CD40 stimulates a "feed-forward" NF-κB-driven molecular pathway that regulates IFN-β expression in carcinoma cells

IFN-β and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-β upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-κB and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-κB, IRF1, and Viperin-dependent IRF7 binding to the IFN-β promoter largely controls its activity. However, full activation of IFN-β also requires the parallel engagement of noncanonical NF-κB2 signaling leading to p52 recruitment to the IFN-β promoter. These data define a novel link between CD40 signaling and IFN-β expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.     

ABIN1 protein cooperates with TAX1BP1 and A20 proteins to inhibit antiviral signaling

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.     

HSV-2 induces TLRs and NF-κB-dependent cytokines in cervical epithelial cells

Herpes simplex virus type 2 (HSV-2) is one of the most common sexually transmitted pathogens worldwide. The host immune response induced by viral infection is cell-type specific. Little is known about the innate immune response to this virus in its natural host cells. In this study, we established an in vitro HSV-2 infection model with human cervical epithelial (HCE) cells. The viral infection was sufficient to induce expression of Toll-like receptors (TLRs), and Western blot and reporter assays suggest that HSV-2 infection leads to dramatic activation of the NF-κB signaling pathway. More importantly, our data provide direct evidence that the activation of NF-κB is required for the production of both IL-6 and IFN-β induced by HSV-2 in HCE cells. Taken together, our results suggest the potential contributions of TLRs and a critical role of NF-κB in the innate immune response to HSV-2 in HCE cells.     

A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB

The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.     

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.     

NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.     

Inducible microRNA-155 feedback promotes type I IFN signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-κB-dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.     

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.