GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins.

Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins have diverse biological functions and many retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, the PIWI proteins-a specialized class of Argonaute proteins-use 21-35 nucleotide PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis1. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalysed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2) and MIWI22-4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm5,6. piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function7-12. Here we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 is thus an example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

House dust mite sensitization and exposure affects bronchial epithelial anti-microbial response to viral stimuli in patients with asthma.

INTRODUCTION: Allergen exposure worsens viral-triggered asthma exacerbations and could predispose the host to secondary bacterial infections. We have previously demonstrated that exposure to house dust mite (HDM) reduced TLR-3-induced IFN-ß in human bronchial epithelial cells (HBECs) from healthy donors. We hypothesize that HDM sensitization in different ways may be involved in both viral and bacterial resistance of HBECs in asthma. In this study, the role of HDM sensitization and effects of HDM exposure on viral stimulus-challenged HBECs from asthmatic donors have been explored with regard to expression and release of molecules involved in anti-viral and anti-bacterial responses, respectively. METHODS: HBECs from HDM-sensitized (HDM+) and unsensitized (HDM-) patients with asthma were used. HBECs were exposed to HDM or heat inactivated (hi)-HDM (20 µg/ml) for 24 h prior to stimulation with the viral infection mimic, Poly(I:C), for 3 or 24 h. Samples were analyzed with ELISA and RT-qPCR for ß-defensin-2, IFN-ß, TSLP, and neutrophil-recruiting mediators: IL-8 and TNF-⍺. NFκB signaling proteins p105, p65, and IκB-⍺ were analyzed by Western blot. RESULTS: Poly(I:C)-induced IFN-ß expression was reduced in HBECs from HDM + compared to HDM- patients (p = 0.05). In vitro exposure of HBECs to HDM furthermore reduced anti-microbial responses to Poly(I:C) including ß-defensin-2, IL-8, and TNF-⍺, along with reduced NFκB activity. This was observed in HBECs from asthma patients sensitized to HDM, as well as in non-sensitized patients. By contrast, Poly (I:C)-induced release of TSLP, a driver of T2 inflammation, was not reduced with exposure to HDM. CONCLUSION: Using HBECs challenged with viral infection mimic, Poly(I:C), we demonstrated that allergic sensitization to HDM was associated with impaired anti-viral immunity and that HDM exposure reduced anti-viral and anti-bacterial defense molecules, but not TSLP, across non-allergic as well as allergic asthma. These data suggest a role of HDM in the pathogenesis of asthma exacerbations evoked by viral infections including sequential viral-bacterial and viral-viral infections.

Direct effects of mast cell proteases, tryptase and chymase, on bronchial epithelial integrity proteins and anti-viral responses.

BACKGROUND: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). METHOD: Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 µg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 µg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 µg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. RESULTS: Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. CONCLUSION: MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

Mast Cell Proteases Tryptase and Chymase Induce Migratory and Morphological Alterations in Bronchial Epithelial Cells.

Chronic respiratory diseases are often characterized by impaired epithelial function and remodeling. Mast cells (MCs) are known to home into the epithelium in respiratory diseases, but the MC-epithelial interactions remain less understood. Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function. Bronchial epithelial cells were stimulated with MC tryptase and/or chymase. Morphology and epithelial function were performed using cell tracking analysis and holographic live-cell imaging. Samples were also analyzed for motility-associated gene expression. Immunocytochemistry was performed to compare cytoskeletal arrangement. Stimulated cells showed strong alterations on gene, protein and functional levels in several parameters important for maintaining epithelial function. The most significant increases were found in cell motility, cellular speed and cell elongation compared to non-stimulated cells. Also, cell morphology was significantly altered in chymase treated compared to non-stimulated cells. In the current study, we show that MC proteases can induce cell migration and morphological and proliferative alterations in epithelial cells. Thus, our data imply that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.

Impaired airway epithelial cell wound-healing capacity is associated with airway remodelling following RSV infection in severe preschool wheeze.

