RESUMO
Klebsiella pneumoniae is a common cause of Gram-negative pneumonia. The spread of antibiotic-resistant and hypervirulent strains has made treatment more challenging. This study sought to determine the immunomodulatory, antibacterial, and therapeutic potential of purified murine stem cell Ag-1+ (Sca-1+) lung mesenchymal stem cells (LMSCs) using in vitro cell culture and an in vivo mouse model of pneumonia caused by K pneumoniae. Sca-1+ LMSCs are plastic adherent, possess colony-forming capacity, express mesenchymal stem cell markers, differentiate into osteogenic and adipogenic lineages in vitro, and exhibit a high proliferative capacity. Further, these Sca-1+ LMSCs are morphologically similar to fibroblasts but differ ultrastructurally. Moreover, Sca-1+ LMSCs have the capacity to inhibit LPS-induced secretion of inflammatory cytokines by bone marrow-derived macrophages and neutrophils in vitro. Sca-1+ LMSCs inhibit the growth of K pneumoniae more potently than do neutrophils. Sca-1+ LMSCs also possess the intrinsic ability to phagocytize and kill K. pneumoniae intracellularly. Whereas the induction of autophagy promotes bacterial replication, inhibition of autophagy enhances the intracellular clearance of K. pneumoniae in Sca-1+ LMSCs during the early time of infection. Adoptive transfer of Sca-1+ LMSCs in K. pneumoniae-infected mice improved survival, reduced inflammatory cells in bronchoalveolar lavage fluid, reduced inflammatory cytokine levels and pathological lesions in the lung, and enhanced bacterial clearance in the lung and in extrapulmonary organs. To our knowledge, these results together illustrate for the first time the protective role of LMSCs in bacterial pneumonia.
Assuntos
Infecções por Klebsiella , Células-Tronco Mesenquimais , Pneumonia Bacteriana , Animais , Klebsiella , Klebsiella pneumoniae , Pulmão , CamundongosRESUMO
While all forms of tobacco exposure have negative health effects, the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here, we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM, GCLC, GPX2, NQO1 and HO-1. Vaporization of, and/or the presence of nicotine in, eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE, and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A, 140, 126, 374A, 26A-2, 147B, 941 and 589) for further study. We validated increased expression of multiple miRNAs, including miR126, following eCig exposure. We also found significant reduction in the expression of two miR126 target genes, MYC and MRGPRX3, following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells, some of which are epigenetically programmed at the level of miRNA regulation.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , MicroRNAs/análise , Mucosa Respiratória/efeitos dos fármacos , Fumar , Células Cultivadas , Humanos , MicroRNAs/genética , Estresse Oxidativo , Estresse FisiológicoRESUMO
Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15. Conversely, treatment of mice with a pharmacological PPARγ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro, CS increased lung epithelial cell chemokine expression in a PPARγ activation-dependent fashion. The ability of PPARγ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPARγ-mediated transrepression of NF-κB activity. Pharmacological or genetic activation of PPARγ activity abrogated CS-dependent induction of NF-κB activity. Regulation of NF-κB activity involved direct PPARγ-NF-κB interaction and PPARγ-mediated effects on IKK activation, IκBα degradation, and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-κB-dependent, CS-induced chemokine-mediated regulation of inflammatory cell accumulation.
