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Background: The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma., Methods: We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance., Results: PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding., Conclusions: In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair., Competing Interests: The authors have declared that no competing interests exist.

Background: House dust mite (HDM) acts on the airway epithelium to induce airway inflammation in asthma. We previously showed that the ability of HDM to induce allergic sensitization in mice is related to airway epithelial CCL20 secretion., Objective: As a disintegrin and metalloprotease (ADAM)s have been implicated in chemokine shedding, we sought to determine their involvement in HDM-induced release of chemokines, including CCL20, by airway epithelial cells., Methods: We studied the effects of pharmacological ADAM inhibitors as well as ADAM10 and ADAM17 siRNA downregulation on chemokine release using (multiplex) ELISA in supernatants from HDM-exposed human bronchial epithelial 16HBE cells and primary normal human bronchial epithelial cells (NHBE) at 4-24 h., Results: House dust) mite markedly increased CCL20 levels in both 16HBE and NHBE cells (16-24 h). In 16HBE cells, the HDM-induced increase was observed as early as 4 h upon exposure and the use of specific inhibitors indicated the involvement of ADAM10/17-mediated shedding. siRNA knockdown of ADAM10, but not of ADAM17, significantly reduced the HDM-induced release of CCL20 in both 16HBE and NHBE cells. A similar effect was observed for HDM-induced CCL2, CCL5, and CXCL8 release in NHBE cells. The HDM-induced increase in CCL20 levels was not affected by protein synthesis inhibitor cycloheximide nor protein transport inhibitor monensin, indicating that HDM induces surface shedding of chemokines., Conclusion: Our data show for the first time that ADAM10 activity contributes to HDM-induced shedding of chemokines, including CCL20. The ADAM10/CCL20 axis may be a target for novel therapeutic strategies in asthma., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The integrity of the airway epithelium in patients with asthma is often disrupted, with loss of epithelial cell-cell contacts. Airway epithelial barrier dysfunction may have important implications for asthma, because structural epithelial barrier function is tightly interwoven with the ability of the epithelium to regulate the immune system. We propose that changes at the airway epithelial barrier play a central role in the sensitisation to allergens and pathogenesis of allergic asthma. Many of the recently identified susceptibility genes for asthma are expressed in airway epithelium. However, the exact mechanisms by which the expression of epithelial susceptibility genes translates into a functionally altered response to aeroallergens in asthma are still unknown. In this review, we will focus on the role of airway epithelial barrier function in the susceptibility to develop allergic asthma and discuss therapeutic strategies aimed at the epithelial barrier., (© 2014 John Wiley & Sons Ltd.)

Background: House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation., Objectives: The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo., Methods: Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model., Results: The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity., Conclusions: This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

A subset of asthma patients suffer from glucocorticoid (GC) insensitivity. T-helper cell type 17 cells have an emerging role in GC insensitivity, although the mechanisms are still poorly understood. We investigated whether interleukin (IL)-17A induces GC insensitivity in airway epithelium by studying its effects on responsiveness of tumour necrosis factor (TNF)-α-induced IL-8 production to budesonide in human bronchial epithelial 16HBE cells. We unravelled the underlying mechanism by the use of specific pathway inhibitors, reporter and overexpression constructs and a histone deacetylase (HDAC) activity assay. We demonstrated that IL-17A-induced IL-8 production is normally sensitive to GCs, while IL-17A pre-treatment significantly reduced the sensitivity of TNF-α-induced IL-8 production to budesonide. IL-17A activated the p38, extracellular signal-related kinase (ERK) and phosphoinositide-3-kinase (PI3K) pathways, and the latter appeared to be involved in IL-17A-induced GC insensitivity. Furthermore, IL-17A reduced HDAC activity, and overexpression of HDAC2 reversed IL-17A-induced GC insensitivity. In contrast, IL-17A did not affect budesonide-induced transcriptional activity of the GC receptor, suggesting that IL-17A does not impair the actions of the ligated GC receptor. In conclusion, we have shown for the first time that IL-17A induces GC insensitivity in airway epithelium, which is probably mediated by PI3K activation and subsequent reduction of HDAC2 activity. Thus, blockade of IL-17A or downstream signalling molecule PI3K may offer new strategies for therapeutic intervention in GC-insensitive asthma.

