1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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Reactive oxygen species oxidize STING and suppress interferon production

  1. Lili Tao
  2. Andrew Lemoff
  3. Guoxun Wang
  4. Christina Zarek
  5. Alexandria Lowe
  6. Nan Yan
  7. Tiffany A Reese  Is a corresponding author
  1. Department of Immunology, University of Texas Southwestern Medical Center, United States
  2. Department of Biochemistry, University of Texas Southwestern Medical Center, United States
  3. Department of Microbiology, University of Texas Southwestern Medical Center, United States
Research Article
Cite this article as: eLife 2020;9:e57837 doi: 10.7554/eLife.57837
7 figures, 1 table and 2 additional files

Figures

ROS promote herpesvirus replication in macrophages.

(A) Fully differentiated bone marrow-derived macrophages (BMDMs) were treated with vehicle or different concentrations of menadione as indicated. Cell viability was determined at 0.5 hr, 2 hr and 16 hr after treatment. n = 2 with two technical repeats each time. (B) BMDMs were treated with vehicle, 4 μM or 8 μM menadione for 16 hr. Transcripts of Gsr and Gclm1 were determined using qRT-PCR. n = 6. (C) BMDMs were treated with vehicle or different concentrations of menadione (mena) for 16 hr and then infected with MHV68 at multiplicity of infection (MOI) = 5. Virus titer was determined by plaque assay at 0 hr, 24 hr, 48 hr, 72 hr and 96 hr after infection. n = 3 with three technical repeats each time. (D) BMDMs were treated with vehicle or different concentrations of H2O2 for 16 hr in culture medium containing 10% fetal bovine serum, then infected with MHV68 at MOI = 5. Twenty-four hours after infection, cells were fixed and cells expressing virus lytic proteins were determined by flow cytometry. n = 3 with two technical repeats each time. Data are shown as mean ± SE, an ordinary one-way ANOVA was performed followed by Dunnett’s multiple comparison test, only the p value for the most relevant comparisons are shown for simplicity. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 2 with 3 supplements
ROS inhibit interferon response upon STING activation.

(A) BMDMs isolated from WT or Ifnar-/- mice were treated with vehicle or 8 μM menadione for 16 hr, then infected with MHV68 at MOI = 5. Virus titer was determined by plaque assay at 0 hr, 10 hr, 24 hr, 48 hr and 72 hr after infection. n = 1 with three technical repeats. (B, C) BMDMs were treated with vehicle, 4 μM or 8 μM menadione for 16 hr, then infected with MHV68 at MOI = 5. Transcripts of Ifnb (B) or ISGs (Cxcl10, Isg20, Ccl5, Isg15) (C) were determined at 6 hr after infection. n = 6. (D) BMDMs were treated with vehicle or different concentrations of menadione as indicated for 30 mins, then stimulated with DMXAA at 1 μg/ml. Transcripts of Ifnb were determined at 2 hr after stimulation. n = 3. (E) BMDMs were treated with vehicle, 25 μM menadione or 25 μM menadione and 2 mM NAC for 30 mins, then stimulated with 1 μg/ml DMXAA. Transcripts of Ifnb were determined at 2 hr after stimulation. n = 3. (F) BMDMs were treated with vehicle or different concentrations of H2O2 in serum free medium for 10 mins, then stimulated with 1 μg/ml DMXAA. Transcripts of Ifnb were determined 2 hr after stimulation. n = 3. (G) BMDMs were treated with vehicle, 200 μM H2O2 for 10 mins or 200 μM H2O2 for 10 mins followed by 5 mM NAC for 30 mins, then stimulated with 1 μg/ml DMXAA. Transcripts of Ifnb were determined 2 hr after stimulation. n = 4. Data are shown as mean ± SE, statistical analysis was conducted using two-way ANOVA followed by Tukey’s multiple comparison test, only the p value for the most relevant comparisons are shown for simplicity. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 2—figure supplement 1
ROS regulate interferon response in macrophages upon STING activation.

