A Dual Functional HBx-3p-siRNA Reverses HBV-mediated Immune Tolerance And Its Molecular Mechanisms

Object:RNA interference (RNAi) of is a conservative mechanism to defense exogenous nucle acid and transposon in plants and lower animals. Generally, endogenous RNAi pathway is usually guided mainly in two ways by short interfering RNA (siRNA) and microRNA (miRNA), the latter was derived from non-perfect matched non-coding RNA that are transcribed from the genome. With the development of RNAi, scientists gradually investigated the mechanism of RNAi.Recently, the use of RNA interference (RNAi) technology has provided insights into HBV pathogenesis through inhibition of viral gene expression and replication in vitro and in vivo. Moreover, use of short interfere RNAs (siRNAs), which are double-stranded RNA molecules and the ligands of intercellular innate immune receptors such as TLR-3 and RIG-I, may activate innate immunity by "off-target" immunostimulatory effects. The exact mechanism of how the innate immune system recognizes siRNA and induces production of IFN and inflammatory cytokines is not clearly defined. Early studies showed that the activation of TLR pathways is especially important, among which TLR7 and TLR8, existing in the endosome, recognize ssRNA and certain siRNA sequences; and their activation subsequently stimulates nuclear factor-KB (NF-κB), mitogen-activated protein Kinase (MAPK) and other signal pathways.The HBx protein is a multifunctional protein that co-activates transcription of viral and cellular genes and coordinates the balance between cellular proliferation and apoptosis. Therefore, the HBx gene is an ideal target for RNA silencing of chronic HBV infection and HBV-related hepatocellular caicinoma. HBV can escape from the host innate and adaptive immunity. Thus, determining how to reverse HBV-induced immune tolerance has become an important approach in treatment of CHB.It was suggested that decreasing the HBV load could potentially benefit a patient by reversing the virally-induced immune tolerance. Recently, the use of RNA interference (RNAi) technology has provided insights into HBV pathogenesis through inhibition of viral gene expression and replication in vitro and in vivoIn this study, we compared the ability of conventional HBx-siRNAs with in vitro transcribed 5’-end triphosphate HBx-siRNAs (3p-HBx-siRNAs) in inducing anti-HBV innate immunity in addition to their ability to inhibit HBV replication. Our results demonstrate that 3p-HBx-siRNA has a strong dual function on inhibiting HBV replication and on triggering innate immunity in a RIG-I-dependent manner which synergistically benefits the reverse of HBV-induced immune tolerance. And we aalso concluded that PKR is involved in the innate immune effects mediated by HBx-siRNAs and further contributes to HBV inhibition. The bifunctional siRNAs with both gene silencing and innate immune activation properties may represent a new potential strategy for treatment of HBV.Methods:Firstly, we observed that HBV RNA sequence-unrelated 3p-scramble-siRNA or 3p-GAPDH-siRNA transfection significantly increased the expression of RIG-I mRNA in HepG2.2.15 cells after 24 h by Q-RT-PCR. Supernatant HBsAg and HBeAg contents were measured by ELISA. We then combined the immunostimulatory functions of 3p-scramble and HBx silencing function of HBx-siRNA by producing 3p-HBx-siRNA by Q-RT-PCR and ELISA. In order to further see if 3p-HBx-siRNA reverse the HBV-induced immunotolerance, we compared the innate immune response of HepG2 cells with that of HBV-transfected HepG2.2.15 after stimulation with immunostimulatory HBV RNA sequence-unrelated siRNA or immunostimulatory HBx RNAi Q-RT-PCR and ELISA.Secondly, quantitative real-time PCR was used to analyze the mRNA expression of PKR, after HBx-siRNAs treatment. Western Blotting analysis of the activation of the protein kinase R (PKR) and PKR substrate, translation initiation factor 2 (eIF2)a, from HepG2.2.15 cells. HepG2.2.15 cells were treated with 2-aminopurine (2-AP) or transfected with full-length PKR plasmid, levels of HBx RNA mRNA and HBsAg proteins in supernatants were examined as described previously.At last, HBV-carrier mice were obtained by hydrodynamic tail vein injection with HBV plasmid DNA. Primary mouse hepatocytes were isolated from HBV-carrier mice and then transfected with LF, HBx-siRNA or 3p-HBx-siRNA, respectively. HBx, HBs/p and HBc/p, and IFN-a, IFN-βand RIG-I were examined by Q-RT-PCR. HBV inhibiting and immunostimulatory effects of 3p-HBx-siRNA on HBV-carrier