Anti-Inflammatory Activity of Glabralactone, a Coumarin Compound from Angelica sinensis, via Suppression of TRIF-Dependent IRF-3 Signaling and NF-κB Pathways.

The dried root of Angelica sinensis (A. sinensis) has been widely used in Chinese traditional medicine for various diseases such as inflammation, osteoarthritis, infections, mild anemia, fatigue, and high blood pressure. Searching for the secondary metabolites of A. sinensis has been mainly conducted. However, the bioactivity of coumarins in the plant remains unexplored. Therefore, this study was designed to evaluate the anti-inflammatory activity of glabralactone, a coumarin compound from A. sinensis, using in vitro and in vivo models, and to elucidate the underlying molecular mechanisms of action. Glabralactone effectively inhibited nitric oxide production in lipopolysaccharide- (LPS-) stimulated RAW264.7 macrophage cells. The downregulation of LPS-induced mRNA and protein expression of iNOS, TNF-α, IL-1ß, and miR-155 was found by glabralactone. The activation of NF-κB and TRIF-dependent IRF-3 pathway was also effectively suppressed by glabralactone in LPS-stimulated macrophages. Glabralactone (5 and 10 mg/kg) exhibited an in vivo anti-inflammatory activity with the reduction of paw edema volume in carrageenan-induced rat model, and the expressions of iNOS and IL-1ß proteins were suppressed by glabralactone in the paw soft tissues of the animal model. Taken together, glabralactone exhibited an anti-inflammatory activity in in vitro and in vivo models. These findings reveal that glabralactone might be one of the potential components for the anti-inflammatory activity of A. sinensis and may be prioritized in the development of a chemotherapeutic agent for the treatment of inflammatory diseases.

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways.

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/ß and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.

TTP488 ameliorates NLRP3-associated inflammation, viability, apoptosis, and ROS production in an Alzheimer's disease cell model by mediating the JAK1/STAT3/NFκB/IRF3 pathway.

Alzheimer's disease (AD), the most prevalent dementia, is identified as a neurodegenerative disease arising from a degenerative disturbance in the central nervous system. A previous study reported that TTP488 could ameliorate symptoms in patients with mild AD, but the underlying mechanisms need to be studied further. Therefore, the objective of this study was to explore the role of TTP488 in the development of an AD cell model. Administration of TTP448 in an AD cell model reduced the expression of pro-inflammatory cytokines [interleukin (IL)-1ß, IL-6, and TNF-α], reversed the inhibitory role of Aß on cell proliferation and viability, and decreased Aß-triggered cell apoptosis and reactive oxygen species (ROS) production. Furthermore, Aß treatment induced activation of JAK1/STAT3/NFκB/IRF3 pathway as well as NLRP3 expression, and TTP488 administration partially reversed the activation of this pathway and NLRP3 expression. Use of WP1160, a STAT3 agonist, re-activated the downstream STAT3/NFκB/IRF3 pathway and NLRP3 expression. Moreover, we found that WP1160 counteracted the role of TTP488 in Aß-induced SH-SY5Y cells' viability, inflammation, apoptosis, and ROS production. SIGNIFICANCE OF THE STUDY: This study explores the role of TTP488 in the development of an Alzheimer's disease (AD) cell model and confirms that TTP488 administration notably promotes cell proliferation and reduces apoptosis, inflammatory factor expression, and reactive oxygen species generation. Further, this study suggests that the NLRP3-relevant JAK1/STAT3/P65/IRF3 signalling pathway is related to AD pathogenesis.

Statins attenuate antiviral IFN-ß and ISG expression via inhibition of IRF3 and JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages.

