Cytosolic Immunostimulatory DNA Ligands and DNA Damage Activate the Integrated Stress Response, Stress Granule Formation, and Cytokine Production

The presence of aberrant double-stranded DNA (dsDNA) in the cytoplasm of cells is sensed by unique pattern recognition receptors (PRRs) to trigger innate immune response. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is activated by the presence of non-self or mislocalized self-dsDNA from nucleus or mitochondria released in response to DNA damage or cellular stress in the cytoplasm. Activation of cGAS leads to the synthesis of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates STING, triggering downstream signaling cascades that result in the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we show that diverse immunostimulatory dsDNA ligands and chemotherapy agents like Doxorubicin and Taxol trigger the integrated stress response (ISR) by activating endoplasmic reticulum (ER) stress kinase, protein kinase RNA-like ER kinase (PERK), in addition to the canonical IFN pathways. PERK-mediated phosphorylation and inactivation of the alpha subunit of eukaryotic translation initiation factor-2 (eIF2α) result in the formation of stress granules (SGs). SG formation by dsDNA was significantly reduced in PERK knockout cells or by inhibiting PERK activity. Transcriptional induction of IFNβ and cytokines, ISR signaling, and SG formation by dsDNA was dampened in cells lacking PERK activity, STING, or key stress-granule nucleating protein, Ras-GAP SH3 domain-binding protein 1 (G3BP1), demonstrating an important role of the signal transduction pathway mediated by STING and SG assembly. Lastly, STING regulates reactive oxygen species (ROS) production in response to DNA damage, highlighting the crosstalk between DNA sensing and oxidative stress pathways. Together, our data identify STING-PERK-G3BP1 signaling axis that couples cytosolic DNA sensing to stress response pathways in maintaining cellular homeostasis.