BACKGROUND: Respiratory syncytial virus (RSV) causes exacerbations of asthma and preschool wheeze (PSW). However, the anti-viral and repair responses of the bronchial epithelium in children with severe therapy-resistant asthma (STRA) and PSW are poorly understood. METHODS: Children with STRA (age 12 [6-16] years), PSW (age 2 [1-5] years) and non-asthmatic controls (age 7 [2-14] years) underwent bronchoscopy with endobronchial brushings and biopsies. Anti-viral, wound injury responses were quantified in biopsies and primary bronchial epithelial cells (PBECs) in response to RSV, poly(I:C), house dust mite (HDM) or IL-33 using RT-qPCR, Luminex and live cell imaging. Collagen deposition and tissue expression of epithelial growth factor receptor (EGFR), IL-33 and receptor ST2 were investigated in bronchial biopsies. RESULTS: PBECs from STRA and PSW had increased TLR3 gene expression and increased secretion of anti-viral and pro-inflammatory cytokines (IFN-γ, IL-6 and IL-13) in response to RSV compared to controls. Exposure of PBECs to concomitant TLR3 agonist poly(I:C) and HDM resulted in a significant reduction in epithelial cell proliferation in PSW compared to controls. Wound-healing was also impaired in PSW compared to controls at baseline and following IL-33 stimulation. In addition, tissue EGFR expression was significantly reduced in PSW and correlated with collagen deposition in endobronchial biopsies. CONCLUSIONS: Despite increased anti-viral responses, preschool children with severe wheeze had impaired airway epithelial proliferative responses following damage. This might be connected to the low expression of EGFR in PSW which may affect epithelial function and contribute to asthma pathogenesis.

Intraepithelial neutrophils in pediatric severe asthma are associated with better lung function.

BACKGROUND: Neutrophils and IL-17A have been linked mechanistically in models of allergic airways disease and have been associated with asthma severity. However, their role in pediatric asthma is unknown. OBJECTIVES: We sought to investigate the role of neutrophils and the IL-17A pathway in mediating pediatric severe therapy-resistant asthma (STRA). METHODS: Children with STRA (n = 51; age, 12.6 years; range, 6-16.3 years) and controls without asthma (n = 15; age, 4.75 years; range, 1.6-16 years) underwent clinically indicated fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), endobronchial brushings, and biopsy. Neutrophils, IL-17A, and IL-17RA-expressing cells and levels of IL-17A and IL-22 were quantified in BAL and biopsies and related to clinical features. Primary bronchial epithelial cells were stimulated with IL-17A and/or IL-22, with and without budesonide. RESULTS: Children with STRA had increased intraepithelial neutrophils, which positively correlated with FEV1 %predicted (r = 0.43; P = .008). Neutrophilhigh patients also had better symptom control, despite lower dose maintenance inhaled steroids. Submucosal neutrophils were not increased in children with STRA. Submucosal and epithelial IL-17A-positive cells and BAL IL-17A and IL-22 levels were similar in children with STRA and controls. However, there were significantly more IL-17RA-positive cells in the submucosa and epithelium in children with STRA compared with controls (P = .001). Stimulation of primary bronchial epithelial cells with IL-17A enhanced mRNA expression of IL-17RA and increased release of IL-8, even in the presence of budesonide. CONCLUSIONS: A proportion of children with STRA exhibit increased intraepithelial airway neutrophilia that correlated with better lung function. STRA was also characterized by increased airway IL-17RA expression. These data suggest a potential beneficial rather than adverse role for neutrophils in pediatric severe asthma pathophysiology.

Revisiting the role of the mast cell in asthma.

PURPOSE OF REVIEW: In humans, mast cells are ubiquitously present in tissues adjacent to external environment and consequently have an important sentential role in host defence, homeostasis and repair. Their key role in allergen-mediated conditions has been recognized for many decades already. So far, therapies targeting mast cells offered clinical efficacy in allergic conditions except for asthma. More recently, sophisticated sampling and detection techniques revealed pleiotrophic immunological and functional properties of mast cells in and beyond asthma with potential clinical and management implications. These findings bring back the mast cell as a key player in the field of asthma and warrant a review of the recent literature. RECENT FINDINGS: The heterogeneity of human mast cells has been recognized: MCTC expressing both tryptase and chymase and MCT expressing tryptase only. Apart from this subphenotyping, mast cells may comprise and produce several other mediators and cytokines. Their immunological and functional properties depend on their (co)localization within the human body and can alter under changing conditions (e.g. pathogens, allergens, etc). Recent data revealed a novel mast cell phenotype within the alveolar tissue of patients with asthma. Increasing evidence shows a key role for alveolar mast cells in the pathophysiology of viral respiratory infections and in the development of allergen sensitization and asthma. SUMMARY: Increasing evidence points toward a key role of mast cells in the pathophysiology and pathogenesis of asthma and this warrants further investigation and the development of effective targeted therapies.

