Interferon Regulatory Factor 3 Signaling Pathways Research Articles

Antigen B from Echinococcus granulosus regulates macrophage phagocytosis by controlling TLR4 endocytosis in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is characterized by a reduction in platelet counts, stemming from an autoimmune-mediated process where platelets are excessively cleared by macrophages. This enhanced phagocytosis is a cardinal pathogenic mechanism in ITP. Antigen B (AgB), a principal component of the Echinococcus granulosus cyst fluid, plays a pivotal role in safeguarding the parasite from host immune defenses by modulating macrophage activation. In this study, we explored the potential of AgB to regulate macrophage activation in the context of ITP. Our observations indicated a diminished presence of M1 macrophages and a reduced phagocytic capacity in patients infected with E. granulosus sensu stricto. We isolated AgB from E. granulosus cyst fluid (EgCF) and discovered that it could suppress the polarization of M1 macrophages and weaken their phagocytic activity via Fcγ receptors, consequently alleviating thrombocytopenia in an ITP mouse model. At the molecular level, AgB was found to suppress the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3) by impeding their nuclear translocation, leading to a reduction in the generation of inflammatory cytokines. Furthermore, AgB was shown to inhibit Toll-like receptor 4 (TLR4) endocytosis and the recycling of CD14. In aggregate, our findings uncover a novel immunomodulatory mechanism of AgB, which suppresses macrophage phagocytosis by regulating TLR4 endocytosis and the subsequent activation of NF-κB and IRF3 signaling pathways. These insights shed new light on the molecular intricacies of E. granulosus-induced immune evasion and suggest that AgB may serve as a promising therapeutic agent for ITP.

467 Stimulator of Interferon Genes Protein (STING) Agonist Incited Sustained Antitumor Immunity in Murine Models of Glioblastoma

INTRODUCTION: Introduction: Stimulator of interferon genes (STING) is an innate pattern recognition receptor natively localized to the endoplasmic reticulum that detects both exogenous and endogenous pathogenic or damage-associated cytosolic cyclic dinucleotides (CDNs), activating IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce a potent type I interferon and proinflammatory cytokine responses. Activation of the STING pathway has been recognized as a critical component of the innate immune sensing of tumors that is required for optimal cytotoxic T-cell priming against tumor antigens. Recently a highly potent cyclic dinucleotide STING agonist, IACS-8803, showed robust activation of the STING pathway resulting in systemic anti-tumor response in the B16 murine model of melanoma. Additionally, the STING agonists have been shown to achieve robust tumor regression at both injected and distal lesions and are reported to facilitate generation of long-lived immunologic memory. METHODS: Methods: We established murine gliomas with stereotactic implantation of GL261, a murine glioma cell line. Mice were assessed with luciferase imaging to ensure baseline tumor size and then randomized into treatment arms (IgG, IACS-8803, CD47, PD-1 and combinations thereof). Treatment was initiated one week after the tumor implantation. RESULTS: Results: All mice that received IACS-8803 had no detectable luciferase activity. Tumor regression was additionally verified via MR Imaging. Utilization of IACS-8803 led to long-term tumor-free survival (p < 0.01). Re-challenge with GL261 tumors ensured long term memory was sustained with a single dose of agonist. CONCLUSIONS: Conclusion: Engaging the STING pathway may be an ideal targeting strategy for GBM. Ongoing studies are delineating the mechanisms of activating this pathway with specific attention being paid to reprogramming and education of innate immunity.

Knockdown of lncRNA TapSAKI alleviates LPS-induced injury in HK-2 cells through the miR-205/IRF3 pathway.

Sepsis is a common and lethal syndrome. Long non-coding RNA (lncRNA) transcript predicting survival in AKI (TapSAKI) has recently been found to serve as an important regulator in sepsis. However, the underlying mechanism of TapSAKI in sepsis pathogenesis remains largely unknown. Our data demonstrated that lipopolysaccharide (LPS)-induced HK-2 cell injury by weakening cell viability and enhancing cell apoptosis and inflammation. TapSAKI was upregulated and miR-205 was downregulated in LPS-induced HK-2 cells. TapSAKI knockdown or miR-205 overexpression alleviated LPS-induced cytotoxicity in HK-2 cells. TapSAKI sequestered miR-205 via acting as a miR-205 sponge. Moreover, the mitigating effect of TapSAKI silencing on LPS-induced HK-2 cell injury was mediated by miR-205. Additionally, the interferon regulatory factor 3 (IRF3) signaling was involved in the regulation of the TapSAKI/miR-205 axis on LPS-induced HK-2 cell damage. Our current study suggested that TapSAKI silencing relieved LPS-induced injury in HK-2 cells at least in part by sponging miR-205 and regulating the IRF3 signaling pathway, highlighting a novel understanding for sepsis pathogenesis and a promising target for this disease treatment.

Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages.

Nickel (Ni), an environmental hazard, widely causes allergic contact hypersensitivity worldwide. Despite that Ni-stimulated pro-inflammatory response is vital in allergy, the underlying molecular mechanisms remain largely unclear. Here, we demonstrated that NiCl2 activated nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and interferon regulatory factor 3 (IRF3) signaling pathways in primary bone marrow-derived macrophages (BMDMs), leading to the altered transcription levels of interleukin-1β (IL-1β), -6, -8, -18, tumor necrosis factor-α (TNF-α) and interferon β (INF-β). We also found that nickel chloride (NiCl2) activated Nod-like receptor 3 (NLRP3) inflammasome pathway, resulting in the proteolytic cleavage and release of IL-1β. NiCl2 induced the accumulation of mitochondrial reactive oxygen species (mtROS) and the release of mitochondrial DNA (mtDNA), thus activating NLRP3 inflammasome pathway. Additionally, NiCl2-induced apoptosis was dependent on the generation of mtROS, and caspase-1 activation might also partly contribute to the apoptotic process. Altogether, abovementioned results indicate that NiCl2 induces inflammatory activation in BMDMs via NF-κB, MAPKs, IRF3 signaling pathways as well as NLRP3 inflammasome pathway, which provides a mechanism to improve the efficiency of treatment against Ni-induced allergic reactions.

IRF3 signaling pathway serves an important role in poly(I:C)-induced procollagen reduction in human skin fibroblasts.

Pattern recognition receptors (PRRs) are part of the immune system. They can recognize pathogen‑associated molecular patterns (PAMPs). Toll‑like receptors (TLRs) and retinoic acid‑inducible gene1 (RIG‑1)‑like receptors (RLRs) are 2types of PRR in the innate immune system. Double‑stranded RNA (dsRNA) can exist as a PAMP, including dsRNA viruses. dsRNA is known as a ligand not only for TLR3 but also for RLRs, including melanoma differentiation‑associated gene5 and RIG‑1. Collagen is the main structural protein in the extracellular space in the skin. Recently, it was reported that treatment of a synthetic dsRNA, poly(I:C), decreases procollagen expression in skin fibroblasts. However, signaling pathways involved in this process have not yet been fully elucidated. The present study further explored the underlying signaling pathways involved in the processes. It was demonstrated by western blotting that treatment of poly(I:C), but not another PAMP, Pam3CSK4, inhibited procollagen expression in cultured human skin fibroblasts. Treatment of poly(I:C)and Pam3CSK4 induced activation of the mitogen‑activated protein kinases and the nuclear factor‑κB pathways. However, only poly(I:C), but not Pam3CSK4, induced the activation of the interferon regulatory factor 3 (IRF3) pathway. By using specific inhibitors, it was demonstrated that inhibition of IRF3 pathway relieved poly(I:C)‑induced procollagen reduction. In conclusion, IRF3 signaling pathway serves an important role in poly(I:C)‑induced procollagen reduction in skin fibroblasts. This suggests that the IRF3 signaling pathway may be a key target for collagen regulation in the skin.

Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway.

TRAF family member-associated NF-κB activator (TANK)-binding kinase1 (TBK1) is a serine-threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling pathways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tissues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFNβ in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFNβ induction in response to viral stimulation in chicken.

