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Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response.As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production.Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFNbeta and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively.dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFNbeta and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38-82) pg/ml vs 106 (57-214) pg/ml for IFNbeta (p0.05) and 114 (86-143) pg/ml vs 65 (32-119) pg/ml for TSLP (p0.05) in response to 10 microg/ml dsRNA for 24 h). Induction of TSLP mRNA by dsRNA was blocked by Toll-like receptor 3 or protein kinase inhibitors or by preventing de novo protein synthesis, but not by neutralisation of type I IFN receptors.BECs from subjects with asthma are biased towards higher TSLP and lower IFNbeta production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.

Background: Transforming growth factor beta (TGFβ) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFβ 2 favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. Methods: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGFβ 2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 “housekeeping” genes. Expression of α-smooth muscle actin (αSMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and γ-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. Results: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGFβ 2 induced mRNA expression for all five smooth muscle related transcripts; αSMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). Conclusions: Although TGFβ 2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFβ 2 is unable to induce a bona fide smooth muscle cell phenotype, it may “prime” (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

Background: In adult asthma the bronchial epithelium shows high expression of the epidermal growth factor receptor (EGFR) and the cyclin dependent kinase inhibitor, p21waf, linked to ongoing stress and injury. Methods: To determine if these are early markers of disease, sections of bronchial specimens obtained post mortem or by bronchoscopy from non-asthmatic (n = 7), moderate (n = 7), or severe (n = 9) asthmatic children aged 5–15 years were examined immunohistochemically. All severe and one moderately asthmatic children were receiving inhaled corticosteroids. Results: The lamina reticularis of the asthmatic biopsy sections was found to be thicker (p = 0.01) than normal with increased deposition of collagen III (p = 0.007); submucosal eosinophil numbers did not differ between groups. As in adults, there was an asthma-related increase in epithelial EGFR (p

Background: Considerable research has been conducted into the nature of airway inflammation in chronic obstructive pulmonary disease (COPD) but the relationship between proximal airways inflammation and both dynamic collapse of the peripheral airways and HRCT determined emphysema severity remains unknown. A number of research tools have been combined to study smokers with a range of COPD severities classified according to the GOLD criteria. Methods: Sixty five subjects (11 healthy smokers, 44 smokers with stage 0–IV COPD, and 10 healthy non-smokers) were assessed using lung function testing and HRCT scanning to quantify emphysema and peripheral airway dysfunction and sputum induction to measure airway inflammation. Results: Expiratory HRCT measurements and the expiratory/inspiratory mean lung density ratio (both indicators of peripheral airway dysfunction) correlated more closely in smokers with the severity of airflow obstruction ( r = −0.64, p

Asthma is a complex disease associated with both genetic and environmental factors such as allergens, respiratory tract infections, and atmospheric pollutants. Like rheumatoid arthritis and inflammatory bowel disease, asthma can be regarded as a chronic relapsing inflammatory disorder. Its inflammatory component involves mast cells and eosinophils as the principal effector cells, with dendritic and T cells orchestrating the response.1 2 Disease chronicity is associated with tissue destruction and airway wall remodelling which is characterised by epithelial disruption, smooth muscle and microvascular proliferation, and altered matrix deposition.2 Most asthma is associated with atopy, a predisposition to generate immunoglobulin E (IgE) against environmental allergens.3However, only a proportion of atopic individuals develop lower airways symptoms consistent with an asthmatic phenotype, although histological examination of the lower airways of atopic non-asthmatics shows these individuals to have an intermediate degree of eosinophilia and collagen deposition.4 It is therefore tempting to speculate that the development of asthma requires combined inheritance of genes which alter the immune cell response to the environment and, at the same time, render the airways structural and neural regulation susceptible to injury caused by inflammation. Several asthma/atopy associated genes have been identified from linkage and association studies3 5 and, as can be seen from table 1, many of the candidate genes are cytokines or cytokine receptors—that is, molecules capable of controlling cell function through regulation of gene expression. Similarly, environmental factors such as ozone are also known to induce gene expression6 7 through activation of transcription factors such as NfκB.8 9 Thus, it is likely that the primary genetic and environmental causes of asthma have downstream effects on expression of many other genes, and that these ultimately co-operate to determine the asthmatic phenotype (fig1). View this table: Table 1 Summary of candidate asthma susceptibility genes Figure 1 Interaction …

