PolyI:C attenuates transforming growth factor-ß signaling to induce cytostasis of surrounding cells by secreted factors in triple-negative breast cancer.

The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-ß that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.

Grass carp (Ctenopharyngodon idella) interferon regulatory factor 8 down-regulates interferon1 expression via interaction with interferon regulatory factor 2 in vitro.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

ORMDL3 regulates poly I:C induced inflammatory responses in airway epithelial cells.

BACKGROUND: Oroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection. METHODS: To investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants,  mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement. RESULTS: ORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells. CONCLUSIONS: ORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

The RNA binding protein Quaking represses host interferon response by downregulating MAVS.

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNß transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNß activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNß induction. Consistently, ectopic expression of RIG-I activates stronger IFNß induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

Bacterial and viral pathogen-associated molecular patterns induce divergent early transcriptomic landscapes in a bovine macrophage cell line.

BACKGROUND: Pathogens stimulate immune functions of macrophages. Macrophages are a key sentinel cell regulating the response to pathogenic ligands and orchestrating the direction of the immune response. Our study aimed at investigating the early transcriptomic changes of bovine macrophages (Bomacs) in response to stimulation with CpG DNA or polyI:C, representing bacterial and viral ligands respectively, and performed transcriptomics by RNA sequencing (RNASeq). KEGG, GO and IPA analytical tools were used to reconstruct pathways, networks and to map out molecular and cellular functions of differentially expressed genes (DE) in stimulated cells. RESULTS: A one-way ANOVA analysis of RNASeq data revealed significant differences between the CpG DNA and polyI:C-stimulated Bomac. Of the 13,740 genes mapped to the bovine genome, 2245 had p-value ≤0.05, deemed as DE. At 6 h post stimulation of Bomac, poly(I:C) induced a very different transcriptomic profile from that induced by CpG DNA. Whereas, 347 genes were upregulated and 210 downregulated in response to CpG DNA, poly(I:C) upregulated 761 genes and downregulated 414 genes. The topmost DE genes in poly(I:C)-stimulated cells had thousand-fold changes with highly significant p-values, whereas in CpG DNA stimulated cells had 2-5-fold changes with less stringent p-values. The highest DE genes in both stimulations belonged to the TNF superfamily, TNFSF18 (CpG) and TNFSF10 (poly(I:C)) and in both cases the lowest downregulated gene was CYP1A1. CpG DNA highly induced canonical pathways that are unrelated to immune response in Bomac. CpG DNA influenced expression of genes involved in molecular and cellular functions in free radical scavenging. By contrast, poly(I:C) highly induced exclusively canonical pathways directly related to antiviral immune functions mediated by interferon signalling genes. The transcriptomic profile after poly(I:C)-stimulation was consistent with induction of TLR3 signalling. CONCLUSION: CpG DNA and poly(I:C) induce different early transcriptional landscapes in Bomac, but each is suited to a specific function of macrophages during interaction with pathogens. Poly(I:C) influenced antiviral response genes, whereas CpG DNA influenced genes important for phagocytic processes. Poly(I:C) was more potent in setting the inflammatory landscape desirable for an efficient immune response against virus infection.

OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.

Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferonresponsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in OAS1-/- and OAS3-/- macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages. [BMB Reports 2019; 52(2): 133-138].

c-Jun N-terminal kinases differentially regulate TNF- and TLRs-mediated necroptosis through their kinase-dependent and -independent activities.

Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.

A haplotype variant of porcine IFIT2 increases poly(I:C)-induced activation of NF-κB and ISRE-binding factors.

Interferon-induced protein with tetratricopeptide repeats (IFIT) 2 is associated with various viral infections and pathogenesis in humans and mice. However, there are few reports on IFIT2 in pigs and the polymorphic information remains unclear. Here, by using a direct PCR sequencing method, we identified four single nucleotide polymorphisms (SNPs), c.259G>A (p.Gly87Ser), c.520T>G (p.Phe174Val), c.571C>T (p.Pro191Ser), and c.879A>G (p.Glu293Glu), for the first time in the coding sequence of the porcine (p) IFIT2 gene from a Chinese local breed (Hebao pig), Western commercial pig breeds (Yorkshire and Landrace), and a Chinese developed breed (Beijing Black pig). SNP c.520T>G (p.Phe174Val) leads to the addition of a tetratricopeptide repeat motif, characteristic structure of the IFIT family. SNPs c.259G>A and c.520T>G are medium polymorphic loci (0.25 < polymorphic information content < 0.5) and distributed differently in Western pig breeds and the Chinese local pig, Hebao, which is well known for its strong resistance to disease. Additionally, they are completely linked. The four SNPs constituted five haplotypes with GTCA and AGCA as dominant. The haplotype variant AGCA, which is mainly present in Hebao pigs, significantly synergized the poly(I:C)-induced activation of transcription factors, including NF-κB and IFN-stimulated response element (ISRE)-binding factors, and the expression of interferon ß, indicating that the variant contributes to the induction or magnitude of the immune response upon viral infection. The data showed that variant AGCA might be useful in improving the resistance of pigs to viruses through marker-assisted selection.

