Endoplasmic Reticulum Stress Regulates the Innate Immunity Critical IRF3

This information is current as Yi-Ping Liu, Ling Zeng, Austin Tian, Ashley Bomkamp, of September 25, 2021. Daniel Rivera, Delia Gutman, Glen N. Barber, Julie K. Olson and Judith A. Smith J Immunol 2012; 189:4630-4639; Prepublished online 1 October 2012;

doi: 10.4049/jimmunol.1102737 Downloaded from http://www.jimmunol.org/content/189/9/4630

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Endoplasmic Reticulum Stress Regulates the Innate Immunity Critical Transcription Factor IRF3

Yi-Ping Liu,* Ling Zeng,* Austin Tian,* Ashley Bomkamp,* Daniel Rivera,* Delia Gutman,† Glen N. Barber,† Julie K. Olson,‡ and Judith A. Smith*

IFN 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 produce synergistically augmented IFN-b when stimulated with pattern recognition 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 used murine cells to demonstrate that both oxygen–glucose deprivation and pharmacologic unfolded protein response inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors used distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, Downloaded from oxygen–glucose deprivation) critically depended upon stimulator of IFN gene, 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 stimulator of IFN gene and TANK-binding kinase 1. Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor that blocks activating transcription factor 6 processing. Interfering with ER stress- induced IRF3 activation abrogated IFN-b synergy. Together, these data suggest ER stress primes cells to respond to innate http://www.jimmunol.org/ 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. The Journal of Immunology, 2012, 189: 4630–4639. ype I IFNs (IFN-a/b) play diverse roles in adaptive and helicases (3). Interestingly, a recently identified molecule stimula- innate immunity; type I IFNs activate macrophages and tor of IFN gene (STING, also known as MPYS/MITA/TMEM173/ T NK cells, promote T cell survival and dendritic cell ERIS), located in the endoplasmic reticulum (ER) membrane, ap- maturation, and increase the production of Th1-polarizing cyto- pears to play a critical role in the induction of IFN-b by cyto- kines (1). Innate immune cells such as macrophages and dendritic plasmic dsDNA and RNA, though STING does not directly bind by guest on September 25, 2021 cells produce large amounts of type I IFN following the ligation nucleic acids (4–7). of diverse pattern recognition receptors (PRRs). PRRs recognize Signaling by these various pathogen sensors converges at the conserved molecular structural motifs on pathogens, as well as activation of the TANK-binding kinase 1 (TBK1) family of kinases endogenous products released by tissue damage (2). The PRRs (8). TBK1 is a serine/threonine kinase that phosphorylates the that mediate IFN-b induction in macrophages include the LPS transcription factor IFN regulatory factor 3 (IRF3) (9). IRF3 is receptor TLR4, the endosomal dsRNA sensor TLR3, and the cy- constitutively expressed and resides in the cytoplasm in latent toplasmic dsRNA-responsive retinoic acid-inducible gene-I family form. Upon phosphorylation, IRF3 dimerizes and translocates from the cytoplasm into the nucleus (10). At the ifnb1 locus, IRF3 cooperatively binds with other transcription factors including NF- *Department of Pediatrics, School of Medicine and Public Health, University of kB, AP-1, and IRF7 to form a multimolecular that Wisconsin, Madison, WI 53792; †Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, University of Miami, promotes ifnb1 transcription (11). IRF3 is absolutely required for Miami, FL 33136; and ‡Department of Neurological Surgery, School of Medicine the induction of IFN-b and certain IFN-a species early during and Public Health, University of Wisconsin, Madison, WI 53792 viral infections and by LPS (12–14). IRF3-regulated early type I Received for publication September 22, 2011. Accepted for publication August 31, IFN production primes cells for greater IFN responses during viral 2012. infections by inducing IRF7 (15). IRF3 also regulates other This work was supported by National Institutes of Health Grant K08 AI081045, the University of Wisconsin-Madison Graduate School, School of Medicine and Public inflammatory mediators such as the chemokines CXCL10 and Health, and Department of Pediatrics. RANTES (16–18). In a murine model of hepatic ischemia-reper- Address correspondence and reprint requests to Dr. Judith A. Smith, University of fusion injury, damage is significantly decreased in both type I IFN Wisconsin-Madison, H4/472 CSC, 600 Highland Avenue, Madison, WI 53792-4108. receptor and in IRF3-deficient animals (19, 20). In addition to its E-mail address: [email protected] transcriptional role, IRF3 promotes apoptosis in virus-infected Abbreviations used in this article: AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluo- cells through association with Bax (21). ride hydrochloride; ATF, activating transcription factor; CBP, CREB-binding protein; 2DG, 2-deoxyglucose; ER, endoplasmic reticulum; F, forward; HA, hemagglutinin; Even as innate immune cells are poised to counter external IKK, IkB kinase; IRE-1, inositol-requiring enzyme-1; IRF3, IFN regulatory factor 3; threats, conserved stress responses respond to intracellular derange- MEF, murine embryonic fibroblast; OGD, oxygen–glucose deprivation; PERK, pro- tein kinase receptor-like endoplasmic reticulum kinase; PRR, pattern recognition ments. We and others (22–25) have shown that type I IFN responses receptor; R, reverse; RNAi, RNA interference; TBK1, TANK-binding kinase 1; Tg, to PRR ligands are dramatically enhanced by an intracellular stress thapsigargin; Tm, tunicamycin; UPR, unfolded protein response; WT, wild-type; response originating in the ER called the unfolded protein response XBP1, X-box binding protein 1. (UPR). The UPR represents a final common pathway in the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 response to a broad variety of stresses perturbing ER function, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102737 The Journal of Immunology 4631 including oxygen and nutrient deprivation, calcium dysregulation, (not heat-inactivated; Invitrogen) and 13 antibiotic-antimycotic solution 2/2 misfolded proteins, and N-linked glycosylation inhibition (26). (4). XBP1 MEFs were kindly provided by Laurie Glimcher (Harvard The three major signaling cascades of the UPR stem from acti- University, Boston, MA). For RNA interference (RNAi) studies, RAW 264.7 cells were transiently transfected with STING or control RNAi vation of ER-resident molecules: protein kinase receptor-like ER (Dharmacon) using AMAXA (Lonza). kinase (PERK), the proto-transcription factor activating tran- To induce ER stress, cells were treated with 10 mg/ml tunicamycin (Tm), scription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE-1). 