Raman Agrawal

Airway Mucus Obstruction Triggers Macrophage Activation and Matrix Metalloproteinase 12–Dependent Emphysema

Whereas cigarette smoking remains the main risk factor for emphysema, recent studies in β-epithelial Na(+) channel-transgenic (βENaC-Tg) mice demonstrated that airway surface dehydration, a key pathophysiological mechanism in cystic fibrosis (CF), caused emphysema in the absence of cigarette smoke exposure. However, the underlying mechanisms remain unknown. The aim of this study was to elucidate mechanisms of emphysema formation triggered by airway surface dehydration. We therefore used expression profiling, genetic and pharmacological inhibition, Foerster resonance energy transfer (FRET)-based activity assays, and genetic association studies to identify and validate emphysema candidate genes in βENaC-Tg mice and patients with CF. We identified matrix metalloproteinase 12 (Mmp12) as a highly up-regulated gene in lungs from βENaC-Tg mice, and demonstrate that elevated Mmp12 expression was associated with progressive emphysema formation, which was reduced by genetic deletion and pharmacological inhibition of MMP12 in vivo. By using FRET reporters, we show that MMP12 activity was elevated on the surface of airway macrophages in bronchoalveolar lavage from βENaC-Tg mice and patients with CF. Furthermore, we demonstrate that a functional polymorphism in MMP12 (rs2276109) was associated with severity of lung disease in CF. Our results suggest that MMP12 released by macrophages activated on dehydrated airway surfaces may play an important role in emphysema formation in the absence of cigarette smoke exposure, and may serve as a therapeutic target in CF and potentially other chronic lung diseases associated with airway mucus dehydration and obstruction.

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o− tracheal, CFBE41o− and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases. Overexpression of miR-17 in cystic fibrosis airway epithelial cells decreases interleukin-8 protein production http://ow.ly/MZbXB

Airway mucus, inflammation and remodeling: emerging links in the pathogenesis of chronic lung diseases

Airway mucus obstruction is a hallmark of many chronic lung diseases including rare genetic disorders such as cystic fibrosis (CF) and primary ciliary dyskinesia, as well as common lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), which have emerged as a leading cause of morbidity and mortality worldwide. However, the role of excess airway mucus in the in vivo pathogenesis of these diseases remains poorly understood. The generation of mice with airway-specific overexpression of epithelial Na+ channels (ENaC), exhibiting airway surface dehydration (mucus hyperconcentration), impaired mucociliary clearance (MCC) and mucus plugging, led to a model of muco-obstructive lung disease that shares key features of CF and COPD. In this review, we summarize recent progress in the understanding of causes of impaired MCC and in vivo consequences of airway mucus obstruction that can be inferred from studies in βENaC-overexpressing mice. These studies confirm that mucus hyperconcentration on airway surfaces plays a critical role in the pathophysiology of impaired MCC, mucus adhesion and airway plugging that cause airflow obstruction and provide a nidus for bacterial infection. In addition, these studies support the emerging concept that excess airway mucus per se, probably via several mechanisms including hypoxic epithelial necrosis, retention of inhaled irritants or allergens, and potential immunomodulatory effects, is a potent trigger of chronic airway inflammation and associated lung damage, even in the absence of bacterial infection. Finally, these studies suggest that improvement of mucus clearance may be a promising therapeutic strategy for a spectrum of muco-obstructive lung diseases.

Hypoxic Epithelial Necrosis Triggers Neutrophilic Inflammation via IL-1 Receptor Signaling in Cystic Fibrosis Lung Disease

