Implicación de las mucinas 1 y 4 en el asma y la polipósis nasal resistentes a corticoides

El Asma y la Poliposis Nasal son enfermedades inflamatorias crónicas que implican acciones del epitelio respiratorio y el sistema inmune, causando la obstrucción de las vías aéreas. Actualmente los corticoides son el tratamiento de primera línea para estas enfermedades. Sin embargo, existen pacientes insensibles a los efectos anti-inflamatorios de los corticoides, que suelen determinarse si tras 2 semanas de tratamiento no se ha observado mejora clínica. Estos pacientes suelen presentar un perfil clínico más severo debido a la ineficacia del tratamiento. Las mucinas MUC1 y MUC4 son proteínas de membrana cuya expresión parece estar modulada por el uso de estos fármacos y que poseen capacidad para interactuar con otras proteínas citoplasmáticas. Así pues y en base a trabajos anteriores, nuestra hipótesis se centró en que las mucinas MUC1 y MUC4 pueden participar en la señalización del receptor de glucocorticoides α y de este modo influir en la eficacia anti-inflamatoria de los corticoides. Para este estudio se reclutaron 27 pacientes asmáticos y 73 pacientes con Poliposis Nasal que se clasificaron como respondedores o corticorresistentes, según si se presentaba mejora clínica o no tras el tratamiento con corticoides, respectivamente. Se analizó la expresión de MUC1 y la respuesta a fluticasona en neutrófilos de sangre periférica procedente los pacientes asmáticos; así como la expresión de MUC4 en pólipos nasales de los pacientes con Poliposis Nasal y la respuesta a dexametasona de sus células epiteliales derivadas por explante. Se realizaron estudios in vitro de silenciamiento génico para ambas mucinas en líneas celulares de epitelio respiratorio (BEAS-2B), endotelio (HMVEC-L) y tipo monocítico (U937); además de la sobre-expresión de MUC4 en la línea HEK293, con el fin de determinar su efecto sobre la eficacia de los corticoides frente a estímulos inflamatorios y la posible interacción entre las mucinas y el receptor de corticoides α. Finalmente, se evaluó la respuesta al tratamiento con dexametasona en un modelo murino de Asma agudo MUC1-Knock Out. Los neutrófilos de pacientes asmáticos resistentes a corticoides mostraron insensibilidad a la dexametasona ex vivo y una correlación positiva entre la expresión de MUC1 y la mejora clínica tras el tratamiento con corticoides. El silenciamiento génico de MUC1 en células endoteliales HMVEC-L y monocitos U937 redujo la eficacia inhibitoria de la dexametasona sobre la adhesión y migración celular, respectivamente. El modelo murino MUC1-KO de asma agudo mimetizó la fisiopatología del asma alérgico humano y presentó insensibilidad a dexametasona in vivo y ex vivo en base a la ausencia únicamente de la proteína MUC1. Los pacientes con Poliposis Nasal que no respondieron a corticoides mostraron una mayor expresión de MUC4 comparada con el tejido de pólipo nasal de pacientes respondedores. El silenciamiento y la sobre-expresión de MUC4 in vitro, aumentó y redujo la eficacia anti-inflamatoria de la dexametasona, respectivamente. Se observó que MUC4β forma un complejo con GRα en el citoplasma que se disgrega en respuesta a dexametasona, permitiendo la translocación al núcleo de GRα. De este modo, los resultados obtenidos demostraron como MUC1 y MUC4 modulan la translocación nuclear de GRα, positiva y negativamente, respectivamente, y por tanto el efecto de los corticoides sobre la inflamación asociada al Asma y la Poliposis nasal tanto en humanos, como en modelos in vitro e in vivo

Papel de la expresión de MUC1 en la rinosinusitis crónica asociada a poliposis nasal corticorresistente

