Rhinoviruses are the major trigger of acute asthma exacerbations and asthmatic subjects are more susceptible to these infections. To investigate the underlying mechanisms of this increased susceptibility, we examined virus replication and innate responses to rhinovirus (RV)-16 infection of primary bronchial epithelial cells from asthmatic and healthy control subjects.Viral RNA expression and late virus release into supernatant was increased 50- and 7-fold, respectively in asthmatic cells compared with healthy controls. Virus infection induced late cell lysis in asthmatic cells but not in normal cells. Examination of the early cellular response to infection revealed impairment of virus induced caspase 3/7 activity and of apoptotic responses in the asthmatic cultures. Inhibition of apoptosis in normal cultures resulted in enhanced viral yield, comparable to that seen in infected asthmatic cultures. Examination of early innate immune responses revealed profound impairment of virus-induced interferon-β mRNA expression in asthmatic cultures and they produced >2.5 times less interferon-β protein. In infected asthmatic cells, exogenous interferon-β induced apoptosis and reduced virus replication, demonstrating a causal link between deficient interferon-β, impaired apoptosis and increased virus replication. These data suggest a novel use for type I interferons in the treatment or prevention of virus-induced asthma exacerbations.

Epithelial damage and airway remodeling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. To examine the mechanisms that control bronchial epithelial repair, we investigated expression of the epidermal growth factor receptor (c-erbB1, EGFR) in asthmatic bronchial mucosa and studied repair responses in vitro. In biopsies from asthmatic subjects, areas of epithelial damage were frequently observed and exhibited strong EGFR immunostaining. EGFR expression was also high in morphologically intact asthmatic epithelium. Using image analysis, EGFR immunoreactivity (% of total epithelial area, median (range) was found to increase from 9.4 (4.1-20.4) in normal subjects (n=10) to 18.4 (9.3-28.9) in mild asthmatics (P<0.01, n=13) and 25.4 (15.4-31.8) in severe asthmatics (P<0.00, n=5). Epithelial EGFR immunoreactivity remained elevated in patients treated with corticosteroids and was positively correlated with subepithelial reticular membrane thickening. Using 16HBE 14o- bronchial epithelial cells, we found that EGF accelerated repair of scrape-wounded monolayers and that the EGFR-selective inhibitor, tyrphostin AG1478, inhibited both EGF-stimulated and basal wound closure whereas dexamethasone was without effect. Intrinsic activation of the EGFR was confirmed by analysis of tyrosine phosphorylated proteins, which revealed a rapid, damage-induced phosphorylation of the EGFR, irrespective of the presence of exogenous EGF. To assess the relationship between EGFR-mediated repair and tissue remodeling, release of the profibrogenic mediator TGF-beta2 was also measured. Scrape wounding increased release of TGF-beta2 from epithelial monolayers and EGF had no additional stimulatory effect. However, when repair was retarded with AG1478, the amount of TGF-beta2 increased significantly. These data indicate that the EGFR may play an important role in bronchial epithelial repair in asthma and that impairment of this function may augment airway remodeling.

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Asthma is a complex disease involving gene and environment interactions (Holgate). Although atopy is a strong predisposing Rationale risk factor for asthma, local tissue susceptibilities also contribute to disease expression (Moffitt). The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction complexes (Swindle et al, 2009). To explain the link between environment exposures and airway vulnerability, our previous studies based on a small number of volunteers have shown that tight junctions are abnormal in asthma. To confirm the previous findings and strengthen the concept of defective epithelial barrier function in asthma, we studied a much bigger population of asthmatics to assess their epithelial barrier integrity and permeability.Non-asthmatic normal and asthmatic subjects were recruited and clinically characterised in accordance with GINA guidelines. Methods:Bronchial brushings were obtained by fibreoptic bronchoscopy following ethical approval and informed consent. Primary bronchial epithelial cells (pBECs) were expanded from brushings then seeded onto collagen I coated porous transwells and differentiated at in vitro an air-liquid interface (ALI) for 3-4 weeks. Transepithelial electrical resistance (TER) was monitored as an indication of tight junction formation and epithelial integrity using a set of chopstick electrodes and a voltohmmeter. Paracellular permeability was quantified by FITC-dextran flux assay.We studied differentiated bronchial epithelial cultures (n=83) grown from bronchial brushings obtained from normal (40) Results:in vitro or asthmatic (n=43) volunteers. Tight junction function assessed by TER measurement was significantly lower in cultures from asthmatic donors compared to normals (median (interquartile range) = 346 (248-536) Ω·cm 428 (340-593) -analysis of the data revealed that 2 versus TER was significantly decreased as a function of disease severity (p<0.05). TER measurements were inversely correlated with permeability to fluorescently conjugated 20 or 4 kDa dextrans indicating alterations to both ionic and macromolecular permeability in asthma.Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of low Conclusions: molecular weight allergens and other agents into the airway tissue leading to immune activation and may thus contribute to the end organ expression of asthma. This work suggests that addressing the barrier defect in asthma may offer a novel therapeutic approach for difficult-to-treat asthmatic subjects who fail to respond to conventional therapy. This abstract is funded by:

