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Abstract / Cytokine 70 (2014) 28–79
Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-b promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1 / > IRF3/7 / > IRF3 / , but not IRF7 / , mice compared with wild-type mice. IFN-b and IFN-k2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-k2/3 was greater in IRF3/7 / mice at 5 days after infection. IRF7 / and IRF3/7 / mice presented with airway eosinophilia, whereas only IRF3/7 / mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1 / mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-b, but not IFN-k2/3, produced via an IPS-1-IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-a/b response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence. http://dx.doi.org/10.1016/j.cyto.2014.07.124
118 Small molecule RIG-I agonists as vaccine adjuvants and antiviral therapy Yueh-Ming Loo a, Courtney R. Wilkins a, Ran Dong a, Sowmya Pattabhi b, Myra L. Wang c, Ernesto J. Muñoz c, Kerry W. Fowler c, John B. Grigg c, Reneé C. Ireton a, Shawn P. Iadonato c, Kristin M. Bedard c, Michael Gale Jr. a,b, a Department of Immunology, University of Washington, Seattle, WA, USA, b Department of Pathobiology, University of Washington, Seattle, WA, USA, c KINETA, Inc., Seattle, WA, USA RIG-I is a pathogen recognition receptor that upon RNA virus infections serves to trigger innate immunity to program the adaptive immune response and is essential for the control of infection. RIG-I signaling initiates antiviral programs that restrict virus infection and activate many cell types including dendritic cells and macrophages for antigen presentation and cytokine production. To develop novel small molecule immune adjuvants, we conducted a high-throughput screen to identify RIG-I agonist molecules from which we derived a lead compound, KIN1148. KIN1148 associates with and activates recombinant RIG-I protein in cell-free assays. Binding to endogenous RIG-I in whole cell extracts promoted KIN1148 association with known RIG-I signaling partners including TRIM25, 14-3-3e, DHX15, TBK1 and NEMO and their assembly on the MAVS adaptor protein. Genomics analyses revealed that KIN1148 activation of RIG-I promotes an IRF3- and NF-kB-dependent transcriptional signature that features the expression of innate immune genes, chemokines and cytokines including CCL2, CCL3, CCL4, CCL7, CXCL10, IL-1b and IL-8 from macrophage-like THP-1 cells and in vivo in mouse tissues. KIN1148 treatment promoted the maturation of primary human dendritic cells to enhance antigen-driven activation of CD8 + T cells in cell culture models. Importantly, when administered to mice in combination with either an experimental influenza A virus or West Nile virus vaccine, KIN1148 enhanced the humoral immune response and protection of mice over vaccine alone in subsequent virus challenge. Thus, we have identified a novel small molecule RIG-I agonist that activates the innate immune response to promote enhanced vaccine immunity against RNA viruses. In parallel work, we have identified a distinct family of compounds that exhibit potent antiviral activity against a broad range of RNA viruses including West Nile, hepatitis C and dengue viruses. Collectively, our work demonstrates the strong potential for developing small molecule agonists that activate RIG-like receptor signaling for prophylactic and therapeutic control of pathogenic RNA viruses. This work is supported by funding from NIH/NIAID (HHSN272200900035C, HHSN272201300023C and 1R01AI098943). http://dx.doi.org/10.1016/j.cyto.2014.07.125
Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kd) controls intracellular TNF trafficking in macrophages [1] and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kd inhibition confers protection in ischemia/reperfusion (I/R) models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia – an effect that is sensitive to PI3Kd inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kd (p110dD910A/D910A) or wild-type mice pre- or post-treated with thePI3Kd inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kd as a potential therapeutic target in ischemic stroke [2].
