BACKGROUND

During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear.

RESULTS

We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria.

CONCLUSIONS

These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction.

Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.

Infections by coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SCoV) and mouse hepatitis virus A59 (MHV-A59) result in very little type I interferon (IFN) production by host cells, which is potentially responsible for the rapid viral growth and severe immunopathology associated with SARS. However, the molecular mechanisms for the low IFN production in cells infected with coronaviruses remain unclear. Here, we provide evidence that Papain-like protease domain 2 (PLP2), a catalytic domain of the nonstructural protein 3 (nsp3) of MHV-A59, can bind to IRF3, cause its deubiquitination and prevent its nuclear translocation. As a consequence, co-expression of PLP2 strongly inhibits CARDIF-, TBK1- and IRF3-mediated IFNbeta reporter activities. In addition, we show that wild-type PLP2 but not the mutant PLP2 lacking the deubiquitinase (DUB) activity can reduce IFN induction and promote viral growth in cells infected with VSV. Thus, our study uncovered a viral DUB which coronaviruses may use to escape from the host innate antiviral responses.

Type I interferon (IFN) is crucial during infection through its antiviral properties and by coordinating the immunocompetent cells involved in antiviral or antibacterial immunity. Type I IFN (IFN-α and IFN-β) is produced after virus or bacteria recognition by cytosolic receptors or membrane-bound TLR receptors following the activation of the transcription factors IRF3 or IRF7. IFN-β production after fungal infection was recently reported, although the underlying mechanism remains controversial. Here we describe that IFN-β production by dendritic cells (DCs) induced by Candida albicans is largely dependent on Dectin-1- and Dectin-2-mediated signaling. Dectin-1-induced IFN-β production required the tyrosine kinase Syk and the transcription factor IRF5. Type I IFN receptor-deficient mice had a lower survival after C. albicans infection, paralleled by defective renal neutrophil infiltration. IFN-β production by renal infiltrating leukocytes was severely reduced in C. albicans-infected mice with Syk-deficient DCs. These data indicate that Dectin-induced IFN-β production by renal DCs is crucial for defense against C. albicans infection.

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

Virus infection induces host antiviral responses including induction of type I IFNs. Transcription factor IFN regulatory factor 3 (IRF3) plays an essential role and is tightly regulated in this process. Herein we report that TRIM21 (tripartite motif-containing 21) is significantly induced and interacts with IRF3 upon RNA virus infection. Ectopic expression or knockdown of TRIM21 could respectively enhance or impair IRF3-mediated gene expression. Mechanistically, TRIM21 interferes with the interaction between Pin1 (peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1) and IRF3, thus preventing IRF3 ubiquitination and degradation. A conserved motif in the B 30.2 domain of TRIM21 is critical for its modulation of IRF3 function, while the RING finger is dispensable. Host antiviral responses are significantly boosted or crippled in the presence or absence of TRIM21. Our results identify TRIM21 as an essential modulator of IRF3 stability and demonstrate that it positively regulates the strength and duration of primary antiviral response, thus further strengthening the notion that the TRIM family is evolutionarily integrated with innate immunity.

'Detoxification' of gut-derived toxins and microbial products from gut-derived microbes is a major role of the liver. While the full repertoire of gut-derived microbial products that reach the liver in health and disease is yet to be explored, the levels of bacterial lipopolysaccharide (LPS), a component of Gram-negative bacteria, is increased in the portal and/or systemic circulation in several types of chronic liver diseases. Increased gut permeability and LPS play a role in alcoholic liver disease where alcohol impairs the gut epithelial integrity through alterations in tight junction proteins. In addition, non-alcoholic fatty liver disease is also associated with increased serum LPS levels and activation of the pro-inflammatory cascade plays a central role in disease progression. Microbial danger signals are recognized by pattern recognition receptors such as the Toll-like receptor 4 (TLR4). Increasing evidence suggests that TLR4-mediated signaling via the MyD88-dependent or MyD88-independent pathways may play different roles in liver diseases associated with increased LPS exposure of the liver as a result of gut permeability. For example, we showed that in alcoholic liver disease, the MyD88-independent, IRF3-dependent TLR4 cascade plays a role in steatosis and inflammation. Our recent data demonstrate that chronic alcohol exposure in the liver leads to sensitization of Kupffer cells to LPS via a mechanism involving upregulation of microRNA-155 in Kupffer cells. Thus, understanding the cell-specific recognition and intracellular signaling events in sensing gut-derived microbes will help to achieve an optimal balance in the gut-liver axis and ameliorate liver diseases.

Expression of type I interferons (IFNs) can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

Induction of type I interferons by the transcription factor IRF3 is essential in the initiation of antiviral innate immunity. Activation of IRF3 requires C-terminal phosphorylation by the upstream kinases TBK1-IKKi, where IRF3 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFNbeta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1-IKKi; however, the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFNbeta production upon viral challenge compared with WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1-IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting Lys(63)-linked polyubiquitination of TBK1-IKKi independently of the A20 deubiquitination domain. Furthermore, TAX1BP1 was required for A20 effector function because A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1(-)(/)(-) MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1-IKKi ubiquitination. Collectively, our results demonstrate TBK1-IKKi to be novel substrates for A20 and further identify a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3-TBK1-IKKi signaling complex.

The tripartite motif (TRIM) family member, TRIM21, is an E3 ubiquitin ligase for IFN regulatory factor (IRF)3 and IRF8 that functions in both innate and acquired immunity. It is also an autoantigen known as Ro52/SS-A. The function of TRIM21 in vivo, however, has remained elusive. We generated Trim21(-/-) mice with the Trim21 gene replaced by an enhanced GFP (EGFP) reporter. EGFP expression analyses showed that Trim21 was widely expressed in many tissues, with the highest levels in immune cells. Studies of Trim21(-/-) embryonic fibroblasts demonstrated that TLR-mediated induction of proinflammatory cytokines, including IL-1beta, IL-6, TNF-alpha, and CXCL10, was consistently up-regulated relative to wild-type cells. Reporter analyses demonstrated that TLR-mediated NF-kappaB activation was higher in Trim21(-/-) cells than in wild-type cells, most likely accounting for their enhanced cytokine expression. In contrast, functional analyses of immune cells from Trim21(-/-) mice revealed no abnormalities in their composition or function, even though ubiquitylation of IRF3 and IRF8 was impaired. These results suggested possible redundancies in activities mediated by TRIM21. In keeping with this concept, we found that a number of TRIM family members were up-regulated in Trim21(-/-) cells. Taken together, these findings demonstrate that TRIM21 plays a previously unrecognized role in the negative regulation of NF-kappaB-dependent proinflammatory cytokine responses, and suggest that multiple TRIM proteins contribute to the maintenance of functional equilibrium in inflammatory responses, in part through functional redundancy.

Adenovirus (Ad) infection triggers a cell-specific antiviral response following exposure of viral DNA to the intracellular compartment. A variety of DNA sensors (DAI, AIM2, DDx41, RNA polymerase [Pol] III, and IFI16 [p204]) have been identified in recent years; however, the DNA sensor involved in detection of adenovirus has not been established. Cyclic GMP-AMP synthase (cGAS), a DNA sensor that produces a cyclic guanine-adenine dinucleotide (cGAMP) inducer of STING, has been examined to determine its role in generating an antiadenoviral response. Short hairpin RNA (shRNA) lentiviral vectors targeting TBK1, STING, and cGAS were established in murine MS1 endothelial and RAW 264.7 macrophage cell lines. Knockdown of TBK1, STING, and cGAS results in a dramatic reduction in the activation of the primary antiviral response marker phosphorylated interferon (IFN) response factor 3 (IRF3) following exposure to adenovirus. Furthermore, activation of secondary type I IFN signaling targets ((ptyr)STAT1 and (ptyr)STAT2 [(ptyr)STAT1/2]) was also compromised. Consistent with compromised activation of primary and secondary response markers, transcriptional activation of IRF3-responsive genes (beta IFN [IFN-β], ISG15, ISG54) and secondary response transcripts were diminished in cells knocked down in cGAS, STING, or TBK1. These data establish cGAS as the dominant cytosolic DNA sensor responsible for detection of internalized adenovirus leading to induction of the type I interferon antiviral cascade.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

In periodontitis, a common chronic inflammatory condition, gram-negative-rich bacterial biofilms trigger, in susceptible individuals, perpetuating inflammation that results in extensive tissue damage of tooth supporting structures. To delineate immune cell-dependent mechanisms whereby bacterial challenge drives persistent destructive inflammation in periodontitis and other inflammatory diseases, we studied involved tissues ex vivo and investigated host cell responses to the periodontal pathogen Porphyromonas gingivalis, in vitro. Diseased lesions were populated by abundant Th17 cells, linked to infection, chronic inflammation/autoimmunity and tissue pathology. In vitro, P. gingivalis, particularly the more virulent strain W83, stimulated myeloid antigen presenting cells (APC) to drive Th17 polarization. Supernatants from myeloid APC exposed to P. gingivalis were capable of enhancing Th17 but not Th1 polarization. P. gingivalis favored the generation of Th17 responses by stimulating the production of Th17 related cytokines IL-1β, IL-6 and IL-23, but not Th1 related IL-12. By inducing NFκB activation, P. gingivalis promoted IL-1β, IL-6 and IL-12p40 production, but not IRF3 phosphorylation, connected to generation of the IL-12p35 chain, ultimately restricting formation of the intact IL-12 molecule. Promotion of Th17 lineage responses was also aided by P. gingivalis proteases, which appeared to differentially degrade pivotal cytokines. In this regard, IL-12 was largely degraded by P. gingivalis, whereas IL-1β was more resistant to proteolysis. Our data unveil multiple pathways by which P. gingivalis may orchestrate chronic inflammation, providing insights into interventional strategies.

Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8⁺ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8⁺ T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565.

The Yersinia pestis virulence factor YopJ is a potent inhibitor of the NF-kappaB and MAPK signalling pathways, however, its molecular mechanism and relevance to pathogenesis are the subject of much debate. In this report, we characterize the effects of this type III effector protein on bone fide signalling events downstream of Toll-like receptors (TLRs), critical sensors in innate immunity. YopJ inhibited TLR-mediated NF-kappaB and MAP kinase activation, as suggested by previous studies. In addition, induction of the TLR-mediated interferon response was blocked by YopJ, indicating that YopJ also inhibits IRF3 signalling. Examination of the NF-kappaB signalling pathway in detail suggested that YopJ acts at the level of TAK1 (MAP3K7) activation. Further studies revealed a YopJ-dependent decrease in the ubiquitination of TRAF3 and TRAF6. These data support the hypothesis that YopJ is a deubiquitinating protease that acts on TRAF proteins to prevent or remove the K63-polymerized ubiquitin conjugates required for signal transduction. Our data do not directly address the alternative hypothesis that YopJ is an acetyltransferase that acts on the activation loop of IKK and MKK proteins, but support the conclusion that the critical function of YopJ is to deubiquinate TRAF proteins.

The expression of antiviral genes during infection is controlled by inducible transcription factors such as IRF3 (interferon regulatory factor). Activation of IRF3 requires its phosphorylation by TBK1 (TANK-binding kinase) or IKKi (inhibitor of nuclear factor kappaB kinase, inducible). We have identified a new and essential component of this pathway, the adaptor protein SINTBAD (similar to NAP1 TBK1 adaptor). SINTBAD constitutively binds TBK1 and IKKi but not related kinases. Upon infection with Sendai virus, SINTBAD is essential for the efficient induction of IRF-dependent transcription, as are two further TBK1 adaptors, TANK and NAP1. We identified a conserved TBK1/IKKi-binding domain (TBD) in the three adaptors, predicted to form an alpha-helix with residues essential for kinase binding clustering on one side. Isolated TBDs compete with adaptor binding to TBK1 and prevent poly(I:C)-induced IRF-dependent transcription. Our results suggest that efficient signal transduction upon viral infection requires SINTBAD, TANK and NAP1 because they link TBK1 and IKKi to virus-activated signalling cascades.

The type I interferon (IFN) system mediates a wide variety of antiviral effects and represents an important first barrier to virus infection. Consequently, viruses have developed an impressive diversity of tactics to circumvent IFN responses. Evasion strategies can involve preventing initial virus detection, via the disruption of the Toll-like receptors or the retinoic acid inducible gene I (RIG-I) -like receptors, or by avoiding the initial production of the ligands recognized by these receptors. An alternative approach is to preclude IFN production by disarming or degrading the transcription factors involved in the expression of IFN, such as interferon regulatory factor 3 (IRF3)/IRF7, nuclear factor-κB (NF-κB), or ATF-2/c-jun, or by inducing a general block on host cell transcription. Viruses also oppose IFN signalling, both by disturbing the type I IFN receptor and by impeding JAK/STAT signal transduction upon IFN receptor engagement. In addition, the global expression of IFN-stimulated genes (ISGs) can be obstructed via interference with epigenetic signalling, and specific ISGs can also be selectively targeted for inhibition. Finally, some viruses disrupt IFN responses by co-opting negative regulatory systems, whereas others use antiviral mechanisms to their own advantage. Here, we review recent developments in this field.

The innate immune system recognizes nucleic acids during viral infection and stimulates cellular antiviral responses. Intracellular detection of RNA virus infection is mediated by the RNA helicases RIG-I (retinoic acid inducible gene I) and MDA-5, which recognize viral RNA and signal through the adaptor molecule MAVS (mitochondrial antiviral signaling) to stimulate the phosphorylation and activation of the transcription factors IRF3 (interferon regulatory factor 3) and IRF7. Once activated, IRF3 and IRF7 turn on the expression of type I interferons, such as beta interferon. Interestingly, unlike other signaling molecules identified in this pathway, MAVS contains a C-terminal transmembrane (TM) domain that is essential for both type I interferon induction and localization of MAVS to the mitochondrial outer membrane. However, the role the MAVS TM domain plays in signaling remains unclear. Here we report the identification of a function for the TM domain in mediating MAVS self-association. The activation of RIG-I/MDA-5 leads to the TM-dependent dimerization of the MAVS N-terminal caspase recruitment domain, thereby providing an interface for direct binding to and activation of the downstream effector TRAF3 (tumor necrosis factor receptor-associated factor 3). Our results reveal a role for MAVS self-association in antiviral innate immunity signaling and provide a molecular mechanism for downstream signal transduction.

Varicella-zoster virus (VZV) infection of differentiated cells within the host and establishment of latency likely requires evasion of innate immunity and limits secretion of antiviral cytokines. Here we report that its immediate-early protein ORF61 antagonizes the beta interferon (IFN-β) pathway. VZV infection down-modulated the Sendai virus (SeV)-activated IFN-β pathway, including mRNA of IFN-β and its downstream interferon-stimulated genes (ISGs), ISG54 and ISG56. Through a primary screening of VZV genes, we found that ORF61 inhibited SeV-mediated activation of IFN-β and ISRE (IFN-stimulated response element) promoter activities but only slightly affected NF-κB promoter activity, implying that the IFN-β pathway may be blocked in the IRF3 branch. An indirect immunofluorescence assay demonstrated that ectopic expression of ORF61 abrogated the detection of IRF3 in SeV-infected cells; however, it did not affect endogenous dormant IRF3 in noninfected cells. Additionally, ORF61 was shown to be partially colocalized with activated IRF3 in the nucleus upon treatment with MG132, an inhibitor of proteasomes, and the direct interaction between ORF61 and activated IRF3 was confirmed by a coimmunoprecipitation assay. Furthermore, Western blot analysis demonstrated that activated IRF3 was ubiquitinated in the presence of ORF61, suggesting that ORF61 degraded phosphorylated IRF3 via a ubiquitin-proteasome pathway. Semiquantitative reverse transcription-PCR (RT-PCR) analysis demonstrated that the level of ISG54 and ISG56 mRNAs was also downregulated by ORF61. Taken together, our results convincingly demonstrate that ORF61 down-modulates the IRF3-mediated IFN-β pathway by degradation of activated IRF3 via direct interaction, which may contribute to the pathogenesis of VZV infection.

Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-alpha/beta) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-alpha/beta induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein N(pro). Central players in the IFN-alpha/beta induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of N(pro), while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV N(pro) induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, N(pro) coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with N(pro). Finally, we show that N(pro) does not downregulate IRF7 expression.

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4(-/-), Myd88(-/-), Trif(-/-), and Nod2(-/-) mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.

OBJECTIVE

The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.