Introduction and objectives ADAM33 is an asthma susceptibility gene associated with bronchial hyperresponsiveness (BHR). It encodes a membrane-anchored protein with metalloprotease (MP) activity whose ectodomain can be shed from the cell surface as a soluble protein (sADAM33-MP). sADAM33-MP levels are increased in asthmatic airways and inversely correlated with FEV1. We have previously generated a pulmonary epithelium-specific, doxycycline (DOX)-inducible double transgenic (dTg) mouse expressing human (h)sADAM33-MP and found that the transgene caused airway remodelling in the absence of inflammation or BHR. Therefore, as asthma involves gene-environment interactions, we postulated that there is a synergistic relationship between ADAM33-remodelled airways and responses to the common aeroallergen, house dust mite (HDM). Methods DOX was administered to dTg mice to induce hsADAM33 expression and airway remodelling for up to 6 weeks; single transgenic (sTg) littermate controls were similarly treated. Mice were then sensitised to HDM and challenged with HDM or saline. Airway resistance was measured in response to increasing concentrations of methacholine using the forced oscillation technique in anesthetised mice. Inflammatory cell counts were performed on bronchoalveolar lavage fluid (BALF) and indices of inflammation measured by RTqPCR and Luminex ELISA. Results We first performed a concentration-response experiment with HDM extract with a standard sensitisation protocol to determine the amount of HDM extract (6.25 μg), which elicited minimal BHR and eosinophilia. This low-dose allergen challenge protocol was then applied to dTg Ccsp/ADAM33 and sTg control mice. Allergen challenge of dTg mice resulted in a significant increase in methacholine-induced airway resistance and eosinophilic airway inflammation compared to HDM-challenged sTg controls. The dTg mice also showed a significant increase in airway inflammatory mediators IL-5, IL-13 and eotaxin, in addition to markers of remodelling. Conclusions This study demonstrates that hsADAM33-MP driven airway remodelling enhances susceptibility to HDM with increases in BHR and inflammation. These functional studies demonstrate, for the first time, a gene-environment interaction involving ADAM33 to cause remodelling and the disproportional inflammatory responses seen in the asthmatic airway. sADAM33 might be a potential target for novel disease-modifying therapies.

Introduction Asthma is an inflammatory disease of the conducting airways which is exacerbated by environmental exposures, such as viral infections. Bronchial epithelial cells (BECs) together with underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) that maintains normal tissue homeostasis. In asthma the EMTU is dysregulated. Recent evidence suggests that viral infections activate the epithelial barrier resulting in mediator release which could potentially activate fibroblasts. Therefore, we hypothesised that exposure to viruses activates inflammatory and anti-viral responses in the EMTU. Methods The EMTU was modelled using a co-culture system of polarised BECs (16HBE14o-) and fibroblasts (MRC5s) maintained on the apical and basolateral surface of a nanoporous membrane respectively. After 6 days the model was challenged apically with poly (I:C) (a viral mimetic) and barrier responses were determined by measuring transepithelial resistance (TER) while cytokine release was determined by ELISA. Results Following poly (I:C) stimulation a significant reduction in TER was observed in both the EMTU model and BEC monocultures. However, the EMTU model maintained a higher TER at 6–24 h after challenge. With regards to cytokine secretion, poly (I:C) stimulation significantly induced pro-inflammatory (IL-6, IL-8, GM-CSF and IL-1α) and anti-viral (IP-10) mediator release from BEC but not fibroblast monocultures. In the EMTU model, basolateral IL-6, IL-8, GM-CSF and IP-10 responses to poly (I:C) were significantly enhanced compared to BEC monocultures. In addition, basolateral pro-inflammatory (IL-6, IL-8 and GM-CSF) but not antiviral (IP-10) responses to poly (I:C) were abrogated in the EMTU model after pre-incubation with IL-1 receptor antagonist (IL-1ra). Furthermore, direct stimulation with IL-1α induced IL-6 and IL-8 release in the EMTU model and fibroblast monocultures but not in BEC monocultures. Conclusions Poly (I:C) activates inflammatory and anti-viral responses in BEC monocultures and fibroblast co-culture models of the EMTU. These responses were enhanced in the co-culture model suggesting that the EMTU is activated. Inflammatory but not anti-viral responses were mediated by epithelial-derived IL-1α acting on the underlying fibroblasts. This may have important consequences in promotion of inflammation and airway remodelling in viral-induced exacerbations of asthma.