RATIONALE In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via IL-1R signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis caused by airway mucus obstruction precedes neutrophilic inflammation in Scnn1b-transgenic (Scnn1b-Tg) mice with CF-like lung disease. OBJECTIVES To determine the role of epithelial necrosis and IL-1R signaling in the development of neutrophilic airway inflammation, mucus obstruction, and structural lung damage in CF lung disease. METHODS We used genetic deletion and pharmacologic inhibition of IL-1R in Scnn1b-Tg mice and determined effects on airway epithelial necrosis; levels of IL-1α, keratinocyte chemoattractant, and neutrophils in bronchoalveolar lavage; and mortality, mucus obstruction, and structural lung damage. Furthermore, we analyzed lung tissues from 21 patients with CF and chronic obstructive pulmonary disease and 19 control subjects for the presence of epithelial necrosis. MEASUREMENTS AND MAIN RESULTS Lack of IL-1R had no effect on epithelial necrosis and elevated IL-1α, but abrogated airway neutrophilia and reduced mortality, mucus obstruction, and emphysema in Scnn1b-Tg mice. Treatment of adult Scnn1b-Tg mice with the IL-1R antagonist anakinra had protective effects on neutrophilic inflammation and emphysema. Numbers of necrotic airway epithelial cells were elevated and correlated with mucus obstruction in patients with CF and chronic obstructive pulmonary disease. CONCLUSIONS Our results support an important role of hypoxic epithelial necrosis in the pathogenesis of neutrophilic inflammation independent of bacterial infection and suggest IL-1R as a novel target for antiinflammatory therapy in CF and potentially other mucoobstructive airway diseases.

Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease

Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

miRNA-221 is elevated in cystic fibrosis airway epithelial cells and regulates expression of ATF6

BackgroundMicroRNA (miRNA) and messenger RNA (mRNA) expression differs in cystic fibrosis (CF) versus non-CF bronchial epithelium. Here, the role of miRNA in basal regulation of the transcription factor ATF6 was investigated in bronchial epithelial cells in vitro and in vivo.MethodsUsing in silico analysis, miRNAs predicted to target the 3′untranslated region (3′UTR) of the human ATF6 mRNA were identified.ResultsThree of these miRNAs, miR-145, miR-221 and miR-494, were upregulated in F508del-CFTR homozygous CFBE41o- versus non-CF 16HBE14o- bronchial epithelial cells and also in F508del-CFTR homozygous or heterozygous CF (n = 8) versus non-CF (n = 9) bronchial brushings. ATF6 was experimentally validated as a molecular target of these miRNAs through the use of a luciferase reporter vector containing the full-length 3′UTR of ATF6. Expression of ATF6 was observed to be decreased in CF both in vivo and in vitro. miR-221 was also predicted to regulate murine ATF6, and its expression was significantly increased in native airway tissues of 6-week-old βENaC-overexpressing transgenic mice with CF-like lung disease versus wild-type littermates.ConclusionsThese results implicate miR-145, miR-221 and miR-494 in the regulation of ATF6 in CF bronchial epithelium, with miR-221 demonstrating structural and functional conservation between humans and mice. The altered miRNA expression evident in CF bronchial epithelial cells can affect expression of transcriptional regulators such as ATF6.

Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair

Abstract The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE−/−), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Impaired mucus clearance exacerbates allergen-induced type 2 airway inflammation in juvenile mice

Background Type 2 airway inflammation plays a central role in the pathogenesis of allergen‐induced asthma, but the underlying mechanisms remain poorly understood. Recently, we demonstrated that reduced mucociliary clearance, a characteristic feature of asthma, produces spontaneous type 2 airway inflammation in juvenile &bgr;‐epithelial Na+ channel (Scnn1b)‐transgenic (Tg) mice. Objective We sought to determine the role of impaired mucus clearance in the pathogenesis of allergen‐induced type 2 airway inflammation and identify cellular sources of the signature cytokine IL‐13. Methods We challenged juvenile Scnn1b‐Tg and wild‐type mice with Aspergillus fumigatus and house dust mite allergen and compared the effects on airway eosinophilia, type 2 cytokine levels, goblet cell metaplasia, and airway hyperresponsiveness. Furthermore, we determined cellular sources of IL‐13 and effects of genetic deletion of the key type 2 signal‐transducing molecule signal transducer and activator of transcription 6 (STAT6) and evaluated the effects of therapeutic improvement of mucus clearance. Results Reduced mucociliary allergen clearance exacerbated Stat6‐dependent secretion of type 2 cytokines, airway eosinophilia, and airway hyperresponsiveness in juvenile Scnn1b‐Tg mice. IL‐13 levels were increased in airway epithelial cells, macrophages, type 2 innate lymphoid cells, and TH2 cells along with increased Il33 expression in the airway epithelium of Scnn1b‐Tg mice. Treatment with the epithelial Na+ channel blocker amiloride, improving airway surface hydration and mucus clearance, reduced allergen‐induced inflammation in Scnn1b‐Tg mice. Conclusion Our data support that impaired clearance of inhaled allergens triggering IL‐13 production by multiple cell types in the airways plays an important role in the pathogenesis of type 2 airway inflammation and suggests therapeutic improvement of mucociliary clearance as a novel treatment strategy for children with allergen‐induced asthma. Graphical abstract Figure. No Caption available.

