THP1-Dual suspension cells (InvivoGen) were cultivated in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete RPMI). Adherent HEK293FT cells (Invitrogen) were cultivated in DMEM medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete DMEM).

The Alt-R CRISPR-Cas9 system from Integrated DNA Technologies was utilized to edit genes of interest, resulting in loss of protein expression from these genes. Different protocols were used for THP1-Dual cells and HEK293FT cells.

For THP1-Dual suspension cells, 0.4 µl of both gene-specific crRNA and tracrRNA (both 100 µM, see Table 1 ) were mixed, heat-denatured at 95°C for 5 min, and slowly cooled to room temperature (RT) to generate an RNA duplex serving as guideRNA. For double KOs, 0.2 µl of each crRNA was annealed with 0.4 µl tracrRNA. The RNA duplexes were then incubated for 20 min at RT with 1 volume of 36 µM Cas9 enzyme to facilitate complex formation. After washing THP1-Dual cells (5×10⁵ per electroporation reaction) once with PBS, they were resuspended in buffer R (Neon Transfection System kit, Thermo Fisher Scientific, 13.5 µl per electroporation reaction). 1.5 µl of the crRNA:tracrRNA: Cas9 complex and 3 µl of electroporation enhancer (10.8 µM) were added to the cell suspension and the complex was delivered into the cells using the Neon Transfection System device (Thermo Fisher Scientific, 1600 V, 3 pulses, 10 ms pulse width). The cells were transferred to prewarmed complete RPMI and were allowed to recover and expand for 3 days before single cell cloning was performed by limiting dilution seeding.

For HEK293FT cells, 0.3 µl of both TBK1-specific crRNA and tracrRNA (both 100 µM, see Table 1 ) were mixed with 29.4 µl nuclease-free duplex buffer and were then heated at 95°C for 5 min, followed by a slow cool-down to RT. After combining 9 µl of the formed RNA duplex with 9 µl of 2 µM Cas9 and 132 µl Opti-MEM, the mixture was incubated for 5 min at RT to allow complex formation. 7.2 µl Lipofectamine RNAiMAX (Thermo Fisher Scientific) diluted in Opti-MEM to a final volume of 150 µl were then combined with the RNA : Cas9 complex and incubated for 20 min at RT. In the meantime, HEK293FT cells (2.4×10⁵ per electroporation reaction) were washed once with PBS, detached from the cell culture flask, resuspended in 300 µl DMEM without antibiotics supplemented with 10% FCS, and were then combined with the transfection mixture in a 24-well plate. After 2 days of recovery, single cell cloning was performed.

After expansion, monoclonal cell lines were analyzed for loss of protein expression by SDS-PAGE and immunoblotting. Furthermore, genome editing was confirmed by Sanger sequencing of PCR-amplified genomic loci using TIDE (26). Genotypes are shown in Table 2 .

The studies involving primary human monocytes were approved by the local ethics committee (Ethikkommission der Medizinischen Fakultät Bonn) according to the ICH-GCP guidelines. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy, voluntary donors by Ficoll-Paque (Cytiva) density gradient centrifugation. Residual erythrocytes were removed by incubation in red blood cell lysis buffer (Roche). Isolation of primary monocytes from PBMCs was achieved by positive magnetic selection using CD14 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions.

Primary monocytes freshly isolated from human blood were differentiated to macrophages by cultivation in complete RPMI supplemented with 40 ng/ml M-CSF (Peprotech) at a cell density of 1.1×10⁶/ml in cell culture dishes. After three days, macrophages were detached by incubation for 10 min with ice-cold 5 mM EDTA/PBS, resuspended, and then used for CRISPR-Cas9 genome editing.

To reduce the expression of TBK1 in primary monocyte-derived macrophages, polyclonal gene KOs were created. 0.6 µl of gene-specific crRNA and 0.6 µl tracrRNA (Integrated DNA Technologies, both 100 µM, see Table 1 ) were combined and were hybridized by incubation at 95°C for 5 min, followed by a slow cool down. Then, the RNA duplex was incubated with 1 volume of Cas9 (36 µM) for 20 min at RT. A non-targeting crRNA (Integrated DNA Technologies) was used as a negative control. After washing primary monocyte-derived macrophages (3.6×10⁶ per electroporation reaction) with PBS once, they were resuspended in buffer R (Neon electroporation kit, 21.6 µl per electroporation reaction) and combined with 2.4 µl of RNA: Cas9 complex and 4.8 µl electroporation enhancer (10.8 µM). Complex delivery into macrophages was achieved by electroporation using the Neon Transfection System device (Thermo Fisher Scientific, 1400 V, 2 pulses, 20 ms pulse width). Then, the cells were transferred to prewarmed RPMI supplemented with 10% FCS and 40 ng/ml M-CSF without antibiotics. After three days, the medium was exchanged to complete RPMI containing 40 ng/ml M-CSF and the cells were incubated for three further days before lysates were prepared for SDS-PAGE and immunoblot analysis, or qRT-PCR.

