- Email: [email protected]
ID: 58
Abstract / Cytokine 76 (2015) 66–112 fibroblasts displayed enhanced antiviral protection and transduction of ISG15 restored viral replication to control levels. Difference in antiviral immunity in absence of ISG15 in humans vs. mice is mechanistically explained by ISG15 stabilizing USP18 in humans but not in mice. Thus, ISG15-deficient individuals present with gain-of-function in antiviral immunity at the expense of increased susceptibility to mycobacteria and auto-immunity.
http://dx.doi.org/10.1016/j.cyto.2015.08.085
ID: 56 Moderate chronic levels of IFN-g induced an effective anti-melanoma response with limited autoimmune effects Howard Young *, Julio Valencia, Heekyong Bae, Diana Haines, Stephen Lockett, Megan Karwan, Charlotte Hanson, Alec Barlow, National Institute of Health, United States * Corresponding author. The success of immunotherapies against melanoma involves the development of autoimmune-type responses due to increases of interferon-gamma (IFN-g). However, attempts to use IFN-g in melanoma have been unsuccessful due to low response rate and significant toxicity. Here, we used the unique features of our mouse model to investigate the anti-melanoma effects of IFN-g on a phenotype based autoimmune background. Syngeneic B16-F10 melanoma cells were injected intravenously (iv.) or subcutaneously (sc) into C57BL/6 mice with deletion of the IFN-g 30 UTR AU-Rich element (ARE-DEL) region. We found that moderate sustained levels of IFN-g increased significantly (p = 0.02; ANOVA) overall survival (OS) and decreased tumor growth rate (p = 0.0154, ANOVA) compared to wild-type (WT) mice. Necropsy after clinical sacrifice showed significantly less (p < 0.05; ANOVA) organ colonization and ulceration of the skin overlaying the graft in ARE-DEL mice compared to WT control mice. However, evidence of neoplastic emboli causing micro and macro infarctions (such as in salivary glands) more likely affected OS in ARE-DEL mice. Interestingly, melanoma tumors exhibited increased grade of necrosis and macrophage infiltration in ARE-DEL mice suggesting that moderate increases of IFN-g exert anti- metastatic immune response. Live cell imaging and spheroid invasion assays on Matrigel confirmed that pre-treatment with IFN-g for 48 h limited directional movement, dendrite formation, and metastatic ability of B16-F10 cells. Given the lack of consensus on the right dose of IFN-g, our data suggest that sustained moderately high levels of IFN-g hamper B16-F10 melanoma metastasis but enhance the risk of neoplastic emboli.
http://dx.doi.org/10.1016/j.cyto.2015.08.086
ID: 57 C-type lectin receptor MCL stabilizes Mincle through complex formation for enhances inflammatory cytokine production upon mycobacterial infection Yasunobu Miyake 1, Sho Yamasaki 2, Hiroki Yoshida 1,*, 1 Div. Immunology, Dept. Biomolecular Sciences, Faculty of Medicine, Saga University, Japan, 2 Div. Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Japan * Corresponding author. Tuberculosis is a life-threatening infectious disease caused by Mycobacterial tuberculosis. C-type lectin receptor Mincle (macrophage-inducible C-type lectin) recognizes mycobacterial glycolipid, TDM (trehalose-dimycolate), and induces the production of inflammatory cytokines such as TNF-a and MIP-2 to elicit anti-mycobacterial responses. We recently reported that MCL, which shares high homology with Mincle, is also indispensable for efficient TDM responses. MCL has low affinity to TDM, which is substantiated by structural analysis. We therefore speculated that MCL has an important function that is distinct from one as a direct receptor for TDM. Here we show that MCL forms a complex with Mincle and stabilizes it on the cell surface. The stalk region that connects transmembrane domain and carbohydrate recognition domain is necessary and sufficient for the heterodimer formation and the stabilization of Mincle. Mutagenesis of hydrophobic residues in the stalk region of MCL disrupted the efficient expression of Mincle and compromised the production of TNF-a and MIP-2 upon TDM stimulation. In conclusion, we have identified a novel function of MCL as a positive regulator of Mincle expression through heterodimer formation via its stalk region. MCL thereby magnifies the Mincle-mediated anti-mycobacterial responses including production of TNF-a and MIP-2.