20 mM 2-deoxyglucose (2DG), 10 mM A23187, or 1 mM Tg (all from IRE-1 is both a kinase and endonuclease that cleaves 26 bp from Sigma-Aldrich). For OGD, cells were washed three times with a glucose- free isotonic salt solution (OGD buffer [pH 7.4]: 20 mM NaHCO , 120 the X-box binding protein 1 (XBP1) transcription factor mRNA. 3 mM NaCl, 5.36 mM KCl, 0.33 Na2HPO 4 mM, 0.44 mM KH2PO4, 1.27 This atypical splicing eliminates a premature stop codon and thus mM CaCl2, and 0.81 mM MgSO4) and then incubated in OGD buffer in enables translation of full-length active XBP1 (26). XBP1 is es- hypoxic conditions containing 94% N2,5%CO2, and 1%O2 (33). Fol- sential for synergistic type I IFN responses to PRR agonists (22). lowing OGD, cells were provided with growth media with or without LPS We have shown that XBP1 binds an enhancer element 6 kb and transferred to a standard 5% CO2 incubator. For LPS stimulations, RAW 264.7 macrophage cells were treated with 100 ng/ml and primary downstream of the ifnb1 gene (23). Interestingly, during con- bone marrow macrophages and MEFs treated with 10 ng/ml for 3 h unless comitant UPR and LPS stimulation, IRF3 recruitment increased at otherwise indicated. For and chemokine protein assessment, IFN-a both the ifnb1 promoter and enhancer (23). IRF3 binding was only and IFN-b ELISA kits were from PBL InterferonSource and eBioscience k ε observed in the presence of LPS (23). Although XBP1, a member (RANTES). For TBK1/I B kinase (IKK) inhibition, cells were pretreated with 2 mM MRT67307 (generous gift from Dr. Philip Cohen, University of of the CREB family of transcription factors, is known to hetero- Dundee, Dundee, Scotland) (34). The serine protease inhibitor AEBSF was dimerize with other transcription factors, IRF3 is not a known from Sigma-Aldrich. binding partner (27, 28). Downloaded from Investigations of in macrophages from rats Abs overexpressing the misfolding protein HLA-B27 reveal both UPR b-actin, mouse mAb, NF-kB rabbit polyclonal Ab, and USF2 rabbit and IFN signature, including several IRF3-regulated genes (IFN-b, polyclonal Ab were from Santa Cruz Biotechnology; hemagglutinin (HA)- OAS, and IP-10) (29). However, the relationship between ER Tag mouse mAb, IRF3 rabbit mAb (detects one IRF3 band), and phospho- stress and IRF3 activation was unclear. A previous study had IRF3 (pS396) rabbit mAb from Technology; phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects two demonstrated activation of IRF3, evident by phosphorylation, IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; http://www.jimmunol.org/ nuclear translocation, and CREB-binding protein (CBP) associa- TBK1 rabbit mAb from Abcam; and normal rabbit IgG from Millipore. tion in response to DNA-damaging agents such as doxorubicin in Secondary Abs were goat anti-mouse IgG-Alexa Fluor 594 and anti-rabbit HeLa cells (30). However, a later study employing a wider range IgG-Alexa Fluor 488 (Invitrogen). of stressors failed to validate these findings in HEK293 cells (31). Immunofluorescence microscopy In a recent study by Hu et al. (25), the UPR inducer thapsigargin (Tg) augmented polyinosinic-polycytidylic acid-dependent IRF3 Cells were plated on coverslips in 60-mm dishes for 24 h prior to treatment. For STING–TBK1 colocalization, MEFs were transiently transfected with phosphorylation, supporting the involvement of UPR in IRF3 MPYS-HA (generous gift of John Cambier, University of Colorado, regulation. This study also supported a role for XBP1 in pro- Denver, CO) using Fugene HD (Roche) 24 h prior to stimulation (5). After moting antiviral activity. treatment, cells were washed (3 3 5minPBS)andthenfixedin4% by guest on September 25, 2021 In this study, we sought to determine whether ER stress exerts paraformaldehyde 30 min at room temperature. Cells were then washed a direct effect on IRF3 activation. We found that multiple pharma- with PBS, Tris A buffer (0.1 M [pH 7.6] Tris and 0.1% Triton X-100), and Tris B buffer (0.1 M [pH 7.6] Tris, 0.1% Triton X-100, and 0.2% BSA) cologic ER stress inducers and oxygen–glucose deprivation (OGD) 3 3 5mineachandincubatedwith10%goatseruminTrisBbufferfor triggered activation of IRF3, as indicated by IRF3 phosphorylation 1 h. Primary Abs were added in Tris B buffer and cells incubated at 4˚C and nuclear translocation. Mechanism of IRF3 activation varied by overnight. After washing the cells with Tris A 3 3 5 min, secondary type of ER stressor: some forms of ER stress, in particular those that fluorescence-conjugated Ab was added and samples incubated 1 h at room temperature. Cells were washed with PBS 3 3 5 min and the coverslips also involve calcium mobilization, require TBK1 and STING for mounted on slides with ProLong Gold antifade reagent with DAPI nuclear phosphorylation and nuclear translocation of IRF3. Indeed, STING stain (Invitrogen). For negative controls, the same concentration of primary was essential for synergistic IRF3 phosphorylation and optimal mouse IgG (Sigma-Aldrich) or rabbit IgG (Sigma-Aldrich) was added. IFN-b induction by concurrent Tg and LPS. These data suggest Images were acquired on a Nikon Eclipse 50i fluorescence microscope m intracellular stress responses may use innate immune pathogen- (Nikon). Scale bars in images are 50 M. sensing pathways to augment IRF3-regulated cytokine and che- Quantitative PCR mokine production. In contrast, other ER stress inducers activated IRF3 by a pathway inhibited by 4-(2-aminoethyl)-benzenesulfonyl Cells were lysed with TRIzol (Invitrogen) and processed according to the manufacturer’s instructions. Briefly, RNA was extracted with chloroform fluoride hydrochloride (AEBSF), a serine protease inhibitor that and precipitated with isopropanol. Total RNA was purified with and treated prevents UPR-dependent processing of ATF6. Thus, at least two with DNase I (Invitrogen) prior to reverse transcription using random distinct pathways link ER stress to IRF3 activation. primers (Promega). Gene expression level in cDNA was quantitated by SYBR Green (Bio-Rad) fluorescence, using an iCycler or MyiQ (Bio-Rad). Relative mRNA expression was normalized to 18S rRNA housekeeping Materials and Methods gene. Primers were designed using Beacon design software (Premier Cells, reagents, and stimulations Biosoft): 18S rRNA, forward (F) 59-GGA CAC GGA CAG GAT TGA CAG-39 and reverse (R) 59-ATC GCT CCA CCA ACT AAG AAC G-39; The RAW264.7 macrophage cell line (American Type Culture Collection) IFN-b,F59-ACT AGA GGA AAA GCA AGA GGA AAG-39 and R 59- was maintained in DMEM/high glucose (Mediatech) with 10% FBS CCA CCA TCC AGG CGT AGC-39; IFN-a4, F 59-CAC AAT GGC TAG (Hyclone) and 13 antibiotic-antimycotic solution (Mediatech). Murine GCT CTG-39 and R 59-TGT TGG TTA TCC ACC TTC TC-39; IL-6, F 59- bone marrow macrophages were isolated from C57BL/6 femurs with CTT CCA TCC AGT TGC CTT C-39 and R 59-ATT TCC ACG ATT TCC Histopaque 1083 (Sigma-Aldrich) and plated 3 d in non–tissue-culture CAG AG-39; IL-1b,F59-CTC GCA GCA GCA CAT CAA C-39 and R 59- petri dishes in DMEM (as above) supplemented with 5% M-CSF–con- ACG GGA AAG ACACAG GTA GC-39; STING, F 59-TGC CGC CTC taining conditioned supernatant from CMG-14-12 cells (23, 32). Adherent ATT GTC TAC-39 and R 59-GCT GAT CCA TAC CAC TGA TG-39; BiP, cells were detached by 10 mM EDTA and replated in tissue-culture dishes F59-AGG ATG CGG ACA TTG AAG AC-39 and R 59-AGG TGA AGA with CMG-14-12 supernatant 3 more d. IRF32/2 mice were previously TTC CAA TTA CAT TCG-39; spliced XBP1, F 59-GAG TCC GCA GCA described (13). Wild-type (WT) and STING2/2 murine embryonic fibro- GGT G-39 and R 59-GTG TCA GAG TCC ATG GGA-39; ifit2, F 59-GAT blasts (MEFs) were maintained in DMEM supplemented with 10% FBS TCT GTC TAC CAC CAT-39 and R 59-CAA GCA TCA ATC CAA GTT- 4632 ENDOPLASMIC RETICULUM STRESS REGULATES IRF3

