- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314 latory effects on NK cells and T cells in HCV-infected patients. We synthesized all human IFN-a subtypes and tested their antiviral properties against VSV, HIV-1, Influenza, HCV, HBV, EMCV and HSV-1 in vitro. We stimulated PBMCs from HCV-infected patients and healthy individuals and analyzed the modulation of immune cells by the different IFN-a subtypes. Differences in the antiviral activity of the distinct IFN-a subtypes against the tested viruses were detected. IFN-a21 was very effective in inhibiting VSV and EMCV in vitro, whereas the highest antiviral activity against HSV-1 was mediated by IFN-a8 and IFN-a4. Human IFN-a16 and IFN-a8 were the most potent inhibitors of HCV replication in vitro. Using a transgenic luciferase reporter cell line we observed that human IFN-a2, IFN-a8, IFN-a16 and IFN-a21 showed the highest transcriptional activity under the control of the ISRE promotor. Stimulation of human PBMCs with the tested IFN-a subtypes revealed differences in the modulation of effector functions of T and NK cells. These results demonstrate a unique function of each IFN-a subtype in their direct inhibition of viral infections and their modulation of the innate and adaptive immune response.
http://dx.doi.org/10.1016/j.cyto.2013.06.086
84 Extensive collaboration of immune master regulators IRF3 and NFjB in RNA Pol II recruitment and pause-release in human innate antiviral transcription Jonathan E. Freaney, Rebecca Kim, Curt M. Horvath, Department of Molecular Biosciences, Northwestern University, Evanston, IL 60201, USA The eukaryotic transcription factors, IRF3 and NFjB, regulate immunity and inflammatory responsive genes. Both factors are activated by external stimuli that evoke strong immune and/or inflammatory responses, including virus infection, to translocate to the nucleus and bind their genomic target sites. These two proteins are critical regulators of immunity and inflammatory responses, control diverse normal cellular functions, and aberrations in their activity contribute to pathologies including inflammatory diseases and cancer. Despite the wealth of information regarding the ability of IRF3 and NFjB to regulate the expression of IFNB1 and other individual target genes, their relative contributions to overall virus-activated transcription, their degree of overlap within the antiviral gene regulation network, and their breadth of target sites throughout the genome are under-investigated. The mechanisms used in regulating antiviral Pol II recruitment, initiation, and elongation at specific targets, as well as their abilities to regulate non-coding and protein-coding genes remain poorly understood. To investigate the mechanisms used to mediate RNA Polymerase II (Pol II) recruitment, initiation, and elongation that contribute to the human antiviral gene regulatory network, a comprehensive genome-wide ChIP-seq analysis was conducted during the initial phase of virus infection to provide a detailed and quantitative genome-wide analysis of transcriptional regulation of the cellular antiviral response. Results reveal extensive integration of IRF3 and NFjB with Pol II and its associated machinery, and implicate new transcriptional partners for antiviral gene regulation. In addition to mRNA-encoding loci, a majority of IRF3 and NFjB target sites stimulate RNA production at novel genomic regions not previously associated with antiviral transcription through a combination of de novo polymerase recruitment and release of paused Pol II. These factors work together to tightly regulate gene activation essential to cellular innate immunity. http://dx.doi.org/10.1016/j.cyto.2013.06.087
85 A novel role for the oligoadenylate synthetase-like protein in viral recognition and interferon induction Hans Henrik Gad a, Mikkel S. Ibsen a, Line L. Andersen a, Veit Hornung b, Saumendra N. Sarka c, Rune Hartmann a, a Aarhus University, Aarhus, Denmark, b University Hospital of Bonn, Bonn, Nordrhein-Westfalen, Germany, c University of Pittsburgh, Pittsburgh, PA, USA One of the most important pattern-recognition receptors in terms of recognition of viral RNAs in the cytoplasm is the retinoic acid inducible gene I (RIG-I). Following viral infection, RIG-I predominantly recognizes short double-stranded RNAs (dsRNA) containing triphosphate at their 50 end and triggers a distinct signal transduction pathway leading to induction of cytokines such as interferon (IFN) as well