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The Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative Gram-negative bacterium that causes acute gastroenteritis and food poisoning. S. Typhimurium can survive within macrophages that are able to initiate the innate immune response after recognizing bacteria via various pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs). In this study, we investigated the effects and molecular mechanisms by which agonists of endosomal TLRs-especially TLR3-contribute to controlling S. Typhimurium infection in murine macrophages. Treatment with polyinosinic:polycytidylic acid (poly(I:C))-an agonist of TLR3-significantly suppressed intracellular bacterial growth by promoting intracellular ROS production in S. Typhimurium-infected cells. Pretreatment with diphenyleneiodonium (DPI)-an NADPH oxidase inhibitor-reduced phosphorylated MEK1/2 levels and restored intracellular bacterial growth in poly(I:C)-treated cells during S. Typhimurium infection. Nitric oxide (NO) production increased through the NF-κB-mediated signaling pathway in poly(I:C)-treated cells during S. Typhimurium infection. Intracellular microtubule-associated protein 1A/1B-light chain 3 (LC3) levels were increased in poly(I:C)-treated cells; however, they were decreased in cells pretreated with 3-methyladenine (3-MA)-a commonly used inhibitor of autophagy. These results suggest that poly(I:C) induces autophagy and enhances ROS production via MEK1/2-mediated signaling to suppress intracellular bacterial growth in S. Typhimurium-infected murine macrophages, and that a TLR3 agonist could be developed as an immune enhancer to protect against S. Typhimurium infection.
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BACKGROUND: The intrinsic immuno-ge7nomic characteristics of colorectal cancer cells that affect tumor biology and shape the tumor immune microenvironment (TIM) are unclear. METHODS: We developed a patient-derived colorectal cancer organoid (CCO) model and performed pairwise analysis of 87 CCOs and their matched primary tumors. The TIM type of the primary tumor was classified as immuno-active, immuno-exhausted, or immuno-desert. RESULTS: The gene expression profiles, signaling pathways, major oncogenic mutations, and histology of the CCOs recapitulated those of the primary tumors, but not the TIM of primary tumors. Two distinct intrinsic molecular subgroups of highly proliferative and mesenchymal phenotypes with clinical significance were identified in CCOs with various cancer signaling pathways. CCOs showed variable expression of cancer-specific immune-related genes such as those encoding HLA-I and HLA-II, and molecules involved in immune checkpoint activation/inhibition. Among these genes, the expression of HLA-II in CCOs was associated with favorable patient survival. K-means clustering analysis based on HLA-II expression in CCOs revealed a subgroup of patients, in whom cancer cells exhibited Intrinsically Immunogenic Properties (Ca-IIP), and were characterized by high expression of signatures associated with HLA-I, HLA-II, antigen presentation, and immune stimulation. Patients with the Ca-IIP phenotype had an excellent prognosis, irrespective of age, disease stage, intrinsic molecular type, or TIM status. Ca-IIP was negatively correlated with intrinsic E2F/MYC signaling. Analysis of the correlation between CCO immuno-genotype and TIM phenotype revealed that the TIM phenotype was associated with microsatellite instability, Wnt/ß-catenin signaling, APC/KRAS mutations, and the unfolded protein response pathway linked to the FBXW7 mutation in cancer cells. However, Ca-IIP was not associated with the TIM phenotype. CONCLUSIONS: We identified a Ca-IIP phenotype from a large set of CCOs. Our findings may provide an unprecedented opportunity to develop new strategies for optimal patient stratification in this era of immunotherapy.
