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  • br Material and methods br Results br Discussion HBV


    Material and methods
    Discussion HBV develops several strategies to suppress antiviral immune responses. Here, we used a bifunctional siRNA molecule to silence HBx and activate RIG-I, which induced HBV inhibition partly mediated by RIG-I in HBV-carrier mice. Furthermore, this 3p-siHBx could induce DC maturation and NK cell activation, and reverse HBV-induced CD8+ T cell exhaustion. These data clearly indicate that RNAi-mediated sequence-specific degradation of HBV RNA and RIG-I responses are major antiviral mechanisms in HBV-carrier mice. A major hurdle to treating HBV is the profound immunosuppression, both systemically and locally, within the liver immune microenvironment through different pathways (Chyuan and Hsu, 2018; Shin et al., 2016). For example, HBx disrupts the interaction between RIG-I and TRAF3 (TNF receptor–associated factor 3) and finally dampens type I IFN induction. HBV polymerase inhibits RIG-I–induced IFN-α/β induction by interfering with IRF-3 phosphorylation, dimerization, and nuclear translocation, and suppressing TBK1/IKKε (TANK-binding kinase 1/inhibitor of NF-κB kinase) and DDX3 (DEAD-box helicase 3, X-linked) interaction in human hepatocytes (Jiang and Tang, 2010; Wang et al., 2010). RIG-I activation leads to type I IFN responses, triggering the innate and adaptive immune responses. We previously demonstrated that 3p-siHBx exerted potent anti–HBV efficacy via HBx silencing and RIG-I activation (Han et al, 2011). We found HBx-siRNA has obvious HBx silencing but weak RIG-I activation effect. On the contrary, 3p-scramble siRNA has week HBV silencing but strong RIG-I activation effect by their 5′-triphosphate structure. In addition to its direct HBx silencing, the bifunctional siRNA (3p-siHBx) exerts more inhibitory effect on HBV replication, which derived from its arousal of IFN response by activation of RIG-I pathway. The potential of bifunctional 3p-siRNAs has also been studied in several other models, including influenza A Virus, coxsackievirus, Nodamura zcl infection (Fan et al., 2015; Lin et al., 2012). Also, several studies reported bifunctional 3p-siRNA (Exp:targeting Bcl 2/TGF-β/Survivin/Glutaminase/IDO/VEGF/MDR1) with target silence and stimulated innate immunity by way of RIG-I activation, could confer potent antitumor efficacy (Li et al., 2017; Meng et al., 2014; Wang et al., 2013; Yuan et al., 2015). Therefore, bifunctional 3p-siRNA is extremely attractive as a candidate for tumor or antiviral therapy. NK cells and CD8+ T cells are important effector cells in anti-HBV immune responses; however, both are impaired in the presence of chronic HBV infection. TGF-β, IL-10, MDSCs, and Tregs play immunosuppressive roles during HBV infection (Hong and Bertoletti, 2017; Li et al., 2015; Tan et al., 2015). Here, we confirmed that 3p-siHBx markedly decreased HBV load (Fig. 1). Significantly, we observed that 3p-siHBx could promote the activation of NK cells and CD8+ T cells, and induce the expression of CD80 and CD86 on DCs in naïve C57BL/6 mice (sFig. 4). Subsequently, we found NK cells, particularly CD11bhighCD27low mature NK cells, were recruited to the liver after 3p-siHBx treatment (Fig. 2). Meanwhile, the frequency of CD69highKLRG1low hepatic NK cells was increased in 3p-siHBx–treated HBV-carrier mice, accompanied by increased IFN-γ–producing NK cells. Furthermore, 3p-siHBx activated CD8+ T cells, exhibited augmented ability to produce TNF-α and IFN-γ and decreased PD-1 expression, which is similar to the effect of irrelevant 3p-scramble treatment. Interestingly, 3p-siHBx but not 3p-scramble inhibited Treg generation and TGF-β and IL-10 production effectively (Fig. 3). More importantly, 3p-siHBx significantly induced DC maturation (Fig. 4) and reduced CD11b+Gr-1+ MDSC numbers, while siHBx treatment only slightly inhibited MDSC, and there is no difference in MDSC and suppressive cytokines between 3p-scramble and control group (Fig. 5). To date, there has been no report that RIG-I activation can activate B cells and promote antibody secretion. In our study, we found that 3p-siHBx did not increase anti-HBs antibodies in HBV-carrier mice (sTable.1). We surmise that the cytokines produced by the 3p-siHBx–treated hepatocytes were not sufficient to activate B cells and T follicular helper (Tfh) cells. Nevertheless, this study presents an interesting issue to be resolved in future studies. These findings all suggest that the bifunctional 3p-siHBx can reverse HBV infection–induced immune dysfunction by improving NK cell and CD8+ T cell function.