RESUMO
IL-33 and its receptor ST2 play important roles in airway inflammation and contribute to asthma onset and exacerbation. The IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. MyD88 forms a complex with IL-R-associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88-IRAK4-IRAK2). The myddosome subsequently activates downstream NF-κB and MAPKs p38 and JNK. We established an asthma-like mouse model by intratracheal administration of IL-33. The IL-33 model has a very similar phenotype compared with the OVA-induced mouse asthma model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knockout mice, which were protected from the IL-33-induced asthma. We synthesized small molecule mimetics of the α-helical domain of IRAK2 with drug-like characteristics based on the recent advances in the designing of α-helix compounds. The mimetics can competitively interfere in the protein-protein interaction between IRAK2 and IRAK4, leading to disruption of Myddosome formation. A series of small molecules were screened using an NF-κB promoter assay in vitro. The lead compound, 7004, was further studied in the IL-33-induced and OVA-induced asthma mouse models in vivo. Compound 7004 can inhibit the IL-33-induced NF-κB activity, disrupt Myddosome formation, and attenuate the proinflammatory effects in asthma-like models. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases.
Assuntos
Asma/tratamento farmacológico , Inflamação/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Asma/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fenótipo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Chronic obstructive pulmonary disease (COPD) is a worldwide threat. Cigarette smoke (CS) exposure causes cardiopulmonary disease and COPD and increases the risk for pulmonary tumors. In addition to poor lung function, patients with COPD are susceptible to bouts of dangerous inflammation triggered by pollutants or infection. These severe inflammatory episodes can lead to additional exacerbations, hospitalization, further deterioration of lung function, and reduced survival. Suitable models of the inflammatory conditions associated with CS, which potentiate the downward spiral in patients with COPD, are lacking, and the underlying mechanisms that trigger exacerbations are not well understood. Although initial CS exposure activates a protective role for vascular endothelial growth factor (VEGF) functions in barrier integrity, chronic exposure depletes the pulmonary VEGF guard function in severe COPD. Thus, we hypothesized that mice with compromised VEGF production and challenged with CS would trigger human-like severe inflammatory progression of COPD. In this model, we discovered that CS exposure promotes an amplified IL-33 cytokine response and severe disease progression. Our VEGF-knockout model combined with CS recapitulates severe COPD with an influx of IL-33-expressing macrophages and neutrophils. Normally, IL-33 is quickly inactivated by a post-translational disulfide bond formation. Our results reveal that BAL fluid from the CS-exposed, VEGF-deficient cohort promotes a significantly prolonged lifetime of active proinflammatory IL-33. Taken together, our data demonstrate that with the loss of a VEGF-mediated protective barrier, the CS response switches from a localized danger to an uncontrolled long-term and long-range, amplified, IL-33-mediated inflammatory response that ultimately destroys lung function.
Assuntos
Inflamação/metabolismo , Interleucina-33/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumar/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Citocinas/líquido cefalorraquidiano , Citocinas/metabolismo , Humanos , Inflamação/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , Nicotiana/efeitos adversosRESUMO
NEW FINDINGS: What is the central question of this study? Non-invasive, quantitative methods to assess right cardiac function in mice with pulmonary hypertension have not been demonstrated. What is the main finding and its importance? This study shows the potential of magnetic resonance imaging to estimate right ventricular ejection fraction and measure spatial, dynamic changes in cardiac structure in the Sugen 5416/hypoxia mouse model of pulmonary hypertension. Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery pressures and right heart failure. Mouse models of PAH are instrumental in understanding the disease pathophysiology. However, few methods are available to evaluate right cardiac function in small animals. In this study, magnetic resonance imaging was used to measure in vivo cardiac dimensions in the Sugen 5416/hypoxia mouse model. Pulmonary hypertension (PH) was induced in C57BL/6 mice by 3 weeks of exposure to 10% oxygen and vascular endothelial growth factor receptor inhibition (20 mg kg-1 SU5416). Control mice were housed in room air and received vehicle (DMSO). Right ventricular pressures were recorded with a pressure-conductance transducer. Short-axis contiguous 1-mm-thick slices were acquired through the heart and great vessels using a fast low-angle shot (FLASH)-cine sequence. Thirteen images were collected throughout each cardiac cycle. Right ventricular systolic pressure was elevated in PH mice (23.6 ± 6 versus 41.0 ± 11 mmHg, control versus PH, respectively; P < 0.001, n = 5-11). Right ventricular wall thickness was greater in PH than in control mice at end diastole (0.30 ± 0.05 versus 0.48 ± 0.06 mm, control versus PH, respectively; P < 0.01, n = 6), but measurements were not different at end systole (control versus PH, 0.59 ± 0.11 versus 0.70 ± 0.11 mm, respectively). Right ventricular ejection fraction was decreased in PH mice (72 ± 3 versus 58 ± 5%, control versus PH, respectively; P < 0.04, n = 6). These data demonstrate that magnetic resonance imaging is a precise method to monitor right ventricular remodelling and cardiac output longitudinally in mouse models of PH.
Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Animais , Pressão Sanguínea/fisiologia , Débito Cardíaco/fisiologia , Diástole/fisiologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Artéria Pulmonar/metabolismo , Volume Sistólico/fisiologia , Sístole/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Direita/fisiologia , Remodelação Ventricular/fisiologiaRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that leads to right ventricular failure. Skeletal muscle maladaptations limit physical activity and may contribute to disease progression. The role of alarmin/inflammatory signaling in PAH respiratory muscle dysfunction is unknown. We hypothesized that diaphragm mitochondrial and contractile functions are impaired in SU5416/hypoxia-induced pulmonary hypertension due to increased systemic IL-33 signaling. We induced pulmonary hypertension in adult C57Bl/6 J (WT) and ST2 (IL1RL1) gene ablated mice by SU5416/hypoxia (SuHx). We measured diaphragm fiber mitochondrial respiration, inflammatory markers, and contractile function ex vivo. SuHx reduced coupled and uncoupled permeabilized myofiber respiration by â¼40 %. During coupled respiration with complex I substrates, ST2-/- attenuated SuHx inhibition of mitochondrial respiration (genotype × treatment interaction F[1,67] = 3.3, p = 0.07, η2 = 0.04). Flux control ratio and coupling efficiency were not affected by SuHx or genotype. A higher substrate control ratio for succinate was observed in SuHx fibers and attenuated in ST2-/- fibers (F[1,67] = 5.3, p < 0.05, η2 = 0.07). Diaphragm TNFα, but not IL-33 or NFkB, was increased in SuHx vs. DMSO in both genotypes (F[1,43] = 4.7, p < 0.05, η2 = 0.1). Diaphragm force-frequency relationships were right-shifted in SuHx vs. WT (F[3,440] = 8.4, p < 0.05, η2 = 0.0025). There was no effect of ST2-/- on the force-frequency relationship. Force decay during a fatigue protocol at 100 Hz, but not at 40 Hz, was attenuated by SuHx vs. DMSO in both genotypes (F[1,41] = 5.6, p < 0.05, η2 = 0.11). SuHx mice exhibit a modest compensation in diaphragm contractility and mitochondrial dysfunction during coupled respiration; the latter partially regulated through ST2 signaling.
Assuntos
Diafragma/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Proteína 1 Semelhante a Receptor de Interleucina-1/fisiologia , Mitocôndrias/fisiologia , Doenças Mitocondriais/fisiopatologia , Contração Muscular/fisiologia , Hipertensão Arterial Pulmonar/fisiopatologia , Animais , Modelos Animais de Doenças , Hipóxia/induzido quimicamente , Indóis/farmacologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Mitocondriais/genética , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologiaRESUMO
Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL-33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL-33 receptor gene deleted (ST2-/- ) and MYD88 gene deleted (MYD88-/- ) mice by exposure to 10% O2 and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL-33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p < 0.0001) and this SUHX response was attenuated in ST2-/- mice (29% male p = 0.003; 30% female p = 0.001) and absent in MYD88-/- mice. Wall thickness was increased in SUHX C57Bl/6J mice (p = 0.005), but not in ST2-/- or MYD88-/- mice. Proliferating cells were detected in C57Bl/6J mice by flow cytometry (CD31+ /BrDU+ ; p = 0.02) and immunofluorescence methods (Ki-67+). IL-33 was increased by SUHX (p = 0.03) but a genotype effect was not observed (p = 0.76). We observed that in hPAECs, IL-33 expression is regulated by both IL-33 and DLL4. These data suggest IL-33/ST2 signaling is essential for the endothelial cell proliferative response in PH.