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Neoplasias Colorrectales/inmunología , Organoides/inmunología , Neoplasias Colorrectales/mortalidad , Femenino , Humanos , Masculino , Pronóstico , Análisis de SupervivenciaRESUMEN
Epithelial barrier dysfunction is involved in allergic inflammation and asthma, due to increased exposure of sub-epithelial tissues to inhaled allergens and air pollutants. The tight junction proteins claudins (CLDNs) are important regulators of paracellular permeability. CLDN7 is expressed in the alveolar epithelium; however, its contribution to airway barrier function remains unclear. The aim of this study was to assess the effects of TiO2 on epithelial barrier function in asthma. Mice were sensitized and challenged with OVA or exposed to TiO2 on days 21-23. The effect of TiO2 on CLDN7 was assessed by ELISA, immunoblotting, and immunohistochemical analysis. The levels of CLDN7 in the plasma of patients with asthma and healthy individuals were also examined. CLDN7 levels were lower in plasma from patients with asthma compared with healthy individuals. CLDN7 levels were associated with FEV1/FVC and the blood eosinophils (%) in patients with asthma. Although CLDN7 expression was elevated in the lungs of mice with asthma and in NHBE cells treated with HDM extracts, its expression was suppressed by exposure to TiO2. p-AKT and p-ERK was increased in asthmatic mice and decreased in mice with TiO2 treatment. p-AKT and p-ERK was decreased in NHBE cells treated with TiO2 and HDM extracts. Trans-epithelial electrical resistance (TEER) was higher in NHBE cells treated with TiO2 or HDM extracts; however, this was decreased by concurrent TiO2 and HDM extracts treatment. Our data suggest that particulate matter contributes to airway epithelial barrier dysfunction and results in airway inflammation and responsiveness.
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Contaminantes Atmosféricos/efectos adversos , Asma/metabolismo , Claudinas/metabolismo , Células Epiteliales/efectos de los fármacos , Material Particulado/efectos adversos , Titanio/efectos adversos , Animales , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Pruebas de Función Respiratoria/métodos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismoRESUMEN
BACKGROUND/AIM: Although molecular targeting therapy is an attractive treatment for cancer, resistance eventually develops in most cases. Here, we evaluated chemotherapeutic efficacy on non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor inhibitors mechanistically. MATERIALS AND METHODS: Antitumor effects of taxotere were evaluated using multiple models, including xenograft, and patient-derived models developed from adenocarcinoma cancer patients. Protein expressions were analyzed after drug treatment. RESULTS: Taxotere inhibited tumor growth of NSCLC cells harboring drug resistance, and reduced the expression of phosphorylated MET proto-oncogene, receptor tyrosine kinase (MET). A tumor-inhibitory effect of taxotere was also demonstrated in vivo in xenografts in mice, patient-derived primary lung tumor cells and patient-derived xenograft with concomitant repression of phosphorylated MET expression. Chemotherapeutic and MET-targeting drug exhibited a synergistic cell growth-inhibitory effect. CONCLUSION: These results suggest that the anticancer drug taxane may be an adjuvant for lung tumors exhibiting enhanced signaling of MET networks.
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Antineoplásicos/farmacología , Docetaxel/farmacología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The interaction between chronic inflammation and neural dysfunction points to a link between the nervous and immune systems in the airways. In particular, environmental exposure to nanoparticles (NPs), defined as particulate matter having one dimension <100 nm, is associated with an enhanced risk of childhood and adult asthma. However, the impact of NPs on the neural response in asthma remains to be determined. This study determined the impact of NPs on neuroinflammation in a mouse model of allergic asthma. Ovalbumin (OVA) sensitized mice were treated with saline (Sham), OVA challenged and exposed to 200 µg/m3 NPs 1 h a day for 3 days on days 21-23 in a closed-system chamber attached to a ultrasonic nebulizer. The effect of NPs on the levels of neuropeptides, transient receptor potential vanilloid 1 (TRPV1), TRPV4, P2 × 4, and P2 × 7 was assessed by enzyme-linked immunosorbent assays, immunoblotting, and immunohistochemistry. NP exposure increased airway inflammation and responsiveness in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. The lung tissue levels of TRPV1, TRPV4, P2 × 4, and P2 × 7 were increased in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. The substance P, adenosine triphosphate (ATP), and calcitonin gene-related peptide (CGRP) levels in bronchoalveolar lavage fluid were increased in OVA mice, and these increases were augmented in OVA plus NP-exposed mice. Bradykinin, ATP, and CGRP were dose dependently increased in NP-exposed normal human bronchial epithelial (NHBE) cells. The calcium concentration was increased in NHBE cells exposed to NPs for 8 h. These results indicate that neuroinflammation can be involved in the pathogenesis of bronchial asthma and that NPs can exacerbate asthma via neuromediator release.