39; and RANTES, F 59-GAA TAC ATC AAC TAT TTG GAG AT-39 and R59-TAG AGC AAG CAA TGA CAG-39. Western blotting and coimmunoprecipitations Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and whole-cell lysates or cytoplasmic and nuclear fractions (Nuclear extraction kit; Sigma-Aldrich) resolved by 10% SDS-PAGE. Samples were transferred to polyvinylidene difluoride membrane (Amersham Biosciences) and immunoblotted with primary Ab followed by HRP-conjugated secondary Ab (Bio-Rad). Proteins were vi- sualized by ECL (Amersham Biosciences) and film exposure. MEF lysates (150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM Tris [pH 7.5], DTT, and protease inhibitor mixture) were incubated 1 h with anti- STING at 4˚C and then 1% BSA/PBS suspended protein A/G agarose (Santa Cruz Biotechnology). Samples were rotated at 4˚C overnight. Beads were washed two times with PBS, added to loading buffer, and then samples were denatured and resolved by SDS-PAGE. Statistics FIGURE 1. OGD synergistically augments LPS-induced IFN-b and Statistical significance between different groups of data were determined by causes IRF3 nuclear translocation. (A) MEFs were subjected to 0, 0.5, 1, or two-tailed Student t test. In all figures, error bars represent SEs of the mean 2 h OGD, followed by media with or without 10 ng/ml LPS for 3 h in from combined experiments or SD within a representative experiment. Downloaded from standard culture conditions. IFN-b expression was determined by quanti- tative PCR with normalization to 18S rRNA (relative mRNA). Bars rep- Results resent combined results from three independent experiments with error OGD synergistically augments LPS-induced IFN-b and bars showing SEM. p , 0.0005 comparing OGD pretreated conditions and activates IRF3 no OGD. Comparable results have been obtained in five independent B In previous studies, we determined that multiple pharmacologic experiments. ( ) MEFs were subjected to 1 h OGD followed by standard culture conditions as above. Expression of BiP and spliced XBP1 was inducers of the UPR, or an overexpressed misfolding MHC allele http://www.jimmunol.org/ b detected by quantitative PCR and normalized to untreated controls for fold HLA-B27, greatly augment LPS-dependent IFN- mRNA and induction (NT = 1). Results are representative of two individual experi- protein production (22, 23). This robust synergy was observed in ments, and error bars denote SD. p , 0.0004 comparing OGD-treated and primary macrophages, macrophage cell lines, MEFs, and LPS NT cells. (C) MEFs were subjected to OGD for 1 h followed by 2 h receptor-transfected HEK293 cells (22, 23). However, it was un- reoxygenation (Re-ox) in standard culture as indicated. Fixed cells were clear if more physiologic stress, such as the oxygen and nutrient incubated with anti-IRF3 followed by anti-rabbit IgG Alexa Fluor 488 and deprivation that might occur with ischemia, would enhance IFN-b visualized by immunofluorescence microscopy. Results are representative production. MEFs were subjected to OGD for various times and of three independent experiments. (D) Nuclear and cytoplasmic lysates b then stimulated with LPS for 3 more h (Fig. 1A). OGD alone did were resolved by SDS-PAGE and immunoblotted with anti-IRF3, -actin, not induce IFN-b mRNA; however, OGD dramatically augmented or the nuclear protein anti-USF2. Results are representative of two separate by guest on September 25, 2021 experiments. Scale bars, 50 mM. LPS-induced IFN-b, with maximal enhancement of a log-fold occurring after 1 h of OGD pretreatment. Thus, in vitro ische- 2+ mia augmented IFN-b mRNA levels to a similar degree as the coplasmic/ER Ca -ATPase pump inhibitor frequently used to pharmacologic UPR inducers. In vitro OGD has been shown to study the UPR in vitro (37). By 2 h of Tg treatment, IRF3 levels induce early UPR events within 15 min (35). We confirmed BiP increased in nuclear extracts as detected by Western blot (Fig. induction and increase in spliced XBP1 mRNA during the culture 2A). Differentially migrating forms of IRF3 have been reported in time frame (Fig. 1B). OGD also induced synergistic IFN-b and unstimulated cells (referred to as forms I and II) and stimulated BiP in RAW 264.7 macrophages (data not shown). cells (forms III and IV); these forms may represent variably In dissecting the mechanism behind UPR–TLR4 synergistic IFN- phosphorylated or alternatively spliced forms of IRF3 detected b production, we observed increased IRF3 recruitment to both the by polyclonal Ab preparations (31, 38, 39). Similar results were ifnb1 promoter and a newly described enhancer in the context of observed in RAW 264.7 cells (data not shown). The kinetics ob- ER stress (23). Although ER stress alone was not sufficient for served by immunofluorescence microscopy correlated well with recruitment of IRF3 to DNA, these findings raised the possibility these findings (Fig. 2B). Previous studies had identified a critical that ER stress might contribute to the activation of IRF3. In role for the UPR-regulated transcription factor XBP1 in syner- unstimulated cells, IRF3 resides in the cytoplasm as monomers. gistic IFN induction (22, 23, 25). To determine if XBP1 was re- Upon stimulation through PRRs, serine-threonine kinases such quired for IRF3 activation, nuclear translocation was examined 2/2 as TBK1 and IKKε/IKKi phosphorylate IRF3 at multiple sites, in XBP1 MEFs (Fig. 2C). Tg-induced nuclear translocation resulting in dimerization of IRF3 and nuclear translocation (10). of IRF3 was intact, suggesting that the UPR activates IRF3 inde- We tested whether OGD would affect IRF3 localization in MEFs pendently of the XBP1 pathway. Similar results were obtained in the absence of exogenous LPS (Fig. 1C, 1D). After 1 h of OGD, with other ER stressors (data not shown). IRF3 resided in the cytoplasm. However, after 2 h of reoxygena- Nuclear translocation of IRF3 theoretically implies preceding tion, IRF3 appeared predominantly nuclear, colocalizing with phosphorylation. To directly test if the UPR elicits IRF3 phos- DAPI by immunofluorescence microscopy. Nuclear translocation phorylation, MEFs were stimulated with multiple commonly used was also evident as assessed by Western blot (Fig. 1D). pharmacologic UPR inducers, including Tg, Tm (N-linked gly- cosylation inhibitor), 2DG (glucose deprivation simulator), and ER stress results in the phosphorylation and nuclear OGD, with LPS as a positive control (Fig. 2C). Treatment times translocation of IRF3 were based upon mechanism of action, time required to induce OGD induces various stress responses including the UPR (35, 36). XBP1 splicing (data not shown), and required pretreatment times To determine if pharmacologic UPR induction stimulates the for synergistic IFN-b induction (23). All UPR agents induced nuclear translocation of IRF3, MEFs were treated with Tg, a sar- detectable IRF3 phosphorylation at S386, comparable with LPS The Journal of Immunology 4633

