CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

miR-26a exerts broad-spectrum antiviral effects via the enhancement of RIG-I-mediated type I interferon response by targeting USP15.

IMPORTANCE: miR-26a serves as a potent positive regulator of type I interferon (IFN) responses. By inhibiting USP15 expression, miR-26a promotes RIG-I K63-ubiquitination to enhance type I IFN responses, resulting in an active antiviral state against viruses. Being an intricate regulatory network, the activation of type I IFN responses could in turn suppress miR-26a expression to avoid the disordered activation that might result in the so-called "type I interferonopathy." The knowledge gained would be essential for the development of novel antiviral strategies against viral infection.

In vitro cell culture models to study hepatitis B and D virus infection.

Chronic infection with the hepatitis B virus (HBV) and hepatitis D virus (HDV) can cause a major global health burden. Current medication regimens can repress viral replication and help to control disease progression, but a complete cure is hardly achieved due to the difficulties to eradicate viral templates (cccDNA and integrates). To develop novel curative antiviral therapies for HBV/HDV infection, it is vital to precisely understand the details of the molecular biology of both viruses and the virus-host interactions. One important prerequisite for gaining this aim is the availability of suitable in vitro models that support HBV/HDV infection, replicate both viruses via their authentic template and allow to adequately study host cell responses. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) receptor as the most crucial host factor promoted HBV/HDV research to a new era. Recently, the structure of human NTCP was solved, gaining a deeper understanding of HBV recognition as the bona fide receptor. After decades of continuous efforts, new progress has been achieved in the development of cell culture models supporting HBV/HDV study. This review summarizes the cell culture models currently available, discusses the advantages and disadvantages of each model, and highlights their future applications in HBV and HDV research.

Advanced Cell Therapies for Glioblastoma.

The sheer ubiquity of Gioblastoma (GBM) cases would lead you to believe that there should have been many opportunities for the discovery of treatments to successfully render it into remission. Unfortunately, its persistent commonality is due in large part to the fact that it is the most treatment-resistant tumors in adults. That completely changes the treatment plan of attack. Long established and accepted treatment therapies such as surgical resection, radiation, and aggressive chemotherapy, (and any combination thereof) have only confirmed that the disease lives up to its treatment-resistant reputation. To add to the seemingly insurmountable task of finding a cure, GBM has also proven to be a very stubborn and formidable opponent to newer immunotherapies. Across the board, regardless of the therapy combination, the five-year survival rate of GBM patients is still very poor at a heartbreaking 5.6%. Obviously, the present situation cannot be tolerated or deemed acceptable. The grave situation calls for researchers to be more innovative and find more efficient strategies to discover new and successful strategies to treat GBM. Inspired by researchers worldwide attempting to control GBM, we provide in this review a comprehensive overview of the many diverse cell therapies currently being used to treat GBM. An overview of the treatments include: CAR T cells, CAR NK cells, gamma-delta T cells, NKT cells, dendritic cells, macrophages, as well stem cell-based strategies. To give you the complete picture, we will discuss the efficacy, safety, and developmental stages, the mechanisms of action and the challenges of each of these therapies and detail their potential to be the next-generation immunotherapeutic to eliminate this dreadful disease.

LGR5 marks targetable tumor-initiating cells in mouse liver cancer.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5+ compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer.

MDA5 against enteric viruses through induction of interferon-like response partially via the JAK-STAT cascade.

Enteric viruses including hepatitis E virus (HEV), human norovirus (HuNV), and rotavirus are causing global health issues. The host interferon (IFN) response constitutes the first-line defense against viral infections. Melanoma Differentiation-Associated protein 5 (MDA5) is an important cytoplasmic receptor sensing viral infection to trigger IFN production, and on the other hand it is also an IFN-stimulated gene (ISG). In this study, we investigated the effects and mode-of-action of MDA5 on the infection of enteric viruses. We found that MDA5 potently inhibited HEV, HuNV and rotavirus replication in multiple cell models. Overexpression of MDA5 induced transcription of important antiviral ISGs through IFN-like response, without triggering of functional IFN production. Interestingly, MDA5 activates the expression and phosphorylation of STAT1, which is a central component of the JAK-STAT cascade and a hallmark of antiviral IFN response. However, genetic silencing of STAT1 or pharmacological inhibition of the JAK-STAT cascade only partially attenuated the induction of ISG transcription and the antiviral function of MDA5. Thus, we have demonstrated that MDA5 effectively inhibits HEV, HuNV and rotavirus replication through provoking a non-canonical IFN-like response, which is partially dependent on JAK-STAT cascade.

