Background Asthma is a chronic inflammatory disease with structural changes in the lungs defined as airway remodelling. Mast cell responses are important in asthma as they, upon activation, release mediators inducing bronchoconstriction, inflammatory cell recruitment, and often remodelling of the airways. As guinea pigs exhibit anatomical, physiological, and pharmacological features resembling human airways, including mast cell distribution and mediator release, we evaluated the effect of extracts from two common allergens, house dust mite (HDM) and cat dander (CDE), on histopathological changes and the composition of tryptase- and chymase-positive mast cells in the guinea pig lungs. Methods Guinea pigs were exposed intranasally to HDM or CDE for 4, 8, and 12 weeks, and airway histology was examined at each time point. Hematoxylin and eosin, Picro-Sirius Red, and Periodic Acid-Schiff staining were performed to evaluate airway inflammation, collagen deposition, and mucus-producing cells. In addition, Astra blue and immunostaining against tryptase and chymase were used to visualize mast cells. Results Repetitive administration of HDM or CDE led to the accumulation of inflammatory cells into the proximal and distal airways as well as increased airway smooth muscle mass. HDM exposure caused subepithelial collagen deposition and mucus cell hyperplasia at all three time points, whereas CDE exposure only caused these effects at 8 and 12 weeks. Both HDM and CDE induced a substantial increase in mast cells after 8 and 12 weeks of challenges. This increase was primarily due to mast cells expressing tryptase, but not chymase, thus indicating mucosal mast cells. Conclusions We here show that exposure to HDM and CDE elicits asthma-like histopathology in guinea pigs with infiltration of inflammatory cells, airway remodelling, and accumulation of primarily mucosal mast cells. The results together encourage the use of HDM and CDE allergens for the stimulation of a clinically relevant asthma model in guinea pigs.

Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth–death regulation.

Introduction: Epithelial remodelling and dysfunction are characteristics for asthmatic disease. In asthma, mast cells (MCs) infiltrate the epithelium and alveolar parenchyma. However, the MC-epithelium crosstalk is not known. We aim to study if MC proteases tryptase and chymase induce bronchial and alveolar remodelling in a viral environment. Methods: Human bronchial and alveolar epithelial cells were treated with MC tryptase and/or chymase and co-stimulated with toll-like receptor agonists to mimic a pro-inflammatory/viral setting. Cell growth were assessed using holographic live cell imaging and a profiler gene array associated with cell survival. Data was confirmed with qPCR, western blot and immunocytochemistry. The epithelial release of vascular epithelial growth factor (VEGF) and fibroblastic growth factors (FGF) was analysed. Results: Tryptase enhanced cell growth and release of VEGF and FGF in bronchial and alveolar cells when compared to non-stimulated cells, both in normal and pro-inflammatory/viral condition. Anti-apoptotic proteins BIRC3 and BFL-1 was increased in tryptase treated cells, both at gene and protein level. No alterations was found in chymase treated cells compared to non-stimulated cells. Conclusion: We show that MC tryptase, but not chymase, increases cell growth, anti-apoptotic gene and protein expression as well as release of growth factors in bronchial and alveolar epithelial cells. These effects remained elevated in a pro-inflammatory and viral milieu. Thus, our data implies that MC release of tryptase may play a critical role in airway epithelial and alveolar homeostasis by disturbing cell growth-death regulations.

Mast cells (MCs) are distributed in tissues throughout the body and are highly involved in many physiological and pathophysiological processes. The potential and involvement of different MC phenotypes are still not well understood. MCs are present in blood vessel walls, but their specific phenotypic features are unknown. We aimed at characterizing MCs from human saphenous veins for localization, mediator content, and receptor expression. This was done in MCs from both healthy and varicose human saphenous veins (hSV and vSV, respectively). For both vSV and hSV, we found that vein MCs are mainly present in the tunica adventitia (99% MCs in adventitia) and that the population consists of both MCT and MCTC phenotypes (vSV: 55% MCT, hSV: 64% MCT). The vein MCs contained high levels of histamine (vSV: 27 pg/MC, hSV: 55 pg/MC) and tryptase (vSV: 98 pg/MC, hSV: 111 pg/MC), indicating a strong potential for regulatory effects on blood vessels. The receptor expression of FcεRI, MRGPRX2, PTAFR, C3aR, and C5aR was found, even though the percentage of positive cells differed between vSV and hSV MCs. We conclude that vein MCs from the blood vessel wall have a high potential to affect the tissue around them.