Alveolar T-helper type-2 immunity in atopic asthma is associated with poor clinical control.

Real-world evaluation studies have shown that many patients with asthma remain symptomatic despite treatment with inhaled corticosteroids (ICSs). As conventional ICSs have poor access to the peripheral airways, the aim of the present paper was to study the relationship between peripheral airway inflammation and clinical control in allergic asthma. Consequently, bronchial and transbronchial biopsies were obtained from patients with poorly controlled asthma [n=12, asthma control test (ACT) score<20], patients with well-controlled asthma (n=12, ACT score≥20) and healthy controls (n=8). Tissue sections were immunostained to assess multiple leucocyte populations. To determine the degree of T-helper type-2 (Th2) immunity, the logarithmic value of the ratio between Th2 cells/mm2 and Th1 cells/mm2 was used as a surrogate score for Th2-skewed immunity. In the bronchi, the leucocyte infiltration pattern and the Th2-score were similar between patients with well-controlled asthma and those with poorly controlled asthma. In contrast, in the alveolar parenchyma, the expression of T-helper cells was significantly higher in patients with poorly controlled asthma than in patients with well-controlled asthma (P<0.01). Furthermore, the alveolar Th2-score was significantly higher in patients with poorly controlled asthma (median 0.4) than in the controlled patients (median -0.10, P<0.05). In addition, in contrast with bronchial Th2-score, the alveolar Th2-score correlated significantly with ACT score (rs=-0.62, P<0.01) in the pooled asthma group. Collectively, our data reveal an alveolar Th2-skewed inflammation, specifically in asthmatic patients who are poorly controlled with ICSs, and suggest that pharmacological targeting of the peripheral airways may be beneficial in this large patient category.

Controlled and uncontrolled asthma display distinct alveolar tissue matrix compositions.

OBJECTIVE: Whether distal inflammation in asthmatics also leads to structural changes in the alveolar parenchyma remains poorly examined, especially in patients with uncontrolled asthma. We hypothesized that patients who do not respond to conventional inhaled corticosteroid therapy have a distinct tissue composition, not only in central, but also in distal lung. METHODS: Bronchial and transbronchial biopsies from healthy controls, patients with controlled atopic and patients with uncontrolled atopic asthma were processed for immunohistochemical analysis of fibroblasts and extracellular matrix molecules: collagen, versican, biglycan, decorin, fibronectin, EDA-fibronectin, matrix metalloproteinase (MMP)-9 and tissue-inhibitor of matrix metalloproteinase (TIMP)-3. RESULTS: In central airways we found increased percentage areas of versican and decorin in patients with uncontrolled asthma compared to both healthy controls and patients with controlled asthma. Percentage area of biglycan was significantly higher in both central airways and alveolar parenchyma of patients with uncontrolled compared to controlled asthma. Ratios of MMP-9/TIMP-3 were decreased in both uncontrolled and controlled asthma compared to healthy controls. In the alveolar parenchyma, patients with uncontrolled asthma had increased percentage areas of collagen, versican and decorin compared to patients with controlled asthma. Patients with uncontrolled asthma had significantly higher numbers of myofibroblasts in both central airways and alveolar parenchyma compared to patients with controlled asthma. CONCLUSIONS: Tissue composition differs, in both central and distal airways, between patients with uncontrolled and controlled asthma on equivalent doses of ICS. This altered structure and possible change in tissue elasticity may lead to abnormal mechanical properties, which could be a factor in the persistent symptoms for patients with uncontrolled asthma.