75
ID: 58 Involvement of MAPK- and PI3K-pathway in Gab1 translocation and phosphorylation Wiebke Hessenkemper 1,*, Hannes Bongartz 1, Johannes Fritsch 1, Alexandra Wolf 1, René Eulenfeld 1, Philip C. Simister 2, Stephan M. Feller 2,3, Fred Schaper 1, 1 Otto-vonGuericke University, Magdeburg, Germany, 2 University of Oxford, John Radcliffe Hospital, Oxford, UK, 3 Martin-Luther-University Halle-Wittenberg, Halle, Germany * Corresponding author. The timely orchestration of multiple signalling pathways is crucial for the integrity of organisms and therefore tightly controlled. Gab family proteins coordinate signal transduction at the plasma membrane by acting as docking platforms for signalling components involved in various pathways such as MAP kinase (MAPK) and PI3 kinase (PI3K) cascade. The interaction with these components as well as the targeting of the docking platform to the plasma membrane underlies complex spatial and temporal regulatory mechanisms. In this study, we focus on Gab1, which is known to be activated and regulated by PI3K- and MAPK-cascades, but is also regulating these pathways. Previously, we could demonstrate that the presence of PIP3 in the plasma membrane alone, which is generated by PI3K, is not sufficient for constitutive Gab1 membrane recruitment. In addition, MAPK-dependent phosphorylation of Gab1 at serine 552 (Ser552) is vital for Gab1 membrane binding. Here, we confirm our hypothesis that in the absence of MAPK activity or by substituting Ser552 in Gab1 for an alanine an intramolecular interaction within Gab1 prevents binding to the plasma membrane. This interaction is mediated by the Gab1 PH domain and an epitope of Gab1, which encompasses Ser552 as well as two arginine residues (Arg556/ Arg560) that are essential for the inhibition of membrane recruitment of Gab1 in the absence of a stimulus. Our data document that Gab1 recruitment to the plasma membrane is highly dynamic and continuous activation of both, PI3K and MAPK are essential for sustained Gab1 membrane localisation and phosphorylation of Gab1. http://dx.doi.org/10.1016/j.cyto.2015.08.088
ID: 59 Combination of classical genetics, reverse genetics and transcriptomics to study reovirus’ interferon sensitivity and host-cell response Simon Boudreault 2, Stéphanie Côté 1, Delphine Lanoie 1, Mathieu Durand 2, Elvy Lapointe 2, Philippe Thibault 2, Véronique Sandekian 1, Martin Bisaillon 2, Guy Lemay 1,*, 1 Département de microbiologie, infectiologie et immunologie, Université de Montréal, Québec, Canada, 2 Département de biochimie, Université de Sherbrooke, Québec, Canada * Corresponding author. Mammalian reovirus is an oncolytic virus that discriminates between parental and cancer cells. This is due in part to altered interferon-induced antiviral response in cancer cells. However, understanding of viral and cellular determinants involved remains largely incomplete. In our current work, two different approaches were used to identify reovirus variants exhibiting increased interferon sensitivity. (1) Chemical mutagenesis followed by selection of small plaques mutants in the presence of interferon; (2) Selection on interferon-deficient cells in order to relieve the pressure to keep interferon resistance. Virus variants were sequenced and plasmid-based reverse genetics was used to examine the effect of each mutation. A single amino acid substitution in the 20 -O methyltransferase of viral mRNA capping enzyme was responsible for 10–100 fold increased interferon sensitivity for one variant; work is under progress for additional viruses. In parallel, we took advantage of these well genetically-defined viruses to investigate host-cell response using RNA-Seq. A large number of induced genes was observed in wild-type virus-infected cells, to an extent that was not revealed by previously-used approaches. Most induced genes encode sensors or antiviral response determinants (ISG, IFIT, IRF, Mx). They were similarly induced by the wild-type and interferon-sensitive virus. This suggests that altered RNA capping increases virus’ sensitivity to interferon-induced antiviral factors rather than induction of interferon signaling. Similar analysis with other viruses of different interferon sensitivity, in parental and transformed cells, should allow to better comprehend the antiviral response during reovirus infection and could contribute to the design of better oncolytic viruses. http://dx.doi.org/10.1016/j.cyto.2015.08.089
ID: 60 Novel intrinsic/innate pathways that sense the ‘‘stress” of virus infection Karen Mossman, David Hare, Susan Collins, McMaster University, Canada
http://dx.doi.org/10.1016/j.cyto.2015.08.087 Directly sensing virus particles is critical as a first line of antiviral defense, because once allowed to replicate, most viruses will prevent induction of a response.