Viral infection is a significant burden to health care worldwide. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, are widely used as cholesterol-lowering drugs. Recently, long-term statin therapy was shown to reduce the antiviral immune response; however, the underlying molecular mechanisms are unclear. Here, we found that simvastatin decreased polyinosinic-polycytidylic acid [poly(I:C)]-induced expression of antiviral interferon (IFN)-ß and IFN-stimulated genes (ISGs) in the bronchoalveolar lavage fluid (BALF) and lungs of mice with high-fat diet-induced hyperlipidemia. Macrophages were the dominant cell type in the BALF of poly(I:C)-treated mice. We examined the effects of simvastatin in primary lung macrophages and found that simvastatin suppressed poly(I:C)-induced expression of IFN-ß and ISGs. We examined the molecular mechanisms of statin-mediated inhibition of antiviral gene expression using murine macrophage-like cell line, J774.1/JA-4. Simvastatin and pitavastatin decreased poly(I:C)-induced expression of IFN-ß and ISGs. Moreover, they repressed poly(I:C)-induced phosphorylation of IFN regulatory factor (IRF) 3 and signal transducers and activators of transcription (STAT) 1, which is involved in Janus kinase (JAK)/STAT signaling. Mevalonate and geranylgeranyl pyrophosphate (GGPP), but not cholesterol, counteracted the negative effect of statins on IFN-ß and ISG expression and phosphorylation of IRF3 and STAT1. The geranylgeranyltransferase inhibitor suppressed poly(I:C)-induced expression of IFN-ß and ISGs and phosphorylation of IRF3 and STAT1. These results suggest that statins suppressed the expression of IFN-ß and ISGs in poly(I:C)-treated hyperlipidemic mice and murine macrophages and that these effects occurred through the inhibition of IRF3 and JAK/STAT signaling in macrophages. Furthermore, GGPP recovered the statin-suppressed IRF3 and JAK/STAT signaling in poly(I:C)-treated macrophages.

Dual TBK1/IKKε inhibitor amlexanox mitigates palmitic acid-induced hepatotoxicity and lipoapoptosis in vitro.

The common causes of Non-alcoholic fatty liver disease (NAFLD) are obesity, dyslipidemia, and insulin resistance. Metabolic disorders and lipotoxic hepatocyte damage are hallmarks of NAFLD. Even though amlexanox, a dual inhibitor of TRAF associated nuclear factor κB (NF-κB) activator-binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), has been reported to effectively improve obesity-related metabolic dysfunctions in mice models, its molecular mechanism has not been fully investigated. This study was designed to investigate the effects of amlexanox on in vitro nonalcoholic steatohepatitis (NASH) model induced by treatment of palmitic acid (PA, 0.4 mM), using a trans-well co-culture system of hepatocytes and Kupffer cells (KCs). Stimulation with PA significantly increased the phosphorylation levels of TBK1 and IKKε in both hepatocytes and KCs, suggesting a potential role of TBK1/IKKε in PA-induced NASH progression. Treatment of amlexanox (50 µM) showed significantly reduced phosphorylation of TBK1 and IKKε and hepatotoxicity as confirmed by decreased levels of lactate dehydrogenase released from hepatocytes. Furthermore, PA-induced inflammation and lipotoxic cell death in hepatocytes were significantly reversed by amlexanox treatment. Intriguingly, amlexanox inhibited the activation of KCs and induced polarization of KCs towards M2 phenotype. Mechanistically, amlexanox treatment decreased the phosphorylation of interferon regulator factor 3 (IRF3) and NF-κB in PA-treated hepatocytes. However, decreased phosphorylation of NF-κB, not IRF3, was found in PA-treated KCs upon amlexanox treatment. Taken together, our findings show that treatment of amlexanox attenuated the severity of PA-induced hepatotoxicity in vitro and lipoapoptosis by the inhibition of TBK1/IKKε-NF-κB and/or IRF3 pathway in hepatocytes and KCs.

Zika Virus Proteins NS2A and NS4A Are Major Antagonists that Reduce IFN-ß Promoter Activity Induced by the MDA5/RIG-I Signaling Pathway.

Zika virus (ZIKV) is a mosquito-transmitted, emerging Flavivirus that causes Guillain-Barré syndrome and microcephaly in adults and fetuses, respectively. Since ZIKV was first isolated in 1947, severe outbreaks have occurred at various places worldwide, including Yap Island in 2007, French Polynesia in 2013, and Brazil in 2015. Although incidences of ZIKV infection and dissemination have drastically increased, the mechanisms underlying the pathogenesis of ZIKV have not been sufficiently studied. In addition, despite extensive research, the exact roles of individual ZIKV genes in the viral evasion of the host innate immune responses remain elusive. Besides, it is still possible that more than one ZIKV-encoded protein may negatively affect type I interferon (IFN) induction. Hence, in this study, we aimed to determine the modulations of the IFN promoter activity, induced by the MDA5/RIG-I signaling pathway, by over-expressing individual ZIKV genes. Our results show that two nonstructural proteins, NS2A and NS4A, significantly down-regulated the promoter activity of IFN-ß by inhibiting multiple signaling molecules involved in the activation of IFN-ß. Interestingly, while NS2A suppressed both full-length and constitutively active RIG-I, NS4A had inhibitory activity only on full-length RIG-I. In addition, while NS2A inhibited all forms of IRF3 (full-length, regulatory domain-deficient, and constitutively active), NS4A could not inhibit constitutively active IRF3-5D. Taken together, our results showed that NS2A and NS4A play major roles as antagonists of MDA5/RIG-I-mediated IFN-ß induction and more importantly, these two viral proteins seem to inhibit induction of the type I IFN responses in differential mechanisms. We believe this study expands our understanding regarding the mechanisms via which ZIKV controls the innate immune responses in cells and may pave the way to development of ZIKV-specific therapeutics.