Introduction: Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma. The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated. Aim: To investigate the effect of tryptase on epithelial cell migration and proliferation in a wound gap model. Methods: Human bronchial epithelial cells (HBECs) were treated with tryptase and/or PAR2 inhibitor I-191. Gap closure, migration, migratory direction, cell speed and cell length were studied using a novel live cell imaging technique. Proliferation and migration markers were analyzed using qPCR, ELISA, Luminex and immunocytochemistry. Results: Stimulation of HBECs with tryptase promoted gap closure (P<0.01), migration (P<0.05) and cellular speed (P<0.05) compared to controls. Stimulated HBECs had higher expression of migration marker CD151 compared to controls (P<0.05). Proliferation marker KI67 was upregulated in tryptase stimulated HBECs compared to controls (P<0.01). Treatment with I-191 reduced gap closure (P<0.05), migration (P<0.0001) and cell speed (P<0.001) compared to HBECs stimulated with tryptase. Proliferation was however not significantly altered by PAR2 inhibition. Conclusion: We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2. Our data suggests that tryptase might be beneficial in tissue repair under baseline conditions. However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodelling and loss of function.

Abstract 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.

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.

Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma. The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated. Stimulation of bronchial epithelial cells (BECs) with tryptase promoted gap closure, migration and cellular speed compared to controls. Stimulated BECs had higher expression of migration marker CD151 compared to controls. Proliferation marker KI67 was upregulated in tryptase-stimulated BECs compared to controls. Treatment with PAR2 antagonist I-191 reduced gap closure, migration and cell speed compared to BECs stimulated with tryptase. We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2. Our data suggest that tryptase might be beneficial in tissue repair under baseline conditions. However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodeling and loss of function.

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.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.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.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.

Background: The inflammatory response in asthma is a complex interaction between structural cells and inflammatory cells. Increased mast cell (MC) densities in the airway epithelium is a hallmark o ...

Background: Increased mast cell (MC) densities in the airway epithelium is a hallmark of asthma. However, little is known regarding the role and effect of MC mediators on epithelium damaged by respiratory viruses. Aim: To investigate the density of MCs in biopsies in patients with different asthma phenotypes and the effect of MC mediator tryptase on wound-healing in bronchial epithelial cells. Methods: Intraepithelial MCs (IEMCs) were stained for tryptase (MCT) and tryptase/chymase (MCTC) in bronchial biopsies. In a BEAS-2B cell line, the effect of tryptase and the TLR3 agonist polyI:C on wound healing was studied using live cell imaging (Phase Holographic Imaging). Results: MCT, but not MCTC, were increased in the epithelium in ICS-naïve mild asthma and ICS-responsive asymptomatic moderate and severe asthma when compared to ICS-unresponsive symptomatic moderate and severe asthmatics (p=0.03). Within the asthma groups, patients with atopy had the highest numbers of IEMCs compared to non-atopic asthmatics and controls (p=0.002). Stimulation of BEAS-2B with polyI:C resulted in impaired wound gap closure compared to untreated cells (p=0.05). However, MC tryptase enhanced cell proliferation and wound healing responses when compared to cells stimulated with polyI:C (p<0.0001) and untreated control (p=0.0007). Conclusion: We showed that asymptomatic patients with different severities of asthma had a higher density of IEMCs regardless of treatment with ICS. Further, the MC mediator tryptase promoted epithelial proliferation and wound healing responses. Hence, IEMCs may have beneficial roles and MC localization may be an important determinant in asthma pathology.