RESUMO
Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death1-3. Oxidative stress is believed to be critical in this disease pathogenesis4-6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)7. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.
Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Proteínas/metabolismo , Animais , Feminino , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , OxirreduçãoRESUMO
BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.
Assuntos
Asma/metabolismo , Hipersensibilidade/metabolismo , Interleucina-1beta/metabolismo , Pulmão/metabolismo , Nariz/patologia , Mucosa Respiratória/metabolismo , Escarro/metabolismo , Animais , Antígenos de Dermatophagoides/imunologia , Células Cultivadas , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Glicólise , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Ácido Láctico/metabolismo , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Pyroglyphidae , RNA Interferente Pequeno/genética , Mucosa Respiratória/patologia , Transdução de SinaisRESUMO
Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFß-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp-/- mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFß-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.
RESUMO
Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKß), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKß and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKß. SiRNA-mediated knockdown of GSTP decreased IKKß-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKß-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.
Assuntos
Asma/genética , Glutationa S-Transferase pi/genética , Quinase I-kappa B/genética , Inflamação/genética , Pulmão/metabolismo , Animais , Asma/induzido quimicamente , Asma/patologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glutarredoxinas/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/patologia , Camundongos , NF-kappa B/genética , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/genética , Transdução de SinaisRESUMO
Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.
Assuntos
Glutarredoxinas/metabolismo , Hipersensibilidade/patologia , Hipersensibilidade/parasitologia , Pulmão/patologia , Pulmão/parasitologia , Pyroglyphidae/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Glutationa/metabolismo , Hiperplasia , Hipersensibilidade/sangue , Hipersensibilidade/complicações , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Muco/metabolismo , Pneumonia/sangue , Pneumonia/complicações , Pneumonia/parasitologia , Pneumonia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hipersensibilidade Respiratória/sangue , Hipersensibilidade Respiratória/parasitologia , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/fisiopatologia , Mecânica Respiratória , Células Th2/imunologiaRESUMO
Selective glucocorticoid receptor agonists (SEGRAs) are a new class of compounds under clinical evaluation for treatment of ocular inflammation. Widely prescribed therapeutics, such as glucocorticoids, are effective at reducing ocular inflammation, but their long term use predisposes to undesirable side effects. The purpose of this study was to investigate a novel SEGRA, mapracorat (BOL-303242-X), and the differences in mapracorat's mechanism of action compared with traditional steroids (i.e. dexamethasone). Keratocytes from three different humans were cultured and treated with mapracorat or dexamethasone, with and without a strong provoking agent, interleukin (IL)-1ß. The effects of mapracorat compared to dexamethasone were determined by measuring protein levels (Western blotting) and DNA binding (ELISA) for two nuclear factor-kappaB (NF-κB) family members, RelA and RelB. Cytokine production (i.e. IL-6, IL-8, prostaglandin E2 (PGE2)) was characterized by immunoassay. Our findings reveal mechanistic differences between mapracorat and traditional steroid therapies. Mapracorat showed partial attenuation of the classical NF-κB pathway, consistent with traditional steroids. However, mapracorat uniquely potentiated a novel anti-inflammatory mechanism through rapid upregulation of RelB, an anti-inflammatory member of the NF-κB alternative pathway. Mapracorat potently inhibits ocular inflammation in vitro and is a promising new treatment for ocular inflammatory disease. Mapracorat acts, in part, by a novel mechanism via upregulation of RelB in the NF-κB alternative pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Benzofuranos/farmacologia , Ceratócitos da Córnea/efeitos dos fármacos , NF-kappa B/metabolismo , Pentanóis/farmacologia , Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Fator de Transcrição RelB/metabolismo , Western Blotting , Células Cultivadas , Ceratócitos da Córnea/metabolismo , Citocinas/metabolismo , Dexametasona/farmacologia , Ensaio de Imunoadsorção Enzimática , Glucocorticoides/farmacologia , Humanos , Fator de Transcrição RelA/metabolismo , Regulação para CimaRESUMO