Transcriptome analysis of Macrobrachium rosenbergii intestines under the white spot syndrome virus and poly (I:C) challenges.

Intestine is a primary site of the white spot syndrome virus (WSSV) infection in most crustaceans. To date, little is known about its role in the anti-viral immune response in the freshwater prawn Macrobrachium rosenbergii. In this study, next-generation sequencing was employed to investigate the M. rosenbergii intestine transcriptomes following WSSV or poly I:C challenges. A total of 41.06 M, 39.58 M and 47.00 M clean reads were generated and assembled into 65,340, 71,241 and 70,614 transcripts from the negative control group (NG), WSSV challenge group (WG) and poly I:C treatment group (PG) respectively. Based on homology searches, functional annotation with 7 databases (NR, NT, GO, COG, KEGG, Swissprot and Interpro) for 88,412 transcripts was performed. After WSSV or poly (I:C) challenge, the numbers of up-regulated differentially expressed genes (DEGs) were greater than the down-regulated DEGs. Gene Ontology (GO) classification of the DEGs also distributed similarly, with the same top 10 annotations and were all assigned to the signaling pathways, including spliceosome, Rap1 signaling pathway, proteoglycans, PI3K-Akt signaling pathway, ECM receptor interaction. Results could contribute to a better understanding of the intestinal immune response to viral pathogens.

Phosphatase Cdc25A Negatively Regulates the Antiviral Immune Response by Inhibiting TBK1 Activity.

The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-ß) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-ß stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-ß. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-ß reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity.IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-ß transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.

MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2.

MicroRNAs (miRNAs) are small noncoding RNAs that are crucial posttranscriptional regulators for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA virus infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we showed that expression of cellular miRNA miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses H5N1, A/PR8/H1N1, and Newcastle disease virus (NDV) and transfection with poly(I·C). We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of type I interferon, type III interferon, and interferon-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of the JAK1-STAT3 pathway. Together, these findings highlight that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003 to 2017, H5N1 HPAIV caused 453 human deaths, giving it a high mortality rate (52.74%). This work shows that miR-324-5p suppresses H5N1 HPAIV replication by directly targeting the viral genome (thereby inhibiting viral gene expression) and cellular CUEDC2 gene, the negative regulator of the interferon pathway (thereby enhancing antiviral genes). Our study enhances the knowledge of the role of microRNAs in the cellular response to viral infection. Also, the study provides help in understanding how the host cells utilize small RNAs in controlling the viral burden.

Inhibition of Microprocessor Function during the Activation of the Type I Interferon Response.

Type I interferons (IFNs) are central components of the antiviral response. Most cell types respond to viral infections by secreting IFNs, but the mechanisms that regulate correct expression of these cytokines are not completely understood. Here, we show that activation of the type I IFN response regulates the expression of miRNAs in a post-transcriptional manner. Activation of IFN expression alters the binding of the Microprocessor complex to pri-miRNAs, reducing its processing rate and thus leading to decreased levels of a subset of mature miRNAs in an IRF3-dependent manner. The rescue of Microprocessor function during the antiviral response downregulates the levels of IFN-ß and IFN-stimulated genes. All these findings support a model by which the inhibition of Microprocessor activity is an essential step to induce a robust type I IFN response in mammalian cells.

Genomic structure and molecular characterization of Toll-like receptors 1 and 2 from golden pompano Trachinotus ovatus (Linnaeus, 1758) and their expression response to three types of pathogen-associated molecular patterns.