mice. Three days after HBx-siRNA or 3p-HBx-siRNA injection, HBV-carrier mice were sacrificed and the serum contents of HBsAg or serum IFN-α/βlevel were measured by radioimmunoassay or ELISA. Intrahepatic HBcAg expression was assayed by immunohistochemical staining. Hepatic lymphocytes were labeled with anti-NK1.1, anti-CD3, anti-CD69 and NKG2D antibodies and were analyzed by FACS. Representative photographs for H&E-stained, paraffin-embedded liver sections at the indicated days after siRNA or 3p-siRNA injection were shown, and serum ALT/AST levels were measured. Results:Part 1:Reverse of HBV-induced immune tolerance by an immunostimulatory 3p-HBx-siRNA in a retinoic acid inducible gene I (RIG-I)-dependent manner1. The in vitro transcription of siRNAThree chemically synthesized siRNAs targeting HBx gene and their corresponding in vitro transcribed 3p-siRNAs using T7 RNA polymerase were designed and produced. 3p-GAPDH (specific for GAPDH) and 3p-scramle (a random sequence without any target RNA sequence) were also designed and produced. The feature of 3p-siRNAs was confirmed by using 4% agarose gel electrophoresis.2. Immunostimulatory effect of sequence-unrelated 3p-siRNA on HBV-induced HepG2.2.15 cellsBy quantitative real-time PCR analysis, we found that relative to LF-treated cells, HBV RNA sequence-unrelated 3p-scramble-siRNA or 3p-GAPDH-siRNA transfection significantly increased the expression of RIG-I mRNA in HepG2.2.15 cells after 24 h. The activation of innate immune responses by 3p-scramble-siRNA or 3p-GAPDH-siRNA might partially inhibit HBV replication through RNA interference-unrelated but extrinsic innate immune pathway.3. The antiviral effect and innate immune response induced by chemically synthesized HBx-siRNAsChemically synthesized HBx-siRNA were transfected into HepG2.2.15 cells. As expected, HBx-siRNAs significantly inhibited HBx and HBs/p mRNA expression at 24 h post transfection, and the secretion of HBsAg and HBeAg was significantly decreased at 48 h post transfection with a weak effect on RIG-I-related typeⅠIFN response.4. Innate immune responses induced by 3p-HBx-siRNAWe observed that all these 3p-HBx-siRNAs obviously activated innate immune responses by markedly increasing type I IFN production, IFN-(3 promoter function and inflammatory secretion, and obviously decreasing the production of immunosuppressive cytokines. We also observed that RIG-I expression was augmented and the expressions of typeⅠIFN-induced genes (MxA and ISG15) were increased at 24h post transfection with 3p-HBx-siRNAs.5. Immunostimulatory 3p-HBx-siRNA exerted stronger inhibitory effect on HBV replication3p-HBx-siRNA (3p-siRNA3) was more effective in inhibiting HBV replication than HBx-siRNA, showing significantly lower expression of HBx mRNA and HBs/p mRNA of cells at 24 h post transfection. Importantly, treatment with 3p-HBx-siRNAs resulted in a greater reduction in HBsAg and HBeAg at 48 h.6. Immunostimulatory 3p-HBx-siRNA improved the inversion of HBV-induced innate immune toleranceIt was noted that 3p-scramble exerted a similar effect on typeⅠIFNs production as 3p-scramble+scramble, indicating scramble-siRNA itself exerted a very weak stimulation to RIG-I.3p-HBx-siRNA alone had a most strong stimulation to IFNs production, a little higher than 3p-scramble+HBx-siRNA, demonstrating that 3p-HBx-siRNA has a dual function which is beneficial to reversing of HBV-induced immunotolerance.7. Immunostimulatory function of 3p-HBx-RNA correlates to RIG-I-dependent typeⅠIFN productionOver-expression of RIG-I further promoted the inhibition of HBV replication by 3p-HBx-siRNAs via activation of the RIG-I pathway. RIG-I activation correlates to the stronger inhibition of HBV replication by 3p-HBx-siRNAs, and over-expression of RIG-I promotes antiviral effects by 3p-HBx-siRNAs. The results demonstrated that blockade of IFN receptors significantly attenuated the inhibitory effect of 3p-HBx-siRNAs on expression of HBx mRNA and the secretion of HBsAg and HBeAg. Part 2:Involvement of Activation of PKR in HBx-siRNA-mediated Innate Immune Effects on HBV Inhibition1. HBx-siRNAs inhibit HBV expression in HepG2.2.15 cellsWe observed that treatment with HBx-siRNAs resulted in decrease in HBx expression, By Western Blotting analysis, we also observed the significant silencing effect of HBx-siRNAs on HBx expression at protein level, A dose-dependent effect was observed.2. Innate immune responses induced by HBx-siRNAs in HepG2.2.15 cellsSynthesis of mRNA for IFNa, IFNβ, and interferon-stimulated genes (ISG) were