Assuntos
Quimiocinas/metabolismo , PPAR gama/fisiologia , Enfisema Pulmonar/metabolismo , Fumar/efeitos adversos , Animais , Linhagem Celular , Suscetibilidade a Doenças , Feminino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/imunologia , Transdução de Sinais , Fumar/imunologia , Fumar/metabolismo , Ativação TranscricionalRESUMO
Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2(-/-) mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB-driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2(-/-) DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2(-/-) mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Lisofosfolipídeos/imunologia , NF-kappa B/imunologia , Receptores de Ácidos Lisofosfatídicos/imunologia , Administração por Inalação , Transferência Adotiva , Alérgenos/imunologia , Animais , Asma/patologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Deleção de Genes , Células HEK293 , Humanos , Inflamação/imunologia , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Receptores de Ácidos Lisofosfatídicos/deficiência , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais , Transcrição GênicaRESUMO
Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress-dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke-induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress-dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-kappaB (NF-kappaB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-kappaB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke-induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke-induced lung injury.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Enfisema Pulmonar/induzido quimicamente , Fumar/efeitos adversos , Fatores de Transcrição/fisiologia , Animais , Ativação Enzimática , Homeostase , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Enfisema Pulmonar/genética , Serina-Treonina Quinases TOR , Fatores de Transcrição/genéticaRESUMO
Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4(+) T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4(+) T-cell cytokines, especially IFN-gamma. The enhancing effects of hypoxia on IFN-gamma secretion were independent of mouse strain, and were also unaffected using CD4(+) T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1alpha. Using T cells from IFN-gamma receptor-deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-gamma expression were not due to effects on IFN-gamma consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45-related factor 2 attenuated the enhancing effect of hypoxia on IFN-gamma secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4(+) T cells.
Assuntos
Hipóxia , Interferon gama/metabolismo , Ativação Linfocitária , Linfócitos T/citologia , Animais , Complexo CD3/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxigênio/metabolismoRESUMO
Oxidative stress plays an important role in immune regulation and dendritic cell (DC) maturation. Recent studies indicate that allergens, including ragweed extract (RWE), possess prooxidant activities, but how RWE interacts with DCs is not well understood. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a key transcription factor that regulates constitutive and coordinated induction of a battery of antioxidant genes. We hypothesized that RWE would activate DCs and that this response would be augmented in the absence of Nrf2. We generated bone marrow-derived DCs (BM-DCs) and isolated lung DCs from Nrf2(+/+) and Nrf2(-/-) mice and studied the effects of RWE on DCs in vitro. Under resting conditions, Nrf2(-/-) BM-DCs exhibited constitutively greater levels of inflammatory cytokines and costimulatory molecules than Nrf2(+/+) BM-DCs. Exposure to RWE impaired endocytic activity, significantly induced oxidative stress, and enhanced the expression of CD80, CD86, and MHCII in Nrf2(-/-) BM-DCs when compared with Nrf2(+/+) BM-DC, in association with reduced expression of Nrf2-regulated antioxidant genes. RWE significantly induced the secretion of inflammatory cytokines IL-6 and TNF-alpha in BM-DCs and lung DCs from Nrf2(-/-) mice than Nrf2(+/+) mice and significantly inhibited the secretion of IL-12 in Nrf2(+/+) BM-DCs and IL-18 in Nrf2(+/+) and Nrf2(-/-) BM-DCs. The stimulatory effects of RWE on DC activation were inhibited to varying degrees by the antioxidant N-acetyl cysteine. Our findings indicate that a defect in Nrf2-mediated signaling mechanisms alters the response of DCs to a common environmental allergen, which may contribute to the susceptibility to allergic diseases.
Assuntos
Ambrosia , Células Dendríticas/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/farmacologia , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Citometria de Fluxo , Pulmão/citologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Estresse OxidativoRESUMO
Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.
