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. 2024 Aug 7;27(9):110693.
doi: 10.1016/j.isci.2024.110693. eCollection 2024 Sep 20.

IKKɛ induces STING non-IFN immune responses via a mechanism analogous to TBK1

Rajan Venkatraman  1   2 , Katherine R Balka  1 , Wilson Wong  3   4 , Jananipriya Sivamani  1 , Zoe Magill  1 , Kirsteen M Tullett  1 , Rachael M Lane  1 , Tahnee L Saunders  5   2 , Maximilien Tailler  6 , Peter J Crack  7 , Linda M Wakim  8 , Mireille H Lahoud  1 , Kate E Lawlor  3   4 , Benjamin T Kile  6 , Meredith O'Keeffe  1 , Dominic De Nardo  1
Affiliations

IKKɛ induces STING non-IFN immune responses via a mechanism analogous to TBK1

Rajan Venkatraman et al. iScience. .

Abstract

The cGAS-STING pathway responds to cytosolic DNA to elicit host immunity to infection. The activation of stimulator of interferon genes (STING) can trigger a number of critical cellular responses including inflammation, noncanonical autophagy, lipid metabolism, senescence, and cell death. STING-mediated immunity through the production of type I interferons (IFNs) and nuclear factor kappa B (NF-κB)-driven proinflammatory cytokines is primarily driven via the effector protein TBK1. We have previously found that IκBα kinase epsilon (IKKε), a homolog of TBK1, can also facilitate STING-NF-κB responses. Therefore, a thorough understanding of how IKKε participates in STING signaling is essential. Here, we used a combination of genetic and biochemical approaches to provide mechanistic details into how IKKε confers non-IFN (e.g., NF-κB and MAPK) STING responses in macrophages, including in the absence of TBK1. We demonstrate a conserved mechanism of STING binding between TBK1 and IKKε. These findings strengthen our understanding of cGAS-STING signaling and the preservation of host immunity in cases of TBK1-deficiency.