Assuntos
Hipertensão Pulmonar/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Feminino , Deleção de Genes , Hipertensão Pulmonar/etiologia , Indóis/toxicidade , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Pirróis/toxicidadeRESUMO
Sodium nitroprusside is used to treat hypertensive emergencies and acute heart failure. It acts by releasing nitric oxide (NO), a highly potent vasodilator, but unfortunately, for each NO molecule released, five cyanide ions are released. Thus, nitroprusside therapy is limited by cyanide toxicity. Therefore, a cyanide scavenger could be beneficial when administering nitroprusside. Hydroxocobalamin, which has a relatively high binding affinity for cyanide, has been shown to reduce cyanide levels in nitroprusside-treated patients. Cobinamide, the penultimate precursor in hydroxocobalamin biosynthesis, has a much greater affinity for cyanide than cobalamin, and binds two cyanide ions. We now show that cobinamide is highly effective in neutralizing cyanide ions released by nitroprusside in cultured mammalian cells, Drosophila melanogaster, and mice. Cobinamide also binds NO, but at molar concentrations 2.5-5 times that of nitroprusside, it did not decrease NO concentrations or the physiological effectiveness of nitroprusside. We conclude that cobinamide could be a valuable adjunct to nitroprusside therapy.
Assuntos
Anti-Hipertensivos/farmacocinética , Cobamidas/farmacologia , Cianetos/metabolismo , Óxido Nítrico/metabolismo , Nitroprussiato/farmacocinética , Trifosfato de Adenosina/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Células Cultivadas , Drosophila melanogaster , Frequência Cardíaca/efeitos dos fármacos , Masculino , Túbulos de Malpighi/efeitos dos fármacos , Túbulos de Malpighi/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Nitroprussiato/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Artéria Pulmonar/citologia , Ratos , Tiocianatos/sangue , Tiocianatos/urina , Vitamina B 12RESUMO
A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi-stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.
Assuntos
Cobamidas/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Compostos Nitrosos/farmacologia , Vasodilatadores/farmacologia , Angina Pectoris/tratamento farmacológico , Angina Pectoris/metabolismo , Animais , Aorta/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Cobamidas/síntese química , Cobamidas/química , Circulação Coronária/efeitos dos fármacos , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Túbulos de Malpighi/metabolismo , Camundongos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Doadores de Óxido Nítrico/síntese química , Doadores de Óxido Nítrico/química , Compostos Nitrosos/síntese química , Compostos Nitrosos/química , Técnicas de Cultura de Órgãos , Ratos , Vasodilatadores/síntese química , Vasodilatadores/químicaRESUMO
Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.
Assuntos
Intubação Intratraqueal/métodos , Intubação Intratraqueal/veterinária , Laringoscopia/métodos , Laringoscopia/veterinária , Otoscópios/veterinária , Animais , Intubação Intratraqueal/instrumentação , Laringoscopia/instrumentação , Camundongos , Modelos AnimaisRESUMO
CONTEXT: Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties. OBJECTIVE: We are developing two new cyanide antidotes--cobinamide, a vitamin B(12) analog, and sulfanegen, a 3-mercaptopyruvate prodrug. Both drugs can be given by intramuscular administration, and therefore could be used to treat a large number of people quickly. We now asked if the two drugs would have an augmented effect when combined. MATERIALS AND METHODS: We used a non-lethal and two different lethal models of cyanide poisoning in mice. The non-lethal model assesses neurologic recovery by quantitatively evaluating the innate righting reflex time of a mouse. The two lethal models are a cyanide injection and a cyanide inhalation model. RESULTS: We found that the two drugs are at least additive when used together in both the non-lethal and lethal models: at doses where all animals died with either drug alone, the combination yielded 80 and 40% survival in the injection and inhalation models, respectively. Similarly, drug doses that yielded 40% survival with either drug alone, yielded 80 and 100% survival in the injection and inhalation models, respectively. As part of the inhalation model, we developed a new paradigm in which animals are exposed to cyanide gas, injected intramuscularly with an antidote, and then re-exposed to cyanide gas. This simulates cyanide exposure of a large number of people in a closed space, because people would remain exposed to cyanide, even after receiving an antidote. CONCLUSION: The combination of cobinamide and sulfanegen shows great promise as a new approach to treating cyanide poisoning.
