- Email: [email protected]
PS1-69 Inducible STAT1 protein in mice
Abstracts / Cytokine 52 (2010) 17–34 sequences) and associated with a type-1 IFN response. Induction of these genes by autocrine type-I and type-III IFN signaling was ruled out using both neutralizing antibodies to these IFNs in biological assays and qRT-PCR. Despite the absence of type-I or type-III IFNs, IFN-c treatment induced ISGF3 formation and ISRE binding, as shown by STAT2 co-immunoprecipitation and ChIP analysis of the PKR promoter. STAT2 and IRF9 knockdown in A549 cells reversed IFN-c-mediated ISRE induction and antiviral activity – implicating ISGF3 formation as a significant component of the cellular response and biological activity of IFN-c. doi:10.1016/j.cyto.2010.07.136
PS1-65 Investigating the effect of the FHA region of Pellino 3 on TRL4 signaling proteins Lisa S. Tang, Antonio Campos-Torres, Fiachara E. Humphries, Paul Moynagh, Institute of Immunology, Biology Department, National University of Ireland, Maynooth, Maynooth, Kildare, Ireland Pellino 3 has been shown to be a negative regulator of TLR4 signalling. The process by which the protein does this has yet to be elucidated. However, the phosphorylation of Pellino 3 by IRAK-1 has been shown to initiate the E3 ligase activity of Pellino 3. This E3 ligase activity in turn leads to the polyubiquitination and subsequent degradation of IRAK-1. The Pellino Family also share a conserved forkheaded associated (FHA) domain that binds to phosphorylated proteins. This FHA region has already been shown to be crucial in Pellino 2, IRAK-1 binding and processing. This study will examine other proteins within the TLR4 signalling cascade that interact with Pellino 3 via the FHA region which then leads to their degradation. This will then provide a mechanism by which the Pellino protein family regulates TLR4 signalling.
doi:10.1016/j.cyto.2010.07.137
PS1-66 A phosphomimetic substitution of STAT2 serine-287 negatively regulates STAT2 function and type I interferon signaling Håkan C. Steen 1, Suresh H. Basagoudanavar 2, Roshan Thapa 2, Siddharth Balachandran 2, Ana M. Gamero 1, 1 Department of Biochemistry, Temple University, Philadelphia, PA USA, 2 Fox Chase Cancer Center, Philadelphia, PA USA Type I interferons (IFN-a and -b) are cytokines that activate primarily the JAK/ STAT pathway to induce an anti-proliferative, pro-apoptotic or antiviral response in cells. We previously identified a motif in the SH2 domain of STAT2 that, when mutated, prolonged nuclear retention of the STAT1/STAT2 heterocomplex during IFN treatment and induced apoptosis in certain tumor cell lines. With the exception of STAT2, other members in the STAT family of transcription factors have been shown to be phosphorylated on tyrosine and serine residues for biological activity. Therefore, we searched for additional phosphorylation sites in STAT2 by a combination of mass spectrometry analysis and prediction software (NetPhos 2.0 and Motif Scan). Serine-287, located in the coiled-coil domain of STAT2, was found to be phosphorylated in untreated STAT2 null U6A cells reconstituted with wild type STAT2 but not in cells treated with IFN-a for 20 minutes. To determine the biological consequence of this putative phosphorylation event, a phosphomimetic mutant (S287D-STAT2) of STAT2 or a phospho inert mutant (S287A–STAT2) was expressed in U6A cells. In response to IFN-a, S287D-STAT2 poorly induced ISG expression and conferred no protection against vesicular stomatitis virus (VSV) infection, whereas the S287A-STAT2 mutant showed prolonged ISG expression and lengthened VSV protection. The molecular mechanism behind these phenotypical changes remains unknown. Yet we observed that STAT2-S287A phosphorylation on Tyrosine-690 was prolonged whereas S287D-STAT2 activation was defective in response to type I IFN, resulting in less STAT2 translocating to the nucleus. Therefore, our study shows that phosphorylation of STAT2 on Serine287 negatively impacts STAT2 function in the type I IFN signaling pathway and suggests that other signaling pathways modulate STAT2 function and the cellular response to type I IFNs.
doi:10.1016/j.cyto.2010.07.138
PS1-67 TYK2 is required for IL-17 production by innate immune cells in response to IPS Rita Stiefvater 1, Elisabeth Hofmann 1, Thomas Kolbe 2,3, Ursula Reichart 1, Caroline Lassnig 1,2, Claus Vogl 1, Valeria Poli 4, Mathias Müller 1,2, Birgit Strobl 1, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine, Vienna, Austria,
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Institute of Biotechnology in Animal Production, Department FA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Turin, Turin, Italy The Janus kinase (JAK) family member tyrosine kinase 2 (Tyk2) is an integral part of various cytokine and growth hormone signaling pathways. We have reported previously that macrophages from Tyk2-deficient mice exhibit selective defects in response to lipopolysaccharide (LPS). We demonstrate now, that LPS stimulation induces interleukin-17 (IL-17) production via a Tyk2-dependent pathway in thioglycolate-elicited peritoneal macrophages. IL-17 and IL-17F were upregulated upon LPS treatment with similar kinetics and both mRNAs were considerably reduced in the absence of Tyk2. Interestingly, signal transducer and activator of transcription 3 (STAT3) was not required for LPS-induced IL-17 production in macrophages. In the absence of STAT3, IL-17/IL-17F mRNA and IL-17 protein expression were strongly increased upon LPS treatment and, to a lower extend, produced constitutively. Thus, in contrast to its essential role in the differentiation/maintenance of IL-17 producing T cells (Th17), STAT3 exerts inhibitory rather than stimulatory effects on LPS-induced IL-17 production in macrophages. Of note, we also prove that Tyk2 is indispensable for IL-17 production following LPS challenge in vivo. We could exclude mature T cells as main source of IL-17 in spleens following intraperitoneal administration of LPS. Currently, we are investigating the contribution of Tyk2 to innate IL-17 production in specific cell types in vivo.
doi:10.1016/j.cyto.2010.07.139
PS1-68 STAT1a and STAT1b knockin mice: Initial findings and some surprises Christian Semper 1, Nicole R. Leitner 1, Michael Rammerstorfer 1, Thomas Kolbe 2,3, Thomas Rülicke 2,4, Birgit Strobl 1, Mathias Müller 1,2, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine Vienna, Vienna, Austria, 3 Institute of Biotechnology in Animal Production, Department IFA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Institute of Laboratory Animal Sciences, University of Veterinary Medicine Vienna, Vienna, Austria Signal transducer and activator of transcription 1 (Stat1) participates in Jak-Stat signalling pathways which (co-) regulate immunity to infection, inflammatory processes and carcinogenesis. As a result of alternative splicing Stat1 exists in two isoforms, the full length Stat1a and the C-terminal truncated Stat1b isoform. Stat1b lacks 38 amino acids of the transactivation domain including the serine 727 phosphorylation site, which is essential for full transcriptional activation. Previously, it has been reported that in vitro only Stat1a is transcriptionally active, therefore Stat1b was considered to act in a dominant negative manner. In order to investigate the Stat1 isoform functions in vivo we generated mice, which are deficient for either Stat1a or Stat1b (i.e. Stat1Da/Da and Stat1Db/Db mice, respectively). Protein expression levels of each isoform are similar to wildtype Stat1a/b expression levels in primary embryonic fibroblasts (PEFs), bone marrow derived macrophages (BMMUs) and organs. Upon interferon (IFN) stimulation cells derived from Stat1Db/Dbfnmice show phosphorylation at tyrosine 701 and serine 727. As expected, in Stat1Da/Da cells only tyrosine 701 of the Stat1b isoform is phosphorylated. Analysis of respective cells show that either Stat1 isoform is able to translocate to the nucleus and to bind to DNA response elements. IFN type I and II induced gene expression of selected target genes in Stat1Db/Dbfncells is similar to wildtype cells. Unexpectedly, Stat1Da/Da cells also show gene induction upon IFN type II stimulation. Thus, in contrast to what has been shown before in cell lines, these data demonstrate that Stat1b alone is transcriptionally active. Further analysis will define detailed function of Stat1a and Stat1b in vivo.