Estimating the Global Prevalence, Disease Progression, and Clinical Outcome of Hepatitis Delta Virus Infection.

BACKGROUND: Hepatitis delta virus (HDV) coinfects with hepatitis B virus (HBV) causing the most severe form of viral hepatitis. However, its exact global disease burden remains largely obscure. We aim to establish the global epidemiology, infection mode-stratified disease progression, and clinical outcome of HDV infection. METHODS: We conducted a meta-analysis with a random-effects model and performed data synthesis. RESULTS: The pooled prevalence of HDV is 0.80% (95% confidence interval [CI], 0.63-1.00) among the general population and 13.02% (95% CI, 11.96-14.11) among HBV carriers, corresponding to 48-60 million infections globally. Among HBV patients with fulminant hepatitis, cirrhosis, or hepatocellular carcinoma, HDV prevalence is 26.75% (95% CI, 19.84-34.29), 25.77% (95% CI, 20.62-31.27), and 19.80% (95% CI, 10.97-30.45), respectively. The odds ratio (OR) of HDV infection among HBV patients with chronic liver disease compared with asymptomatic controls is 4.55 (95% CI, 3.65-5.67). Hepatitis delta virus-coinfected patients are more likely to develop cirrhosis than HBV-monoinfected patients with OR of 3.84 (95% CI, 1.79-8.24). Overall, HDV infection progresses to cirrhosis within 5 years and to hepatocellular carcinoma within 10 years, on average. CONCLUSIONS: Findings suggest that HDV poses a heavy global burden with rapid progression to severe liver diseases, urging effective strategies for screening, prevention, and treatment.

Pregnane X receptor activation constrains mucosal NF-κB activity in active inflammatory bowel disease.

BACKGROUND: The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. METHODS: We stimulate a total of 106 colonic biopsies from 19 Crohn's disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-κB activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) mRNA levels by qPCR and in cell culture models by NF-κB reporter-driven luciferase activity and Western blot for signal transduction elements. RESULTS: We observe a strict inverse correlation between colonic epithelial PXR levels and NF-κB target gene expression in colonic biopsies from Crohn's disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-κB activation in IBD. The correlation between colonic epithelial PXR levels and NF-κB target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-κB-dependent signal transduction whereas conversely NF-κB signaling reduces levels of PXR expression. CONCLUSIONS: Our data indicate that the PXR is a major and clinically relevant antagonist of NF-κB activity in the intestinal epithelial compartment during inflammatory bowel disease.

CADM2 inhibits human glioma proliferation, migration and invasion.

Malignant glioma is one of the most common malignant tumors in the brain parenchyma with a poor prognosis. Cell adhesion molecules (CADMs) immunoglobulin super family is involved in the maintenance of cell adhesion, polarity and tumor suppression. However, the role and mechanisms of CADM2 in human glioma have yet to be elucidated. Therefore, the present study evaluated the expression level of CADM2 and demonstrated that CADM2 was markedly downregulated in human glioma tissues compared with normal brain tissue and glioma cell lines, and the CADM2 expression level was significantly decreased in high­grade glioma tissues. Overexpression of CADM2 inhibited the proliferation of glioma cell proliferation in vitro and in vivo. CADM2 also inhibited the migration and invasion of U87 and U251 cells. Furthermore, overexpression of CADM2 induced a significant decrease in the expression of G1/S transition key regulators, cyclin D1, cyclin E, cyclin­dependent kinase (CDK)2 and CDK4. Additionally, CADM2 expression was associated with alterations in epithelial­mesenchymal transition (EMT) markers, including E­cadherin and ß­catenin. Taken together, the results of the present study demonstrated that CADM2 inhibits glioma tumorigenesis by regulating the cell cycle and the EMT process, suggesting that CADM2 may be a novel potential therapeutic target in human glioma.

Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Hepatitis E virus (HEV) infection has emerged as a global health problem. However, no approved medication is available, and the infection biology remains largely elusive. Electron transport chain (ETC), a key component of the mitochondria, is the main site that produces ATP and reactive oxygen species (ROS). By profiling the role of the different complexes of the mitochondrial ETC, we found that pharmacological inhibition of complex III, a well-defined drug target for the treatment of malaria and Pneumocystis pneumonia, potently restricts HEV replication. This effect demonstrated in our HEV models is equivalent to the anti-HEV potency of ribavirin, a widely used off-label treatment for patients with chronic HEV. Mechanistically, we found that this effect is independent of ATP production, ROS level, and pyridine depletion. By using pharmacological inhibitors and genetic approaches, we found that mitochondrial permeability transition pore (MPTP), a newly identified component of ETC, provides basal defense against HEV infection. HEV interferes with the opening of the MPTP. Furthermore, inhibition of the MPTP attenuated the anti-HEV effect of complex III inhibitors, suggesting that the MPTP mediates the antiviral effects of these inhibitors. These findings reveal new insights on HEV-host interactions and provide viable anti-HEV targets for therapeutic development.-Qu, C., Zhang, S., Wang, W., Li, M., Wang, Y., van der Heijde-Mulder, M., Shokrollahi, E., Hakim, M. S., Raat, N. J. H., Peppelenbosch, M. P., Pan, Q. Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC. KEY MESSAGES: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent. U-STAT1 sustains but p-STAT1 inhibits HCC growth. The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment.

Gene expression profile analysis of U251 glioma cells with shRNA-mediated SOX9 knockdown.

PURPOSE: In glioma, the sex-determining region Y-box 9 gene (SOX9) is overexpressed and its downregulation leads to inhibition of cell proliferation, invasion and increased cell apoptosis. To further evaluate the molecular and signal pathways associated with the function of SOX9 and SOX9 target genes, a global gene expression profile of the established SOX9-knockdown U251 cells was investigated. METHODS: The molecular function and biological pathways of differentially expressed genes (DEGs) were identified by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The interactome networks of DEGs were constructed using the STRING online tool. The genes were further validated by RT-qPCR. RESULTS: GO analysis revealed that a set of 194 DEGs was shared in both the SOX9 KD-1 and SOX9 KD-2 U251 cells. GO analysis and KEGG pathway analysis showed that the DEGs were associated with biological processes involving cellular responses to hypoxia, osteoblast differentiation and angiogenesis, and special biological pathways, such as a TGF-beta signaling pathway and a HIF-1 signaling pathway. In addition, computational network of novel identified potential target genes linked to SOX9, including TGFB2, VEGFA, EGLN3 (PHD3), CA9 and HIF-1a. All of these genes were downregulated in the SOX9 knockdown U251 cells. CONCLUSIONS: SOX9 may be a key regulator impacting the glioma cellular processes by influencing the cellular response to hypoxia and HIF-1 signaling pathway. TGFB2, VEGFA, EGLN3 (PHD3), CA9, and HIF-1a may be the target genes of SOX9.

6-Thioguanine inhibits rotavirus replication through suppression of Rac1 GDP/GTP cycling.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational.

TNF-α exerts potent anti-rotavirus effects via the activation of classical NF-κB pathway.

Active virus-host interactions determine the outcome of pathogen invasions. It has been shown that in isolated dendritic cells (DCs), rotavirus can induce the expression of tumor necrosis factor α (TNF-α), a vital cytokine mediating host immune responses. However, the role of TNF-α in rotavirus infection is unknown. In this study, we demonstrated that TNF-α has potent anti-rotavirus effects, independent of type I interferon production. Blocking of TNF-α by infliximab, a clinically available TNFα antibody, totally abrogated this effect. Mechanistic studies revealed that the anti-rotavirus effect of TNF-α was achieved by NFκB-regulated genes via the activation of classical nuclear factor κB (NF-κB) signaling. Our study reveals the pivotal role and the mechanism-of-actions of TNF-α in the host defense against rotavirus. Thus, this knowledge may contribute to the better understanding of the complexity of rotavirus-host interactions.