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Asma/inducido químicamente , Asma/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Nanopartículas/toxicidad , Animales , Células Cultivadas , Femenino , Humanos , Exposición por Inhalación/efectos adversos , Ratones , Ratones Endogámicos BALB C , Material Particulado/efectos adversos , Titanio/toxicidadRESUMEN
Acrolein, an α/ß-unsaturated aldehyde, is volatile at room temperature. It is a respiratory irritant found in environmental tobacco smoke, which can be generated during cooking or endogenously at sites of injury. An acute high concentration of uncontrolled irritant exposure can lead to an asthma-like syndrome known as reactive airways dysfunction syndrome (RADS). However, whether acrolein can induce RADS remains poorly understood. The aim of study is to develop a RADS model of acrolein inhalation in mice and to clarify the mechanism of RADS. Mice were treated with ovalbumin (OVA) and exposed to acrolein (5 ppm/10 min). Airway hyper-responsiveness (AHR) was measured on days 24 and 56, and samples were collected on days 25 and 57. Tight junction protein, antioxidant-associated protein, and vascular endothelial growth factor (VEGF) levels were estimated by Western blotting and immunohistochemical staining. Reactive oxygen species (ROS) was calculated using enzyme linked immunosorbent assays. Acrolein or OVA groups exhibited an increase in airway inflammatory cells and AHR compared to a sham group. These effects were further increased in mice in the OVA + acrolein exposure group than in the OVA exposure group and persisted in the acrolein exposure group for 8 weeks. CLDNs, carbonyls, VEGF, Nrf2, and Keap1 were observed in the lungs. Our data demonstrate that acrolein induces RADS and that ROS, angiogenesis, and tight junction proteins are involved in RADS in a mouse model.
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Acroleína/efectos adversos , Alérgenos/efectos adversos , Asma Ocupacional/inducido químicamente , Exposición a Riesgos Ambientales/efectos adversos , Ovalbúmina/efectos adversos , Hipersensibilidad Respiratoria/inducido químicamente , Acroleína/administración & dosificación , Administración por Inhalación , Alérgenos/administración & dosificación , Animales , Asma Ocupacional/diagnóstico , Claudinas/análisis , Claudinas/metabolismo , Femenino , Proteína 1 Asociada A ECH Tipo Kelch/análisis , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/análisis , Factor 2 Relacionado con NF-E2/metabolismo , Ovalbúmina/administración & dosificación , Hipersensibilidad Respiratoria/diagnóstico , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: Annexin A5 (ANXA5) has a potential role in cellular signal transduction, inflammation, and fibrosis. However, the exact role of ANXA5 in asthma remains to be clarified. The aims of the present study were to investigate ANXA5 protein expression in a mouse model of asthma and pollutant exposure and to elucidate the relationships between clinical variables and plasma ANXA5 levels in patients with asthma. METHODS: A murine model of asthma induced by ovalbumin (OVA) and titanium dioxide (TiO2) nanoparticles has been established using BALB/c mice, and we examined ANXA5 expression and lung fibrosis using this model. Moreover, we also compared ANXA5 plasma levels in patients with controlled vs. exacerbated asthma. RESULTS: ANXA5 protein levels were lower in lung tissue from OVA + OVA mice than in control mice. Lung ANXA5, connective tissue growth factor (CTGF), and transforming growth factor ß1 (TGF-ß1) protein levels were higher in OVA + TiO2-exposed mice than in control or OVA + OVA mice. Although Dermatophagoides pteronyssinus (Derp1) treatment increased lung ANXA5 protein levels in MRC-5 cells and A549 epithelial cells, it decreased lung ANXA5 levels in NHBE cells. Treatment with TiO2 nanoparticles increased lung ANXA5, CTGF, and TGF-ß1 protein levels in MRC-5 cells, A549 epithelial cells, and NHBE cells. Plasma ANXA5 levels were lower in asthmatic patients than in healthy controls, and they were significantly enriched in patients with exacerbated asthma compared with those with controlled asthma (P < 0.05). ANXA5 levels were correlated with pulmonary function as assessed by spirometry. CONCLUSION: Our results imply that ANXA5 plays a potential role in asthma pathogenesis and may be a promising marker for exacerbated bronchial asthma and exposure to air pollutants.
