- Email: [email protected]
ID: 88
Abstract / Cytokine 76 (2015) 66–112 These studies were supported by Federal Target Program ‘‘The development of the pharmaceutical and medical industry of the Russian Federation for the period up to 2020 and beyond”. http://dx.doi.org/10.1016/j.cyto.2015.08.112
ID: 86 Defining the functional binding sites of IL-12Rb1 and IL-23R to Janus kinases Doreen Manuela Floss *, Tobias Klöcker, Jürgen Scheller, Heinrich-Heine-University, Medical Faculty, Institute of Biochemistry and Molecular Biology II, Germany * Corresponding author. The Interleukin (IL)-12-type cytokines IL-12 and IL-23 show structural similarities but have different functions in the immune system. They are involved in T helper (Th) 1 or Th17 immunity, respectively. Both cytokines are heterodimers composed of the shared IL-12p40 subunit and IL-12p35 or IL-23p19, respectively, and share the IL12Rb1 as one component of their receptor signaling complexes. The second receptor for IL-12 signaling is IL-12Rb2 and for IL-23 signal transduction IL-23R. Stimulation of cells containing the appropriate receptors with IL-12 or IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase Tyrosine kinase (Tyk) 2 was shown to associate with IL-12Rb1, whereas Jak2 binds to the IL-23R and also to the IL-12Rb2. Association of Janus kinases to receptors is mediated by so-called Box1 and Box2 motifs within the intracellular domain of the receptor chains. We have defined the hypothesized Box1 and Box2 binding sites in the IL-12Rb1 and an unusual Jak2 binding site in the IL-23R by the use of deletion variants and site-directed mutagenesis. Non-functional box motifs abrogate IL-12 and IL-23 induced STAT phosphorylation and cytokine-dependent proliferation of respective generated Ba/F3 cells. Co-immunoprecipitation of Tyk2 by IL-12Rb1 and Jak2 by IL 23R support our findings. In summary, we characterize the functional association of IL 12Rb1 with Tyk2 and IL-23R with Jak2, which are mandatory for IL-12 and/or IL-23 signaling. http://dx.doi.org/10.1016/j.cyto.2015.08.113
ID: 87 Anti-proliferative and cell death-inducing effects of interferon lambda-4 (IFNL4) may contribute differentially to HCV pathogenesis and cancer Olusegun Onabajo *, Ludmila Prokunina-Olsson, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, United States * Corresponding author. IFN-k4 is a novel type-III interferon with strong clinical significance in humans. Only individuals who carry the DG allele of a genetic variant rs368234815-TT/DG are genetically able to produce IFN-k4 protein. Carriers of the DG allele have impaired ability to clear hepatitis C virus (HCV) infection (Prokunina-Olsson et al., 2013). At the same time, recent findings suggest that carriers of the DG allele are protected from mucinous type of ovarian cancer (Kelemen et al., 2015). We explored the function of IFN-k4 in an inducible cell line HepG2- IFN-k4 -GFP, which produces a partially secreted IFN-k4. We observed two main phenotypes caused by IFN-k4 expression: induction of interferon-stimulated genes (ISGs) both in the expressing and bystander cells, and increased cell death with reduced proliferation in the expressing cells. Both effects were inhibited by a blocking antibody for IFN-k4, suggesting that they might be related and caused by secreted IFN-k4. Our results suggest that chronic induction of IFN-k4 in HCV-infected hepatocytes of individuals with rs368234815-DG allele could contribute to pre-activation of interferon signaling competing with effects of type-I interferons (IFN-aÞ. IFN-k4-induced death of hepatic cells may contribute to development of chronic hepatitis and eventual liver failure. At the same time, we suggest that the anti-proliferative effects of IFN-k4 might be beneficial in other circumstances, and result in prevention of excessive proliferation and elimination of tumor cells. The pathogenic or beneficial effect of IFN-k4 might depend on cell type specificity and triggers of its expression. http://dx.doi.org/10.1016/j.cyto.2015.08.114
ID: 88 Regulation of strawberry notch homolog 2 – A novel inflammatory response factor in astroglia and other CNS cells Taylor Edward Syme 1,*, Magdalena Grill 1,2, Aline Lara Noçon 1, Stefan Rose-John 3, Iain Leslie Campbell 1, 1 School of Molecular Bioscience, University of Sydney, Australia,
2 3
81
Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria, Department of Biochemistry, University of Kiel, Germany * Corresponding author.