as other antiviral proteins. Activation of RIG-I is regulated at several levels by cellular proteins in order to both prevent undue activation of the innate immune response and to increase the efficiency of RIG-I-mediated signalling. In recent years, it has become clear that ubiquitination plays a key role in regulating RIG-I-mediated signalling and in order to become activated, RIG-I needs not only to bind viral RNA but also free unanchored K63-linked polyubiquitin chains. However, our collaborator Saumendra N. Sarkar from the University of Pittsburgh Cancer Institute, USA, has recently discovered that another IFN-stimulated protein can act as a substitute for polyubiquitin in
263
activation of RIG-I. This protein is known as the oligoadenylate synthetase-like protein (OASL) and interacts with the RIG-I caspase recruitment domains (CARD) through its two C-terminal ubiquitin-like repeats. Furthermore, the C-terminal domain of RIGI and the N-terminal domain of OASL, which both binds dsRNA, were shown to interact with each other. We aim to characterize the mechanism by which OASL induces RIG-I signalling and understand the functional consequences of dsRNA recognition by OASL and I will present recent work on this topic. http://dx.doi.org/10.1016/j.cyto.2013.06.088
86 IFN-c, ER-stress, autophagy and antimicrobial defense: A novel ATF6/C/EBP-b centric signaling pathway Padmaja Gade a, Srikatha Belagihalli a, Girish Ramachandran a, Alan S. Cross a, Kazutoshi Mori b, Dhan V. Kalvakolanu a, a Departments of Microbiology and Immunology, and Greenebaum Cancer, University of Maryland School of Medicine, Baltimore, MD 21201, USA, b Department of Biophysics, Kyoto University, Kyoto, Japan The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The DeathAssociated Protein Kinase 1 (DAPK1), an IFN-g induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis; and its expression is frequently down regulated in a number of human tumors. Previously, we have shown that transcription factor C/EBP-b is required for the basal and IFN-g-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/ EBP-b in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing and p38 MAPK dependent phosphorylation of ATF6; and ERK1/2-mediated phosphorylation of C/EBP-b are necessary for these interactions. The activation of this pathway is independent from the IFN-induced JAK-STAT pathways. More importantly, IFN-g-failed to activate autophagy in cells lacking either ATF6 or C/EBP-b. Consistent with these observations, the Atf6-/- mice were highly susceptible to lethal bacterial infections compared to the wildtype mice. These studies, for the first time, not only unravel a novel IFN signaling pathway that controls cell growth, autophagy and antibacterial defense but also expand the role of ATF6 beyond ER-stress. http://dx.doi.org/10.1016/j.cyto.2013.06.089
87 Caspase-8 regulates b-glucan induced IL-1b production and cell death Sandhya Ganesan a, Vijay A. K. Rathinam a, Lukas Bossaller a, William J. Kaiser b, Edward S. Mocarski b, Douglas R. Green c, Neal S. Silverman a,1, Katherine A. Fitzgerald a,1, a Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA, b Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA, c Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA Inflammasomes play a crucial role in host defense against a variety of microbial infections through regulating the maturation of the IL-1 family of cytokines as well as pyroptotic cell death. We have previously identified an essential role for the NLRP3-ASC-Caspase-1 inflammasome in IL-1b production triggered by the fungal pathogen, C. albicans and the importance of this pathway in the clearance of C. albicans in an in vivo mouse model of Oropharyngeal Candidiasis (OPC). Here, we uncovered an important role for the pro-apoptotic caspase, Caspase-8 in IL-1b production induced by b-glucan, a potent immunostimulatory component of fungal cell walls. We found that b-glucan induced IL-1b production requires Caspase-8, in addition to the NLRP3-ASC-Caspase-1 inflammasome. We treated bone marrow derived macrophages and dendritic cells from RIP3/Caspase-8/ mice with various b-glucan related molecules, such as curdlan, fungal whole glucan particles or heat killed C. albicans, and found that Caspase-8 was required for IL-1b production with these stimuli but not with canonical inflammasome activators such as silica, nigericin or poly (dA:dT). Of note, Caspase-8 deficiency did not affect b-glucan induced synthesis of pro-IL1b. In addition, RIP3/Caspase-8/cells were protected from b-glucan induced cell death, whereas RIP3/ deficiency did not affect b-glucan induced IL-1b production or cell death. In contrast to these observations, we found that IL-1b production and cell death in response to live C. albicans infection was not Caspase-8 dependent, raising the possibility that b-glucan may not be exposed by C. albicans to engage this pathway. This study establishes a key link between Caspase-8 and Caspase-1 in coordinating cytokine secretion and cell death in response to fungal pathogenic patterns,
