RESUMO
IL-33 and its receptor ST2 are contributing factors to airway inflammation and asthma exacerbation. The IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma. In this study, we address the role of endogenous IL-33 and its autoamplification of the IL-33/ST2 pathway in Ag-dependent and Ag-independent asthma-like models. Wild-type, IL-33 knockout, ST2 knockout mice were either intratracheally administrated with 500 ng of rIL-33 per day for four consecutive days or were sensitized and challenged with OVA over 21 d. In wild-type mice, IL-33 or OVA induced similar airway hyperresponsiveness and eosinophilic airway inflammation. IL-33 induced its own mRNA and ST2L mRNA expression in the lung. IL-33 autoamplified itself and ST2 protein expression in airway epithelial cells. OVA also induced IL-33 and ST2 protein expression. In IL-33 knockout mice, the IL-33- and OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation were both significantly attenuated, whereas IL-33-induced ST2L mRNA expression was preserved, although no autoamplification of IL-33/ST2 pathway was observed. In ST2 knockout mice, IL-33 and OVA induced airway hyperresponsiveness and eosinophilic airway inflammation were both completely diminished, and no IL-33/ST2 autoamplification was observed. These results suggest that endogenous IL-33 and its autoamplification of IL-33/ST2 pathway play an important role in the induction of asthma-like phenotype. Thus an intact IL-33/ST2 pathway is necessary for both Ag-dependent and Ag-independent asthma-like mouse models.
Assuntos
Asma/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Asma/sangue , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/administração & dosagem , Interleucina-33/genética , Camundongos , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Transdução de Sinais/genéticaRESUMO
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
The pathophysiology of chronic kidney disease (CKD) is driven by alterations in surviving nephrons to sustain renal function with ongoing nephron loss. Oxygen supply-demand mismatch, due to hemodynamic adaptations, with resultant hypoxia, plays an important role in the pathophysiology in early CKD. We sought to investigate the underlying mechanisms of this mismatch. We utilized the subtotal nephrectomy (STN) model of CKD to investigate the alterations in renal oxygenation linked to sodium (Na) transport and mitochondrial function in the surviving nephrons. Oxygen delivery was significantly reduced in STN kidneys because of lower renal blood flow. Fractional oxygen extraction was significantly higher in STN. Tubular Na reabsorption was significantly lower per mole of oxygen consumed in STN. We hypothesized that decreased mitochondrial bioenergetic capacity may account for this and uncovered significant mitochondrial dysfunction in the early STN kidney: higher oxidative metabolism without an attendant increase in ATP levels, elevated superoxide levels, and alterations in mitochondrial morphology. We further investigated the effect of activation of hypoxia-inducible factor-1α (HIF-1α), a master regulator of cellular hypoxia response. We observed significant improvement in renal blood flow, glomerular filtration rate, and tubular Na reabsorption per mole of oxygen consumed with HIF-1α activation. Importantly, HIF-1α activation significantly lowered mitochondrial oxygen consumption and superoxide production and increased mitochondrial volume density. In conclusion, we report significant impairment of renal oxygenation and mitochondrial function at the early stages of CKD and demonstrate the beneficial role of HIF-1α activation on renal function and metabolism.
Assuntos
Aminoácidos Dicarboxílicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Oxigênio/sangue , Insuficiência Renal Crônica/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Hipóxia Celular , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/metabolismo , Rim/ultraestrutura , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Reabsorção Renal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , Superóxidos/metabolismo , Fatores de TempoRESUMO
Renal hypoxia contributes to chronic kidney disease (CKD) progression, as validated in experimental and human CKD. In the early stages, increased oxygen consumption causes oxygen demand/supply mismatch, leading to hypoxia. Hence, early targeting of the determinants and regulators of oxygen consumption in CKD may alter the disease course before permanent damage ensues. Here, we focus on hypoxia inducible factor-1α (HIF-1α) and AMP-activated protein kinase (AMPK) and on the mechanisms by which they may facilitate cellular hypoxia adaptation. We found that HIF-1α activation in the subtotal nephrectomy (STN) model of CKD limits protein synthesis, inhibits apoptosis, and activates autophagy, presumably for improved cell survival. AMPK activation was diminished in the STN kidney and was remarkably restored by HIF-1α activation, demonstrating a novel role for HIF-1α in the regulation of AMPK activity. We also investigated the independent and combined effects of HIF-1α and AMPK on cell survival and death pathways by utilizing pharmacological and knockdown approaches in cell culture models. We found that the effect of HIF-1α activation on autophagy is independent of AMPK, but on apoptosis it is partially AMPK dependent. The effects of HIF-1α and AMPK activation on inhibiting protein synthesis via the mTOR pathway appear to be additive. These various effects were also observed under hypoxic conditions. In conclusion, HIF-1α and AMPK appear to be linked at a molecular level and may act as components of a concerted cellular response to hypoxic stress in the pathophysiology of CKD.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adaptação Fisiológica/fisiologia , Hipóxia Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Modelos Animais de Doenças , Masculino , Nefrectomia , Ratos , Ratos Wistar , Circulação Renal/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
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
Fatty acids have been implicated in having both anti- or pro-inflammatory actions, which may contribute to the progression and severity of atherosclerosis. Linoleic acid has been shown by others to decrease CD18 expression and leukocyte adhesion under static conditions. We investigated the effect of steric acid (18:0), oleic acid (18:1), and linoleic acid (18:2) on the cortical tension (a measure of cell membrane deformability) and adhesion characteristics of the monocytic cell line Mono Mac 6 (MM6) cells to TNF-alpha activated HUVEC under fluid flow. Linoleic acid concentrations up to 23 microM decreased cortical tension and increased adhesion frequencies. Increased adhesion was not due to altered cell morphology or adhesion kinetics and occurred despite decreases in receptor expression (CD18 and CD11a). At higher levels of linoleic acid (> or = 46 microM), cell dissociation constants significantly increased. Results show that decreasing cortical tension increased the probability that contact between MM6 cells and endothelium would produce an adhesive interaction, possibly due to increased deformation of the microvilli and the cell membrane cortex. However, more deformable cells rolled more erratically at low shear rates. The different behavior during initial contact and rolling suggest that adhesion is influenced by two force-dependent mechanisms, deformation of microvilli and a steric barrier. Incubation of MM6 with 23 microM steric or oleic acid did not significantly affect cortical tension. However, cells incubated with steric acid greatly increased their adherence to HUVEC and cells incubated with oleic acid showed no significant effect, indicating factors other than deformability may dominate.