Publikationsserver der Universitätsbibliothek Marburg

Titel: Die Rolle der microRNA-15b in der Pathogenese des experimentellen allergischen Asthma bronchiale
Autor: Müllers, Charlotte
Weitere Beteiligte: Renz, Harald (Prof. Dr. med.)
Veröffentlicht: 2016
URI: https://archiv.ub.uni-marburg.de/diss/z2016/0573
DOI: https://doi.org/10.17192/z2016.0573
URN: urn:nbn:de:hebis:04-z2016-05736
DDC: Medizin
Titel(trans.): The role of microRNA-15b in experimental atopic asthma
Publikationsdatum: 2016-08-24
Lizenz: https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Bronchialasthma, microRNA, miRNS, Atopie, Allergie, Asthma

Zusammenfassung:
Das allergische Asthma bronchiale zeichnet sich durch eine von T-Helferzellen gesteuerte, komplexe und bis heute nicht vollständig verstandene chronische Entzündungsreaktion aus. Auch wenn im Rahmen des Krankheitsbildes zunehmend von einer Heterogenität und Plastizität der T-Helferzellen insgesamt ausgegangen wird, so nimmt die Gruppe der TH2-Zellen im Speziellen weiterhin eine zentrale Rolle in der Pathogenese der Erkrankung ein. Mit ihrer charakteristischen Zytokinproduktion unterhalten sie die lokale Immunreaktion des respiratorischen Epithels und führen so zum typischen remodeling der Atemwege. Die Neigung des Immunsystems nach Allergenkontakt eine TH2-dominierte Reaktion auszubilden, wird unter anderem auf epigenetische und posttranskriptionelle Regulationsmechanismen zurückgeführt. Die genauen Umstände, die zur Ausbildung dieses Ungleichgewichts zwischen TH1- und TH2-Zellen in der Immunantwort führen, sind jedoch noch in weiten Bereichen ungeklärt. Ein wesentlicher Teil der posttranskriptonellen Regulation einer Zelle oder eines Gewebes wird durch ihr miRNA-Expressionsmuster gesteuert. In den letzten Jahren konnte gezeigt werden, dass miRNAs auch im Bereich der Differenzierung, Heterogenität und Ausübung von Effektorfunktionen der Zellen des Immunsystems eine wichtige Rolle spielen. In miRNA-Arrays von murinen T-Helferzellen aus Modellen der akut allergisch entzündeten Lunge konnte eine erhöhte Expression der miRNA-15b in TH2-Zellen im Vergleich zu TH1- und naiven T Helferzellen nachgewiesen werden. Basierend auf diesen Erkenntnissen wurde in der vorliegenden Arbeit die Fragestellung untersucht, ob Expressionsunterschiede der miRNA-15b Einfluss auf die Entwicklung der TH2 dominanten Entzündungsreaktion beim allergischen Asthma bronchiale haben. Hierfür wurde sowohl die Expression der miRNA-15b in vitro während der Differenzierungsphase von TH1- und TH2 Zellen als auch der Einfluss der miRNA 15b auf das TH1-Leitzytokin IFN-γ näher analysiert. Zunächst konnte IFN-γ in silico per Datenbankanalyse und experimentell im Reportergenassay als Zielgen der miRNA-15b bestätigt werden. Des Weiteren wurde eine Hochregulation der miRNA-15b in der Differenzierungsphase von TH1- und TH2 Zellen in vitro im Vergleich zu naiven T-Helferzellen gezeigt. Eine Hochregulation der miRNA-15b in vitro speziell in TH2-Zellen konnte im Kontrast zu den vorausgegangenen miRNA-Arrays nicht bestätigt werden. Bezüglich des Einflusses der untersuchten miRNA auf IFN-γ wurde gezeigt, dass eine Transfektion von anti-miRNA-15b auf in vitro-Ebene keinen Effekt auf die Expression von IFN-γ-mRNA zeigt, in ex vivo isolierten T-Helferzellen aus Modellen der akut allergisch entzündeten Lunge jedoch zu einer vermehrten Expression der IFN-γ-mRNA führt. Methodisch wurde in dieser Dissertation die Lipofektion mit HiPerFect als Alternative für die Transfektion von T-Helferzellen präsentiert. Diese Ergebnisse zeigen, dass IFN-γ einer Regulation durch die miRNA-15b unterliegt und seine Expression im Modell des allergischen Asthma bronchiale durch die miRNA 15b beeinflusst wird. Die Arbeit konnte somit ihr Ziel, einen Beitrag zum besseren Verständnis des Einflusses von miRNAs auf die Pathogenese des Asthma bronchiale zu leisten, erfüllen. Die gefundenen Erkenntnisse machen die miRNA-15b zu einem interessanten Kandidaten für weitere Untersuchungen im Forschungsfeld Asthma bronchiale.

Summary:
Atopic asthma is a chronic inflammatory disease of the airways. Even though T cell plasticity is becoming more important in the context of atopic asthma, TH2 cells still count as one of the main contributing factors in the pathophysiology of the disease. With their specific cytocine production, they orchestrate and maintain the chronic respiratory inflammation which can ultimately lead to a remodeling of the airways. Why it is that atopic indiviuals react with a TH2 cell dominated immune response upon allergen contact is still not fully understood, but it is believed to be caused by a combination of genetic, epigenetic, and posttranscriptional regulation. An important part of posttranscriptional regulation is the large group of microRNAs (miRNAs) that was discovered only about twenty years ago. Changes in miRNA expression can have numerous effects on gene expression levels in both physiological and pathological conditions, including differentiation, activation, and key effector functions of immune cells. miRNA expression arrays of T helper cells isolated from lungs of mice with acute asthma phenotype showed higher miRNA-15b levels in TH2 cells compared to TH1 and naïve T helper cells. These results demanded further research and are the subject of this dissertation. The research objective for this thesis was to contribute to a better understanding of the role of miRNA in the pathogenesis of atopic asthma. This was achieved by analyzing the expression of miRNA-15b during the differentiation of TH1 and TH2 cells from naïve T helper cells in vitro. Furthermore, the influence of the miRNA-15b on the expression of IFN-γ in T helper cells was investigated in vitro and in isolated CD4+ cells from mouse models of atopic asthma. As a result, it could be shown that miRNA-15b is increasingly expressed during T helper cell differentiation compared to naïve T helper cells, but in contrast to the miRNA arrays mentioned above, no difference in expression levels between TH1 and TH2 cells was found in vitro. In addition to this finding, IFN-γ was shown to be a predicted target of miRNA-15b in silico and could be experimentally validated with luciferase reporter assays. Furthermore, it was demonstrated that transfection of anti miRNA-15b had no effect on IFN-γ expression in vitro but increased IFN-γ-mRNA in ex vivo isolated T helper cells from models of acute asthma. Methodically, lipofection with HiPerFect could be presented as an alternative method for transfecting primary T helper cells. These findings show that miRNA-15b targets IFN γ and that it seems to have an effect on IFN-γ expression in T helper cells in atopic asthma. This makes it an interesting topic for further research.

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