Keywords: Cell biology; Immunology; Molecular biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
IKKε functions downstream of STING in human and murine myeloid cells (A) Primary BMDMs were left untreated or challenged with 20 μg/mL 2′3′-cGAM(PS)2 for 1, 2, or 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 5 independent experiments. (B) THP-1 monocytes were left untreated or challenged with 20 μg/mL 2′3′-cGAM(PS)2 for 1 or 2 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (C) Primary BMDMs isolated from Sting+/+ and Sting−/− mice were left uninfected or infected with HSV-1 with the indicated multiplicity of infections (MOI) for 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (D) STINGWT and STINGR284S THP-1 monocytes were left untreated or treated with doxycycline as indicated for 6 h. Cells were lysed and immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. See also Figure S1.
Figure 2
Figure 2
TBK1 and IKKε redundantly mediate MAP kinase activation downstream of STING in a similar fashion to NF-κB (A) Tbk1fl/fl × RosaCre (TBK1 KO) mice were treated with tamoxifen (+) or not (−) before primary BMDMs were left untreated or stimulated with 20 μg/mL 2′3-cGAM(PS)2 for 1.5 and 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (B) WT, TBK1KO, IKKεKO, and TBK1/IKKεKO iBMDMs were left untreated or stimulated with 20 μg/mL 2′3′-cGAM(PS)2 for 1.5 and 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (C) WT, TBK1/IKKεKO, and TBK1/IKKεKO expressing HA-IKKε iBMDMs were left untreated or stimulated with 20 μg/mL 2′3′-cGAM(PS)2 for 1.5 and 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (D) Primary BMDMs were pre-treated with either a DMSO vehicle or 20 μM 5z-7-oxozeaenol (5z-7; TAK1 inhibitor) for 30 min. BMDMs were then left unstimulated or stimulated with 20 μg/mL 2′3′-cGAM(PS)2 for 1 and 2 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (E) Primary BMDMs were pre-treated with either a DMSO vehicle or 50 nM MRT67307 (MRT: TBK1/IKKε kinase inhibitor) for 30 min. BMDMs were then left unstimulated or stimulated with 20 μg/mL 2′3′-cGAM(PS)2 for 1.5 and 3 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. See also Figure S2.
Figure 3
Figure 3
STING recruits IKKε following activation (A) Primary BMDMs from Sting+/+ and Sting−/− mice were left unstimulated or stimulated with 50 μg/mL DMXAA for 90 min. Following stimulation, cells were lysed to generate whole cell lysate (WCL) for immunoblot while the remaining lysate underwent IP with an anti-STING antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (B) THP-1 monocytic-like cells were left untreated or treated with 20 μg/mL 2′3′-cGAM(PS)2 for 1 and 2 h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-phospho-STING (S366) antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (C) Sting−/− iBMDMs reconstituted with mCitrine-fused murine STING (mCit-STING) were treated with 1 μg/mL doxycycline overnight. The cells were then left untreated or treated with 50 μg/mL DMXAA for 2 h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-GFP antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (D–E) Sting−/− iBMDMs reconstituted with mCit-STING were treated with 1 μg/mL doxycycline overnight. The cells were then left untreated or treated with 50 μg/mL DMXAA for 1 h. Cells were fixed and underwent immunofluorescent staining for (D) P-IKKε or (E) P-TBK1. Images were acquired on the LSM980 confocal microscope. Images are displayed as merged images for a single Z slice. P-IKKε or P-TBK1 is shown in magenta, mCit-STING is in green, while nuclear staining (DAPI) is shown in blue. White boxes indicate magnified regions of interest. Scale bar, 10 μm. Data shown are representative of 3 independent experiments. See also Figure S3.
Figure 4
Figure 4
Understanding the composition of STING complexes with TBK1 and IKKε (A) Sting−/− iBMDMs reconstituted with mCit-STING were left untreated or treated with 1 μg/mL doxycycline overnight before cells were left untreated or stimulated with 50 μg/mL DMXAA as indicated. Following stimulation, cells were lysed to generate whole cell lysate WCL for immunoblot while the remaining lysate underwent IP with an anti-GFP antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (B) Primary BMDMs were left untreated or treated with 50 μg/mL DMXAA for 90 min. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-TBK1 antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (C) IKKεKO iBMDMs and IKKεKO iBMDMs reconstituted with HA-IKKε were left untreated or treated with 50 μg/mL DMXAA for 1 and 2 h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-HA antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (D) WT and TBK1KO iBMDMs were left untreated or stimulated with 50 μg/mL DMXAA for 90 min. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-STING antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. See also Figure S4.
Figure 5
Figure 5
STING recruits IKKε via the TBK1-binding motif within its C-terminal tail (A) Sting−/− iBMDMs reconstituted with either wild-type (WT) mCit-STING or mCit-STING L373A were treated with 1 μg/mL doxycycline overnight. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-GFP antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (B) Following overnight doxycycline treatment (1 μg/mL) of mCit-STING WT or mCit-STING L373A iBMDMs were left untreated or stimulated with 50 μg/mL DMXAA for 1 h. Cells were fixed before images were acquired on the LSM980 confocal microscope. Images are displayed as a single Z slice. Scale bar, 10 μm. Data shown are representative of 3 independent experiments. (C) Sting−/− iBMDMs and those reconstituted with either HA-STING WT or HA-STING L373A were left untreated or stimulated with 50 μg/mL DMXAA for 1 and 2 h. Cells were lysed, and lysate was immunoblotted with indicated antibodies. Data shown are representative of 3 independent experiments. (D and E) Sting−/− iBMDMs and those reconstituted with either HA-STING WT or HA-STING L373A were left untreated or stimulated with 50 μg/mL DMXAA or 10 μM diABZi for 4 h before measuring IFNβ (D) and TNF (E) secretion in cell supernatants by ELISA. Data shown as mean ± SEM combined from 3 independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, compared to HA-STING WT iBMDMs as determined by unpaired Student’s t test. See also Figure S5.
Figure 6
Figure 6
TBK1 residues responsible for interaction with STING conserved in IKKε (A) Multiple sequence alignment of human and murine TBK1 and IKKε. Green shading indicates conserved residues. Red line highlights the STING binding interface within human TBK1 that was previously identified to mediate the interaction with STING. (B and C) Alignment of the cryo-EM structure of chicken STING C-terminal tail (shown in blue; line representation) in complex with: (B) human TBK1 (shown in green; cartoon representation); or (C) the Alpha-Fold predicted model of human IKKε (shown in yellow; cartoon representation). (D) IKKεKO iBMDMs and IKKεKO iBMDMs reconstituted with HA-IKKε WT, HA-IKKε Y568A or HA-IKKε Q572A were left untreated or treated with 50 μg/mL DMXAA for 1 h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-HA antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. See also Figure S6.
Figure 7
Figure 7
IKKε forms additional contact points with STING in comparison to TBK1 (A and B) Alignment of the cryo-EM structure of chicken STING C-terminal tail (line representation) in complex with: (A) human TBK1; or (B) the Alpha-Fold predicted model of human IKKε. The dotted line indicates a potential hydrophobic residue interaction. Amino acids are colored based on their hydrophobicity (red represents hydrophobic residue). (C) Sting−/− iBMDMs reconstituted with HA-STING WT or HA-STING I364A were left untreated or stimulated with 50 μg/mL DMXAA for 1 h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-HA antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (D and E) Sting−/− iBMDMs and those reconstituted with HA-STING WT or HA-STING I364A were left untreated or stimulated with 50 μg/mL DMXAA for 4 h before measuring IFNβ (D) and TNF (E) secretion in cell supernatants by ELISA. Data shown as mean ± SEM combined from 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, compared to HA-STING WT iBMDMs as determined by unpaired Student’s t test. (F) IKKεKO iBMDMs reconstituted with HA-IKKε WT or HA-IKKε V403A were left untreated or treated with 50 μg/mL DMXAA for 1h. Following stimulation, cells were lysed to generate WCL for immunoblot while the remaining lysate underwent IP with an anti-HA antibody. Samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (G) TBK1KO/IKKεKO iBMDMs reconstituted with HA-IKKε WT, HA-IKKε V403A or HA-IKKε Q572A were left untreated or treated with 50 μg/mL DMXAA for 1 and 2 h. WCL samples then underwent immunoblot with the indicated antibodies. Data are representative of 3 independent experiments. (H) TBK1KO/IKKεKO iBMDMs reconstituted with HA-IKKε WT, HA-IKKε V403A, or HA-IKKε Q572A were left untreated or treated with 50 μg/mL DMXAA for 4 h before measuring TNF secretion in cell supernatants by ELISA. Data shown as mean ± SEM combined from 3 independent experiments. ∗∗∗p < 0.001, compared to HA-IKKε WT iBMDMs as determined by unpaired Student’s t test. See also Figure S6.
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