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Anexina A5/metabolismo , Antígenos Dermatofagoides/farmacología , Asma/diagnóstico , Asma/fisiopatología , Células A549/metabolismo , Anciano , Contaminantes Atmosféricos/efectos adversos , Animales , Asma/etiología , Asma/patología , Biomarcadores/sangre , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Dermatophagoides pteronyssinus , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Nanopartículas/efectos adversos , Ovalbúmina , Fibrosis Pulmonar/patología , Titanio/efectos adversos , Factor de Crecimiento Transformador beta1/metabolismo , Capacidad VitalRESUMEN
PURPOSE: The tight junction protein claudin-5 (CLDN5) is critical to the control of endothelial cellular polarity and pericellular permeability. The role of CLDN5 in chronic obstructive pulmonary disease (COPD) remains unclear. The aim of this study was to investigate the association between CLDN5 levels and clinical variables in patients with COPD. METHODS: In total, 30 patients with COPD and 30 healthy controls were enrolled in the study. The plasma CLDN5 level was checked in patients with stable or exacerbated COPD and in healthy controls. RESULTS: The mean plasma CLDN5 level of patients with COPD was 0.63 ± 0.05 ng/mL and that of healthy controls was 6.9 ± 0.78 ng/mL (P = 0.001). The mean plasma CLDN5 level was 0.71 ± 0.05 ng/mL in exacerbated COPD patients and 0.63 ± 0.04 ng/mL in patients with stable COPD (P < 0.05). The plasma CLDN5 level among COPD subjects was correlated with the smoking amount (r = -0.530, P = 0.001). The plasma CLDN5 level in stable COPD patients was correlated with forced expiratory volume in one second (FEV1, %pred.) (r = -0.481, P = 0.037). CONCLUSIONS: The plasma CLDN5 level was not correlated with age. CLDN5 may be involved in the pathogenesis of COPD. Further studies having a larger sample size will be needed to clarify CLDN5 in COPD.
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Claudins (CLDNs) are a major transmembrane protein component of tight junctions (TJs) in endothelia and epithelia. CLDNs are not only essential for sustaining the role of TJs in cell permeability but are also vital for cell signaling through protein-protein interactions. Ozone induces oxidative stress and lung inflammation in humans and experimental models, but the impact of ozone on claudins remains poorly understood. This study was to determine the expression of TJ proteins, such as claudin 3, 4, 5, and 14 following ozone exposure. Mice were exposed to 0.1, 1, or 2 ppm of ozone or ambient air for 6 h for 3 days. The impact of ozone on CLDNs, Nrf2, Keap1, and reactive oxygen species (ROS) were estimated using immunoblotting, immunohistochemical staining, confocal imaging, and ELISA analysis in mice and bronchial epithelial cells. Mice exposed to ozone experienced increased airway inflammatory cell infiltration and bronchial hyper-responsiveness compared to control mice. Additionally, CLDN3, CLDN4, ROS, Nrf2, and Keap1 protein expression increased, and lung CLDN14 protein expression decreased, in mice exposed to ozone compared with control mice. These results indicate that CLDNs are involved in airway inflammation following ozone exposure, suggesting that ozone affects TJ proteins through oxidative mechanisms.