Toll-like receptors (TLRs) play an essential role in the immune response. Here two Toll-like receptors from golden pompano (Trachinotus ovatus), ToTLR1 and ToTLR2, were characterized, the full-length cDNAs were 3126 bp and 7430 bp, and the deduced proteins consisted of 801 and 825 amino acids, respectively. ToTLR1 and ToTLR2 both contained the typical TLR domain architecture including signal peptide, leucine rich repeat (LRR), C-terminal LRR domain at the extracellular region and Toll/interleukin (IL)-1 receptor (TIR) domain in the cytoplasmic region. ToTLR1 only had one intron and two exons, but ToTLR2 consisted of twelve introns and thirteen exons. The promoters of ToTLR1 and ToTLR2 contained several putative transcription factor binding sites. Phylogenetic analysis showed that ToTLR1 and ToTLR2 were clustered into the clade of TLR1 and TLR2, respectively. Tissues distribution analysis indicated that both genes were ubiquitously expressed in all examined tissues, with higher expression levels observed in blood, head-kidney and spleen. After injection with poly inosinic:cytidylic [poly(I:C)], flagellin and lipopolysaccharides (LPS), ToTLR1 and ToTLR2 mRNAs were significantly up-regulated in the immune related tissues, indicating the possible the role of ToTLR1 and ToTLR2 in defense against pathogenic microbes. Further research should be carried out to identify ligands of fish TLR1 and TLR2 in order to understand the function of these receptors.

A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR.

Bats are known to harbor many zoonotic viruses, some of which are pathogenic to other mammals while they seem to be harmless in bats. As the interferon (IFN) response represents the first line of defense against viral infections in mammals, it is hypothesized that activation of the IFN system is one of the mechanisms enabling bats to co-exist with viruses. We have previously reported induction of type I IFN in a cell line from the common vampire bat, Desmodus rotundus, upon polyinosinic:polycytidylic acid (poly(I:C)) stimulation. To deepen our knowledge on D. rotundus' IFN-I antiviral response, we molecularly characterized three interferon-stimulated genes (ISGs), OAS1, PKR and ADAR1, closely implicated in the IFN-I antiviral response, and tested their functionality in our cellular model. We first found that D. rotundus encoded two OAS1 paralogs, OAS1a and OAS1b, and that the functional domains of the four ISGs characterized were highly conserved with those of other mammals. Despite their significant transcription level in the absence of stimulation, the transcription of the four ISGs characterized was enhanced by poly(I:C). In addition, the transcription of OAS1a and OAS1b appears to be differentially regulated. These findings demonstrate an active ISG antiviral response in D. rotundus in which OAS1b may play an important role.

TLR3 Activation of Hepatic Stellate Cell Line Suppresses HBV Replication in HepG2 Cells.

There is limited information about the role of hepatic stellate cells (HSCs) in the liver innate immunity against hepatitis B virus (HBV) infection. We thus examined whether hepatic stellate cell line (LX-2) can be immunologically activated and produce antiviral factors that inhibit HBV replication in HepG2 cells. We found that LX-2 cells expressed the functional Toll-like receptor 3 (TLR3), activation of which by PolyI:C resulted in the selective induction of interferon-ß (IFN-ß) and IFN-λs, the phosphorylation of IFN regulatory factor 3 (IRF3) and IRF7. When HepG2 cells were treated with supernatant (SN) from PolyI:C-activated LX-2 cells, HBV replication was significantly inhibited. IFN-ß and IFN-λ appeared to contribute to LX-2 SN-mediated HBV inhibition, as the antibodies to IFN-ß and IFN-λ receptors could largely block the LX-2 SN action. Mechanistically, LX-2 SN treatment of the HepG2 cells induced a number of antiviral IFN-stimulated genes (ISGs: ISG20, ISG54, ISG56, OAS-1, Trim22, and Trim25) and facilitated the phosphorylation of STATs. These observations support further studies on the role of HSCs in the liver innate immunity against HBV infection.

Interferon induces interleukin 8 and bone marrow stromal cell antigen 2 expression, inhibiting the production of hepatitis B virus surface antigen from human hepatocytes.

