Analog Of Double-stranded RNA Research Articles

Promoting proteasomal degradation of the viral RNA sensor RIG-I

The p97 complex attenuates antiviral signaling by recruiting the E3 ubiquitin ligase RNF125 to RIG-I.

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes.

BackgroundToll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes.MethodsIn the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent l-leucyl-l-leucine methyl ester (L-LME).ResultsFlow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor.ConclusionsTLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0458-6) contains supplementary material, which is available to authorized users.

PHF11 expression and cellular distribution is regulated by the Toll-Like Receptor 3 Ligand Polyinosinic:Polycytidylic Acid in HaCaT keratinocytes.

BackgroundInflammatory skin diseases such as atopic dermatitis and psoriasis represent a complex interaction between the skin and infiltrating immune cells, resulting in damage to the skin barrier and increased inflammation. Polymorphisms in PHF11 have been associated with dermatitis and allergy and PHF11 regulates the transcription of T-cell cytokines as well as class switching to IgE in activated B-cells. The importance of skin barrier homeostasis in the context of inflammatory skin diseases, together with reports identifying PHF11 as an interferon-induced gene, have led us to examine its role in the innate immune response of keratinocytes.ResultsWe developed a cell culture model that allowed us to analyze the effects of the double-stranded RNA analogue poly(I:C) on a confluent cell monolayer immediately after a 24-h treatment, as well as three days after withdrawal of treatment. Immediately after treatment with poly(I:C), PHF11, IL8, and interferon-dependent ISG15 RNA expression was increased. This was accompanied by nuclear localization of PHF11 as well the tight junction protein claudin-1. Knock-down of PHF11 resulted in increased interleukin-8 expression and secretion immediately following treatment with poly(I:C), as well as changes in the cellular distribution of membrane-bound and increased nuclear claudin-1 that was observed up to 3 days after the withdrawal of poly(I:C). This was associated with lower cell density and a decrease in the number of cells in the G1 phase of the cell cycle.ConclusionsIn addition to a role for PHF11 in lymphocyte gene expression, we have now shown that PHF11 was part of the keratinocyte innate immune response by poly(I:C). As knock-down of PHF11 was associated with increased expression of the pro-inflammatory chemokine IL-8 and changes in the cellular distribution of claudin-1, a change normally associated with increased proliferation and migration, we suggest that PHF11 may contribute to epidermal recovery following infection or other damage.

Interferon (IFN) and Cellular Immune Response Evoked in RNA-Pattern Sensing During Infection with Hepatitis C Virus (HCV).

Hepatitis C virus (HCV) infects hepatocytes but not dendritic cells (DCs), but DCs effectively mature in response to HCV-infected hepatocytes. Using gene-disrupted mice and hydrodynamic injection strategy, we found the MAVS pathway to be crucial for induction of type III interferons (IFNs) in response to HCV in mouse. Human hepatocytes barely express TLR3 under non-infectious states, but frequently express it in HCV infection. Type I and III IFNs are induced upon stimulation with polyI:C, an analog of double-stranded (ds)RNA. Activation of TLR3 and the TICAM-1 pathway, followed by DC-mediated activation of cellular immunity, is augmented during exposure to viral RNA. Although type III IFNs are released from replication-competent human hepatocytes, DC-mediated CTL proliferation and NK cell activation hardly occur in response to the released type III IFNs. Yet, type I IFNs and HCV-infected hepatocytes can induce maturation of DCs in either human or mouse origin. In addition, mouse CD8+ DCs mature in response to HCV-infected hepatocytes unless the TLR3/TICAM-1 pathway is blocked. We found the exosomes containing HCV RNA in the supernatant of the HCV-infected hepatocytes act as a source of TLR3-mediated DC maturation. Here we summarize our view on the mechanism by which DCs mature to induce NK and CTL in a status of HCV infection.

Maternal intranasal immunization with HTLV-1 Env induces Env specific antibody responses in breast milk