[EN] Chronic Rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease of the nasal mucosa and upper airways. The main treatment of CRSwNP with topic or oral corticosteroids presents a degree of resistance and moderately high therapeutic failure. The mucin MUC1 is a mucosal receptor with anti-inflammatory properties that appears to be modulated by those drugs. In this regard, the present work analyses the expression of MUC1 in different cohorts of CRSwNP patients and in vitro models of cell culture. This approximation evaluates how the expression of MUC1 can alter the response to oral corticosteroids. Our results suggest that a cytoplasmic region of MUC1 mediates the anti-inflammatory effect of corticosteroids by modulating nuclear translocation of Glucocorticoid Receptor α. Thus, the lower expression of MUC1 observed in some CRSwNP patients could explain their corticosteroid resistance. PALABRAS CLAVE[ES] La Rinosinusitis Crónica asociada a Pólipos Nasales (RSC-PN) es una enfermedad inflamatoria que afecta a la mucosa nasal y a las vías aéreas altas, cuyo tratamiento, corticosteroides tópicos o sistémicos, presenta un grado de resistencia y fracaso terapéutico moderadamente elevado. La mucina MUC1 es un receptor de las mucosas con propiedades anti-inflamatorias que parece estar modulado por el uso de estos fármacos. En este sentido el presente trabajo se centra en analizar la expresión de MUC1 en diferentes cohortes de pacientes con Rinosinusitis Crónica asociada a Poliposis Nasal así como en modelos in vitro de cultivo celular, para evaluar de qué manera la expresión de MUC1 puede alterar la respuesta a corticosteroides orales. Nuestros resultados sugieren que una región citoplasmática de MUC1 media la respuesta anti-inflamatoria de los corticosteroides al participar en la translocación nuclear del receptor de glucocortidoides α. De esta manera, la menor expresión de MUC1 observada en determinados pacientes de RSC-PN podría explicar la corticorresistencia que los caracteriza. ChronicMorell Garcia, A. (2014). Papel de la expresión de MUC1 en la rinosinusitis crónica asociada a poliposis nasal corticorresistente. http://hdl.handle.net/10251/52567Archivo delegad

MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps

Background Current evidence suggests that membrane-tethered mucins could mediate corticosteroid efficacy, interacting with glucocorticoid receptor (GR) in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Mucin 4 (MUC4)–tethered mucin is expressed in nasal polyp (NP) epithelial cells and upregulated under inflammatory conditions. Moreover, MUC4β has the capacity to interact with other intracellular proteins. We hypothesized that MUC4 modulates corticosteroid efficacy of patients with CRSwNP. Objective We sought to analyze the role of MUC4 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. Methods Eighty-one patients with CRSwNP took oral corticosteroids for 15 days. Corticosteroid resistance was evaluated by using nasal endoscopy. Expression of MUC4 and MUC4β was evaluated by means of real-time PCR, Western blotting, and immunohistochemistry. BEAS-2B knockdown with RNA interference for MUC4 (small interfering RNA [siRNA]–MUC4) was used to analyze the role of MUC4 in the anti-inflammatory effects of dexamethasone. Results Twenty-two patients had NPs resistant to oral corticosteroids. MUC4 expression was upregulated in these patients. In siRNA-MUC4 BEAS-2B airway epithelial cells dexamethasone produced higher anti-inflammatory effects, increased inhibition of phospho–extracellular signal-regulated kinase 1/2, increased mitogen-activated protein kinase phosphatase 1 expression, and increased glucocorticoid response element activation. Immunoprecipitation and immunofluorescence experiments revealed that MUC4β forms a complex with GRα in the nuclei of NP epithelial cells from corticosteroid-resistant patients. Conclusion MUC4β participates in the corticosteroid resistance process, inhibiting normal GRα nuclear function. The high expression of MUC4 in patients with CRSwNP might participate in corticosteroid resistance.Supported by grants SAF2014-55322-P (to J.C.), FISPI14/01733 (to J.M.), FISPI11/02618 (to M.A.), SAF2015-65368-R (to E.M.), CIBERES (CB06/06/0027), TRACE (TRA2009-0311; Spanish Government), and research grants from Regional Government Prometeo II/2013/014 (to J.C., E.M., and J.M.) Generalitat Valenciana