FIGURE 2. ER stress induces IRF3 nuclear translocation and phosphorylation. (A) MEFs were treated with 1 mM Tg for the times indicated. Nuclear Downloaded from lysates were resolved by SDS-PAGE and immunoblotted with anti-IRF3 or b-actin control. Results are representative of four independent experiments. (B) MEFs were treated with 1 mM Tg for 1 or 2 h, fixed, incubated with anti-IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immu- nofluorescence microscopy. Results are representative of three independent experiments. (C) XBP12/2 MEFs were treated with Tg for 0, 60, or 90 min and Western blots incubated with anti-IRF3 or anti-USF2. Results are representative of three independent experiments. (D) MEFs were left untreated or subjected to OGD for 1 h, 10 mg/ml Tm for 6 h, 20 mM 2DG for 6 h, 1 mM Tg for 2 h, or LPS 10 ng/ml for 2 h. Fixed cells were incubated with anti-pS386 IRF3 followed by anti-rabbit IgG Alexa Fluor 488 and visualized by immunofluorescence microscopy. Results are representative of four independent experiments. (E) WT or IRF32/2 primary bone marrow-derived macrophages were treated with 1 mM Tg for 2 h prior to fixation and staining for p-IRF3 http://www.jimmunol.org/ (S386). Results are representative of two independent experiments. Scale bars, 50 mM. treatment (Fig. 2D). IRF3 phosphorylation was not detected in 16). Within the past few years, STING has been identified as an IRF32/2 primary macrophages, confirming immunofluorescence ER-resident transmembrane protein that plays a critical role in specificity (Fig. 2E). Synergistic IRF3 phosphorylation with dual the induction of IRF3 and thus IFN-b by cytoplasmic nucleic Tg and LPS treatment was not evident by immunofluorescence (data not shown) in comparison with Western blot (see below). The pattern of phospho-IRF3 localization differed between LPS by guest on September 25, 2021 and UPR inducers; LPS treatment resulted in qualitatively higher order clustering (dots arranged in small circular clusters, compare OGD versus LPS). ER stress does not augment all IRF3-regulated genes Our previous studies examining UPR–TLR synergy had focused on IFN-b production (23). However, the activation of IRF3 by ER stress raised the possibility that ER stress might augment the production of other known IRF3-regulated genes. Martinon et al. (24) reported log-fold synergistic induction of the IRF3 regulated gene ISG15 in Tm plus LPS-stimulated cells. IFN-a4 transcription is an early type I IFN, for which expression is highly dependent upon IRF3 (15, 40). In addition to increased IFN-b production, primary bone marrow-derived mouse macrophages treated con- currently with Tg and LPS produced increased levels of IFN-a4 (Fig. 3). Thus, the effect of ER stress on IRF3 activation has consequences for other antiviral and inflammatory mediators be- sides IFN-b. As previously described, synergistic IFN-b mRNA induction correlated well with protein production (23). IFN-a was produced at such a low level; only Tg plus LPS stimulated cyto- kine was present above the limit of detection (data not shown). However, synergistic induction of IRF3-regulated genes was not FIGURE 3. ER stress augments LPS induction of some, but not all, IRF3- A universal: neither Ifit2/ISG54 nor RANTES (as noted by others) regulated genes. ( ) Bone marrow-derived macrophages were treated with 1 mM Tg, 10 ng/ml LPS, or 1 mMTgplus10ng/mlLPSfor3h.Relativeex- were significantly augmented by UPR induction (24). pression of IFN-b,IFN-a4, RANTES, and ifit2 was determined by quanti- Activation of IRF3 by Tg requires STING and TBK1 tative PCR with normalization to 18S rRNA. Bars depict combined means of six (IFN-b,**p = 0.001), three (IFN-a4, **p , 0.006), and four (RANTES To begin elucidating the mechanism of UPR-induced IRF3 and ifit2) independent experiments with error bars showing the SEM. To com- phosphorylation, we sought to determine the relevant IRF3 kinase. bine IFN-a4, RANTES, and ifit2 experiments, relative expression was nor- TBK1 is an upstream serine/threonine kinase that serves as the malized (fold induction versus LPS = 1). (B) Macrophages were treated as final point of convergence for multiple innate immune-sensing above for 8 h and supernatants assessed for cytokine and chemokine protein by pathways culminating in the phosphorylation of IRF3 (8, 9, ELISA. Bars represent SE of triplicate determinations. **p , 0.00002 (IFN-b). 4634 ENDOPLASMIC RETICULUM STRESS REGULATES IRF3 acids (4). The direct nucleic acid-binding molecule(s) upstream over that observed with LPS alone (Fig. 4E). These results are of STING have not yet been well defined, but may include the consistent with those observed by Hu et al. (25), in which Tg helicase DDX41 (41). Upon activation by nucleic acids, STING augmented polyinosinic-polycytidylic acid-dependent IIRF3 associates with and phosphorylates IRF3 via TBK1 (4, 6). phosphorylation but did not appear to induce IRF3 on its own. The ER-resident location of STING raised the possibility that However, in the STING2/2 MEFs, no p-IRF3 was observed by ER stress-induced activation of IRF3 might proceed through this immunoblot. The lack of p-IRF3 was not because of IRF3 defi- pathway. However, it was not clear if ER stress-induced IRF3 ciency or generally defective LPS signaling in the MEFs, as LPS- phosphorylation required TBK1 or STING or induced TBK1– induced NF-kB nuclear translocation was intact in STING2/2 STING association. To determine the requirement for TBK1, MEFs. Together, these results support the idea that Tg uses STING MEFs were pretreated with MRT67307, a TBK1/IKKε family and downstream TBK1 to phosphorylate IRF3 and that synergistic kinase inhibitor, prior to Tg stimulation (34). MRT67307 abro- p-IRF3 induction by LPS and Tg requires STING. gated Tg-dependent IRF3 phosphorylation (Fig. 4A). To deter- b mine if ER stress induced association between TBK1 and STING, Optimal synergistic induction of IFN- by Tg and LPS requires coimmunoprecipitation was performed. TBK1 and STING asso- STING ciated even before stimulation, and the association continued with STING appeared to be required for phosphorylation of IRF3 Tg treatment (Fig. 4B). Similar results were obtained in primary during ER stress and synergistically induced p-IRF3 during Tg and macrophages (data not shown). However, by immunofluorescence LPS stimulation. However, the consequences for an IRF3-regu- microscopy, a striking ER stress-induced relocalization of STING lated gene, such as IFN-b, were not clear. Thus, synergistic and TBK1 was evident, with association into larger order clusters IFN-b induction was evaluated in RNAi-transfected RAW 264.7 Downloaded from around the nucleus (Fig. 4C). Together, these data suggested that macrophages and STING2/2 bone marrow-derived macrophages. Tg-induced IRF3 phosphorylation requires TBK1 and that Tg Even moderate knockdown of STING with RNAi (Fig. 5A, 5B) mobilizes STING and TBK1. However, it was not clear if STING decreased IFN-b induction by combined Tg and LPS stimulation. was required for Tg-induced IRF3 phosphorylation via TBK1. There was no significant effect of STING knockdown on LPS- To determine if STING was specifically required for ER stress- induced IFN-b alone or the induction of two IRF3-independent 2/2 induced IRF3 phosphorylation, STING MEFs were stimulated , IL-1b and IL-6, by any of the stimuli. The effect of http://www.jimmunol.org/ with Tg and assessed by immunofluorescence for IRF3 phos- STING deficiency was more evident in the primary STING2/2 phorylation (4). Neither IRF3 phosphorylation nor nuclear trans- bone marrow macrophages in comparison with the RNAi knock- location was evident in STING2/2 MEFs (Fig. 4D, IRF3 immuno- down (Fig. 5C). Again, IL-6 induction was intact. Synergy was fluorescence not shown). By immunoblot, in WT MEFs, p-IRF3 not completely abrogated in the STING2/2 macrophages or in (S396) is detectable in LPS-stimulated nuclear lysates, although STING2/2 MEFs (data not shown). However, the magnitude of we did not detect Tg-induced p-IRF3 nuclear phosphorylation. In synergistic IFN-b expression was decreased by about a log-fold in the WT cells, Tg and LPS cotreatment increased levels of p-IRF3 primary macrophages. by guest on September 25, 2021

