TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

RACK1 attenuates RLR antiviral signaling by targeting VISA-TRAF complexes

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-β (IFN-β) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-β production in the RIG-I/VISA signaling pathway.     

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN‐I) during viral infections, in response to triggering of endosomal Toll‐like receptors (TLRs) 7 or 9 by viral single‐stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN‐I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN‐I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN‐I downstream of the TLR9‐to‐MyD88‐to‐IRF7 signaling pathway without requiring IFN‐I positive feedback, high IRF7 expression, or AP3‐driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN‐I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.     

TLR3 signaling in a hepatoma cell line is skewed towards apoptosis

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double-stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNbeta) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kappaB and IRF3 (interferon regulatory factor 3) and induced IFNbeta (interferon beta) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNbeta expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies.     

IRAK-2 participates in multiple toll-like receptor signaling pathways to NFkappaB via activation of TRAF6 ubiquitination

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFkappaB signaling axis, recent data have shown that it is dispensable for NFkappaB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFkappaB activation pathway. The poxviral TLR antagonist A52 inhibited NFkappaB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFkappaB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFkappaB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFkappaB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFkappaB.     

WDFY1 mediates TLR3/4 signaling by recruiting TRIF

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR–TRIF interaction, which is necessary for TLR signaling.     

A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3β (MAP1LC3β). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3β fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.     

Ubiquitin-Specific Proteases 25 Negatively Regulates Virus-Induced Type I Interferon Signaling

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.     

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-beta) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-alpha 3 and karyopherin-alpha 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.     

Phospholipase Cγ-2 and intracellular calcium are required for lipopolysaccharide-induced Toll-like receptor 4 (TLR4) endocytosis and interferon regulatory factor 3 (IRF3) activation

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.