Human T cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia and Lymphoma (ATLL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus prevalent worldwide with foci of high prevalence. However, effective vaccines or drugs against HTLV-1 infection are not available. Because of the severity of the diseases and the absence of a satisfactory treatment, there is indeed an urgent need for an efficient vaccine against HTLV-1 in endemic regions. HTLV-1 is associated with persistent and lifelong infections and a low incidence of lymphomas within its hosts, and can be spread through the contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. These previous observations suggest that HTLV-1 has common features in infection and protective immune mechanisms with Bovine Leukemia virus (BLV). Imm une protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. Interestingly, previous study revealed that antibodies from the maternal colostrum protect from BLV infection. On the basis of the above reports, we have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and / or lactation. In the present study, we investigated the effectiveness of intranasal vaccine using HTLV-1 Env protein as an antigen in combination with a synthetic double-stranded RNA analog for the induction of specific antibody in serum and breast milk in mother mice. Full-length Env protein was synthesized using the wheat germ cell-free protein production system. Intranasal administration for mother mice with the Env protein and the synthetic double-stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid [poly(I:C)] generated significant Env-specific systemic and mucosal immune responses as evidenced by the prominent induction of serum and milk antibodies. Our results provide the first evidence that intranasal co-administration of HTLV-1 Env protein with the poly(I:C) adjuvant augments the viral-specific immunity against HTLV-1.

The ASK family kinases differentially mediate induction of type I interferon and apoptosis during the antiviral response.

Viral infection activates host defense mechanisms, including the production of type I interferon (IFN) and the apoptosis of infected cells. We investigated whether these two antiviral responses were differentially regulated in infected cells. We showed that the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) apoptosis signal-regulating kinase 1 (ASK1) was activated in cells by the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid [poly(I:C)] and by RNA viruses, and that ASK1 played an essential role in both the induction of the gene encoding IFN-β (IFNB) and apoptotic cell death. In contrast, we found that the MAPKKK ASK2, a modulator of ASK1 signaling, was essential for ASK1-dependent apoptosis, but not for inducing IFNB expression. Furthermore, genetic deletion of either ASK1 or ASK2 in mice promoted the replication of influenza A virus in the lung. These results indicated that ASK1 and ASK2 are components of the antiviral defense mechanism and suggested that ASK2 acts as a key modulator that promotes apoptosis rather than the type I IFN response. Because ASK2 is selectively present in epithelium-rich tissues, such as the lung, ASK2-dependent apoptosis may contribute to an antiviral defense in tissues with a rapid repair rate in which cells could be readily replaced.

Polyinosinic-Polycytidylic Acid Perturbs Ovarian Functions Through Toll-Like Receptor 3-Mediated Tumor Necrosis Factor A Production in Female Mice.

Viral infections may perturb ovarian functions and female fertility. Mechanisms underlying viral perturbation of ovarian functions are incompletely understood. This study found that intraperitoneal injection of polyinosinic-polycytidylic acid [poly (I:C)] in female mice inhibits estradiol synthesis and induces ovarian granulosa cell apoptosis. Poly (I:C) is a synthetic viral double-stranded RNA analog, which induces innate antiviral responses mimicking a viral infection through activation of pattern recognition receptors, including toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated gene 5. Injection of poly (I:C) significantly induced granulosa cell apoptosis in antral follicles and reduced antral follicle numbers. These effects were significantly diminished in Tlr3 knockout or tumor necrosis factor-alpha (Tnfa) knockout mice. We demonstrated that poly (I:C) induced TNFA production at a relatively high level in wild-type mice compared with that in Tlr3 knockout mice. Notably, TNFA neutralizing antibody significantly reduced poly (I:C)-induced ovarian dysfunction. In vitro assays confirmed that TNFA inhibits estradiol synthesis and induces granulosa cell apoptosis. Results provide novel insights into the mechanisms by which a mimicked viral infection perturbs ovarian functions in mice.

Immune responses of porcine airway epithelial cells to poly(I:C), a synthetic analogue of viral double-stranded RNA

Yang, X.-q., Wang, L., Li, H.-t. and Liu, D. 2015. Immune responses of porcine airway epithelial cells to poly(I:C), a synthetic analogue of viral double-stranded RNA. Can. J. Anim. Sci. 95: 13–20. Swine respiratory disease (SRD) is one of the most economically important diseases affecting the pig industry. The main infectious agents that cause SRD are viruses, but the molecular pathogenesis of viral SRD has not been extensively studied. Here, using digital gene expression tag profiling, the global transcriptional responses to poly(I:C), a synthetic analogue of viral double-stranded RNA, was analyzed in porcine airway epithelial cells (PAECs). The profiling analysis revealed numerous differentially expressed genes (DEGs), including unknown sequences in the porcine nucleotide databases. Gene ontology enrichment analysis showed that DEGs were mainly enriched in response to stress (GO: 0006950), of which, defense response is one sub-process. Poly(I:C) challenge induced a general inflammation response as indicated by marked upregulation of a variety of pathogen recognition receptors, interferon-stimulated genes, proinflammatory cytokines, and chemokines, together with the significant downregulation of anti-inflammatory molecules. Furthermore, the antiapoptotic pathway was triggered, as demonstrated by the significant suppression of molecules involved in the induction of apoptosis, together with the significant stimulation of putative inhibitor of apoptosis. The results indicate that PAECs initiated defense against poly(I:C) challenge through the inflammation responses, whereas poly(I:C) can utilize antiapoptotic pathway to evade host defense.