Assuntos
Antídotos/administração & dosagem , Cobamidas/administração & dosagem , Cianetos/intoxicação , Cisteína/análogos & derivados , Pró-Fármacos/administração & dosagem , Animais , Cisteína/administração & dosagem , Modelos Animais de Doenças , Quimioterapia Combinada , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
CONTEXT: Cyanide is a rapidly acting cellular poison, primarily targeting cytochrome c oxidase, and is a common occupational and residential toxin, mostly via smoke inhalation. Cyanide is also a potential weapon of mass destruction, with recent credible threats of attacks focusing the need for better treatments, as current cyanide antidotes are limited and impractical for rapid deployment in mass casualty settings. OBJECTIVE: We have used mouse models of cyanide poisoning to compare the efficacy of cobinamide (Cbi), the precursor to cobalamin (vitamin B(12)), to currently approved cyanide antidotes. Cbi has extremely high affinity for cyanide and substantial solubility in water. MATERIALS AND METHODS: We studied Cbi in both an inhaled and intraperitoneal model of cyanide poisoning in mice. RESULTS: We found Cbi more effective than hydroxocobalamin, sodium thiosulfate, sodium nitrite, and the combination of sodium thiosulfate-sodium nitrite in treating cyanide poisoning. Compared to hydroxocobalamin, Cbi was 3 and 11 times more potent in the intraperitoneal and inhalation models, respectively. Cobinamide sulfite (Cbi-SO(3)) was rapidly absorbed after intramuscular injection, and mice recovered from a lethal dose of cyanide even when given at a time when they had been apneic for over 2 min. In range-finding studies, Cbi-SO(3) at doses up to 2000 mg/kg exhibited no clinical toxicity. DISCUSSION AND CONCLUSION: These studies demonstrate that Cbi is a highly effective cyanide antidote in mouse models, and suggest it could be used in a mass casualty setting, because it can be given rapidly as an intramuscular injection when administered as Cbi-SO(3). Based on these animal data Cbi-SO(3) appears to be an antidote worthy of further testing as a therapy for mass casualties.
Assuntos
Antídotos/uso terapêutico , Cobamidas/uso terapêutico , Cianetos/intoxicação , Administração por Inalação , Animais , Cobamidas/administração & dosagem , Cianetos/farmacocinética , Relação Dose-Resposta a Droga , Hidroxocobalamina/uso terapêutico , Injeções Intramusculares , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos C57BLAssuntos
Asma/genética , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/genética , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Asma/enzimologia , Linhagem Celular , Humanos , Técnicas In Vitro , Monócitos/enzimologiaAssuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/genética , Fagócitos/enzimologia , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genes Reporter , Células HeLa , Humanos , Luciferases/genética , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Fatores de Transcrição/química , Fatores de Transcrição/genética , TransfecçãoRESUMO
Asthma remains a major health problem worldwide that has increased in developed countries. Much of the focus in asthma research in the past has been on adaptive, antigen-dependent immune responses. Recent work suggests that the innate, non-antigen-dependent immune system plays a critical role in asthma pathogenesis. Here we will highlight innate receptors and cells in the context of allergic responses. Reviewing animal models and human studies, we focus on interactions of innate and adaptive immunity.
Assuntos
Asma/imunologia , Inflamação/imunologia , Animais , Asma/fisiopatologia , Humanos , Imunidade Inata , Inflamação/fisiopatologia , Modelos AnimaisRESUMO
Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacterium's virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.