High molecular weight polyinosinic-polycytidylic acid (polyI:C) was obtained from InvivoGen. Hybridization of G3-YSD ( Table 3 ) was achieved by heating the combined single-stranded DNA oligonucleotides in NEB2 buffer (New England Biolabs) at 95°C for 5 min and subsequent cooling to RT at a rate of 1°C/min. Plasmid DNA (pDNA) was isolated from pBluescript-transformed E. coli K12 using a PureLink HiPure Plasmid Filter Midiprep Kit (Life Technologies) according to the manufacturer’s instructions.

THP1-Dual cells (6×10⁴/well of a 96-well plate, 3.6×10⁵/well of a 24-well plate, or 7.2×10⁵/well of a 12-well plate) were seeded in 150 µl, 450 µl, or 900 µl complete RPMI for type I IFN reporter assay, qRT-PCR, or immunoblotting readout, respectively, and were stimulated with RIG-I/MDA5 (polyI:C) or cGAS (pDNA, G3-YSD) agonists. For type I IFN reporter assays, cells were stimulated in duplicates. Stimuli were complexed with the vehicle Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and were then added to the cells, resulting in a final volume of 200 µl (96-well plate), 1200 µl (24-well plate), or 2400 µl (12-well plate) with stimuli concentrations ranging from 10 ng/ml to 500 ng/ml, as indicated in the figure legends. IFNα2a (Miltenyi Biotec) was used as a control for equal IFN-stimulated response element (ISRE) reporter responsiveness of the different cell lines.

For lentiviral vector production and reconstitution of TBK1-deficient THP1-Dual cells, human TBK1 variants (WT and mutants) with C-terminal Flag tag were cloned into a pLVX-puro-EF1α vector via Gibson assembly using the primers listed in Table 4 . Similarly, Flag-tagged human IKKϵ was cloned into the same vector for IKKϵ overexpression (see Table 4 ). For identification of IKKϵ ubiquitination by immunoprecipitation, GFP-tagged human IKKϵ was cloned into a pLVX-puro-EF1α vector via Gibson assembly (see Table 4 ).

Lentiviral vectors were produced by transfection of HEK293FT cells with lentiviral transfer plasmid (1.6 µg of pLVX-puro-EF1α-TBK1-Flag (WT or mutant), pLVX-puro-EF1α-IKKϵ-Flag, pLVX-puro-EF1α-GFP, or pLVX-puro-EF1α-IKKϵ-GFP) and packaging plasmids (0.4 µg, 0.6 µg, and 1.1 µg of pRSV-rev, pMD2.G, and pMDL.g, respectively) using calcium phosphate transfection. Virus-containing cell supernatants were harvested 72 h post transfection. After resuspension of THP1-Dual cells in virus supernatant supplemented with polybrene (8 µg/ml), spin infection was performed for 1 h at 600×g and 32°C. 24 h post transduction, the medium was replaced with puromycin-containing medium (1 µg/ml) and selection of transduced cells was performed for 3 days.

L. monocytogenes (wildtype strain EGD) was cultivated in BHI medium until reaching the log phase and was then stored at -80°C until infection was performed. THP1-Dual cells (3.6×10⁵/well of a 24-well plate) were washed with PBS twice and seeded in RPMI-1640 supplemented with 10% FCS but devoid of antibiotics. Infection with L. monocytogenes was performed in a final volume of 600 µl at a multiplicity of infection (MOI) of 1. After 2 h, cells were washed with antibiotic-free RPMI-1640 once and seeded in 600 µl RPMI-1640 supplemented with 10% FCS and 50 µg/ml gentamycin. After 24 h, the supernatant was harvested to determine type I IFN reporter activation and the cells were lysed for RNA isolation followed by qRT-PCR analysis.

Lucia luciferase secretion of THP1-Dual cells, controlled by an ISRE, was utilized as a surrogate parameter for IRF activation and type I IFN production. To determine type I IFN reporter activity, cell-free supernatant was harvested 16 or 24 h after stimulation, as indicated in the figure legend, and 30 µl supernatant were mixed with 30 µl coelenterazine solution (1 µg/ml in H₂O) in a white 96-well F-bottom plate. Luciferase activity was determined immediately after coelenterazine addition with an EnVision 2104 Multilabel Reader device.

