RESUMEN
PURPOSE: Alpha-melanocyte stimulating hormone (α-MSH) is known to have anti-inflammatory effects. However, the anti-inflammatory properties of α-MSH on normal bronchial epithelial cells are largely unknown, especially in the context of in vitro sarcoidosis models. METHODS: We evaluated the anti-inflammatory effects of α-MSH on two different in vitro sarcoidosis models (lung-on-membrane model; LOMM and three-dimensional biochip pulmonary sarcoidosis model; 3D-BSGM) generated from NBECs and an in vivo sarcoidosis mouse model. RESULTS: Treatment with α-MSH decreased inflammatory cytokine levels and downregulated type I interferon pathway genes and related proteins in LOMM and 3D-BSGM models. Treatment with α-MSH also significantly decreased macrophages and cytotoxic T-cells counts in a sarcoidosis mice model. CONCLUSION: Our results confirm the direct role of type I IFNs in the pathogenesis of sarcoid lung granulomas and highlight α-MSH as a potential novel therapeutic agent for treating pulmonary sarcoidosis.
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Sarcoidosis Pulmonar , Sarcoidosis , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Granuloma/tratamiento farmacológico , Inflamación/metabolismo , Ratones , Sarcoidosis/tratamiento farmacológico , Sarcoidosis Pulmonar/tratamiento farmacológico , alfa-MSH/metabolismo , alfa-MSH/farmacología , alfa-MSH/uso terapéuticoAsunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Células Epiteliales/microbiología , Pulmón/microbiología , Pulmón/patología , Mycobacterium abscessus/crecimiento & desarrollo , Anciano , Animales , Citocinas/metabolismo , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Introduction:Mycobacteria are aerobic non-motile organisms with lipid rich, hydrophobic cell walls that render them resistant to antibiotics. While there are over 150 different species of NTM, Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are two of the most common culprits of pulmonary infection. MAB has been found to be most common in southeastern United States (Florida to Texas) and the third most rapidly growing NTM infection. It is responsible for chronic lung infections. Mycobacterial cell wall components initiate the interaction between bacteria and host. The reaction between bronchial epithelia and components in the envelope of mycobacterial cell wall is poorly understood. Methods: A lung-on-membrane model was developed with normal human bronchial epithelial (NHBE) cells re-differentiated at the air-liquid interface (ALI) and human endothelial cells on a transwell® polyester membrane. Microparticles from MAB cell walls were developed by an inhouse protocol and added to the ALI side of lung model. NHBE cells were harvested at day 3. RNA was isolated and analyzed with RNASeq. NHBE cells were lysed and protein assay was performed with western blot. We tested whether lung INF-alpha expression would increase in mice treated with intratracheal MAB cell wall particles. A paired t-test is used to compare two population means using GraphPad Prism 7 software. Results: RNAseq analysis identified 1759 differentially expressed genes between NHBE cells challenged with and without MAB microparticles with FDR < 0.5. 410 genes had a 2.5-fold change (FC) or greater. NHBE cells exposure to MAB microparticles significantly enriched the IFN I signaling pathway. Protein overexpression of IFN I family (2'-5'-Oligoadenylate Synthetase 1, Interferon-induced GTP-binding protein Mx1, Interferon-stimulated gene 15) was found in bronchial epithelial cells following exposure to MAB cell wall microparticles. IFN-α protein and gene expressions were significantly increased in mice lung challenged with microparticles in comparison with controls. Conclusion: These data strongly support the role of Type I IFN in cross-talk between NHBE cells and MAB. They also suggest that initiating immune response by NHBE cells may play a central role in innate immunity. Furthermore, this study underscores the importance of mycobacterial cell wall in initiating innate immune response.
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Interferón Tipo I/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/fisiología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Transducción de Señal , Adulto , Anciano , Animales , Citocinas/genética , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mucosa Respiratoria/inmunología , Adulto JovenRESUMEN
Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases.
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Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Mucosa Respiratoria/efectos de los fármacos , Fumar/efectos adversos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cilios/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Gefitinib , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/efectos de los fármacos , Humo/efectos adversosRESUMEN
In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.
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Células Epiteliales/virología , Vectores Genéticos , Lentivirus/crecimiento & desarrollo , Transducción Genética , Cultivo de Virus/métodos , Bronquios/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , HumanosRESUMEN
Loss of ciliated cells and increases in goblet cells are seen in respiratory diseases such as asthma. These changes result in part from reduced differentiation of basal progenitor cells to ciliated cells during injury and repair. The T helper 2 cytokine, IL-13, has been shown to inhibit ciliated cell differentiation, but the mechanism is not clearly understood. We recently showed that Notch signaling inhibits ciliated cell differentiation in submerged culture by repressing multicilin and forkhead box J1 (FOXJ1) expression, genes required for ciliogenesis. Using a novel method to study ciliated cell differentiation, we investigated the relationship between IL-13 and Notch signaling pathways. We found that IL-13 inhibits ciliated cell differentiation by repressing multicilin and FOXJ1 expression but does so independent of Notch signaling. In addition, we show that pharmacological inhibition of Janus kinase/signal transducer and activator of transcription, but not mitogen activated protein kinase kinase, signaling rescues multicilin and FOXJ1 expression and ciliated cell differentiation in the presence of IL-13. These findings indicate that regulation of multicilin expression by two distinct signaling pathways affects ciliated cell differentiation. In addition, the requirement for Janus kinase activation in IL-13-induced inhibition of ciliogenesis provides a potential therapeutic target for the treatment of respiratory disease.
