Analyse der antiviralen Aktivität und Bedeutung von Interferon-stimulierten Genen in Cyclophilin-Inhibitor-behandelten Epithelzellen aus verschiedenen Abschnitten des Respirationstraktes
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Philipps-Universität Marburg
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The aim of this work was to characterize the antiviral activity of the cyclophilin inhibitor cyclosporin A and its derivative alisporivir. This activity is mediated in part by interferon-λ induction dependent on the interferon regulatory factor 1 and has already been demonstrated for various viruses.
In the present study, these mechanisms were also confirmed for primary murine lung epithelial cells. It was also shown that mainly epithelial cells of the upper respiratory tract responded with interferon-λ induction to treatment with cyclophilin inhibitors, while no IFN response was detected in alveolar epithelial cells. Furthermore, differential expression of interferon regulatory factor 1 was detected in these cells, which could explain the different IFN response. Finally, it was shown that especially those cells were protected from infection by cyclophilin inhibitor treatment that responded to this treatment with an interferon-λ response. These results illustrate that cyclosporin A leads to a protective interferon-λ response, particularly in epithelial cells of the upper respiratory tract, which correlates with the expression of interferon-regulating factor 1. This response is consistent with the physiological interferon response of the human lung to viral pathogens, in which interferon-λ in bronchial epithelial cells is particularly important for the early control of viral infections. In addition, pro-inflammatory proteins are less expressed in alveolar epithelial cells to prevent inflammatory damage to the sensitive tissue. Cyclosporin A thus appears to initiate a natural and tissue-sparing interferon-λ response and is therefore a promising candidate for the treatment of respiratory viral infections.
In the second part of this thesis, the mechanism leading to the activation of the interferon response was investigated in more detail in order to gain a better understanding of the mode of action of cyclophilin inhibitors. First, it was shown that the retinoic acid inducible gene I and the mitochondrial antiviral signaling protein are responsible for the cyclophilin inhibitor induced interferon response. This signaling cascade is normally induced during viral infections as soon as the retinoic acid inducible gene I recognizes viral double-stranded RNA. In this study, it was shown that treatment of bronchial Calu3 cells with Cyclophilin inhibitors leads to an accumulation of endogenous double-stranded RNA that activates this signaling pathway in the absence of viral infection. This illustrates the ability of the immunosuppressant cyclosporin A to induce an antiviral signaling cascade by mimicking infection of the cells with a viral pathogen. The utilization of this conserved cellular signaling pathway could also explain the broad antiviral spectrum of cyclosporin A. Furthermore, the mechanism leading to this accumulation of endogenous double-stranded RNA was deciphered in this work. The double-stranded RNAs were identified as transcripts of the mitochondrial genome. These RNAs probably originate from damaged mitochondria, which could be detected in increased numbers in cyclosporine A-treated cells. Indeed, mitochondrial double-stranded RNA is able to induce an interferon response. Finally, this study showed that cyclosporin A leads to a reduction in autophagy, which is important for the removal of damaged mitochondria and immunostimulatory molecules. In addition, external activation of autophagy counteracted all cyclosporin A induced processes. These data thus show that cyclosporin A, via the inhibition of autophagy, leads to an accumulation of damaged mitochondria and immunostimulatory double-stranded RNA in the cells, which is recognized by the retinoic acid inducible gene I.
In the last part of this work, individual interferon stimulated genes were to be examined for their significance for the antiviral activity of cyclosporin A. To this end, knockdown experiments were carried out with the Z-DNA binding protein 1 and the cytidine/uridine monophosphate kinase 2. The latter was identified for the first time as a new restriction factor for infection with the vesicular stomatitis virus. In addition, it was essential for the antiviral activity of cyclosporin A against vesicular stomatitis virus. In contrast, this effect was not detectable in infection with the Middle East respiratory syndrome coronavirus. Furthermore, Z-DNA binding protein 1 had no effect on replication in either of the two viruses examined. These results make it clear that the induction of interferon stimulated genes in cyclosporin A treated cells is essential for its antiviral activity. Furthermore, it became clear that even single interferon stimulated genes can be important for the restriction of certain viruses and that the significance of these genes differs in infections with different viruses.
Similar, albeit weaker, effects were confirmed for the cyclosporin A derivative alisporivir. Due to the lack of immunosuppressive effects, alisporivir could offer potential advantages over cyclosporin A, for example in the treatment of people with already weakened immune systems. In summary, the results of this work have contributed to a better understanding of the mode of action of cyclosporin A. Since cyclosporin A is already approved as an immunosuppressant and has a broad antiviral spectrum of activity, it represents a promising option as an emergency drug against emerging zoonotic viral diseases.
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Created: 2025Issued: 2025-12-01Updated: 2025-06-24
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Medizin
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Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
respiratorische VirenCyclosporine AdsRNAISGsRIG-IInterferoneInterferonsdsRNACyclosporin AISGsRIG-IRespiratory viruses
DFG-subjects
ISGsrespiratorische VirenInterferonedsRNACyclosporin ARIG-I
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610
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Meier, Lars: Analyse der antiviralen Aktivität und Bedeutung von Interferon-stimulierten Genen in Cyclophilin-Inhibitor-behandelten Epithelzellen aus verschiedenen Abschnitten des Respirationstraktes. : Philipps-Universität Marburg 2025-12-01. DOI: https://doi.org/10.17192/z2025.0333.