Interferon regulatory factor 3 (IRF3) regulates early type I IFNs and other genes involved in innate immunity. We have previously shown that cells undergoing an endoplasmic reticulum (ER) stress response called the “Unfolded Protein Response” (UPR) produce synergistically augmented IFN-β when stimulated with pattern recognition receptor agonists such as LPS Concomitant ER stress and LPS stimulation resulted in greater recruitment of the IRF3 transcription factor to ifnb1 gene regulatory elements. In this study, we utilized murine cells to demonstrate that both oxygen-glucose deprivation and pharmacologic UPR inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors utilized distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, oxygen-glucose deprivation) critically depended upon Stimulator of interferon gene (STING), an ER-resident nucleic acid-responsive molecule. However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. In contrast, other forms of ER stress (e.g., tunicamycin treatment) promote IRF3 phosphorylation independently of STING and Tank binding kinase 1 (TBK1). Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by AEBSF, a serine protease inhibitor that blocks ATF6 processing. Interfering with ER stress-induced IRF3 activation abrogated IFN-β synergy. Together, these data suggest ER stress primes cells to respond to innate immune stimuli by activating the IRF3 transcription factor. Our results also suggest certain types of ER stress accomplish IRF3 phosphorylation by co-opting existing innate immune pathogen response pathways. These data have implications for diseases involving ER stress and type I IFN.
STING (TMEM173, MPYS,MITA) located in the endoplasmic reticulum (ER), plays a critical role in type I IFN responses to intracellular DNA. STING activates TBK1, resulting in phosphorylated IRF3 that regulates IFN-β transcription. Our previous work showing markedly enhanced LPS-induced IFN-β during ER stress implicated increased IRF3 recruitment to the ifnb1 promoter. In this study, utilizing STING-/- mouse embryonic fibroblasts (MEFs) and RNAi (in MEFs and macrophages) we determined that STING was essential for synergistic induction of IFN-β mRNA in response to LPS and ER stress (thapsigargin/TPG treatment). TPG treatment alone induced re-localization and clustering of STING and TBK1 in MEFs. Although ER stress resulted in nuclear translocation of IRF3 in a STING-independent manner, TPG and TPG+LPS-induced IRF3 phosphorylation (as detected by immunofluorescence and western blot respectively) depended upon STING. In comparison, STING was not required for LPS or LPS+TPG induced NF-κB phosphorylation. These results indicate that ER stress alone results in IRF3 nuclear translocation and phosphorylation. In mice, treatment with the ER stress inducer tunicamycin was sufficient for induction of serum-detectable IFN-β, comparable with low dose LPS. Our results also suggest that the synergistic IFN-β responses during LPS and ER stress involve STING-dependent IRF3 phosphorylation.
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