Patrick Dennison

Barrier responses of human bronchial epithelial cells to grass pollen exposure

The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3&agr;, CCL22/monocyte-derived chemokine and tumour necrosis factor-&agr; was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-&ggr;-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.

Transforming Growth Factor-Beta Promotes Rhinovirus Replication in Bronchial Epithelial Cells by Suppressing the Innate Immune Response

Rhinovirus (RV) infection is a major cause of asthma exacerbations which may be due to a deficient innate immune response in the bronchial epithelium. We hypothesized that the pleiotropic cytokine, TGF-β, influences interferon (IFN) production by primary bronchial epithelial cells (PBECs) following RV infection. Exogenous TGF-β2 increased RV replication and decreased IFN protein secretion in response to RV or double-stranded RNA (dsRNA). Conversely, neutralizing TGF-β antibodies decreased RV replication and increased IFN expression in response to RV or dsRNA. Endogenous TGF-β2 levels were higher in conditioned media of PBECs from asthmatic donors and the suppressive effect of anti-TGF-β on RV replication was significantly greater in these cells. Basal SMAD-2 activation was reduced when asthmatic PBECs were treated with anti-TGF-β and this was accompanied by suppression of SOCS-1 and SOCS-3 expression. Our results suggest that endogenous TGF-β contributes to a suppressed IFN response to RV infection possibly via SOCS-1 and SOCS-3.

Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.

The Effects on Bronchial Epithelial Mucociliary Cultures of Coarse, Fine, and Ultrafine Particulate Matter From an Underground Railway Station

We have previously shown that underground railway particulate matter (PM) is rich in iron and other transition metals across coarse (PM10–2.5), fine (PM2.5), and quasi-ultrafine (PM0.18) fractions and is able to generate reactive oxygen species (ROS). However, there is little knowledge of whether the metal-rich nature of such particles exerts toxic effects in mucus-covered airway epithelial cell cultures or whether there is an increased risk posed by the ultrafine fraction. Monolayer and mucociliary air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) were exposed to size-fractionated underground railway PM (1.1–11.1 µg/cm2) and release of lactate dehydrogenase and IL-8 was assayed. ROS generation was measured, and the mechanism of generation studied using desferrioxamine (DFX) and N-acetylcysteine (NAC). Expression of heme oxygenase-1 (HO-1) was determined by RT-qPCR. Particle uptake was studied by transmission electron microscopy. Underground PM increased IL-8 release from PBECs, but this was diminished in mucus-secreting ALI cultures. Fine and ultrafine PM generated a greater level of ROS than coarse PM. ROS generation by ultrafine PM was ameliorated by DFX and NAC, suggesting an iron-dependent mechanism. Despite the presence of mucus, ALI cultures displayed increased HO-1 expression. Intracellular PM was observed within vesicles, mitochondria, and free in the cytosol. The results indicate that, although the mucous layer appears to confer some protection against underground PM, ALI PBECs nonetheless detect PM and mount an antioxidant response. The combination of increased ROS-generating ability of the metal-rich ultrafine fraction and ability of PM to penetrate the mucous layer merits further research.

Buckwheat allergy: a potential problem in 21st century Britain

Buckwheat is commonly consumed in many parts of the world and has recently become more available in the UK. Buckwheat allergy is well recognised in parts of mainland Europe and Asia, typically associated with consumption of specific regional foods. No adult cases of buckwheat allergy in the UK have been reported in the literature. The authors present two cases of buckwheat allergy that presented to our UK allergy service recently. A 57-year-old man presented with anaphylaxis after eating home-baked bread prepared using buckwheat flour bought in France. In the second case, a 63-year-old lady presented with bronchospasm and urticaria after consuming health-food muesli. Sensitisation was confirmed in both cases by positive skin prick testing and specific IgE to buckwheat. Given the growing popularity of foods that may contain buckwheat, including ethnic and health-food ranges, buckwheat allergy is likely to become increasingly common in the UK.

IL-1α mediates cellular cross-talk in the airway epithelial mesenchymal trophic unit

ABSTRACT The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection.