Assuntos
Cianetos/isolamento & purificação , Cianetos/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Drosophila melanogaster/classificação , Frequência Cardíaca/efeitos dos fármacos , Humanos , Nitratos/farmacologia , Nitritos/farmacologia , Pseudomonas aeruginosa/fisiologia , Tiocianatos/isolamento & purificação , Tiocianatos/metabolismo , Tiocianatos/farmacologiaRESUMO
We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. We conclude that LPS enhances FLAP gene expression via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteínas Ativadoras de 5-Lipoxigenase , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Genes Reporter/genética , Genes Reporter/fisiologia , Humanos , Leucotrienos/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genéticaRESUMO
We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Fator de Necrose Tumoral alfa/metabolismo , Regiões 5' não Traduzidas , Proteínas Ativadoras de 5-Lipoxigenase , Sítios de Ligação , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desoxirribonuclease I/metabolismo , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , Mutação , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.
Assuntos
Regulação para Baixo/imunologia , Proteínas de Drosophila , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Lipopolissacarídeos/farmacologia , Monócitos/enzimologia , Monócitos/imunologia , Anticorpos Bloqueadores/farmacologia , Calcimicina/farmacologia , Linhagem Celular , Relação Dose-Resposta Imunológica , Glutationa Transferase/biossíntese , Glutationa Transferase/metabolismo , Humanos , Soros Imunes/farmacologia , Ionóforos/farmacologia , Leucotrieno C4/antagonistas & inibidores , Leucotrieno C4/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Subunidade p50 de NF-kappa B , Regiões Promotoras Genéticas/imunologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/imunologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/biossíntese , Fatores de Tempo , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Transcrição RelA , Transfecção , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Compostos de Vanádio/farmacologiaRESUMO
While effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty-four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS-ODN or control oligodeoxynucleotide (C-ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS-ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS-ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C-ODN. In addition, splenocytes from ISS-ODN but not C-ODN treated mice displayed attenuated OVA-specific interleukin (IL)-4, IL-5, and IL-13 but increased interferon-gamma responses. Finally, ISS-ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2-biased immune parameters towards Th1 dominance. As ISS-ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS-ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.
Assuntos
Oligodesoxirribonucleotídeos/imunologia , Hipersensibilidade Respiratória/prevenção & controle , Rinite/prevenção & controle , Administração Intranasal , Alérgenos/imunologia , Animais , Células da Medula Óssea/imunologia , Citocinas/biossíntese , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Rinite/imunologia , Rinite/patologia , Células Th2/imunologia , Vacinas de DNA/imunologiaRESUMO
Significant glomerular vasoconstriction and production of reactive oxygen species has been known to occur with exposure to anti-glomerular basement membrane antibody (AGBM-Ab) in the rat model. Previously published studies have demonstrated that such effects can be reduced by therapy with phentolamine, an alpha-adrenergic antagonist. It was hypothesized that antioxidant pretreatment with water-soluble probucol would improve glomerular hemodynamics 60 to 90 min after the administration of AGBM-Ab. These relationships were examined with both in vivo renal micropuncture and in vitro studies in rats. Single-nephron GFR (SNGFR) decreased markedly in untreated rats after AGBM-Ab as a result of afferent and efferent arteriolar vasoconstriction with consequent reductions in nephron plasma flow (SNPF) and decreases in the glomerular ultrafiltration coefficient (LpA). Basal SNGFR was increased, and SNGFR was significantly higher after AGBM-Ab in probucol-treated versus untreated rats. This finding was due solely to higher values for SNPF and prevention of afferent arteriolar constriction. A reduction in LpA after AGBM-Ab was not prevented by probucol treatment. In vitro analyses of glomeruli revealed reduced myeloperoxidase activity in antioxidant-treated rats. Lipoxygenase activity and leukotriene products, however, were not changed by antioxidant therapy, yet vasoconstriction was prevented. H(2)O(2) generation before and after formyl-methionyl-leucyl-phenylalanine stimulation was significantly reduced before and after AGBM-Ab in glomeruli harvested from rats that were treated with the antioxidant. Antioxidant therapy in this model of AGBM-Ab injury did not prevent reductions in LpA, an index of glomerular membrane damage, but did prevent afferent arteriolar vasoconstriction. Reactive oxygen species generation was reduced by probucol. The specific mechanisms whereby antioxidant therapy ameliorates glomerular hemodynamic effects will be defined in additional studies and is likely to involve either enhanced vasodilator or diminished vasoconstrictor activity.