Untersuchung der Relevanz unterschiedlicher Asthma-Suszeptibilitätsgene im Modellorganismus Drosophila melanogaster

Asthma ist eine chronisch-entzündliche Erkrankung der Atemwege, die weltweit etwa 300 Millionen Menschen betrifft. Die Pathogenese beruht auf einem Zusammenspiel von Umweltfaktoren mit einer genetischen Prädisposition und sie ist noch unzu¬reichend verstanden. Diese genetische Prädisposition wird durch zahlreiche Gene vermittelt, deren Relevanz für die Asthma-Pathogenese teilweise noch unverstanden ist. Eine Analyse dieser Asthma-Suszeptibilitätsgene kann zum Verständnis der Asthma-Pathogenese beitragen und damit neue Therapie- und Präventionsmöglich¬keiten eröffnen. Traditionell wird Asthma als eine Erkrankung betrachtet, deren Ursprünge in den aller¬gischen Mechanismen des adaptiven Immunsystems liegen. In den letzten Jah-ren hat sich dieser Fokus verschoben und das Epithel steht nunmehr im Fokus der Asthma-Pathogenese. Dabei schafft das Epithel in Asthmatikern infolge einer abnor¬men Antwort auf Umweltfaktoren womöglich eine Mikroumgebung, die Entzündungs¬reaktionen fördert und eine allergische Sensibilisierung ermöglicht. Basierend auf dieser Hypothese wurde ein Drosophila-Asthmamodell zur Unter-suchung von Asthma-Suszeptibilitätsgenen im Atemwegsepithel angewendet. Dieses Modell hat zwei wesentliche Vorteile, eine Fokussierung auf das Atemwegsepithel ohne Einflüsse einer adaptiven Immunantwort sowie eine Einschränkung des zeit¬lichen Aspektes. In der vorliegenden Arbeit wurde die Relevanz der Asthma-Suszeptibilitätsgene ORMDL3, STAT3 und STAT6 im Atemwegsepithel des Modellorganismus Drosophila melanogaster untersucht. Eine erhöhte Expression von ormdl, dem Drosophila Homolog zum humanen ORMDL3, in den Atemwegen und im Darm von Drosophila führte zu einer erhöhten Antwort gegenüber Umweltstressfaktoren. Diese war von einer Modulation der zellulären Stressantwort, Veränderungen beim Vorkommen von Asthma-relevanten Lipiden und der reduzierten Expression von Reparatur-Signal¬wegen begleitet. Die Aktivierung des JAK/STAT-Signalweges im Atemwegsepithel von Drosophila hatte eine Beeinträchtigung der epithelialen Barriere zur Folge. An der Entstehung dieser Barrierestörung war der Wnt-Signalweg mutmaßlich beteiligt. In beiden Fällen kam es zusätzlich zu einer transkriptionellen Anreicherung von Ent¬wicklungs-assoziierten Genen. Diese Resultate unterstützen die Hypothese, dass eine genetische Prädisposition das asthmatische Epithel bei der Bewältigung von umweltbedingtem Stress und Verletzungen einschränkt. Eine Kombination aus mehreren fehlregulierten Genen und Umweltfaktoren beeinträchtigen die Stressantwort und die Erneuerungsvorgänge des Epithels möglicherweise so stark, dass eine allergische Sensibilisierung ermög¬licht wird. Darüber hinaus existiert eine Schnittmenge der Resultate mit den funktionellen Eigen¬schaften des asthmatischen Epithels, das unter anderem gekennzeichnet ist durch eine beeinträchtigte Barriere, einen erhöhten Stress-Status und eine ver-ringerte Reparatur. Diese Schnittmenge verdeutlicht die Qualität des Drosophila-Asthma-Modells bei der initialen Analyse der funktionellen Relevanz von Asthma-Suszeptibilitätsgenen im respiratorischen Epithel.Asthma is a chronic inflammatory disease of the airways that affects 300 million people world-wide. The pathogenesis is not completely understood. However, an interaction of environmental and genetic factors is thought to cause asthma development. This genetic predisposition is caused by many asthma susceptibility genes. For some of these genes, the relevance for asthma pathogenesis, in general or regarding particular aspects, is unknown. Studying the functional relevance of these genes can provide a better understanding of asthma pathogenesis and thus contribute to the development of new therapies. Asthma has long been thought to be a disease of the adaptive immune system. In contrast, an opposing view puts the epithelium in the center of the pathogenesis. Through its abnormal response to environmental stimuli, the asthmatic epithelium is assumed to create a microenvironment that facilitates allergic sensitization and pro-motes inflammation. Taking this in account, a Drosophila asthma model for initial studies on asthma susceptibility genes in the airway epithelium was used. This model has two major advantages. First, a focus on the airway epithelium with no influence of the adaptive immune response is possible. Second, the temporal aspect of the studies can be strongly reduced. In this study, the relevance of the asthma susceptibility genes ORMDL3, STAT3 and STAT6 was analyzed using the model organism Drosophila melanogaster. Overexpression of ormdl, the Drosophila representative of human ORMDL3, in the airway epithelium and in the gut resulted in increased susceptibility to environmental stress. This enhanced susceptibility was accompanied by an increased cellular stress status, changes in lipid amounts and reduced expression of repair pathways. Further¬more, activation of the JAK/STAT pathway in the airway epithelium of Drosophila led to an epithelial barrier disruption, which was likely to depend on Wnt signaling. In addition, a transcriptional enrichment of developmental genes was present in both cases. These results support the hypothesis that a genetic predisposition compromises the ability of the asthmatic epithelium to cope with stress and injury resulting from environ¬mental stress. Thus, a combination of environmental factors and several de-regulated asthma susceptibility genes might impair the stress response and the re-newal of the epithelium in a manner that facilitates allergic sensitization. Moreover, the results obtained in Drosophila share an intersection with the functional characteristics of the asthmatic epithelium. In asthma, the epithelial barrier is im¬paired, the cellular stress status is enhanced and repair is reduced. This intersection emphasizes the quality of the Drosophila asthma model in the initial analysis of the functional relevance of asthma susceptibility genes in the respiratory epithelium