Assuntos
Elasticidade , Matriz Extracelular/fisiologia , Contração Muscular/fisiologia , Contração Muscular/efeitos da radiação , Miócitos de Músculo Liso/fisiologia , Traqueia/fisiologia , Animais , Masculino , Camundongos , Camundongos Mutantes , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Mecânico , Traqueia/citologiaRESUMO
Cigarette smoking is the major risk factor for developing chronic obstructive pulmonary disease, the fourth leading cause of deaths in the United States. Despite recent advances, the molecular mechanisms involved in the initiation and progression of this disease remain elusive. We used Affymetrix Gene Chip arrays to determine the temporal alterations in global gene expression during the progression of pulmonary emphysema in A/J mice. Chronic cigarette smoke (CS) exposure caused pulmonary emphysema in A/J mice, which was associated with pronounced bronchoalveolar inflammation, enhanced oxidative stress, and increased apoptosis of alveolar septal cells. Microarray analysis revealed the upregulation of 1,190, 715, 260, and 246 genes and the downregulation of 1,840, 730, 442, and 236 genes in the lungs of mice exposed to CS for 5 h, 8 days, and 1.5 and 6 mo, respectively. Most of the genes belong to the functional categories of phase I genes, Nrf2-regulated antioxidant and phase II genes, phase III detoxification genes, and others including immune/inflammatory response genes. Induction of the genes encoding multiple phase I enzymes was markedly higher in the emphysematous lungs, whereas reduced expression of various cytoprotective genes constituting ubiquitin-proteasome complex, cell survival pathways, solute carriers and transporters, transcription factors, and Nrf2-regulated antioxidant and phase II-responsive genes was noted. Our data indicate that the progression of CS-induced emphysema is associated with a steady decline in the expression of various genes involved in multiple pathways in the lungs of A/J mice. Many of the genes discovered in this study could rationally play an important role in the susceptibility to CS-induced emphysema.
Assuntos
Apoptose/fisiologia , Enfisema , Perfilação da Expressão Gênica , Estresse Oxidativo/fisiologia , Fumar/efeitos adversos , Animais , Doença Crônica , Enfisema/genética , Enfisema/metabolismo , Enfisema/patologia , Pulmão/patologia , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos A , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Oxidantes/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2(+/+) and Nrf2(-/-) mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2(+/+) mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2(-/-) mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD.
Assuntos
Cardiopatias/prevenção & controle , Fator 2 Relacionado a NF-E2/fisiologia , Ácido Oleanólico/análogos & derivados , Enfisema Pulmonar/prevenção & controle , Fumaça/efeitos adversos , Animais , Apoptose , Sistemas de Liberação de Medicamentos , Cardiopatias/tratamento farmacológico , Hipertensão Pulmonar , Imidazóis , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico/antagonistas & inibidores , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/etiologia , Enfisema Pulmonar/tratamento farmacológicoRESUMO
Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2(-/-) DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2(-/-) DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2(+/+) DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2(+/+) DCs, coincubation of Nrf2(-/-) DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/imunologia , Material Particulado/toxicidade , Células Th2/imunologia , Animais , Baltimore , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
In infants, smoke exposure is associated with more respiratory illnesses and decreased lung function. We hypothesized that perinatal lung is particularly susceptible to the damaging effects of cigarette smoke (CS) and that exposure to CS during this period may alter expression of immune response genes and adversely affect lung growth. To test this, we exposed neonatal mice to 14 days of CS. Immediately after exposure to CS, pulmonary gene expression profiling was performed on 2-week-old CS-exposed lung and age-matched control lung. Nitrotyrosine, TUNEL, MAC3, and phospho-SMAD-2 (p-SMAD2) staining was also performed. At 8 weeks of age, lung volume measurements were determined and mean linear intercept measurements were calculated. Pulmonary gene expression profiling revealed that CS exposure significantly inhibited type 1 and type 2 interferon pathway genes in neonatal lung, compared with age-matched control lung. Neonatal CS-exposed lung also had a significant increase in n-tyrosine, TUNEL, and p-SMAD2 staining when compared with adult CS-exposed lung and age-matched control lung. Lung volumes at 8 weeks of age were modestly but significantly decreased in mice exposed to CS in the neonatal period compared with age-matched controls, consistent with impaired lung growth. The results of this study indicate that exposure to CS during the neonatal period inhibits expression of genes involved in innate immunity and mildly impairs postnatal lung growth. These findings may in part explain the increased incidence of respiratory symptoms in infants and children exposed to CS.
Assuntos
Homeostase/efeitos dos fármacos , Exposição por Inalação , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Nicotiana , Fumaça/efeitos adversos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Contagem de Células , Morte Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Imunidade/efeitos dos fármacos , Imunidade/genética , Interferons/farmacologia , Pulmão/citologia , Pulmão/enzimologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Disruption of NF-E2-related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke-induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2-/- mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2'deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5' promoter-flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter-reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress-inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.