Cells were washed once with PBS, resuspended in RIPA lysis buffer supplemented with cOmplete Protease Inhibitor (Roche), and incubated on ice for 30 min. For the analysis of IRF3 phosphorylation, PhosSTOP phosphatase inhibitor cocktail (Roche) was additionally included in the lysis buffer. After clearing the lysates by centrifugation at 21,000×g for 10 min at 4°C, protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 10–30 µg protein were loaded on an 8% SDS-PAGE gel and gel electrophoresis was performed for 90 min at 110 V. Proteins were transferred to a nitrocellulose membrane by blotting at 450 mA for 90 min. Total protein staining was performed by incubating the membrane for 5 min in Ponceau S solution (0.1% (w/v) Ponceau S in 5% acetic acid). After blocking the membranes with Intercept blocking buffer (LI-COR Biosciences), TBK1 and IKKϵ were detected with primary antibodies D1B4 and D20G4 (Cell Signaling Technology, 1:1000), respectively. Primary antibodies #2141, #8605 (both Cell Signaling Technology, 1:1000), and 15042-1-AP (Proteintech, 1:1000), were used to stain TANK, SINTBAD, and NAP1, respectively. Phosphorylation of IRF3 at S396 was detected with primary antibody 4D4G, while total IRF3 protein was stained with D6I4C (both Cell Signaling Technology, 1:1000). The housekeeping protein β-actin was detected with primary antibody 926-42212 (LI-COR Biosciences, 1:3000). Goat anti-rabbit IRDye800CW, goat anti-mouse IRDye800CW, and goat anti-mouse IRDye680 (LI-COR Biosciences, 1:10,000) were used as secondary antibodies. Blots were imaged using an Odyssey FC Dual imaging platform (LI-COR Biosciences). Quantification of signal intensities was performed using Image Studio Lite.

THP1-Dual cells or primary monocyte-derived macrophages were lysed in 350 µl RLT buffer (Qiagen) and were frozen at -80°C for at least 5 min. After thawing at RT, 1 volume of 70% ethanol was added and the sample was transferred to a Zymo III column (Zymo Research). After washing the column sequentially with 1 volume RW1 buffer (Qiagen) and 1 volume RNA wash buffer (Zymo Research), the column was dried by centrifugation for 2 min at maximum speed. The RNA was then eluted with RNase-free distilled H₂O. DNase I digest was performed at 37°C for 30 min to destroy residual genomic DNA before the enzyme was inactivated at 70°C for 10 min in the presence of 2.5 mM EDTA. Reverse transcription was performed with RevertAid Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions using random hexamer primers (Integrated DNA Technologies). qRT-PCR reactions were performed in a QuantStudio 5 Real-Time PCR cycler (Thermo Fisher Scientific) using 5× EvaGreen QPCR Mix II (ROX) (Bio-Budget). Gene-specific primers used for quantification of mRNA levels ( Table 5 ) were tested for efficiency by cDNA dilution.

THP1-Dual cells (3.6×10⁵ per well of a 24-well plate) were seeded in 450 µl complete RPMI. Then, 150 µl of a cycloheximide dilution in complete RPMI were added, resulting in a final concentration of 10 µg/ml. After 4, 8, 12, 16, and 20 h, the cells were lysed and protein expression of IKKϵ was determined by SDS-PAGE and immunoblotting.

