Comparison of the cytokines levels in aqueous humor in vitrectomized eyes versus non-vitrectomized eyes with diabetic macular edema.

BACKGROUND: This study was conducted to compare concentrations of VEGF family growth factors, inflammation-related factors, and adhesion molecules in the aqueous humor of eyes with diabetic macular edema (DME), with and without prior vitrectomy. METHODS: A total of 31 eyes were included, 11 with DME that had undergone vitrectomy, 9 with DME but without vitrectomy, and 11 from age-related cataract patients as controls. The concentrations of cytokines including TNF-α, IL-6, IL-8, IP-10, MCP-1, IFN-γ, MIP-1 α, MIP-1 ß, PECAM-1, MIF, VCAM-1, ICAM-1, PIGF were quantified using Luminex Human Discovery Assay. Central macular thickness (CMT) values of all eyes were measured using optical coherence tomography (OCT). RESULTS: (1) Vitrectomized DME eyes exhibited significantly higher levels of IL-6 and IL-8 compared to non-vitrectomized eyes (P < 0.05). (2) In vitrectomized group, after Benjamini-Hochberg correction, there was a significant positive correlation between the levels of VEGF and PlGF (rs = 0.855, P < 0.05), as well as the levels of TNF-α and IFN-γ (rs = 0.858, P < 0.05). In non-vitrectomized group, significant positive correlations were found between VEGF and PlGF levels after correcting for multiple comparisons (rs = 0.9, P < 0.05). (3) In non-vitrectomized group, the concentrations of VEGF and PlGF in aqueous humor were significantly positively correlated with CMT values (rs = 0.95, P < 0.05; rs = 0.9, P < 0.05, respectively). CONCLUSIONS: The concentrations of IL-6 and IL-8 in the aqueous humor were significantly higher in vitrectomized DME eyes compared to nonvitrectomized DME eyes and the levels of VEGF were similar in the two groups, suggesting that inflammation after vitrectomy may be a key factor in the occurrence and development of DME.

Normalization of Tumor Vessels by Lenvatinib-Based Metallo-Nanodrugs Alleviates Hypoxia and Enhances Calreticulin-Mediated Immune Responses in Orthotopic HCC and Organoids.

Immunocheckpoint inhibitors combined with Lenvatinib is the first line treatment for hepatocellular carcinoma (HCC), but their potency is hampered by the low response rate and adverse events. Herein, a targeted therapeutic strategy through the coassembly of Lenvatinib, Adriamycin, Fe3+ ion, and a natural polyphenol (metallo-nanodrugs) is presented by coordination effect for potentiating tumor vascular normalization and systematic chemo-immunotherapy to effectively inhibit the progression of HCC in both orthotopic model and patients-derived organoids. In mice with orthotopic HCC, the obtained metallo-nanodrugs efficiently increase the drug accumulation in orthotopic tumors and can respond to acidic tumor environment. The promotion of tumor vascular normalization by metallo-nanodrugs is observed, which enhances the infiltrating T lymphocytes in tumor, and reinforces the calreticulin-mediated antitumor immunity through alleviating hypoxia, reducing regulatory T cells, and down-regulating PDL1 expression of tumors. The excellent therapeutic efficiency with complete remission of orthotopic tumors (3/6) and long-term survival of mice (4/6, 42 days) are also achieved. Furthermore, the excellent therapeutic effect of metallo-nanodrugs is also validated in 5 patient-derived organoids, and hence can provide a marvelous systemic chemo-immunotherapy strategy for enhancing HCC treatment.

Cell phenotypic plasticity requires autophagic flux driven by YAP/TAZ mechanotransduction.