References [1] Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Teasdale RD, et al.. Phosphoinositide 3-kinase d regulates membrane fission of Golgi carriers for selective cytokine secretion. J Cell Biol 2010;190:1053–65. [2] Low PC, Manzanero S, Mohannak N, Narayana VK, Nguyen TH, Kvaskoff D, et al.. PI3Kd inhibitor reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model. Nature Commun 2014;5:3450. http://dx.doi.org/10.1016/j.cyto.2014.07.126
120 PRMT5 dimethylates R30 of the p65 subunit to activate NF-jB Han Wei 1, Benlian Wang 2, Masaru Miyagi 2, Yun She 1, Banu Gopalan 3, De-bin Huang 4, Gourisankar Ghosh 4, George Stark 5, Tao Lu 1, 1 Pharmacology and Toxicology, Indiana University, Indianapolis, IN, USA, 2 Proteomics Center, Case Western Reserve University, Cleveland, OH, USA, 3 Bioinformtic Center, Cleveland Clinic, Cleveland, OH, USA, 4 Biochemistry, University of California, San Diego, CA, USA, 5 Molecular Genetics, Cleveland Clinic, Cleveland, OH, USA The ubiquitous inducible transcription factor NF-jB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-jB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-jB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1b, the p65 subunit of NF-jB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-jB to bind to jB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-jB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that 85% of the NF-jB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-jB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-jB-induced gene expression.
http://dx.doi.org/10.1016/j.cyto.2014.07.127 119 PI3Kd inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model Pei Ching (Regine) Low 1,2, Silvia Manzanero 2, Vinod K. Narayana 2, Tam H. Nguyen 2, David Kvaskoff 2, Faith H. Brennan 2, Marc J. Ruitenberg 2, Mathias Gelderblom 3, Tim Magnus 3, Hyun Ah Kim 4, Brad R.S. Broughton 4, Christopher G. Sobey 4, Bart Vanhaesebroeck 1, Jennifer L. Stow 2, Thiruma V. Arumugam 5, Frédéric A. Meunier 2, 1 UCL Cancer Institute, London, UK, 2 The University of Queensland, Brisbane, Queensland, Australia, 3 Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia, 5 Department of
121 The effect of Salsola tuberculata extracts and Compound A against intracellular interleukin-1alpha (icIL-1a) in the UVB in vitro model for inflammation and chemoprevention in skin Tandeka Magcwebeba, Andrea Van Zyl, Amanda Swart, Pieter Swart, Biochemistry, Stellenbosch University, Stellenbosch, Western Cape Province, South Africa Interleukin-1a is a primary cytokine that is constitutively produced by keratinocytes in skin where it facilitates cell proliferation and inflammation [1–3]. The chronic
Abstract / Cytokine 70 (2014) 28–79
Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-b promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1 / > IRF3/7 / > IRF3 / , but not IRF7 / , mice compared with wild-type mice. IFN-b and IFN-k2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-k2/3 was greater in IRF3/7 / mice at 5 days after infection. IRF7 / and IRF3/7 / mice presented with airway eosinophilia, whereas only IRF3/7 / mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1 / mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-b, but not IFN-k2/3, produced via an IPS-1-IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-a/b response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence. http://dx.doi.org/10.1016/j.cyto.2014.07.124
118 Small molecule RIG-I agonists as vaccine adjuvants and antiviral therapy Yueh-Ming Loo a, Courtney R. Wilkins a, Ran Dong a, Sowmya Pattabhi b, Myra L. Wang c, Ernesto J. Muñoz c, Kerry W. Fowler c, John B. Grigg c, Reneé C. Ireton a, Shawn P. Iadonato c, Kristin M. Bedard c, Michael Gale Jr. a,b, a Department of Immunology, University of Washington, Seattle, WA, USA, b Department of Pathobiology, University of Washington, Seattle, WA, USA, c KINETA, Inc., Seattle, WA, USA RIG-I is a pathogen recognition receptor that upon RNA virus infections serves to trigger innate immunity to program the adaptive immune response and is essential for the control of infection. RIG-I signaling initiates antiviral programs that restrict virus infection and activate many cell types including dendritic cells and macrophages for antigen presentation and cytokine production. To develop novel small molecule immune adjuvants, we conducted a high-throughput screen to identify RIG-I agonist molecules from which we derived a lead compound, KIN1148. KIN1148 associates with and activates recombinant RIG-I protein in cell-free assays. Binding to endogenous RIG-I in whole cell extracts promoted KIN1148 association with known RIG-I signaling partners including TRIM25, 14-3-3e, DHX15, TBK1 and NEMO and