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Mol Med ; 26(1): 16, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32013888


The Editors-in-Chief would like to alert readers that this article (Sitapara et al. 2014) is part of an investigation being conducted by the journal following the conclusions of an institutional enquiry at the University of Liverpool with respect to the quantitative mass spectrometry-generated results regarding acetylated and redox-modified HMGB1.

Am J Respir Cell Mol Biol ; 52(2): 171-82, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24992505


The prolonged exposure to hyperoxia can compromise macrophage functions and contribute to the development of ventilator-associated pneumonia. High levels of extracellular high-mobility group box-1 (HMGB1) in the airways of mice exposed to hyperoxia can directly cause macrophage dysfunction. Hence, inhibition of the release of nuclear HMGB1 into the extracellular milieu may help to maintain macrophage functions under hyperoxic conditions. The present study investigates whether ethacrynic acid (EA) affects hyperoxia-induced HMGB1 release from macrophages and improves their functions. Macrophage-like RAW 264.7 cells and bone marrow-derived macrophages were exposed to different concentrations of EA for 24 hours in the presence of 95% O2. EA significantly decreased the accumulation of extracellular HMGB1 in cultured media. Importantly, the phagocytic activity and migration capability of macrophages were significantly enhanced in EA-treated cells. Interestingly, hyperoxia-induced NF-κB activation was also inhibited in these cells. To determine whether NF-κB plays a role in hyperoxia-induced HMGB1 release, BAY 11-7082, an inhibitor of NF-κB activation, was used. Similar to EA, BAY 11-7082 significantly inhibited the accumulation of extracellular HMGB1 and improved hyperoxia-compromised macrophage migration and phagocytic activity. Furthermore, 24-hour hyperoxic exposure of macrophages caused hyperacetylation of HMGB1 and its subsequent cytoplasmic translocation and release, which were inhibited by EA and BAY 11-7082. Together, these results suggest that EA enhances hyperoxia-compromised macrophage functions by inhibiting HMGB1 hyperacetylation and its release from macrophages, possibly through attenuation of the NF-κB activation. Therefore, the activation of NF-κB could be one of the underlying mechanisms that mediate hyperoxia-compromised macrophage functions.

Ácido Etacrínico/farmacologia , Proteína HMGB1/metabolismo , Hiperóxia/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Lipopolissacarídeos/farmacologia , Camundongos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia
Methods Mol Biol ; 1172: 137-45, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908301


Phagocytosis is the process by which phagocytes, including macrophages, neutrophils and monocytes, engulf and kill invading pathogens, remove foreign particles, and clear cell debris. Phagocytes and their ability to phagocytose are an important part of the innate immune system and are critical for homeostasis of the host. Impairment in phagocytosis has been associated with numerous diseases and disorders. Different cytokines have been shown to affect the phagocytic process. Cytokines including TNFα, IL-1ß, GM-CSF, and TGF-ß1 were found to promote phagocytosis, whereas high mobility group box-1 (HMGB1) inhibited the phagocytic function of macrophages. Here, we describe two commonly used methods to assess the phagocytic function of cultured macrophages, which can easily be applied to other phagocytes. Each method is based on the extent of engulfment of FITC-labeled latex minibeads by macrophages under different conditions. Phagocytic activity can be assessed either by counting individual cells using a fluorescence microscope or measuring fluorescence intensity using a flow cytometer.

