Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells.

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression.

UNLABELLED: Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE: Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Optimal preservation of liver biopsy samples for downstream translational applications.

OBJECTIVE: Molecular analysis of liver biopsy samples from patients requires ideal tissue preservation and handling to yield suitable material for laboratory analysis. Biopsy size, tissue handling and preservation method all may affect the quality and quantity of DNA, RNA and protein that can be extracted from liver biopsy samples. METHOD: In the present study, murine liver biopsies were performed and stored under various conditions: snap-freezing, RNAlater and Allprotect. Yield was compared to fresh biopsy tissue. RESULTS: Fresh tissue generated the highest yield of RNA while samples subjected to the snap-freezing generated the lowest yield of RNA. Preservation in RNAlater yielded higher quantities of RNA than storage in Allprotect, particularly with larger biopsy samples. There was a non-significant trend toward improved RNA quality with RNAlater (p = 0.35). DNA and protein yield were similar with RNAlater and Allprotect under a number of handling condition. Errors in tissue handling such as delays in tissue submersion or freezing did not significantly affect tissue yields in either preservation solution. Tissue yield was unchanged with up to three freeze-thaw cycles in both solutions. Biopsy size (5 vs 2 mm) and width (15 vs 18 g) had a marked effect on tissue yield. CONCLUSION: Ideally 5-mm biopsies with 15-gauge needles should be used to maximize yield. RNAlater provided higher RNA yield with similar yields of DNA and protein and was notably cheaper and easier to handle.

S-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis C nonresponders.

BACKGROUND & AIMS: Less than half of patients infected with hepatitis C virus (HCV) achieve sustained viral clearance after pegylated interferon (peginterferon) and ribavirin therapy. S-adenosyl methionine (SAMe) improves interferon signaling in cell culture. We assessed the effect of SAMe on the kinetics of the early antiviral response and interferon signaling in nonresponders to previous antiviral therapy and investigated the mechanisms involved. METHODS: Nonresponders with HCV genotype 1 were given peginterferon alfa-2a and ribavirin for 2 weeks (course A, baseline/control). After 1 month, patients received SAMe (1600 mg daily) for 2 weeks and then peginterferon and ribavirin for 48 weeks (course B; completed by 21 of 24 patients). Viral kinetics and interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) were compared between courses. RESULTS: The decrease in HCV RNA from 0 to 48 hours (phase 1) was similar with and without SAMe. However, the second phase slope of viral decline was improved with SAMe (course A, 0.11 ± 0.04 log(10) IU/mL/wk; course B, 0.27 ± 0.06; P = .009); 11 patients (53%) achieved an early virological response, and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater during course B. In cultured cells, SAMe increased induction of ISGs and the antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding. CONCLUSIONS: The addition of SAMe to peginterferon and ribavirin improves the early viral kinetics and increases ISG induction in nonresponders to previous therapy. SAMe might be a useful adjunct to peginterferon-based therapies in chronic HCV infection.

Human neutrophil peptides and phagocytic deficiency in bronchiectatic lungs.

RATIONALE: A well-known clinical paradox is that severe bacterial infections persist in the lungs of patients with cystic fibrosis (CF) despite the abundance of polymorphonuclear neutrophils (PMN) and the presence of a high concentration of human neutrophil peptides (HNP), both of which are expected to kill the bacteria but fail to do so. The mechanisms remain unknown. OBJECTIVES: This study examined several possible mechanisms to understand this paradox. METHODS: PMN were isolated from sputum and blood of subjects with and without CF or non-CF bronchiectasis for phagocytic assays. HNP isolated from patients with CF were used to stimulate healthy PMN followed by phagocytic tests. MEASUREMENTS AND MAIN RESULTS: PMN isolated from the sputum of the bronchiectatic patients display defective phagocytosis that correlated with high concentrations of HNP in the lung. When healthy PMN were incubated with HNP, decreased phagocytic capacity was observed in association with depressed surface Fc gamma RIII, actin-filament remodeling, enhanced intracellular Ca(2+), and degranulation. Treatment of PMN with an intracellular Ca(2+) blocker or alpha1-proteinase inhibitor to attenuate the activity of HNP largely prevented the HNP-induced phagocytic deficiency. Intratracheal instillation of HNP in Pallid mice (genetically deficient in alpha1-proteinase inhibitor) resulted in a greater PMN lung infiltration and phagocytic deficiency compared with wild-type mice. CONCLUSIONS: HNP or PMN alone exert antimicrobial ability, which was lost as a result of their interaction. These effects of HNP may help explain the clinical paradox seen in patients with inflammatory lung diseases, suggesting HNP as a novel target for clinical therapy.

CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages.

Diverse receptors, including Fcgamma receptors and beta(2) integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg(2+) but not Ca(2+). In addition, buffering of intracellular Ca(2+) did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.

Abnormalities in the pulmonary innate immune system in cystic fibrosis.

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.

Tyrosine phosphatase MEG2 modulates murine development and platelet and lymphocyte activation through secretory vesicle function.

MEG2, a protein tyrosine phosphatase with a unique NH2-terminal lipid-binding domain, binds to and is modulated by the polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3. Recent data implicate MEG2 in vesicle fusion events in leukocytes. Through the genesis of Meg2-deficient mice, we demonstrate that Meg2-/- embryos manifest hemorrhages, neural tube defects including exencephaly and meningomyeloceles, cerebral infarctions, abnormal bone development, and >90% late embryonic lethality. T lymphocytes and platelets isolated from recombination activating gene 2-/- mice transplanted with Meg2-/- embryonic liver-derived hematopoietic progenitor cells showed profound defects in activation that, in T lymphocytes, was attributable to impaired interleukin 2 secretion. Ultrastructural analysis of these lymphocytes revealed near complete absence of mature secretory vesicles. Taken together, these observations suggest that MEG2-mediated modulation of secretory vesicle genesis and function plays an essential role in neural tube, vascular, and bone development as well as activation of mature platelets and lymphocytes.

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity.

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

Leukocyte elastase induces epithelial apoptosis: role of mitochondial permeability changes and Akt.

During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.

Protein-tyrosine phosphatase MEG2 is expressed by human neutrophils. Localization to the phagosome and activation by polyphosphoinositides.

Signaling pathways involving reversible tyrosine phosphorylation are essential for neutrophil antimicrobial responses. Using reverse transcriptase PCR, expression of the protein-tyrosine phosphatase MEG2 by peripheral neutrophilic polymorphonuclear leukocytes (PMN) was identified. Polyclonal antibodies against MEG2 were developed that confirmed expression of MEG2 protein by PMN. Through a combination of immunofluorescence and cell fractionation followed by immunoblotting, we determined that MEG2 is predominantly cytosolic with components present in secondary and tertiary granules and secretory vesicles. MEG2 activity, as determined by immunoprecipitation and in vitro phosphatase assays, is inhibited after exposure of cells to the particulate stimulant opsonized zymosan or to phorbol 12-myristate 13-acetate but largely unaffected by the chemoattractant N-formyl-methionyl-leucyl-phenyalanine. Studies using bacterially expressed glutathione S-transferase MEG2 fusion protein indicate that cysteine 515 is essential for catalytic activity, whereas the noncatalytic (N-terminal) domain of MEG2 negatively regulates the enzymatic activity of the C-terminal phosphatase domain. The activity of MEG2 is enhanced by specific polyphosphoinositides with the order of potency being phosphatidylinositol (PI) 4,5-diphosphate > PI 3,4,5-triphosphate > PI 4-phosphate. MEG2 associates at an early stage with nascent phagosomes. Taken together, our results indicate that MEG2 is a polyphosphoinositide-activated tyrosine phosphatase that may be involved in signaling events regulating phagocytosis, an essential antimicrobial function in the innate immune response.