FIGURE 4. Tg induces colocalization of TBK1 and STING and requires STING and TBK1 family kinases for IRF3 phosphorylation. (A) MEFs were pretreated with 2 mM MRT67307 for 30 min prior to 1 mM Tg for 2 h. Fixed cells were incubated with anti–p-IRF3 (pS386) followed by Alexa Fluor 488- coupled secondary Ab and immunofluorescence detected by microscopy. Results are representative of four independent experiments. (B) MEFs were untreated or treated with 1 mM Tg for 2 h. Samples were immunoprecipitated with control IgG or anti-STING. Whole-cell lysate (input) or immuno- precipitations were resolved by SDS-PAGE and immunoblotted with anti-TBK1 or anti-STING. Results are representative of four independent experiments. (C) MEFs were transfected with STING/MPYS-HA 24 h prior to stimulation with 1 mM TPG for 2 h. Fixed cells were incubated with rabbit anti-TBK1 and mouse anti-HA followed by anti-rabbit Alexa Fluor 488 (green) or anti-mouse Alexa Fluor 594 (red) secondary Abs. Results are representative of three independent experiments. (D) WT or STING2/2 MEFs were treated with 1 mM Tg for 2 h as indicated. Cells were then fixed, incubated with anti-pS386 IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immunofluorescence microscopy. Results are representative of four independent experiments. (E) WT and STING2/2 MEFs were stimulated with 1 mM Tg, 10 ng/ml LPS, or 1 mM Tg + 10 ng/ml LPS for 2 h. Nuclear lysates were resolved by SDS-PAGE and immunoblotted with Abs specific for p-IRF3 (S396), total IRF3, NF-kB, or USF2. Results are representative of three inde- pendent experiments. Scale bars, 50 mM. The Journal of Immunology 4635