their assembly on the MAVS adaptor protein. Genomics analyses revealed that KIN1148 activation of RIG-I promotes an IRF3- and NF-kB-dependent transcriptional signature that features the expression of innate immune genes, chemokines and cytokines including CCL2, CCL3, CCL4, CCL7, CXCL10, IL-1b and IL-8 from macrophage-like THP-1 cells and in vivo in mouse tissues. KIN1148 treatment promoted the maturation of primary human dendritic cells to enhance antigen-driven activation of CD8 + T cells in cell culture models. Importantly, when administered to mice in combination with either an experimental influenza A virus or West Nile virus vaccine, KIN1148 enhanced the humoral immune response and protection of mice over vaccine alone in subsequent virus challenge. Thus, we have identified a novel small molecule RIG-I agonist that activates the innate immune response to promote enhanced vaccine immunity against RNA viruses. In parallel work, we have identified a distinct family of compounds that exhibit potent antiviral activity against a broad range of RNA viruses including West Nile, hepatitis C and dengue viruses. Collectively, our work demonstrates the strong potential for developing small molecule agonists that activate RIG-like receptor signaling for prophylactic and therapeutic control of pathogenic RNA viruses. This work is supported by funding from NIH/NIAID (HHSN272200900035C, HHSN272201300023C and 1R01AI098943). http://dx.doi.org/10.1016/j.cyto.2014.07.125
Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kd) controls intracellular TNF trafficking in macrophages [1] and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kd inhibition confers protection in ischemia/reperfusion (I/R) models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia – an effect that is sensitive to PI3Kd inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kd (p110dD910A/D910A) or wild-type mice pre- or post-treated with thePI3Kd inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kd as a potential therapeutic target in ischemic stroke [2].
References [1] Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Teasdale RD, et al.. Phosphoinositide 3-kinase d regulates membrane fission of Golgi carriers for selective cytokine secretion. J Cell Biol 2010;190:1053–65. [2] Low PC, Manzanero S, Mohannak N, Narayana VK, Nguyen TH, Kvaskoff D, et al.. PI3Kd inhibitor reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model. Nature Commun 2014;5:3450. http://dx.doi.org/10.1016/j.cyto.2014.07.126
120 PRMT5 dimethylates R30 of the p65 subunit to activate NF-jB Han Wei 1, Benlian Wang 2, Masaru Miyagi 2, Yun She 1, Banu Gopalan 3, De-bin Huang 4, Gourisankar Ghosh 4, George Stark 5, Tao Lu 1, 1 Pharmacology and Toxicology, Indiana University, Indianapolis, IN, USA, 2 Proteomics Center, Case Western Reserve University, Cleveland, OH, USA, 3 Bioinformtic Center, Cleveland Clinic, Cleveland, OH, USA, 4 Biochemistry, University of California, San Diego, CA, USA, 5 Molecular Genetics, Cleveland Clinic, Cleveland, OH, USA The ubiquitous inducible transcription factor NF-jB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-jB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-jB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1b, the p65 subunit of NF-jB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-jB to bind to jB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-jB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that 85% of the NF-jB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-jB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-jB-induced gene expression.
http://dx.doi.org/10.1016/j.cyto.2014.07.127 119 PI3Kd inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model Pei Ching (Regine) Low 1,2, Silvia Manzanero 2, Vinod K. Narayana 2, Tam H. Nguyen 2, David Kvaskoff 2, Faith H. Brennan 2, Marc J. Ruitenberg 2, Mathias Gelderblom 3, Tim Magnus 3, Hyun Ah Kim 4, Brad R.S. Broughton 4, Christopher G. Sobey 4, Bart Vanhaesebroeck 1, Jennifer L. Stow 2, Thiruma V. Arumugam 5, Frédéric A. Meunier 2, 1 UCL Cancer Institute, London, UK, 2 The University of Queensland, Brisbane, Queensland, Australia, 3 Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia, 5 Department of
121 The effect of Salsola tuberculata extracts and Compound A against intracellular interleukin-1alpha (icIL-1a) in the UVB in vitro model for inflammation and chemoprevention in skin Tandeka Magcwebeba, Andrea Van Zyl, Amanda Swart, Pieter Swart, Biochemistry, Stellenbosch University, Stellenbosch, Western Cape Province, South Africa Interleukin-1a is a primary cytokine that is constitutively produced by keratinocytes in skin where it facilitates cell proliferation and inflammation [1–3]. The chronic