doi:10.1016/j.cyto.2010.07.140
PS1-69 Inducible STAT1 protein in mice Nicole R. Leitner 1, Thomas Kolbe 2,3, Caroline Lassnig 1,2, Konrad Hochedlinger 4, Thomas Rülicke 2,5, Mathias Müller 1,2, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine Vienna, Austria, 3 Institute of Biotechnology in Animal Production, Department IFA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Harvard Stem Cell Institute, Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Boston, USA, 5 Institute of Laboratory Animal Sciences, University of Veterinary Medicine Vienna, Austria Signal transducer and activator of transcription 1 (Stat1) is an integral constituent of the Jak-Stat signaling network mediating cellular responses including pro-
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Abstracts / Cytokine 52 (2010) 17–34
liferation, differentiation and apoptosis. Stat1 deficiency in mice leads to high susceptibility to viruses and other pathogens. Depending on the tumor model, Stat1 contributes to tumor surveillance or tumorigenesis. Interestingly, mice deficient for several components of the Jak-Stat pathway and other innate immune signaling cascades show reduced Stat1 protein levels. So far, the phenotypes of these gene targeted mice have been attributed mainly to the loss of function, however, little is known about the reduced Stat1 level contribution to the phenotype. In order to reconstitute Stat1 levels in relevant mice and to identify time- and dose-dependent Stat1 functions, we generated mice with inducible Stat1 protein (i.e. Stat1ind mice). First, Stat1ind mice were crossed back to mice deficient for endogenous Stat1 (Stat1 / Stat1ind) to be able to show that inducible Stat1 protein can fulfill wildtype Stat1 functions. Inducibility of Stat1 protein was shown in organs and bone-marrow derived macrophages (BMMU). Stat1 protein levels were strictly dependent on time and dose of doxycycline (dox) treatment and could be expressed at lower, equal or higher levels as compared to wildtype Stat1. In BMMUs, dox induced Stat1 protein showed interferon induced phosphorylation, DNA binding and activation of target genes comparable to wildtype cells. Furthermore, dox-induced Stat1 protein was sufficient to convert cells from Vesicular Stomatitis Virus (VSV) susceptible to VSV resistant. Preliminary in vivo experiments showed that Stat1 / Stat1ind mice are resistant against VSV only in the presence of dox, proving functionality of inducible Stat1 protein in vivo. The Stat1ind mice will enable us to study requirement and function of Stat1 during onset, progression and defeat of disease. doi:10.1016/j.cyto.2010.07.141
PS1-70 An USP18-based negative feedback control induced by Type I and Type III interferons specifically inactivates interferon a response Véronique Francois-Newton 1, Zhi Li 1, Béatrice Payelle-Brogard 1, Gabriel Magno de Freitas Almeida 2, Danièle Monneron 2, Gilles Uzé 2, Sandra Pellegrini 1, 1 Institut Pasteur, Cytokine Signaling Unit, CNRS URA 1961, Paris, 2 CNRS UMR 5235, Montpellier, France Type I Interferons (IFN) are helical cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 IFN subtypes exist, of which IFN a2 and IFN b are the best studied and are widely used in the clinic. Their binding to the IFNAR1/2 receptor complex activates the associated Janus tyrosine kinases Tyk2 and Jak1, leading, via Stat activation, to induction of hundreds of IFN-stimulated genes. Despite common biological activities, the nonredundant function of IFN a2 and IFN b is suggested by their different potency for given bioactivities. These differential activities are determined by receptor binding parameters and by cell intrinsic mechanisms, e.g. receptor density on the target cell. We describe a new type of ‘‘a/b differential”. The prolonged exposure of various cell types to type I IFN interferes with their subsequent ability to respond to a IFNs. Interestingly, primed cells retain sensitivity to IFN b. We refer to this state as differential desensitization the mechanism of which we have dissected. We found that this refractory state, observed towards all IFN a subtypes tested (a1, a2, a8, a21, x), is induced not only by a/b IFNs but also by IFN k (type III IFNs). Differential desensitization is not consequent to surface receptor down-regulation but is dependent of protein synthesis. We show that the isopeptidase USP18/UBP43 is necessary and sufficient to induce differential desensitization and, accordingly, the USP18 gene is transcriptionally induced by IFN a/b as well as IFN k. USP18 was described to dampen type I IFN signaling by binding to IFNAR2. Importantly, using 125I-radiolabeled ligands, we found that desensitized cells, ie expressing active USP18, loose high affinity binding sites for IFN a2. Our data suggests that, via its isopeptidase activity, USP18 loosens the assembly of functional IFN a binding sites. doi:10.1016/j.cyto.2010.07.142