different UPR inducers activate IRF3 through a variety of sig- naling pathways. One profound difference between Tg and Tm is that Tg induces the UPR by inhibiting the sarcoplasmic/ER Ca2+-ATPase pump, severely depleting ER calcium stores (37). Thus, it was possible that mobilization of STING reflected altered calcium metabolism and not the UPR at all. To test this hypothesis, cells were stimu- lated with ionomycin, a calcium ionophore that mobilizes calcium by influx, but does not trigger the UPR at 1 mM (data not shown and 42, 43). Even at higher doses, ionomycin is much less ef- fective at upregulating the UPR gene BiP as compared with an- other ionophore A23187 (44). In contrast to Tg, ionomycin did not induce significant IRF3 phosphorylation (Fig. 6B). However, A23187, an agent widely used to induce the UPR, did induce IRF3 phosphorylation (45). These results indicate that collapsing the calcium gradient is not sufficient for IRF3 phosphorylation and that some component of the ER stress response, or ER calcium depletion, is required as well. Furthermore, A23187 also required

STING for IRF3 phosphorylation. Thus, Tg is not the only UPR Downloaded from inducer dependent upon STING for IRF3 activation. It was not clear if the STING–TBK1 pathway would be involved in a more physiologic setting. During ischemia-reperfusion injury, ATP de- pletion disrupts sequestration of calcium in the ER, resulting in excess cytosolic calcium, mitochondrial calcium uptake, and ER

dysfunction (46, 47). Intracellular disruptions in calcium handling http://www.jimmunol.org/ are reproduced by in vitro OGD (48). As seen in Fig. 6C, OGD- dependent nuclear IRF3 translocation was impaired in STING2/2 cells. Together, these data suggest that ER stressors that dys- FIGURE 5. STING is required for optimal synergistic induction of regulate calcium metabolism phosphorylate IRF3 through the IFN-b by Tg and LPS. (A) RAW264.7 macrophages were transfected with STING–TBK1 pathway. 100–200 nM control or STING-specific RNAi. After 24 h, cells were treated with 1 h 1 mM Tg followed by 100 ng/ml LPS for 3 h. Relative expression A site 1 protease inhibitor of ATF6 processing prevents of STING, IFN-b, IL-1b, and IL-6 was determined by quantitative PCR Tm-induced activation of IRF3 with normalization to 18S rRNA. Results were combined from three We sought to determine which of the three major UPR pathways, by guest on September 25, 2021 (STING and IL-1b) or four (IFN-b and IL-6) independent experiments stemming from IRE1/XBP1, PERK, and ATF6, might be playing with error bars representing the SEM. *p # 0.032, **p = 0.011. (B) RAW264.7 macrophages were transfected with control or STING-specific a role in IRF3 activation in response to ER stressors. As mentioned RNAi and lysed at 24, 48, or 72 h. Lysates were resolved by SDS-PAGE above (Fig. 2), XBP1 is not absolutely required for nuclear trans- and immunoblotted with anti-STING. Western blot was performed twice. location. PERK was not obligatory for synergistic IFN-b mRNA (C) WT or STING2/2 bone marrow-derived macrophages were treated induction by either Tg or Tm (22). In response to ER stress, ATF6 with 1 mM Tg, 10 ng/ml LPS, or TPG+LPS for 3 h. Cells were processed transits to the Golgi, where it is cleaved to an active transcription as in (A) by quantitative PCR to assess IFN-b and IL-6 mRNA expression. factor by site 1- and site 2-specific proteases. AEBSF, a serine 2/2 Results were combined from three (WT) and five (STING ) independent protease inhibitor, has been shown to block this processing event experiments, with error bars representing the SEM. **p = 6.1e-6. by inhibiting the site 1 protease (49). We confirmed this activity (Fig. 7). AEBSF prevented Tm- and 2DG- but not Tg-dependent Not all ER stressors require TBK1 and STING for IRF3 IRF3 phosphorylation, as visualized by immunofluorescence (Fig. phosphorylation; ER stressors mobilizing calcium depend upon 7A). By Western blot, the presence of AEBSF prevented syn- STING for IRF3 phosphorylation ergistic p-IRF3 in dually treated LPS+Tm cells, reducing p-IRF3 Although all of the frequently used pharmacologic ER stress- to the level of LPS alone (Fig. 7B); however, AEBSF did not inducing agents activate the three primary signaling pathways prevent LPS+Tg-enhanced p-IRF3. Consistent with these results, encompassed by the UPR and have final common end points, their AEBSF significantly diminished Tm, but not Tg-dependent IFN-b mechanisms of action vary. Ultimately, the quality and magnitude mRNA synergy (Fig. 7C). The lack of effect on Tg-induced syn- of all the various UPR stress pathways induced by these agents are ergy argues against nonspecific toxicity. Together, these results likely to differ as well. To determine if the dependence on STING indicate that AEBSF sensitivity distinguishes between IRF3 acti- was a general property of UPR induction, STING+/+ and 2/2 MEFs vation by Tg and Tm/2DG and is consistent with a possible role for were stimulated with Tm, an agent that induces the UPR inde- ATF6 in the latter. Furthermore, ER stressors activate IRF3, lead- pendently of calcium (Fig. 6A). There were no evident differences ing to synergistic IFN-b mRNA induction, by at least two distin- in Tm-induced IRF3 phosphorylation in the presence or absence guishable pathways. of STING. Likewise, 2DG-induced IRF3 phosphorylation oc- curred in the absence of STING (data not shown). Consistent with Discussion these findings, Tm-induced IRF3 phosphorylation also did not This study describes the novel observation that ER stress, even require TBK1 family kinases, because MRT67307 did not prevent in absence of PRR stimulation, activates IRF3. Different forms of Tm-induced IRF3 phosphorylation (Fig. 6A). Thus, although Tg ER stress accomplish this through at least two distinct pathways, and Tm treatment may lead to similar common end points, the ER requiring either TBK1/STING or AEBSF-sensitive signaling. The stress responses are qualitatively different. These data suggest that synergistic induction of several IRF3-regulated inflammatory 4636 ENDOPLASMIC RETICULUM STRESS REGULATES IRF3 Downloaded from