Equine herpesvirus-1 infection disrupts interferon regulatory factor-3 (IRF-3) signaling pathways in equine endothelial cells

Equine herpesvirus-1 (EHV-1) is a major respiratory viral pathogen of horses, causing upper respiratory tract disease, abortion, neonatal death, and neurological disease that may lead to paralysis and death. EHV-1 replicates initially in the respiratory epithelium and then spreads systemically to endothelial cells lining the small blood vessels in the uterus and spinal cord leading to abortion and EHM in horses. Like other herpesviruses, EHV-1 employs a variety of mechanisms for immune evasion including suppression of type-I interferon (IFN) production in equine endothelial cells (EECs). Previously we have shown that the neuropathogenic T953 strain of EHV-1 inhibits type-I IFN production in EECs and this is mediated by a viral late gene product. But the mechanism of inhibition was not known. Here we show that T953 strain infection of EECs induced degradation of endogenous IRF-3 protein. This in turn interfered with the activation of IRF-3 signaling pathways. EHV-1 infection caused the activation of the NF-κB signaling pathways, suggesting that inhibition of type-I IFN production is probably due to interference in IRF-3 and not NF-κB signal transduction.

Molecular cloning and characterization of porcine interferon-induced protein with tetratricopeptide repeats (IFIT) 5

Zhang, J., Shao, S.-y., Li, L.-z., Liu, D. and Yang, X.-q. 2015. Molecular cloning and characterization of porcine interferon-induced protein with tetratricopeptide repeats (IFIT) 5. Can. J. Anim. Sci. 95: 551–556. Interferon-induced protein with tetratricopeptide repeats (IFIT) family members play important roles in host defense against viral infection. In the present study, the complete coding sequence (CDS) of porcine IFIT5 gene was cloned using molecular biology techniques, and the genomic structure was determined using the bioinformatic method. The porcine IFIT5 is located on chromosome 14 containing 2 exons. Quantitative real-time PCR revealed that the transcripts of IFIT5 gene were unevenly distributed in all tissues studied, including heart, bladder, liver, large intestine, spleen, small intestine, lung, kidney, stomach, muscle, and lymph. Only one synonymous single nucleotide polymorphism was found in the complete CDS except for the first five nucleotides. IFIT5 is induced by poly(I:C) in a dose- and time-dependent manner, as revealed by using dual-luciferase analysis and reverse transcription-quantitative PCR methods. Furthermore, ectopic expression of porcine IFIT5 had no effect on the activation of interferon regulatory factor 3 (IRF3) significantly (P>0.05), suggesting it might not be a regulator of IRF3 signaling pathway.

Molecular cloning and characterization of porcine interferon-induced protein with tetratricopeptide repeats (IFIT) 5

Zhang, J., Shao, S.-y., Li, L.-z., Liu, D. and Yang, X.-q. 2015. Molecular cloning and characterization of porcine interferoninduced protein with tetratricopeptide repeats (IFIT) 5. Can. J. Anim. Sci. 95: 551� 556. Interferon-induced protein with tetratricopeptide repeats (IFIT) family members play important roles in host defense against viral infection. In the present study, the complete coding sequence (CDS) of porcine IFIT5 gene was cloned using molecular biology techniques, and the genomic structure was determined using the bioinformatic method. The porcine IFIT5 is located on chromosome 14 containing 2 exons. Quantitative real-time PCR revealed that the transcripts of IFIT5 gene were unevenly distributed in all tissues studied, including heart, bladder, liver, large intestine, spleen, small intestine, lung, kidney, stomach, muscle, and lymph. Only one synonymous single nucleotide polymorphism was found in the complete CDS except for the first five nucleotides. IFIT5 is induced by poly(I:C) in a dose- and time-dependent manner, as revealed by using dual-luciferase analysis and reverse transcription-quantitative PCR methods. Furthermore, ectopic expression of porcine IFIT5 had no effect on the activation of interferon regulatory factor 3 (IRF3) significantly (P� 0.05), suggesting it might not be a regulator of IRF3 signaling pathway.