PS1-71 Characterization of a novel STAT5-regulated ubiquitin ligase in human T cells Marie-Jose Bijlmakers, Sang-Mi Kim, Ravneet K. Jandu, Francesca Eddy, LemlemTewolde Berhan, Aradhana Rani, Susan John, Dept. of Immunobiology, Kings College London, Guys Hospital, Great Maze Pond, London SE1 9RT. UK Signal transducers and activators of transcription (STAT) are activated by cytokines and growth factors and are critical regulators of cell proliferation, differentiation and survival. Of the seven known mammalian STAT proteins, STAT5a and STAT5b (STAT5) are the main effectors of IL-2 signaling. We recently identified in vivo binding sites of STAT5 in human peripheral T cells by chromatin immunoprecipitation (ChIP). Validation of one of these sites revealed coordinate IL-2 –regulation of three tandemly linked genes on chromosome 2. One of these, RNF144A is a RING-between RING (RBR) protein of unknown function. We show that expression of this gene is regulated by T cell receptor activation, and more potently by interferon-gamma stimulation in addi-
tion to IL-2. Furthermore RNF144A expression is down-regulated during cell-cycle progression following T-cell activation. Our characterization of the RNF144A protein by immunofluorescence microscopy and cellular fractionation experiments, demonstrates that RNF144A is a membrane-associated protein with a C-terminal tail-anchor, which predicts that the RING domains are oriented towards cytoplasm. Moreover, in vitro ubiquitination assays verified that RNF144A functions as a ubiquitin ligase. Studies are on-going to evaluate the role of RNF144A during T cell activation. Thus, we have identified a novel STAT5-regulated ubiquitin ligase. doi:10.1016/j.cyto.2010.07.143
PS1-72 Chronic and acute inflammatory conditions deterine MEK1 or MEK2 usage as regulators of IL1b and sILRa expression Karim J. Brandt, Nicolas Molnarfi, Lyssia Gruaz, Danielle Burger, Division of Immunology and Allergy, Hans Wilsdorf Laboratory, IARG, Department of Internal Medicine, Faculty of Medicine, University Hospital and University of Geneva, Switzerland Deregulation of the production of IL-1b and its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra), plays an important role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Relevant to these conditions direct cellular contact with stimulated T cells potently trigger cytokine production in human monocytes. Preliminary experiments suggested the differential involvement of MAPK pathway elements MEK1 and MEK2 in the control of IL-1b/sIL-1Ra balance in conditions underlying chronic and acute inflammation. To investigate further the implication of MEK1 and MEK2 in the control of IL1b and sIL-1Ra production by human monocytes, we used two different stimuli: (i) soluble extracts of plasma membranes from stimulated T cells (CEsHUT), mimicking cellular contact with T cells which is relevant to chronic/sterile inflammation; and (ii) LPS that is relevant to acute/infectious inflammation. Cytokine expression was assessed by ELISA and qPCR. The MEK1/2 (U0126) and MEK1 (PD98059) specific inhibitors diminished the expression (protein and mRNA) of sIL-1Ra in both CEsHUTand LPS-activated monocytes. In contrast, although U0126 inhibited IL-1b production in both CEsHUT- and LPS-activated monocytes, PD98059 did not affected IL-1b expression induced by LPS. Similar results were obtained in monocytes knocked-down with siRNA specific to MEK1 and MEK2. These results suggest that MEK1 and MEK2 are differentially involved in the induction of IL-1b production upon chronic/sterile and acute/infectious inflammatory conditions. MEK1 represent a potential therapeutic target which could participate in the restoration of IL-1b/sIL-1Ra balance in chronic/sterile inflammation without affecting normal response to infectious agents. doi:10.1016/j.cyto.2010.07.144
PS1-73 Hypoxia-inducible factor 1a and the glucose-regulated protein 78 are up-regulated by oncostatin M in human hepatocytes and hepatoma cells Stefan Vollmer 1, Valérie Kappler 1, Jakub Kaczor 1, Daniela Flügel 3, Catherine Rolvering 1, Nobuyuki Kato 2, Thomas Kietzmann 3, Iris Behrmann 1, Claude Haan 1, 1 Life Sciences Research Unit, University of Luxembourg, Luxembourg, Luxembourg, 2 Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 3 Chemistry Department, University of Kaiserslautern, Kaiserslautern, Rheinland-Pfalz, Germany The interleukin-6-type cytokine oncostatin M (OSM) is known to regulate processes like inflammation, hematopoiesis as well as angiogenesis. Angiogenesis is also induced in hypoxic tumors via the expression of the hypoxia-inducible factor-1a (HIF-1a). HIF-1a forms a heterodimer with the b subunit of the transcription factor HIF-1 which is a key regulator of the hypoxic response. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF-1a under normoxic and hypoxic conditions. Further, the OSM-dependent HIF1a increase is mediated via Jak/STAT3- and the MEK/Erk1/2-pathway. OSM-mediated HIF1a up-regulation did not result from an increase in HIF1a protein stability but from increased transcription from the HIF1a gene. Additionally, we show that the OSM-induced HIF-1a protein levels are important for the OSM-dependent vascular endothelial growth factor (VEGF) - and plasminogen activator inhibitor-1 (PAI1)-gene induction. Since VEGF and PAI-1 are also crucial players in liver development and regeneration we further investigated the properties of OSM on hepatocytes and hepatoma cells. We could observe enhanced levels of Glucose-regulated protein 78 (Grp78) upon OSM treatment. Interestingly, we cannot detect this up-regulation in any other tested cell line derived from other tissues (breast, prostate, kidney cancer and melanoma). Similar to HIF-1a, the increase of Grp78 on the protein level is due to OSM-induced Grp78 gene transcription. Our results indicate that this rather depends on the activation of MAPK pathways (MEK/Erk1/2) than on STAT3 activation. In conclusion, OSM treatment of hepatocytes and hepatoma cells leads to the up-regulation of HIF-1a and Grp78. Both are known markers for tumor progression and sur-
PS1-65 Investigating the effect of the FHA region of Pellino 3 on TRL4 signaling proteins Lisa S. Tang, Antonio Campos-Torres, Fiachara E. Humphries, Paul Moynagh, Institute of Immunology, Biology Department, National University of Ireland, Maynooth, Maynooth, Kildare, Ireland Pellino 3 has been shown to be a negative regulator of TLR4 signalling. The process by which the protein does this has yet to be elucidated. However, the phosphorylation of Pellino 3 by IRAK-1 has been shown to initiate the E3 ligase activity of Pellino 3. This E3 ligase activity in turn leads to the polyubiquitination and subsequent degradation of IRAK-1. The Pellino Family also share a conserved forkheaded associated (FHA) domain that binds to phosphorylated proteins. This FHA region has already been shown to be crucial in Pellino 2, IRAK-1 binding and processing. This study will examine other proteins within the TLR4 signalling cascade that interact with Pellino 3 via the FHA region which then leads to their degradation. This will then provide a mechanism by which the Pellino protein family regulates TLR4 signalling.
doi:10.1016/j.cyto.2010.07.137
PS1-66 A phosphomimetic substitution of STAT2 serine-287 negatively regulates STAT2 function and type I interferon signaling Håkan C. Steen 1, Suresh H. Basagoudanavar 2, Roshan Thapa 2, Siddharth Balachandran 2, Ana M. Gamero 1, 1 Department of Biochemistry, Temple University, Philadelphia, PA USA, 2 Fox Chase Cancer Center, Philadelphia, PA USA Type I interferons (IFN-a and -b) are cytokines that activate primarily the JAK/ STAT pathway to induce an anti-proliferative, pro-apoptotic or antiviral response in cells. We previously identified a motif in the SH2 domain of STAT2 that, when mutated, prolonged nuclear retention of the STAT1/STAT2 heterocomplex during IFN treatment and induced apoptosis in certain tumor cell lines. With the exception of STAT2, other members in the STAT family of transcription factors have been shown to be phosphorylated on tyrosine and serine residues for biological activity. Therefore, we searched for additional phosphorylation sites in STAT2 by a combination of mass spectrometry analysis and prediction software (NetPhos 2.0 and Motif Scan). Serine-287, located in the coiled-coil domain of STAT2, was found to be phosphorylated in untreated STAT2 null U6A cells reconstituted with wild type STAT2 but not in cells treated with IFN-a for 20 minutes. To determine the biological consequence of this putative phosphorylation event, a phosphomimetic mutant (S287D-STAT2) of STAT2 or a phospho inert mutant (S287A–STAT2) was expressed in U6A cells. In response to IFN-a, S287D-STAT2 poorly induced ISG expression and conferred no protection against vesicular stomatitis virus (VSV) infection, whereas the S287A-STAT2 mutant showed prolonged ISG expression and lengthened VSV protection. The molecular mechanism behind these phenotypical changes remains unknown. Yet we observed that STAT2-S287A phosphorylation on Tyrosine-690 was prolonged whereas S287D-STAT2 activation was defective in response to type I IFN, resulting in less STAT2 translocating to the nucleus. Therefore, our study shows that phosphorylation of STAT2 on Serine287 negatively impacts STAT2 function in the type I IFN signaling pathway and suggests that other signaling pathways modulate STAT2 function and the cellular response to type I IFNs.
