Qiuju Han

Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I-dependent manner.

It is extensively accepted that hepatitis B virus (HBV) escapes from innate immunity by inhibiting type I interferon (IFN) production, but efficient intervention to reverse the immune tolerance is still not achieved. Here, we report that 5′‐end triphosphate hepatitis B virus X gene (HBx)‐RNAs (3p‐HBx‐short interfering [si]RNAs) exerted significantly stronger inhibitory effects on HBV replication than regular HBx‐siRNAs in stably HBV‐expressing hepatoplastoma HepG2.2.15 cells through extremely higher expression of type I IFNs, IFN‐induced genes and proinflammatory cytokines, and retinoic acid inducible gene I (RIG‐I) activation. Also, 3p‐HBx‐siRNA were more efficient to stimulate type I IFN response than HBx sequence‐unrelated 3p‐scramble‐siRNA in HepG2.2.15 cells, indicating that a stronger immune‐stimulating effect may partly result from the reversal of immune tolerance through decreasing HBV load. In RIG‐I‐overexpressed HepG2.2.15 cells, 3p‐HBx‐siRNAs exerted stronger inhibitory effects on HBV replication with greater production of type I IFNs; on the contrary, in RIG‐I‐silenced HepG2.2.15 cells or after blockade of IFN receptor by monoclocnal antibody, inhibitory effect of 3p‐HBx‐siRNAs on HBV replication was largely attenuated, indicating that immunostimulatory function of 3p‐HBx‐siRNAs was RIG‐I and type I IFN dependent. Moreover, in HBV‐carrier mice, 3p‐HBx‐siRNA more strongly inhibited HBV replication and promoted IFN production than HBx‐siRNA in primary HBV+ hepatocytes and, therefore, significantly decreased serum hepatitis B surface antigen and increased serum IFN‐β. Conclusion: 3p‐HBx‐siRNAs may not only directly inhibit HBV replication, but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV‐induced immune tolerance. (HEPATOLOGY 2011;)

Therapeutic recovery of hepatitis B virus (HBV)-induced hepatocyte-intrinsic immune defect reverses systemic adaptive immune tolerance†‡

Hepatitis B virus (HBV) persistence aggravates hepatic immunotolerance, leading to the failure of cell‐intrinsic type I interferon and antiviral response, but whether and how HBV‐induced hepatocyte‐intrinsic tolerance influences systemic adaptive immunity has never been reported, which is becoming the major obstacle for chronic HBV therapy. In this study, an HBV‐persistent mouse, established by hydrodynamic injection of an HBV‐genome‐containing plasmid, exhibited not only hepatocyte‐intrinsic but also systemic immunotolerance to HBV rechallenge. HBV‐specific CD8+ T‐cell and anti‐HBs antibody generation were systemically impaired by HBV persistence in hepatocytes. Interestingly, HBV‐induced hepatocyte‐intrinsic immune tolerance was reversed when a dually functional vector containing both an immunostimulating single‐stranded RNA (ssRNA) and an HBx‐silencing short hairpin RNA (shRNA) was administered, and the systemic anti‐HBV adaptive immune responses, including CD8+ T‐cell and anti‐HBs antibody responses, were efficiently recovered. During this process, CD8+ T cells and interferon‐gamma (IFN‐γ) secreted play a critical role in clearance of HBV. However, when IFN‐α/β receptor was blocked or the Toll‐like receptor (TLR)7 signaling pathway was inhibited, the activation of CD8+ T cells and clearance of HBV was significantly impaired. Conclusion: These results suggest that recovery of HBV‐impaired hepatocyte‐intrinsic innate immunity by the dually functional vector might overcome systemic adaptive immunotolerance in an IFN‐α‐ and TLR7‐dependent manner. The strategy holds promise for therapeutic intervention of chronic persistent virus infection and associated cancers. (Hepatology 2013;)

The role of innate immunity in HBV infection

Hepatitis B virus (HBV) infection is one of the main causes of chronic liver diseases. Whether HBV infection is cleared or persists is determined by both viral factors and host immune responses. It becomes clear that innate immunity is of importance in protecting the host from HBV infection and persistence. However, HBV develops strategies to suppress the antiviral immune responses. A combined therapeutic strategy with both viral suppression and enhancement of antiviral immune responses is needed for effective long-term clearance and cure for chronic HBV infection. We and others confirmed that bifunctional siRNAs with both gene silencing and innate immune activation properties are beneficial for inhibition of HBV and represent a potential approach for treatment of viral infection. Understanding the nature of liver innate immunity and their roles in chronic HBV progression and HBV clearance may aid in the design of novel therapeutic strategies for chronic HBV infection.

miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression

MicroRNAs (miRNAs) play an important role in tumorigenesis, but their role in tumor-induced immune suppression is largely unknown. STAT3 signaling, a key pathway mediating immune suppression in the tumor microenvironment, is responsible for the transcription of several important miRNAs. In this study, we observed that miR-146a, a known important regulator of immune responses, was downregulated by blocking activated STAT3 in hepatocellular carcinoma (HCC) cells. Furthermore, miR-146a inhibition in HCC cells not only altered the STAT3 activation–associated cytokine profile but also reversed HCC-induced NK cell dysfunction in vitro and improved the anti-tumor effect of lymphocytes in vivo. Importantly, ChIP and luciferase reporter assays confirmed that STAT3 directly bound to the miR-146a promoter and induced miR-146a expression. These findings indicated that miR-146a expression was regulated by aberrantly activated STAT3 in HCC cells and exerted negative effects on anti-tumor immune response, which resulted in the upregulation of cytokines such as TGF-β, IL-17, VEGF and downregulation of type I IFN to create an immunosuppressive microenvironment. This further insight into understanding the mechanism responsible for tumor-induced immune suppression highlights the potential application of miR-146a as a novel immunotherapeutic target for HCC.

Involvement of Activation of PKR in HBx-siRNA-Mediated Innate Immune Effects on HBV Inhibition

RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-hepatitis B virus (anti-HBV) therapy. Recent studies have revealed that siRNAs can induce an innate immune response in vitro and in vivo. Here, HBVx (HBx) mRNA expression and HBV replication were significantly inhibited, followed by the enhancement of expression of type I interferons (IFNs), IFN-stimulated genes (ISG15 and ISG56) and proinflammatory cytokines after HepG2.2.15 cells were transfected with chemically synthesized HBx-siRNAs. Transfection with HBx-siRNAs also significantly increased expression of dsRNA-dependent protein kinase R (PKR) in HepG2.2.15 cells, followed by activation of downstream signaling events such as eukaryotic initiation factor 2α (eIF2-α). In PKR-over-expressing HepG2.2.15 cells, HBx-siRNAs exerted more potent inhibitory effects on HBV replication and greater production of type I IFNs. By contrast, the inhibitory effect of HBx-siRNAs on HBV replication was attenuated when PKR was inhibited or silenced, demonstrating that HBx-siRNAs greatly promoted PKR activation, leading to the higher production of type I IFN. Therefore, we 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.

NK Cells Are the Crucial Antitumor Mediators When STAT3-Mediated Immunosuppression Is Blocked in Hepatocellular Carcinoma

STAT3 is highly activated in a wide variety of cancers and functions to promote tumor survival. We previously reported that blocking STAT3 activation inhibited human hepatocellular carcinoma (HCC) growth in vitro, but whether this treatment also triggered antitumor immune responses in vivo remained unknown. In this study, we found that blocking the STAT3 pathway in HCC cells dramatically inhibited murine HCC growth in vivo and prolonged survival of tumor-bearing mice. Importantly, the presence of STAT3-blocked HCC augmented NK cell cytotoxicity against HCC and increased expression of molecules associated with NK cell activation and cytotoxicity. In T cell–deficient nude mice, a unique NK cell–mediated antitumor function against STAT3-blocked HCC was suggested. NK cells were shown to be necessary and sufficient in NK or T cell depletion experiments, or by adoptively transferring NK cells. Furthermore, regulatory T cells and immunosuppressive IL-10 and TGF-β cytokines were reduced in mice bearing STAT3-blocked HCC cells, suggesting that these factors may be involved in HCC-induced NK cell suppression. These findings indicate that blocking STAT3 in HCC cells can initiate innate immunity in vivo.

HBV inhibits LPS-induced NLRP3 inflammasome activation and IL-1β production via suppressing the NF-κB pathway and ROS production

BACKGROUND & AIMS Hepatitis B virus (HBV) has developed strategies to evade immune responses. However, the mechanisms involved remain unclear. The NLRP3 inflammasome plays crucial roles in antiviral host defense and its downstream factor IL-1β has been shown to inhibit HBV infection in vivo. This study aims to assess whether HBV can affect the NLRP3 inflammasome signaling pathways and shed light on the underlying mechanisms HBV utilizes to evade host innate immune responses. METHODS HBV inhibition of the lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation was evaluated by Western blot, quantitative RT-PCR, flow cytometry and immunofluorescence. RESULTS Kupffer cells expressed significantly more NLRP3 and IL-1β after LPS stimulation; whereas, chronic HBV infection suppressed LPS-induced NLRP3 and pro-IL-1β expression as well as IL-1β maturation. This inhibitory activity is mediated by HBeAg, and is involved in the inhibition of NF-κB signal pathway and reactive oxygen species (ROS) production. The inhibitory effect of HBeAg was confirmed in patients with chronic hepatitis B (CHB) and hepatocellular carcinoma by comparing the levels of IL-1β and NLRP3-related proteins in para-carcinoma tissues from HBeAg-positive or negative patients. Moreover, chronic HBV infection increases the susceptibility of mice to S. typhimurium infection, possibly via inhibiting the NLRP3 inflammasome activation and IL-1β production. CONCLUSIONS HBeAg inhibits LPS-induced NLRP3 inflammasome activation and IL-1β production via suppressing NF-κB pathway and ROS production. This finding provides a novel mechanism for HBV-mediated suppression of innate immune responses, and identifies new therapeutic targets for chronic HBV infection and related diseases. LAY SUMMARY HBeAg suppresses LPS-induced NLRP3 inflammasome activation and IL-1β production in two ways, one is to repress NLRP3 and pro-IL-1β expression via inhibiting NF-κB phosphorylation, and the other is to repress caspase-1 activation and IL-1β maturation via inhibiting ROS production. This effect contributes to the HBV persistence and immune tolerance.

miR-146a negatively regulates NK cell functions via STAT1 signaling.

