Robert M. Powell

Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus

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.

Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells

Claudin proteins comprise a recently described family of tight junction proteins that differentially regulate paracellular permeability. Since other tight junction proteins show alterations in distribution or expression in inflammatory bowel disease (IBD) we assessed expression of claudins (CL) 2, 3 and 4 in IBD. CL 2 was strongly expressed along the inflamed crypt epithelium, whilst absent or barely detectable in normal colon. In contrast, CL 3 and 4 were present throughout normal colonic epithelium and were reduced or redistributed in the diseased surface epithelium. In a T84-cell culture model of the gut barrier, paracellular permeability decreased with time after plating and correlated with a marked decrease in the expression of CL 2. Addition of IFNγ/TNFα led to further decreases in CL 2 and 3, the redistrbution of CL 4 and a marked increase in paracellular permeability. Conversely, IL-13 dramatically increased CL 2, with little effect on CL 3 or 4, but also resulted in increased paracellular permeability. Expression of CL 2 did not correlate with proliferation or junctional reorganisation after calcium ion depletion. Re-expression of CL 2 in response to IL-13 was inhibited by phophatidylinositol 3 kinase inhibitor, LY294002, which also restored the ion permeability to previous levels. CL 2 expression could be stimulated in the absence of IL-13 by activation of phospho-Akt in the phophatidylinositol 3 kinase pathway. These results suggest that INFγ/TNFα and IL-13 have differential effects on CL 2, 3 and 4 in tight junctions, which may lead to increased permeability via different mechanisms.

Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms

While asthma is an inflammatory disorder of the airways usually associated with atopy, an important additional component is involvement of the epithelium and underlying mesenchyme acting as a trophic unit (EMTU). In addition to allergens, a wide range of environmental factors interact with the EMTU, such as virus infections, environmental tobacco smoke and pollutants, to initiate tissue damage and aberrant repair responses that are translated into remodelling of the airways. While candidate gene association studies have revealed polymorphic variants that influence asthmatic inflammation, positional cloning of previously unknown genes is identifying a high proportion of novel genes in the EMTU. Dipeptidyl peptidase (DPP) 10 and disintegrin and metalloproteinase (ADAM)33 are newly identified genes strongly associated with asthma that are preferentially expressed in the airway epithelium and underlying mesenchyme, respectively. Also of increasing importance is the recognition that genes associated with asthma and atopy have important interactions with the environment through epigenetic mechanisms that influence their expression. This type of research will not only identify biomarkers of different types of asthma across the full range of phenotypic expression, but will also identify novel therapeutic targets that could influence the natural history of the heterogenes lung disease.

The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis : Implications for airway remodeling in asthma

BACKGROUND A disintegrin and metalloprotease (ADAM)-33 is a susceptibility gene for asthma and chronic obstructive pulmonary disease whose function remains unknown. OBJECTIVE Because asthmatic bronchoalveolar lavage fluid contains high levels of soluble ADAM33 (sADAM33), which includes the catalytic domain, we postulated that its release from cell membranes might play functional roles in airway remodeling by promoting angiogenesis. METHODS The proangiogenic activity of the highly purified catalytic domain of ADAM33 or a catalytically inactive mutant was studied in vitro (Matrigel assay), ex vivo (human embryonic/fetal lung explants) and in vivo (chorioallantoic membrane assay). The regulation of sADAM33 release from cells overexpressing full-length ADAM33 and its biological activity were characterized. RESULTS We show that the purified catalytic domain of ADAM33, but not its inactive mutant, causes rapid induction of endothelial cell differentiation in vitro, and neovascularization ex vivo and in vivo. We also show that TGF-beta(2) enhances sADAM33 release from cells overexpressing full-length ADAM33 and that this truncated form is biologically active. CONCLUSION The discovery that sADAM33 promotes angiogenesis defines it as a tissue remodeling gene with potential to affect airflow obstruction and lung function independently of inflammation. As TGF-beta(2) enhances sADAM33 release, environmental factors that cause epithelial damage may synergize with ADAM33 in asthma pathogenesis, resulting in a disease-related gain of function. This highlights the potential for interplay between genetic and environmental factors in this complex disease.

Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells

BACKGROUND A disintegrin and metalloprotease 33 (ADAM33) polymorphism is strongly associated with asthma and bronchial hyperresponsiveness. Although considered to be a mesenchymal cell-specific gene, recent reports have suggested epithelial expression of ADAM33 in patients with severe asthma. OBJECTIVES Because dysregulated expression of ADAM33 can contribute to disease pathogenesis, we characterized the mechanism or mechanisms that control its transcription and investigated ADAM33 expression in bronchial biopsy specimens and brushings from healthy and asthmatic subjects. METHODS The ADAM33 promoter and CpG island methylation were analyzed by using bioinformatics, luciferase reporters, and bisulfite sequencing of genomic DNA. Epithelial-mesenchymal transition was induced by using TGF-beta1. ADAM33 mRNA was scrutinized in bronchial biopsy specimens and brushings by using reverse transcriptase-quantitative polymerase chain reaction, melt-curve analysis, and direct sequencing. RESULTS The predicted ADAM33 promoter (-550 to +87) had promoter transcriptional activity. Bisulfite sequencing showed that the predicted promoter CpG island (-362 to +80) was hypermethylated in epithelial cells but hypomethylated in ADAM33-expressing fibroblasts. Treatment of epithelial cells with 5-aza-deoxycytidine caused demethylation of the CpG island and induced ADAM33 expression. In contrast, phenotypic transformation of epithelial cells through a TGF-beta-induced epithelial-mesenchymal transition was insufficient to induce ADAM33 expression. ADAM33 mRNA was confirmed in bronchial biopsy specimens, but no validated signal was detected in bronchial brushings from healthy or asthmatic subjects. CONCLUSION The ADAM33 gene contains a regulatory CpG island within its promoter, the methylation status of which tightly controls its expression in a cell type-specific manner. ADAM33 repression is a stable feature of airway epithelial cells, irrespective of disease.

Identification of novel expressed sequences, up‐regulated in the leucocytes of chronic fatigue syndrome patients

Background Chronic fatigue syndrome (CFS) is an increasing medical phenomenon of unknown aetiology leading to high levels of chronic morbidity. Of the many hypotheses that purport to explain this disease, immune system activation, as a central feature, has remained prominent but unsubstantiated. Supporting this, a number of important cytokines have previously been shown to be over‐expressed in disease subjects. The diagnosis of CFS is highly problematic since no biological markers specific to this disease have been identified. The discovery of genes relating to this condition is an important goal in seeking to correctly categorize and understand this complex syndrome.

Enhanced upregulation of smooth muscle related transcripts by TGFβ2 in asthmatic (myo) fibroblasts

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 TGFβ2 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 TGFβ2 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. TGFβ2 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 TGFβ2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFβ2 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.

The role of ADAM33 in the pathogenesis of asthma

While asthma is a disorder of the conducting airways characterised by Th2-directed inflammation, a second set of mechanisms is being increasingly recognised as fundamental to disease chronicity and severity, for which the term “remodelling” has been used. The cellular and mediator responses underpinning airway remodelling involve aberrant communication between the airway epithelium and underlying mesenchyme, involving the generation of growth factors that lead to proliferation of fibroblasts and smooth muscle and the deposition of matrix proteins to cause airway wall thickening linked to bronchial hyperresponsiveness and fixed airflow obstruction. The identification of ADAM33 on chromosome 20p13 from positional cloning as a novel candidate gene involved in the pathogenesis of these structural and functional changes has opened the way to further insight into these processes that contribute to corticosteroid refractoriness. The preferential expression of ADAM33 in mesenchymal cells and its multiple molecular actions provide ample opportunity for incriminating this molecule in chronic asthma. Its association with progressive asthma and in predicting reduced lung function in young children suggest that ADAM33 has an important role in the natural history and possibly the origins of asthma, a disease unique to humans.

ADAM 33: just another asthma gene or a breakthrough in understanding the origins of bronchial hyperresponsiveness?

ADAM 33, the latest of the ADAM proteins to be described, has been identified as a major susceptibility gene in asthma linked to bronchial hyperresponsiveness. It provides an important breakthrough in our understanding of this complex disorder and its variable clinical and physiological presentations. Asthma is a disorder of the conducting airways in which Th2 mediated inflammation interacts with structural changes to cause variable airflow obstruction. Fundamental to disordered function is the concept of bronchial hyperresponsiveness (BHR) in which the airways constrict too much and too easily. In chronic severe asthma the inflammation and structural changes both become more intense1 and are paralleled by an increase in BHR that is only partially or non-responsive to treatment with corticosteroids.2 Explanations for BHR include mucosal and adventitial swelling causing a disproportionate reduction in airway calibre for a given degree of airways smooth muscle (ASM) shortening,3 excessive ASM shortening,4 an increase in ASM mass causing greater force generation,5 and an excessive velocity of contraction linked to altered crossbridge cycling.6 Morphometric studies have shown a graded increase in ASM mass in proportion to disease severity, and computer modelling has revealed that this and altered contractility are the most plausible explanations for BHR.5,7 Asthma has a high heritability of up to 79%,8 involving a few genes with moderate effects rather than many genes with small effects.9 We have recently reported the first novel asthma related gene identified by positional cloning.10 A genome wide screen was undertaken using phenotypic data and DNA from 362 families in Wessex and 98 in the USA with at least two siblings with asthma. Using 401 microsatellite markers at a density of 9 cM and multipoint linkage analysis, suggestive evidence for linkage (maximum lod score (MLS) 2.24) was found on …

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-β(2) to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β(2) affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β(2) caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β(2) also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β(2) 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-β(2) 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-β(2)-induced ectodomain shedding of ADAM33.