doi:10.1016/j.cyto.2010.07.138
PS1-67 TYK2 is required for IL-17 production by innate immune cells in response to IPS Rita Stiefvater 1, Elisabeth Hofmann 1, Thomas Kolbe 2,3, Ursula Reichart 1, Caroline Lassnig 1,2, Claus Vogl 1, Valeria Poli 4, Mathias Müller 1,2, Birgit Strobl 1, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine, Vienna, Austria,
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Institute of Biotechnology in Animal Production, Department FA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Turin, Turin, Italy The Janus kinase (JAK) family member tyrosine kinase 2 (Tyk2) is an integral part of various cytokine and growth hormone signaling pathways. We have reported previously that macrophages from Tyk2-deficient mice exhibit selective defects in response to lipopolysaccharide (LPS). We demonstrate now, that LPS stimulation induces interleukin-17 (IL-17) production via a Tyk2-dependent pathway in thioglycolate-elicited peritoneal macrophages. IL-17 and IL-17F were upregulated upon LPS treatment with similar kinetics and both mRNAs were considerably reduced in the absence of Tyk2. Interestingly, signal transducer and activator of transcription 3 (STAT3) was not required for LPS-induced IL-17 production in macrophages. In the absence of STAT3, IL-17/IL-17F mRNA and IL-17 protein expression were strongly increased upon LPS treatment and, to a lower extend, produced constitutively. Thus, in contrast to its essential role in the differentiation/maintenance of IL-17 producing T cells (Th17), STAT3 exerts inhibitory rather than stimulatory effects on LPS-induced IL-17 production in macrophages. Of note, we also prove that Tyk2 is indispensable for IL-17 production following LPS challenge in vivo. We could exclude mature T cells as main source of IL-17 in spleens following intraperitoneal administration of LPS. Currently, we are investigating the contribution of Tyk2 to innate IL-17 production in specific cell types in vivo.
doi:10.1016/j.cyto.2010.07.139
PS1-68 STAT1a and STAT1b knockin mice: Initial findings and some surprises Christian Semper 1, Nicole R. Leitner 1, Michael Rammerstorfer 1, Thomas Kolbe 2,3, Thomas Rülicke 2,4, Birgit Strobl 1, Mathias Müller 1,2, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine Vienna, Vienna, Austria, 3 Institute of Biotechnology in Animal Production, Department IFA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Institute of Laboratory Animal Sciences, University of Veterinary Medicine Vienna, Vienna, Austria Signal transducer and activator of transcription 1 (Stat1) participates in Jak-Stat signalling pathways which (co-) regulate immunity to infection, inflammatory processes and carcinogenesis. As a result of alternative splicing Stat1 exists in two isoforms, the full length Stat1a and the C-terminal truncated Stat1b isoform. Stat1b lacks 38 amino acids of the transactivation domain including the serine 727 phosphorylation site, which is essential for full transcriptional activation. Previously, it has been reported that in vitro only Stat1a is transcriptionally active, therefore Stat1b was considered to act in a dominant negative manner. In order to investigate the Stat1 isoform functions in vivo we generated mice, which are deficient for either Stat1a or Stat1b (i.e. Stat1Da/Da and Stat1Db/Db mice, respectively). Protein expression levels of each isoform are similar to wildtype Stat1a/b expression levels in primary embryonic fibroblasts (PEFs), bone marrow derived macrophages (BMMUs) and organs. Upon interferon (IFN) stimulation cells derived from Stat1Db/Dbfnmice show phosphorylation at tyrosine 701 and serine 727. As expected, in Stat1Da/Da cells only tyrosine 701 of the Stat1b isoform is phosphorylated. Analysis of respective cells show that either Stat1 isoform is able to translocate to the nucleus and to bind to DNA response elements. IFN type I and II induced gene expression of selected target genes in Stat1Db/Dbfncells is similar to wildtype cells. Unexpectedly, Stat1Da/Da cells also show gene induction upon IFN type II stimulation. Thus, in contrast to what has been shown before in cell lines, these data demonstrate that Stat1b alone is transcriptionally active. Further analysis will define detailed function of Stat1a and Stat1b in vivo.
doi:10.1016/j.cyto.2010.07.140
PS1-69 Inducible STAT1 protein in mice Nicole R. Leitner 1, Thomas Kolbe 2,3, Caroline Lassnig 1,2, Konrad Hochedlinger 4, Thomas Rülicke 2,5, Mathias Müller 1,2, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Austria, 2 University Center Biomodels Austria, University of Veterinary Medicine Vienna, Austria, 3 Institute of Biotechnology in Animal Production, Department IFA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, 4 Harvard Stem Cell Institute, Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Boston, USA, 5 Institute of Laboratory Animal Sciences, University of Veterinary Medicine Vienna, Austria Signal transducer and activator of transcription 1 (Stat1) is an integral constituent of the Jak-Stat signaling network mediating cellular responses including pro-