Autophagy, besides ensuring energy metabolism and organelle renewal, is crucial for the biology of adult normal and cancer stem cells. However, it remains incompletely understood how autophagy connects to stemness factors and the nature of the microenvironmental signals that pattern autophagy in different cell types. Here we advance in these directions by reporting that YAP/TAZ transcriptionally control autophagy, being critical for autophagosomal degradation into autolysosomes. YAP/TAZ are downstream effectors of cellular mechanotransduction and indeed we found that cell mechanics, dictated by the physical property of the ECM and cytoskeletal tension, profoundly impact on autophagic flux in a YAP/TAZ-mediated manner. Functionally, by using pancreatic and mammary organoid cultures, we found that YAP/TAZ-regulated autophagy is essential in normal cells for YAP/TAZ-mediated dedifferentiation and acquisition of self-renewing properties. In tumor cells, the YAP/TAZ-autophagy connection is key to sustain transformed traits and for acquisition of a cancer stem cell state by otherwise more benign cells. Mechanistically, YAP/TAZ promote autophagic flux by directly promoting the expression of Armus, a RAB7-GAP required for autophagosome turnover and whose add-back rescues autophagy in YAP/TAZ-depleted cells. These findings expand the influence of YAP/TAZ mechanotransduction to the control of autophagy and, vice versa, the role of autophagy in YAP/TAZ biology, and suggest a mechanism to coordinate transcriptional rewiring with cytoplasmic restructuring during cell reprogramming.

Moesin facilitates metastasis of hepatocellular carcinoma cells by improving invadopodia formation and activating ß-catenin/MMP9 axis.

Moesin has been proved to be implicated in invasiveness and metastasis in many other cancers, but unclear in HCC. Thus, this study was performed to investigate the clinical significance of moesin and its biological functions in HCC. The results showed that moesin was significantly up-regulated in HCC tissues and was an independent prognostic factor for predicting the recurrence of HCC patients, postoperatively. Furthermore, we also demonstrated that moesin promoted the migration and invasion of HCC cells in vitro and in vivo. And the mechanism studies indicated that moesin overexpression increased the formation of invadopodia and improved the activation of ß-catenin/MMP9 axis. Taken together, our findings revealed that moesin acted as an important onco-protein participating in the metastasis of HCC.

Lnc-CCNH-8 promotes immune escape by up-regulating PD-L1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis. In recent years, immune checkpoint inhibitors (ICIs) have enabled breakthroughs in the clinical treatment of patients with HCC, but the overall response rate to ICIs in HCC patients is still low, and no validated biomarker is available to guide clinical decision making. Here, we demonstrated that the long non-coding RNA Lnc-CCNH-8 is highly expressed in HCC and correlates with poor prognosis. Functionally, elevated Lnc-CCNH-8 inactivated co-cultured T cells in vitro and compromised antitumor immunity in an immunocompetent mouse model. Mechanistically, up-regulated Lnc-CCNH-8 can sponge microRNA (miR)-217 to regulate the expression of PD-L1. In addition, Lnc-CCNH-8 can also stabilize PD-L1 through miR-3173/PKP3 axis. Furthermore, mice bearing tumors with high Lnc-CCNH-8 expression had significant therapeutic sensitivity to anti-PD-L1 monoclonal antibody treatment. More important, HCC patients with high levels of plasma exosomal Lnc-CCNH-8 had a better therapeutic response to ICIs. Taken together, our results reveal the function of Lnc-CCNH-8 in inducing immune escape from CD8+ T-cell-mediated killing by up-regulating PD-L1 in a miR-217/miR-3173-dependent manner, which also reveals a novel mechanism of PD-L1 regulation in HCC, and exosomal Lnc-CCNH-8 can serve as a predictive marker for immunotherapy response in HCC.

RNA Methyltransferase FTSJ3 Regulates the Type I Interferon Pathway to Promote Hepatocellular Carcinoma Immune Evasion.

