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Redox homeostasis maintained by GPX4 facilitates STING activation

Abstract

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS–STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress–induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

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Fig. 1: GPX4 inhibition selectively attenuates the cGAS–STING pathway.
Fig. 2: GPX4 deficiency specifically inhibits the cGAS–STING pathway.
Fig. 3: Lipid peroxidation inhibits cGAS signaling.
Fig. 4: GPX4 deficiency enhances the cellular level of lipid peroxide and attenuates the cGAS pathway.
Fig. 5: GPX4 is required for STING trafficking at the Golgi and promotes STING activation.
Fig. 6: 4-HNE-induced carbonylation blocks palmitoylation of STING at Cys 88.
Fig. 7: RSL3 attenuates HSV-1-induced IFN-β and promotes HSV-1 replication in vivo.
Fig. 8: GPX4 deficiency inhibits innate immune responses against HSV-1 in vivo.

Data availability

All data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. A Nature Research Reporting Summary for this article is available as a Supplementary Information file. Source data for Figs. 1–8 and Extended Data Figs. 1–9 are presented with the paper.

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Acknowledgements

We thank F. Y. Liew for expertise and advice. This work was supported by grants from the National Key Research and Developmental Program of China (grant no. 2017YFC1001100 to W.Z.), the National Natural Science Foundation of China (grant nos. 81622030, 31870866 and 81861130369 to W.Z.). W.Z. is a Newton Advanced Fellow of the Academy of Medical Sciences (NAF\R1\180232 to W.Z.).

Author information

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W.Z. and M.J. designed the experiments, analyzed the data and wrote the manuscript. M.J. and D.Q. performed most of the experiments. C.Z., L.C., Z.Y., W.W., L.T., L.L. and Y.W. assisted with experiments and provided technical help. J.R. and J.Y. provided expertise and advice. W.Z. conceived the project and provided overall direction.

Corresponding author

Correspondence to Wei Zhao.

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The authors declare no competing interests.

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Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 GPX4 inhibitors specifically inhibit cGAS–STING signaling.

a-f, Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay and cell viability analysis of PMs treated with indicated doses of RSL3 or FIN56. The dose used in the following experiments was 0.5 μM for RSL3 and 1 μM for FIN56. g and h, MTT analysis of PMs treated with RSL3 (0.5 µM) or FIN56 (1 µM) for indicated time periods. i and j, qPCR analysis of Ifnb and Cxcl10 mRNA level in PMs pretreated with increasing concentrations of RSL3 (0, 0.25, 0.5, 0.75, 1, 2 μM), followed by ISD transfection. k-l, qPCR analysis of Ifnb and Cxcl10 mRNA level in PMs pretreated with DMSO or RSL3 (0.5 µM) and then transfected with ISD, cGAMP or poly (I:C). m-p, Immunoblot assays of p-TBK1, p-IRF3 and p-STAT1 in PMs pretreated with DMSO or RSL3 (0.5 µM), followed by stimulation as indicated. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in i-l. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 2 Gpx4 deficiency specifically inhibits cGAS–STING signaling.

a, Protein levels of GPX4 in wild-type (WT) or Gpx4CKO PMs. b, qPCR analysis of Ifnb and Ifna4 (Ifn-a) mRNA level in PMs from WT or Gpx4CKO mice, plus transfected with ISD, cGAMP or poly (I:C). c, qPCR analysis of Ifnb, Ifna4 (Ifn-a) and Cxcl10 mRNA level in PMs from WT or Gpx4CKO mice plus stimulation with DMXAA or CMA. d-f, Immunoblot assays of indicated proteins in PMs from WT or Gpx4CKO mice plus stimulation with DMXAA, CMA or poly (I:C). g, qPCR analysis of Rantes mRNA level in PMs pretreated with DMSO or RSL3(0.5 µM) and then infected with HSV-1. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in b, c, g. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 3 Gpx4 knockdown specifically inhibits cGAS–STING signaling.

a, Protein levels of GPX4 in PMs transfected with Control siRNA (siCtrl) or GPX4 siRNAs (siGPX4-1, 2 or 3) for 48 h. b-e, IFN-β expression (b-d) and phosphorylated IRF3 level (e) in PMs transfected with siCtrl or siGPX4, followed by stimulation as indicated. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in b, c, d. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 4 Ferrous ions, but not rastin and hydrogen peroxide, inhibit HSV-1-induced IFN-β expression.

a, Cell viability analysis of PMs from WT or Gpx4CKO mice, plus HSV-1 infection for indicated time periods. b, Schematic representation of GPX4 inactivation leads to lipid ROS production and ferroptosis. c, ELISA analysis of IFN-β secretion in PMs pretreated with DMSO or Erastin(10 µM) plus stimulation as indicated. d-f, qPCR analysis of Ifnb mRNA level in PMs pretreated with increasing concentrations of Erastin (0, 5, 10, 15 μM), H2O2 (0, 1, 2, 5, 10 μM), or Fe2+ (0, 5, 10, 15, 20 μM), followed by HSV-1 infection. g, p-IRF3 level in PMs retreated with increasing concentrations of Fe2+, followed by HSV-1 infection. h-j, qPCR analysis of Ifnb mRNA level in PMs pretreated with L-α-phosphatidylcholine (PC, 20 μM), phosphatidylinositol (PI, 20 μM) or linoleic acid (20 μM). k,l, qPCR analysis of Ifnb mRNA level in PMs pretreated with DTT (k) or NAC (1 mM)(l), followed by RSL3(0.5 µM) treatment and DMXAA stimulation. m, Protein levels of ACSL4 in ACSL4+/+ or ACSL4-/- U2OS cells. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in c-f, h-l. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 5 The effects of RSL3 targets on cGAS signaling.

a, List of selenoproteins covalently modified by RSL3. b-g, qPCR analysis of Ifnb mRNA level in PMs transfected with indicated siRNAs for 48 h, followed by HSV-1 infection. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in b-g. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. *P < 0.05, ***P < 0.001. Source data

Extended Data Fig. 6 GPX4 has no effect on cGAS activity.

a,b, ChIP analysis of HSV-1 genomic DNA binding to cGAS in PMs from WT or Gpx4CKO mice (a) or PMs treated with DMSO or RSL3 (0.5 µM) (b). c, ELISA analysis of cGAMP production in PMs treated with DMSO or RSL3 (0.5 µM), followed by HSV-1 infection or ISD transfection. d, cGAMP activity was analyzed in PMs treated with DMSO or RSL3 (0.5 µM), followed by HSV-1 infection. e, cGAMP activity was analyzed in PMs from WT or Gpx4CKO mice, followed by HSV-1 infection. Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in a-e. Data are shown as mean ± SD. Source data

Extended Data Fig. 7 GPX4 has no effect on cGAMP binding to STING and STING dimerization.

a, Luciferase activity assays of IFN-β activation in 293-STING A162 cells pretreated with increasing concentrations of RSL3 (0, 0.25, 0.5, 1 μM) or FIN56 (0, 0.2, 0.5 μM), followed by stimulation with DMXAA. b, Immunoblot assays of Gpx4+/+ MEFs transfected with empty vector (CTRL) and Gpx4-/- MEFs transfected with empty vector (Gpx4-/-), wild type GPX4 (Gpx4-/- plus GPX4 plasmid), or GPX4 mutants (Gpx4-/- plus U73A mutant). c, Immunofluorescence analysis of the colocalization of STING (red) and cGAMP-FITC (green) in PMs treated with DMSO or RSL3(0.5 µM). Scale bar, 20 μm. (c) Immunofluorescence analysis of the colocalization of STING (red) and cGAMP-FITC (green) in PMs treated with DMSO or RSL3(0.5 µM). Scale bar, 20 μm. f, Immunoblot assays of phosphorylated and total STING in DMSO or RSL3(0.5 µM) treated PMs followed by DMXAA stimulation. g, Confocal analysis of the colocalization of STING (green) and cis-Golgi (GM130, red) in mouse embryonic fibroblasts (MEFs) pretreated with water or 4-HNE (5 µM) and then stimulated with HSV-1. Scale bar, 10 μm. Manders split coefficients values as indicated. h, Immunoblot assays of Sting1 expression in Sting1+/+ and Sting1-/- MEFs. i, Luciferase activity assays of IFN-β activation in Sting1-/- MEFs transfected with empty vector (CTRL), STING wild type (STING), STING mutants (V147L and N154S) or cGAS plus STING, followed by treatment with DMSO or RSL3 (0.5 µM). Statistical significance was determined by unpaired two-sided multiple Student’s t-tests in a, i. Data are shown as mean ± SD or typical photographs of one representative from three independent experiments. **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 8 Identification of STING carbonylation.

a, Staining of STING (red) and lipid peroxidation sensor C11 (green) (i, Scale bar 5μm) in PMs treated with RSL3(0.5 μM) and then infected with HSV-1. b, List of identified carbonylation sites by of STING by LC-MS. c, Conserved amino acid residues of STING in mammals. d, LC-MS spectra of the HNE modification of STING at C257. e, In vitro carbonylation and oxy-immunoblot assay analysis of STING carbonylation. f, Immunoblot assays of carbonylation of STING by m-APA selectively labeled in PMs infected with VSV. g, Oxy-immunoblot analysis of total carbonylation in PMs treated with DMSO or RSL3, followed by HSV-1 infection. h, Immunoblot assays of carbonylation of MAVS by m-APA selectively labeled in PMs treated with 4-HNE (6.4 µM). Data are shown as typical photographs of one representative from three independent experiments. Source data

Extended Data Fig. 9 Schematic representation of the role of GPX4 in STING activation.

a, GPX4 suppresses lipid peroxidation caused by stress, such as DNA viral infection, and thus maintains cellular redox homeostasis, which is required for STING activation. b, GPX4 deficiency promotes lipid peroxidation and enhances STING carbonylation, resulting in the inhibition of STING trafficking from the ER to the Golgi and suppression of STING activation.

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Jia, M., Qin, D., Zhao, C. et al. Redox homeostasis maintained by GPX4 facilitates STING activation. Nat Immunol 21, 727–735 (2020). https://doi.org/10.1038/s41590-020-0699-0

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