We have identified a novel putative transcriptional regulator called strawberry notch homolog 2 (Sbno2) as a prominent IL-6-stimulated gene in astrocytes. The aim of this study was to: (i) examine the regulation of Sbno2 in astrocytes in vitro and in vivo, and (ii) establish lentiviral transduction of cells with a Sbno2 transgene for the functional characterization of Sbno2. In murine cultured astrocytes Sbno2 gene expression was significantly up-regulated in a dose- and time-dependent fashion following treatment with hyper-IL-6 (IL-6 + soluble IL-6 receptor). These changes were reflected by corresponding alterations in the level of the SBNO2 protein. The level of Sbno2 mRNA was up-regulated significantly in murine astrocytes by other glycoprotein130 cytokine-family members and the pro-inflammatory cytokines IL-1b and TNF-a. Cycloheximide treatment resulted in markedly higher Sbno2 mRNA and did not abolish the up-regulation of Sbno2 mRNA mediated by hyper-IL-6. Following intra-peritoneal LPS injection in mice, Sbno2 mRNA levels in the brain increased significantly and was localized predominantly to astrocytes and the choroid plexus, and to a lesser extent to microglia, endothelial cells, and neurons. Characterisation of transduced NIH-3T3 fibroblasts revealed transgenic SBNO2 localised exclusively to the nucleus but not the nucleoli. Our findings indicate that the Sbno2 gene is broadly regulated in astrocytes and likely encodes a nuclear factor involved in the innate host response. A mouse model for the conditional deletion of the Sbno2 gene in astrocytes developed by us will provide an invaluable tool for evaluating function of Sbno2 in mammalian cells. Funding: NHMRC APP1047265; FWF J3081-B09. http://dx.doi.org/10.1016/j.cyto.2015.08.115
ID: 89 Comparison of IFNL4-DG/TT and IFNL3 rs4803217 for association with hepatitis C virus clearance Thomas O’Brien 1, Ruth Pfeiffer 1, Ashley Paquin 1, Krystle Lang Kuhs 1, Sabrina Chen 2, Charles Howell 3, Ludmila Prokunina-Olsson 1,*, 1 National Cancer Institute, Bethesda, MD, USA, 2 Information Management Services, Calverton, MD, USA, 3 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA * Corresponding author. Genetic polymorphisms within the interferon lambda region are strongly associated with hepatitis C virus (HCV) clearance; the rs368234815 (IFNL4-DG/TT) polymorphism, which controls generation of the IFNL4 protein, is more strongly associated with HCV clearance than rs12979860 (the ‘IL28B marker’). A polymorphism rs4803217 within the 3’ untranslated region of IFNL3 has been proposed as a causal variant affecting HCV clearance by altering IFNL3 mRNA stability. We compared response to peg-IFN-a/ ribavirin treatment in the VIRAHEP-C and HALT-C trials, and spontaneous HCV clearance in the ALIVE, UHS and WIHS sets in relation to genotypes and haplotypes of rs368234815 and rs4803217. In European Americans, linkage disequilibrium between rs368234815 and rs4803217 was strong (r2 = 0.89–0.99) and there were no significant differences between the variants. Decreased linkage disequilibrium between these variants in African Americans (r2 = 0.74–0.80), allowed comparisons between these candidates. In individuals enrolled in VIRAHEP-C, HCV RNA at treatment day 28 was more strongly associated with rs368234815 than rs4803217 (p = 0.003); the rs368234815-DG: rs4803217-G haplotype, which includes the putatively favorable IFNL3 allele, was actually associated with the poorest day 28 response (p = 0.03, compared to rs368234815DG:rs4803217-T haplotype). Associations with undetectable HCV RNA at week 24 in VIRAHEP-C (p = 0.03) and week 20 in HALT-C (p = 0.03), as well as for spontaneous HCV clearance (p = 0.048) were also stronger for rs368234815 than rs4803217. Our results provide evidence that rs3682348150 is associated with HCV clearance stronger than rs4803217 and suggest a possible negative role for the IFNL3 rs4803217-G allele in individuals who can produce IFNL4 protein. http://dx.doi.org/10.1016/j.cyto.2015.08.116
ID: 90 The pestiviral IFN antagonist E(rns) cleaves dsRNA as nicking endoribonuclease Carmela Lussi *, Matthias Schweizer, Institute of Virology and Immunology (IVI), Federal Food Safety and Veterinary Office and Vetsuisse Faculty University of Bern, Länggass-Str. 122, CH-3001 Bern, Switzerland * Corresponding author. Bovine virus diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, causes persistent infection (PI) in cattle by entering the fetus early in gestation prior to development of adaptive immunity. In addition, the inhibition of interferon (IFN) type I synthesis plays an important role in escaping innate immunity and is a prerequisite