Case series reporting the effectiveness of mycophenolate mofetil in treatment-resistant asthma

To the Editor: Treatment-resistant asthma represents a significant unmet clinical need. Due to lack of therapeutic options many patients receive treatment with long-term oral steroids despite appreciation of the associated systemic adverse effects. While anti-IgE therapy with omalizumab affords benefit and there have been encouraging outcomes with monoclonal antibodies against interleukin (IL)-5 and IL-13 [1, 2], all such treatments target T-helper cell type 2 (Th2) orientated disease in a sub-population of patients. Mycophenolate mofetil (MMF) is a reversible inhibitor of inosine monophosphate dehydrogenase, an enzyme required by T- and B-cells for guanine synthesis [3]. It is most commonly used to prevent transplanted organ rejection, but is increasingly prescribed to treat inflammatory disorders such as connective tissue disorders and systemic vasculitides. In January 2009, a patient in our institution with lifelong severe treatment-resistant asthma, poorly controlled despite high-dose inhaled corticosteroids, regular oral steroids and nebulised bronchodilators, was prescribed MMF for co-existent undifferentiated vasculitis. Over the following year his disease improved dramatically with a reduction in hospital admissions, enabling a reduction in oral steroids with a concurrent decrease in weight, improvement in quality of life and exercise capacity. In view of this experience, another individual with severe asthma resistant to all standard therapies was placed on MMF. This patient also showed a dramatic clinical improvement. Subsequently we treated a further 20 patients with MMF with varying results to give a case series of 22, which we report here. All patients started on MMF had been under specialist asthma care for at least 12 months and despite step 4 or 5 of asthma guidelines management had poor control, as measured by the asthma control questionnaire (ACQ), and …

S116 MicroRNA Regulation of Toll-Like Receptor 7 Function in Severe Asthma: Relevance to Viral Responses

Background and Aims Viral-induced disease exacerbation is common in asthma and studies have identified that both bronchial epithelial cells and alveolar macrophages (AM) from asthmatics have a reduced interferon (IFN) response to rhinovirus infection. The mechanism behind this defect is unclear. As asthmatic peripheral blood mononuclear cells have been reported to have defective toll-like receptor (TLR) 7 function, we investigated the expression of microRNAs (miRNAs) in AM from healthy control (HC) and severe asthma (SA) volunteers with relevance to TLR7-viral interactions. MicroRNAs are non-coding RNAs that down-regulate gene expression by suppressing translation. We identified and focused on 3 miRNAs that could target TLR7. Additionally, we investigated if manipulating the expression of these miRNAs can ameliorate the defective IFN response in AM. Methods 26 HC and 30 patients with SA (BTS Step 4/5) were recruited for bronchoscopy. AM were isolated from bronchoalveolar lavage using the adherence to plastic technique. Expression of miRNAs and TLR7 was determined by qRT-PCR and western blotting. AM were transfected with a combination of antagomirs, specifically directed against the 3 miRNAs, and then treated with imiquimod (5ug/ml), a TLR7 agonist, or human rhinovirus-16 (HRV16) and IFN-β expression was determined after 24 hours using qRT-PCR and ELISA. Results Expression of all three miRNAs was significantly increased in SA compared to HC TLR7 mRNA was found to be significantly reduced in AM from volunteers with SA compared to HC. Western blotting confirmed reduced expression of TLR7 protein in AM from SA compared to HC. Compared to mock transfected AM, AM transfected with the 3 antagomirs showed significantly increased imiquimod-induced IFN-β mRNA and protein expression and significantly increased HRV16-induced IFN-β mRNA production. Conclusion TLR7 expression is significantly reduced in SA compared to HC. The differential expression of the miRNAs identified may lead to impaired viral sensing by asthmatic AM and contribute to the defective IFN response to rhinovirus. Importantly, TLR7 induced IFN-β production by human AM can be significantly augmented by inhibition of these miRNAs. The identification of these miRNAs and our ability to manipulate their expression in human AM offers the potential for future miRNA-based therapies in asthma.

In vitro, in silico and in vivo study challenges the impact of bronchial thermoplasty on acute airway smooth muscle mass loss

Bronchial thermoplasty is a treatment for asthma. It is currently unclear whether its histopathological impact is sufficiently explained by the proportion of airway wall that is exposed to temperatures necessary to affect cell survival. Airway smooth muscle and bronchial epithelial cells were exposed to media (37–70°C) for 10 s to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution post-thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 patients with severe asthma. In vitro airway smooth muscle and epithelial cell number decreased significantly following the addition of media heated to ≥65°C. In silico simulations showed a heterogeneous heat distribution that was amplified in larger airways, with <10% of the airway wall heated to >60°C in airways with an inner radius of ∼4 mm. In vivo at 6 weeks post-thermoplasty, there was an improvement in asthma control (measured via Asthma Control Questionnaire-6; mean difference 0.7, 95% CI 0.1–1.3; p=0.03), airway smooth muscle mass decreased (absolute median reduction 5%, interquartile range (IQR) 0–10; p=0.03) and epithelial integrity increased (14%, IQR 6–29; p=0.007). Neither of the latter two outcomes was related to improved asthma control. Integrated in vitro and in silico modelling suggest that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating, and nor did this reduction confer a greater improvement in asthma control. Bronchial thermoplasty treatment for asthma has unexpected possible mechanisms of action http://ow.ly/ZcuE30jsaSa