Die immunregulativen Funktionen von adenosine deaminase acting on RNA 1 (ADAR1)

Goal of this study was to characterize the immunoregulatory functions of ADAR1. The special domain structure as well as the IFN inducibility of ADAR1 supposes a function beyond the editing of known neuronal specific substrates. Some studies already associated ADAR1 with signaling processes within the type I IFN response, but the underlying mechanisms are largely unknown. Therefore the ADAR1-dependent involvement in the type I IFN response in the context of contact with various PRR ligands and viruses was investigated and potential cell type specific effects were characterized. For this purpose murine ADAR1-deficient macrophages and dendritic cells were established using the Cre/loxP recombination system (ADAR1f7-9/ LysMCre or CD11cCre). For the human system an ADAR1 knockdown in human monocytes was developed by the use of siRNAs. The analysis of the type I IFN activation potential by different PRR ligands and viruses showed in ADAR1-deficient in comparison to ADAR1-competent murine macrophages as well as in human monocytes a strongly modified release of type I IFNs in dependency on ADAR1. While stimulation with TRIF-dependent PRR ligands led to an increased IFNβ release, type I IFN response upon stimulation with viral PAMPs or infection with viruses, that trigger the signaling pathway in a TRIF-independent manner, was strongly decreased. Further investigations of signaling molecules, important for the induction of type I IFNs, revealed a remarkable pronounced type I IFN signature in ADAR1-deficient macrophages upon TRIF-dependent stimulation consistent with the increased IFNβ levels. Surprisingly ADAR1 deficiency increased even in context of TRIF-independent stimulation the type I IFN signature, however secretion of type I IFNs was strongly decreased. Notably IRF3 as well as the activated phosphorylated form were increased upon TRIF-dependent as well as TRIF-independent stimulation. Immunofluorescence data showed that in case of TRIF-independent signaling despite elevated IRF3 level the nuclear translocation of IRF3 was abolished in ADAR1-deficient macrophages, while TRIF-dependent stimulation still led to an efficient IRF3 translocalization. These findings indicate for ADAR1 a newly so far unknown function as an important modulator of type I IFN responses to PRR ligands and viral infections. In this context ADAR1 exerted on the one hand inhibitory functions on the TRIF-IRF3 axis in the type I IFN induction pathway, while in contrast to that IRF3 translocation downstream of TRIF independent signaling was strongly promoted by ADAR1 and hence revealed for ADAR1 in this case an activator function in IRF3-dependent type I IFN response. Consistently a significant ADAR1 dependent effect on the type I IFN activation potential of pDCs, which trigger the type I IFN response independently of IRF3, could not be observed. Further experiments have to elucidate the underlying mechanisms of the modulatory functions of ADAR1 on the IRF3 translocation as well as on the TRIF activity. Additionally in the course of the analyses of different ADAR1 deficient immune cells a critical role for ADAR1 function in the differentiation of certain cDC populations was observed in vitro as well as in vivo. The in vitro differentiation of mDCs in ADAR1-deficient cultures was completely lost, while in vivo the CD103-positive cDC population in different peripheral organs of conditionally ADAR1-deficient mice was not detectable. Since in vitro generated cDCs as well as the CD103-positive cDC population in vivo show a dependency on GM-CSF signaling, a defect in this signaling pathway mediated by the ADAR1 deficiency suggested itself. Further investigations will clarify this issue and moreover a potential functional relation between the dysregulated cDC differentiation and a possibly increased type I IFN signature.