Immunotherapies such as immune checkpoint blockade have achieved remarkable success in treating cancer. Unfortunately, response rates have been limited in multiple cancers including hepatocellular carcinoma (HCC). The critical function of epigenetics in tumor immune evasion and antitumor immunity supports harnessing epigenetic regulators as a potential strategy to enhance the efficacy of immunotherapy. Here, we discovered a tumor-promoting function of FTSJ3, an RNA 2'-O-methyltransferase, in HCC by suppressing antitumor immune responses. FTSJ3 was upregulated in hepatocellular carcinoma, and high FTSJ3 expression correlated with reduced patient survival. Deletion of FTSJ3 blocked HCC growth and induced robust antitumor immune responses. Mechanistically, FTSJ3 suppressed double-stranded RNA (dsRNA)-induced IFNß signaling in a 2'-O-methyltransferase manner. Deletion of RNA sensors in HCC cells or systemic knockout of type I IFN receptor IFNAR in mice rescued the in vivo tumor growth defect caused by FTSJ3 deficiency, indicating that FTSJ3 deletion suppresses tumor growth by activating the RNA sensor-mediated type I IFN pathway. Furthermore, FTSJ3 deletion significantly enhanced the efficacy of programmed cell death protein 1 (PD-1) immune checkpoint blockade. The combination of FTSJ3 deficiency and anti-PD-1 antibody treatment effectively eradicated tumors and increased the survival time. In conclusion, this study reveals an epigenetic mechanism of tumor immune evasion and, importantly, suggests FTSJ3-targeting therapies as potential approach to overcome immunotherapy resistance in patients with HCC. SIGNIFICANCE: Hepatocellular carcinoma cells use 2'-O-methylation catalyzed by FTSJ3 for immune evasion by suppressing abnormal dsRNA-mediated type I IFN responses, providing a potential target to activate antitumor immunity and enhance immunotherapy efficacy.

Ku70 Binding to YAP Alters PARP1 Ubiquitination to Regulate Genome Stability and Tumorigenesis.

YAP is a central player in cancer development with functions extending beyond its recognized role in cell growth regulation. Recent work has identified a link between YAP/TAZ and the DNA damage response. Here, we investigated the mechanistic underpinnings of the crosstalk between DNA damage repair and YAP activity. Ku70, a key component of the non-homologous end joining pathway to repair DNA damage, engaged in a dynamic competition with TEAD4 for binding to YAP, limiting the transcriptional activity of YAP. Depletion of Ku70 enhanced interaction between YAP and TEAD4 and boosted YAP transcriptional capacity. Consequently, Ku70 loss enhanced tumorigenesis in colon cancer and hepatocellular carcinoma (HCC) in vivo. YAP impeded DNA damage repair and elevated genome instability by inducing PARP1 degradation through the SMURF2-mediated ubiquitin-proteasome pathway. Analysis of HCC patient samples substantiated the link between Ku70 expression, YAP activity, PARP1 levels, and genome instability. In conclusion, this research provides insight into the mechanistic interactions between YAP and key regulators of DNA damage repair, highlighting the role of a Ku70-YAP-PARP1 axis in preserving genome stability.

Natural killer cells modified with a Gpc3 aptamer enhance adoptive immunotherapy for hepatocellular carcinoma.

INTRODUCTION: Natural killer cells can attack cancer cells without prior sensitization, but their clinical benefit is limited owing to their poor selectivity that is caused by the lack of specific receptors to target tumor cells. In this study, we aimed to endow NK cells with the ability to specifically target glypican-3+ tumor cells without producing cell damage or genetic alterations, and further evaluated their therapeutic efficiency. METHODS: NK cells were modified with a Gpc3 DNA aptamer on the cell surface via metabolic glycoengineering to endow NK cells with specific targeting ability. Then, the G-NK cells were evaluated for their specific targeting properties, cytotoxicity and secretion of cytokines in vitro. Finally, we investigated the therapeutic efficiency of G-NK cells against glypican-3+ tumor cells in vivo. RESULTS: Compared with NK cells modified with a random aptamer mutation and unmodified NK cells, G-NK cells induced significant apoptosis/necrosis of GPC3+ tumor cells and secreted cytokines to preserve the intense cytotoxic activities. Moreover, G-NK cells significantly suppressed tumor growth in HepG2 tumor-bearing mice due to the enhanced enrichment of G-NK cells at the tumor site. CONCLUSIONS: The proposed strategy endows NK cells with a tumor-specific targeting ability to enhance adoptive therapeutic efficiency in GPC3+ hepatocellular carcinoma.

Hypoxia induces hepatocellular carcinoma metastasis via the HIF-1α/METTL16/lnc-CSMD1-7/RBFOX2 axis.

