Nivenka Jayasekera

TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation

BACKGROUND Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. OBJECTIVES We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. METHODS Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. RESULTS TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. CONCLUSIONS Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.

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.

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.

Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

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 …

S42 Airway epithelial toll receptor expression in asthma and its relationship to disease severity

Introduction Asthma is classically considered a Th2 mediated disease. However, severe and treatment-resistant disease is more heterogeneous and often associated with airway neutrophil recruitment. This may be related to an altered airway bacterial colonisation. Bacteria express pathogen associated molecular patterns (PAMPs) that are recognised as non-self by pattern recognition receptors (PRRs). These PRRs represent an essential component of the innate immunity and an important family of PPRs are the Toll-like receptors (TLR). These are expressed on a range of innate immune cells including epithelial cells. TLR-1, -2, -4, -5 and -6 are located on the cell surface membrane and respond to bacterial cell wall components. This study has investigated the expression of mRNA for TLR-2, -4 and -5 in airway epithelial cells in asthmatics and healthy volunteers. Methods Epithelial brushings were obtained from the large central airways at fibre-optic bronchoscopy from 18 healthy non-asthmatic volunteers (8 female and mean age 26 years) and 34 asthmatic volunteers (25 female, mean age 43 years). The asthmatic group comprised 7 non-steroid treated and 27 steroid treated asthmatics. The brushings were placed in Trizol and the RNA subsequently extracted and converted to cDNA, prior to real time TaqMan RT-qPCR analysis for the Toll receptors, TLR2, TLR4 and TLR5, as well as IL-8. Results Gene expression for TLR-2 (p=0.008) and TLR-4 (p=0.012) was significantly increased within the epithelial brushing sample from the asthmatics compared to the healthy control subjects whilst that for TLR-5 did not differ significantly. Interleukin 8 mRNA was also increased within the epithelial brushing sample in the asthmatics (p=0.007) compared to that in the healthy control subjects. These significant differences from the healthy population were also individually present in both the mild and severe asthmatic groups, with no significant difference being evident between mild and severe asthma. Conclusions These findings reveal up regulation of epithelial gene expression for members of the Toll receptor family relevant to bacterial responses within the airways. Additionally there is enhanced IL-8 gene expression. These features are indicative of on-going innate immune airway responses in asthma. The relevance of this to clinical disease expression requires understanding.

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.