http://dx.doi.org/10.1016/j.cyto.2015.08.085
ID: 56 Moderate chronic levels of IFN-g induced an effective anti-melanoma response with limited autoimmune effects Howard Young *, Julio Valencia, Heekyong Bae, Diana Haines, Stephen Lockett, Megan Karwan, Charlotte Hanson, Alec Barlow, National Institute of Health, United States * Corresponding author. The success of immunotherapies against melanoma involves the development of autoimmune-type responses due to increases of interferon-gamma (IFN-g). However, attempts to use IFN-g in melanoma have been unsuccessful due to low response rate and significant toxicity. Here, we used the unique features of our mouse model to investigate the anti-melanoma effects of IFN-g on a phenotype based autoimmune background. Syngeneic B16-F10 melanoma cells were injected intravenously (iv.) or subcutaneously (sc) into C57BL/6 mice with deletion of the IFN-g 30 UTR AU-Rich element (ARE-DEL) region. We found that moderate sustained levels of IFN-g increased significantly (p = 0.02; ANOVA) overall survival (OS) and decreased tumor growth rate (p = 0.0154, ANOVA) compared to wild-type (WT) mice. Necropsy after clinical sacrifice showed significantly less (p < 0.05; ANOVA) organ colonization and ulceration of the skin overlaying the graft in ARE-DEL mice compared to WT control mice. However, evidence of neoplastic emboli causing micro and macro infarctions (such as in salivary glands) more likely affected OS in ARE-DEL mice. Interestingly, melanoma tumors exhibited increased grade of necrosis and macrophage infiltration in ARE-DEL mice suggesting that moderate increases of IFN-g exert anti- metastatic immune response. Live cell imaging and spheroid invasion assays on Matrigel confirmed that pre-treatment with IFN-g for 48 h limited directional movement, dendrite formation, and metastatic ability of B16-F10 cells. Given the lack of consensus on the right dose of IFN-g, our data suggest that sustained moderately high levels of IFN-g hamper B16-F10 melanoma metastasis but enhance the risk of neoplastic emboli.
http://dx.doi.org/10.1016/j.cyto.2015.08.086
ID: 57 C-type lectin receptor MCL stabilizes Mincle through complex formation for enhances inflammatory cytokine production upon mycobacterial infection Yasunobu Miyake 1, Sho Yamasaki 2, Hiroki Yoshida 1,*, 1 Div. Immunology, Dept. Biomolecular Sciences, Faculty of Medicine, Saga University, Japan, 2 Div. Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Japan * Corresponding author. Tuberculosis is a life-threatening infectious disease caused by Mycobacterial tuberculosis. C-type lectin receptor Mincle (macrophage-inducible C-type lectin) recognizes mycobacterial glycolipid, TDM (trehalose-dimycolate), and induces the production of inflammatory cytokines such as TNF-a and MIP-2 to elicit anti-mycobacterial responses. We recently reported that MCL, which shares high homology with Mincle, is also indispensable for efficient TDM responses. MCL has low affinity to TDM, which is substantiated by structural analysis. We therefore speculated that MCL has an important function that is distinct from one as a direct receptor for TDM. Here we show that MCL forms a complex with Mincle and stabilizes it on the cell surface. The stalk region that connects transmembrane domain and carbohydrate recognition domain is necessary and sufficient for the heterodimer formation and the stabilization of Mincle. Mutagenesis of hydrophobic residues in the stalk region of MCL disrupted the efficient expression of Mincle and compromised the production of TNF-a and MIP-2 upon TDM stimulation. In conclusion, we have identified a novel function of MCL as a positive regulator of Mincle expression through heterodimer formation via its stalk region. MCL thereby magnifies the Mincle-mediated anti-mycobacterial responses including production of TNF-a and MIP-2.
