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1.  CaMKII Is Essential for the Proasthmatic Effects of Oxidation 
Science translational medicine  2013;5(195):195ra97.
Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl− current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyper-reactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent pro-asthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
PMCID: PMC4331168  PMID: 23884469
2.  A MicroRNA Network Dysregulated in Asthma Controls IL-6 Production in Bronchial Epithelial Cells 
PLoS ONE  2014;9(10):e111659.
MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.
PMCID: PMC4216117  PMID: 25360780
3.  Potentially Pathogenic Airway Bacteria and Neutrophilic Inflammation in Treatment Resistant Severe Asthma 
PLoS ONE  2014;9(6):e100645.
Molecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches.
We aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers.
Induced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis.
In 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67–83) vs 43% (29–67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count.
Airway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies.
PMCID: PMC4067344  PMID: 24955983
4.  Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors 
PLoS ONE  2013;8(8):e71278.
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.
PMCID: PMC3751915  PMID: 24009658
5.  Regulation of A Disintegrin And Metalloprotease-33 Expression by Transforming Growth Factor-β 
The asthma susceptibility gene, a disintegrin and metalloprotease-33 (ADAM33), is selectively expressed in mesenchymal cells, and the activity of soluble ADAM33 has been linked to angiogenesis and airway remodeling. Transforming growth factor (TGF)-β is a profibrogenic growth factor, the expression of which is increased in asthma, and recent studies show that it enhances shedding of soluble ADAM33. In this study, we hypothesized that TGF-β also affects ADAM33 expression in bronchial fibroblasts in asthma. Primary fibroblasts were grown from bronchial biopsies from donors with and those without asthma, and treated with TGF-β2 to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β2 affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β2 caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β2 also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β2 down-regulated ADAM33 mRNA expression by causing chromatin condensation around the ADAM33 promoter with deacetylation of histone H3, demethylation of H3 on lysine-4, and hypermethylation of H3 on lysine-9. However, the methylation status of the ADAM33 promoter did not change. Together, these data suggest that TGF-β2 suppresses expression of ADAM33 mRNA in normal or asthmatic fibroblasts. This occurs by altering chromatin structure, rather than by gene silencing through DNA methylation as in epithelial cells. This may provide a mechanism for fine regulation of levels of ADAM33 expression in fibroblasts, and may self-limit TGF-β2–induced ectodomain shedding of ADAM33.
PMCID: PMC3359905  PMID: 22227561
a disintegrin and metalloprotease-33; myofibroblast; transforming growth factor-β; histone modification
6.  Transforming Growth Factor-Beta Promotes Rhinovirus Replication in Bronchial Epithelial Cells by Suppressing the Innate Immune Response 
PLoS ONE  2012;7(9):e44580.
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.
PMCID: PMC3435262  PMID: 22970254
7.  Airway Epithelial Transcription Factor NK2 Homeobox 1 Inhibits Mucous Cell Metaplasia and Th2 Inflammation 
Rationale: Airway mucous cell metaplasia and chronic inflammation are pathophysiological features that influence morbidity and mortality associated with asthma and other chronic pulmonary disorders. Elucidation of the molecular mechanisms regulating mucous metaplasia and hypersecretion provides the scientific basis for diagnostic and therapeutic opportunities to improve the care of chronic pulmonary diseases.
Objectives: To determine the role of the airway epithelial–specific transcription factor NK2 homeobox 1 (NKX2-1, also known as thyroid transcription factor-1 [TTF-1]) in mucous cell metaplasia and lung inflammation.
Methods: Expression of NKX2-1 in airway epithelial cells from patients with asthma was analyzed. NKX2-1+/− gene targeted or transgenic mice expressing NKX2-1 in conducting airway epithelial cells were sensitized to the aeroallergen ovalbumin. In vitro studies were used to identify mechanisms by which NKX2-1 regulates mucous cell metaplasia and inflammation.
Measurements and Main Results: NKX2-1 was suppressed in airway epithelial cells from patients with asthma. Reduced expression of NKX2-1 in heterozygous NKX2-1+/− gene targeted mice increased mucous metaplasia in the small airways after pulmonary sensitization to ovalbumin. Conversely, mucous cell metaplasia induced by aeroallergen was inhibited by expression of NKX2-1 in the respiratory epithelium in vivo. Genome-wide mRNA analysis of lung tissue from ovalbumin-treated mice demonstrated that NKX2-1 inhibited mRNAs associated with mucous metaplasia and Th2-regulated inflammation, including Spdef, Ccl17, and Il13. In vitro, NKX2-1 inhibited SPDEF, a critical regulator of airway mucous cell metaplasia, and the Th2 chemokine CCL26.
Conclusions: The present data demonstrate a novel function for NKX2-1 in a gene network regulating mucous cell metaplasia and allergic inflammation in the respiratory epithelium.
PMCID: PMC3175541  PMID: 21562130
asthma; goblet cell; respiratory epithelium; NK2 homeobox 1
8.  Pathways Activated during Human Asthma Exacerbation as Revealed by Gene Expression Patterns in Blood 
PLoS ONE  2011;6(7):e21902.
Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations.
Methodology/Principal Findings
Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations.
This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.
PMCID: PMC3136489  PMID: 21779351
10.  A new look at the pathogenesis of asthma 
Asthma is an inflammatory disorder of the conducting airways that has strong association with allergic sensitization. The disease is characterized by a polarized Th-2 (T-helper-2)-type T-cell response, but in general targeting this component of the disease with selective therapies has been disappointing and most therapy still relies on bronchodilators and corticosteroids rather than treating underlying disease mechanisms. With the disappointing outcomes of targeting individual Th-2 cytokines or manipulating T-cells, the time has come to re-evaluate the direction of research in this disease. A case is made that asthma has its origins in the airways themselves involving defective structural and functional behaviour of the epithelium in relation to environmental insults. Specifically, a defect in barrier function and an impaired innate immune response to viral infection may provide the substrate upon which allergic sensitization takes place. Once sensitized, the repeated allergen exposure will lead to disease persistence. These mechanisms could also be used to explain airway wall remodelling and the susceptibility of the asthmatic lung to exacerbations provoked by respiratory viruses, air pollution episodes and exposure to biologically active allergens. Variable activation of this epithelial–mesenchymal trophic unit could also lead to the emergence of different asthma phenotypes and a more targeted approach to the treatment of these. It also raises the possibility of developing treatments that increase the lung's resistance to the inhaled environment rather than concentrating all efforts on trying to suppress inflammation once it has become established.
PMCID: PMC2805922  PMID: 20025610
allergen; asthma; inflammation; remodelling; T-cell; virus infection; BHR, bronchial hyper-responsiveness; CT, computed tomography; DC, dendritic cell; ADC, airway DC; EBUS, endobronchial ultrasound; EMTU, epithelial–mesenchymal trophic unit; ETS, environmental tobacco smoke; IFN, interferon; IL, interleukin; IoW, Isle of Wight; LT, leukotriene; mAb, monoclonal antibody; RV, rhinovirus; TGF-β, transforming growth factor-β; Th-2, T-helper-2; TJ, tight junction; TSLP, thymic stromal lymphopoietin
11.  Proof of concept pilot study: prevalence of grass virus infection and the potential for effects on the allergenic potency of pollen 
Environmental Health  2009;8(Suppl 1):S10.
Wild plants harbour a variety of viruses and these have the potential to alter the composition of pollen. The potential consequences of virus infection of grasses on pollen-induced allergic disease are not known.
We have collected pollen from Dactylis glomerata (cocksfoot; a grass species implicated as a trigger of allergic rhino-conjunctivitis) from Wytham Wood, Oxfordshire UK. Extracts were prepared from pollen from uninfected grass, and from grass naturally infected by the Cocksfoot streak potyvirus (CSV). Preparations of pollen from virus-infected and non-infected grasses were employed in skin testing 15 grass pollen-allergic subjects with hayfever. Allergen profiles of extracts were investigated by Western blotting for IgE with sera from allergic subjects.
The prevalence of CSV infection in cocksfoot grasses sampled from the study site varied significantly over an eight-year period, but infection rates of up to 70% were detected. Virus infection was associated with small alterations in the quantities of pollen proteins detected by polyacrylamide gel electrophoresis, and in the patterns of allergens identified by Western blotting with IgE from grass pollen allergic subjects. For individual subjects there were differences in potencies of standardised extracts of pollen from virus-free and virus-infected plants as assessed by skin testing, though a consistent pattern was not established for the group of 15 subjects.
Infection rates for CSV in cocksfoot grass can be high, though variable. Virus-induced alterations in components of grass pollen have the potential to alter the allergenic potency.
PMCID: PMC2796488  PMID: 20102577
13.  A review of the tolerability and safety of levocabastine eye drops and nasal spray. Implications for patient management 
Mediators of Inflammation  1995;4(7):S26-S30.
Levocabastine is a highly potent and selective H1-receptor antagonist specifically developed for topical administration by ocular and nasal routes. The clinical effects of levocabastine occur rapidly and are predominantly due to local antihistaminic effects at the site of application. Clinically, levocabastine is well tolerated with an adverse effect profile comparable with that of sodium cromoglycate and placebo. As might be expected from the route of drug administration, local irritation is the most frequent adverse event seen with levocabastine eye drops and nasal spray with an incidence comparable with that in placebo-treated controls. Intranasal application of levocabastine has been shown to have no adverse effect on ciliary activity both in vitro and in vivo, while ocular administration has not been shown to have any significant or consistent adverse effect in both animal and human studies. At therapeutic doses, levocabastine appears to be devoid of significant systemic activity producing no apparent effects on cardiovascular, psychomotor and cognitive function. Since levocabastine undergoes little hepatic metabolism, and only low plasma levels of the drug are attained following topical administration, drug interactions are unlikely.
PMCID: PMC2365682  PMID: 18475686

Results 1-13 (13)