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. 2013 May 15;13(5):558-569.
doi: 10.1016/j.chom.2013.03.011.

The unfolded protein response element IRE1α senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling

Jin A Cho  1 , Ann-Hwee Lee  2 , Barbara Platzer  1 , Benedict C S Cross  3 , Brooke M Gardner  4 , Heidi De Luca  1 , Phi Luong  1 , Heather P Harding  3 , Laurie H Glimcher  5 , Peter Walter  6 , Edda Fiebiger  7 , David Ron  3 , Jonathan C Kagan  7 , Wayne I Lencer  8
Affiliations

The unfolded protein response element IRE1α senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling

Jin A Cho et al. Cell Host Microbe. .

Retraction in

Abstract

The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cytosolic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.

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Figures

Figure 1
Figure 1. The CT A Subunit Induces IL-6 and IL-8 from within the ER
(A) Polarized T84 cells were intoxicated with 3 nM toxin for 4 hr, and IL-6 and IL-8 were assessed by real-time qPCR. The TLR4 agonist LPS (applied basolaterally, bLPS) and the ER stress-inducing agent thapsigargin (Thap) were used as positive controls. (B) Polarized Caco-2 cells treated as in (A). (C) Polarized T84 cells treated as in (A) with or without brefeldin A (BFA) pretreatment and assayed for IL-6 by real-time qPCR. Open bars represent negative controls. Gray bars represent positive controls. wt CT, wild-type CT; E/E, E110D/E112D mutant; R192G, R192G mutant; CTB, B subunit alone. Data are shown as means ± SEM. *p < 0.05 by ANOVA. See also Figure S1.
Figure 2
Figure 2. The Inflammatory Response Induced by CT A Subunit Is NF-κB Dependent
(A and B) Lysates of T84 cells were analyzed for nuclear translocation of p65 by immunoblot (A) and for phosphorylation of IKKα and lκBα (B). TATA-binding protein (TBP), total IKKα/β, and total lκBα are loading controls. (C) T84 cells pretreated with NF-κB inhibitor Bay11 were intoxicated and analyzed for IL-8 by realtime PCR. Data are shown as means ± SEM. Nomenclature is as described in Figure 1. See also Figure S2.
Figure 3
Figure 3. The CT A Subunit Activates the IRE1α Pathway, but XBP1 and PERK Are Dispensable
(A–C) T84 cells were intoxicated as indicated and analyzed for phospho-IRE1 α (A), the spliced form of XBP1 mRNA (B), and ERdj4 mRNA (C). T84 cells treated with thapsigargin or apically applied LPS (aLPS) (or buffer alone) provided positive and negative controls, respectively. (D–F) IRE1 α or XBP1 was silenced in HeLa cells (D′ and E′). HeLa cells lacking IRE1 α or XBP1 were intoxicated and analyzed for IL-6 mRNA expression by real-time PCR (D and E) and nuclear translocation of p65 by immunoblot (F). TBP and β-actin are loading controls. (G–I) MEF cells lacking IRE1 α (IRE1 α−/−; G), XBP1 (XBP1−/−; H), or PERK (PERK−/−; I) were intoxicated and analyzed for IL-6 mRNA expression by real-time PCR. Data are shown as means ± SEM. Nomenclature is as described in Figure 1. In (E′), * indicates nonspecific protein bands. See also Figure S3.
Figure 4
Figure 4. The Inflammatory Response Depends on the RNase Activity of IRE1α and on RIG-I, a Cytosolic Sensor of RNA Fragments
(A and B) T84 cells pretreated with 5 µM 4µ8C for 5 min were intoxicated and analyzed for transcription of IL-8 (A) and nuclear translocation of p65 (B). TBP is a loading control. (C) Wild-type IRE1 α rescued and K907A RNase domain-dead transfected IRE1 α knockout MEFs were intoxicated and analyzed for transcription of IL-6. (D) Huh 7.0 (WT) and Huh 7.5 (mutant RIG-I) cells were intoxicated and IL-8 mRNA was measured. (E) Purified RNA from IRE1 knockout cells was digested with purified cytosolic domain of human IRE1 α in the presence or absence of 4µ8C. (F) Low-molecular-weight fragments of mRNA produced in vitro were transfected in Huh 7.0 (WT) and Huh 7.5 (mutant RIG-I) cells for 16 hr and IL-8 mRNA was assessed. (G) Wild-type primary MEFs (RIG-I+/+) or immortalized MEFs lacking RIG-I (RIG-I−/−) were intoxicated, and IL-6 mRNA was measured. (H) MEF cells lacking the scaffolding protein MAVS (MAVS−/−) and the same cells rescued by transfection with wild-type MAVS (wt MAVS rescue) were intoxicated, and IL-6 mRNA was measured. (I) Caco-2 and HeLa cells were intoxicated and analyzed for nuclear translocation of IRF3 by immunoblot. TBP is a loading control. Poly(I:C) and TNFα were used as positive controls. Data are shown as means ± SEM. Nomenclature is as in Figure 1. See also Figure S4.
Figure 5
Figure 5. The IRE1-RIDD Pathway Defines a General Rule for Innate Immune Surveillance by the ER
(A) Wild-type MEFs or MEFs lacking IRE1α (IRE1α−/−) or RIG-I (RIG-I−/−) were intoxicated with an enzymatically inactive Shiga toxin mutant (StxE) or its B subunit alone (STxB), and IL-6 mRNA was quantified. (B and C) T84 or Caco-2 cells pretreated or not with 4µ8C were infected with SV40 virus (B) or Anthrax-fused Diphtheria (C) for 4h and immune responses were assessed by qPCR. (D) Wild-type macrophages or MAVS knockout macrophages pretreated or not with 4µ8C were intoxicated with toxins for 4 hr and analyzed for IL-6 by qPCR. Data are shown as means ± SEM. PA, protective antigen; LFN, amino-terminal 255 residues of lethal factor; LFNDTA*, fusion protein composed of LFN and the inactive catalytic domain of diphtheria toxin; LFNDTA, fusion protein composed of LFN and the catalytic domain of diphtheria toxin. Other nomenclature is as in Figures 1 and 4.
Figure 6
Figure 6. The Luminal Domain of Mouse IRE1α Binds Two Motifs in the CT A1 Chain
(A) An array of 18-mer peptides derived from the CT A subunit was incubated with purified mlrel α tagged with GST, and binding was detected with anti-GST antibodies. (B) The contribution of each amino acid to binding mlre1α was plotted along the sequence of the CT A subunit from the N to C terminus, revealing two distinct GST-mlre1α binding sites. (C) The cholera toxin crystal structure. The motifs that appear to bind IRE1α are on the external surface of the A1 chain (residues highlighted in yellow and orange). (D) HeLa cells expressing FLAG-tagged IRE1α were intoxicated with WT CT in the presence or absence of BFA. IRE1α was immunoprecipitated and analyzed by immunoblot with antibodies against the CT A and B subunits (upper panel) and FLAG (middle panel). Total cell lysates were also immunoblotted for the A1 chain to provide additional controls (lower panel).
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Comment in

  • Did cholera toxin finally get caught?
    Martinon F, van der Goot FG. Martinon F, et al. Cell Host Microbe. 2013 May 15;13(5):501-503. doi: 10.1016/j.chom.2013.04.014. Cell Host Microbe. 2013. PMID: 23684299

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