BMDMs were treated with 20 μM menadione for 30 min, then stimulated with 10 μg/ml ISD, 1 μg/ml poly(I:C), or 1 μg/ml poly(dA:dT). Ifnb transcripts were determined 4 hr after stimulation. n = 4. Bars represent mean ± SE. p value was calculated using two-way ANOVA followed by Tukey’s multiple comparison test, only the p value for the most relevant comparisons are shown for simplicity. ***, p<0.001, n.s., p>0.05.

Figure 2—figure supplement 2
ROS regulate interferon response in macrophages upon STING activation with 2’,3’-cGAMP.

BMDMs were treated with 20 μM menadione for 30 min, then stimulated with 10 μg/ml 2’,3’-cGAMP. Ifnb transcripts were determined 4 hr after stimulation. n = 4. Bars represent mean ± SE. p value was calculated using two-way ANOVA followed by Tukey’s multiple comparison test, only the p value for the most relevant comparisons are shown for simplicity. ***, p<0.001, n.s., p>0.05.

Figure 2—figure supplement 3
ROS do not suppress interferon in primary fibroblasts.

Primary fibroblast cells were treated with 200 μM H2O2 in serum free medium, then stimulated with 2 μg/ml DMXAA. Ifnb transcript levels were determined 2 hr after stimulation. Bars represent mean ± SE. n = 2. Statistical analysis was not performed due to limited repeats.

Figure 3 with 1 supplement
Endogenous ROS regulate interferon response upon STING activation.

(A, B) BMDMs were treated with 2 mM NAC for 30 min, then infected with MHV68 at MOI = 5. Transcripts of Ifnb (A) or ISGs (Cxcl10, Isg20, Ccl5, Isg15) (B) were determined 6 hr after infection. n = 6. (C) BMDMs were treated with 2 mM NAC for 30 min, then stimulated with 1 μg/ml DMXAA. Transcripts of Ifnb were determined 2 hr after stimulation. n = 4. (D) BMDMs isolated from Acox1-/- or WT littermate control were stimulated with 1 μg/ml DMXAA. Transcripts of Ifnb were determined 2 hr after stimulation. n = 4. Data are shown as mean ± SE, statistical analysis was conducted using two-way ANOVA followed by Tukey’s multiple comparison test, only the p value for the most relevant comparisons are shown for simplicity. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 3—figure supplement 1
Inhibition of endogenous ROS has no effect on virus replication.

(A) BMDMs isolated from Acox1-/- or WT littermate control were infected with MHV68 at MOI = 5. n = 1 with three technical repeats. (B) BMDMs were treated with vehicle or 5 mM NAC for 1 hr, then infected with MHV68 at MOI = 5. Virus titer was determined by plaque assay at 0 hr, 10 hr, 24 hr, 48 hr and 72 hr after infection. n = 1 with three technical repeats.

Figure 4 with 2 supplements
ROS regulate interferon response by inhibiting STING dimerization.

(A) BMDMs were treated with vehicle or 200 μM H2O2 for 10 min in serum free culture medium, then stimulated with 1 μg/ml DMXAA. Western blots of TBK1, IRF3, STING, pTBK1 and pIRF3 were performed at 0 min, 30 min, 60 min and 90 min after stimulation. Data shown are representative of 2 independent experiments. (B) BMDMs were treated with vehicle or different concentrations of H2O2 in serum free culture medium for 10 mins, then stimulated with 1 μg/ml DMXAA. Level of pTBK1 was determined at 60 min after stimulation. n = 1 (C) BMDMs were treated with vehicle or 25 μM menadione for 30 min, then stimulated with 1 μg/ml DMXAA. Western blot of TBK1, IRF3, STING, pTBK1 and pIRF3 was performed at 0 min, 30 min, 60 min and 90 min after stimulation. Data shown are representative results of two independent experiments. (D) BMDMs isolated from WT control or Stinggt/gt mice were treated with vehicle or 8 μM menadione for 16 hr, then infected with MHV68 at MOI = 5. Virus titer was determined by plaque assay at 0 hr, 10 hr, 24 hr, 48 hr and 72 hr after infection. n = 3 with three technical repeats each time. (E) BMDMs were treated with vehicle, 25 μM menadione, 25 μM menadione and 2 mM NAC for 30 min, then stimulated with 1 μg/ml DMXAA. STING polymerization was determined by non-reducing SDS-PAGE. M: STING monomer; D: STING dimer. Data shown are representative of 2 experiments.