The poly(I:C)-induced maternal immune activation model in preclinical neuropsychiatric drug discovery.

Increasing epidemiological and experimental evidence implicates gestational infections as one important factor involved in the pathogenesis of several neuropsychiatric disorders. Corresponding preclinical model systems based upon maternal immune activation (MIA) by treatment of the pregnant female have been developed. These MIA animal model systems have been successfully used in basic and translational research approaches, contributing to the investigation of the underlying pathophysiological mechanisms at the molecular, cellular and behavioral levels. The present article focuses on the application of a specific MIA rodent paradigm, based upon treatment of the gestating dam with the viral mimic polyinosinic-polycytidilic acid (Poly(I:C)), a synthetic analog of double-stranded RNA (dsRNA) which activates the Toll-like receptor 3 (TLR3) pathway. Important advantages and constraints of this animal model will be discussed, specifically in light of gestational infection as one vulnerability factor contributing to the complex etiology of mood and psychotic disorders, which are likely the result of intricate multi-level gene×environment interactions. Improving our currently incomplete understanding of the molecular pathomechanistic principles underlying these disorders is a prerequisite for the development of alternative therapeutic approaches which are critically needed in light of the important drawbacks and limitations of currently available pharmacological treatment options regarding efficacy and side effects. The particular relevance of the Poly(I:C) MIA model for the discovery of novel drug targets for symptomatic and preventive therapeutic strategies in mood and psychotic disorders is highlighted in this review article.

Poly (I:C) downregulates platelet production and function through type I interferon.

Thrombocytopenia is a frequent complication of viral infections; the underlying mechanisms appear to depend on the identity of the virus involved. Previous research, including reports from our group, indicates that as well as having antiviral activity type I interferons (IFN I) selectively downregulate platelet production. In this study we extended understanding of the role of endogenous IFN I in megakaryo/thrombopoiesis by evaluating platelet and megakaryocyte physiology in mice treated with polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analogue of double-stranded RNA, Toll-like receptor-3 ligand and strong IFNβ inducer. Mice-treated with poly (I:C) showed thrombocytopaenia, an increase in mean platelet volume and abnormal haemostatic and inflammatory platelet-mediated functionality, indicated by decreased fibrinogen binding and platelet adhesion, prolonged tail bleeding times and impaired P-Selectin externalisation, RANTES release and thrombin-induced platelet-neutrophil aggregate formation. These changes were associated with an increase in size and an abnormal distribution of bone marrow megakaryocytes within the vascular niche and were directly correlated with the plasmatic and bone marrow IFNβ levels. All these effects were absent in genetically modified mice lacking the IFN I receptor. Our results suggest that IFN I is the central mediator of poly (I:C)-induced thrombocytopenia and platelet dysfunction and indicate that these abnormalities are due to changes in the last stages of megakaryocyte development. These data provide new evidence for the role of IFN I in megakaryocyte distribution in the bone marrow niches and its influence on thrombopoiesis and haemostasis.

TLR3 engagement induces IRF-3-dependent apoptosis in androgen-sensitive prostate cancer cells and inhibits tumour growth in vivo.

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.

Induction of interferon-induced transmembrane protein 3 gene expression by lipopolysaccharide in astrocytes

Astrocytes are important modulators of the immune and inflammatory reactions in the central nervous system. We have recently demonstrated the role of interferon-induced transmembrane protein 3 (IFITM3) in long-lasting neuronal impairments in mice following neonatal immune challenge by injections of the double-stranded RNA analog polyriboinosinic polyribocytidylic acid. Here, we show that IFITM3 is induced after lipopolysaccharide (LPS) treatment in cultured astrocytes. The induction of IFITM3 by LPS was completely suppressed by the addition of anti-interferon-β (IFN-β) antibody. In addition, neutralization of tumor necrosis factor-α (TNF-α) with its antibody partially inhibited the induction of IFITM3, suggesting that LPS induces IFITM3 through autocrine secretion of IFN-β and TNF-α. Furthermore, experiments using pharmacological inhibitors suggest that LPS induces IFITM3 through activation of TANK-binding kinase 1, p38 mitogen-activated protein kinase, and nuclear factor-κB pathways. Together, these findings may provide new insight into the role of IFITM3 in the pathogenesis of neurodevelopmental diseases associated with immune activation.

