Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.     

Interferon‐α curbs production of interleukin‐22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi

Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)‐α, either endogenously produced after exposure of cells to toll‐like receptor‐9‐activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti‐bacterial interleukin (IL)‐1/IL‐22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN‐α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

Influence of type‐I Interferon receptor expression level on the response to type‐I Interferons in human pancreatic cancer cells

Pancreatic cancer is a highly aggressive malignancy with limited treatment options. Type‐I interferons (e.g. IFN‐α/‐β) have several anti‐tumour activities. Over the past few years, clinical studies evaluating the effect of adjuvant IFN‐α therapy in pancreatic cancer yielded equivocal results. Although IFN‐α and ‐β act via the type‐I IFN receptor, the role of the number of receptors present on tumour cells is still unknown. Therefore, this study associated, for the first time, in a large panel of pancreatic cancer cell lines the effects of IFN‐α/‐β with the expression of type‐I IFN receptors. The anti‐tumour effects of IFN‐α or IFN‐β on cell proliferation and apoptosis were evaluated in 11 human pancreatic cell lines. Type‐I IFN receptor expression was determined on both the mRNA and protein level. After 7 days of incubation, IFN‐α significantly reduced cell growth in eight cell lines by 5–67%. IFN‐β inhibited cell growth statistically significant in all cell lines by 43–100%. After 3 days of treatment, IFN‐β induced significantly more apoptosis than IFN‐α. The cell lines variably expressed the type‐I IFN receptor. The maximal inhibitory effect of IFN‐α was positively correlated with the IFNAR‐1 mRNA (P < 0.05, r = 0.63), IFNAR‐2c mRNA (P < 0.05, r = 0.69) and protein expression (P < 0.05, r = 0.65). Human pancreatic cancer cell lines variably respond to IFN‐α and ‐β. The expression level of the type‐I IFN receptor is of predictive value for the direct anti‐tumour effects of IFN‐α treatment. More importantly, IFN‐β induces anti‐tumour effects already at much lower concentrations, is less dependent on interferon receptor expression and seems, therefore, more promising than IFN‐α.     

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN‐I) during viral infections, in response to triggering of endosomal Toll‐like receptors (TLRs) 7 or 9 by viral single‐stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN‐I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN‐I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN‐I downstream of the TLR9‐to‐MyD88‐to‐IRF7 signaling pathway without requiring IFN‐I positive feedback, high IRF7 expression, or AP3‐driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN‐I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.     

Role of autophagy in inhibiting the proliferation of A549 cells by type III interferon

Interferons (IFNs) have anti‐viral and anti‐tumour effects. Type III interferon, as a member of the recently discovered interferon family, has been proved to inhibit tumour proliferation and promote the apoptosis of various tumour cells. However, whether type III IFN could inhibit the proliferation of lung cancer was not clear. In this study, we found that interferon λ (IFN λ) could inhibit the proliferation of A549 cells and induce autophagy and apoptosis of A549 cells. IFN λ could promote the expression of autophagy gene Beclin1 and interfere the expression of autophagy gene Beclin1 with small interfering RNA, thus inhibiting the effect of type III interferon on anti‐proliferation and promoting apoptosis of lung cancer cell. These results suggested that IFN λ could inhibit the proliferation of A549 cells by activating autophagy pathway, and IFN λ might be one of the potential therapeutic drugs for lung cancer.     

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1‐caspase‐1 axis

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.     

Identification of lncRNA‐155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection

Long noncoding RNAs (lncRNAs) are single‐stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)‐155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA‐155. Here, we observed that expression of lncRNA‐155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA‐155 was also induced by infections with several other viruses. Disruption of lncRNA‐155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA‐155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA‐155 in human cells suppressed IAV replication, suggesting that lncRNA‐155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA‐155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA‐155 resulted in higher production of interferon‐beta (IFN‐β) and several critical interferon‐stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA‐155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B‐mediated interferon response.     

Super‐infection with Staphylococcus aureus inhibits influenza virus‐induced type I IFN signalling through impaired STAT1‐STAT2 dimerization

Bacterial super‐infections are a major complication in influenza virus‐infected patients. In response to infection with influenza viruses and bacteria, a complex interplay of cellular signalling mechanisms is initiated, regulating the anti‐pathogen response but also pathogen‐supportive functions. Here, we show that influenza viruses replicate to a higher efficiency in cells co‐infected with Staphylococcus aureus (S. aureus). While cells initially respond with increased induction of interferon beta upon super‐infection, subsequent interferon signalling and interferon‐stimulated gene expression are rather impaired due to a block of STAT1‐STAT2 dimerization. Thus, S. aureus interrupts the first line of defence against influenza viruses, resulting in a boost of viral replication, which may lead to enhanced viral pathogenicity.     

The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV‐1 Tat

The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double‐stranded RNA (dsRNA) as a common inducer molecule. The non‐structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules. Here, we show that this plant viral RSS lacks IFN antagonistic activity, yet it is able to substitute the RSS function of the Tat protein of human immunodeficiency virus type 1. An NS3 mutant that is deficient in RNA binding and its associated RSS activity is inactive in this complementation assay. This cross‐kingdom suppression of RNAi in mammalian cells by a plant viral RSS indicates the significance of the antiviral RNAi response in mammalian cells and the usefulness of well‐defined RSS proteins.     

Application of Concanavalin A during immune responsiveness skin‐swelling tests facilitates measurement interpretation in mammalian ecology

The skin‐swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro‐inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin‐swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin‐swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro‐inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72‐hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro‐inflammatory cytokine (interleukin 6 [IL‐6] and interferon gamma[IFN‐γ]) expression than PHA during peak response (24‐h post‐treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin‐swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin‐swelling test results.     

Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors

Background

Interferon‐α2 (IFNα2) is routinely used for anti‐hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half‐life, relatively low local concentration and strong side‐effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector‐delivered IFNα1 was tested for its anti‐HBV effects.

Methods

Adeno‐associated viral vector (AAV‐IFNα1) was generated to deliver the IFNα1 gene into hepatocytes. IFNα1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme‐linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real‐time polymerase chain reaction.

Results

AAV‐IFNα1 effectively transduced HBV‐producing cells (HepAD38) and mouse hepatocytes, where IFNα1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV‐producing mice, the concentration of IFNα1 in the liver was eight‐fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten‐fold from day 1–5, and dropped to an undetectable level on day 9 in the AAV‐IFNα1 group. Concurrently, the level of viral DNA decreased over 30‐fold for several weeks.

Conclusions

A single dose administration of AAV‐IFNα1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV‐IFNα1 might be a potential alternative strategy for anti‐HBV therapy. Copyright © 2008 John Wiley & Sons, Ltd.

     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.     

A chloroplast‐derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti‐Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34‐kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN‐γ, IL‐4 and IL‐10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4‐specific serum antibodies and secretion of IFN‐γ, IL‐4 and IL‐10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.