Citometria de Fluxo/métodos , Macrófagos/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Fagocitose/efeitos dos fármacos , Animais , Linhagem Celular , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interleucina-1beta/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Microesferas , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
Mol Med ; 20: 238-47, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24664237


Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with respiratory distress. However, prolonged exposure to hyperoxia compromises the ability of the macrophage to phagocytose and clear bacteria. Previously, we showed that the exposure of mice to hyperoxia elicits the release of the nuclear protein high mobility group box-1 (HMGB1) into the airways. Extracellular HMGB1 impairs macrophage phagocytosis and increases the mortality of mice infected with Pseudomonas aeruginosa (PA). The aim of this study was to determine whether GTS-21 [3-(2,4 dimethoxybenzylidene)-anabaseine dihydrochloride], an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, could inhibit hyperoxia-induced HMGB1 release into the airways, enhance macrophage function and improve bacterial clearance from the lungs in a mouse model of ventilator-associated pneumonia. GTS-21 (0.04, 0.4 and 4 mg/kg) or saline was systemically administered via intraperitoneal injection to mice that were exposed to hyperoxia (≥99% O2) and subsequently challenged with PA. We found that systemic administration of 4 mg/kg GTS-21 significantly increased bacterial clearance, decreased acute lung injury and decreased accumulation of airway HMGB1. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophagelike cell line, were incubated with different concentrations of GTS-21 in the presence of 95% O2. The phagocytic activity of macrophages was significantly increased by GTS-21 in a dose-dependent manner. In addition, hyperoxia-induced hyperacetylation of HMGB1 was significantly reduced in macrophages incubated with GTS-21. Furthermore, GTS-21 significantly inhibited the cytoplasmic translocation and release of HMGB1 from these macrophages. Our results indicate that GTS-21 is effective in improving bacterial clearance and reducing acute lung injury by enhancing macrophage function via inhibiting the release of nuclear HMGB1. Therefore, the α7nAChR represents a possible pharmacological target to improve the clinical outcome of patients on ventilators by augmenting host defense against bacterial infections.

Compostos de Benzilideno/farmacologia , Hiperóxia/metabolismo , Macrófagos/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Pneumonia Associada à Ventilação Mecânica/metabolismo , Piridinas/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Proteína HMGB1/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Pneumonia Associada à Ventilação Mecânica/microbiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
Redox Biol ; 2: 314-22, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24563849


Prolonged exposure to hyperoxia results in acute lung injury (ALI), accompanied by a significant elevation in the levels of proinflammatory cytokines and leukocyte infiltration in the lungs. However, the mechanisms underlying hyperoxia-induced proinflammatory ALI remain to be elucidated. In this study, we investigated the role of the proinflammatory cytokine high mobility group box protein 1 (HMGB1) in hyperoxic inflammatory lung injury, using an adult mouse model. The exposure of C57BL/6 mice to ≥99% O2 (hyperoxia) significantly increased the accumulation of HMGB1 in the bronchoalveolar lavage fluids (BALF) prior to the onset of severe inflammatory lung injury. In the airways of hyperoxic mice, HMGB1 was hyperacetylated and existed in various redox forms. Intratracheal administration of recombinant HMGB1 (rHMGB1) caused a significant increase in leukocyte infiltration into the lungs compared to animal treated with a non-specific peptide. Neutralizing anti-HMGB1 antibodies, administrated before hyperoxia significantly attenuated pulmonary edema and inflammatory responses, as indicated by decreased total protein content, wet/dry weight ratio, and numbers of leukocytes in the airways. This protection was also observed when HMGB1 inhibitors were administered after the onset of the hyperoxic exposure. The aliphatic antioxidant, ethyl pyruvate (EP), inhibited HMGB1 secretion from hyperoxic macrophages and attenuated hyperoxic lung injury. Overall, our data suggest that HMGB1 plays a critical role in mediating hyperoxic ALI through the recruitment of leukocytes into the lungs. If these results can be translated to humans, they suggest that HMGB1 inhibitors provide treatment regimens for oxidative inflammatory lung injury in patients receiving hyperoxia through mechanical ventilation.

Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Anticorpos/administração & dosagem , Líquido da Lavagem Broncoalveolar/imunologia , Proteína HMGB1/metabolismo , Piruvatos/administração & dosagem , Acetilação , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/terapia , Animais , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Proteína HMGB1/antagonistas & inibidores , Injeções Espinhais , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo