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. 2014 May;88(10):5328-41.
doi: 10.1128/JVI.00037-14. Epub 2014 Mar 5.

Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-κB activation through TBK1

Takayuki Abe  1 , Glen N Barber
Affiliations

Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-κB activation through TBK1

Takayuki Abe et al. J Virol. 2014 May.

Abstract

STING (stimulator of interferon genes) is known to control the induction of innate immune genes in response to the recognition of cytosolic DNA species, including the genomes of viruses such as herpes simplex virus 1 (HSV-1). However, while STING is essential for protection of the host against numerous DNA pathogens, sustained STING activity can lead to lethal inflammatory disease. It is known that STING utilizes interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways to exert its effects, although the signal transduction mechanisms remain to be clarified fully. Here we demonstrate that in addition to the activation of these pathways, potent induction of the Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) pathway was similarly observed in response to STING activation by double-stranded DNA (dsDNA). Furthermore, TANK-binding kinase 1 (TBK1) associated with STING was found to facilitate dsDNA-mediated canonical activation of NF-κB as well as IRF3 to promote proinflammatory gene transcription. The triggering of NF-κB function was noted to require TRAF6 activation. Our findings detail a novel dsDNA-mediated NF-κB activation pathway facilitated through a STING-TRAF6-TBK1 axis and suggest a target for therapeutic intervention to plausibly stimulate antiviral activity or, alternatively, avert dsDNA-mediated inflammatory disease.

Importance: The IKK complex, which is composed of two catalytic subunits, IKKα and IKKβ, has been suggested to be essential for the activation of canonical NF-κB signaling in response to various stimuli, including cytokines (e.g., interleukin-1α [IL-1α] and tumor necrosis factor alpha [TNF-α]), Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide [LPS]), and dsRNAs derived from viruses, or a synthetic analog. STING has been identified as a critical signaling molecule required for the detection of cytosolic dsDNAs derived from pathogens and viruses. However, little is known about how cytosolic dsDNA triggers NF-κB signaling. In the present study, we demonstrate that TBK1, identified as an IKK-related kinase, may predominantly control the activation of NF-κB in response to dsDNA signaling via STING through the IKKαβ activation loop. Thus, our results establish TBK1 as a downstream kinase controlling dsDNA-mediated IRF3 and NF-κB signaling dependent on STING.

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Figures

FIG 1
FIG 1
STING ligand-mediated signaling response in MEFs. Primary MEFs (1 × 105 cells/well) derived from wild-type (STING+/+) or STING-deficient (STING−/−) mice were stimulated with 10 μg/ml of dsDNA90, 5 μg/ml of poly(dA-dT), 5 μg/ml of poly(I·C), or 200 μM DMXAA for the indicated times. The expression levels of IκBα, STING, IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, ERK1/2 phosphorylated at Thr202/Tyr204 (p-ERK1/2), ERK1/2, SAPK/JNK phosphorylated at Thr183/Tyr185 (p-SAPK/JNK), JNK1/2, p38 phosphorylated at Thr180/Tyr182 (p-p38), p38, c-Jun phosphorylated at Ser63 (p-cJun), and β-actin were determined by immunoblotting. The phosphorylation state of STING is indicated by an arrowhead.
FIG 2
FIG 2
IKKαβ is involved in dsDNA-mediated NF-κBp65 activation in MEFs. (A) Immortalized MEFs derived from wild-type (WT) and IKKα- or IKKβ-deficient mice were stimulated with 10 μg/ml of dsDNA90 for the indicated times. The expression levels of STING, TBK1, TBK1 phosphorylated at Ser172 (p-TBK1), IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, IKKα, IKKβ, and β-actin were determined by immunoblotting. (B) WT and IKKα- and IKKβ-deficient MEFs were stimulated with 10 μg/ml of dsDNA90 for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IL-6 and IFN-β were determined by real-time PCR. Real-time PCR data were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. *, P < 0.05 (Student's t test). Error bars indicate standard deviations (SD). (C) IKKα-deficient MEFs subjected to RNAi by use of nonspecific (NS) and IKKβ siRNAs were stimulated with 5 μg/ml of dsDNA90 for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IL-6, CXCL10, and IKKβ were determined by real-time PCR. Real-time PCR data were normalized to the amount of GAPDH mRNA. *, P < 0.05 (Student's t test). Error bars indicate SD. (D) IKKα-deficient MEFs subjected to RNAi by use of NS and IKKβ siRNAs were stimulated with 5 μg/ml of dsDNA90 for the indicated times, and then the signaling response was determined by immunoblotting using specific antibodies for STING, IRF3 phosphorylated at Ser396 (p-IRF3), TBK1 phosphorylated at Ser172 (p-TBK1), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, and TBK1, with β-actin serving as a loading control.
FIG 3
FIG 3
TBK1 regulates dsDNA-mediated NF-κB activation in MEFs. (A) Immortalized MEFs derived from wild-type (TBK1+/+) or TBK1-deficient (TBK1−/−) mice were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for the indicated times. The expression levels of TBK1, STING, IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, and β-actin were determined by immunoblotting. (B) TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then the cells were stained with antibodies against NF-κBp65 or IRF3. (C) Detection of nuclear translocation of NF-κBp65 by fractionation assay. TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then cell lysates were separated into cytosolic and nuclear fractions. Each fraction was concentrated and subjected to immunoblotting with the indicated antibodies. (D) Quantitative analysis of NF-κBp65 phosphorylated at Ser536 by ELISA. TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 3 h, and then endogenous levels of NF-κBp65 phosphorylated at Ser536 were determined by ELISA. *, P < 0.05 (Student's t test). Error bars indicate SD. OD, optical density.
FIG 4
FIG 4
Regulation of dsDNA-mediated gene expression by TBK1 in MEFs. (A) TBK1+/+ and TBK1−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IFN-β, IL-6, CXCL10, Ccl5, and Ccl2 were determined by qRT-PCR. (B) TBK1−/− MEFs reconstituted by transduction with retroviral vectors encoding Myc-tagged human TBK1 (hTBK1) or mock-transduced cells (Mock) were stimulated with 10 μg/ml of dsDNA90 for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IFN-β, IL-6, CXCL10, and Ccl5 were determined by qRT-PCR. Data were normalized to the amount of GAPDH mRNA. Error bars indicate SD. The expression levels of TBK1 and STING were determined by immunoblotting using specific antibodies, with β-actin serving as a loading control.
FIG 5
FIG 5
TBK1 regulates DMXAA-mediated NF-κB activation in MEFs. (A) TBK1+/+ and TBK1−/− MEFs were stimulated with 200 μM DMXAA for the indicated times. The expression levels of TBK1, STING, IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, and β-actin were determined by immunoblotting. (B) Detection of nuclear translocation of NF-κBp65 by fractionation assay. TBK1+/+ and TBK1−/− MEFs were stimulated with 200 μM DMXAA for 3 h, and then cell lysates were separated into cytosolic and nuclear fractions. Each fraction was concentrated and subjected to immunoblotting with the indicated antibodies. (C) TBK1+/+ and TBK1−/− MEFs were stimulated with 200 μM DMXAA for 3 h, and then cells were stained with antibodies against NF-κBp65. (D) TBK1+/+ and TBK1−/− MEFs were stimulated with 200 μM DMXAA for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNA for CXCL10 were determined by qRT-PCR. Data were normalized to the levels of GAPDH mRNA. (E) MEFs subjected to RNAi by use of nonspecific (NS) and TBK1 siRNAs were stimulated with 200 μM DMXAA or 5 μg/ml of poly(I·C) for the indicated times. The cell lysates were subjected to immunoblotting with the indicated antibodies. (F) MEFs subjected to RNAi by use of NS, TBK1, and IKKβ siRNAs were stimulated with 200 μM DMXAA for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNA for CXCL10 were determined by qRT-PCR. Data were normalized to the levels of GAPDH mRNA.
FIG 6
FIG 6
Role of NF-κBp65 in dsDNA-mediated IFN production in MEFs. (A) MEFs subjected to RNAi by use of nonspecific (NS) and NF-κBp65 (RelA/p65) siRNAs were stimulated with 5 μg/ml of dsDNA90, and the expression levels of IFN-β mRNA and production of IFN-β in the supernatant were determined by real-time PCR and ELISA, respectively. Real-time PCR data were normalized to the amount of GAPDH mRNA. Silencing of RelA/p65 expression was demonstrated by immunoblotting, with β-actin serving as a loading control. (B) MEFs subjected to RNAi by use of NS and RelA/p65 siRNAs were stimulated with 5 μg/ml of dsDNA90 for 3 h, and the expression levels of IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65, and β-actin were determined by immunoblotting. (C) MEFs subjected to RNAi by use of NS and RelA/p65 siRNAs were inoculated with HSV-luc at a multiplicity of infection (MOI) of 1, 0.2, or 0.04 PFU/ml for 24 h, and then cell lysates were analyzed by luciferase assay. RLU, relative light units. (D) Following silencing of RelA/p65, the expression levels of viral proteins, such as ICP4 and gD, or of endogenous STING were demonstrated by immunoblotting, with β-actin serving as a loading control. **, P < 0.01; *, P < 0.05 (Student's t test). Error bars indicate SD.
FIG 7
FIG 7
TRAF3 and TRAF6 may contribute to the STING pathway upstream of TBK1. (A) 293T cells were transfected with a plasmid for HA-tagged STING (STING-HA) in combination with empty vector (EV) or a plasmid encoding His-tagged TRAF3 (TRAF3-His) or TRAF6 (TRAF6-His), together with a reporter plasmid carrying the luciferase gene under the control of the IFN-β, NF-κB, pRDIII, or ISRE promoter. Luciferase activity was determined at 24 h posttransfection. (B) Structure of murine TRAF3 and a deletion mutant of the TRAF domain (TRAF3 mut). The E3 ring finger ubiquitin ligase domain, zinc finger domain, and C-terminal TRAF-C domain (also known as a meprin and TRAF homology domain) are indicated. 293T cells were transfected with a STING-HA vector in combination with EV or a TRAF3-His or TRAF3 mut vector, together with a reporter plasmid carrying the luciferase gene under the control of the IFN-β or NF-κB promoter, and luciferase activity was determined at 24 h posttransfection. TRAF3, TRAF3 mut, or STING expression was demonstrated by immunoblotting, with β-actin serving as a loading control. (C) 293T cells subjected to RNAi by use of nonspecific (NS) or TBK1 siRNA were cotransfected with the STING-HA vector in combination with EV or a TRAF3-His or TRAF6-His vector, together with a reporter plasmid carrying the luciferase gene under the control of the IFN-β or NF-κB promoter, and luciferase activity was determined at 24 h posttransfection. **, P < 0.01 (Student's t test). Error bars indicate SD. IB, immunoblotting.
FIG 8
FIG 8
Distinct roles of TRAF3 and TRAF6 in the dsDNA-mediated signaling response in MEFs. (A) MEFs subjected to RNAi by use of nonspecific (NS), TRAF3, or TRAF6 siRNA were stimulated with 5 μg/ml of dsDNA90 for the indicated times. The expression levels of STING, IRF3 phosphorylated at Ser396 (p-IRF3), TBK1 phosphorylated at Ser172 (p-TBK1), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, NF-κB2p52, TRAF3, TRAF6, ERK1/2 phosphorylated at Thr202/Tyr204 (p-ERK1/2), ERK1/2, SAPK/JNK phosphorylated at Thr183/Tyr185 (p-SAPK/JNK), JNK1/2, p38 phosphorylated at Thr180/Tyr182 (p-p38), p38, c-Jun phosphorylated at Ser63 (p-cJun), and β-actin were determined by immunoblotting. (B) STING-deficient MEFs reconstituted with HA-tagged murine STING and knocked down for TRAF3 or TRAF6 by RNAi were stimulated with 5 μg/ml of dsDNA90 for 3 h, and then cells were costained with antibodies against NF-κBp65 and HA. (C) Levels of TRAF3 and TRAF6 in MEFs treated with NS, TRAF3 (T3), TRAF6 (T6), or STING (ST) siRNA were determined by qRT-PCR as shown in panel D. qRT-PCR data were normalized to the amount of GAPDH mRNA. (D) MEFs treated with NS, TRAF3 (T3), TRAF6 (T6), TRAF3 and TRAF6 (T3/T6), or STING (ST) siRNA were stimulated with 5 μg/ml of dsDNA90 or 5 μg/ml of poly(I·C) for 24 h. The production of IL-6 (left) and IFN-β (right) in the supernatants was determined by ELISA. *, P < 0.05 (Student's t test). Error bars indicate SD.
FIG 9
FIG 9
STING ligands activate the noncanonical NF-κB signaling pathway in a STING-dependent manner in MEFs. (A) STING+/+ and STING−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 10 μg/ml of poly(I·C) for the indicated times. The expression levels of STING, NF-κBp52, NF-κBp100 phosphorylated at Ser866/870 (p-NF-κBp100), and β-actin were determined by immunoblotting. (B) Immortalized MEFs derived from wild-type (WT) and IKKα-deficient (IKKα−/−) mice (top panels) or wild-type (TBK1+/+) and TBK1-deficient (TBK1−/−) mice (bottom panels) were stimulated with 10 μg/ml of dsDNA90 for the indicated times. The expression levels of IKKα, TBK1, NF-κBp52, NF-κBp100 phosphorylated at Ser866/870 (p-NF-κBp100), and β-actin were determined by immunoblotting. The phosphorylation state of NF-κBp100 is indicated by an arrowhead. (C) Model of dsDNA-mediated canonical and noncanonical NF-κB activation triggered by STING. Upon dsDNA stimulation, STING is activated and traffics with TBK1 as a signaling complex from the ER to a perinuclear endosomal compartment to activate IRF3 (right arm) and NF-κB. STING is also activated by cGAMP produced by cGAS, which was recently identified as a candidate DNA sensor. TRAF6 may be recruited to the signaling complexes with STING and TBK1, which in turn activates the canonical NF-κBp65 signaling pathway through the IKKαβ activation loop (center arm). STING-mediated NF-κBp65 activation may also contribute to dsDNA-mediated IFN-β production. On the other hand, STING may also activate the noncanonical NF-κB signaling pathway through the TRAF3-IKKα axis, leading to modulation of the dsDNA-mediated canonical NF-κB signaling pathway (left arm). Thus, TRAF3 may be involved in the modulation of canonical and noncanonical dsDNA-mediated NF-κB activation triggered upon STING activation.
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