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Claudinas/metabolismo , Pulmón/efectos de los fármacos , Ozono/toxicidad , Uniones Estrechas/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Citocinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Background/Aims: The methacholine bronchial provocation test (MBPT) is used to detect and quantify airway hyper-responsiveness (AHR). Since improvements in the severity of asthma are associated with improvements in AHR, clinical studies of asthma therapies routinely use the change of airway responsiveness as an objective outcome. The aim of this study was to assess the relationship between serial MBPT and clinical profiles in patients with asthma. METHODS: A total of 323 asthma patients were included in this study. The MBPT was performed on all patients beginning at their initial diagnosis until asthma was considered controlled based on the Global Initiative for Asthma guidelines. A responder was defined by a decrease in AHR while all other patients were considered non-responders. RESULTS: A total of 213 patients (66%) were responders, while 110 patients (34%) were non-responders. The responder group had a lower initial PC20 (provocative concentration of methacholine required to decrease the forced expiratory volume in 1 second by 20%) and longer duration compared to the non-responder group. Members of the responder group also had superior qualities of life, compared to members of the non-responder group. Whole blood cell counts were not related to differences in PC20; however, eosinophil concentration was. No differences in sex, age, body mass index, smoking history, serum immunoglobulin E, or frequency of acute exacerbation were observed between responders and non-responders. Conclusions: The initial PC20, the duration of asthma, eosinophil concentrations, and quality-of-life may be useful variables to identify improvements in AHR in asthma patients.
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Asma , Pruebas de Provocación Bronquial , Asma/diagnóstico , Hiperreactividad Bronquial , Broncoconstrictores , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Cloruro de Metacolina , Persona de Mediana Edad , Valores de Referencia , Estudios RetrospectivosRESUMEN
BACKGROUND: Acrolein (allyl Aldehyde) as one of smoke irritant exacerbates chronic airway diseases and increased in sputum of patients with asthma and chronic obstructive lung disease. But underlying mechanism remains unresolved. The aim of study was to identify protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to acrolein. METHODS: A proteomic approach was used to determine the different expression of proteins at 8 h and 24 h after treatment of acrolein 30 nM and 300 nM to HMVEC-L. Treatment of HMVEC-L with acrolein 30 nM and 300 nM altered 21 protein spots on the two-dimensional gel, and these were then analyzed by MALDI-TOF MS. RESULTS: These proteins included antioxidant, signal transduction, cytoskeleton, protein transduction, catalytic reduction. The proteins were classified into four groups according to the time course of their expression patterns such as continually increasing, transient increasing, transient decreasing, and continually decreasing. For validation immunohistochemical staining and Western blotting was performed on lung tissues from acrolein exposed mice. Moesin was expressed in endothelium, epithelium, and inflammatory cells and increased in lung tissues of acrolein exposed mice compared with sham treated mice. CONCLUSIONS: These results indicate that some of proteins may be an important role for airway disease exacerbation caused by acrolein exposure.
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BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = -â0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.
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Anexina A1/sangre , Asma/sangre , Pulmón/fisiopatología , Adulto , Anciano , Animales , Anexina A1/análisis , Asma/inducido químicamente , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Estudios de Casos y Controles , Femenino , Volumen Espiratorio Forzado , Humanos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Ovalbúmina , Brote de los Síntomas , Capacidad VitalRESUMEN
PURPOSE: Claudin-4 has been reported to function as a paracellular sodium barrier and is one of the 3 major claudins expressed in lung alveolar epithelial cells. However, the possible role of claudin-4 in bronchial asthma has not yet been fully studied. In this study, we aimed to elucidate the role of claudin-4 in the pathogenesis of bronchial asthma. METHODS: We determined claudin-4 levels in blood from asthmatic patients. Moreover, using mice sensitized and challenged with OVA, as well as sensitized and challenged with saline, we investigated whether claudin-4 is involved in the pathogenesis of bronchial asthma. Der p1 induced the inflammatory cytokines in NHBE cells. RESULTS: We found that claudin-4 in blood from asthmatic patients was increased compared with that from healthy control subjects. Plasma claudin-4 levels were significantly higher in exacerbated patients than in control patients with bronchial asthma. The plasma claudin-4 level was correlated with eosinophils, total IgE, FEV1% pred, and FEV1/FVC. Moreover, lung tissues from the OVA-OVA mice showed significant increases in transcripts and proteins of claudin-4 as well as in TJ breaks and the densities of claudin-4 staining. When claudin-4 was knocked down by transfecting its siRNA, inflammatory cytokine expressions, which were induced by Der p1 treatment, were significantly increased. CONCLUSIONS: These findings thus raise the possibility that regulation of lung epithelial barrier proteins may constitute a therapeutic approach for asthma.