Hypoxic microenvironment is clinically associated with metastasis and poor prognosis of numerous cancers. The mechanisms by which intratumoral hypoxia regulates metastasis are not fully understood. Our study identifies a downregulation of Lnc-CSMD1-7 in hepatocellular carcinoma (HCC) and correlated with poor prognosis of HCC patients. Lnc-CSMD1-7 negatively regulated HCC cell migration and invasion in vitro and suppressed lung metastasis in vivo. Mechanistically, Lnc-CSMD1-7 directly binds to RBFOX2, thereby affecting RBFOX2-regulated alternative splicing in epithelial and mesenchymal-specific events. More importantly, hypoxic microenvironment and m6A methylation mediate the downregulation of Lnc-CSMD1-7 expression. Specifically, hypoxia transcriptionally upregulates the expression of the m6A methyltransferase METTL16 via HIF-1α, and METTL16 directly binds to Lnc-CSMD1-7 and downregulates the RNA stability of Lnc-CSMD1-7 via m6A methylation, ultimately promoting HCC metastasis. Our findings highlight the regulatory function of the METTL16/Lnc-CSMD1-7/RBFOX2 axis in modulating hypoxia-induced HCC progression, which may provide potential prognostic and therapeutic targets for HCC treatment.

Systemic delivery of glycosylated-PEG-masked oncolytic virus enhances targeting of antitumor immuno-virotherapy and modulates T and NK cell infiltration.

Rationale: Immuno-virotherapy has emerged as a promising approach for cancer treatment, as it directly and cytotoxically eliminates tumors with systemic immune stimulation. However, the clinical efficacy of this approach remains limited by inappropriate delivery routes, robust antiviral responses, and the tumor immunosuppressive microenvironment. Methods: To address these challenges, we propose a surface engineering strategy that masks oncolytic herpes simplex virus (oHSV) with a galactose-polyethylene-glycol (PEG) polymer chain to minimize host antiviral responses and selectively targets tumors by limiting exposure to circulation upon systemic administration. We evaluated the antitumor efficacy of glycosylated-PEG-oHSV by examining tumor growth in animal models and analyzing tumor-infiltrating CD8+T cells and NK cells in the tumor microenvironment (TME). To assess the neutralizing antibody levels after systemic administration of glycosylated-PEG-oHSV, we utilized a mouse model and measured oHSV-specific IgG. Results: We demonstrate that the glycosylated-PEG modified oHSV does not affect the replication of oHSV yet exhibits high specificity to the asialoglycoprotein receptor (ASGPR) overexpressed in hepatocellular carcinoma cells. This results in selectively targeting cancer cells and deep penetration into tumors while avoiding spreading into the brain. Our approach also effectively reduces oHSV-specific neutralizing antibody levels to mitigate host antiviral immune response. Notably, our glycosylated-PEG-oHSV alleviates the immunosuppressive microenvironment within tumors by reducing regulatory T cells, augmenting the infiltration of activated CD8+T cells and NK cells with increasing release of anti-tumor cytokines, to impede tumor progression. Conclusion: Our findings offer a widely applicable and universal strategy to enhance cancer immuno-virotherapy through systemic administration of non-genetically engineered oncolytic viruses. This approach has the potential to overcome the limitations of current immune-virotherapy strategies and may improve clinical outcomes for cancer patients.

Navoximod modulates local HSV-1 replication to reshape tumor immune microenvironment for enhanced immunotherapy via an injectable hydrogel.

Oncolytic virotherapy can lead to tumor lysis and systemic anti-tumor immunity, but the therapeutic potential in humans is limited due to the impaired virus replication and the insufficient ability to overcome the immunosuppressive tumor microenvironment (TME). To solve the above problems, we identified that Indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitor Navoximod promoted herpes simplex virus type 1 (HSV-1) replication and HSV-1-mediated oncolysis in tumor cells, making it a promising combination modality with HSV-1-based virotherapy. Thus, we loaded HSV-1 and Navoximod together in an injectable and biocompatible hydrogel (V-Navo@gel) for hepatocellular carcinoma (HCC) virotherapy. The hydrogel formed a local delivery reservoir to maximize the viral replication and distribution at the tumor site with a single-dose injection. Notably, V-Navo@gel improved the disease-free survival time of HCC- bearing mice and protects the mice against tumor recurrence. What's more, V-Navo@gel also showed an effective therapeutic efficacy in the rabbit orthotopic liver cancer model. Mechanistically, we further discovered that our combination strategy entirely reprogramed the TME through single-cell RNA sequencing. All these results collectively indicated that the combination of Navoximod with HSV-1 could boost the viral replication and reshape TME for tumor eradication through the hydrogel reservoir.