Rationale The asthma susceptibility gene ADAM33/Adam33 is associated with bronchial hyperresponsiveness(BHR) in humans and mice. Soluble ADAM33 is increased in bronchoalveolar lavagefluid (BALF) of allergic asthma patients (Lee JY et al, AJRCCM 2006 Apr1; 173(7):729–35). Its levels correlate with declining FEV 1 %, suggesting a role in airway remodelling in asthma. Maternal allergy orexogenous IL-13 suppresses Adam33/ADAM33 mRNA expression but enhances ADAM33 protein processing in human embryonic and juvenile mouse lungs (Haitchi HM et al, JACI. 2009 Sep; 124(3):590–7, 597). We hypothesise thatconditional expression of IL-13 in mouse lungs induces the enzymatically active, soluble form of ADAM33 in BALF, which is associated with BHR. Methods IL-13 expression wasinduced using Doxycycline in CCSP-rtTA/Otet-Il-13 double-transgenic (dTg) mice. Methacholine challenge and lung function measurements were performed and lungswere harvested for mRNA extraction and immunohistochemistry (IHC). BALF was obtained forWestern-blotting for ADAM33 and testing of ADAM33 enzymatic activity using a fluorescenceresonance energy transfer (FRET) peptide assay. Results There was asignificant increase in BHR to Methacholine in IL-13 expressing double transgenicmice. IHC showed an increase inbronchial smooth muscle in lungs of double transgenic mice. Similar to the RTqPCR findings in humanembryonic and juvenile mouse lungs, IL-13 suppressed Adam33 mRNA but no difference in a-smooth muscle actin ( aSma) was evident. Immunoblotting for ADAMA33 in BALF demonstrateda 76kDa band, consistent with the ADAM33 ectodomain and processed forms at38/44kDa in dTg animals. ADAM33 enzymatic activity was also significantly increased. Conclusion The data suggest thatallergic inflammation induced by IL-13 suppresses Adam33 mRNA expression but induces the release of soluble forms ofADAM33, yielding enzymatically active forms. The release of soluble forms may play a role in airway remodelling, potentiallyleading to BHR. We next propose to test the effect of specific ADAM33inhibitors on airway remodelling in this allergic mouse model to assess theirpotential as novel treatments for asthma.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease for which there are no effective treatments. As a result the prognosis is poor, with a median survival of 3 years from the onset of symptoms. The key feature of IPF is formation of ‘fibroblastic foci’, accumulations of highly active, proliferative fibroblasts and myofibroblasts that lay down vast quantities of collagen and other extracellular matrix proteins. Lysyl oxidase (LOX) is a secreted enzyme involved in cross-linking of collagen and implicated in several fibrotic conditions. Increased cross-linking due to LOX upregulation in IPF may contribute to decreased lung compliance. Further, LOX may represent a biomarker that can be used to detect early responses or ‘proof-of-mechanism’ in clinical trials for new IPF drugs. We hypothesized that levels of LOX protein were higher in bronchoalveolar lavage (BAL) fluid from IPF patients compared to healthy controls. Methods BAL fluid was collected from patients with IPF or volunteers following ethical approval and informed consent. A quantity of BAL fluid determined to contain 20μg protein was concentrated with Strataclean resin and resuspended in 2x sample buffer. LOX protein content was determined by SDS-PAGE and western blotting followed by densitometry. Results LOX can be detected in BAL fluid from patients with IPF. Both pro and active forms of the LOX enzyme are significantly elevated in IPF compared to healthy controls, with active LOX detected in 12/25 IPF patients compared to 1/9 healthy controls. Conclusions There is increasing evidence for a role of LOX in fibrosis. Here we suggest LOX may be involved in IPF pathogenesis, and demonstrate that it is possible to detect secreted LOX in BAL fluid from patients with this condition. LOX may therefore represent a biomarker that could be used in clinical trials for IPF . Further work would be required to validate and optimise assays for clinical use.

