Autophagy is a highly conserved process that maintains homeostasis by clearing damaged organelles and long-lived proteins. The consequences of deficiency in autophagy manifest in a variety of pathological states including neurodegenerative diseases, inflammatory disorders, and cancer. Here, we studied the role of autophagy in the homeostatic regulation of innate antiviral defense. Single-stranded RNA viruses are recognized by the members of the RIG-I-like receptors (RLRs) in the cytosol. RLRs signal through IPS-1, resulting in the production of the key antiviral cytokines, type I IFNs. Autophagy-defective Atg5 ^−/− cells exhibited enhanced RLR signaling, increased IFN secretion, and resistance to infection by vesicular stomatitis virus. In the absence of autophagy, cells accumulated dysfunctional mitochondria, as well as mitochondria-associated IPS-1. Reactive oxygen species (ROS) associated with the dysfunctional mitochondria were largely responsible for the enhanced RLR signaling in Atg5 ^−/− cells, as antioxidant treatment blocked the excess RLR signaling. In addition, autophagy-independent increase in mitochondrial ROS by treatment of cells with rotenone was sufficient to amplify RLR signaling in WT cells. These data indicate that autophagy contributes to homeostatic regulation of innate antiviral defense through the clearance of dysfunctional mitochondria, and revealed that ROS associated with mitochondria play a key role in potentiating RLR signaling.

Location Matters: Plasmacytoid dendritic cells (pDCs), an immune cell specialized to respond to viral infections, use Toll-like receptors (TLRs) 7 and 9 expressed in endosomes to sense viral nucleic acids. Triggering of TLR7 or 9 results in the induction of two distinct signaling pathways, one that leads to the production of proinflammatory cytokines and another that induces the expression of antiviral type I interferons. How one receptor can trigger two distinct signaling pathways, however, is not clear. Sasai et al. (p. 1530 ) now show that subcellular localization is key. Cells from mice deficient in the Adapter Protein 3 (AP-3) complex, which regulates protein sorting to intracellular vesicles, did not produce type I interferons in response to TLR9 ligand, but proinflammatory cytokine production remained intact. AP-3 was required for trafficking of TLR9 from early endosomes, where proinflammatory signaling can occur, to lysosome-related organelles, where the signaling machinery required for type I interferon induction is located. Such spatial segregation may represent a common mechanism whereby activation of one receptor can result in the induction of multiple independent signaling cascades.

Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-κB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-β genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-β induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-κB and the IFN-β promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-β, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.