Lnc-PLA2G4A-4 facilitates the progression of hepatocellular carcinoma by inducing versican expression via sponging miR-23b-3p.

Aberrant expression of long non-coding RNAs (lncRNAs) is associated with progression of multiple human cancers including hepatocellular carcinoma (HCC). However, the role of lncRNAs in HCC is not been fully understood. Our study aimed to investigate the biological function and potential molecular mechanism of Lnc-PAL2G4A-4 in HCC. In the current study, we show that Lnc-PLA2G4A-4 was significantly up-regulated in HCC tissues and high Lnc-PLA2G4A-4 expression was remarkably associated with tumor size, microvascular invasion and poor prognosis of HCC patients. Functionally, Lnc-PLA2G4A-4 positively regulated cell proliferation, invasion and migration in vitro, and facilitated lung metastasis of HCC in vivo. Mechanistically, Lnc-PLA2G4A-4 functioned as a competing endogenous RNA (ceRNA) to bind to miR-23b-3p and subsequently facilitate miR-23b-3p's target gene versican (VCAN) expression in HCC cells. Over-expression of miR-23b-3p could reverse Lnc-PLA2G4A-4 induced cell phenotypes in HCC and suppress versican expression of by rescue analysis. Collectively, Lnc-PLA2G4A-4 promotes HCC progression by targeting the miR-23b-3p/versican axis, which may be a potential biomarker and therapeutic target for HCC.

LncRNA TIALD contributes to hepatocellular carcinoma metastasis via inducing AURKA lysosomal degradation.

The N6-methyladenosine (m6A) RNA methyltransferase METTL16 is an emerging player in RNA modification landscape and responsible for the deposition of m6A in a few transcripts. AURKA (aurora kinase A) has been confirmed as an oncogene in cancer development including hepatocellular carcinoma (HCC). Nevertheless, it remains unclear whether METTL16 mediated m6A modification of lncRNAs can regulate AURKA activation in cancer progression. Here we aimed to investigate the functional links between lncRNAs and the m6A modification in AURKA signaling and HCC progression. Here we show that LncRNA TIALD (transcript that induced AURKA Lysosomal degradation) was down-regulated in HCC tissues by METTL16 mediated m6A methylation to facilitate its RNA degradation, and correlates with poor prognosis. Functional assays reveal that TIALD inhibits HCC metastasis both in vitro and in vivo. Mechanistically, TIALD directly interacts with AURKA and facilitate its degradation through the lysosomal pathway to inhibited EMT and metastasis of HCC. AURKA's specific inhibitor alisertib exerts effective therapeutic effect on liver cancer with low TIALD expression, which might provide a new insight into HCC therapy. Our study uncovers a negative functional loop of METTL16-TIALD-AURKA axis, and identifies a new mechanism for METTL16 mediated m6A-induced decay of TIALD on AURKA signaling in HCC progression, which may provide potential prognostic and therapeutic targets for HCC.

Synthetic Biology in Chimeric Antigen Receptor T (CAR T) Cell Engineering.

Synthetic biology is a novel interdisciplinary research area following engineering principles to redesign and construct biological systems for useful purposes. As one of the most notable clinically relevant application of synthetic biology, chimeric antigen receptor (CAR) T cells have demonstrated tremendous success for the treatment of advanced hematological malignancies in recent years. However, various unsolved obstacles limit the widespread application of CAR T cell therapies, including treatment-associated toxicities, antigen heterogeneity, antigen escape, poor CAR T cell persistence and expansion, and particularly inefficient homing, infiltrating into, and surviving within solid tumors. Accordingly, to improve therapeutic efficacy and minimize side effects, innovative CAR design becomes urgently necessary, and researchers are developing numerous methods to overcome the limitations. Here we summarize currently available bioengineering strategies and discuss the future development from a viewpoint of synthetic biology.

Remodeling Tumor-Associated Neutrophils to Enhance Dendritic Cell-Based HCC Neoantigen Nano-Vaccine Efficiency.