FIGURE 6. STING-dependent IRF3 phosphorylation requires both calcium mobilization and ER stress. (A) WT or STING2/2 MEFs were pretreated with 2 mM MRT67307 for 30 min then 10 mg/ml Tm for 6 h as indicated. Cells were incubated with anti–p-IRF3 (S386) followed by Alexa Fluor 488 secondary Ab and visualized by immunofluorescence microscopy. Results are representative of four independent experiments. (B) WT or STING2/2 MEFs were untreated (NT) or treated with 1 mM ionomycin or 2 mM A23187 as indicated. Fixed cells were incubated with anti–p-IRF3 followed by Alexa Fluor http://www.jimmunol.org/ 488 secondary Ab and visualized by immunofluorescence microscopy. Results are representative of three independent experiments. (C) WT or STING2/2 MEFs were subjected to OGD (as in Fig. 1) for 1 h followed by 2 h reoxygenation. Cells were fixed (C) or lysed (D) and IRF3 nuclear translocation detected by immunofluorescence or Western blot, respectively. Scale bars, 50 mM. mediators by concurrent UPR and LPS stimulation suggest the the dramatic IFN-b synergism observed with LPS. In evidence activation of IRF3 by ER stress may have a wider impact in innate of this requirement, interfering with Tg-dependent IRF3 phos- immunity, beyond augmenting IFN-b production. Even though ER phorylation through the modulation of STING severely impacts stress alone is not sufficient to trigger the induction of an IRF3- the magnitude of Tg-induced synergy. Similarly, disrupting Tm- by guest on September 25, 2021 regulated gene, activation of IRF3 by ER stress is necessary for dependent IRF3 phosphorylation with AEBSF significantly di-

FIGURE 7. AEBSF, a site 1 protease inhibitor, blocks Tm- and 2DG-induced IRF3 phosphorylation and Tm-dependent synergistic IFN-b induction. (A) RAW cells were pretreated with 300 mM AEBSF for 1 h, then stimulated with 1 mM Tg for 2 h, 20 mM 2DG for 5 h, or 10 mg/ml Tm for 5 h. Cells were fixed and then stained with anti–p-IRF3 (S386) plus secondary anti-rabbit Alexa Fluor 488. Results are representative of two independent experiments. (B) RAW cells were pretreated with AEBSF as in (A) and then untreated (NT), stimulated with Tg 1 h or Tm 5 h, and followed by an additional 3 h media or LPS as indicated. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted with anti–p-IRF3 (S396), IRF-3, or actin. Results are representative of two independent experiments. (C) RAW cells were pretreated with AEBSF as in (A) and then stimulated with 1 h Tg or 5 h Tm followed by an additional 3 h LPS as indicated. Relative IFN-b mRNA was quantified by quantitative PCR with normalization to 18S rRNA. Results were combined from four to five independent experiments, and error bars represent the SEM. *p , 0.007. (D) Cells were stimulated as in (B). Whole-cell lysates were resolved by SDS PAGE and immunoblotted for precursor (P) and mature (M) cleaved forms of ATF6. Results are representative of three independent experiments. Scale bar, 50 mM. The Journal of Immunology 4637 minished synergistic IFN-b expression. Residual IFN-b induction formation requires binding of multiple elements including critical in the LPS+Tg-treated STING2/2 MEFs may relate to IRF3 scaffolding molecules (HMGA1) and histone acetyltransferases phosphorylation below our limits of detection or compensation by (e.g., CBP/p300) (11). LPS stimulation may be required to recruit another IRF3 serine (e.g., S339) for which we did not assay (50). these other molecules. Another possibility is that a stronger NF-kB This study supports the novel concept that intracellular stress signal may be required than that generated during ER stress alone. responses may co-opt innate immune-signaling pathways previ- Finally, there could be a cell-type issue, because our studies are ously thought to be dedicated to pathogen sensing. The proximity conducted in macrophages and MEFs. When mice are treated in vivo of STING to the mitochondria-associated membrane a site of with Tm alone, we observed detectable serum IFN-b (preliminary interorganelle calcium transport and regulation may suggest why data not shown), suggesting that an unidentified cell type is capable UPR inducers that affect calcium also mobilize STING (4, 51). Our of producing IFN during a UPR. findings suggest that, at least in MEFs and macrophages, STING In this study and others, ER stress has been noted to augment and TBK1 associate even prior to stimulation. Upon ER stress transcription of select IRF3-regulated genes (e.g., IFN-b but not induction, STING and TBK1 dramatically reorganize into larger RANTES) (24). IRF3 binds similar DNA sequences within gene macroscopic collections. This mobilization may be a result of promoters designated as IFN-stimulated response elements or reorganization of the ER membranes themselves (containing positive regulatory domains (PRD I and III in the IFN-b promoter) STING), translocation to another organelle (e.g., Golgi), or as- (67). The selectivity in synergism may relate to promoter com- sociation with other unidentified molecules in a multimolecular plexity and requirement for multiple transcription factors, as complex. The augmentation of type I IFN responses by ER stress/ mentioned above. Constitutively activated IRF3 (an aspartate calcium dysregulation and STING may be of particular relevance containing phosphomimetic) is sufficient to activate only a small Downloaded from to viral infections, such as hepatitis C, that induce ER stress and subset of IFN-stimulated response elements containing genes, cause calcium leak (52). Our results obtained with in vitro OGD including ifit2/ISG54, ISG56, ISG60, CIG5, and PMA inducible (Fig. 6) have direct implications for in vivo ischemia-reperfusion protein 1 (68). However, we did not detect robust activation of injury: they suggest the dysregulation of calcium, ER stress, and ISG54 by Tg alone. This failure may reflect suboptimal IRF3 type I IFN-dependent inflammatory injury may be critically in- activation at specific serines. Alternatively, given the indepen-