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Obesity is associated with consumption of energy-dense diets and development of systemic inflammation. Gut microbiota play a role in energy harvest and inflammation and can influence the change from lean to obese phenotypes. The nucleus of the solitary tract (NTS) is a brain target for gastrointestinal signals modulating satiety and alterations in gut-brain vagal pathway may promote overeating and obesity. Therefore, we tested the hypothesis that high-fat dietinduced changes in gut microbiota alter vagal gut-brain communication associated with increased body fat accumulation. Sprague-Dawley rats consumed a low energydense rodent diet (LFD; 3.1 kcal/g) or high energydense diet (HFD, 5.24 kcal/g). Minocycline was used to manipulate gut microbiota composition. 16S Sequencing was used to determine microbiota composition. Immunofluorescence against IB4 and Iba1 was used to determine NTS reorganization and microglia activation. Nodose ganglia from LFD rats were isolated and co-cultured with different bacteria strains to determine neurotoxicity. HFD altered gut microbiota with increases in Firmicutes/Bacteriodetes ratio and in pro-inflammatory Proteobacteria proliferation. HFD triggered reorganization of vagal afferents and microglia activation in the NTS, associated with weight gain. Minocycline-treated HFD rats exhibited microbiota profile comparable to LFD animals. Minocycline suppressed HFDinduced reorganization of vagal afferents and microglia activation in the NTS, and reduced body fat accumulation. Proteobacteria isolated from cecum of HFD rats were toxic to vagal afferent neurons in culture. Our findings show that dietinduced shift in gut microbiome may disrupt vagal gutbrain communication resulting in microglia activation and increased body fat accumulation.
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Tejido Adiposo/metabolismo , Dieta Alta en Grasa , Microbioma Gastrointestinal/fisiología , Núcleo Solitario/fisiología , Nervio Vago/fisiología , Vías Aferentes/fisiología , Animales , Antibacterianos/farmacología , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Heces/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Bacterias Gramnegativas/aislamiento & purificación , Lectinas/metabolismo , Lipopolisacáridos/sangre , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Minociclina/farmacología , Ganglio Nudoso/metabolismo , Ganglio Nudoso/microbiología , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Ratas , Ratas Sprague-Dawley , Núcleo Solitario/efectos de los fármacos , Núcleo Solitario/metabolismo , Factores de Tiempo , Nervio Vago/efectos de los fármacosRESUMEN
PURPOSE: Nanoparticles (NPs) may cause cell and tissue damage, leading to local and systemic inflammatory responses and adverse effects on health due to the inhalation of particulate matter. The inflammasome is a major regulator of inflammation through its activation of pro-caspase-1, which cleaves pro-interleukin-1ß (pro-IL-1ß) into its mature form and may induce acute and chronic immune responses to NPs. However, little is known about the response of the inflammasome to NP exposure via the airways in asthma. The aim of this study was to identify the impact of titanium dioxide (TiO2) NPs on inflammasome in a mouse model of allergic asthma. METHODS: Mice were treated with ovalbumin (OVA) or TiO2 NPs. IL-1ß, IL-18, NAIP, CIITA, HET-E, TP-2 (NACHT), leucine-rich repeat (LRR), pyrin domain-containing protein 3 (NLRP3), and caspase-1 were assessed by Western blotting. Caspase-1 was assessed by immunohistochemistry (IHC). Levels of reactive oxygen species (ROS)-as markers of oxidative damage-and the mediators 8-isoprostane and carbonyl were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Airway hyperresponsiveness (AHR) and inflammation were increased in OVA-sensitized/challenged mice, and these responses were exacerbated by exposure to TiO2 NPs. NP treatment increased IL-1ß and IL-18 expression in OVA-sensitized/challenged mice. NPs augmented the expression of NLRP3 and caspase-1, leading to production of active caspase-1 in the lung. Caspase-1 expression was increased and exacerbated by TiO2 NP exposure in OVA-sensitized/challenged mice. ROS levels tended to be increased in OVA-sensitized/challenged and OVA-sensitized/challenged-plus-TiO2 NP-exposed mice. CONCLUSIONS: Our data demonstrated that inflammasome activation occured in asthmatic lungs following NP exposure, suggesting that targeting the inflammasome may assist in controling NP-induced airway inflammation and hyperresponsiveness.