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Asthma is regarded as an inflammatory disorder of the conducting airways characterized by a mast cell, eosin­ophil and T lymphocyte inflammatory response that is responsive to anti‐inflammatory therapy, such as corticosteroids. In more severe and chronic disease, corticosteroids become less effective. As in other chronic inflammatory diseases, the tissue in which the cellular and mediator processes occur plays a major role in maintaining the response and creating a basis for disease persistence. Herein, we describe evidence that the airway epithelium interacting with the underlying mesenchymal cells recapitulates branching morphogenesis, as observed in the developing lung, to create airway wall remodeling. The reciprocal signaling between the susceptible epithelium and responsive mesenchyme (epithelial mesenchymal trophic unit) offers a new paradigm for asthma and creates new opportunities for developing therapeutics based on reversing the ‘chronic wound’ phenotype of asthmatic airways.

Interleukin (IL)-4 and IL-13 are key proinflammatory cytokines in asthma. Studies in transgenic mice show that both cytokines cause inflammation, but only IL-13 causes subepithelial fibrosis, a characteristic feature of asthma. We compared the in vitro profibrogenic effects of IL-4 and IL-13 using bronchial fibroblasts from asthmatic subjects. In the presence of transforming growth factor (TGF)-beta the cells transformed into contractile myofibroblasts and expressed alpha-smooth muscle actin and procollagen I. IL-4 and IL-13 also stimulated proliferation, but were relatively ineffective in promoting myofibroblast transformation. TGF-beta was more potent than the cytokines in stimulating release of endothelin-1 and vascular endothelial growth factor, whereas IL-4 and IL-13 were more potent stimuli for eotaxin release. Although neither IL-4 nor IL-13 induced profibrotic responses, both cytokines caused a corticosteroid-insensitive stimulation of TGF-beta2 release from primary bronchial epithelial cells. These data indicate that epithelial activation by IL-13 or IL-4 plays a critical role in initiating remodeling through release of TGF-beta2. TGF-beta2 then activates the underlying myofibroblasts to secrete matrix proteins and smooth muscle and vascular mitogens to propagate remodeling changes into the submucosa. In contrast, direct activation of submucosal fibroblasts by IL-4 and IL-13 has a proinflammatory effect via eotaxin release and recruitment of eosinophils into the airways.

Abnormal apoptotic mechanisms are associated with disease pathogenesis. Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis. Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining [median (range) ‫؍‬ 10.5 (1.4-24.5) versus 0.4 (0.0-9.7)]; P Ͻ 0.001). Because we were unable to determine whether this difference was due to ongoing inflammation in vivo , cultures of normal and asthmatic bronchial epithelial cells were used to study apoptosis in vitro . In complete growth medium, these cells showed no difference in their rate of proliferation or viability. However, cells from subjects with asthma were more susceptible to the apoptotic effects of H 2 O 2 than cells from normal control subjects (% apoptotic cells ‫؍‬ 32.2 [8.8-54.9] versus 14.3 [6.4-24.7]; P Ͻ 0.05), even though both were similarly affected by treatment with actinomycin D. These data indicate that the susceptibility of asthmatic bronchial epithelium to oxidants is greater than normal. This susceptibility may contribute to the rising trends in asthma associated with air pollution and diets low in antioxidants.