For the analysis of TBK1-dependent IKKϵ ubiquitination, stable IKKϵ-GFP-expressing cells were generated by transducing IKKϵ-deficient THP1-Dual cells with IKKϵ-GFP-encoding lentivirus. Then, TBK1 protein expression was depleted by CRISPR-Cas9 genome editing. Ubiquitination of IKKϵ in TBK1 polyclonal KO cells was compared to IKKϵ-GFP-expressing WT cells by GFP-based immunoprecipitation (IP): Cells (1.5×10⁷ per condition, seeded at 10⁶ cells/ml in complete RPMI) were treated with the proteasome inhibitor MG-132 (10 µM, Cell Signaling Technologies) for 6 h to induce the accumulation of ubiquitinated proteins. Then, the cells were washed with PBS twice before they were lysed in TAP lysis buffer (100 mM NaCl, 50 mM Tris/HCl, 1.5 mM MgCl₂, 5% (v/v) glycerol, 0.5% (v/v) IGEPAL CA-630, pH 7.5) supplemented with cOmplete Mini EDTA-free protease inhibitor (Roche), PhosSTOP phosphatase inhibitor cocktail (Roche), and the deubiquitinase inhibitor N-ethylmaleimide (10 mM) for 30 min on ice. After sonicating the samples to mechanically disrupt the nuclei (VialTweeter Sonication device, Hielscher Ultrasonics), lysates were cleared by centrifugation at 21,000×g for 20 min at 4°C. Then, the protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 40 µl equilibrated GFP-trap magnetic agarose beads (Chromotek) were incubated with 1500 µg protein in 750 µl TAP lysis buffer supplemented with protease inhibitor, phosphatase inhibitor, and N-ethylmaleimide (10 mM) overnight on a tube rotator at 4°C. The next morning, beads were washed three times with TAP lysis buffer, followed by two washes with TAP wash buffer (100 mM NaCl, 50 mM Tris/HCl, 1.5 mM MgCl₂, 5% (v/v) glycerol, pH 7.5). Then, the beads were incubated in 60 µl 2x Laemmli buffer for 10 min at 95°C, shaking at 350 rpm, to elute and denature the bound proteins. Input controls (2% of lysate used for IP) and eluates were analyzed by SDS-PAGE (30 µl sample per gel) and immunoblotting.

To detect non-covalent interactions of TBK1 with TANK, NAP1, SINTBAD, and IKKϵ, tag-based co-IP was performed. Flag-tagged TBK1 WT, TBK1 L693A, TBK1 K694E, or TBK1 L704A was stably expressed in TBK1-deficient THP1-Dual cells by lentiviral transduction followed by puromycin selection. Cells (1.5×10⁷) were washed with PBS twice and lysed in 700 µl TAP lysis buffer supplemented with protease and phosphatase inhibitor by incubation on ice for 30 min with regular vortexing. The lysates were subjected to sonication and cleared by centrifugation, as described above, before protein concentrations were determined by BCA assay. After equilibrating anti-Flag M2 magnetic agarose beads (50 µl per condition, Sigma-Aldrich) in TAP lysis buffer, 2000 µg protein diluted in 1000 µl TAP lysis buffer (supplemented with protease and phosphatase inhibitor) were added to the beads and incubated for 4 h at 4°C, shaking at 900 rpm. The beads were washed three times with TAP lysis buffer and twice with TAP wash buffer, before elution of the precipitated proteins was achieved by incubation with 3× Flag peptide (Bachem, 150 ng/µl in TAP wash buffer) for 30 min at 4°C, shaking at 700 rpm. After adding Laemmli buffer to a final volume of 80 µl, the input controls (2% of lysate used for IP) and eluates were heat-denatured at 95°C for 5 min, before they were analyzed by SDS-PAGE (40 µl sample per gel) and immunoblotting.

GraphPad Prism 7 was used to perform statistical analysis. Individual values are visualized as dots, whereas columns and error bars represent the mean and standard deviation (SD) of three or more independent experiments or donors, respectively. The statistical significance of differences between groups was determined using the appropriate statistical test, as described in the figure legends. * indicates a p-value ≤ 0.05, ** a p-value ≤ 0.01, *** a p-value ≤ 0.001, and **** a p-value ≤ 0.0001.

Although TBK1 is well-known for its essential function in innate immune responses in mice, loss of human TBK1 surprisingly is not associated with an increased susceptibility to severe infections (24). Based on the observation from this recent study, we aimed at further investigating the consequences of TBK1 deletion for antiviral defense of human monocytic cells, essential players in innate immune recognition and important inducers of type I IFN upon viral infection. We decided to study the type I IFN responses initiated by key sensors of viral nucleic acids: the DNA sensor cGAS and the RLRs RIG-I and MDA5. Although TBK1 is described as important kinase for IRF activation downstream of these receptors, its depletion in THP1 monocytes by CRISPR-Cas9 genome editing ( Supplementary Figure 1A ) did not result in a prominent reduction of type I IFN reporter activity after RIG-I and MDA5 activation with polyI:C and after cGAS activation with the short Y-form DNA G3-YSD (27) or plasmid DNA (pDNA) ( Figure 1A ). Since functional redundancies between TBK1 and IKKϵ have been suggested, we also analyzed IKKϵ-depleted cells and cells deficient for both kinases ( Supplementary Figure 1A ). While knockout (KO) of IKKϵ did not result in a pronounced reduction of type I IFN induction after activation of RLRs and cGAS, depletion of both TBK1 and IKKϵ (TBK1/IKKϵ KO) abrogated the type I IFN response induced by these receptors ( Figure 1A ), suggesting that IKKϵ can substitute the function of TBK1 in these pathways, and vice versa. In contrast, TBK1/IKKϵ KO did not affect IFNAR signaling induced by IFNα, as expected ( Figure 1A ). Notably, overexpression of IKKϵ in TBK1/IKKϵ KO cells completely rescued the cGAS-induced type I IFN response, demonstrating that IKKϵ is capable to induce type I IFN independently of TBK1 ( Supplementary Figure 1B, C ).

