with polyethylene glycol. This filler contains sodium hyaluronate at different concentrations, poly(ethylene glycol) diglycidyl ether cross-linked, glycine, and l-prolyne. Assessing any effects of N-Gel on immunity and inflammation is of crucial importance. The aim of the study was to characterize the ability of N-Gel to modulate human polymorphonuclear leukocyte (PMN) functions, including migration, oxidative metabolism, and production of proinflammatory mediators. N-Gel was tested on isolated human PMN. Spontaneous and N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated migration were examined using the Boyden Chamber technique, whereas the oxidative metabolism was assessed through spectrofluorometric measurement of reactive oxygen species (ROS) production under resting conditions and after stimulation with fMLP. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 mRNA levels were measured by real-time PCR after stimulation with fMLP or Escherichia coli lipopolysaccharide. This study showed that N-Gel reduced fMLP-induced migration and ROS production without affecting these functions in resting cells. In addition, incubation of PMN with N-Gel effectively reduced both TNF-α and IL-8 mRNA levels. N-Gel modulates critical functions of human PMN such as migration and oxidative metabolism, indicating its potential as an anti-inflammatory agent.


Assuntos
Ácido Hialurônico , Neutrófilos , Humanos , Ácido Hialurônico/farmacologia , Hidrogéis , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Polietilenoglicóis/farmacologia
16.
FASEB J ; 34(5): 7127-7143, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32275103

RESUMO

Human neutrophils have a vital role in host defense and inflammatory responses in innate immune systems. Growing evidence shows that the overproduction of reactive oxygen species and granular proteolytic enzymes from activated neutrophils is linked to the pathogenesis of acute inflammatory diseases. However, adequate therapeutic targets are still lacking to regulate neutrophil functions. Herein, we report that MVBR-28, synthesized from the Mannich bases of heterocyclic chalcone, has anti-neutrophilic inflammatory effects through regulation of intracellular pH. MVBR-28 modulates neutrophil functions by attenuating respiratory burst, degranulation, and migration. Conversely, MVBR-28 has no antioxidant effects and fails to alter elastase activity in cell-free systems. The anti-inflammatory effects of MVBR-28 are not seen through cAMP pathways. Significantly, MVBR-28 potently inhibits extracellular Ca2+ influx in N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and thapsigargin-activated human neutrophils. Notably, MVBR-28 attenuates fMLF-induced intracellular alkalization in a K+ -dependent manner, which is upstream of Ca2+ pathways. Collectively, these findings provide new insight into Mannich bases of heterocyclic chalcone regarding the regulation of neutrophil functions and the potential for the development of MVBR-28 as a lead compound for treating neutrophilic inflammatory diseases.


Assuntos
Chalconas/farmacologia , Morfolinas/farmacologia , Neutrófilos/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Chalconas/síntese química , Chalconas/química , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Morfolinas/síntese química , Morfolinas/química , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/patologia , Neutrófilos/fisiologia , Potássio/metabolismo , Explosão Respiratória/efeitos dos fármacos
17.
J Leukoc Biol ; 107(5): 819-831, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303121

RESUMO

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Canais Iônicos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Formilmetionina Leucil-Fenilalanina/farmacologia
18.
J Vis Exp ; (156)2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32091008

RESUMO

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.


Assuntos
Movimento Celular , Neutrófilos/citologia , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Movimento Celular/efeitos dos fármacos , Separação Celular , Modelos Animais de Doenças , Articulações/patologia , Camundongos Endogâmicos C57BL , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia
19.
Protein Pept Lett ; 27(3): 236-242, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31746288

RESUMO

BACKGROUND: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis. METHODS: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis. RESULTS: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data. CONCLUSION: The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs.


Assuntos
Glutamato-Amônia Ligase/química , Isoniazida/administração & dosagem , Mycobacterium tuberculosis/enzimologia , N-Formilmetionina Leucil-Fenilalanina/administração & dosagem , Rifampina/administração & dosagem , Tuberculose/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Glutamato-Amônia Ligase/imunologia , Isoniazida/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Camundongos , Mycobacterium tuberculosis/imunologia , N-Formilmetionina Leucil-Fenilalanina/imunologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rifampina/farmacologia , Baço/efeitos dos fármacos , Baço/microbiologia , Tuberculose/imunologia
20.
Mol Immunol ; 116: 80-89, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31630079

RESUMO

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10-9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.


Assuntos
Galectina 1/farmacologia , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de Formil Peptídeo/metabolismo , Adulto Jovem
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