Hepatocellular carcinoma (HCC) commonly emerges in an immunologically "cold" state, thereafter protects it away from cytolytic attack by tumor-infiltrating lymphocytes, resulting in poor response to immunotherapy. Herein, an acidic/photo-sensitive dendritic cell (DCs)-based neoantigen nano-vaccine has been explored to convert tumor immune "cold" state into "hot", and remodel tumor-associated neutrophils to potentiate anticancer immune response for enhancing immunotherapy efficiency. The nano-vaccine is constructed by SiPCCl2 -hybridized mesoporous silica with coordination of Fe(III)-captopril, and coating with exfoliated membrane of matured DCs by H22-specific neoantigen stimulation. The nano-vaccines actively target H22 tumors and induce immunological cell death to boost tumor-associated antigen release by the generation of excess 1 O2 through photodynamic therapy, which act as in situ tumor vaccination to strengthen antitumor T-cell response against primary H22 tumor growth. Interestingly, the nano-vaccines are also home to lymph nodes to directly induce the activation and proliferation of neoantigen-specific T cells to suppress the primary/distal tumor growth. Moreover, the acidic-triggered captopril release in tumor microenvironment can polarize the protumoral N2 phenotype neutrophils to antitumor N1 phenotype for improving the immune effects to achieve complete tumor regression (83%) in H22-bearing mice and prolong the survival time. This work provides an alternative approach for developing novel HCC immunotherapy strategies.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

OBJECTIVE: To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. METHODS: Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). RESULTS: PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. CONCLUSION: PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

ZBP1 mediates interferon-induced necroptosis.

Interferons (IFNs) play an important role in immunomodulatory and antiviral functions. IFN-induced necroptosis has been reported in cells deficient in receptor-interacting protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8, but the mechanism is largely unknown. Here, we report that the DNA-dependent activator of IFN regulatory factors (ZBP1, also known as DAI) is required for both type I (ß) and type II (γ) IFN-induced necroptosis. We show that L929 fibroblast cells became susceptible to IFN-induced necroptosis when RIPK1, FADD, or Caspase-8 was genetically deleted, confirming the antinecroptotic role of these proteins in IFN signaling. We found that the pronecroptotic signal from IFN stimulation depends on new protein synthesis and identified ZBP1, an IFN-stimulated gene (ISG) product, as the de novo synthesized protein that triggers necroptosis in IFN-stimulated cells. The N-terminal domain (ND) of ZBP1 is important for ZBP1-ZBP1 homointeraction, and its RHIM domain in the C-terminal region interacts with RIPK3 to initiate RIPK3-dependent necroptosis. The antinecroptotic function of RIPK1, FADD, and caspase-8 in IFN-treated cells is most likely executed by caspase-8-mediated cleavage of RIPK3, since the inhibitory effect on necroptosis was eliminated when the caspase-8 cleavage site in RIPK3 was mutated. ZBP1-mediated necroptosis in IFN-treated cells is likely physiologically relevant, as ZBP1 KO mice were significantly protected against acute systemic inflammatory response syndrome (SIRS) induced by TNF + IFN-γ.

RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice.

Necroptosis is a form of programmed necrosis that is mediated by signaling complexes containing the receptor-interacting protein 3 (RIP3) and RIP1 kinases. We show that RIP3 and its interaction with the herpes simplex virus type 1 (HSV-1) protein ICP6 triggers necroptosis in infected mouse cells and limits viral propagation in mice. ICP6 interacts with RIP1/RIP3 through its RHIM domain and forms dimers/oliogmers by its C-terminal R1 domain. These binding events result in RIP1-RIP3 hetero- and RIP3-RIP3 homo-interactions and subsequent necroptosis of HSV-1-infected mouse cells. However, ICP6 RHIM cannot trigger necroptosis and even inhibits TNF-induced necroptosis in human cells. As the RHIM domain in murine cytomegalovirus protein vIRA can inhibit necroptosis in both human and mouse cells, these data suggest that both viral and host RHIM sequences determine whether the virus-host RHIM interaction is pro- or anti-necroptotic and that some viruses may evolve to escape this restriction.

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP.

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.