75
ID: 58 Involvement of MAPK- and PI3K-pathway in Gab1 translocation and phosphorylation Wiebke Hessenkemper 1,*, Hannes Bongartz 1, Johannes Fritsch 1, Alexandra Wolf 1, René Eulenfeld 1, Philip C. Simister 2, Stephan M. Feller 2,3, Fred Schaper 1, 1 Otto-vonGuericke University, Magdeburg, Germany, 2 University of Oxford, John Radcliffe Hospital, Oxford, UK, 3 Martin-Luther-University Halle-Wittenberg, Halle, Germany * Corresponding author. The timely orchestration of multiple signalling pathways is crucial for the integrity of organisms and therefore tightly controlled. Gab family proteins coordinate signal transduction at the plasma membrane by acting as docking platforms for signalling components involved in various pathways such as MAP kinase (MAPK) and PI3 kinase (PI3K) cascade. The interaction with these components as well as the targeting of the docking platform to the plasma membrane underlies complex spatial and temporal regulatory mechanisms. In this study, we focus on Gab1, which is known to be activated and regulated by PI3K- and MAPK-cascades, but is also regulating these pathways. Previously, we could demonstrate that the presence of PIP3 in the plasma membrane alone, which is generated by PI3K, is not sufficient for constitutive Gab1 membrane recruitment. In addition, MAPK-dependent phosphorylation of Gab1 at serine 552 (Ser552) is vital for Gab1 membrane binding. Here, we confirm our hypothesis that in the absence of MAPK activity or by substituting Ser552 in Gab1 for an alanine an intramolecular interaction within Gab1 prevents binding to the plasma membrane. This interaction is mediated by the Gab1 PH domain and an epitope of Gab1, which encompasses Ser552 as well as two arginine residues (Arg556/ Arg560) that are essential for the inhibition of membrane recruitment of Gab1 in the absence of a stimulus. Our data document that Gab1 recruitment to the plasma membrane is highly dynamic and continuous activation of both, PI3K and MAPK are essential for sustained Gab1 membrane localisation and phosphorylation of Gab1. http://dx.doi.org/10.1016/j.cyto.2015.08.088
ID: 59 Combination of classical genetics, reverse genetics and transcriptomics to study reovirus’ interferon sensitivity and host-cell response Simon Boudreault 2, Stéphanie Côté 1, Delphine Lanoie 1, Mathieu Durand 2, Elvy Lapointe 2, Philippe Thibault 2, Véronique Sandekian 1, Martin Bisaillon 2, Guy Lemay 1,*, 1 Département de microbiologie, infectiologie et immunologie, Université de Montréal, Québec, Canada, 2 Département de biochimie, Université de Sherbrooke, Québec, Canada * Corresponding author. Mammalian reovirus is an oncolytic virus that discriminates between parental and cancer cells. This is due in part to altered interferon-induced antiviral response in cancer cells. However, understanding of viral and cellular determinants involved remains largely incomplete. In our current work, two different approaches were used to identify reovirus variants exhibiting increased interferon sensitivity. (1) Chemical mutagenesis followed by selection of small plaques mutants in the presence of interferon; (2) Selection on interferon-deficient cells in order to relieve the pressure to keep interferon resistance. Virus variants were sequenced and plasmid-based reverse genetics was used to examine the effect of each mutation. A single amino acid substitution in the 20 -O methyltransferase of viral mRNA capping enzyme was responsible for 10–100 fold increased interferon sensitivity for one variant; work is under progress for additional viruses. In parallel, we took advantage of these well genetically-defined viruses to investigate host-cell response using RNA-Seq. A large number of induced genes was observed in wild-type virus-infected cells, to an extent that was not revealed by previously-used approaches. Most induced genes encode sensors or antiviral response determinants (ISG, IFIT, IRF, Mx). They were similarly induced by the wild-type and interferon-sensitive virus. This suggests that altered RNA capping increases virus’ sensitivity to interferon-induced antiviral factors rather than induction of interferon signaling. Similar analysis with other viruses of different interferon sensitivity, in parental and transformed cells, should allow to better comprehend the antiviral response during reovirus infection and could contribute to the design of better oncolytic viruses. http://dx.doi.org/10.1016/j.cyto.2015.08.089
ID: 60 Novel intrinsic/innate pathways that sense the ‘‘stress” of virus infection Karen Mossman, David Hare, Susan Collins, McMaster University, Canada
http://dx.doi.org/10.1016/j.cyto.2015.08.087 Directly sensing virus particles is critical as a first line of antiviral defense, because once allowed to replicate, most viruses will prevent induction of a response.