Histone deacetylase (HDAC) 1 controls the expression of beta defensin 1 in human lung epithelial cells.

Deregulation of the expression human beta defensin 1 (DEFB1), an antimicrobial peptide, has been implicated in the pathogenesis of COPD and asthma. Since the molecular mechanisms that regulate DEFB1 gene expression are widely unknown, the epigenetic processes involved in the regulation of the constitutive expression of DEFB1 in lung epithelial cells (A549) were investigated. The data demonstrate that histone deacetylases (HDACs) participate in the regulation of DEFB1 gene expression. Inhibition of the class I HDACs, HDACs 1-3, increases DEFB1 gene expression in A549 cells. Chromatin immunoprecipitation (ChIP) assays revealed that the inhibition of the class I HDACs also results in modifications of the chromatin at the DEFB1 promoter. Histone modifications, histone H3 acetylation and H3K4 trimethylation, that are associated with transcriptional activation, were found to increase after inhibition of HDACs 1-3. Finally, RNAi knockdown experiments identified HDAC1 as the sole HDAC responsible for maintaining the constitutive level of DEFB1 transcription. Taken together, our data reveal epigenetic mechanisms which are the basis of the maintenance of the constitutive gene expression of human beta defensin 1

Knockdown of HDAC1 increases DEFB1 gene expression.

<p>A549 cells were transfected with HDAC and control siRNAs for 48 to 96 h. (A) Cells were lysed 48 h after transfection and Western Blot analysis was performed with an anti-HDAC1 antibody. Data are representative of three independent experiments. (B–F) mRNA expression of <i>Cyclophilin B</i> (B), <i>HDAC1</i> (C), <i>HDAC2</i> (D), <i>HDAC3</i> (E) and <i>DEFB1</i> (F) was analyzed using quantitative Real-Time-PCR. The measured levels of mRNA were normalized to <i>TBP</i> mRNA levels. Bars represent means with SEM of three independent experiments (*: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001).</p

The HDAC inhibitor MS-275 increases H3 acetylation and H3K4 trimethylation at the <i>DEFB1</i> promoter. (A) DEFB1 gene with promoter region including analysis sites I-IV. I: −186 – −250, II: −347 – −406, III: −1002 – −1061, IV: −2436 – −2499.

<p>(B–C) A549 cells were treated with HDAC inhibitor MS-275 for 36 h. ChIP analysis was performed using anti-acetyl-Histone H3 (B) and anti-trimethyl-H3K4 (C) antibodies. Isolated DNA was quantified using quantitative RT-PCR. Bars represent means with SEM of three independent experiments. LINE: long interspersed nuclear element.</p

Modulation of DEFB1 gene expression and histone H3 acetylation by the HDAC inhibitor TSA.

