This study describes the novel observation that ER stress, even in absence of pattern recognition receptor stimulation, activates IRF3. Different forms of ER stress accomplish this through at least 2 distinct pathways, requiring either TBK1/STING or AEBSF-sensitive signaling. The synergistic induction of several IRF3-regulated inflammatory mediators by concurrent UPR and LPS stimulation suggest the activation of IRF3 by ER stress may have a wider impact in innate immunity, beyond augmenting IFN-β production. Even though ER stress alone is not sufficient to trigger the induction of an IRF3-regulated gene, activation of IRF3 by ER stress is necessary for the dramatic IFN-β synergism observed with LPS. In evidence of this requirement, interfering with thapsigargin-dependent IRF3 phosphorylation through the modulation of STING severely impacts the magnitude of thapsigargin-induced synergy. Similarly, disrupting tunicamycin-dependent IRF3 phopshorylation with AEBSF significantly diminished synergistic IFN-β expression. Residual IFN-β induction in the LPS+Tg treated STING-/- MEFs may relate to IRF3 phosphorylation below our limits of detection, or compensation by another IRF3 serine (e.g. Ser 339) for which we did not assay(50
This study supports the novel concept that intracellular stress responses may coopt innate immune signaling pathways previously thought to be dedicated to pathogen sensing. The proximity of STING to the mitochondria-associated membrane (MAMs) a site of inter-organelle calcium transport and regulation may suggest why UPR inducers that affect calcium also mobilize STING(4
). Our findings suggest that at least in MEFs and macrophages, STING and TBK1 associate even prior to stimulation. Upon ER stress induction, STING and TBK1 dramatically reorganize into larger macroscopic collections. This mobilization may be a result of reorganization of the ER membranes themselves (containing STING), translocation to another organelle (e.g. Golgi), or association with other unidentified molecules in a multi-molecular complex. The augmentation of type I IFN responses by ER stress/calcium dysregulation and STING may be of particular relevance to viral infections, such as Hepatitis C, that induce ER stress and cause calcium leak(52
). Our results obtained with in vitro
OGD () have direct implications for in vivo
ischemia-reperfusion injury: they suggest the dysregulation of calcium, ER stress and type I IFN-dependent inflammatory injury may be critically interrelated (20
The UPR has been shown to activate both NF-κB and AP-1 family member transcription factors(58
). Thus it is unclear why UPR-induced IRF3 phosphorylation is not sufficient to induce IFN-β expression in vitro
. Even though the UPR induces nuclear translocation of IRF3, translocation does not automatically confer transcriptional activity: Dissociation between translocation and transcriptional activity has been noted in multiple models of viral IRF3 inhibition (59
). IRF3 has 2 activation clusters comprising 7 potentially phosphorylated serines and threonines (S385, S386, and S396, S398, S402, S405, T404). Some controversy remains regarding serine phosphorylation requirements for IRF3 activity: Phosphorylation of S396 has been proposed as an essential minimal acceptor site for responses to Sendai virus, and may be critical for homodimerization (50
). More recently S396 has been shown to promote higher order oligomerization(64
). However, others have identified S386 as the critical site for homodimerization and nuclear translocation(65
). Unfortunately, it is not yet clear exactly which sites on IRF3 correspond with optimal transcriptional activity in response to LPS and other specific pathogens. Ultimately, both may act cooperatively to bind CBP/p300 with higher affinity (64
). Thus one possibility is that even though ER stress induces S386 phosphorylation, ER stress in isolation does not induce strong enough phosphorylation at S396, as suggested by western blot (e.g. ); the UPR may only induce partial phosphorylation of IRF3 and LPS remains necessary for additional phosphorylation at other serines/threonines. Alternatively, as suggested by our western blot data, UPR induced phosphorylation at S386 may facilitate or enhance LPS dependent S396 phosphorylation. The requirement for multiple-site IRF3 phosphorylation to promote oligomerization may explain the qualitative differences in immunofluorescence between LPS and ER stressors such as OGD ()(64
). Our data would suggest that ultimate phosphorylation at S396 correlates best with IRF3 DNA binding by chromatin immunoprecipitation and transcriptional activation of IFN-β(23
Apart from suboptimal IRF3 activation, there are other possible explanations: IRF3 alone is not sufficient for IFN gene transcription; the enhanceosome also contains NF-kB and AP-1 transcription factors. Transcriptional activation following enhanceosome formation requires binding of multiple elements including critical scaffolding molecules (HMGA1) and histone acetyltransferases (e.g. CBP/p300)(11
). LPS stimulation may be required to recruit these other molecules. Another possibility is that a stronger NF-κB signal may be required than that generated during ER stress alone. Finally, there could be a cell type issue, since our studies are conducted in macrophages and MEFs. When mice are treated in vivo
with tunicamycin alone, we observed detectable serum IFN-β (preliminary data not shown), suggesting that an unidentified cell type is capable of producing IFN during a UPR.
