Therefore, and due to nonspecific inhibition by all inhibitors that we tested (data not shown), we were unable to show a direct effect of TLR3 and RIG-I. However, we have demonstrated that both TLR3 and RIG-I show cross-talk with NOD2. At this moment, we do not know which of these two receptors contributes most to the response to costimulation with MDP and RSV. A previous study into the host receptors involved in the induction of IFN-β by RSV reported that neither TLR3 nor Toll-IL-1R homology

domain-containing adapter molecule 1 (TICAM-1) were essential for IFN-β induction by RSV [[29]]. In contrast, mitochondria antiviral signaling (MAVS), TANK-binding kinase 1 (TBK1), and IκB kinase-related kinase(IKK) were all involved in IFN-β induction [[29]], which would argue for a role for RIG-I. However, other, more recently AZD2014 purchase described cytosolic receptors that can recognize viral RNA, such as the DDX1-DDX21-DHX36 complex [[30]], cannot be excluded. This new receptor associates with Ku-0059436 in vitro TICAM-1 in the cytosol and also induces type I

IFNs. Further research is needed to identify the specific viral RNA receptor. As viral RNA is recognized by either TLR3, RIG-I, or both, we investigated the mechanism by which these two receptors affect signaling through NOD2. In this study, we show that RSV and Poly(I:C) induce transcriptional upregulation of IFN-β. Type I IFNs are generally regarded as fast responders [[31]]. Indeed, stimulation with LPS resulted in the typical fast response that has previously been described, with IFN-β upregulated after 4 h and abolished after 24 h. In contrast,

RSV only showed a modest upregulation of IFN-β transcription after 4 h. However, after 24 h, IFN-β expression was strongly induced. A potential explanation for this delayed response might be the involvement of the NS1/2 genes, known to suppress type I IFN production [[32, 33]] or the newly described viral receptor, the DDX1-DDX21-DHX36 complex [[30]]. This receptor complex is constitutively expressed and not regulated by type I IFNs, in contrast to RIG-I and Acetophenone MDA-5, and to a lower extent TLR3, which are all type I IFN-induced genes [[22]]. It was suggested that this receptor may represent an early sensor of viral infection that triggers an initial IFN response. In turn, this IFN response will upregulate RIG-I, MDA-5, and TLR3, which will then further amplify the type I IFN response. Although we have not specifically focused on the DDX1/DDX21/DHX36 complex in this study, this model would also fit with our observations. Our experiments show that viral infection, Poly(I:C) and IFN-β all induce a comparable upregulation of RIG-I, TLR3 and NOD2 mRNA. Similar findings were reported by Kim et al. (2011), who showed that both viral infection and IFN-β upregulated NOD2 transcription, and Ueta et al. (2010), who showed that RIG-I and TLR3 are type I IFN inducible genes.