Figure 2.

Putative intracellular cascades activated by cyclic di‐nucleotides. In this schematic representation, TLRs are separated in two major groups, those associated to the membrane and those located in the endosomal compartment. For the sake of clarity, in this scheme there is no discrimination between the different TLR at either the membrane or endosomal compartment (for details in TLRs pathways see review by Kawai and Akira, 2011). The membrane‐bound TLRs (TLR‐1, TLR‐2/1, TLR‐2/6, TLR‐4 and TLR‐5) detect PAMPs and DAMPs on the cell surface and bind to specific TIR domain containing adapters, such as TRIF, MyD88, TIRAP and TRAM. Other TLRs, such as TLR‐3, TLR‐7 and TLR‐8, are localized in intracellular vesicles and recognize RNA, whereas the intracellular TLR‐9 recognizes DNA. The important players downstream in these signalling cascades are TRAF6, TAK1 and TBK1, which in turn phosphorylates IRF‐3 and IRF‐7, leading to their homo‐dimerization and translocation into the nucleus, where they drive transcription of IFNs. This signalling cascades result in the activation of NF‐κB and MAPK, which in turn are leading to the production of pro‐inflammatory cytokines and type I IFN. On the other hand, TLR‐independent recognition of PAMPs is mediated by the intracellular receptors NLRs (NODs) and RLRs (RIG‐I, MDA‐5) present in the cytosol, which after activation trigger a subset of responses, which are similar to those promoted by TLRs. Exogenous or viral dsRNA is recognized by the RNA helicase RIG‐I (or MDA‐5), and signals through the mitochondrial antiviral signalling adaptor MAVS (also known as IPS‐1), which activates TBK1, thereby leading to phosphorylation of IRF‐3, NF‐κB release, translocation of the transcriptional regulators and gene induction. DNA is sensed by DAI, leading to activation of the same TBK1/IRF pathway as RIG‐I/MDA‐5. The ER‐localized STING protein was shown to be critical for regulating the production of IFN in response to cytoplasmic DNA virus. In vitro and in vivo studies suggest that c‐di‐GMP and c‐di‐AMP are sensed through a cytosolic pathway leading to type I IFN induction. The induction of type I IFNs by c‐di‐nucleotides is dependent on TBK1/IRF‐3 signalling, although it is independent of known cytosolic receptors. The adaptor molecule STING also seems to be required for the type I IFN responses induced by c‐di‐nucleotides. Preliminary studies suggest that the adjuvanticity of c‐di‐GMP relies on activation by IRF‐3/IRF‐7. However, it is still unclear if c‐di‐nucleotides need to reach the cytosol to exert their activity or they are acting via up‐to‐now uncharacterized surface receptors. It is also unknown to which extend the induction of type I IFN is sufficient to explain the complex and pleiotropic adjuvant properties of these molecules. Additional information is also needed, to assert the molecular processes responsible for the observed differences between the biological activities of different c‐di‐nucleotides. Red lines: putative c‐di‐nucleotide driven pathways for which strong experimental evidence exists. Green lines: Presumptive pathway for which preliminary data is available. Black lines: non c‐di‐nucleotide driven pathways.