Hepatitis B virus (HBV) surface antigen (HBsAg) loss is one of the treatment goals of chronic HBV infection. Bone marrow stromal cell antigen 2 (BST2) is one of the interferon (IFN)-stimulated genes (ISGs) and inhibits the release of various enveloped viruses. Here we examined the effects of antiviral treatment on HBsAg levels and its intracellular mechanism in HBsAg-producing hepatocytes. In PLC/PRF/5 and Huh1, IFNα-2a treatment decreased HBsAg levels in their conditioned media. Upregulation of interleukin 8 (IL8), toll-like receptor 2 (TLR2) and interferon gamma-induced protein 10 (IP10) mRNAs was associated with the reduction of HBsAg in both PLC/PRF/5 and Huh1. The HBsAg level was upregulated by knockdown of IL8, TLR2 or IP10. Exogenous addition of IL8 enhanced BST2 promoter activity and BST2 mRNA expression. Additionally, knockdown of IL8 could lead to the downregulation of BST2 mRNA. Transfection of poly(I-C) enhanced IL8 and BST2 mRNA expression and inhibited HBsAg secretion from PLC/PRF/5 cells. In conclusion, IL8 might play an important role in the enhancement of BST2 and be involved in HBsAg eradication.

Epidermal keratinocytes sense dsRNA via the NLRP3 inflammasome, mediating interleukin (IL)-1ß and IL-18 release.

Skin epidermis, in addition to its barrier function, is able to actively sense harmful pathogens using pattern recognition receptors. In immune cells, the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome can mediate innate immunity against viral infection via a mechanism involving viral dsRNA recognition. Epidermal keratinocytes express NLRP3 inflammasome, which can sense contact sensitizers and mite allergens, leading to pro-interleukin (IL)-1ß and pro-IL-18 cleavage into their active forms. Skin often faces viral infection. However, it is unknown whether viral dsRNA can be detected by the keratinocyte NLRP3 inflammasome. We transfected polyinosinic:polycytidylic acid (poly I:C), a synthetic viral dsRNA analogue, into cultured primary human keratinocytes at the aid of Lipofectamine 2000, and found that transfected poly I:C activated caspase-1 and induced caspase-1-dependent release of IL-1ß and IL-18, which were suppressed on transfection with NLRP3 siRNA. The activation of keratinocyte NLRP3 inflammasome by transfected poly I:C was dependent on dsRNA-induced protein kinase (PKR) activation, and priming with type I interferons upregulated NLRP3 inflammasome activation through promoting PKR activation in poly I:C-transfected keratinocytes. In conclusion, the NLRP3 inflammasome can act as a sensor of dsRNA in epidermal keratinocytes, which may be important in both skin innate immune defense against viral infection and skin inflammation.

PolyICLC Exerts Pro- and Anti-HIV Effects on the DC-T Cell Milieu In Vitro and In Vivo.

Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4ß7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity.

Poly (I:C) transfection induces mitochondrial-mediated apoptosis in cervical cancer.

Polyinosinic acid:polycytidylic acid, known as poly (I:C), is an analogue of double­stranded RNA, which exhibits direct antitumor effects against several types of cancer. The present study aimed to evaluate the role of poly (I:C) in the apoptosis of cervical cancer cells. The HeLa human cervical cancer cell line was used in the present study, and cell apoptosis was determined following poly (I:C) transfection. Furthermore, the mRNA levels of interferon (IFN)­ß, the production of reactive oxygen species (ROS), DNA damage, mitochondrial membrane potential (∆Ψm) and the release of cytochrome c, as well as caspase activation, were determined. The effect of IFN­ß on poly (I:C) transfection­mediated apoptosis was also examined by IFN­ß knockdown. The results showed that poly (I:C) transfection markedly induced HeLa apoptosis, increased the protein levels of pro­apoptotic B cell lymphoma­2 (Bcl­2)­associated X protein (Bax) and BH3 interacting­domain death agonist (Bid), and suppressed the protein expression levels of anti­apoptotic Bcl­2 and Survivin. However, poly (I:C) transfection increased the mRNA levels of IFN­ß, induced ROS production and increased the levels of phosphorylated γH2A.X, an indicator of DNA damage. In addition, poly (I:C) transfection decreased ∆Ψm, triggered the release of cytochrome c from the mitochondria to the cytosol, and induced caspase­9 and ­3 activation. IFN­ß knockdown decreased the poly (I:C)­induced production of ROS and DNA damage, restored ∆Ψm and cytochrome c release, and suppressed caspase­9 and ­3 activation, thereby suppressing poly (I:C)­mediated apoptosis in the HeLa cells. Together, the results of the present study demonstrated that poly (I:C) transfection induced IFN­ß, contributing to ROS production, DNA damage, and caspase­9 and ­3 activation in the HeLa cervical cancer cell line, leading to mitochondrial­mediated apoptosis.