Effect of Oroxylin A on Hydrogen Peroxide Production in Polyinosinic-Polycytidylic acid-induced TM4 Mouse Testis Sertoli Cells

The purpose of this study is to investigate the modulatory effect of oroxylin A on hydrogen peroxide production in TM4 mouse testis sertoli cells induced by the synthetic analog of double-stranded RNA [polyinosinic-polycytidylic acid]. Hydrogen peroxide production was measured by dihydrorhodamine 123 (DHR) assay. Oroxylin A significantly inhibited the polyinosinic-polycytidylic acid (PIC)-induced production of hydrogen peroxide for 0.5, 2, 12, 18, and 24 hr incubation at the concentrations of 5, 10, 25, and 50 μM in TM4 (P < 0.05) in dose dependent manner. These results suggest that oroxylin A has a protective effect against PIC-induced cellular toxicity with its inhibition of hydrogen peroxide production in PIC-induced sertoli cells. 

Revealing interactions between polyaza pyridinophane compounds and DNA/RNA polynucleotides by SERS spectroscopy

Surface‐enhanced Raman scattering spectroscopy (SERS) in the near‐infrared region had been applied to study interactions of polyaza pyridinophanes with single stranded RNA and double stranded DNA and RNA polynucleotides. Studied compounds, PYPOD and PHENPOD, differed in the central aromatic moiety, pyridine and phenanthroline, respectively, which linked two cyclic amines. An intense scattering was obtained from molecules adsorbed onto the silver nanoparticles, showing nonlinear concentration dependence in the 6.5 × 10−8−6.5 × 10−5 M range. New bands in spectra of PYPOD/polynucleotide and PHENPOD/polynucleotide mixtures were assigned to vibrational modes of polynucleotide moieties involved in interactions. SERS spectra of both polyaza pyridinophanes with single stranded RNA polynucleotides indicated hydrogen bonding between the small molecules and the nucleic bases of poly A, poly C and poly U, whereas an interaction of only PYPOD with poly G was suggested. SERS spectra implied that both compounds bound into the minor groove of the helical poly dAdT–poly dAdT polynucleotide, while none of polyaza pyridinophanes interacted with the grooves of the poly dGdC–poly dGdC polynucleotide. Nevertheless, intensity ratios suggested intercalation of the phenanthroline moiety of the PHENPOD molecules into the double helix of the guanine‐cytosine polynucleotide. Spectral features also indicated binding of the PYPOD molecules within the major groove of the double stranded RNA analog. Copyright © 2014 John Wiley &amp; Sons, Ltd.

AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

BackgroundThe aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).ResultsCells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.ConclusionAhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Enhancement of interferon induction by ORF3 product of hepatitis E virus.

Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.

Interleukin-1α released from HSV-1 infected skin keratinocytes initiates protective immunity against systemic infection (VIR1P.964)

Abstract Herpes simplex virus-1 (HSV-1) is a common human pathogen that typically causes self-limiting disease. However, neonates and patients with compromised immune systems are at risk of developing potentially fatal systemic disease. Previously we reported that IL-1α is released by keratinocytes treated with poly(I:C), a double stranded RNA analogue often utilized to simulate virus infections. The objective of this study was to examine whether IL-1 signaling plays a role in initiating protective immunity against HSV-1 infections. Extracellular release of IL-1α protein could be detected 6-24 hours after HSV-1 infection of primary human keratinocytes (4-5-fold increase, p&amp;lt;0.01). This release was not associated with a correlating change in mRNA levels suggesting that the cells released a pre-formed pool of protein. HSV-1 triggered activation of inflammatory caspases-1/4 as determined using fluorescence labeled peptides (6-fold, p&amp;lt;0.001); however, the cellular release of IL-1α was independent of caspases-1/4. IL-1R knockout (KO) mice exhibited an increased mortality following cutaneous HSV-1 infection (60 versus 25%, p&amp;lt;0.01). Caspase-1/4 deficiency did not affect survival rates; hence, the protective function of IL-1R must be exclusively due to IL-1α signaling. Recruitment of leukocytes to the infected epidermis was delayed in IL-1R KO mice. Hence, HSV-1 triggered IL-1α release from infected cells appears important for initiating protective immunity against systemic HSV-1 infections.

Innate immune responses and neuroepithelial degeneration and regeneration in the mouse olfactory mucosa induced by intranasal administration of Poly(I:C).