To understand why patients with TBK1 kinase mutation but not TBK1 deletion suffer from hyper-susceptibility to viral infections, we aimed at comparing the type I IFN response of TBK1-deficient with that of TBK1-mutated monocytic cells. Therefore, we transduced TBK1-deficient cells with lentiviral particles encoding the kinase-dead TBK1 mutants K38A or S172A, which are unable to phosphorylate target proteins, or TBK1 wildtype (WT) as a control, and assessed the capability of these cell lines to induce type I IFN in response to RLR or cGAS ligands. Strikingly, while expression of TBK1 WT did not affect the induction of type I IFN after stimulation with polyI:C, pDNA, or G3-YSD, TBK1 K38A or TBK1 S172A expression resulted in significantly impaired type I IFN responses ( Figure 1B, C ). In contrast, IFNAR signaling triggered by IFNα treatment was unaffected by the expression of these mutants ( Figure 1B, C ). Of note, TBK1 K38A or TBK1 S172A overexpression in WT cells with endogenous TBK1 was not associated with reduced type I IFN induction after RLR-MAVS and cGAS-STING activation ( Supplementary Figure 2 ), excluding a general dominant-negative impact of these kinase-deficient mutants on these pathways.

Downstream of RLRs or cGAS, type I IFN is induced through activation of the transcription factors IRF3 and IRF7 (4). To understand whether TBK1 loss and TBK1 mutation also differentially affect transcription factor activation, we next analyzed IRF3 phosphorylation at S396 as a measure of its activation. Both cGAS activation by G3-YSD and RLR activation by polyI:C resulted in substantial induction of IRF3 phosphorylation ( Figure 2A ), as expected. As observed for type I IFN induction, expression of TBK1 K38A or TBK1 S172A significantly reduced IRF3 phosphorylation induced by G3-YSD or polyI:C compared to TBK1-deficient cells ( Figures 2A–C ). However, in contrast to type I IFN induction, IRF3 phosphorylation was more pronounced in cells expressing TBK1 WT than in TBK1-deficient cells ( Figures 2A–C ), indicating that IRF3 phosphorylation is more dependent on TBK1 than type I IFN expression. This suggests that type I IFN induction downstream of RLR or cGAS activation is not exclusively driven by IRF3 in THP1 monocytes but that other transcription factors, e.g. IRF7, play a role as well.

We additionally measured the cGAS- or RLR-triggered induction of IFNB1 mRNA in THP1 monocytes. Expression of TBK1 K38A or TBK1 S172A resulted in a strong reduction of induced IFNB1 compared to cells devoid of TBK1 ( Figure 2D ), and mRNA expression from the IFN-stimulated gene IFIT1 was impaired by TBK1 mutant expression as well ( Figure 2E ). In contrast, expression from both genes was unaffected by TBK1 WT expression in TBK1-depleted cells ( Figures 2D, E ), corroborating the findings obtained from previous type I IFN reporter assays.

In summary, the data show that kinase activity-abolishing mutations but not depletion of TBK1 in human monocytic cells result in impaired RLR- and cGAS-induced type I IFN responses. The inhibited type I IFN production in monocytes correlates with the insufficient virus control observed in patients with mutation-induced loss of TBK1 kinase activity but not in individuals with complete absence of TBK1 expression (24, 25), suggesting that dysfunction in these PRR pathways contributes to the susceptibility of these patients to severe viral infection.