Lung inflammation can result from exposure to multiple types of inflammatory stimuli. Fibroblasts, key structural cells in the lung that are integral to inflammation and wound healing, produce inflammatory mediators after exposure to stimuli such as IL-1ß. We and others have shown that the NF-κB member RelB has anti-inflammatory properties in mice. Little is known, however, about the anti-inflammatory role of RelB in human cells and how it functions. MicroRNAs (miRNAs), a novel class of small, noncoding RNAs, can mediate inflammatory signaling pathways, including NF-κB, through regulation of target gene expression. Our goal was to analyze the anti-inflammatory properties of RelB in human lung fibroblasts. We hypothesized that RelB regulates inflammatory mediator production in lung fibroblasts in part through a mechanism involving miRNAs. To accomplish this, we transfected human lung fibroblasts with a plasmid encoding RelB and small interfering (si)RNA targeting RelB mRNA to overexpress and downregulate RelB, respectively. IL-1ß, a powerful proinflammatory stimulus, was used to induce NF-κB-driven inflammatory responses. RelB overexpression reduced IL-1ß-induced cyclooxygenase (Cox)-2, PGE2, and cytokine production, and RelB downregulation increased Cox-2 expression and PGE2 production. Furthermore, RelB overexpression increased IL-1ß-induced expression of miRNA-146a, an NF-κB-dependent miRNA with anti-inflammatory properties, whereas RelB downregulation reduced miRNA-146a. miR-146a overexpression ablated the effects of RelB downregulation on IL-1ß-induced Cox-2, PGE2, and IL-6 production, suggesting that RelB mediates IL-1ß-induced inflammatory mediator production in lung fibroblasts through miRNA-146a. RelB and miRNA-146a may therefore be new therapeutic targets in the treatment of lung inflammation caused by various agents and conditions.
Assuntos
Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , MicroRNAs/fisiologia , Fator de Transcrição RelB/metabolismo , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Regulação para Baixo , Fibroblastos/metabolismo , Humanos , Pulmão/efeitos dos fármacos , MicroRNAs/biossíntese , Fator de Transcrição RelB/biossínteseRESUMO
Acute lung inflammation can be caused by a variety of respirable agents, including cigarette smoke. Long-term cigarette smoke exposure can cause chronic obstructive pulmonary disease (COPD), a serious illness that affects >10 million Americans. Cigarette smoke is a known inducer of inflammation and is responsible for approximately 90% of all COPD cases. RelB, a member of the NF-κB family, attenuates cigarette smoke-induced inflammatory mediator production in mouse lung fibroblasts in vitro. We hypothesized that overexpression of RelB in the airways of mice would dampen acute smoke-induced pulmonary inflammation. Mice received a recombinant adenovirus encoding RelB by intranasal aspiration to induce transient RelB overexpression in the lungs and were subsequently exposed to mainstream cigarette smoke. Markers of inflammation were analyzed after smoke exposure. Neutrophil infiltration, normally increased by smoke exposure, was significantly and potently decreased after RelB overexpression. Cigarette smoke-induced proinflammatory cytokine and chemokine production, cyclooxygenase-2 expression, and prostaglandin E(2) production were also significantly decreased in the context of RelB overexpression. The expression of intercellular adhesion molecule 1, an NF-κB-dependent protein, was decreased, indicating a potential mechanism through which RelB can regulate inflammatory cell migration. Therefore, increased expression and/or activation of RelB could be a novel therapeutic strategy against acute lung inflammation caused by respirable agents and possibly against chronic injury, such as COPD.
Assuntos
Terapia Genética , NF-kappa B/metabolismo , Pneumonia/prevenção & controle , Fumar/efeitos adversos , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Adenoviridae/genética , Administração Intranasal , Animais , Western Blotting , Lavagem Broncoalveolar , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas Imunoenzimáticas , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Pneumonia/induzido quimicamente , Pneumonia/genéticaRESUMO
PURPOSE: Various ocular and orbital tissues differ in their manifestations of inflammation, although the reasons for this are unclear. Such differences may be due to behaviors exhibited by resident cell types, including fibroblasts. Fibroblasts mediate immune function and produce inflammatory mediators. Chronic stimulation of ocular fibroblasts can lead to prolonged inflammation and, in turn, to impaired vision and blindness. Interleukin (IL)-1ß, which is produced by various cells during inflammation, is a potent activator of fibroblasts and inducer of the expression of inflammatory mediators. The hypothesis for this study was that that human fibroblasts derived from distinct ocular tissues differ in their responses to IL-1ß and that variations in the IL-1 signaling pathway account for these differences. METHODS: Human fibroblasts were isolated from the lacrimal gland, cornea, and Tenon's capsule and treated with IL-1ß in vitro. Cytokine and prostaglandin (PG)E(2) production were measured by ELISA and EIA. Cyclooxygenase (Cox)-2 expression was detected by Western blot. Components of the IL-1 signaling pathway were detected by flow cytometry, ELISA, Western blot, and immunofluorescence. RESULTS: Cytokine and PGE(2) production and Cox-2 expression were greatest in corneal fibroblasts. VEGF production was greatest in Tenon's capsule fibroblasts. Variations in IL-1 receptor and receptor antagonist expression, IκBα degradation and p65 nuclear translocation, however, did not account for these differences, but overexpression of the NF-κB member RelB dampened Cox-2 expression in all three fibroblast types. CONCLUSIONS: The results highlight the inherent differences between ocular fibroblast strains and provide crucial insight into novel, tissue-specific treatments for ocular inflammation and disease, such as RelB overexpression.
Assuntos
Córnea/patologia , Citocinas/biossíntese , Endoftalmite/patologia , Fibroblastos/metabolismo , Aparelho Lacrimal/patologia , Cápsula de Tenon/patologia , Cicatrização , Western Blotting , Células Cultivadas , Córnea/metabolismo , Citocinas/imunologia , Endoftalmite/imunologia , Endoftalmite/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/imunologia , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Imunidade Celular , Aparelho Lacrimal/metabolismo , Microscopia de Fluorescência , Cápsula de Tenon/metabolismoRESUMO
OBJECTIVE: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules. METHODS AND RESULTS: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels. CONCLUSIONS: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , NF-kappa B/metabolismo , Adulto , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Humanos , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patologia , Masculino , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/metabolismo , Nitrilas/farmacologia , Sulfonas/farmacologia , Fator de Transcrição RelA/metabolismoRESUMO
N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (alpha-SNAP) are thought to be soluble factors that transiently bind and disassemble SNAP receptor complex during exocytosis in neuronal and endocrine cells. Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. However, the secretion of lung surfactant is a relatively slow process, and involvement of SNAP receptor and its cofactors (NSF and alpha-SNAP) in this process has not been demonstrated. In this study, we investigated a possible role of alpha-SNAP in surfactant secretion. alpha-SNAP was predominantly associated with the membranes in alveolar type II cells as determined by Western blot and immunocytochemical analysis using confocal microscope. Membrane-associated alpha-SNAP was not released from the membrane fraction when the cells were lyzed in the presence of Ca2+ or Mg2+ATP. The alkaline condition (0.1 M Na2CO3, pH 12), known to extract peripheral membrane proteins also failed to release it from the membrane. Phase separation using Triton X-114 showed that alpha-SNAP partitioned into both aqueous and detergent phases. NSF had membrane-bound characteristics similar to alpha-SNAP in type II cells. Permeabilization of type II cells with beta-escin resulted in a partial loss of alpha-SNAP from the cells, but cellular NSF was relatively unchanged. Addition of exogenous alpha-SNAP to the permeabilized cells increased surfactant secretion in a dose-dependent manner, whereas exogenous NSF has much less effects. An alpha-SNAP antisense oligonucleotide decreased its protein level and inhibited surfactant secretion. Our results suggest a role of alpha-SNAP in lung surfactant secretion.