A novel biosensor for the spatiotemporal analysis of STING activation during innate immune responses to dsDNA
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ABSTRACT:
SUBMITTER: Steve Smarduch
PROVIDER: S-SCDT-10_1038-S44318-025-00370-Y | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.
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Project description:Dysregulated sensing of self-nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type I IFNs by innate cells. Here we show that immune complexes of self-DNA and autoantibodies (DNA-ICs) contribute to elevated IFN levels via activation of the cGAS/STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp caused a delay in endolysosomal maturation and prolonged the transit time of ingested DNA-ICs. Stalling in maturation-defective organelles facilitated leakage of DNA-ICs into the cytosol, promoting activation of the TBK1/STING pathway. Genetic deletion of STING and STING and cGAS chemical inhibitors abolished IFN production and rescued systemic activation of IFN-stimulated genes in vivo. These data unveil the contribution of cytosolic self-nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodeling in preventing innate activation.
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Project description:Sensing of cytosolic DNA of microbial or cellular/mitochondrial origin by cGAS initiates innate immune responses via the adaptor protein STING. It remains unresolved how the activity of STING is balanced between a productive innate immune response and induction of autoimmunity. Here we show that interferon regulatory factor 8 (IRF8) is essential for efficient activation of STING-mediated innate immune responses in monocytes. This function of IRF8 is independent of its transcriptional role in monocyte differentiation. In uninfected cells, IRF8 remains inactive via sequestration of its IRF-associated domain by its N- and C-terminal tails, which reduces its association with STING. Upon triggering the DNA sensing pathway, IRF8 is phosphorylated at Serine 151 to allow its association with STING via the IRF-associated domain. This is essential for STING polymerization and TBK1-mediated STING and IRF3 phosphorylation. Consistently, IRF8-deficiency impairs host defense against the DNA virus HSV-1, and blocks DNA damage-induced cellular senescence. Bone marrow-derived mononuclear cells which have an autoimmune phenotype due to deficiency of Trex1, respond to IRF-8 deletion with reduced pro-inflammatory cytokine production. Peripheral blood mononuclear cells from systemic lupus erythematosus patients are characterized by elevated phosphorylation of IRF8 at the same Serine residue we find to be important in STING activation, and in these cells STING is hyper-active. Taken together, the transcription-independent function of IRF8 we describe here appears to mediate STING activation and represents an important regulatory step in the cGAS/STING innate immune pathway in monocytes. The transcription factor IRF8 has been shown to regulate monocyte differentiation via its DNA-binding activity. Here authors show that IRF8 is also involved in cytosolic DNA sensing via its phosphorylation-dependent association to the adaptor protein STING, thus representing an important checkpoint between immune response and autoimmunity in monocytes.
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Project description:The stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that is a target of therapeutics for infectious diseases and cancer. However, early-phase clinical trials of small-molecule STING agonists have shown limited antitumour efficacy and dose-limiting toxicity. Here, we show that a polyvalent STING agonist-a pH-sensitive polymer bearing a seven-membered ring with a tertiary amine (PC7A)-activates innate-immunity pathways through the polymer-induced formation of STING-PC7A condensates. In contrast to the natural STING ligand 2',3'-cyclic-GMP-AMP (cGAMP), PC7A stimulates the prolonged production of pro-inflammatory cytokines by binding to a non-competitive STING surface site that is distinct from the cGAMP binding pocket. PC7A induces antitumour responses that are dependent on STING expression and CD8+ T-cell activity, and the combination of PC7A and cGAMP led to synergistic therapeutic outcomes (including the activation of cGAMP-resistant STING variants) in mice bearing subcutaneous tumours and in resected human tumours and lymph nodes. The activation of the STING pathway through polymer-induced STING condensation may offer new therapeutic opportunities.
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