Inhibition of TLR4/TRIF/IRF3 Signaling Pathway by Curcumin in Breast Cancer Cells.

PURPOSE: Toll-like receptor 4 (TLR4) is over-expressed in breast tumors and thus contributing to the tumor progression and metastasis. Natural products have drawn attention in cancer immunotherapy due to their various biological activities. Curcumin is well investigated in different types of cancer. However, the mechanisms underlying its anti-inflammatory actions have not been extensively elucidated.  For this purpose, we explored the inhibitory effects of curcumin on lipopolysaccharide (LPS)-induced TLR4 dependent TRIF signaling pathway in two subtypes of breast cancer cell lines (MCF-7 and MDA-MB-231) in this study. METHODS: In this context, the cytotoxicity of curcumin and LPS alone and the combination of curcumin with LPS on these cells was evaluated by WST-1 assay.  The expression level of TLR4 and the release of type I interferon (IFN) levels were determined after treatment with curcumin and/or LPS by RT-PCR and ELISA analysis, respectively. Furthermore, the subcellular localization of TLR4 and interferon regulatory factor 3 (IRF3) were detected by immunofluorescence analysis. RESULTS: Curcumin treatment suppressed breast cancer cells viabilities and the activation of TLR4-mediated TRIF signaling pathway by the downregulation of TLR4 and IRF3 expression levels and the inhibition of type I IFN (IFN-α/ß) levels induced by LPS. However, curcumin was more efficient in MDA- MB-231 cells than MCF-7 cells owing to its greater inhibitory efficacy in the LPS- enhanced TLR4 signaling pathway. Furthermore, IFN-α/ß levels induced by TLR4 and IRF3 were decreased in these cells following curcumin treatment. CONCLUSIONS: Consequently, these results demonstrated that the activation of LPS stimulated TLR4/TRIF/IRF3 signaling pathway was mediated by curcumin in breast cancer cells, in vitro. However, more studies are necessary to examine the curcumin's anti-inflammatory activities on TLR4/MyD88/NF-κB as well as other signaling pathways downstream of TLRs in breast cancer.

Establishment and Characterization of a New Cell Line Permissive for Hepatitis C Virus Infection.

Hepatitis C virus (HCV) cell culture systems have facilitated the development of efficient direct-acting antivirals against HCV. Huh-7.5, a subline of the human hepatoma cell line Huh-7, has been used widely to amplify HCV because HCV can efficiently replicate in these cells due to a defect in innate antiviral signalling. Recently, we established a novel cell line, KH, derived from human hepatocellular carcinoma, which showed atypical uptake of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in a Gd-EOB-DTPA-enhanced magnetic resonance imaging study. KH cells expressed hepatocyte markers including microRNA-122 (miR-122) at a lower level than Huh-7.5 cells. We demonstrated that KH cells could support the entire life cycle of HCV; however, HCV replicated at a lower rate in KH cells compared to Huh-7.5 cells, and virus particles produced from KH cells seemed to have some disadvantages in viral assembly compared with those produced from Huh-7.5 cells. KH cells had more robust interferon-stimulated gene expression and induction upon HCV RNA transfection, interferon-α2b addition, and HCV infection than Huh-7.5 cells. Interestingly, both miR-122 supplementation and IRF3 knockout in KH cells boosted HCV replication to a similar level as in Huh-7.5 cells, suggesting that intact innate antiviral signalling and lower miR-122 expression limit HCV replication in KH cells. KH cells will enable a deeper understanding of the role of the innate immune response in persistent HCV infection.

Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-ß production by impairing dsRNA and PACT binding to RIG-I.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-ß production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-ß production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-ß production and transcription factor IRF3 activation, but did not block IFN-ß production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.

TRIM27 Promotes Hepatitis C Virus Replication by Suppressing Type I Interferon Response.

Type I interferon (IFN) response is central for host defense against viral infection. Tripartite motif 27 (TRIM27) is implicated in antiviral innate immune response; however, whether it affects the replication of hepatitis C virus (HCV) and the underlying mechanisms remain uncharacterized. Here, we show that TRIM27 expression is induced in Huh7.5 human hepatoma cells infected with HCV or stimulated with type I IFNs in vitro. In addition, TRIM27 overexpression increases and its knockdown decreases viral RNA and protein levels, suggesting that TRIM27 positively regulates HCV replication. Mechanistically, TRIM27 inhibits type I IFN response against HCV infection through inhibiting IRF3 and NF-κB pathways, since TRIM27 mutant unable to inhibit these two inflammatory pathways fails to promote HCV replication. Taken together, this study identifies TRIM27 as a novel positive regulator of HCV replication, and also implicates that targeting TRIM27 may serve as a therapeutic strategy for controlling HCV replication.

Identification of an IKBKE inhibitor with antitumor activity in cancer cells overexpressing IKBKE.

BACKGROUND: The serine/threonine kinase IKBKE is frequently overexpressed or activated in a variety of human cancers. Ectopic expression of IKBKE induces malignant transformation, cell migration, invasion and chemoresistance. Thus, IKBKE is an attractive target for anti-cancer drug development. METHODS: By screening of NCI Diversity Set and Clinical Collection I and II compound libraries using cell-based assay, we identified several candidates of IKBKE inhibitors, which directly inhibited IKBKE kinase activity in vitro and in vivo. One of them, malachite green oxalate (MCCK1), was further characterized. The mechanism was examined by western blot, immunoprecipitation (IP) and Immunofluorescence. We also evaluated in a mouse xenograft model. In vitro kinase assay and luciferase reporter assay were also performed in our experiments. RESULTS: MCCK1 inhibits IKBKE kinase as well as its downstream targets such as IκBα, p65 and IRF3. MCCK1 is a selective inhibitor for IKBKE, with moderate effect on TBK1, but does not inhibit the activation of IKKα/ß, STAT3, Erk-1/2, p38 or JNK. The inhibition of IKBKE by MCCK1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor aberrant expression of IKBKE. Furthermore, MCCK1 inhibits tumor growth in nude mice of human cancer cells in which IKBKE is elevated but not of those cancer cells in which it is not. CONCLUSION: These data indicate that MCCK1 is an IKBKE inhibitor with anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients with tumors over expressing IKBKE.

Role of miR-148a in Mitigating Hepatic Ischemia-Reperfusion Injury by Repressing the TLR4 Signaling Pathway via Targeting CaMKIIα in Vivo and in Vitro.

BACKGROUND/AIMS: Hepatic ischemia-reperfusion (I/R) injury, which is mainly induced by inflammation and unstable intracellular ions, is a major negative consequence of surgery that compromises hepatic function. However, the exact mechanisms of liver I/R injury have not been determined. Positive crosstalk with the Ca2+/CaMKII pathway is required for complete activation of the TLR4 pathway and inflammation. We previously found that miR-148a, which decreased in abundance with increasing reperfusion time, targeted and repressed the expression of CaMKIIα. In the present study, we examined the role of the miR-148a machinery in I/R-induced Ca2+/CaMKII and TLR4 signaling changes, inflammation, and liver dysfunction in vivo and in vitro. METHODS: Liver function was evaluated by serum aminotransferase levels and hematoxylin-eosin (HE) staining. Inflammatory factors were detected by enzyme-linked immunosorbent assay. Gene and protein expression were assessed by RT-PCR and western blot. Small interfering RNA was used to silence target gene expression. HE staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to measure hepatic tissue apoptosis. These assays were performed to identify factors upregulated in hepatic I/R injury and downregulated by miR-148a. RESULTS: We manifested that expression of CaMKIIα and phosphorylation of TAK1 and IRF3 were elevated in hypoxia/reoxygenation (H/R)-treated primary Kupffer cells (KCs) and liver tissue of I/R-treated mice, but these effects were attenuated by treatment with miR-148a mimic and were accompanied by the alleviation of liver dysfunction and hepatocellular apoptosis. Luciferase reporter experiments showed that miR148a suppressed luciferase activity by almost 60%. Moreover, knockdown of CaMKIIα in H/R KCs led to significant deficiencies in p-TAK1, P-IRF3, IL-6, and TNF-α, which was consistent with the effects of miR-148a overexpression. Otherwise, the same trend of activation of TAK1 and IRF3 and inflammatory factors in vitro was observed in the siTAK1 + siIRF3 group compared with the siCaMKIIα group. CONCLUSION: Taken together, we conclude that miR-148a may mitigate hepatic I/R injury by ameliorating TLR4-mediated inflammation via targeting CaMKIIα in vitro and in vivo.

Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells.

BACKGROUND: Dingchuan tang (asthma-relieving decoction), a formula of nine herbs, has been used for treating respiratory inflammatory diseases for >400 years in the People's Republic of China. However, the mechanisms underlying the anti-inflammatory action of dingchuan tang is not fully understood. This study aims to investigate the effects of Dingchuan tang essential oil (DCEO) on inflammatory mediators and the underlying mechanism of action. MATERIALS AND METHODS: DCEO was extracted by steam distillation. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. Production of nitric oxide (NO) was determined by the Griess test. Protein secretion and mRNA levels of inflammatory mediators were measured by the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Protein levels were examined by Western blot. Nuclear localization of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence analyses. RESULTS: DCEO significantly reduced LPS-triggered production of NO and prostaglandin E2 (PGE2) and decreased protein and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). LPS induced upregulation of protein and mRNA levels of cytokines (interleukin-1ß [IL-1ß], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and chemokines (monocyte chemoattractant protein-1 [MCP-1], chemokine [C-C motif] ligand 5 [CCL-5], and macrophage inflammatory protein [MIP]-1α) were suppressed by DCEO treatment. Phosphorylation and nuclear protein levels of transcription factors (activator protein-1 [AP-1], NF-κB, interferon regulatory factor 3 [IRF3]) were decreased by DCEO. Protein levels of phosphorylated IκB-α, IκB kinase α/ß (IKKα/ß), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), TGF ß-activated kinase 1 (TAK1), TANK-binding kinase 1 (TBK1), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) were lowered by DCEO. Moreover, degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 induced by LPS was inhibited by DCEO treatment. CONCLUSION: Suppression of the interleukin-1 receptor-associated kinase (IRAK)/NF-κB, IRAK/AP-1 and TBK1/IRF3 pathways was associated with the inhibitory effects of DCEO on inflammatory mediators in LPS-stimulated RAW264.7 macrophages. This study provides a pharmacological justification for the use of dingchuan tang in managing inflammatory disorders.

C6orf106 is a novel inhibitor of the interferon-regulatory factor 3-dependent innate antiviral response.

Host recognition of intracellular viral RNA and subsequent induction of cytokine signaling are tightly regulated at the cellular level and are a target for manipulation by viruses and therapeutics alike. Here, we characterize chromosome 6 ORF 106 (C6orf106) as an evolutionarily conserved inhibitor of the innate antiviral response. C6orf106 suppresses the synthesis of interferon (IFN)-α/ß and proinflammatory tumor necrosis factor (TNF) α in response to the dsRNA mimic poly(I:C) and to Sendai virus infection. Unlike canonical inhibitors of antiviral signaling, C6orf106 blocks interferon-regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activity without modulating their activation, nuclear translocation, cellular expression, or degradation. Instead, C6orf106 interacts with IRF3 and inhibits IRF3 recruitment to type I IFN promoter sequences while also reducing the nuclear levels of the coactivator proteins p300 and CREB-binding protein (CBP). In summary, we have defined C6orf106 as a negative regulator of antiviral immunity that blocks IRF3-dependent cytokine production via a noncanonical and poorly defined mechanism. This work presents intriguing implications for antiviral immunity, autoimmune disorders, and cancer.

Feline Herpesvirus 1 US3 Blocks the Type I Interferon Signal Pathway by Targeting Interferon Regulatory Factor 3 Dimerization in a Kinase-Independent Manner.