IRF-1, RIG-I and MDA5 display potent antiviral activities against norovirus coordinately induced by different types of interferons.

Norovirus represents the main cause of acute nonbacterial gastroenteritis worldwide. In immunocompromised patients, it bears high risk of causing chronic infection with significant morbidity and mortality. The lack of specific treatment prompts the development of anti-norovirus agents. In this study, we have investigated the role of interferon (IFN) response and evaluated antiviral activities of different IFNs against human norovirus (HuNoV) replication using a HuNoV replicon model. We found that HuNoV RNA replication was sensitive to all types of IFNs, including IFNα (type I), IFNγ (type II), IFNλ1 and 3 (type III). IFNs canonically induce interferon-stimulated genes (ISGs) to exert their antiviral activities. By profiling a subset of important human ISGs using an overexpression approach, we have identified RTP4 and HPSE as moderate anti-norovirus ISGs, whereas IRF-1, RIG-I (also known as DDX58) and MDA5 (also known as IFIH1) were identified as potent anti-norovirus effectors. Interestingly, type I and III IFNs coordinately induced IRF-1, RIG-I and MDA5; whereas type II IFN predominantly induced IRF-1 to exhibit their anti-norovirus activities. Combination of different IFNs revealed that IFNγ worked cooperatively with type I or type III IFNs to induce ISGs and subsequently inhibit HuNoV replication. Of note, replication of HuNoV did not interfere with antiviral IFN response. In summary, we showed the potent anti-norovirus activities of different types of IFNs and identified the key anti-norovirus effectors. These findings are important for understanding norovirus-host interactions and developing antiviral therapies.

PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target.

Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

The RNA genome of hepatitis E virus robustly triggers an antiviral interferon response.

The outcomes of hepatitis E virus (HEV) infection are diverse, ranging from asymptomatic carrier, self-limiting acute infection, and fulminant hepatitis to persistent infection. This is closely associated with the immunological status of the host. This study aimed to understand the innate cellular immunity as the first-line defense mechanism in response to HEV infection. Phosphorylation of signal transducer and activator of transcription 1, a hallmark of the activation of antiviral interferon (IFN) response, was observed in the liver tissues of the majority of HEV-infected patients but not in the liver of uninfected individuals. In cultured cell lines and primary liver organoids, we found that HEV RNA genome potently induced IFN production and antiviral response. This mechanism is conserved among different HEV strains, including genotypes 1, 3, and 7 as tested. Interestingly, single-stranded HEV RNA is sufficient to trigger the antiviral response, without the requirement of viral RNA synthesis and the generation of an RNA replicative form or replicative intermediate. Surprisingly, the m7 G cap and poly A tail are not required, although both are key features of the HEV genome. Mechanistically, this antiviral response occurs in a retinoic acid-inducible gene-I-independent, melanoma differentiation-associated protein 5-independent, mitochondrial antiviral signaling protein-independent, and ß-catenin-independent but IRF3-dependent and IRF7-dependent manner. Furthermore, the integrity of the Janus kinase-signal transducer and activator of transcription pathway is essentially required. CONCLUSION: HEV infection elicits an active IFN-related antiviral response in vitro and in patients, triggered by the viral RNA and mediated by IFN regulatory factors 3 and 7 and the Janus kinase-signal transducer and activator of transcription cascade; these findings have revealed new insights into HEV-host interactions and provided the basis for understanding the pathogenesis and outcome of HEV infection. (Hepatology 2018;67:2096-2112).

Immunity against hepatitis E virus infection: Implications for therapy and vaccine development.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide and an emerging cause of chronic infection in immunocompromised patients. As with viral infections in general, immune responses are critical to determine the outcome of HEV infection. Accumulating studies in cell culture, animal models and patients have improved our understanding of HEV immunopathogenesis and informed the development of new antiviral therapies and effective vaccines. In this review, we discuss the recent progress on innate and adaptive immunity in HEV infection, and the implications for the devolopment of effective vaccines and immune-based therapies.

