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. 2019 Jul 5;365(6448):eaaw4144.
doi: 10.1126/science.aaw4144.

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling

Mena Abdel-Nour  1 , Leticia A M Carneiro  1 , Jeffrey Downey  2 , Jessica Tsalikis  1 , Ahmed Outlioua  3   4 , Dave Prescott  1   5 , Leandro Silva Da Costa  3 , Elise S Hovingh  1 , Armin Farahvash  1 , Ryan G Gaudet  6 , Raphael Molinaro  1 , Rob van Dalen  1 , Charles C Y Lau  1   5 , Farshad C Azimi  1 , Nichole K Escalante  5 , Aaron Trotman-Grant  5 , Jeffrey E Lee  1 , Scott D Gray-Owen  6 , Maziar Divangahi  2 , Jane-Jane Chen  7 , Dana J Philpott  5 , Damien Arnoult  3 , Stephen E Girardin  8
Affiliations

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling

Mena Abdel-Nour et al. Science. .

Abstract

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.

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Conflict of interest statement

Competing interests: R.G.G. and S.D.G. have filed patents concerning the therapeutic potential of heptose 1-phosphate and heptose 1,7-biphosphate.

Figures

Fig. 1.
Fig. 1.. The HRI/eIF2α axis is required for inflammatory responses during infection.
(A) Cxcl1 secretion from the supernatants of wild-type (WT) and eIF2α S51A knock-in (KI) MEFs after infection with Shigella for the indicated times, as measured by ELISA. (B and C) Expression of Cxcl1 in WT and KI MEFs (B) and WT and Atf4 knockout (Atf4–/–) MEFs (C) left unstimulated (CTR) or after infection with Shigella. (D) HeLa cells infected with Shigella or treated for 1 hour with 300 mM sodium arsenite (Ars) and analyzed by immunoblotting. (E) HeLa cells transduced with lentiviral particles targeting a scrambled sequence (SC), both HRI and GCN2 [double knockdown (DKD)] left untreated (U), infected 1 hour with Shigella (S.f), treated 1 hour with 300 μM Ars or treated with Krebs-Ringer buffer (KRB) for 45 min and analyzed by immunoblotting. (F and G) Expression of ATF3 (F) and IL8 (G) in HeLa cells transduced with lentiviral particles targeting a scrambled sequence (SC), HRI (shHRI), or GCN2 (shGCN2) and infected with Shigella. (H and I) Expression of CXCL1 (H) and IL1α (I) in scrambled, HRI KD, and GCN2 KD HeLa cells infected with Shigella. Data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2.
Fig. 2.. HRI is essential for NOD1- and NOD2-driven inflammatory responses.
(A) Scrambled, HRI KD, and GCN2 KD HeLa cells were infected with Shigella for 30 min; the percentage of cells displaying nuclear NF-κB p65 was quantified by immunofluorescence. (B and C) Expression of Cxcl1 (B) and Il-6 (C) in wildtype (HRI+/+) and HRI knockout (HRI–/–) MEFs infected with Shigella. (D) Expression of IL-8 in scrambled and HCT116 KD cells after 4 hours of stimulation with L18-MDP (10 ng/ml) or C12-iE-DAP (10 μg/ml). (E) Cxcl1 secretion from the supernatants of HRI+/+ and HRI–/– bone marrow–derived macrophages (BMDMs) stimulated for 6 hours with L18-MDP (10 ng/ml) or C12-iE-DAP (10 μg/ml), as measured by ELISA. (F) Expression of Cxcl1 in intestinal organoids derived from HRI+/+ and HRI–/– mice treated for 4 hours with Shigella conditioned medium (CM Shigella). (G) Serum Cxcl1 in HRI+/+, HRI heterozygous (HRI+/–), and HRI–/– mice after intraperitoneal injection with MDP, as measured by ELISA. (H and I) Cxcl1 (H) and CCL2 (I) in the cecum homogenate of HRI+/+ and HRI–/– mice 4 days after infection with Citrobacter rodentium, as measured by ELISA. Graphs represent means ± SD from three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA followed by Tukey multiple-comparisons test).
Fig. 3.
Fig. 3.. The HRI signaling axis controls the folding and solubility of NOD1 oligomers.
