Detection of pathogens by the innate immune system depends on the recognition of microbial components by dedicated receptors. During viral and bacterial infections, pathogen-derived nucleic acids are recognized by innate receptors resulting in the production of type I IFNs and inflammatory cytokines. While it is established that cytoplasmic delivery of viral, bacterial and even host DNA triggers type I IFN production via a pathway depending on STING, TBK1, IKKe and IRF3, the identification of the upstream DNA-sensing molecule(s) remains a key issue to be elucidated. Some proteins (such as DAI and IFI16) have been proposed to play such a role, but available data indicate that additional receptor(s) must exist.The primary goal of the proposed project is to identify new cytoplasmic DNA-sensing proteins mediating type I IFN induction. First, we will identify interferon inducible DNA-binding proteins by combining a systematic proteomics screen for proteins that associate with DNA with a screen for transcripts induced by interferon beta. Using bioinformatic analysis of these datasets, we will then select candidate proteins to perform functional investigation on. The effect of shRNA-mediated silencing or overexpression of these proteins on DNA-induced IFN production will be evaluated. Promising proteins will be selected for detailed characterization. Importantly, our project could also reveal proteins that do not participate in the sensing, but in the effector responses against pathogens, thereby opening other interesting research directions.In addition to the relevance for antiviral and antibacterial immunity, a better understanding of DNA sensing pathways may have broader implications as for example DNA-induced IFN contributes to the immunogenicity of plasmid-based vaccines. Moreover, type I IFN induced by self-DNA has been linked to autoimmune diseases. Therefore, identification of new DNA sensing molecules will uncover new potential therapeutic targets.