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liferation, differentiation and apoptosis. Stat1 deficiency in mice leads to high susceptibility to viruses and other pathogens. Depending on the tumor model, Stat1 contributes to tumor surveillance or tumorigenesis. Interestingly, mice deficient for several components of the Jak-Stat pathway and other innate immune signaling cascades show reduced Stat1 protein levels. So far, the phenotypes of these gene targeted mice have been attributed mainly to the loss of function, however, little is known about the reduced Stat1 level contribution to the phenotype. In order to reconstitute Stat1 levels in relevant mice and to identify time- and dose-dependent Stat1 functions, we generated mice with inducible Stat1 protein (i.e. Stat1ind mice). First, Stat1ind mice were crossed back to mice deficient for endogenous Stat1 (Stat1 / Stat1ind) to be able to show that inducible Stat1 protein can fulfill wildtype Stat1 functions. Inducibility of Stat1 protein was shown in organs and bone-marrow derived macrophages (BMMU). Stat1 protein levels were strictly dependent on time and dose of doxycycline (dox) treatment and could be expressed at lower, equal or higher levels as compared to wildtype Stat1. In BMMUs, dox induced Stat1 protein showed interferon induced phosphorylation, DNA binding and activation of target genes comparable to wildtype cells. Furthermore, dox-induced Stat1 protein was sufficient to convert cells from Vesicular Stomatitis Virus (VSV) susceptible to VSV resistant. Preliminary in vivo experiments showed that Stat1 / Stat1ind mice are resistant against VSV only in the presence of dox, proving functionality of inducible Stat1 protein in vivo. The Stat1ind mice will enable us to study requirement and function of Stat1 during onset, progression and defeat of disease. doi:10.1016/j.cyto.2010.07.141
PS1-70 An USP18-based negative feedback control induced by Type I and Type III interferons specifically inactivates interferon a response Véronique Francois-Newton 1, Zhi Li 1, Béatrice Payelle-Brogard 1, Gabriel Magno de Freitas Almeida 2, Danièle Monneron 2, Gilles Uzé 2, Sandra Pellegrini 1, 1 Institut Pasteur, Cytokine Signaling Unit, CNRS URA 1961, Paris, 2 CNRS UMR 5235, Montpellier, France Type I Interferons (IFN) are helical cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 IFN subtypes exist, of which IFN a2 and IFN b are the best studied and are widely used in the clinic. Their binding to the IFNAR1/2 receptor complex activates the associated Janus tyrosine kinases Tyk2 and Jak1, leading, via Stat activation, to induction of hundreds of IFN-stimulated genes. Despite common biological activities, the nonredundant function of IFN a2 and IFN b is suggested by their different potency for given bioactivities. These differential activities are determined by receptor binding parameters and by cell intrinsic mechanisms, e.g. receptor density on the target cell. We describe a new type of ‘‘a/b differential”. The prolonged exposure of various cell types to type I IFN interferes with their subsequent ability to respond to a IFNs. Interestingly, primed cells retain sensitivity to IFN b. We refer to this state as differential desensitization the mechanism of which we have dissected. We found that this refractory state, observed towards all IFN a subtypes tested (a1, a2, a8, a21, x), is induced not only by a/b IFNs but also by IFN k (type III IFNs). Differential desensitization is not consequent to surface receptor down-regulation but is dependent of protein synthesis. We show that the isopeptidase USP18/UBP43 is necessary and sufficient to induce differential desensitization and, accordingly, the USP18 gene is transcriptionally induced by IFN a/b as well as IFN k. USP18 was described to dampen type I IFN signaling by binding to IFNAR2. Importantly, using 125I-radiolabeled ligands, we found that desensitized cells, ie expressing active USP18, loose high affinity binding sites for IFN a2. Our data suggests that, via its isopeptidase activity, USP18 loosens the assembly of functional IFN a binding sites. doi:10.1016/j.cyto.2010.07.142
PS1-71 Characterization of a novel STAT5-regulated ubiquitin ligase in human T cells Marie-Jose Bijlmakers, Sang-Mi Kim, Ravneet K. Jandu, Francesca Eddy, LemlemTewolde Berhan, Aradhana Rani, Susan John, Dept. of Immunobiology, Kings College London, Guys Hospital, Great Maze Pond, London SE1 9RT. UK Signal transducers and activators of transcription (STAT) are activated by cytokines and growth factors and are critical regulators of cell proliferation, differentiation and survival. Of the seven known mammalian STAT proteins, STAT5a and STAT5b (STAT5) are the main effectors of IL-2 signaling. We recently identified in vivo binding sites of STAT5 in human peripheral T cells by chromatin immunoprecipitation (ChIP). Validation of one of these sites revealed coordinate IL-2 –regulation of three tandemly linked genes on chromosome 2. One of these, RNF144A is a RING-between RING (RBR) protein of unknown function. We show that expression of this gene is regulated by T cell receptor activation, and more potently by interferon-gamma stimulation in addi-
