The classical I-κB kinase complex consisting of the two catalytical subunits IKKα and β and the regulatory subunit IKKγ (NEMO) has been suggested to be an essential player in inflammatory NF-κB activation [
30] and also contributes to inflammatory nociception [
10,
31]. In a recent study, we found that the IKK-related IKKε is also involved in processing of inflammatory pain by regulating NF-κB activity and the expression of NF-κB dependent genes [
13]. TBK1 constitutes another non-canonical IKK which might form a complex with IKKε and is involved in regulation of antiviral responses by modulating IFN Type I and also in inflammatory processes by regulating NF-κB signaling (reviewed in [
7,
32,
33]). The aim of this study was to clarify whether TBK1 is also involved in inflammatory nociceptive processing. We observed TBK1 expression in “pain-relevant” cells in the spinal cord and the DRGs. After peripheral inflammatory nociceptive stimulation, TBK1 was upregulated in the spinal cord but not in the DRGs indicating its main role in the central nervous system. Effects in the formalin model could be detected at earlier time points compared to the zymosan A model which might reflect the fact that the formalin model constitutes a more acute inflammatory nociceptive model leading to early effects while zymosan A-induced paw inflammation provides more information about persistent hyperalgesia and later regulation [
23]. TBK1 knock-out mice were used to assess inflammatory hyperalgesia in the two different mouse models after confirming that similar kinases such as IKKα, β, and ε do not compensate for the TBK1 deletion. Due to embryonic lethality of complete TBK1 knock-out mice as a result of liver degeneration and apoptosis [
16,
34], the TBK1
−/− mice had to be bred under a TNFα-receptor knock-out and were compared to TNF-α receptor knock-out mice (TNFR
−/−) as controls. A knock-out of TNFR in mice has already been repeatedly associated with reduced nociceptive responses in models of inflammatory, cancer, and neuropathic pain [
35‐
37] and might thus cover antinociceptive effects mediated by TBK1 knock-down. Accordingly, we showed that the TNFR knock-out already ameliorates nociception in the formalin test and in the zymosan A-induced paw inflammation. Nevertheless, in TBK1/TNFR double-knock-out mice, this effect was significantly enhanced indicating that TBK1 contributes to inflammatory hyperalgesia. These results are in accordance with studies showing anti-inflammatory effects of TBK1 inhibition in fibroblast-like synoviocytes by reducing the expression of the proinflammatory protein IP-10 [
17]. Furthermore, TBK1 is involved in the inflammatory response in obesity and hypertension and contributes to phosphorylation of the insulin receptor thus impairing its function and supporting insulin resistance [
18]. Treatment of obese mice with the TBK1/IKKε inhibitor amlexanox led to weight loss and improved insulin sensitivity by elevation of energy expenditure [
38,
39], which further confirms that TBK1 contributes to proinflammatory reactions. Our concern was to investigate whether TBK1 effects in nociception are mediated via NF-κB signaling possibly in concert with IKKε or via other pathways. In a recent study, we used BX795 as a pharmacological inhibitor of IKKε and TBK1 and showed that the combined inhibition of both kinases led to a stronger reduction of the noxious response in comparison to single IKKε knock-out [
13]. Together, with the results of this study, it seems that inhibition of both kinases by BX795 leads to an addition of the effects of IKKε- and TBK1-mediated analgesia which might be due to synergistic modulation of the same target genes or to regulation of different targets, respectively. For IKKε, we showed that a complete inhibition of NF-κB p65 phosphorylation at Ser 536 was responsible for inhibition of NF-κB dependent target genes, such as COX-2, iNOS, and MMP-9, while NF-κB independent targets were not affected [
13]. The results of this study revealed that a knock-down of TBK1 also inhibits NF-κB activation by decreasing phosphorylation of p65 Ser536 but to a lesser extent than that shown for IKKε inhibition. From these data and published studies which hint towards divergent functional roles for TBK1 and IKKε [
5,
6], we postulated that TBK1 might affect other target genes in addition. Indeed, we found that the NF-κB-independent marker of neural activity, c-fos, was also differentially regulated in TBK1 knock-out mice in comparison to TNFR
−/− and wild type mice in association with a decreased activation of the MAP kinases ERK1/2 and SAPK/JNK. These MAPKs have been frequently associated with nociceptive responses [
27‐
29] and inhibition of their activation might contribute to antinociceptive effects in TBK1 knock-out mice. A relationship between TBK1 and MAPKs has already been found in a study with TBK1
−/− mouse embryonic fibroblasts (MEFs) which showed reduced TNFα-induced MAPK activation in comparison to TBK1
+/+ cells [
40]. In contrast, TBK1 was not involved in MAPK activation stimulated by double stranded DNA [
41] or TLR-3 activation [
42]. A recent study has further shown that NF-κB activation is capable of participation in c-fos induction, but only in concert with ERK-mediated fos induction [
43], a mechanism which might also have contributed to TBK1 signaling in this study and supports the increase in c-fos. IRF3, which has been frequently described as a prominent TBK1 target, seems to play only a minor role in nociception, since it was not affected by nociceptive stimulation or by deletion of TBK1.