The Roles of Two IκB Kinase-related Kinases in Lipopolysaccharide and Double Stranded RNA Signaling and Viral Infection

Abstract

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-β gene induction. Recently, two IκB kinase (IKK)–related kinases, inducible IκB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-β production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-β and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i–deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-β and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.

TLR signaling

“…77 Fibroblasts or conventional DCs derived from TLR3-deficient mice are capable of producing IFNb following intracellular administration of poly IC and infection with Sendai virus (SeV), Newcastle disease virus (NDV) and VSV. 60,78,79 Together, these observations strongly suggest that host cells express cytoplasmic sensors that can detect actively replicating viruses. In these scenarios, a cytoplasmic protein, namely retinoic acid-inducible gene I (RIG-I), was identified through a functional screening.…”

“…71 Although it is reported that IRF5 is phosphorylated by TBK1 and IKKi in vitro, cells deficient in TBK1 and IKKi show normal inflammatory responses in response to various TLR ligands. 60,72 As mentioned above, an IRAK1 deficiency does not affect production of inflammatory cytokine. Thus, it is possible that as yet-unknown protein kinases that might associate with MyD88 are responsible for IRF5 phosphorylation and activation.…”

TLR signalling and activation of IRFs: revisiting old friends from the NF-κB pathway

“…To study the in vivo function of IKK-i and TBK1, relevant knockout mice were generated [37,40]. Deficiency in TBK1 causes TNF-mediated liver degeneration, resulting in embryonic lethality [37].…”

“…However, embryonic fibroblasts were generated from TBK1 K/K mice, stimulated with TLR3 and TLR4 ligands and characterised relative to wild type cells, in relation to IRF3 activation and induction of type I IFNs and IFN-inducible genes. There was severe impairment of the activation and nuclear translocation of IRF3 and induction of IFN-a, IFN-b and IFN-inducible genes, such as RANTES and IP10 (IFN-g-inducible protein 10), in response to both TLR ligands [39,40]. Such defective TLR3 and TLR4 signalling is also evident in bone marrow-derived macrophages derived from TBK1 K/K TNFR1 K/K mice [41], further emphasizing the crucial role of TBK1 in promoting IRF3-dependent gene expression.…”

“…Embryonic fibroblasts from IKK-i K/K mice showed normal responses to dsRNA and LPS with respect to IRF3 activation and induction of type I IFNs. However, IKKi K/K TBK1 K/K mice fail to express IFN genes or IFN-inducible genes in response to dsRNA, and thus IKK-i might account for the low residual expression of these genes still observed in TBK1 K/K cells [40]. Furthermore, reconstitution of TBK1 K/K cells with IKK-i can compensate partially for the lack of TBK1, with respect to IRF3 activation and induction of IFN-b.…”