ID: 86 Defining the functional binding sites of IL-12Rb1 and IL-23R to Janus kinases Doreen Manuela Floss *, Tobias Klöcker, Jürgen Scheller, Heinrich-Heine-University, Medical Faculty, Institute of Biochemistry and Molecular Biology II, Germany * Corresponding author. The Interleukin (IL)-12-type cytokines IL-12 and IL-23 show structural similarities but have different functions in the immune system. They are involved in T helper (Th) 1 or Th17 immunity, respectively. Both cytokines are heterodimers composed of the shared IL-12p40 subunit and IL-12p35 or IL-23p19, respectively, and share the IL12Rb1 as one component of their receptor signaling complexes. The second receptor for IL-12 signaling is IL-12Rb2 and for IL-23 signal transduction IL-23R. Stimulation of cells containing the appropriate receptors with IL-12 or IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase Tyrosine kinase (Tyk) 2 was shown to associate with IL-12Rb1, whereas Jak2 binds to the IL-23R and also to the IL-12Rb2. Association of Janus kinases to receptors is mediated by so-called Box1 and Box2 motifs within the intracellular domain of the receptor chains. We have defined the hypothesized Box1 and Box2 binding sites in the IL-12Rb1 and an unusual Jak2 binding site in the IL-23R by the use of deletion variants and site-directed mutagenesis. Non-functional box motifs abrogate IL-12 and IL-23 induced STAT phosphorylation and cytokine-dependent proliferation of respective generated Ba/F3 cells. Co-immunoprecipitation of Tyk2 by IL-12Rb1 and Jak2 by IL 23R support our findings. In summary, we characterize the functional association of IL 12Rb1 with Tyk2 and IL-23R with Jak2, which are mandatory for IL-12 and/or IL-23 signaling. http://dx.doi.org/10.1016/j.cyto.2015.08.113
ID: 87 Anti-proliferative and cell death-inducing effects of interferon lambda-4 (IFNL4) may contribute differentially to HCV pathogenesis and cancer Olusegun Onabajo *, Ludmila Prokunina-Olsson, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, United States * Corresponding author. IFN-k4 is a novel type-III interferon with strong clinical significance in humans. Only individuals who carry the DG allele of a genetic variant rs368234815-TT/DG are genetically able to produce IFN-k4 protein. Carriers of the DG allele have impaired ability to clear hepatitis C virus (HCV) infection (Prokunina-Olsson et al., 2013). At the same time, recent findings suggest that carriers of the DG allele are protected from mucinous type of ovarian cancer (Kelemen et al., 2015). We explored the function of IFN-k4 in an inducible cell line HepG2- IFN-k4 -GFP, which produces a partially secreted IFN-k4. We observed two main phenotypes caused by IFN-k4 expression: induction of interferon-stimulated genes (ISGs) both in the expressing and bystander cells, and increased cell death with reduced proliferation in the expressing cells. Both effects were inhibited by a blocking antibody for IFN-k4, suggesting that they might be related and caused by secreted IFN-k4. Our results suggest that chronic induction of IFN-k4 in HCV-infected hepatocytes of individuals with rs368234815-DG allele could contribute to pre-activation of interferon signaling competing with effects of type-I interferons (IFN-aÞ. IFN-k4-induced death of hepatic cells may contribute to development of chronic hepatitis and eventual liver failure. At the same time, we suggest that the anti-proliferative effects of IFN-k4 might be beneficial in other circumstances, and result in prevention of excessive proliferation and elimination of tumor cells. The pathogenic or beneficial effect of IFN-k4 might depend on cell type specificity and triggers of its expression. http://dx.doi.org/10.1016/j.cyto.2015.08.114
ID: 88 Regulation of strawberry notch homolog 2 – A novel inflammatory response factor in astroglia and other CNS cells Taylor Edward Syme 1,*, Magdalena Grill 1,2, Aline Lara Noçon 1, Stefan Rose-John 3, Iain Leslie Campbell 1, 1 School of Molecular Bioscience, University of Sydney, Australia,
2 3
81
Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria, Department of Biochemistry, University of Kiel, Germany * Corresponding author.