In this study and others, ER stress has been noted to augment transcription of select IRF3-regulated genes (e.g. IFN-β but not RANTES)(24
). IRF3 binds similar DNA sequences within gene promoters designated as interferon stimulated response elements (ISRE) or positive regulatory domains (PRD I and III in the IFN-β promoter)(67
). The selectivity in synergism may relate to promoter complexity and requirement for multiple transcription factors, as mentioned above. Constitutively activated IRF3 (an aspartate containing phosphomimetic) is sufficient to activate only a small subset of ISRE containing genes, including ifit2/ISG54, ISG56, ISG60, CIG5 and PMA inducible protein 1(68
). However, we did not detected robust activation of ISG54 by thapsigargin alone. This failure may reflect suboptimal IRF3 activation at specific serines. Alternatively, given the independence of XBP1 and IRF3 translocation (), and the discovery of XBP1 binding sites in cytokine promoters and enhancers, significant synergy might require DNA binding sites for both IRF3 and UPR-dependent transcription factors(22
). The experience with IFN-β would favor this “multi-hit” hypothesis.
It is not clear which aspects of the UPR are necessary for IRF3 phosphorylation and nuclear translocation. The answer may differ depending upon type of ER stress. Our studies would suggest that XBP1 is not required for ER stress-induced IRF3 nuclear translocation. PERK is not necessary for synergistic IFN induction ((22
) and data not shown). AEBSF, a protease inhibitor that prevents ATF6 processing, blocked tunicamycin but not thapsigargin-dependent IRF3 phosphorylation and synergy ()(22
). Thapsigargin may utilize an IRE1 kinase mediated pathway to activate IRF3. Alternatively, thapsigargin and A23187 could mobilize a non-classical UPR ER stress pathway related to calcium flux that has not been described. Another possibility is that IRF3 activation resulting from profound ER calcium depletion, and the UPR are independent outcomes of treatment with these stressors.
Our results are consistent with the hypothesis that tunicamycin and 2-deoxyglucose-induced IRF3 phosphorylation proceed through ATF6 or a related protein. ATF6 belongs to the OASIS family of transcription factors that is processed by the site 1 proteases. However, protein distribution of these other family members is much more restricted than ATF6(69
). AEBSF also inhibits reactive oxygen species generation (ROS) by NADPH(70
). Thapsigargin, tunicamycin and ER stress related to cholesterol loading have all been found to induce oxidative stress via NADPH(71
). ROS potentiate IRF3 activation(74
). However, NADPH oxidase inhibition by AEBSF would not explain the divergent effects on thapsigargin and tunimcamycin, particularly given the involvement of calcium in ER stress mediated NADPH oxidase activation(71
). The specific AEBSF sensitive signaling event remains to be confirmed.
Another outstanding question is the identity of the kinase activated by non-thapsigargin UPR inducers that is responsible for phosphorylating IRF3. In this study, tunicamycin led to IRF3 phosphorylation in the presence of a TBK1/IKKε inhibitor. The kinase cascade involving NIK and IKK-α has been reported to phosphorylate IRF3 independently of TBK1(75
). The MAP-kinase cascade initiated by IRE-1 that includes p38 may also play a role(76
). Dissecting the exact mechanism of IRF3 activation by all ER stressors, at the level of the UPR and subsequent kinase cascades, is outside the scope of this study but will be interesting to tease apart in the future.
Ultimately, our findings raise the possibility that different forms of ER stress/UPR inducers may utilize innate immune sensing pathways including STING. Thus, ER stress is poised to significantly augment type I IFN and innate immune responses in the setting of pathogen challenge, or endogenous damage. These findings have implications for conditions involving type I IFN and ER stress such as viral infections, certain bacterial infections, ischemia-reperfusion injury, and potentially rheumatic inflammatory disease. The recruitment of innate immune sensing molecules such as STING represents a newly described interface between intracellular stress and innate immunity.