Cell Intrinsic Innate Immunity Interactive Signaling Pathway
The intracellular sensing of pathogens operates through detection of foreign molecular components, including viral and bacterial nucleic acids in the cytosol. Once detected, the innate immune system can induce the production of type I interferons (IFNs) and cytokines (TNF-α) through the TBK1-IRF-3 and NF-κB pathways . Adapter proteins localized on the mitochondria (i.e., MAVS) or endoplasmic reticulum (ER) (i.e., STING) respond to RNA or DNA sensing pathways, respectively.
RIG-I-like Receptors (RLRs), including RIG-1 and MDA5, are able to detect double stranded viral RNA and 5’-triphosphate short dsRNA. MDA5 and RIG-1 induce the dimerization of the mitochondrial antiviral-signaling protein (MAVS) through interaction of shared caspase recruitment domains. MAVS dimerization allows TNF receptor-associated factor 3 (TRAF3) binding, via the interaction of a TRAF-interaction motif and TRAF domains, respectively. TRAF3, in turn, recruits the adapter proteins TANK, NAP1 and SINTBAD. TANK serves to link upstream RLR signaling to the TANK binding kinase 1 (TBK1) which induces phosphorylation of Interferon Regulatory Factor 3 (IRF-3). IRF-3 phosphorylation and subsequent dimerization induces nuclear translocation of IRF-3 which then binds to the Interferon-Stimulated Response Element (ISRE) leading to type I Interferon gene expression.
Cytosolic double-stranded DNA detection also feeds into the TBK1-IRF-3 signaling axis through multiple portals. The major cytosolic DNA binding sensor is cyclic GMP-AMP synthase (cGAS) which responds to DNA binding by producing the cyclic dinucleotide c-GMP-AMP (cGAMP). cGAMP binds to STING and activates TBK1-IRF-3-mediated IFN expression. Additional dsDNA sensors such as IFI16, DAI and DDX41 also signal through the STING pathway. In addition, certain pathogenic bacterial infections produce cyclic di-nucleotide second messengers which also activate the STING pathway. Finally, cytosolic DNA can also engage the RIG-I-MAVS pathway through DNA-dependent RNA polymerase III (RNA Pol III) transcribing viral 5′-triphosphate RNA.
The RLR pathway also induces NF-κB mediated transcription. In the case of RNA sensing this occurs through the RLR induced polymerization of MAVS and subsequent binding of TRAF proteins (TRAF2/5/6), the activity of which is mediated by NLRX1. Ubiquitination of TRAF2/5/6 is required for association with MAVS and is negatively regulated by OTUB1/2 deubiquitination. TRAF2/5/6 in turn recruits NEMO and the IκB kinases IKKα/IKKβ which phosphorylate IκB, thereby activating downstream NF-κB signaling.
Ubiquitination plays a significant regulatory role in the STING and MAVS pathways. RIG-1 requires K63-ubiquitination by TRIM25 for activation by RNA binding, but unanchored chains also suffice. TRAF3 undergoes modification with K63-ubiquitin, possibly autocatalytic, to stimulate IRF-3 phosphorylation, while IFN production is inhibited by TRAF3 degradation triggered by DUBA-mediated deubiquitination. The central importance of STING is reflected by the extent of its regulation. TRIM32 (K63), TRIM56 (K63) and AMFR (K27) are all postulated to be involved in the poly-ubiquitination that induces translocation of STING from the ER through the Golgi to microsomes, a process negatively regulated by Atg9a. In addition, RNF5 has been shown to downregulate type I IFN expression by K48 ubiquitin-mediated degradation of STING. DDX41 degradation is also controlled by ubiquitination through the action of TRIM21. Finally, STING activity can also regulated through phosphorylation by the serine-threonine kinase ULK1, which turns off STING pathway activation.
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We would like to thank Dr. Kate Fitzgerald, Professor of Medicine, Director of the Program in Innate Immunity and The Worcester Foundation Chair in Biomedical Sciences at the University of Massachusetts Medical School for reviewing this diagram.