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J McCarthy1, X Gong2, D Nahirney2, M Duszyk2, MW Radomski11School of Pharmacy and Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Physiology, University of Alberta, Edmonton, Alberta, CanadaBackground: Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function.Methods: Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR) and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Cl- channels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches.Results: Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl- channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl- and HCO3- secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl- channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl- channels by the nanoparticles.Conclusion: This is the first study to identify the activation of ion channels in airway cells after exposure to polystyrene-based nanomaterials. Thus, polystyrene nanoparticles cannot be considered as a simple neutral vehicle for drug delivery for the treatment of lung diseases, due to the fact that they may have the ability to affect epithelial cell function and physiological processes on their own.Keywords: CFTR, cystic fibrosis transmembrane conductance regulator, ion channels, K+ channels, lung cells, polystyrene nanoparticle

Background Chemoattractant receptor-homologous molecule expressed on Th2 cells ( CRTh2) is a receptor for PGD2 and expressed by T cells, eosinophils, basophils, and ILC2 cells. CRTh2 expression by CD4+ T cells identifies the Th2 subset, and these cells have been characterized as allergen-specific central memory Th2 cells. Recently, activation of the PGD2- CRTh2 pathway in the lungs was associated with severe asthma. Objective To assess circulating levels of Th2 cells and related mediators in severe asthma and those who experience asthma exacerbations. Methods Peripheral blood cells expressing CRTh2 were characterized by flow cytometry and qRT- PCR. Serum IL-13 and PGD2 were measured by ELISA and compared with asthma severity and tendency to exacerbate. Results Severe asthmatics had more circulating CD4+ CRTh2+ T cells, CRTh2 and GATA3 mRNA, and a higher level of serum IL-13 compared to mild/moderate asthmatics. The proportion of CD4+ CRTh2+ T cells was associated with lower lung function and was highest in severe asthmatics that exacerbated in the last year. Circulating CD4+ CRTh2+ T cells, unlike eosinophils, were positively correlated with inhaled steroid dose. Conclusions and Clinical Relevance Elevated levels of circulating CD4+ CRTh2+ T cells are a feature of severe asthma, despite high-dose corticosteroids. Tracking the systemic level of these cells may help identify type 2 severe asthmatics at risk of exacerbation. [ABSTRACT FROM AUTHOR]

Background: Asthma is a complex disease with variable course. Efforts to identify biomarkers to predict asthma severity, the course of disease and response to treatment have not been very successful so far. We have previous suggested that PAR-2 and CRTh2 expression on specific peripheral blood cell subtypes may be biomarkers of asthma severity. We reasoned that parameters that remain stable when asthma symptoms are controlled would be the most appropriate to evaluate for their utility to predict loss of asthma control and/or severity of the disease., Methods: Nineteen stable asthmatics were recruited from the University of Alberta Asthma clinic and followed in clinic every 3 months for a total of 4 visits. Patients had spirometry and completed the ACQ questionnaire in every visit. Blood was drawn in every visit and analyzed for a number of immune parameters by flow cytometry. These parameters included PAR-2 and CRTh2 expression on monocyte subgroups and T lymphocytes respectively, as well as numbers of eosinophils, innate lymphoid type-2 cells (ILC2) and dendritic cells. Within person stability of immune and physiological parameters was calculated using the intraclass correlation (ICC) using R version 3.4.0., Results: FEV 1 (% predicted), FEV 1 /FVC ratio, ACQ5 and ACQ7 did not differ significantly over the 4 visits, as would be expected for patients with stable asthma. Peripheral blood eosinophil numbers by Kimura stain and by flow cytometry showed ICC scores of 0.44 and 0.52 respectively, indicating moderate stability. The % of ILC2 cells in peripheral blood also showed moderate stability [ICC score of 0.45 (0.14-0.67)]. The stability for all other immune parameters was poor., Conclusion: Among the peripheral blood immune parameters we studied, only numbers of eosinophils and ILC2 in peripheral blood were moderately stable over a year in stable asthmatics. Further studies are required to understand the reasons for the variability of the other cell types., Competing Interests: The authors declare that they have no competing interests.

Background: Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied., Methods: We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry., Results: Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations., Conclusions: PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma.

Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways.

House dust mite (HDM) allergens are the most prevalent allergens associated with asthma and rhinitis around the world. The mechanisms of allergic sensitization and allergic airway inflammation after exposure to HDM have been studied extensively, but many questions remain unanswered. Airway epithelial cells are the first line of defense against external antigens and are considered an important player in the development of allergic airway inflammation. Both genetic susceptibility to allergic sensitization and HDM composition play decisive roles in the outcome of HDM-epithelium interactions, especially regarding airway epithelial dysfunction and allergic inflammation. Interactions between HDM and the airway epithelium have consequences for both development of allergy and asthma and development of allergic airway inflammation. This review will describe in detail these interactions and will identify issues that require more study.

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, enhanced adenosine A(2A) receptor-activated transepithelial short circuit current (I(sc)), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I(sc). Cholesterol depletion had a major effect on the functional selectivity of A(2A) receptors. Under control conditions, adenosine activated I(sc) more potently than the specific A(2A) agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K(+) channels. Following cholesterol depletion, CGS-21680 activated I(sc) more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca(2+)-activated K(+) (IK1) channels. Co-immunoprecipitation experiments revealed that A(2A) receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.

Bacterial resistance to conventional antibiotics is a major challenge in controlling infectious diseases and has necessitated the development of novel approaches in antimicrobial therapy. One such approach is the use of antimicrobial peptides, such as the bacterially produced bacteriocins. Carnocyclin A (CclA) is a 60-amino acid circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 that exhibits potent activity against many Gram-positive bacteria. Lipid bilayer and single channel recording techniques were applied to study the molecular mechanisms by which CclA interacts with the lipid membrane and exerts its antimicrobial effects. Here we show that CclA can form ion channels with a conductance of 35 pS in 150 mM NaCl solution. This channel displays a linear current-voltage relationship, is anion-selective, and its activation is strongly voltage-dependent. The formation of ion channels by CclA is driven by the presence of a negative membrane potential and may result in dissipation of membrane potential. Carnocyclin A's unique functional activities as well as its circular structure make it a potential candidate for developing novel antimicrobial drugs.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel in epithelial cells; recently, we identified it in mast cells. Previous work that we confirmed showed that interferon gamma (IFNgamma) down-regulated CFTR expression in epithelial cells (T84), but by contrast, we found that IFNgamma up-regulated CFTR mRNA and protein expression in rat and human mast cells. IFNgamma up-regulation of CFTR in mast cells was inhibited by p38 and extracellular signal-regulated kinase (ERK) kinase inhibitors but not a Janus tyrosine kinase (JAK)2 inhibitor, whereas in T84 cells IFNgamma-mediated down-regulation of CFTR was JAK2-dependent and ERK- and p38-independent. Furthermore, IFNgamma down-regulation of CFTR in T84 epithelial cells was STAT1-dependent, but up-regulation of CFTR in mast cells was STAT1-independent. Thus, differential regulatory pathways of CFTR expression in mast cells and epithelial cells exist that depend upon either p38/ERK or JAK/STAT pathways, respectively. Surprisingly, IFNgamma treatment of mast cells inhibited Cl(-) efflux, in contrast to up-regulation of CFTR/mRNA and protein expression. However, down-regulation of Cl(-) flux correlated with IFNgamma-mediated inhibition of mediator secretion. This and other work suggests that the effect of IFNgamma on CFTR expression in mast cells is important for their function.

The purpose of this study was to identify Cl- channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl- channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT-PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl- flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl- channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.