Hans Michael Haitchi

Defective epithelial barrier function in asthma

BACKGROUND Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. OBJECTIVES To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. METHODS Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. RESULTS By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower (P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects (P < .01) and protected against cigarette smoke-induced barrier disruption (P < .01). CONCLUSIONS Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.

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.

Rhinovirus Induction of the CXC Chemokine Epithelial-Neutrophil Activating Peptide-78 in Bronchial Epithelium

Epithelial-neutrophil activating peptide-78 (ENA-78) induces neutrophil migration, an early response to viral infection. Rhinovirus serotype 16 (RV16) was used to infect primary bronchial epithelial cells and a cell line (BEAS-2B). Release of ENA-78 protein was measured by enzyme-linked immunosorbent assay, ENA-78 mRNA production was quantified by reverse-transcription polymerase chain reaction, and ENA-78 promoter activity was assessed by use of a promoter construct. After infection with RV16, ENA-78 protein and mRNA increased significantly, and RV16 induced 3-fold increases in ENA-78 gene transcription. Nasal ENA-78 measured in patients with asthma with and without RV infection was more elevated in patients with RV infection present. Our study demonstrates that ENA-78 is produced in bronchial epithelial cells in response to RV16 infection. With other chemokines, it may be an important initiator of neutrophil airway inflammation during RV common colds and thus may play a role in the development of virus-associated airway pathologies.

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.

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.

Intersections between Pulmonary Development and Disease

Recent advances in cellular, molecular, and developmental biology have revolutionized our concepts regarding the process of organogenesis that have important implications for our understanding of both lung formation and pulmonary disease pathogenesis. Pulmonary investigators have long debated whether developmental processes are recapitulated during normal repair of the lung or in the setting of chronic pulmonary diseases. Although the cellular events involved in lung morphogenesis and those causing pulmonary disease are likely to include processes that are distinct, there is increasing evidence that the pathogenesis of many lung disorders involves the same genetic machinery that regulates cell growth,specification, and differentiation during normal lung development.

Foxa3 induces goblet cell metaplasia and inhibits innate antiviral immunity

RATIONALE Goblet cell metaplasia accompanies common pulmonary disorders that are prone to recurrent viral infections. Mechanisms regulating both goblet cell metaplasia and susceptibility to viral infection associated with chronic lung diseases are incompletely understood. OBJECTIVES We sought to identify the role of the transcription factor FOXA3 in regulation of goblet cell metaplasia and pulmonary innate immunity. METHODS FOXA3 was identified in airways from patients with asthma and chronic obstructive pulmonary disease. We produced transgenic mice conditionally expressing Foxa3 in airway epithelial cells and developed human bronchial epithelial cells expressing Foxa3. Foxa3-regulated genes were identified by immunostaining, Western blotting, and RNA analysis. Direct binding of FOXA3 to target genes was identified by chromatin immunoprecipitation sequencing correlated with RNA sequencing. MEASUREMENTS AND MAIN RESULTS FOXA3 was highly expressed in airway goblet cells from patients with asthma and chronic obstructive pulmonary disease. FOXA3 was induced by either IL-13 or rhinovirus. Foxa3 induced goblet cell metaplasia and enhanced expression of a network of genes mediating mucus production. Paradoxically, FOXA3 inhibited rhinovirus-induced IFN production, IRF-3 phosphorylation, and IKKε expression and inhibited viral clearance and expression of genes required for antiviral defenses, including MDA5, RIG-I, TLR3, IRF7/9, and nuclear factor-κB. CONCLUSIONS FOXA3 induces goblet cell metaplasia in response to infection or Th2 stimulation. Suppression of IFN signaling by FOXA3 provides a plausible mechanism that may serve to limit ongoing Th1 inflammation during the resolution of acute viral infection; however, inhibition of innate immunity by FOXA3 may contribute to susceptibility to viral infections associated with chronic lung disorders accompanied by chronic goblet cell metaplasia.

Osteopontin is expressed and functional in human eosinophils.

To cite this article: Puxeddu I, Berkman N, Ribatti D, Bader R, Haitchi HM, Davies DE, Howarth PH, Levi‐Schaffer F. Osteopontin is expressed and functional in human eosinophils. Allergy 2010; 65: 168–174.

Contribution of bronchial fibroblasts to the antiviral response in asthma.

Human rhinoviruses (HRV) are a major cause of asthma exacerbations and hospitalization. Studies using primary cultures suggest that this may be due to impaired production of type I and type III IFNs by asthmatic bronchial epithelial cells. Although epithelial cells are the main target for HRV infection, HRV can be detected in the subepithelial layer of bronchial mucosa from infected subjects by in situ hybridization. Therefore, we postulated that submucosal fibroblasts are also involved in the innate antiviral response to HRV infection in asthma. We found that regardless of subject group, bronchial fibroblasts were highly susceptible to RV1b infection. IL-8 and IL-6 were rapidly induced by either HRV or UV-irradiated virus, suggesting that these responses did not require viral replication. In contrast, RANTES expression was dependent on viral replication. Regardless of disease status, fibroblasts did not respond to HRV infection with significant induction of IFN-β, even though both groups responded to synthetic dsRNA with similar levels of IFN-β expression. Exogenous IFN-β was highly protective against viral replication. Our data suggest that fibroblasts respond to HRV with a vigorous proinflammatory response but minimal IFN-β expression. Their susceptibility to infection may cause them to be a reservoir for HRV replication in the lower airways, especially in asthmatic subjects where there is reduced protection offered by epithelial-derived IFNs. Their ability to support viral replication coupled with their vigorous proinflammatory response following infection may contribute to asthma exacerbations.