Hu Tiancheng, Liu Yiping, Fleck Jeremy, King Cason, Schalk Elaine, Zhang Zhenyu, Mehle Andrew, Smith Judith A
bioRxiv. 2024 May 14:2024.05.10.593557. doi: 10.1101/2024.05.10.593557.
The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.
双链DNA（dsDNA）传感器STING越来越多地参与对“无菌”内源性威胁和病原体的反应，这些病原体没有典型的DNA或环二核苷酸刺激。先前的研究表明，内质网（ER）应激反应，即未折叠蛋白反应（UPR），可激活STING。在此，我们试图确定内质网应激是否产生了STING配体，并确定参与的UPR途径。用UPR诱导剂毒胡萝卜素（TPG）或氧葡萄糖剥夺刺激后，IFN-β表达的诱导需要STING和dsDNA感应环鸟苷酸-腺苷酸合成酶（cGAS）。此外，TPG增加了细胞质线粒体DNA，免疫荧光显示在小鼠和人类细胞中有dsDNA斑点，提供了一种cGAS刺激。N-乙酰半胱氨酸降低了TPG诱导的IFN-β，这与活性氧（ROS）有关。然而，线粒体氧化应激抑制剂mitoTEMPO对TPG诱导的IFN没有影响。另一方面，抑制肌醇需求酶1（IRE1）内质网应激传感器及其靶转录因子XBP1可减少细胞质dsDNA的产生。诱导型一氧化氮合酶（iNOS）的上调依赖于XBP1，iNOS抑制剂可降低细胞质dsDNA和IFN-β，这表明ROS在IRE1-XBP1途径的下游起作用。抑制蛋白激酶R样内质网激酶（PERK）途径也减弱了细胞质dsDNA的释放。PERK调节的凋亡因子Bim对于dsDNA的释放和IFN-β mRNA的诱导都是必需的。最后，XBP1和PERK途径促进了RNA病毒水疱性口炎病毒（VSV）诱导的细胞质dsDNA释放和IFN诱导。总之，我们的研究结果表明，内质网应激源，包括没有典型STING或cGAS配体的病毒病原体，如RNA病毒，触发了多个经典的UPR途径，这些途径通过线粒体dsDNA释放协同激活STING和下游的IFN-β。