tion to IL-2. Furthermore RNF144A expression is down-regulated during cell-cycle progression following T-cell activation. Our characterization of the RNF144A protein by immunofluorescence microscopy and cellular fractionation experiments, demonstrates that RNF144A is a membrane-associated protein with a C-terminal tail-anchor, which predicts that the RING domains are oriented towards cytoplasm. Moreover, in vitro ubiquitination assays verified that RNF144A functions as a ubiquitin ligase. Studies are on-going to evaluate the role of RNF144A during T cell activation. Thus, we have identified a novel STAT5-regulated ubiquitin ligase. doi:10.1016/j.cyto.2010.07.143
PS1-72 Chronic and acute inflammatory conditions deterine MEK1 or MEK2 usage as regulators of IL1b and sILRa expression Karim J. Brandt, Nicolas Molnarfi, Lyssia Gruaz, Danielle Burger, Division of Immunology and Allergy, Hans Wilsdorf Laboratory, IARG, Department of Internal Medicine, Faculty of Medicine, University Hospital and University of Geneva, Switzerland Deregulation of the production of IL-1b and its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra), plays an important role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Relevant to these conditions direct cellular contact with stimulated T cells potently trigger cytokine production in human monocytes. Preliminary experiments suggested the differential involvement of MAPK pathway elements MEK1 and MEK2 in the control of IL-1b/sIL-1Ra balance in conditions underlying chronic and acute inflammation. To investigate further the implication of MEK1 and MEK2 in the control of IL1b and sIL-1Ra production by human monocytes, we used two different stimuli: (i) soluble extracts of plasma membranes from stimulated T cells (CEsHUT), mimicking cellular contact with T cells which is relevant to chronic/sterile inflammation; and (ii) LPS that is relevant to acute/infectious inflammation. Cytokine expression was assessed by ELISA and qPCR. The MEK1/2 (U0126) and MEK1 (PD98059) specific inhibitors diminished the expression (protein and mRNA) of sIL-1Ra in both CEsHUTand LPS-activated monocytes. In contrast, although U0126 inhibited IL-1b production in both CEsHUT- and LPS-activated monocytes, PD98059 did not affected IL-1b expression induced by LPS. Similar results were obtained in monocytes knocked-down with siRNA specific to MEK1 and MEK2. These results suggest that MEK1 and MEK2 are differentially involved in the induction of IL-1b production upon chronic/sterile and acute/infectious inflammatory conditions. MEK1 represent a potential therapeutic target which could participate in the restoration of IL-1b/sIL-1Ra balance in chronic/sterile inflammation without affecting normal response to infectious agents. doi:10.1016/j.cyto.2010.07.144
PS1-73 Hypoxia-inducible factor 1a and the glucose-regulated protein 78 are up-regulated by oncostatin M in human hepatocytes and hepatoma cells Stefan Vollmer 1, Valérie Kappler 1, Jakub Kaczor 1, Daniela Flügel 3, Catherine Rolvering 1, Nobuyuki Kato 2, Thomas Kietzmann 3, Iris Behrmann 1, Claude Haan 1, 1 Life Sciences Research Unit, University of Luxembourg, Luxembourg, Luxembourg, 2 Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 3 Chemistry Department, University of Kaiserslautern, Kaiserslautern, Rheinland-Pfalz, Germany The interleukin-6-type cytokine oncostatin M (OSM) is known to regulate processes like inflammation, hematopoiesis as well as angiogenesis. Angiogenesis is also induced in hypoxic tumors via the expression of the hypoxia-inducible factor-1a (HIF-1a). HIF-1a forms a heterodimer with the b subunit of the transcription factor HIF-1 which is a key regulator of the hypoxic response. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF-1a under normoxic and hypoxic conditions. Further, the OSM-dependent HIF1a increase is mediated via Jak/STAT3- and the MEK/Erk1/2-pathway. OSM-mediated HIF1a up-regulation did not result from an increase in HIF1a protein stability but from increased transcription from the HIF1a gene. Additionally, we show that the OSM-induced HIF-1a protein levels are important for the OSM-dependent vascular endothelial growth factor (VEGF) - and plasminogen activator inhibitor-1 (PAI1)-gene induction. Since VEGF and PAI-1 are also crucial players in liver development and regeneration we further investigated the properties of OSM on hepatocytes and hepatoma cells. We could observe enhanced levels of Glucose-regulated protein 78 (Grp78) upon OSM treatment. Interestingly, we cannot detect this up-regulation in any other tested cell line derived from other tissues (breast, prostate, kidney cancer and melanoma). Similar to HIF-1a, the increase of Grp78 on the protein level is due to OSM-induced Grp78 gene transcription. Our results indicate that this rather depends on the activation of MAPK pathways (MEK/Erk1/2) than on STAT3 activation. In conclusion, OSM treatment of hepatocytes and hepatoma cells leads to the up-regulation of HIF-1a and Grp78. Both are known markers for tumor progression and sur-