Assuntos
Glutationa Peroxidase/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Nicotiana , Fumaça/efeitos adversos , Animais , Sequência de Bases , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Pulmão/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/genética , Proteínas Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Elementos de Resposta/genética , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture-induced septic shock. LPS as well as TNF-alpha stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-kappaB and interferon regulatory factor 3 in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2's regulation of cellular glutathione and other antioxidants is critical for optimal NF-kappaB activation in response to LPS and TNF-alpha. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.
Assuntos
Imunidade Inata/genética , Fator 2 Relacionado a NF-E2/fisiologia , Sepse/imunologia , Animais , Antioxidantes/farmacologia , Ceco , Moléculas de Adesão Celular/metabolismo , Quimiocinas/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Pneumonia/imunologia , Pneumonia/metabolismo , Receptores de Superfície Celular/metabolismo , Sepse/metabolismo , Transdução de Sinais/imunologia , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.
Assuntos
Asma/etiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Transativadores/deficiência , Transativadores/genética , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Asma/metabolismo , Asma/patologia , Sequência de Bases , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CCL11 , Quimiocinas CC/metabolismo , DNA Complementar/genética , Regulação da Expressão Gênica , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2 , NF-kappa B/metabolismo , Ovalbumina/imunologia , Oxirredução , Estresse Oxidativo , Células Th2/imunologiaRESUMO
Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke-induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells--predominantly endothelial and type II epithelial cells--as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke-induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.
Assuntos
Proteínas de Ligação a DNA/genética , Desoxiguanosina/análogos & derivados , Enfisema/genética , Predisposição Genética para Doença , Pulmão/efeitos dos fármacos , Fumar , Transativadores/genética , 8-Hidroxi-2'-Desoxiguanosina , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Apoptose , Lavagem Broncoalveolar , Desoxiguanosina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia de Fluorescência , Fator 2 Relacionado a NF-E2 , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fenótipo , Alvéolos Pulmonares/patologia , Fatores de Tempo , Transcrição Gênica , Regulação para CimaRESUMO
Acrolein, a highly electrophilic alpha,beta-unsaturated aldehyde, is by far the most reactive amongst the aldehydes present in smoke. The relative contribution of acrolein to complex mixture toxicity of smoke at the molecular level remains unknown. The current study examines the ability of acrolein to modulate the effect of benzo[a]pyrene (B[a]P), a major carcinogen found in smoke, on p53. Exposure of human lung adenocarcinoma A549 cells to 1 mM B[a]P for 48 h strongly activated the expression of p53 as seen by western blotting, and its DNA binding as shown by an electrophoretic mobility shift assay. Treatment of A549 cells with a non-lethal dose of acrolein alone (50 fmol/cell for 0.5 h) depleted 80% of total cellular glutathione but had no effect on basal p53 protein levels. When B[a]P-treated cells (48 h) were exposed to acrolein for 0.5 h there was also no effect on B[a]P-induced p53 protein levels. However, acrolein treatments profoundly inhibited the DNA binding of p53 under both basal and B[a]P-induced conditions. Depleting glutathione with buthionine sulfoximine in B[a]P-treated cells to levels similar to those obtained with acrolein decreased p53 DNA binding substantially less than with acrolein. Using a p53 dual luciferase reporter assay, acrolein caused an 83% decrease in the p53 activity induced by B[a]P (1 mM for 24 h post-transfection). The p53 protein that was immunoprecipitated after acrolein treatment was reactive with an anti-acrolein antibody indicating covalent modification. Results from this study suggest that acrolein can inhibit p53 DNA binding and activity by direct covalent modification as well as alteration of intracellular redox status. As both acrolein and B[a]P are found in cigarette smoke, this type of interaction may play an important role in the initiation of lung cancer by altering the tumor suppressor activity of p53.