It is known that natural killer (NK) cell function is downregulated in chronic hepatitis B (CHB)-infected patients and in hepatic carcinoma (HCC) patients, but the mechanisms underlying this functional downregulation are largely unclear. In this study, microRNA (miR)-146a expression increased in NK cells from CHB and HCC patients compared with NK cells from healthy donors, and miR-146a levels were negatively correlated to NK cell functions. Overexpression of miR-146a reduced NK cell-mediated cytotoxicity and the production of interferon (IFN)-γ and tumor necrosis factor-α, which were reversed upon inhibition of miR-146a. In NK cells, miR-146a expression was induced by interleukin (IL)-10 and transforming growth factor-β, but reduced after treatment with interleukin-12, IFN-α and IFN-β. We further revealed that miR-146a regulated NK cell functions by targeting STAT1. Taken together, upregulated miR-146a expression, at least partially, attributes to NK cell dysfunction in CHB and HCC patients. Therefore, miR-146a may become a therapeutic target with great potential to ameliorate NK cell functions in liver disease.

Poly(I:C) exhibits an anti-cancer effect in human gastric adenocarcinoma cells which is dependent on RLRs.

Poly(I:C), an agonist of TLR3 and RLRs, has been used as an immune adjuvant in clinical trials for many years. Although it has been found to trigger apoptosis in a variety of cancers, its mechanisms in human gastric adenocarcinoma is unclear. The purpose of this study was to assess the effect of poly(I:C) on human gastric adenocarcinoma cells. Our observations showed that intracellular delivery of poly(I:C) by liposomes had a pro-apoptotic effect in vitro, and significantly inhibited xenograft growth of human gastric adenocarcinoma in nude mice. Further investigations indicated that RLRs, as intrinsic RNA sensors, contributed to the poly(I:C)-triggered apoptotic effect through upregulation of RIG-I, MDA-5, and most significantly, LGP2, accompanied by increased expression of Bcl-2 family members. Conversely, this apoptotic effect was greatly reduced by silencing RIG-I, MDA-5, or LGP2. Although LGP2 is considered an innate immune negative regulator of RIG-I and MDA-5, it exhibited a positive regulatory effect on poly(I:C)-induced apoptosis in human gastric adenocarcinoma cells. These findings suggested that poly(I:C) may be a promising chemotherapeutic agent against human gastric adenocarcinoma.

Toll-Like Receptor 2 (TLR2) and TLR9 Play Opposing Roles in Host Innate Immunity against Salmonella enterica Serovar Typhimurium Infection

ABSTRACT Toll-like receptors (TLRs) are evolutionarily conserved host proteins that are essential for effective host defense against pathogens. However, recent studies suggest that some TLRs can negatively regulate immune responses. We observed here that TLR2 and TLR9 played opposite roles in regulating innate immunity against oral infection of Salmonella enterica serovar Typhimurium in mice. While TLR9 −/− mice exhibited shortened survival, an increased cytokine storm, and more severe Salmonella hepatitis than wild-type (WT) mice, TLR2 −/− mice exhibited the opposite phenomenon. Further studies demonstrated that TLR2 deficiency and TLR9 deficiency in macrophages both disrupted NK cell cytotoxicity against S. Typhimurium-infected macrophages by downregulating NK cell degranulation and gamma interferon (IFN-γ) production through decreased macrophage expression of the RAE-1 NKG2D ligand. But more importantly, we found that S. Typhimurium-infected TLR2 −/− macrophages upregulated inducible nitric oxide synthase (iNOS) expression, resulting in a lower bacterial load than that in WT macrophages in vitro and livers in vivo as well as low proinflammatory cytokine levels. In contrast, TLR9 −/− macrophages showed decreased reactive oxygen species (ROS) expression concomitant with a high bacterial load in the macrophages and in livers of TLR9 −/− mice. TLR9 −/− macrophages were also more susceptible than WT macrophages to S. Typhimurium-induced necroptosis in vitro, likely contributing to bacterial spread and transmission in vivo. Collectively, these findings indicate that TLR2 negatively regulates anti-S. Typhimurium immunity, whereas TLR9 is vital to host defense and survival against S. Typhimurium invasion. TLR2 antagonists or TLR9 agonists may thus serve as potential anti-S. Typhimurium therapeutic agents.