Figure 4—figure supplement 1
Schematic diagrams of STING monomer, dimer and polymer on different electrophoresis gels.

STING is a dimeric protein at resting state, which is formed by non-covalent bonds and can be disrupted by SDS treatment. Ligand binding alters the conformation of STING and triggers its oligomerization through the formation of covalent disulfide bonds. These disulfide bonds are resistant SDS treatment but sensitive to reducing reagents. As such, STING proteins that are manifested as monomer and dimer on non-reducing SDS PAGE gels are actually dimer and oligomer, respectively.

Figure 4—figure supplement 2
ROS decrease TBK1 recruitment during STING activation.

BMDMs were treated with vehicle or 25 μm menadione for 30 min, then stimulated with 1 μg/ml DMXAA for 1 hr. Cells were lysed and immunoprecipitated with anti-STING antibody, and the protein levels of TBK1 and STING in both total cell lysate and immunoprecipitated samples were determined by western blot.

Figure 5 with 2 supplements
ROS oxidize C148 on STING.

(ASting-/- fibroblasts stably expressing HA-tagged human STING were treated with vehicle or 200 μM H2O2 in serum free medium for 10 min. Cell lysates were incubated with 5 μM 5-IAF for 1 hr at room temperature to label free thiols. Protein levels of STING and FITC were detected after immunoprecipitation for HA-tagged protein. Data shown are representative results of two independent experiments. (B, C) BMDMs were treated with 200 μM diamide for 30 mins. STING polymers (B, n = 2) and Ifnb transcripts (C, n = 4) were determined at 1 hr after 1 μg/ml DMXAA stimulation. M: STING monomer; D: STING dimer. Bars represent the mean ± SE, p value was calculated using two-way ANOVA followed by Tukey’s multiple comparison test. Only the p values for the most relevant comparison are shown for clarity purpose. ***, p<0.001. (D) Schematic of differential alkylation (IAM labeling followed by DTT reducing and NEM labeling) of cysteines for mass spectrometry analysis. (E) Mass spectra of IAM- and NEM-modified STING in vehicle and menadione treated samples. n = 1 (F) Quantification of Cysred and Cysox from mass spectrometry analysis. (G) Vectors with WT STING or C148A mutated STING were transfected into HEK293T cells. Twenty-four hours after transfection, cells were treated with vehicle or 200 μM diamide for 30 min. Polymer of STING was determined with non-reducing SDS-PAGE. M: STING monomer; D: STING dimer. n = 1.

Figure 5—figure supplement 1
Representative MS/MS spectra of SAVC147EEK peptide in STING protein modified by both IAM (CAM) and NEM.

This analysis was performed on the samples shown in Figure 5D.

Figure 5—figure supplement 2
Sequence alignment of STING from multiple species suggested C148 of STING is highly conserved across mammalian species.

C148 residue is highlighted in yellow.

ROS regulate MHV68 replication in vivo.

(A) Schematic of MHV68 replication in mice with menadione treatment or NAC treatment. (B, C) 8–12 weeks old mice were sex-matched and randomly assigned to groups prior to experiment. Mice were treated and infected as shown in (A). Total flux (photons/second) was measured using IVIS bioluminescence imager at day 2 and day 4 after infection to quantitate lytic replication of MHV68. Data shown were the results obtained from a pool of two independent experiments. Bars represent geometric mean ±geometric SD. Each dot represents an individual mouse. (D) Schematic for the quantification of interferon and ISG transcripts in mice with menadione treatment during MHV68 infection. 7–12 weeks old mice were sex-matched and randomly assigned to groups prior to experiment. Mice were treated with either vehicle control (5% DMSO in corn oil) or menadione (10 mg/kg in corn oil) for 3 days, then infected with 106 PFU MHV68 peritoneally. Twelve hours post infection, transcripts of Ifnb and Cxcl10 in peritoneal exudate cells (PECs) were determined from individual mice. (E, F) Transcripts of Ifnb and Cxcl10 in PECs from mock (vehicle treatment, no infection), MHV68 (vehicle treatment, MHV68 infected) and MHV68+Menadione (Menadione treatment, MHV68 infected) mice. Bars represent mean ± SE, each dot represent an individual mouse. Data are collected from one controlled experiment. Statistical analysis was conducted using two-way ANOVA (repeated measures). *p<0.05.

Model of ROS modification of STING.

Increasing ROS leads to oxidation of free thiol on C148 of human STING and C147 of mouse STING. This prevents polymerization of STING upon binding of 2’,3’-cGAMP and leads to reduced interferon production.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6JThe Jackson LaboratoryStock No. 000664Bred at UTSW facility with IACUC approval
Strain, strain background (Mus musculus)C57BL/6J-Tmem173gt/JThe Jackson LaboratoryStock No. 017537Bred at UTSW facility with IACUC approval
Strain, strain background (Mus musculus)B6.129S2-Ifnar1tm1Agt/JThe Jackson LaboratoryStock No.
32045
Bred at UTSW facility with IACUC approval
Strain, strain background (Mus musculus)B6.129P2-Acox1tm1Jkr/JThe Jackson LaboratoryStock No.
029747
Bred at UTSW facility with IACUC approval
Cell line (Herpesviridae, Rhadinovirus)Murine gamma herpesvirus 68 (WUSM stain)ATCCVR-1465
Cell line (Herpesviridae, Rhadinovirus)Murine gamma herpesvirus 68-M3FLHome made
Hwang et al., 2008
Cell line (Mus musculus)3T12ATCCCat# ATCC CCL-164; RRID:CVCL_0637
Cell line (Homo sapiens)293TATCCCat# ATCC CRL-3216; RRID:CVCL_0063
Transfected construct (Homo sapiens)STINGGenBankAVQ94753.1Express STING into 293T cells
AntibodyAnti-STING (Rabbit polyclonal)ProteintechCat# 19851-1-AP; RRID:AB_10665370WB (1:1000);
IP (1 ug/ml)
AntibodyAnti-STING (Rabbit monoclonal)Cell SignalingCat# 50494S; RRID:AB_2799375WB (1:1000)
AntibodyAnti-TBK1/NAK (Rabbit monoclonal)Cell SignalingCat# 3504S; RRID:AB_2255663WB (1:1000)
AntibodyAnti-IRF-3 (Rabbit monoclonal)Cell SignalingCat# 4302S; RRID:AB_1904036WB (1:1000)
AntibodyAnti-Phospho-TBK1 (Ser172) (Rabbit monoclonal)Cell SignalingCat# 5483S; RRID:AB_10693472WB (1:1000)
AntibodyAnti-Phospho-IRF-3 (Ser396) (Rabbit monoclonal)Cell SignalingCat# 4947S; RRID:AB_823547WB (1:1000)
AntibodyMouse monoclonal anti-SDHA antibodyAbcamCat# ab14715; RRID:AB_301433WB (1:5000)
AntibodyAnti-MHV68 (Rabbit polyclonal)Home made
Weck et al., 1997
FACs (1:1000)
AntibodyAnti-Rabbit IgG(H+L) secondary antibody, Alexa Fluor 647 (Goat polyclonal)Thermo Fisher ScientificCat# A-21245; RRID:AB_2535813FACs (1:4000)
AntibodyAnti-Rabbit IgG, Peroxidase (Donkey polyclonal)Jackson ImmunoResearch LaboratoryCat# 711-035-152; RRID:AB_10015282WB (1:5000)
AntibodyAnti-Mouse IgG, Peroxidase (Goat polyclonal)Jackson ImmunoResearch LaboratoryCat# 115-035-174; RRID:AB_2338512WB (1:5000)
AntibodyAnti-FITC (Rabbit polyclonal)Thermo Fisher ScientificCat# 71-1900; RRID:AB_2533978WB (1:2000)
Recombinant DNA reagentpcDNA 3.1(+) Mammalian Expression Vector (plasmid)Thermo Fisher ScientificCat# V79020Vector for the expression of human STING
Sequence-based reagentIfnb forwardThis paperqPCR primersCAGCTCCAAGAAAGGACGAAC
Sequence-based reagentIfnb reverseThis paperqPCR primersGGCAGTGTAACTCTTCTGCAT
Sequence-based reagentCxcl10 forwardThis paperqPCR primersTTAACGTCAGGCCAACAGAG
Sequence-based reagentCxcl10 reverseThis paperqPCR primersGAGGGAAACCAGGAAAGATAGG