Background The asthma and chronic obstructive pulmonary disease (COPD) gene, A Disintegrin And Metalloproteinase (ADAM)33 , is selectively expressed in mesenchymal cells and its metalloprotease activity has been linked to angiogenesis and airway remodelling. A soluble form of ADAM33 (sADAM33) has been identified in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and its levels inversely correlate with lung function and disease severity. Because tissue remodelling also occurs in pulmonary sarcoid, we hypothesised that sADAM33 is elevated in BALF of patients with this disease which, like asthma, is heterogeneous. Methods BALF was obtained from healthy controls (n=11) and patients with sarcoid (n=13) using fibre optic bronchoscopy according to current guidelines. After removal of immunoglobulins using Protein A/G and enrichment using Concanavalin A beads, sADAM33 was identified in BALF by Western blotting. A FRET peptide cleavage assay was used to assess ADAM33-like activity in BALF. Lung function (FVC%) and gas transfer (TLCO%) were measured at time of first diagnostic workup. Results sADAM33 protein in BALF was detected as a 25kDa fragment and levels were significantly increased in samples from sarcoid patients when compared to healthy controls (p Conclusion Release of sADAM33 is increased in sarcoid in association with abnormal lung function. Further studies will be required to determine whether the release of sADAM33 results in dysregulated metalloprotease activity, leading to angiogenesis and pulmonary parenchymal remodelling in pulmonary sarcoid. Since ADAM33 polymorphism is related to reduced lung function in asthma and COPD, this study raises the possibility that there may also be genetic associations between ADAM33 and some forms of pulmonary sarcoid. Finally, the occurrence of sADAM33 in asthma and sarcoidosis and its relation to reduced lung function suggests that it may be a biomarker of pulmonary remodelling in these diseases.

Rationale A Disintegrin And Metalloprotease (ADAM)33 is a susceptibility gene associated with asthma, bronchial hyperresponsiveness (BHR) and reduced lung function in young children. It is selectively expressed in mesenchymal cells, including bronchial smooth muscle and fibroblasts. We have previously shown that ADAM33 expression increases during lung development when spontaneous peristaltic contractions of the airways commence. Therefore we hypothesised that mechanical strain induces ADAM33 expression and affects smooth muscle/myofibroblast differentiation. Methods Primary human embryonic fibroblasts from the pseudoglandular stage of lung development were cultured on flexible collagen-coated membranes and exposed to cyclical mechanical stretch (30% amplitude, 12 cycles per minute) for 48, 96 and 168 h. Control cells were cultured on the same membranes without mechanical stretch. Quantitative RT-PCR was performed for ADAM33 and a-smooth muscle actin (a-SMA), a marker of smooth muscle and myofibroblast differentiation. We also measured collagen III and IL-8 mRNA. Soluble collagen protein levels in culture supernatants were measured using soluble collagen assay. Results ADAM33 and a-SMA mRNA expression were not significantly affected by mechanical strain. In contrast, collagen III mRNA expression was increased fourfold by cyclical mechanical strain and there was a threefold increase in soluble collagen proteins in culture supernatants of stretched cells. Unexpectedly, IL-8 expression was also increased by cyclical mechanical strain. Conclusion Mechanical strain did not appear to influence markers of smooth muscle differentiation. However, the increase in ECM production may indicate a requirement for stiffening of the airways as the tubular structures develop. We postulate that IL-8 is not proinflammatory in the context of airway development, and may be a paracrine growth factor for developing epithelial cells.

As asthma becomes more severe it adopts additional characteristics including corticosteroid refractoriness and a neutrophil-predominant inflammatory response implicating Th1 or Th17 responses involving cytokines such as tumour necrosis factor α, interleukin (IL)-6 and IL-8. We have examined the role of IL-6 and IL-8 in severe asthma. Subjects with severe asthma (GINA stage IV) who were exacerbation-free for ≥4 weeks with a forced expiratory volume in 1 s (FEV1) >30% but