(A) Scrambled and HRI KD HEK293T cells were stimulated for 30 min with C12-iE-DAP (10 ng/ml) and analyzed by blue native PAGE and SDS-PAGE. (B) HEK293T cells were stimulated with C12-iE-DAP (10 ng/ml) for 30 min, and NOD1-HA immunocomplexes were subjected to SYPRO Orange hydrophobicity measurements. RFU, relative fluorescence units. (C) Scrambled or HRI KD HEK293T cells were stimulated for 30 min with C12-iE-DAP (10 ng/ml) and NOD1-HA immunocomplexes were analyzed as in (B). (D) HEK293T cells with or without ISRIB treatment (1 μg/ml) were stimulated with C12-iE-DAP (10 ng/ml) and NOD1-HA immunocomplexes were analyzed as in (B). Graphs represent means ± SD from three or four independent experiments. *P < 0.05, **P < 0.01; ns, not significant.
Fig. 4.
Fig. 4.. The HRI-eIF2α-HSPB8 loop is essential for NOD1 and NOD2 signaling.
(A to C) Expression of Hspb8/HSPB8 in WT MEFs (A), Atf4–/– MEFs (B), and scrambled, HRI KD, and GCN2 KD HeLa cells (C) infected with Shigella or treated with 5 μM thapsigargin for 4 hours. (D) Expression of IL8 in scrambled or HSPB8 KD (shHSPB8) HeLa cells infected with Shigella. (E) Expression of IL8 in scrambled or HRI KD HeLa cells infected 4 hours with Shigella with or without transfection of HSPB8-V5, as measured by qPCR. (F) Coimmunoprecipitation experiments with unstimulated (CTR) or Shigella-infected HeLa cell lysates. (G) Coimmunoprecipitation experiments with the lysates of HeLa cells that were transfected with NOD1-HA and HSPB8-V5 and infected with Shigella. (H) Scrambled or HRI KD HEK293T cells transfected with NOD1-HA with or without HSPB8-V5 and analyzed as in Fig. 3A. (I and J) Scrambled or HRI KD HEK293T cells transfected with or without HSPB8-V5 (I) and scrambled or HSPB8 KD HEK293T cells (J) were stimulated for 30 min with C12-iE-DAP (10 ng/ml) and NOD1-HA complexes were subjected to SYPRO Orange hydrophobicity assays. Graphs display means ± SD from three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Asterisk in (F) denotes a nonspecific band.
Fig. 5.
Fig. 5.. HRI differentially regulates PRM signaling.
(A) Cxcl1 secretion from the supernatant of HRI+/+ or HRI–/– BMDMs stimulated for 4 hours with LPS (1 μg/ml), flagellin (100 ng/ml), or Pam3CSK4 (1 μg/ml), as measured by ELISA. (B and C) Il6 (B) and Ifnβ (C) expression in HRI+/+ or HRI–/– BMDMs treated with LPS (100 ng/ml) for the indicated times, as measured by qPCR. (D) Ifnβ expression in HRI+/+ or HRI–/– BMDMs treated with poly(I:C) (1 μg/ml) for the indicated times, as measured by qPCR. (E and F) Cxcl1 (E) and Ifnβ expression (F) in HRI+/+ and HRI–/– BMDMs after the indicated stimulations, as measured by qPCR. (G to I) Total lung cells (G) and inflammatory monocytes (H) from HRI+/+, HRI+/–, and HRI–/– mice infected with Puerto Rico/8/34 (H1N1) influenza A virus (IAV) at 6 days after infection, as measured by flow cytometry. (I) Representative micrographs (magnification 40×) of H&E-stained lungs from HRI+/+, HRI+/–, and HRI–/– mice infected as in (G) and (H). (J and K) Secretion of IL-1β from the supernatant of HRI+/+ and HRI–/– BMDMs stimulated with LPS (100 ng/ml) for 3 hours alone or subsequently stimulated with 10 μM nigericin or 5mMATP for 45min (J) and BMDMs primed for 4 hours with LPS (100 ng/ml) in Opti-MEM and stimulated with Fugene alone or transfected with poly(dA:dT) for 16 hours (K), as measured by ELISA. (L) HRI+/+ and HRI–/– BMDMs were stimulated for 3 hours with LPS (100 ng/ml) and stimulated with 10 μM nigericin for 45min, then analyzed by blue native PAGE. Bar graphs represent means ± SD from three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Tukey multiple-comparisons test).
Fig. 6.
Fig. 6.. HRI controls the cellular response to prefibrillar PRM adaptors and α-synuclein.
(A and B) IL8 (A) and HSPB8 (B) expression in HEK293T cells transduced with lentiviral particles targeting a scrambled sequence (SC) and HRI (shHRI) and transfected with the indicated plasmids. (C to E) ATF3 (C), CHOP (D), and HSPB8 (E) expression in SC and shHRI HEK293T cells transfected with the indicated plasmids. (F) SC and shHRI SH-SY5Y cells treated with 5 mM MG132 for 4 hours and analyzed by Western blotting. Graphs represent means ± SD from three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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