T helper (Th) 2 lymphocytes play a crucial role in the initiation, progression and persistence of allergic diseases, including asthma. Drugs that interfere with the activation of T-cells or more selectively Th2-specific signaling molecules and drugs that prevent the selective migration into lung tissue are promising novel strategies for the treatment of allergic asthma. Although the mainstay asthma therapy of inhaled glucocorticoids is rather effective, targeting Th2 cells may be an important alternative in childhood. Regulatory T-cells (Treg cells) have a physiological role in protection of unwanted immune responses to auto-antigens and allergens. Literature data indicate that an imbalance between Th2 and Treg cells may underlie development and disease expression of allergic asthma. Drugs or immunotherapies that stimulate these counter-Treg cells may limit aberrant Th2 responses leading to suppression of symptoms. Furthermore, these types of treatments may offer the perspective of disease modification and long-term relief of symptoms.

Background: Airway inflammation in asthma is orchestrated by recruitment of T helper (Th)2 lymphocytes to the lung and subsequent production of Th2-like cytokines upon allergen challenge., Objective: To examine whether allergen-induced dysfunction of the beta2-adrenergic receptor (beta2-AR) contributes to the enhanced T(h2) cell activity in asthma., Methods: Beta2-adrenergic regulation of cytokine mRNA expression was studied in alpha-CD3/alpha-CD28-activated peripheral blood lymphocytes from seven asthma patients before and 6 h after allergen challenge, in conjunction with the effects of beta2-agonist fenoterol on T cell chemotaxis and signalling pathways., Results: A complete loss of beta2-AR control over expression of the Th2 cytokines IL-4, IL-5 and IL-13, but not of the Th1 cytokine IFN-gamma, was observed after allergen challenge. Furthermore, we found impaired beta2-AR regulation of T cell migration as well as signal transduction pathways, i.e. the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein and the inhibition of the mitogen-activated protein kinase pathway. The loss of beta2-AR control was associated with increased beta-adrenergic receptor kinase expression, which might be involved in beta2-AR desensitization. In addition, we demonstrate for the first time that T cells exposed to the chemokine thymus and activation-regulated chemokine show hyporesponsiveness to fenoterol., Conclusion: Our results suggest that allergen-induced loss of beta2-AR control, possibly mediated by chemokine release, plays an important role in enhanced Th2-like activity in asthma.

Background House dust mite ( HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown. Objective Given the implications for adenosine triphosphate ( ATP)-dependent Ca2+ signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca2+ concentration ([ Ca2+]i) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium. Methods We investigated the effect of HDM on accumulation of [ Ca2+]i levels, barrier function, and CCL20 release in human bronchial epithelial 16 HBE cells and primary bronchial epithelial cells ( PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca2+ influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA- AM, and calpain inhibitors. Results Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca2+ influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16 HBE cells and the HDM-induced barrier dysfunction in 16 HBE cells are dependent on [ Ca2+]i accumulation. Additionally, we demonstrate that [ Ca2+]i accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion. Conclusion Our data show for the first time that Ca2+ signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma. [ABSTRACT FROM AUTHOR]

Airway inflammation in asthma is orchestrated by recruitment of T helper (Th)2 lymphocytes to the lung and subsequent production of Th2-like cytokines upon allergen challenge. To examine whether allergen-induced dysfunction of the β2-adrenergic receptor (β2-AR) contributes to the enhanced T(h2) cell activity in asthma. β2-adrenergic regulation of cytokine mRNA expression was studied in α-CD3/α-CD28-activated peripheral blood lymphocytes from seven asthma patients before and 6 h after allergen challenge, in conjunction with the effects of β2-agonist fenoterol on T cell chemotaxis and signalling pathways. A complete loss of β2-AR control over expression of the Th2 cytokines IL-4, IL-5 and IL-13, but not of the Th1 cytokine IFN-γ, was observed after allergen challenge. Furthermore, we found impaired β2-AR regulation of T cell migration as well as signal transduction pathways, i.e. the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein and the inhibition of the mitogen-activated protein kinase pathway. The loss of β2-AR control was associated with increased β-adrenergic receptor kinase expression, which might be involved in β2-AR desensitization. In addition, we demonstrate for the first time that T cells exposed to the chemokine thymus and activation-regulated chemokine show hyporesponsiveness to fenoterol. Our results suggest that allergen-induced loss of β2-AR control, possibly mediated by chemokine release, plays an important role in enhanced Th2-like activity in asthma. [ABSTRACT FROM AUTHOR]