Poly(I:C) induces expressions of MMP-1, -2, and -3 through various signaling pathways including IRF3 in human skin fibroblasts

BackgroundUltraviolet (UV) irradiation can result in premature skin aging (photoaging) which is characterized by decreased expression of collagen and increased expression of matrix metalloproteinases (MMPs). Double-stranded RNAs (dsRNAs) can be generated at various conditions including virally infected cells or UV-damaged skin cells. Recent studies have shown that a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), can reduce procollagen expression in human skin fibroblasts. However, little is known about the effect of poly(I:C) on the expression of MMPs in skin fibroblasts and its underlying mechanisms. ObjectiveWe examined the effect of poly(I:C) on MMP-1, -2, and -3 expressions in human skin fibroblasts. Then, we further explored the underlying signaling pathways involved in the processes. MethodsHuman skin fibroblasts were treated with poly(I:C) for the indicated times in the presence or the absence of various chemical inhibitors or small interfering RNAs (siRNAs) at the indicated concentrations. Protein and mRNA levels of various target molecules were examined by Western blotting and quantitative real-time PCR, respectively. ResultsPoly(I:C) induced MMP-1, -2, and -3 expressions, which were dependent on TLR3. Poly(I:C) also induced activations of the mitogen-activated protein kinases (MAPKs), the nuclear factor-kappaB (NF-κB) and the interferon regulatory factor 3 (IRF3) pathways. By using specific inhibitors, we found that poly(I:C)-induced expressions of MMP-1, -2, and -3 were differentially regulated by these signaling pathways. In particular, we found that the inhibition of IRF3 signaling pathways attenuated poly(I:C)-induced expressions of all the three MMPs. ConclusionOur data show that the expressions of MMP-1, -2, and -3 are induced by poly(I:C) through various signaling pathways in human skin fibroblasts and suggest that TLR3 and/or IRF3 may be good targets for regulating the expressions of MMP-1, -2, and -3 induced by dsRNAs.

Anti-inflammatory effects of benzenediamine derivate FC-98 on sepsis injury in mice via suppression of JNK, NF-κB and IRF3 signaling pathways.

FC-98, a synthesized benzenediamine derivate, was reported to regulate Toll-like receptor 9-induced activation of dendritic cells in our previous study. In this study, we evaluated the anti-inflammatory properties of FC-98 both in macrophages and in septic mouse models. By using enzyme-linked immunosorbent assay and real-time quantitative PCR, we found that FC-98 (6.25, 25 and 100μM) dose-dependently attenuated lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP-1) productions in RAW264.7 and primary mouse peritoneal macrophages. These inhibitory effects were not due to inducing cell cytotoxicity or altering LPS binding or TLR4 expression. Subsequently, western blot, immunofluorescence and luciferase reporter assays were used to investigate the underlying mechanisms of its anti-inflammatory activities. Results showed that FC-98 blocked activation of the c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways. In vivo, FC-98 (30 or 100mg/kg) was intraperitoneally administrated into LPS-induced or CLP-induced sepsis mice. It was observed to enhance the survival rate, inhibit pro-inflammatory mediator production, improve organ injuries and suppress bacterial propagation. In conclusion, FC-98 effectively inhibited macrophage inflammatory responses and ameliorated sepsis in mice through down-regulation of both MyD88 and TRIF-dependent pathways. These results suggest that FC-98 could be a promising therapeutic agent for inflammatory diseases.

Cyanidin-3-O-β-glucoside inhibits lipopolysaccharide-induced inflammatory response in mouse mastitis model

Cyanidin-3-O-β-glucoside (C3G) (CAS number 7084-24-4), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. However, the effect of C3G on lipopolysaccharide (LPS)-induced mastitis and the molecular mechanisms have not been investigated. In this study, we detected the protective effects of C3G on a LPS-induced mouse mastitis model and investigated the molecular mechanisms in LPS-stimulated mouse mammary epithelial cells (MMECs). Our results showed that C3G could attenuate mammary histopathologic changes and myeloperoxidase activity, and inhibit TNF-α, interleukin (IL)-1β, and IL-6 production caused by LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6 in LPS-stimulated MMECs. C3G suppressed LPS-induced nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) activation. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol. Moreover, C3G activated liver X receptor (LXR)-ABCG1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G are associated with upregulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol, thereby suppressing toll-like receptor 4-mediated NF-κB and IRF3 signaling pathways induced by LPS.

The Rab11a GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes.

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.

Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.