The function of TBK1 in activating IRF transcription factors downstream of RLRs and cGAS depends on its kinase activity. However, why expressed but inactive TBK1 inhibits type I IFN induction more than TBK1 loss has so far remained mechanistically unclear. The finding that TBK1 and IKKϵ have redundant functions in RLR and cGAS signaling in human monocytic cells ( Figure 1A , Supplementary Figures 1B, C ) prompted us to hypothesize that IKKϵ might play a role for the observed difference. When analyzing TBK1- and IKKϵ-deficient cells in the course of KO validation, we strikingly realized that TBK1-deficient cells exhibited enhanced protein expression levels of IKKϵ ( Supplementary Figure 1A ). To further examine this observation, we quantified IKKϵ expression of two TBK1-deficient monoclonal cell lines, WT cells, and control cells, which have gone through the CRISPR-Cas9 genome editing procedure with a non-targeting control crRNA. Lack of TBK1 expression was associated with a more than 3-fold induction of IKKϵ protein expression compared to control cells ( Figures 3A, B ), suggesting that a loss of TBK1 results in the upregulation of IKKϵ. While IKKϵ protein expression was strongly affected by the presence of TBK1, IKBKE mRNA levels were found unchanged between WT and TBK1-deficient cells ( Figure 3C ), pointing towards a post-transcriptional control of IKKϵ expression by TBK1.

In addition to the THP1 monocyte model, we investigated the influence of TBK1 depletion on IKKϵ protein expression in human primary monocyte-derived macrophages. Although TBK1 protein expression could be reduced by only approximately 80%, polyclonal TBK1 KO macrophages generated by CRISPR-Cas9 genome editing with either of three different TBK1-specific crRNAs exhibited a significant induction of IKKϵ protein expression compared to macrophages electroporated with a non-targeting control crRNA ( Figures 3D–F ). As in THP1 monocytes, IKBKE mRNA levels were unaffected by TBK1 KO in primary human monocyte-derived macrophages ( Figure 3G ), which indicates that the post-transcriptional regulation of IKKϵ is conserved among monocytic cells. In contrast to monocytes/macrophages, TBK1 depletion in HEK293FT cells did not result in increased protein expression of IKKϵ ( Supplementary Figure 3 ), suggesting that the observed kinase cross-regulation might be a specific feature of myeloid cells, or of immune cells in general.

Overall, the data demonstrate that TBK1 diminishes the protein expression of its related kinase IKKϵ in both THP1 monocytes and primary human monocyte-derived macrophages. The resulting enhanced amounts of IKKϵ protein upon loss of TBK1 can substitute the function of TBK1 in IRF activation due to the functional redundancy of the kinases, which explains why RLR- and cGAS-induced type I IFN production is not substantially impaired in TBK1-deficient cells.

We next aimed at investigating whether the discovered TBK1-mediated control of IKKϵ protein expression is involved in the differential susceptibility to viral infection between patients with TBK1 deficiency and TBK1 mutation. We could show that IKKϵ plays an essential role in type I IFN induction downstream of RLRs and cGAS in TBK1-deficient cells ( Figure 1A ). Moreover, we observed that the expression of TBK1 mutants lacking kinase activity resulted in diminished type I IFN responses compared to TBK1-deficient cells ( Figures 1B, C ). We therefore hypothesized that the expression levels of IKKϵ might differ between these conditions. Indeed, not only re-expression of TBK1 WT but also expression of the TBK1 kinase-dead mutants K38A or S172A in TBK1-deficient THP1 monocytes resulted in significantly reduced levels of IKKϵ protein ( Figure 4 ). These data suggest that TBK1 diminishes the protein expression of its related kinase IKKϵ independent of its kinase activity. Since kinase-dead TBK1 mutants are still capable to downregulate IKKϵ expression but cannot activate IRF3 and IRF7, cells expressing these mutants exhibit an overall reduced activity of IKK-related kinases compared to cells devoid of TBK1, in which IKKϵ protein expression is strongly enhanced. The reduced level of IRF-activating kinases is associated with a weaker type I IFN response downstream of RLR and cGAS activation, which likely explains the enhanced susceptibility of patients with TBK1 mutation to severe viral infection.

We then further examined the mechanism of IKKϵ control by TBK1. Since the unaffected IKBKE mRNA expression suggested a post-transcriptional regulation, we investigated whether the protein stability of IKKϵ was influenced by TBK1. After blocking the synthesis of new proteins with the translation elongation inhibitor cycloheximide, protein degradation of IKKϵ in WT and TBK1-deficient THP1 monocytes was assessed by immunoblotting. Cycloheximide treatment of WT cells resulted in a decrease in IKKϵ protein expression over time, as expected ( Figures 5A, B ). In contrast, IKKϵ protein levels were not reduced throughout 20 h in TBK1-deficient cells ( Figures 5A, B ), indicating a much slower degradation rate of IKKϵ in absence of TBK1. Since cycloheximide treatment caused massive cell death in both WT and TBK1-deficient cells after more than 20 h incubation, the half-life of IKKϵ in presence and absence of TBK1 could not be determined with this method. However, statistical comparison of the two degradation curves revealed a highly significant difference in IKKϵ protein stability between TBK1-competent and -deficient cells ( Figure 5B ).