As a prevalent agent in cats, feline herpesvirus 1 (FHV-1) infection contributes to feline respiratory disease and acute and chronic conjunctivitis. FHV-1 can successfully evade the host innate immune response and persist for the lifetime of the cat. Several mechanisms of immune evasion by human herpesviruses have been elucidated, but the mechanism of immune evasion by FHV-1 remains unknown. In this study, we screened for FHV-1 open reading frames (ORFs) responsible for inhibiting the type I interferon (IFN) pathway with an IFN-ß promoter reporter and analysis of IFN-ß mRNA levels in HEK 293T cells and the Crandell-Reese feline kidney (CRFK) cell line, and we identified the Ser/Thr kinase US3 as the most powerful inhibitor. Furthermore, we found that the anti-IFN activity of US3 depended on its N terminus (amino acids 1 to 75) and was independent of its kinase activity. Mechanistically, the ectopic expression of US3 selectively inhibited IFN regulatory factor 3 (IRF3) promoter activation. Furthermore, US3 bound to the IRF association domain (IAD) of IRF3 and prevented IRF3 dimerization. Finally, US3-deleted recombinant FHV-1 and US3-repaired recombinant FHV-1 (rFHV-dUS3 and rFHV-rUS3, respectively) were constructed. Compared with wild-type FHV-1 and rFHV-rUS3, infection with rFHV-dUS3 induced large amounts of IFN-ß in vitro and in vivo More importantly, US3 deletion significantly attenuated virulence, reduced virus shedding, and blocked the invasion of trigeminal ganglia. These results indicate that FHV-1 US3 efficiently inhibits IFN induction by using a novel immune evasion mechanism and that FHV-1 US3 is a potential regulator of neurovirulence.IMPORTANCE Despite widespread vaccination, the prevalence of FHV-1 remains high, suggesting that it can successfully evade the host innate immune response and infect cats. In this study, we screened viral proteins for inhibiting the IFN pathway and identified the Ser/Thr kinase US3 as the most powerful inhibitor. In contrast to other members of the alphaherpesviruses, FHV-1 US3 blocked the host type I IFN pathway in a kinase-independent manner and via binding to the IRF3 IAD and preventing IRF3 dimerization. More importantly, the depletion of US3 attenuated the anti-IFN activity of FHV-1 and prevented efficient viral replication in vitro and in vivo Also, US3 deletion significantly attenuated virulence and blocked the invasion of trigeminal ganglia. We believe that these findings not only will help us to better understand the mechanism of how FHV-1 manipulates the host IFN response but also highlight the potential role of US3 in the establishment of latent infection in vivo.

A High Content Screen in Macrophages Identifies Small Molecule Modulators of STING-IRF3 and NFkB Signaling.

We screened a library of bioactive small molecules for activators and inhibitors of innate immune signaling through IRF3 and NFkB pathways with the goals of advancing pathway understanding and discovering probes for immunology research. We used high content screening to measure the translocation from the cytoplasm to nucleus of IRF3 and NFkB in primary human macrophages; these transcription factors play a critical role in the activation of STING and other pro-inflammatory pathways. Our pathway activator screen yielded a diverse set of hits that promoted nuclear translocation of IRF3 and/or NFkB, but the majority of these compounds did not cause activation of downstream pathways. Screening for antagonists of the STING pathway yielded multiple kinase inhibitors, some of which inhibit kinases not previously known to regulate the activity of this pathway. Structure-activity relationships (SARs) and subsequent chemical proteomics experiments suggested that MAPKAPK5 (PRAK) is a kinase that regulates IRF3 translocation in human macrophages. Our work establishes a high content screening approach for measuring pro-inflammatory pathways in human macrophages and identifies novel ways to inhibit such pathways; among the targets of the screen are several molecules that may merit further development as anti-inflammatory drugs.

Identification of a small molecule that primes the type I interferon response to cytosolic DNA.