Activating Interferons with Noncanonical IKKs

Toll-like receptors (TLRs) initiate immune responses to bacterial and viral pathogens by recognition of conserved patterns presented by the pathogens. The TLR signaling pathways culminate in activation of inflammatory cytokine genes, including stimulation of the expression of the type 1 interferons (IFNα/β). Although all TLRs can signal through the adaptor MyD88, TLR3 and TLR4 continue to activate gene expression in the absence of MyD88 (see Barton and Medzhitov). Fitzgerald et al. investigated, using overexpression studies and interfering RNA (RNAi) techniques, the roles of the noncanonical inhibitor of κB kinase (IKK) members, IKKε and TANK-binding kinase 1 [(TBK1), also known as NF-κB-activating kinase (NAK) and tumor necrosis factor receptor-associated factor 2-associated kinase (T2K)] in the activation of interferon regulatory factor 3 (IRF3)-mediated gene expression. Overexpression of TBK1 or IKKε stimulated IRF3 translocation to the nucleus (traced by the movement of an IRF3-green fluorescent protein fusion construct) and stimulated IRF3-dependent expression of an IFNβ reporter and a reporter containing the interferon-stimulated regulatory element (IRSE) from the gene IFIT2 . In addition, overexpression of TBK1or IKKε stimulated secretion of the cytokine RANTES. Treatment of HEK293 cells or 293 T cells with RNAi against TBK1 or IKKε inhibited stimulation of expression of IFIT2 and the IRF3-dependent IFNβ and IRSE reporters and blocked secretion of RANTES in response to virus infection or double-stranded RNA. Coprecipitation experiments demonstrated an interaction between TBK1, IKKε, and IRF3 or the adaptor TRIF. Furthermore, increased gene expression in response to overexpression of TRIF was inhibited by kinase inactive mutants of TBK1 or IKKε. Thus, the authors suggest that the pathway from TLR to IRF3 proceeds through the TRIF adaptor and involves the noncanonical IKKs, TBK1 and IKKε. G. M. Barton, R. Medzhitov, Linking Toll-like receptors to IFNα/β expression. Nat. Immunol. 4 , 432-433 (2003). [Online Journal] K.A. Fitzgerald, S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, T. Maniatis, IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4 , 491-496 (2003). [Online Journal]

Activating Interferons with Noncanonical IKKs

Toll-like receptors (TLRs) initiate immune responses to bacterial and viral pathogens by recognition of conserved patterns presented by the pathogens. The TLR signaling pathways culminate in activation of inflammatory cytokine genes, including stimulation of the expression of the type 1 interferons (IFNα/β). Although all TLRs can signal through the adaptor MyD88, TLR3 and TLR4 continue to activate gene expression in the absence of MyD88 (see Barton and Medzhitov). Fitzgerald et al. investigated, using overexpression studies and interfering RNA (RNAi) techniques, the roles of the noncanonical inhibitor of κB kinase (IKK) members, IKKε and TANK-binding kinase 1 [(TBK1), also known as NF-κB-activating kinase (NAK) and tumor necrosis factor receptor-associated factor 2-associated kinase (T2K)] in the activation of interferon regulatory factor 3 (IRF3)-mediated gene expression. Overexpression of TBK1 or IKKε stimulated IRF3 translocation to the nucleus (traced by the movement of an IRF3-green fluorescent protein fusion construct) and stimulated IRF3-dependent expression of an IFNβ reporter and a reporter containing the interferon-stimulated regulatory element (IRSE) from the gene IFIT2. In addition, overexpression of TBK1or IKKε stimulated secretion of the cytokine RANTES. Treatment of HEK293 cells or 293 T cells with RNAi against TBK1 or IKKε inhibited stimulation of expression of IFIT2 and the IRF3-dependent IFNβ and IRSE reporters and blocked secretion of RANTES in response to virus infection or double-stranded RNA. Coprecipitation experiments demonstrated an interaction between TBK1, IKKε, and IRF3 or the adaptor TRIF. Furthermore, increased gene expression in response to overexpression of TRIF was inhibited by kinase inactive mutants of TBK1 or IKKε. Thus, the authors suggest that the pathway from TLR to IRF3 proceeds through the TRIF adaptor and involves the noncanonical IKKs, TBK1 and IKKε.G. M. Barton, R. Medzhitov, Linking Toll-like receptors to IFNα/β expression. Nat. Immunol. 4, 432-433 (2003). [Online Journal]K.A. Fitzgerald, S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, T. Maniatis, IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491-496 (2003). [Online Journal]

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.

The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response.