Selective protein degradation is commonly mediated by the ubiquitin-proteasome system. In a three-step enzymatic process that involves E3 ubiquitin ligases, ubiquitin moieties are transferred to lysine residues of target proteins to mark them for proteasomal degradation (28). To determine whether TBK1 reduces IKKϵ protein levels through proteasomal degradation, we examined IKKϵ protein expression after treatment of WT and TBK1-deficient cells with the proteasome inhibitor MG-132. While proteasome inhibition for 24 h resulted in massive cell death, precluding any analysis, a shorter treatment time of 6 h had no effect on IKKϵ protein levels (data not shown), which may result from the very long half-life of IKKϵ observed before ( Figures 5A, B ). Therefore, no reliable conclusion about the role of proteasomal degradation in TBK1-mediated control of IKKϵ expression could be drawn from these experiments.

Recently, TBK1 has been described to harbor a E3 ubiquitin ligase activity itself, requiring residues C426 and C605 (29). To investigate whether this intrinsic ubiquitin ligase activity of TBK1 is responsible for inducing IKKϵ protein degradation, we analyzed the expression of IKKϵ in TBK1-deficient cells expressing the ubiquitin ligase-deficient TBK1 mutant C426A/C605A. Similar to the kinase-dead mutants K38A and S172A, TBK1 C426A/C605A expression reduced the protein expression of IKKϵ compared to untransduced TBK1-deficient cells ( Figures 5C–E ). Thus, the putative C426/C605-dependent intrinsic E3 ubiquitin ligase activity of TBK1 is not required for the induction of IKKϵ degradation. Instead, another ubiquitin-conjugating enzyme might be recruited to TBK1 to target IKKϵ.

Among the different types of ubiquitin chains, K48-linked polyubiquitin is the most abundant proteasome-targeting signal (30). We therefore raised the question whether the presence of TBK1 enhances K48-linked ubiquitination of IKKϵ. To address this hypothesis, GFP-tagged IKKϵ was stably expressed in IKKϵ-deficient THP1 monocytes by lentiviral transduction to allow specific GFP-based immunoprecipitation. Polyclonal TBK1 KO in these cells resulted in a strong increase of IKKϵ-GFP expression ( Figure 5F ), proving that the regulation of IKKϵ degradation by TBK1 is maintained despite its ectopic expression as a GFP fusion protein. An efficient pulldown of IKKϵ-GFP was observed with GFP-binding magnetic beads in both TBK1-expressing and TBK1-depleted cells ( Figure 5F ). Furthermore, TBK1 co-immunoprecipitated with IKKϵ-GFP, demonstrating the interaction of the two kinases in non-activated myeloid cells ( Figure 5F ). Although pronounced K48-linked polyubiquitination of IKKϵ could be detected, it was not reduced by depletion of TBK1 ( Figure 5F ). Instead, the polyubiquitin signal was even enhanced for TBK1-deficient cells, which is most likely related to the increased amount of IKKϵ protein expressed in TBK1-deficient cells and immunoprecipitated from these cells ( Figure 5F ). Thus, the data suggest that TBK1 regulates IKKϵ degradation through a K48-polyubiquitin-independent mechanism.

Since the enzymatic activities of TBK1 were found to be dispensable for its suppressive effect on IKKϵ protein levels, we hypothesized that instead the interaction of TBK1 with other proteins is essential. To investigate whether TBK1 induces the degradation of IKKϵ via interaction with one of its scaffold proteins, we expressed the TBK1 mutant L704A, described to be unable to interact with TANK, NAP1, and SINTBAD (16), in TBK1-deficient THP1 monocytes ( Figure 6A ). Strikingly, although the expression level of TBK1 L704A was comparable to TBK1 WT expression in WT cells, TBK1 L704A in contrast to TBK1 WT could not induce protein degradation of IKKϵ ( Figures 6A–C ). Thus, the interaction of TBK1 with its scaffold proteins seems to be crucial for the regulation of IKKϵ protein expression. In contrast to the kinase-dead mutants K38A and S172A, expression of TBK1 L704A in TBK1-deficient cells was not associated with a reduced type I IFN induction in response to RLR and cGAS ligands ( Figure 6D ). Since the interaction of TBK1 with scaffold proteins has been shown to be required for TBK1-mediated type I IFN induction (16), these data further demonstrate that the type I IFN response induced by RLRs and cGAS is determined by the level of IKKϵ expression in cells lacking active TBK1.