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cyto.2013.06.086
84 Extensive collaboration of immune master regulators IRF3 and NFjB in RNA Pol II recruitment and pause-release in human innate antiviral transcription Jonathan E. Freaney, Rebecca Kim, Curt M. Horvath, Department of Molecular Biosciences, Northwestern University, Evanston, IL 60201, USA The eukaryotic transcription factors, IRF3 and NFjB, regulate immunity and inflammatory responsive genes. Both factors are activated by external stimuli that evoke strong immune and/or inflammatory responses, including virus infection, to translocate to the nucleus and bind their genomic target sites. These two proteins are critical regulators of immunity and inflammatory responses, control diverse normal cellular functions, and aberrations in their activity contribute to pathologies including inflammatory diseases and cancer. Despite the wealth of information regarding the ability of IRF3 and NFjB to regulate the expression of IFNB1 and other individual target genes, their relative contributions to overall virus-activated transcription, their degree of overlap within the antiviral gene regulation network, and their breadth of target sites throughout the genome are under-investigated. The mechanisms used in regulating antiviral Pol II recruitment, initiation, and elongation at specific targets, as well as their abilities to regulate non-coding and protein-coding genes remain poorly understood. To investigate the mechanisms used to mediate RNA Polymerase II (Pol II) recruitment, initiation, and elongation that contribute to the human antiviral gene regulatory network, a comprehensive genome-wide ChIP-seq analysis was conducted during the initial phase of virus infection to provide a detailed and quantitative genome-wide analysis of transcriptional regulation of the cellular antiviral response. Results reveal extensive integration of IRF3 and NFjB with Pol II and its associated machinery, and implicate new transcriptional partners for antiviral gene regulation. In addition to mRNA-encoding loci, a majority of IRF3 and NFjB target sites stimulate RNA production at novel genomic regions not previously associated with antiviral transcription through a combination of de novo polymerase recruitment and release of paused Pol II. These factors work together to tightly regulate gene activation essential to cellular innate immunity. http://dx.doi.org/10.1016/j.cyto.2013.06.087
85 A novel role for the oligoadenylate synthetase-like protein in viral recognition and interferon induction Hans Henrik Gad a, Mikkel S. Ibsen a, Line L. Andersen a, Veit Hornung b, Saumendra N. Sarka c, Rune Hartmann a, a Aarhus University, Aarhus, Denmark, b University Hospital of Bonn, Bonn, Nordrhein-Westfalen, Germany, c University of Pittsburgh, Pittsburgh, PA, USA One of the most important pattern-recognition receptors in terms of recognition of viral RNAs in the cytoplasm is the retinoic acid inducible gene I (RIG-I). Following viral infection, RIG-I predominantly recognizes short double-stranded RNAs (dsRNA) containing triphosphate at their 50 end and triggers a distinct signal transduction pathway leading to induction of cytokines such as interferon (IFN) as well as other antiviral proteins. Activation of RIG-I is regulated at several levels by cellular proteins in order to both prevent undue activation of the innate immune response and to increase the efficiency of RIG-I-mediated signalling. In recent years, it has become clear that ubiquitination plays a key role in regulating RIG-I-mediated signalling and in order to become activated, RIG-I needs not only to bind viral RNA but also free unanchored K63-linked polyubiquitin chains. However, our collaborator Saumendra N. Sarkar from the University of Pittsburgh Cancer Institute, USA, has recently discovered that another IFN-stimulated protein can act as a substitute for polyubiquitin in