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Bronquios/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Cilios , Interleucina-13/farmacología , Quinasas Janus/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Receptores Notch/metabolismo , Factores de Transcripción STAT/metabolismo , Bronquios/citología , Bronquios/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factores de Transcripción Forkhead/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteolisis , ARN Mensajero/genética , Factores de TranscripciónRESUMEN
The epithelium that lines the conducting airways is composed of several distinct cell types that differentiate from common progenitor cells. The signals that control fate selection and differentiation of ciliated cells, a major component of the epithelium, are not completely understood. Ciliated cell differentiation can be accomplished in vitro when primary normal human bronchial epithelial (NHBE) cells are cultured at an air-liquid interface, but is inhibited when NHBE cells are cultured under submerged conditions. The mechanism by which submersion prevents ciliogenesis is not understood, but may provide clues to in vivo regulation of ciliated cell differentiation. We hypothesized that submersion creates a hypoxic environment that prevents ciliated cell differentiation by blocking the gene expression program required for ciliogenesis. This was confirmed by showing that expression of multicilin and Forkhead box J1, key factors needed for ciliated cell differentiation, was inhibited when NHBE cells were cultured in submerged and hypoxic conditions. Multicilin and Forkhead box J1 expression and ciliated cell differentiation were restored in submerged and hypoxic cells upon treatment with the γ-secretase inhibitor, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which suggested that Notch signaling was involved. Overexpression of Notch intracellular domain inhibited differentiation in the presence of DAPT, confirming the role of Notch signaling. These results indicate that submersion and hypoxia prevent ciliated cell differentiation by maintaining Notch signaling, which represses genes necessary for ciliogenesis. These data provide new insights into the molecular mechanisms that control human bronchial differentiation.
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Diferenciación Celular , Células Epiteliales/metabolismo , Receptores Notch/metabolismo , Mucosa Respiratoria/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula , Células Cultivadas , Cilios/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Inmersión , Movimiento (Física) , Receptores Notch/genética , Mucosa Respiratoria/efectos de los fármacos , Transducción de Señal , TransfecciónRESUMEN
Corticosteroids inhibit organic cation transporters (OCTs) that play an important role in drug absorption, tissue distribution and elimination. Corticosteroid sensitivity of bronchodilator trafficking in the airway tissue, however, is poorly understood. To assess the effects of inhaled corticosteroids on airway absorption and disposal mechanisms of long-acting ß(2)-agonists, human airway epithelial and smooth muscle cell uptake of tritiated formoterol and salmeterol was measured in vitro. Corticosteroids caused a rapid, concentration-dependent inhibition of uptake of the cationic formoterol by airway smooth muscle cells, but not airway epithelial cells. Uptake of the non-charged lipophilic salmeterol was corticosteroid-insensitive in both cell types. In smooth muscle cells, inhaled corticosteroids inhibited formoterol uptake with a novel potency rank order: des-ciclesonide > budesonide > beclomethasone 17-monopropionate > beclomethasone dipropionate > ciclesonide > fluticasone. The inhibitory action was rapidly reversible, and was not enhanced by prolonged corticosteroid exposure or sensitive to a transcription inhibitor. Suppression of OCT3 expression using lentivirus-mediated production of shRNA reduced corticosteroid sensitivity of formoterol uptake by smooth muscle cells. Our data support a corticosteroid insensitive absorption and a corticosteroid-sensitive disposition mechanism for cationic long-acting ß(2)-agonist bronchodilators in the airway. Potency rank order and other 'classical' features of anti-inflammatory effects do not apply to inhaled corticosteroids' rapid drug transport actions.
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Corticoesteroides/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacocinética , Bronquios/metabolismo , Administración por Inhalación , Corticoesteroides/administración & dosificación , Transporte Biológico/efectos de los fármacos , Bronquios/citología , Células Cultivadas , Interacciones Farmacológicas , Células Epiteliales/metabolismo , Humanos , Miocitos del Músculo Liso/metabolismo , Proteínas de Transporte de Catión Orgánico/fisiologíaRESUMEN
Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.
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Membrana Celular/enzimología , Proteínas del Citoesqueleto/metabolismo , Mucosa Intestinal/enzimología , Isoenzimas/metabolismo , Microdominios de Membrana/enzimología , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Sitios de Unión/fisiología , Células CACO-2 , Diferenciación Celular/fisiología , Membrana Celular/ultraestructura , Polaridad Celular/fisiología , Proteínas del Citoesqueleto/química , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , Humanos , Insectos , Mucosa Intestinal/citología , Isoenzimas/genética , Microdominios de Membrana/ultraestructura , Ratones , Microvellosidades/enzimología , Microvellosidades/ultraestructura , Fosforilación , Proteína Quinasa C/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/genética , Tirosina/metabolismoRESUMEN
Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels.