Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV-nsp1 and LDV-nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV-nsp1 remained uncleaved. SHFV-nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV-nsp1αβ and SHFV-nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV-nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV-nsp1β, LDV-nsp1β, EAV-nsp1, and SHFV-nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV-nsp1α, CBP degradation was evident in cells expressing LDV-nsp1α and SHFV-nsp1γ, but no such degradation was observed for EAV-nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other.

The type I interferon (IFN) response plays a critical role in autoimmunity and is induced by innate receptor ligation and activation of IFN-regulatory factors (IRF). The present study investigated the roles and functional hierarchy of IRF3, IRF5, and IRF7 in expression of cytokines, chemokines, and matrix metalloproteinases in human THP1 monocytic cells. Targeted IRF knockdown was followed by evaluation of gene expression, promoter activation, and mRNA stability to determine the role of IRF as potential targets for modulating IFN responses in patients with autoimmune diseases. IRF played a distinct role in regulation of type I IFN gene expression in human monocytic cells and specifically regulated gene expression through the IFN-stimulated response element, with no contribution to transcription of traditionally AP-1 or NF-kB regulated genes. IRF7 regulated IL-6 gene expression by increasing IL-6 mRNA stability. IRF regulation of inflammation and induction of the IFN signature might contribute to the pathogenesis of autoimmune diseases and therefore represent novel therapeutic targets.

The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.

OBJECTIVE

Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.

METHODS

Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting.

RESULTS

In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2'-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction.

CONCLUSIONS

RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.

Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-α, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-κB and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia.

Toll-like receptors (TLRs) mediated signaling is mainly implicated in inflammatory activation which contributes to the initiation and progression of stroke. Using a model of transient global cerebral ischemia (tGCI) in rats, we investigated the changes of pro-inflammation mediators and tested the effects of Chloroquine pretreatment on the expression of pro-inflammation mediators after stroke. Adult Male Sprague-Dawley (SD) rats were subjected to transient global cerebral ischemia (tGCI) and treated without or with Chiloquin pretreatment (60mg/kg) 2h before tGCI. Short-term spatial memory capacity, Western blot assay and semi-quantitive RT-PCR were performed. Compared to sham operated rats, tGCI rats showed worsened learning and memory capacity and increased expression of TLR3, interferon regulatory factor 3 (IRF3), and interferon β (IFN-β) in the Hippocampus after stroke. Chloroquine pretreatment significantly enhanced rats' short-term spatial memory capacity and attenuated the expression of TLR3, IFR3, and IFN-β in the Hippocampus compared to non-treatment control in tGCI rats. Therefore, Chloroquine pretreatment of stroke inhibits inflammatory response and improves short-term spatial memory capacity. The TLR3/IFR3-IFN-β signaling pathway may contribute to the reduced inflammatory response after stroke. Chloroquine warrants further investigation as a therapeutic agent for the treatment of stroke.

Type I interferon (IFN) induces the antiviral response in innate immunity. The type I IFN gene cloned from Japanese flounder (Paralichthys olivaceus) has a length of 1189 bp and consisting of 5 exons and 4 introns. In a phylogenetic tree of type I IFNs, Japanese flounder grouped with other Acanthopterygii. To gain insight into the transcriptional regulation of IFN gene, the 1.36 kb 5'-upstream region including numerous canonical motifs to bind transcription factors [for example, IFN regulatory factor (IRF)] was analyzed. In HINAE cells using a luciferase reporter assay, poly I:C-responsive transcriptional activity was found in the region from -634 to -179 bp. This region includes several IRF motifs. In the presence of poly I:C, overexpression of IRF3 and RLR strongly enhanced transcriptional activity. These results suggest that the transcriptional regulation of Japanese flounder type I IFN is regulated by IRF3 after triggering with dsRNA sensors.

In this study, we examined the capacities of non-antigen-presenting cell types to propagate antiviral signals following infection with recombinant adenovirus or by direct nucleic acid transfection. Three murine cell lines (RAW264.7 macrophages as a positive control, FL83B hepatocytes, and MS1 endothelial cells) were assessed following exposure to adenovirus, DNA, or RNA ligands. Based on primary (interferon response factor 3 [IRF3] phosphorylation) and secondary (STAT1/2 phosphorylation) response markers, we found each cell line presented a unique response profile: RAW cells were highly responsive, MS1 cells were modified in their response, and FL83B cells were essentially nonresponsive. Comparative reverse transcription-quantitative PCR (RT-qPCR) of nucleic acid sensing components revealed major differences between the three cell types. A prominent difference was at the level of adaptor molecules; TRIF, MyD88, MAVS, and STING. TRIF was absent in MS1 and FL83B cells, whereas MyD88 levels were diminished in FL83B hepatocytes. These differences resulted in compromised TLR-mediated activation. While the cytosolic adaptor MAVS was well represented in all cell lines, the DNA adaptor STING was deficient in FL83B hepatocytes (down by nearly 3 log units). The absence of STING provides an explanation for the lack of DNA responsiveness in these cells. This hypothesis was confirmed by acquisition of IRF3 activation in Flag-STING FL83B cells following DNA transfection. To consolidate the central role of adaptors in MS1 endothelial cells, short hairpin RNA (shRNA) knockdown of STING and MAVS resulted in a ligand-specific loss of IRF3 responsiveness. In contrast to the requirement for specific adaptor proteins, a requirement for a specific DNA sensor (AIM2, DDx41, or p204) in the IRF3 activation response was not detected by shRNA knockdown in MS1 cells. The data reveal that cell-specific regulation of nucleic acid sensing cascade components influences antiviral recognition responses, that controlling levels of adaptor molecules is a recurring strategy in regulating antiviral recognition response functions, and that comparative RT-qPCR has predictive value for antiviral/innate response functions in these cells.

The mitochondrial adaptor, IFN-β promoter stimulator-1 (IPS-1), also known as MAVS/VISA/Cardif, plays a key role in the signal transduction of the RIG-1/MDA5 pathway to induce the production of interferons (IFNs) and other cytokines. In the present study, Japanese flounder (Paralichthys olivaceus) IPS-1 cDNA was cloned from Japanese flounder spleen using PCR-based methods. The full-length cDNA has 2235 nucleotides and encodes a polypeptide of 641 amino acids. The putative Japanese flounder IPS-1 protein contains an N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to other teleost counterparts ranging from 20% to 34%. Semi-quantitative RT-PCR showed that Japanese flounder IPS-1 mRNA was expressed in all tissues examined. The expression level of flounder IPS-1 gene was unchanged in viral hemorrhagic septicemia virus (VHSV)-infected kidney as measured by quantitative real-time PCR (Q-PCR). In addition, Japanese flounder IPS-1-overexpressing cells were protected against hirame rhabdovirus (HIRRV) and VHSV infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral titers. Expression of IFN-inducible genes including Mx, ISG15 and IRF3 were also induced in the IPS-1-overexpressing cells. These results suggest that Japanese flounder IPS-1 is involved in the antiviral immunity as a one of the adaptors in fish IFN-activation pathway.

During a viral infection, binding of viral double-stranded RNAs (dsRNAs) to the cytosolic RNA helicase RIG-1 leads to recruitment of the mitochondria-associated Cardif protein, involved in activation of the IRF3-phosphorylating IKKepsilon/TBK1 kinases, interferon (IFN) induction, and development of the innate immune response. The hepatitis C virus (HCV) NS3/4A protease cleaves Cardif and abrogates both IKKepsilon/TBK1 activation and IFN induction. By using an HCV replicon model, we previously showed that ectopic overexpression of IKKepsilon can inhibit HCV expression. Here, analysis of the IKKepsilon transcriptome profile in these HCV replicon cells showed induction of several genes associated with the antiviral action of IFN. Interestingly, IKKepsilon still inhibits HCV expression in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative STAT1alpha mutant. This suggests that good IKKepsilon expression levels are important for rapid activation of the cellular antiviral response in HCV-infected cells, in addition to provoking IFN induction. To determine the physiological importance of IKKepsilon in HCV infection, we then analyzed its expression levels in liver biopsy specimens from HCV-infected patients. This analysis also included genes of the IFN induction pathway (RIG-I, MDA5, LGP2, Cardif, TBK1), and three IKKepsilon-induced genes (IFN-beta, CCL3, and ISG15). The results show significant inhibition of expression of IKKepsilon and of the RNA helicases RIG-I/MDA5/LGP2 in the HCV-infected patients, whereas expression of TBK1 and Cardif was not significantly altered. In conclusion, given the antiviral potential of IKKepsilon and of the RNA helicases, these in vivo data strongly support an important role for these genes in the control of HCV infection.

Sepsis is a highly lethal disorder characterized by systemic inflammation, and Toll-like receptor 4 (TLR4) in macrophages plays a crucial role in modulating innate immune response and outcome of sepsis. During the screening of natural products against inflammation, we identified bis-N-norgliovictin, a small-molecule compound isolated from marine-derived fungus, significantly inhibited lipopolysaccharide (LPS, ligand of TLR4)-induced tumor necrosis factor-α (TNF-α) production in RAW264.7 cells. In this study, we evaluated the effect of bis-N-norgliovictin on TLR4-mediated inflammation in mouse macrophages and LPS-induced sepsis model. In RAW264.7 and mouse peritoneal macrophages, bis-N-norgliovictin dose-dependently inhibited LPS-induced production of TNF-α, interleukin-6 (IL-6), interferon-β (IFN-β) and monocyte chemoattractant protein (MCP-1), but without suppressing cell viability. The anti-inflammatory effect was attributed to the down-regulation of TLR4-triggered myeloid differentiation primary response protein 88 (MyD88)-dependent and TIR-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways, including p38 and c-Jun N-terminal kinase (JNK) of mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) cascades. Importantly, bis-N-norgliovictin also protected mice against LPS-induced endotoxic shock. Intravenous injection of bis-N-norgliovictin 1h before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-α, IL-6, MCP-1 and IL-10, attenuated liver and lung injury and diminished M1 macrophage polarization in liver. Our results demonstrate that bis-N-norgliovictin exhibit potent anti-inflammatory effect both in vitro and in vivo. These findings suggest that bis-N-norgliovictin can be a useful therapeutic candidate for the treatment of sepsis and other inflammatory diseases.

The inducible IkappaB kinase (IKKi/IKKepsilon) is a recently described serine-threonine kinase that activates the transcription factors NFkappaB, interferon regulatory factor-3 (IRF3) and CCAAA/enhancer-binding protein (C/EBPdelta). Several inflammatory agents have been shown to induce the expression of the IKKi gene in macrophages and other cell types but the mechanism is unknown. We have found that the IKKi expression was constitutive in human chondrocytes from OA cartilage and a human chondrocytic cell line C28/I2 but was up-regulated by the inflammatory cytokines TNFalpha or IL-1betain an NFkappaB-dependent manner. To understand the constitutive and inducible expression of the IKKi gene we localized the transcription start site (TSS), cloned and sequenced a 2 kb genomic DNA fragment 5' of the TSS and characterized the putative promoter region (PPR), and identified the motifs therein that are required for basal and cytokine-induced IKKi gene promoter activity. We found that IKKi core promoter was TATA-less and by using PCR generated deletion mutants of the PPR we found that the cis-elements responsible for basal transcriptional activity were located between -51 and -100 bp upstream of the TSS while the cytokine response elements were located distally between -501 and -1000 bp upstream of the TSS. The DNA region containing the cytokine response elements had two kappaB sites as the most relevant regulatory motifs. The results of site-directed mutagenesis revealed that the kappaB site located between -833 and -847 bp upstream of the TSS was biologically functional and required for cytokine-induced IKKi promoter activity in human chondrocytes and HeLa cells. The silence of the other kappaB site (-816/-802) was positional, rather than sequence-specific. Over-expression of NFkappaB p65 mimics the TNFalpha-induced activation of the IKKi promoter. Also the gel shift assay suggested that NFkappaB p65 is responsible for activation of the IKKi promoter. These data for the first time characterize the promoter region and provide further insights into the transcriptional regulation of IKKi in human chondrocytes and other cell types.

Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection.

OBJECTIVE

Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection.

Viral infection activates the transcription factors NF-κB and IRF3, which contribute to the induction of type I interferons (IFNs) and cellular antiviral responses. Protein kinases play a critical role in various signaling pathways by phosphorylating their substrates. Here, we identified dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) as a negative regulator of virus-triggered type I IFN induction. DYRK2 inhibited the virus-triggered induction of type I IFNs and promoted the K48-linked ubiquitination and degradation of TANK-binding kinase 1 (TBK1) in a kinase-activity-dependent manner. We further found that DYRK2 phosphorylated Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for the E3 ubiquitin ligase DTX4 to degrade TBK1. These findings suggest that DYRK2 negatively regulates virus-triggered signaling by targeting TBK1 for phosphorylation and priming it for degradation, and these data provide new insights into the molecular mechanisms that dictate the cellular antiviral response.

Mediator of IRF3 activation (MITA), also known as stimulator of interferon genes (STING), plays a vital role in the innate immune responses to cytosolic dsDNA. The trafficking of MITA from the ER to perinuclear vesicles is necessary for its activation of the downstream molecules, which lead to the production of interferons and pro-inflammatory cytokines. However, the exact mechanism of MITA activation remains elusive. Here, we report that transmembrane emp24 protein transport domain containing 2 (TMED2) potentiates DNA virus-induced MITA signaling. The suppression or deletion of TMED2 markedly impairs the production of type I IFNs upon HSV-1 infection. TMED2-deficient cells harbor greater HSV-1 load than the control cells. Mechanistically, TMED2 associates with MITA only upon viral stimulation, and this process potentiates MITA activation by reinforcing its dimerization and facilitating its trafficking. These findings suggest an essential role of TMED2 in cellular IFN responses to DNA viruses.

The activity and stability of the adapter protein MAVS (also known as VISA, Cardif and IPS-1), which critically mediates cellular antiviral responses, are extensively regulated by ubiquitination. However, the process whereby MAVS is deubiquitinated is unclear. Here, we report that the ovarian tumor family deubiquitinase 4 (OTUD4) targets MAVS for deubiquitination. Viral infection leads to the IRF3/7-dependent upregulation of OTUD4 which interacts with MAVS to remove K48-linked polyubiquitin chains, thereby maintaining MAVS stability and promoting innate antiviral signaling. Knockout or knockdown of OTUD4 impairs RNA virus-triggered activation of IRF3 and NF-κB, expression of their downstream target genes, and potentiates VSV replication in vitro and in vivo. Consistently, Cre-ER Otud4fl/fl or Lyz2-Cre Otud4fl/fl mice produce decreased levels of type I interferons and proinflammatory cytokines and exhibit increased sensitivity to VSV infection compared to their control littermates. In addition, reconstitution of MAVS into OTUD4-deficient cells restores virus-induced expression of downstream genes and cellular antiviral responses. Together, our findings uncover an essential role of OTUD4 in virus-triggered signaling and contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses.

BACKGROUND

Dryopteris crassirhizoma Nakai (Aspiadaceae) has been traditionally used as an herbal medicine for treating various inflammatory and infectious diseases such as tapeworm infestation, colds, and viral diseases. However, no systematic studies on the anti-inflammatory actions of Dryopteris crassirhizoma and its inhibitory mechanisms have been reported. We therefore aimed at exploring the anti-inflammatory effects of 95% ethanol extracts (Dc-EE) of this plant.

METHODS

The anti-inflammatory effect of Dc-EE on the production of inflammatory mediators in RAW264.7 cells and HCl/EtOH-induced gastritis was examined. Inhibitory mechanisms were also evaluated by exploring activation of transcription factors, their upstream signalling, and target enzyme activities. Finally, the active components from this extract were also identified using HPLC system.

RESULTS

Dc-EE diminished the production of nitric oxide (NO) and prostaglandin (PG)E(2) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner. Dc-EE also downregulated the levels of mRNA expression of pro-inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and TNF-α by inhibiting the activation of activator protein (AP-1) and IRF3. Indeed, the extract strongly blocked the activities of their upstream kinases ERK1 and TBK1. This extract also strongly ameliorated gastritis symptoms stimulated by HCl/EtOH in mice. According to HPLC fingerprinting, resveratrol, quercetin, and kampferol were identified from Dc-EE.

CONCLUSIONS

Dc-EE displays strong anti-inflammatory activity by suppressing ERK/AP-1 and TBK1/IRF3 pathways, which contribute to its major ethno-pharmacological role as an anti-inflammatory and anti-infectious disease remedy.

8-Prenylkaempferol (8-PK) is a prenylflavonoid isolated from Sophora flavescens, a Chinese herb with antiviral and anti-inflammatory properties. In this study, we investigated its effect on regulated activation, normal T cell expressed and secreted (RANTES) secretion by influenza A virus (H1N1)-infected A549 alveolar epithelial cells. Cell inoculation with H1N1 evoked a significant induction in RANTES accumulation accompanied with time-related increase in nuclear translocation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF-3), but showed no effect on c-Jun phosphorylation. 8-PK could significantly inhibit not only RANTES production but also NF-κB and IRF-3 nuclear translocation. We had proved that both NF-κB and IRF-3 participated in H1N1-induced RANTES production since NF-κB inhibitor pyrrolidinedithio carbamate (PDTC) and IRF-3 siRNA attenuated significantly RANTES accumulation. H1N1 inoculation also increased PI3K activity as well as Akt phosphorylation and such responsiveness were attenuated by 8-PK. In the presence of wortmannin, nuclear translocation of NF-κB and IRF3 as well as RANTES production by H1N1 infection were all reversed, demonstrating that PI3K-Akt pathway is essential for NF-κB- and IRF-3-mediated RANTES production in A549 cells. Furthermore, 8-PK but not wortmannin, prevented effectively H1N1-evoked IκB degradation. In conclusion, 8-PK might be an anti-inflammatory agent for suppressing influenza A virus-induced RANTES production acts by blocking PI3K-mediated transcriptional activation of NF-κB and IRF-3 and in part by interfering with IκB degradation which subsequently decreases NF-κB translocation.

Influenza A virus non-structural protein 1 (NS1) antagonizes interferon response through diverse strategies, particularly by inhibiting the activation of interferon regulatory factor 3 (IRF3) and IFN-β transcription. However, the underlying mechanisms used by the NS1 C-terminal effector domain (ED) to inhibit the activation of IFN-β pathway are not well understood. In this study, we used influenza virus subtype of H5N1 to demonstrate that the NS1 C-terminal ED but not the N-terminal RNA-binding domain, binds TNF receptor-associated factor 3 (TRAF3). This results in an attenuation of the type I IFN signaling pathway. We found that the NS1 C-terminal ED (named NS1/126-225) inhibits the active caspase activation and recruitment domain-containing form of RIG-I [RIG-I(N)]-induced IFN-β reporter activity, the phosphorylation of IRF3, and the induction of IFN-β. Further analysis showed that NS1/126-225 binds to TRAF3 through the TRAF domain, subsequently decreasing TRAF3 K63-linked ubiquitination. NS1/126-225 binding also disrupted the formation of the mitochondrial antiviral signaling (MAVS)-TRAF3 complex, increasing the recruitment of IKKε to MAVS; ultimately shutting down the RIG-I(N)-mediated signal transduction and cellular antiviral responses. This attenuation of cellular antiviral responses leads to evasion of the innate immune response. Taken together, our findings offer an important insight into the interplay between the influenza virus and host innate immunity.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.

Cytosolic pattern recognition receptors (PRRs) sense intracellular nucleic acids from pathogens such as bacteria and viruses, which leads to the induction of type I interferon (IFN) responses that are essential for an effective immune response. Further, these PRR pathways can be aberrantly activated by self DNA, which leads to autoimmunity. Therefore, understanding the signaling mechanisms that underlie PRR-induced production of IFN is vital to health and disease. A key transcription factor that is involved in these pathways is IFN regulatory factor 3 (IRF3), which is often activated by the kinase TANK-binding kinase 1 (TBK1). STING (stimulator of interferon genes) is a master regulator for the cyto-solic nucleic acid-mediated activation of IRF3 through TBK1 stimulation, but how the STING-TBK1-IRF3 signaling axis operates has been unclear. A study now shows that in response to cytosolic double-stranded DNA, the C-terminal tail of STING provides a scaffold to assemble IRF3 and TBK1, which leads to TBK1-dependent phosphorylation of IRF3. Thus, STING directs TBK1 to activate IRF3 in DNA-sensing pathways.

Respiratory dendritic cells (DC) play a pivotal role in the initiation of adaptive immune responses to influenza virus. To do this, respiratory DCs must ferry viral antigen from the lung to the draining lymph node without becoming infected and perishing en route. We show that respiratory DCs up-regulate the expression of the antiviral molecule, interferon-induced transmembrane protein 3 (IFITM3) in response to influenza virus infection, in a manner dependent on type I interferon signaling and the transcription factors IRF7 and IRF3. Failure of respiratory DCs to up-regulate IFITM3 following influenza virus infection resulted in impaired trafficking to the draining LN and consequently in impaired priming of an influenza-specific CD8+ T cell response. The impaired trafficking of IFITM3-deficient DC correlated with an increased susceptibility of these DC to influenza virus infection. This work shows that the expression of IFITM3 protects respiratory DCs from influenza virus infection, permitting migration from lung to LN and optimal priming of a virus specific T-cell response.

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity.

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites (Plasmodium falciparum, Pf), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf-DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.