The pathogenesis of postviral olfactory disorder (PVOD) has not been fully elucidated. We investigated morphological changes and innate immune responses in the mouse olfactory mucosa induced by intranasal administration of polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA. Mice received three administrations of saline with or without Poly(I:C), once every 24 h. The olfactory mucosa was harvested at various intervals after the first administration (8 h, 3, 9 and 24 days). In the Poly(I:C) group, the number of apoptotic cells in the olfactory neuroepithelium had increased at 8 h. At 9 days, the olfactory neuroepithelium had severely degenerated and behavioral tests demonstrated that the mice showed signs of olfactory deterioration. At 24 days, the structure of the neuroepithelium had regenerated almost completely. Regarding the innate immune responses, many neutrophils had infiltrated the olfactory neuroepithelium at 8 h and had exuded into the nasal cavity by 3 days. Macrophages had also infiltrated the olfactory neuroepithelium at 8 h although to a lesser extent, but they still remained in the neuroepithelium at 24 days. Poly(I:C)-induced neuroepithelial damage was significantly inhibited by a neutrophil elastase inhibitor and was suppressed in neutropenic model mice. These findings suggest that the secondary damage caused by the neutrophil-mediated innate immune response plays an important role in the pathogenesis of PVOD.

T-cell Ig and ITIM domain regulates natural killer cell activation in murine acute viral hepatitis

Uncontrolled natural killer (NK) cell activation during the early response to acute viral infection can lead to severe immunopathology, and the mechanisms NK cells use to achieve self-tolerance in such contexts are currently unclear. Here, NK cells up-regulated a coinhibitory receptor, T-cell Ig and ITIM domain (TIGIT), during challenge with the viral double-stranded RNA (dsRNA) analog poly I:C. Blocking TIGIT by antibody treatment in vivo or a genetic deficiency in Tigit enhanced NK cell activation and aggravated liver injury in a poly I:C/D-GalN-induced model of acute fulminant hepatitis, suggesting that TIGIT is normally required for protecting against NK cell-mediated liver injury. Furthermore, adoptively transferring Tigit(-/-) NK cells into NK cell-deficient Nfil3(-/-) mice also resulted in elevated liver injury. Reconstituting Kupffer cell-depleted mice with poliovirus receptor (PVR/CD155, a TIGIT ligand)-silenced Kupffer cells led to aggravated liver injury in a TIGIT-dependent manner. Blocking TIGIT in an NK-Kupffer cell coculture in vitro enhanced NK cell activation and interferon-gamma (IFN-γ) production in a PVR-dependent manner. We also found that TIGIT was up-regulated selectively on NK cells and protected against liver injury in an acute adenovirus infection model in both an NK cell- and Kupffer cell-dependent manner. Knocking down PVR in Kupffer cells resulted in aggravated liver injury in response to adenovirus infection in a TIGIT-dependent manner. TIGIT negatively regulates NK-Kupffer cell crosstalk and alleviates liver injury in response to poly I:C/D-GalN challenge or acute adenovirus infection, suggesting a novel mechanism of NK cell self-tolerance in liver homeostasis during acute viral infection.

High Degree of Overlap between Responses to a Virus and to the House Dust Mite Allergen in Airway Epithelial Cells

BackgroundAirway epithelium is widely considered to play an active role in immune responses through its ability to detect changes in the environment and to generate a microenvironment for immune competent cells. Therefore, besides its role as a physical barrier, epithelium affects the outcome of the immune response by the production of various pro-inflammatory mediators.MethodsWe stimulated airway epithelial cells with viral double stranded RNA analogue poly(I:C) or with house dust mite in a time course of 24 hours. In order to determine cytokines production by stimulated cells, we performed multiplex enzyme linked immunosorbant assay (ELISA).ResultsWe demonstrate that the temporal pattern of the genes that respond to virus exposure in airway epithelium resembles to a significant degree their pattern of response to HDM. The gene expression pattern of EGR1, DUSP1, FOSL1, JUN, MYC, and IL6 is rather similar after viral (poly(I:C)) and HDM exposure. However, both triggers also induce a specific response (e.g. ATF3, FOS, and NFKB1). We confirmed these data by showing that epithelial cells produce a variety of similar mediators in response to both poly(I:C) and HDM challenge (IL1-RA, IL-17, IFN-α and MIP1-α), sometimes with a quantitative difference in response (IL2-R, IL-6, IL-8, MCP-1, MIG, and HGF). Interestingly, only four mediators (IL-12, IP-10, RANTES and VEGF) where up-regulated specifically by poly(I:C) and not by HDM. Additionally, we report that pre-exposure to HDM deregulates production of cytokines and mediators in response to poly(I:C).ConclusionsEpithelial cells responses to the HDM-allergen and a virus strongly resemble both in gene expression and in protein level explaining why these two responses may affect each other.