Sequence-based reagentIsg15 forwardThis paperqPCR primersCAGGACGGTCTTACCCTTTCC
Sequence-based reagentIsg15 reverseThis paperqPCR primersAGGCTCGCTGCAGTTCTGTAC
Sequence-based reagentIsg20 forwardThis paperqPCR primersCCATGGACTGTGAGATGGTG
Sequence-based reagentIsg20 reverseThis paperqPCR primersCTCGGGTCGGATGTACTTGT
Sequence-based reagentGapdh forwardThis paperqPCR primersGGGTGTGAACCACGAGAAATA
Sequence-based reagentGapdh reverseThis paperqPCR primersGTCATGAGCCCTTCCACAAT
Sequence-based reagentGsr forwardThis paperqPCR primersCACCGAGGAACTGGAGAATG
Sequence-based reagentGsr reverseThis paperqPCR primersATCTGGAATCATGGTCGTGG
Sequence-based reagentGclm forwardThis paperqPCR primersAATCAGCCCCGATTTAGTCAG
Sequence-based reagentGclm reverseThis paperqPCR primersCGATCCTACAATGAACAGTTTTGC
Sequence-based reagentC148 forwardThis paperSite direct mutagenesis PCR primersCTCTGCAGTGCTGAAAAAGGGAATTTCAACGTGGC
Sequence-based reagentC148A reverseThis paperSite direct mutagenesis PCR primersATCTCAGCTGGGGCCAGG
Commercial assay or kitLipofectamine 3000 ReagentThermo Fisher ScientificCat# L3000008
Commercial assay or kitQiagen RNeasy Mini KitQiagenCat# 74104
Commercial assay or kitSuperScript VILO cDNA Synthesis KitThermo Fisher ScientificCat# 11754050
Commercial assay or kitPowerUp SYBR Green Master MixThermo Fisher ScientificCat# A25776
Commercial assay or kitLIVE/DEAD Fixable Dead Cell Stain KitsInvitrogenCat# L34975
Commercial assay or kitQ5 Site-Directed Mutagenesis KitNew England BioLabsCat# E0554S
Chemical compound, drugDMXAAInvivoGenCat# tlrl-dmx(1 ug/ml) for macrophages,
(2 ug/ml) for fibroblasts
Chemical compound, drug2’3’-cGAMPInvivoGenCat# tlrl-nacga23(10 ug/ml)
Chemical compound, drugISDInvivoGenCat# tlrl-isdn(10 ug/ml)
Chemical compound, drugpoly(dA:dT)InvivoGenCat# tlrl-patn-1(1 ug/ml)
Chemical compound, drugpoly(I:C)InvivogenCat# tlrl-picw(1 ug/ml)
Chemical compound, drugD-Luciferin, Potassium SaltGOLDBIOCat# LUCK(150 mg/kg)
Chemical compound, drugMenadioneSigmaCat# M9429(10 mg/kg) for mice, concentration for cells were indicated in each experiment
Chemical compound, drugHydrogen peroxide solutionSigmaCat# 216763Concentration for cells were indicated in each experiment
Chemical compound, drugN-Acetyl-L-cysteineSigmaCat# A72502 mM for macrophages, 5 mM in drinking water with 2% sucrose
Chemical compound, drugIodoacetamideSigmaCat# I1149100 mM
Chemical compound, drugN-ethylmaleimideSigmaCat# E387650 mM
Chemical compound, drugElastaseWorthingtonCat# LS0063631 mg/ml
Chemical compound, drug5-(Iodoacetamido) fluoresceinSigmaCat# I92715 ug/ml
Chemical compound, drugDiamideSigmaCat# D3648200 uM
Software, algorithmGraphPad Prism 8GraphPadwww.graphpad.com; RRID:SCR_002798
Software, algorithmFlowJo softwareFlowJowww.flowjo.com; RRID:SCR_008520
Software, algorithmLive Image SoftwarePerkin Elmerwww.perkinelmer.com; RRID:SCR_014247

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