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Animal model systems are invaluable for examining human diseases. Our laboratory recently established a mouse model of nasal polyps (NPs) and investigated similarities and differences between this mouse model and human NPs. We especially focus on the hypothesis that B cell activation occurs during NP generation in the murine model. After induction of ovalbumin-induced allergic rhinosinusitis, 6% ovalbumin and Staphylococcus aureus enterotoxin B (10 ng) were instilled into the nasal cavity of mice three times per week for 8 weeks. The development of structures that somewhat resemble NPs (which we will refer to as NPs) was confirmed by hematoxylin and eosin staining. The mRNA and protein levels of various inflammatory cell markers and mediators were measured by real-time PCR in nasal tissue and by ELISA in nasal lavage fluid (NLF), respectively. Total Ig isotype levels in NLF were also quantitated using the Mouse Ig Isotyping Multiplex kit (EMD Millipore, Billerica, MA) on a Luminex 200 instrument (Life Technologies, Grand Island, NY). Similar to human NPs, there were significant increases in gene expression of inflammatory cell markers, such as CD19, CD138, CD11c, and mast cell protease-6 in nasal tissue samples of the NP group compared with those of the control group. In further investigations of B cell activation, mRNA expressions of B cell activating factor and a proliferation-inducing ligand were found to be significantly increased in mouse NP tissue. B cell-activating factor protein concentration and IgA and IgG1 levels in NLF were significantly higher in the NP group compared with the control group. In this study, the NP mouse model demonstrated enhanced B cell responses, which are reminiscent of B cell responses in human NPs.
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Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Animales , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Inflamación/patología , Ratones Endogámicos BALB C , Líquido del Lavado Nasal , Pólipos Nasales/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Wnt2 is implicated in various human cancers. However, it remains unknown how Wnt2 is upregulated in human cancer and contributes to tumorigenesis. Here we found that Wnt2 is highly expressed in colorectal cancer (CRC) cells. In addition to co-expression of Wnt2 with Wnt/ß-catenin target genes in CRC, knockdown or knockout of Wnt2 significantly downregulates Wnt/ß-catenin target gene expression in CRC cells. Importantly, depletion or ablation of endogenous Wnt2 inhibits CRC cell proliferation. Similarly, neutralizing secreted Wnt2 reduces Wnt target gene expression and suppresses CRC cell proliferation. Conversely, Wnt2 increases cell proliferation of intestinal epithelial cells. Intriguingly, WNT2 expression is transcriptionally silenced by EZH2-mediated H3K27me3 histone modification in non-CRC cells, However, WNT2 expression is de-repressed by the loss of PRC2's promoter occupancy in CRC cells. Our results reveal the unexpected roles of Wnt2 in complementing Wnt/ß-catenin signaling for CRC cell proliferation.