138 Dysregulation of epithelial miR-148b contributes to goblet cell metaplasia, inflammation and alveolar damage in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is primarily characterized by chronic mucus obstruction, inflammation and structural damage. While mutations in the CFTR gene are an initial trigger, molecular mechanisms underlying CF pathogenesis remains poorly understood. miRNAs are key regulators of diverse biological and pathological processes. Here, we identified association of miR-148b during the in vivo pathogenesis of CF using Scnn1b-overexpressing (Scnn1b-Tg) mice as a model of CF-like lung disease. miR-148b expression was elevated in the lung tissue of Scnn1b-Tg mice and is localized mainly in airway and alveolar epithelial cells. We demonstrate that ERBB receptor feedback inhibitor 1 (Errfi1) is one of the target genes of miR-148b. Antagomir-mediated selective silencing of miR-148b resulted in reduced goblet cell metaplasia, mucus hypersecretion, neutrophilic inflammation, and alveolar damage in the lungs of Scnn1b-Tg mice compared to wild-type. Our human studies demonstrate differential upregulation of miR-148b in the bronchial brushing of CF compared to non-CF individuals and localization in airway and alveolar epithelial cells. Further, we found that the allele distribution at MIR148B-Sat, a polymorphic marker that reflects the genetic variation at the human MIR148B gene, is associated with disease severity in F508del-CFTR homozygous sibling pairs. Together, this study provides a functional link between miR-148b and in vivo pathogenesis of CF and may have a potential for the development of novel therapeutic target.

WS15.6 Upregulation of MicroRNA-148b in cystic fibrosis like lung disease of ßENaC-overexpressing mice

In previous studies we showed that transcomplementation of DF508 by truncated CFTR allows DF508 to progress to the cell surface. Here we study whether D27– 264 CFTR which is missing the first 4 transmembrane segments can also rescue DF508 chloride channel function. Whole cell currents were recorded from CHO cells expressing wt, DF508, D27–264, or both D27–264 and DF508 CFTR. Currents were activated by cpt-cAMP and Forskolin. Importantly no current was generated from unrescued DF508 alone, or from D27–264 alone. Only when both were added did we observe significant whole cell currents which reached to a level near to that of wtCFTR. The currents measured only when both are cotransfected could have originated from either from DF508 or D27–264 CFTR. To determine which one generated the currents, we utilized a CFTR conduction mutant, S341A, which alters the conductance of CFTR. S341A/ wtCFTR showed drastically reduced currents even though the protein was highly expressed. When we studied the double mutant, S341A/DF508 we noted expression of B band but no detectable chloride currents. When we cotransfected S341A /DF508 with D27–264 CFTR we did detect CFTR protein but the choride currents were dramatically reduced showing that the currents observed when DF508 and D27–264 CFTR are cotransfected were generated by rescued DF508. Immunoprecipitation studies showed that D27–264 can bind to DF508 CFTR. Our data show that transcomplementation with D27–264 can rescue both trafficking and chloride channel function of DF508 CFTR most likely via a bimolecular interaction. WS15.6 Upregulation of MicroRNA-148b in cystic fibrosis like lung disease of bENaC-overexpressing mice R. Agrawal1,2,3, M. Castoldi2,4, S. Altamura2,4, M.U. Muckenthaler2,4, M.A. Mall1,2,3. 1University of Heidelberg, Division of Paediatric Pulmonology and Cystic Fibrosis Center, Department of Paediatric III, Heidelberg, Germany; 2European Molecular Biology Laboratory and University of Heidelberg, Molecular Medicine Partnership Unit, Heidelberg, Germany; 3Translational Lung Research Center, Department of Translational Pulmonology, Heidelberg, Germany; 4University of Heidelberg, Department of Paediatric Oncology, Haematology and Immunology, Heidelberg, Germany