To further elucidate the determinants of TBK1-mediated IKKϵ degradation, we studied TBK1 mutants that have lost the ability to interact with individual scaffold proteins: While L693A mutation has been shown to selectively abrogate TANK binding, TBK1 K694E has been described to be unable to interact with TANK and SINTBAD and to only retain binding to NAP1 in HEK293 cells (16). To confirm that these findings also apply to TBK1 in monocytic cells, we expressed TBK1 WT, TBK1 L693A, TBK1 K694E, or TBK1 L704A in TBK1-deficient THP1 cells and analyzed the interaction of these TBK1 variants with the scaffold proteins TANK, NAP1, and SINTBAD by Flag-based co-immunoprecipitation. Unexpectedly, unlike described for HEK293 cells (16), L704A mutation decreased but not abolished NAP1 interaction in THP1 monocytes ( Figure 6E ). However, this mutant was unable to interact with TANK and SINTBAD ( Figure 6E ). Similar to L704A, K694E mutation abolished TBK1 interaction with TANK and SINTBAD, while NAP1 binding of this mutant was unaffected ( Figure 6E ). Of note, endogenous expression of SINTBAD was strongly reduced by expression of TBK1 K694E or TBK1 L704A ( Figure 6E ), suggesting that SINTBAD stability might be regulated by TBK1. Importantly, TBK1 L693A was unable to interact with TANK in human monocytic cells, while SINTBAD and NAP1 binding were maintained ( Figure 6E ), confirming the selective effect of this mutation that has been described in HEK293 cells (16).

Since we hypothesized that scaffold protein interaction of TBK1 is essential for complex formation with IKKϵ, we then compared the TBK1:IKKϵ interaction in cells expressing TBK1 WT, TBK1 L693A, TBK1 K694E, or TBK1 L704A. Strikingly, co-immunoprecipitation revealed that all TBK1 variants are capable of interacting with IKKϵ ( Figure 6E ). Thus, the scaffold proteins do not determine the association of TBK1 with IKKϵ but may rather link TBK1 to a mechanism that induces the degradation of IKKϵ.

To evaluate the involvement of the individual scaffold proteins in regulating IKKϵ protein stability, we next analyzed the effects of the TBK1 mutants L693A and K694E on IKKϵ expression. Although expressed to a similar level as TBK1 in WT cells, the TBK1 mutants L693A and K694E in contrast to TBK1 WT could not reduce IKKϵ protein levels in TBK1-deficient cells ( Figures 6F–H ). Since TBK1 L693A mutation selectively abolishes TANK binding but retains binding to NAP1 and SINTBAD, these data demonstrate that among the three scaffold proteins, TANK is required for TBK1-controlled IKKϵ degradation.

Type I IFN production by innate immune cells is of imminent importance for the defense against invading pathogens. In consequence, viruses have evolved to antagonize type I IFN induction, such as by TBK1 degradation, which is described for SARS-CoV-2, HSV-1 and HIV-1 (18–21). Also bacteria have been found to subvert the production of type I IFNs via degradation of TBK1 (22). Based on our finding that RLR- and cGAS-induced type I IFN production in TBK1-deficient cells is strongly determined by the level of IKKϵ expression, we hypothesized that in case of TBK1 degradation during an infection, IKKϵ upregulation is highly relevant for efficient type I IFN responses and pathogen control. To address this hypothesis, we aimed at comparing infection-induced type I IFN production in 1) a condition of TBK1 deficiency in which IKKϵ is upregulated (TBK1-depleted cells) with 2) a condition of TBK1 deficiency in which IKKϵ is not upregulated (TBK1-depleted cells expressing inactive TBK1 K38A or S172A). We used Listeria monocytogenes (L. monocytogenes) as a model pathogen, as it is known to induce a type I IFN response via RLRs and cGAS/STING (31, 32). Infection of THP1 monocytes resulted in prominent type I IFN induction, as expected, which was found completely dependent on TBK1 and IKKϵ ( Figure 7A ). Strikingly, type I IFN induction after infection was strongly diminished in cells expressing the kinase-dead TBK1 mutants K38A or S172A, in which IKKϵ upregulation does not take place ( Figure 7B ). Moreover, mRNA levels of IFNB1 and of the IFN-stimulated gene IFIT1 were drastically reduced in these cells ( Figures 7C, D ). Overall, these data demonstrate that in conditions of TBK1 deficiency, IKKϵ upregulation is crucial to ensure efficient type I IFN production during infection.