263
activation of RIG-I. This protein is known as the oligoadenylate synthetase-like protein (OASL) and interacts with the RIG-I caspase recruitment domains (CARD) through its two C-terminal ubiquitin-like repeats. Furthermore, the C-terminal domain of RIGI and the N-terminal domain of OASL, which both binds dsRNA, were shown to interact with each other. We aim to characterize the mechanism by which OASL induces RIG-I signalling and understand the functional consequences of dsRNA recognition by OASL and I will present recent work on this topic. http://dx.doi.org/10.1016/j.cyto.2013.06.088
86 IFN-c, ER-stress, autophagy and antimicrobial defense: A novel ATF6/C/EBP-b centric signaling pathway Padmaja Gade a, Srikatha Belagihalli a, Girish Ramachandran a, Alan S. Cross a, Kazutoshi Mori b, Dhan V. Kalvakolanu a, a Departments of Microbiology and Immunology, and Greenebaum Cancer, University of Maryland School of Medicine, Baltimore, MD 21201, USA, b Department of Biophysics, Kyoto University, Kyoto, Japan The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The DeathAssociated Protein Kinase 1 (DAPK1), an IFN-g induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis; and its expression is frequently down regulated in a number of human tumors. Previously, we have shown that transcription factor C/EBP-b is required for the basal and IFN-g-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/ EBP-b in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing and p38 MAPK dependent phosphorylation of ATF6; and ERK1/2-mediated phosphorylation of C/EBP-b are necessary for these interactions. The activation of this pathway is independent from the IFN-induced JAK-STAT pathways. More importantly, IFN-g-failed to activate autophagy in cells lacking either ATF6 or C/EBP-b. Consistent with these observations, the Atf6-/- mice were highly susceptible to lethal bacterial infections compared to the wildtype mice. These studies, for the first time, not only unravel a novel IFN signaling pathway that controls cell growth, autophagy and antibacterial defense but also expand the role of ATF6 beyond ER-stress. http://dx.doi.org/10.1016/j.cyto.2013.06.089
87 Caspase-8 regulates b-glucan induced IL-1b production and cell death Sandhya Ganesan a, Vijay A. K. Rathinam a, Lukas Bossaller a, William J. Kaiser b, Edward S. Mocarski b, Douglas R. Green c, Neal S. Silverman a,1, Katherine A. Fitzgerald a,1, a Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA, b Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA, c Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA Inflammasomes play a crucial role in host defense against a variety of microbial infections through regulating the maturation of the IL-1 family of cytokines as well as pyroptotic cell death. We have previously identified an essential role for the NLRP3-ASC-Caspase-1 inflammasome in IL-1b production triggered by the fungal pathogen, C. albicans and the importance of this pathway in the clearance of C. albicans in an in vivo mouse model of Oropharyngeal Candidiasis (OPC). Here, we uncovered an important role for the pro-apoptotic caspase, Caspase-8 in IL-1b production induced by b-glucan, a potent immunostimulatory component of fungal cell walls. We found that b-glucan induced IL-1b production requires Caspase-8, in addition to the NLRP3-ASC-Caspase-1 inflammasome. We treated bone marrow derived macrophages and dendritic cells from RIP3/Caspase-8/ mice with various b-glucan related molecules, such as curdlan, fungal whole glucan particles or heat killed C. albicans, and found that Caspase-8 was required for IL-1b production with these stimuli but not with canonical inflammasome activators such as silica, nigericin or poly (dA:dT). Of note, Caspase-8 deficiency did not affect b-glucan induced synthesis of pro-IL1b. In addition, RIP3/Caspase-8/cells were protected from b-glucan induced cell death, whereas RIP3/ deficiency did not affect b-glucan induced IL-1b production or cell death. In contrast to these observations, we found that IL-1b production and cell death in response to live C. albicans infection was not Caspase-8 dependent, raising the possibility that b-glucan may not be exposed by C. albicans to engage this pathway. This study establishes a key link between Caspase-8 and Caspase-1 in coordinating cytokine secretion and cell death in response to fungal pathogenic patterns,
1
These authors contributed equally to this work.