terrelated (20, 53–57). dence of XBP1 and IRF3 translocation (Fig. 2) and the discovery http://www.jimmunol.org/ The UPR has been shown to activate both NF-kB and AP-1 of XBP1 binding sites in cytokine promoters and enhancers, sig- family member transcription factors (58). Thus, it is unclear why nificant synergy might require DNA binding sites for both IRF3- UPR-induced IRF3 phosphorylation is not sufficient to induce and UPR-dependent transcription factors (22–24). The experience IFN-b expression in vitro. Even though the UPR induces nuclear with IFN-b would favor this multi-hit hypothesis. translocation of IRF3, translocation does not automatically confer It is not clear which aspects of the UPR are necessary for IRF3 transcriptional activity: dissociation between translocation and phosphorylation and nuclear translocation. The answer may differ transcriptional activity has been noted in multiple models of viral depending upon type of ER stress. Our studies would suggest that IRF3 inhibition (59–62). IRF3 has two activation clusters com- XBP1 is not required for ER stress-induced IRF3 nuclear trans- prising seven potentially phosphorylated serines and threonines location. PERK is not necessary for synergistic IFN induction (22 by guest on September 25, 2021 (S385, S386, S396, S398, S402, S405, and T404). Some contro- and data not shown). AEBSF, a protease inhibitor that prevents versy remains regarding serine phosphorylation requirements for ATF6 processing, blocked Tm but not Tg-dependent IRF3 phos- IRF3 activity: phosphorylation of S396 has been proposed as an phorylation and synergy (Fig. 7) (22). Tg may use an IRE1 kinase- essential minimal acceptor site for responses to Sendai virus and mediated pathway to activate IRF3. Alternatively, Tg and A23187 may be critical for homodimerization (50, 63). More recently, could mobilize a nonclassical UPR ER stress pathway related to S396 has been shown to promote higher-order oligomerization calcium flux that has not been described. Another possibility is (64). However, others have identified S386 as the critical site for that IRF3 activation resulting from profound ER calcium deple- homodimerization and nuclear translocation (65, 66). Unfortu- tion and the UPR are independent outcomes of treatment with nately, it is not yet clear exactly which sites on IRF3 correspond these stressors. with optimal transcriptional activity in response to LPS and other Our results are consistent with the hypothesis that Tm and 2DG- specific pathogens. Ultimately, both may act cooperatively to bind induced IRF3 phosphorylation proceed through ATF6 or a related CBP/p300 with higher affinity (64). Thus, one possibility is that protein. ATF6 belongs to the OASIS family of transcription factors even though ER stress induces S386 phosphorylation, ER stress in that is processed by the site 1 proteases. However, protein distri- isolation does not induce strong enough phosphorylation at S396, bution of these other family members is much more restricted than as suggested by Western blot (e.g., Fig. 4E); the UPR may only ATF6 (69). AEBSF also inhibits reactive oxygen species genera- induce partial phosphorylation of IRF3 and LPS remains neces- tion by NADPH (70). Tg, Tm, and ER stress related to cholesterol sary for additional phosphorylation at other serines/threonines. loading have all been found to induce oxidative stress via NADPH Alternatively, as suggested by our Western blot data, UPR in- (71–73). Reactive oxygen species potentiate IRF3 activation (74). duced phosphorylation at S386 may facilitate or enhance LPS- However, NADPH oxidase inhibition by AEBSF would not ex- dependent S396 phosphorylation. The requirement for multiple- plain the divergent effects on Tg and Tm, particularly given the site IRF3 phosphorylation to promote oligomerization may explain involvement of calcium in ER stress-mediated NADPH oxidase the qualitative differences in immunofluorescence between LPS activation (71). The specific AEBSF-sensitive signaling event and ER stressors such as OGD (Fig.2)(64).Ourdatawould remains to be confirmed. suggest that ultimate phosphorylation at S396 correlates best with Another outstanding question is the identity of the kinase ac- IRF3 DNA binding by chromatin immunoprecipitation and tran- tivated by non-Tg UPR inducers that is responsible for phos- scriptional activation of IFN-b (23). phorylating IRF3. In this study, Tm led to IRF3 phosphorylation in Apart from suboptimal IRF3 activation, there are other possible the presence of a TBK1/IKKε inhibitor. The kinase cascade in- explanations: IRF3 alone is not sufficient for IFN gene transcrip- volving NF-kB–inducing kinase and IKKa has been reported to tion; the enhanceosome also contains NF-kB and AP-1 transcrip- phosphorylate IRF3 independently of TBK1 (75). The MAPK tion factors. Transcriptional activation following enhanceosome cascade initiated by IRE-1 that includes p38 may also play a role 4638 ENDOPLASMIC RETICULUM STRESS REGULATES IRF3

(76, 77). Dissecting the exact mechanism of IRF3 activation by all 19. Zhai, Y., X. D. Shen, R. O’Connell, F. Gao, C. Lassman, R. W. Busuttil, G. Cheng, and J. W. Kupiec-Weglinski. 2004. Cutting edge: TLR4 activation ER stressors, at the level of the UPR and subsequent kinase cas- mediates liver ischemia/reperfusion inflammatory response via IFN regulatory cades, is outside the scope of this study but will be interesting to factor 3-dependent MyD88-independent pathway. J. Immunol. 173: 7115–7119. tease apart in the future. 20. Zhai, Y., B. Qiao, F. Gao, X. Shen, A. Vardanian, R. W. Busuttil, and J. W. Kupiec-Weglinski. 2008. Type I, but not type II, is critical in Ultimately, our findings raise the possibility that different forms liver injury induced after ischemia and reperfusion. Hepatology 47: 199–206. of ER stress/UPR inducers may use innate immune-sensing path- 21. Chattopadhyay, S., J. T. Marques, M. Yamashita, K. L. Peters, K. Smith, ways including STING. 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