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Adenocarcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Mucosa Intestinal/metabolismo , Vía de Señalización Wnt , Proteína wnt2/metabolismo , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Comunicación Autocrina , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Histonas/metabolismo , Humanos , Mucosa Intestinal/patología , Metilación , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , Transcripción Genética , Transfección , Proteína wnt2/genética , beta Catenina/genéticaRESUMEN
The MET receptor tyrosine kinase, the receptor for hepatocyte growth factor (HGF), has been implicated in cancer growth, invasion, migration, angiogenesis, and metastasis in a broad variety of human cancers, including human hepatocellular carcinoma (HCC). Recently, MET was suggested to be a potential target for the personalized treatment of HCC with an active HGF-MET signaling pathway. However, the mechanisms of resistance to MET inhibitors need to be elucidated to provide effective treatment. Here, we show that HCC cells exhibit different sensitivities to the MET inhibitor PHA665752, depending on the phosphorylation status of FGFR. Treatment of cells expressing both phospho-FGFR and phospho-MET with the inhibitor PHA665752 did not cause growth inhibition and cell death, whereas treatment with AZD4547, a pan-FGFR inhibitor, resulted in decreased colony formation and cleavage of caspase-3. Moreover, silencing of endogenous FGFR1 and FGFR2 by RNAi of HCC cells expressing phospho-FGFR, phospho-FGFR2, and phospho-MET overcame the resistance to PHA665752 treatment. Treatment of primary cancer cells from patients with HCC expressing both phospho-FGFR and phospho-MET with PHA665752 did not induce cell death, whereas AZD4547 treatment induced cell death through the cleavage of caspase-3. In addition, treatment of cells resistant to PHA665752 with AZD4547 abrogated the activation of downstream effectors of cell growth, proliferation, and survival. On the basis of these results, we conclude that the FGFR pathway is critical for HCC survival, and that targeting this pathway with AZD4547 may be beneficial for the treatment of patients with HCC-expressing phospho-FGFR and phospho-MET.
Asunto(s)
Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Antineoplásicos/farmacología , Benzamidas/farmacología , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Indoles/farmacología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Microscopía Confocal , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Pirazoles/farmacología , Interferencia de ARN , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonas/farmacologíaRESUMEN
K-Ras mutation is detected in over 30% of human malignancies. In particular, 90% of human pancreatic cancers are initiated by K-Ras mutation. Thus, selective elimination of K-Ras mutated cells would be a plausible strategy to prevent or cure the malignancies. In our previous reports, it has been revealed that oncogenic K-Ras promotes the exocytosis of p53 with Snail. In this study, we have followed the final destination of extracellular p53, which is secreted by the Snail complex. Here we provide evidences that p53, exported from K-Ras-mutated cells, is specifically re-endocytosed by oncogenic K-Ras-containing cancer cells. The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis. Using a serial deletion approach, we revealed that a fragment of human p53 extending from 93-143 amino acids (AA) is responsible for binding with caveolin-1 and for endocytosis. In contrast, p53-Snail binding occurs at the 143-193 aa region. Finally, through in vivo study, we confirmed that injected recombinant p53 could be up-taken by tumor tissues, constructed by oncogenic K-Ras transformed MEF cells. In contrast, the tumors formed by H-Ras mutated MEF cells did not accumulate the injected p53 protein. These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.
Asunto(s)
Caveolina 1/metabolismo , Genes ras , Mutación , Neoplasias Pancreáticas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Caveolina 1/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Endosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: HMLEs (HMLE-SNAIL and Kras-HMLE, Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We had earlier developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using systems biology and pathway network analysis, we show that GN-25 could cause reversal of EMT leading to mesenchymal-to-epithelial transition (MET) in a well-recognized HMLE-SNAIL and Kras-HMLE-SNAIL models. RESULTS: GN-25 induced MET was found to be consistent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. Pathway network analysis of mRNA expression using microarrays from GN-25 treated Kras-HMLE-SNAIL cells showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level (down-regulation of mesenchymal markers such as TWIST1 and TWIST2 that was concurrent with up-regulation of epithelial marker E-Cadherin), and RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 modulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and led to enhancement in the expression of tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as cell death. CONCLUSIONS: Our systems and network investigations provide convincing pre-clinical evidence in support of the clinical application of GN-25 for the reversal of EMT and thereby reducing cancer cell aggressiveness.