We have identified a novel putative transcriptional regulator called strawberry notch homolog 2 (Sbno2) as a prominent IL-6-stimulated gene in astrocytes. The aim of this study was to: (i) examine the regulation of Sbno2 in astrocytes in vitro and in vivo, and (ii) establish lentiviral transduction of cells with a Sbno2 transgene for the functional characterization of Sbno2. In murine cultured astrocytes Sbno2 gene expression was significantly up-regulated in a dose- and time-dependent fashion following treatment with hyper-IL-6 (IL-6 + soluble IL-6 receptor). These changes were reflected by corresponding alterations in the level of the SBNO2 protein. The level of Sbno2 mRNA was up-regulated significantly in murine astrocytes by other glycoprotein130 cytokine-family members and the pro-inflammatory cytokines IL-1b and TNF-a. Cycloheximide treatment resulted in markedly higher Sbno2 mRNA and did not abolish the up-regulation of Sbno2 mRNA mediated by hyper-IL-6. Following intra-peritoneal LPS injection in mice, Sbno2 mRNA levels in the brain increased significantly and was localized predominantly to astrocytes and the choroid plexus, and to a lesser extent to microglia, endothelial cells, and neurons. Characterisation of transduced NIH-3T3 fibroblasts revealed transgenic SBNO2 localised exclusively to the nucleus but not the nucleoli. Our findings indicate that the Sbno2 gene is broadly regulated in astrocytes and likely encodes a nuclear factor involved in the innate host response. A mouse model for the conditional deletion of the Sbno2 gene in astrocytes developed by us will provide an invaluable tool for evaluating function of Sbno2 in mammalian cells. Funding: NHMRC APP1047265; FWF J3081-B09. http://dx.doi.org/10.1016/j.cyto.2015.08.115
ID: 89 Comparison of IFNL4-DG/TT and IFNL3 rs4803217 for association with hepatitis C virus clearance Thomas O’Brien 1, Ruth Pfeiffer 1, Ashley Paquin 1, Krystle Lang Kuhs 1, Sabrina Chen 2, Charles Howell 3, Ludmila Prokunina-Olsson 1,*, 1 National Cancer Institute, Bethesda, MD, USA, 2 Information Management Services, Calverton, MD, USA, 3 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA * Corresponding author. Genetic polymorphisms within the interferon lambda region are strongly associated with hepatitis C virus (HCV) clearance; the rs368234815 (IFNL4-DG/TT) polymorphism, which controls generation of the IFNL4 protein, is more strongly associated with HCV clearance than rs12979860 (the ‘IL28B marker’). A polymorphism rs4803217 within the 3’ untranslated region of IFNL3 has been proposed as a causal variant affecting HCV clearance by altering IFNL3 mRNA stability. We compared response to peg-IFN-a/ ribavirin treatment in the VIRAHEP-C and HALT-C trials, and spontaneous HCV clearance in the ALIVE, UHS and WIHS sets in relation to genotypes and haplotypes of rs368234815 and rs4803217. In European Americans, linkage disequilibrium between rs368234815 and rs4803217 was strong (r2 = 0.89–0.99) and there were no significant differences between the variants. Decreased linkage disequilibrium between these variants in African Americans (r2 = 0.74–0.80), allowed comparisons between these candidates. In individuals enrolled in VIRAHEP-C, HCV RNA at treatment day 28 was more strongly associated with rs368234815 than rs4803217 (p = 0.003); the rs368234815-DG: rs4803217-G haplotype, which includes the putatively favorable IFNL3 allele, was actually associated with the poorest day 28 response (p = 0.03, compared to rs368234815DG:rs4803217-T haplotype). Associations with undetectable HCV RNA at week 24 in VIRAHEP-C (p = 0.03) and week 20 in HALT-C (p = 0.03), as well as for spontaneous HCV clearance (p = 0.048) were also stronger for rs368234815 than rs4803217. Our results provide evidence that rs3682348150 is associated with HCV clearance stronger than rs4803217 and suggest a possible negative role for the IFNL3 rs4803217-G allele in individuals who can produce IFNL4 protein. http://dx.doi.org/10.1016/j.cyto.2015.08.116
ID: 90 The pestiviral IFN antagonist E(rns) cleaves dsRNA as nicking endoribonuclease Carmela Lussi *, Matthias Schweizer, Institute of Virology and Immunology (IVI), Federal Food Safety and Veterinary Office and Vetsuisse Faculty University of Bern, Länggass-Str. 122, CH-3001 Bern, Switzerland * Corresponding author. Bovine virus diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, causes persistent infection (PI) in cattle by entering the fetus early in gestation prior to development of adaptive immunity. In addition, the inhibition of interferon (IFN) type I synthesis plays an important role in escaping innate immunity and is a prerequisite