Mucin 1 deficiency mediates corticosteroid insensitivity in asthma

BACKGROUND: The loss of corticosteroid efficacy is an important issue in severe asthma management and may lead to poor asthma control and deterioration of airflow. Recent data indicate that Mucin 1 (MUC1) membrane mucin can mediate corticosteroid efficacy in chronic rhinosinusitis, but the role of MUC1 in uncontrolled severe asthma is unknown. The objective was to analyze the previously unexplored role of MUC1 on corticosteroid efficacy in asthma. METHODS: Mucin 1 expression was evaluated by real-time PCR in human bronchial epithelial cells (HBEC) and blood neutrophils from uncontrolled severe asthma (n = 27), controlled mild asthma (n = 16), and healthy subjects (n = 13). IL-8, MMP9, and GM-CSF were measured by ELISA in HBEC and neutrophils. An asthma model of ovalbumin (OVA) was used in MUC1 KO and WT C57BL/6 mice according to ARRIVE guidelines. RESULTS: Mucin 1-CT expression was downregulated in bronchial epithelial cells and peripheral blood neutrophils from severe asthma patients compared with mild asthma and healthy subjects (P < 0.05). Daily dose of inhaled corticosteroids (ICS) inversely correlated with MUC1 expression in neutrophils from mild and severe asthma (ρ = -0.71; P < 0.0001). Dexamethasone showed lower anti-inflammatory effects in severe asthma peripheral blood neutrophils and HBECs stimulated with lipopolysaccharide (LPS) than in cells from mild asthma. Glucocorticoid receptor (GR)-α phosphorylated at serine 226 was increased in cells from severe asthma, and the MUC1-CT/GRα complex was downregulated in severe asthma cells. OVA asthma model in MUC1 KO mice was resistant to the anti-inflammatory effects of dexamethasone. CONCLUSION: Mucin 1-CT modulates corticosteroid efficacy in vitro and in vivo asthma models

Bruton’s Tyrosine Kinase Inhibitors Ibrutinib and Acalabrutinib Counteract Anthracycline Resistance in Cancer Cells Expressing AKR1C3

Over the last few years, aldo-keto reductase family 1 member C3 (AKR1C3) has been associated with the emergence of multidrug resistance (MDR), thereby hindering chemotherapy against cancer. In particular, impaired efficacy of the gold standards of induction therapy in acute myeloid leukaemia (AML) has been correlated with AKR1C3 expression, as this enzyme metabolises several drugs including anthracyclines. Therefore, the development of selective AKR1C3 inhibitors may help to overcome chemoresistance in clinical practice. In this regard, we demonstrated that Bruton&rsquo;s tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib efficiently prevented daunorubicin (Dau) inactivation mediated by AKR1C3 in both its recombinant form as well as during its overexpression in cancer cells. This revealed a synergistic effect of BTK inhibitors on Dau cytotoxicity in cancer cells expressing AKR1C3 both exogenously and endogenously, thus reverting anthracycline resistance in vitro. These findings suggest that BTK inhibitors have a novel off-target action, which can be exploited against leukaemia through combination regimens with standard chemotherapeutics like anthracyclines

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists. Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was measured in cell cultures after TLR3 stimulation with poly I:C 10 μg/mL. We found that TLR3 expression was increased by 1.95 fold (protein) and 2.5 fold (mRNA) in lung tissues from smokers with COPD and inversely correlated with lung function. The TLR3 agonist poly I:C 10 μg/mL increased the IL-8 release in HBECs that was poorly inhibited by dexamethasone in smokers (24.5%) and smokers with COPD (21.6%). In contrast, roflumilast showed similar inhibitory effects on IL-8 release in healthy (58.8%), smokers (56.6%) and smokers with COPD (50.5%). The combination of roflumilast N-oxide and dexamethasone showed additive inhibitory effects. Mechanistically, roflumilast N-oxide when combined with dexamethasone increased the expression of MKP1, and enhanced the inhibitory effects on phospho-p38, AP1 and NFκB activities which may explain the additive anti-inflammatory effects. Altogether, our data provide in vitro evidence for a possible clinical utility to add roflumilast on top of inhaled corticosteroid in COPD.3.751 JCR (2015) Q1, 13/58 Respiratory systemUE

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study

Background Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation (V)/perfusion (Q) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. Objective The study of JAK2 as potential target to treat PH in IPF. Methods and results JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P<0.0001) compared with PA from control subjects (n=10). PA remodelling was evaluated in human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BKCa). JAK2 inhibition activated BKCa channels and reduced intracellular Ca2+. JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V/Q mismatching in rats. The animal studies followed the ARRIVE guidelines. Conclusions JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH

Additional file 1: of The JAK2 pathway is activated in idiopathic pulmonary fibrosis

Supplementary data. (DOCX 1218 kb