The type I interferon response plays a pivotal role in host defense against infectious agents and tumors, and promising therapeutic approaches rely on small molecules designed to boost this system. To identify such compounds, we developed a high-throughput screening assay based on HEK-293 cells expressing luciferase under the control of Interferon-Stimulated Response Elements (ISRE). An original library of 10,000 synthetic compounds was screened, and we identified a series of 1H-benzimidazole-4-carboxamide compounds inducing the ISRE promoter sequence, specific cellular Interferon-Stimulated Genes (ISGs), and the phosphorylation of Interferon Regulatory Factor (IRF) 3. ISRE induction by ChX710, a prototypical member of this chemical series, was dependent on the adaptor MAVS and IRF1, but was IRF3 independent. Although it was unable to trigger type I IFN secretion per se, ChX710 efficiently primed cellular response to transfected plasmid DNA as assessed by potent synergistic effects on IFN-ß secretion and ISG expression levels. This cellular response was dependent on STING, a key adaptor involved in the sensing of cytosolic DNA and immune activation by various pathogens, stress signals and tumorigenesis. Our results demonstrate that cellular response to cytosolic DNA can be boosted with a small molecule, and potential applications in antimicrobial and cancer therapies are discussed.

Crude Ecklonia cava Flake Extracts Attenuate Inflammation through the Regulation of TLR4 Signaling Pathway in LPS-Induced RAW264.7 Cells.

We investigated the beneficial effects of the crude Ecklonia cava flake (CEF), which is a residual product after polyphenol extraction from Ecklonia cava, on inflammation in LPS-stimulated RAW264.7 cells. A group of five different CEF extracts was obtained by a preparation process using water, hydrochloric acid or temperature. We observed that large-size (>19 kDa) CEF extract, which was extracted with water at 95 °C (CEF-W, 95 °C), suppressed the production of inflammatory cytokines by inhibiting its mRNA expression in LPS-induced RAW264.7 cells. TLR4 signaling involvements were negatively regulated by CEF-W, 95 °C. CEF-W, 95 °C repressed the translocation of NF-κB from cytoplasm into nucleus in LPS-induced RAW264.7 cells. CEF-W, 95 °C attenuated the phosphorylation of TBK1 and IRF3 by inhibiting the phosphorylation of ERK. Taken together, we demonstrated that large-size CEF-W, 95 °C may act as a negative regulator of inflammation through the suppression of TLR4 signaling constituents in LPS-induced RAW264.7 cells.

Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKKε-IRF3 complex.

TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)-(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK-TBK1-IKKε-IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.

Japanese Encephalitis Virus NS5 Inhibits Type I Interferon (IFN) Production by Blocking the Nuclear Translocation of IFN Regulatory Factor 3 and NF-κB.

The type I interferon (IFN) response is part of the first-line defense against viral infection. To initiate replication, viruses have developed powerful evasion strategies to counteract host IFN responses. In the present study, we found that the Japanese encephalitis virus (JEV) NS5 protein could inhibit double-stranded RNA (dsRNA)-induced IFN-ß expression in a dose-dependent manner. Our data further demonstrated that JEV NS5 suppressed the activation of the IFN transcriptional factors IFN regulatory factor 3 (IRF3) and NF-κB. However, there was no defect in the phosphorylation of IRF3 and degradation of IκB, an upstream inhibitor of NF-κB, upon NS5 expression, indicating a direct inhibition of the nuclear localization of IRF3 and NF-κB by NS5. Mechanistically, NS5 was shown to interact with the nuclear transport proteins KPNA2, KPNA3, and KPNA4, which competitively blocked the interaction of KPNA3 and KPNA4 with their cargo molecules, IRF3 and p65, a subunit of NF-κB, and thus inhibited the nuclear translocation of IRF3 and NF-κB. Furthermore, overexpression of KPNA3 and KPNA4 restored the activity of IRF3 and NF-κB and increased the production of IFN-ß in NS5-expressing or JEV-infected cells. Additionally, an upregulated replication level of JEV was shown upon KPNA3 or KPNA4 overexpression. These results suggest that JEV NS5 inhibits the induction of type I IFN by targeting KPNA3 and KPNA4.IMPORTANCE JEV is the major cause of viral encephalitis in South and Southeast Asia, with high mortality. However, the molecular mechanisms contributing to the severe pathogenesis are poorly understood. The ability of JEV to counteract the host innate immune response is potentially one of the mechanisms responsible for JEV virulence. Here we demonstrate the ability of JEV NS5 to interfere with the dsRNA-induced nuclear translocation of IRF3 and NF-κB by competitively inhibiting the interaction of IRF3 and NF-κB with nuclear transport proteins. Via this mechanism, JEV NS5 suppresses the induction of type I IFN and the antiviral response in host cells. These findings reveal a novel strategy for JEV to escape the host innate immune response and provide new insights into the pathogenesis of JEV.