<p>The human lung epithelial cell lines A549 (A) and NCI-H727 (B–C) were treated with the HDAC inhibitor trichostatin A for 24 h. (A–B) mRNA expression of <i>DEFB1</i> was analyzed using quantitative Real-Time-PCR. The measured levels of mRNA were normalized to <i>PBG-D</i> mRNA levels. (C) ChIP analysis was performed using anti-acetyl-Histone H3 antibody. Isolated DNA was quantified using quantitative RT-PCR. Bars represent means with SD of three independent experiments. LINE: long interspersed nuclear element.</p

Specificity of HDAC inhibitors.

<p>Specificity of HDAC inhibitors.</p

Transcriptional Regionalization of the Fruit Fly’s Airway Epithelium

<div><p>Although airway epithelia are primarily devoted to gas exchange, they have to fulfil a number of different tasks including organ maintenance and the epithelial immune response to fight airborne pathogens. These different tasks are at least partially accomplished by specialized cell types in the epithelium. In addition, a proximal to distal gradient mirroring the transition from airflow conduction to real gas exchange, is also operative. We analysed the airway system of larval <i>Drosophila melanogaster</i> with respect to region-specific expression in the proximal to distal axis. The larval airway system is made of epithelial cells only. We found differential expression between major trunks of the airways and more distal ones comprising primary, secondary and terminal ones. A more detailed analysis was performed using DNA-microarray analysis to identify cohorts of genes that are either predominantly expressed in the dorsal trunks or in the primary/secondary/terminal branches of the airways. Among these differentially expressed genes are especially those involved in signal transduction. <i>Wnt</i>-signalling associated genes for example are predominantly found in secondary/terminal airways. In addition, some G-protein coupled receptors are differentially expressed between both regions of the airways, exemplified by those activated by octopamine or tyramine, the invertebrate counterparts of epinephrine and norepinephrine. Whereas the OAMB is predominantly found in terminal airway regions, the oct3βR has higher expression levels in dorsal trunks. In addition, we observed a significant association of both, genes predominantly expressed in dorsal trunks or in primary to terminal branches branches with those regulated by hypoxia. Taken together, this observed differential expression is indicative for a proximal to distal transcriptional regionalization presumably reflecting functional differences in these parts of the fly’s airway system.</p></div

The airway epithelium of <i>Drosophila</i> larvae is characterized by different expression domains.

<p>The larval airway system is composed of interconnected tubes organized in a hierarchic structure (adapted after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102534#pone.0102534-Ruehle1" target="_blank">[6]</a>, A). Dorsal trunks are connected to the outside world and primary branches originate from them. The blind endings of the tracheal system are made from terminal branches (A). Different promoter Gal4 lines direct expression into different parts of the tracheal system. Driver lines such as <i>ppk4</i>-Gal4 label the entire airway system (B). In contrast, <i>c929</i>-Gal4 is specific for structures in the dorsal trunks (C), <i>ppk10</i>-Gal4 is surprisingly specific for primary branches (D) and <i>DSRF</i>-Gal4 labels exclusively terminal cells (E fluorescent channel, F transmitted light). Scale bars in B, C, D is 50 µm, in E, F 20 µm.</p

qRT-PCR analyses of randomly selected genes with predicted differential expression between dorsal trunks and primary/secondary/terminal branches.

<p>Differential expression of the listed genes was evaluated by qRT-PCR with cDNA derived from dorsal trunks or primary, secondary and terminal airways. Predominant expression was predicted from DNA-microarray studies for 4 transcripts in the dorsal trunks (A) or in the primary, secondary and terminal ones (B). Values are mean results from three different experiments performed in triplicate (±S.D.). Asterisks show statistically significant differences.</p

Venn diagram analysis of genes predominantly expressed in dorsal trunks or primary, secondary and terminal branches with those involved in defence responses (A) or regulated by hypoxia (B).

<p>The sets of genes predominantly expressed in dorsal trunks or primary, secondary and terminal branches were compared with those linked to immunity (A) or responses to hypoxia (B). The numbers of genes found in both sets is listed in the intersecting regions. Statistical analyses were performed with Fisher’s exact test, the corresponding p-values are listed close to the intersections.</p