Abstract
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 lipopolysaccharide (LPS). Previous studies revealed increased IRF3 at ifnb1 genetic regulatory elements during ER stress; however the underlying mechanism was unclear [1]. In this study we sought to determine whether ER stress directly activated IRF3.
This study was conducted in murine macrophages (primary and RAW264.7) and STING−/− or +/+ murine embryonic fibroblasts. IRF3 activation was monitored by immunofluorescence microscopy and western blot detection of S386 and S396 respectively as well as IRF3 nuclear translocation. IRF3 regulated gene expression was detected by quantitative PCR. TBK1 family kinases were inhibited with MRT67307. Site-1 protease cleavage of ATF6 was blocked with AEBSF.
Oxygen-glucose deprivation and multiple pharmacologic UPR inducers triggered the phosphorylation (S386) and nuclear translocation of IRF3, even in the absence of exogenous LPS. UPR induction also enhanced LPS-dependent phosphorylation of IRF3 at S396. Consistent with IRF3 activation, ER stress augmented LPS-dependent induction of several IRF3 regulated genes (IFN-β, IFN-α4, and CXCL10) but not others (ISG54, RANTES). Further mechanistic dissection revealed differences among UPR inducers: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin or A23187) critically depended upon Stimulator of interferon gene (STING) and Tank-binding kinase (TBK1). However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. STING was also necessary for optimal synergistic induction of IFN-βby thapsigargin and LPS. In contrast, non-calcium-dependent forms of ER stress (e.g., tunicamycin treatment, oxygen glucose deprivation, 2-deoxyglucose) promoted IRF3 phosphorylation independently of STING and TBK1. Rather, IRF3 phosphorylation and synergistic IFN-βinduction by these UPR inducers was abrogated by AEBSF, an inhibitor of ATF6 processing. AEBSF had no effect on thapsigargin induced IRF3 phosphorylation or IFN-β synergy.
Discrete IRF3 activating pathways were identified for different types of ER stress that involved either STING/TBK1 or Site-1 specific proteases (e.g. ATF6). The activation of STING by ER stress has not been described. Although ER stress is not sufficient for expression of IRF3-regulated genes, activation of IRF3 by the UPR plays a critical role in augmenting LPS-dependent expression, as interference in this process severely impacted IFN-β induction. These data have implications for diseases involving ER stress and type I IFN.