Epigenome-Wide Association Study Identifies Methylation Sites Associated With Liver Enzymes and Hepatic Steatosis.

BACKGROUND & AIMS: Epigenetic mechanisms might be involved in the regulation of liver enzyme level. We aimed to identify CpG sites at which DNA methylation levels are associated with blood levels of liver enzymes and hepatic steatosis. METHODS: We conducted an epigenome-wide association study in whole blood for liver enzyme levels, including gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), among a discovery set of 731 participants of the Rotterdam Study and sought replication in a non-overlapping sample of 719 individuals. Significant DNA methylation changes were further analyzed to evaluate their relation with hepatic steatosis. Expression levels of the top identified gene were measured in 9 human liver cell lines and compared with expression profiles of its potential targets associated with lipid traits. The candidate gene was subsequently knocked down in human hepatoma cells using lentiviral vectors expressing small hairpin RNAs. RESULTS: Eight probes annotated to SLC7A11, SLC1A5, SLC43A1, PHGDH, PSORS1C1, SREBF1, ANKS3 were associated with GGT and 1 probe annotated to SLC7A11 was associated with ALT after Bonferroni correction (1.0 × 10-7). No probe was identified for AST levels. Four probes for GGT levels including cg06690548 (SLC7A11), cg11376147 (SLC43A1), cg22304262 (SLC1A5), and cg14476101 (PHGDH), and 1 for ALT cg06690548 (SLC7A11) were replicated. DNA methylation at SLC7A11 was associated with reduced risk of hepatic steatosis in participants (odds ratio, 0.69; 95% CI= 0.55-0.93; P value: 2.7 × 10-3). In functional experiments, SLC7A11 was highly expressed in human liver cells; its expression is positively correlated with expression of a panel of lipid-associated genes, indicating a role of SLC7A11 in lipid metabolism. CONCLUSIONS: Our results provide new insights into epigenetic mechanisms associated with markers of liver function and hepatic steatosis, laying the groundwork for future diagnostic and therapeutic applications.

RIG-I is a key antiviral interferon-stimulated gene against hepatitis E virus regardless of interferon production.

Interferons (IFNs) are broad antiviral cytokines that exert their function by inducing the transcription of hundreds of IFN-stimulated genes (ISGs). However, little is known about the antiviral potential of these cellular effectors on hepatitis E virus (HEV) infection, the leading cause of acute hepatitis globally. In this study, we profiled the antiviral potential of a panel of important human ISGs on HEV replication in cell culture models by overexpression of an individual ISG. The mechanism of action of the key anti-HEV ISG was further studied. We identified retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5, and IFN regulatory factor 1 (IRF1) as the key anti-HEV ISGs. We found that basal expression of RIG-I restricts HEV infection. Pharmacological activation of the RIG-I pathway by its natural ligand 5'-triphosphate RNA potently inhibits HEV replication. Overexpression of RIG-I activates the transcription of a wide range of ISGs. RIG-I also mediates but does not overlap with IFN-α-initiated ISG transcription. Although it is classically recognized that RIG-I exerts antiviral activity through the induction of IFN production by IRF3 and IRF7, we reveal an IFN-independent antiviral mechanism of RIG-I in combating HEV infection. We found that activation of RIG-I stimulates an antiviral response independent of IRF3 and IRF7 and regardless of IFN production. However, it is partially through activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) cascade of IFN signaling. RIG-I activated two distinct categories of ISGs, one JAK-STAT-dependent and the other JAK-STAT-independent, which coordinately contribute to the anti-HEV activity. CONCLUSION: We identified RIG-I as an important anti-HEV ISG that can be pharmacologically activated; activation of RIG-I stimulates the cellular innate immunity against HEV regardless of IFN production but partially through the JAK-STAT cascade of IFN signaling. (Hepatology 2017;65:1823-1839).