significantly induced as a consequence of HBx-siRNA transfection, induction of IFN responses is in a concentration-dependent manner, he expression of inflammatory-related chemotactic factors, such as CXCL2, CCL4 and CXCL10, did not change signficantly after HBx-siRNA transfection.3. HBx-siRNAs promote the activation of PKR and its downstream signal pathwayRT-PCR analysis demonstrated that transfection of HepG2.2.15 cells with HBx-siRNA did increase the expression of PKR at the mRNA level. Western Blotting analysis demonstrated that transfection with HBx-siRNA, but not with scramble siRNA, promoted the phosphorylation of PKR, and that the PKR substrate eIF2a is also transiently phosphorylated.4. PKR is involved in the innate immune responses induced by HBx-siRNAs in HepG2.2.15 cellsThe induction of IFN-a and IFN-βmRNA after HBx-siRNA transfection was attenuated significantly, p-Statl activation was also suppressed after 2-AP treatment as assayed by flow cytometry. These results suggested the participation of PKR, which contributed to the immune responses triggered by HBx-siRNAs in HepG2.2.15 cells.5. PKR activation contributes to HBV inhibition in HepG2.2.15 cellsThe suppression of HBx expression was attenuated significantly by treatment with 2-AP. We then detected the levels of HBx and HBs/p in HepG2.2.15 cells that were transfected with PKR followed by stimulation with HBx-siRNAs. The levels of HBx and HBs/p were decreased more obviously in cells with over-expression of PKR. Part 3:Activation of innate immune response by HBx-siRNA and 3p-HBxsiRNA contributed HBV inhibition in HBV mouse1. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infectionHBV-carrier mice were obtained by hydrodynamic tail vein injection with HBV plasmid DNA.The rate of HBV positive mice is around 80%.2. HBV inhibiting and immunostimulatory effects of 3p-HBx-siRNA on freshly isolated primary HBV-containing hepatocytesThe fresh mouse hepatocytes were isolated and transfected with HBx-siRNA and 3p-HBx-siRNAs, respectively. Treatment with 3p-HBx-siRNAs resulted in significantly more reduction of HBx, HBs/p and HBc/p expression, and also significantly higher mRNA levels of IFN-a, IFN-βand RIG-I than HBx-siRNA, indicating that 3p-HBx-siRNA exerts more inhibitory effects of HBV replication and type I IFN production than HBx-siRNA in primary HBV+ hepatocytes of mouse.3. HBV inhibiting and immunostimulatory effects of 3p-HBx-siRNA on HBV-carrier miceWe treated HBV-carrier mice with 3p-HBx-siRNA or HBx-siRNA on days 3,6 and 9, respectively. It was noted that serum HBsAg was significantly decreased in HBV-carrier mice by 3p-HBx-siRNA than by HBx-siRNA. These results suggested that 3p-HBx-siRNAs exerts HBV-specific gene silencing effects.4.3p-HBx-siRNA induces CD69 and NKG2D expression in liver NK cells.3p-siRNA treatment induced high systemic levels of IFN-a and IFN-β.3p-siRNA significantly induced NK cells activation, whereas siRNA was inactive.3p-RNA did not induce CD69 and NKG2D in T cells. Next, we studied 3p-siRNA-induced innate immune responses in C57BL/6 mice in vivo.5. HBx-siRNA or 3p-HBx-siRNA treatment did not induce hepatitis in mice siRNA or 3p-siRNA treatment did not elevate serum ALT levels in mouse serum compared with the levels in untreated mice Such effect was further confirmed by pathology. massive necroses were not been observed in RNA-treated groups. Furthermore, lymphocyte infiltration were observed in RNA-treated groups, and 3p-siRNA conferred enhanced lymphocyte infiltration. ConclusionFirstly,3p-HBx-siRNAs inhibited HBV replication not only by direct silencing of HBx expression but also by enhancing type I IFN production and inflammatory cytokines through RIG-I activation, and the adjuvant effects of inflammatory cytokines could potentially prime the adaptive immunity against HBV.Secondly, we concluded that PKR is involved in the innate immune effects mediated by HBx-siRNAs and further contributes to HBV inhibition.At last, HBx-siRNA and 3p-HBx-siRNAs not only exerts HBV-specific gene silencing effects in fresh mouse hepatocytes and in mouse model, but also activated liver lymphocyte and induced IFN-α/βsecretion.In our research, we discovered the decrease in HBV load, the increase in activation and function of type I IFNs and the priming of the anti-HBV adaptive immunity may finally benefit to reversal of HBV-induced immune tolerance. The bifunctional siRNAs with both gene silencing and innate immune activation properties represent a new potential strategy for treatment of viral infection.