Toll-like receptor 3 triggering renders renal epithelial cells sensitive to FAS-induced apoptosis

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Abstracts / Cytokine 48 (2009) 91–137

moDCs were stimulated with TLR3 ligand polyI:C, TLR4 ligand lipopolysaccharide (LPS) and TLR7/8 ligand R848 alone or in a combination of two. RNA was isolated for semi-quantitative real-time PCR analysis and DNA-binding assay was used to study TLR ligand-induced binding of transcription factors to the IFN-k1 promoter IFN-stimulated response element (ISRE). Stimulation of moDCs with polyI:C or LPS alone induced mRNA expression of IFN-b, IFN-k1 and IFN-k2/3, whereas R848 alone was ineffective. However, simultaneous stimulation with R848 together with polyI:C led to a synergistic IFN-a, -b, -k1 and -k2/3 mRNA expression already at 3 h and the synergistic effect was sustained in the later time points. Interestingly, R848 combined with LPS induced synergistic IFN mRNA expression only at the later time points, when the IFN expression induced with LPS alone had declined. IFN-b priming further enhanced the synergistic effect. To clarify the mechanisms behind the synergistic effect, DNA-binding assay was used to study LPS and/or R848 induced binding of IFN regulatory factors (IRF) to the IFN-k1 promoter ISRE site. LPS and/or R848 stimulation induced the binding of IRF1, IRF7 and IRF8 to the ISRE site. We show that stimulation of moDCs via TLR7/8 ligand R848 together with TLR3 or TLR4 ligands, polyI:C or LPS, respectively, leads to a synergistic and sustained expression of IFN-b, IFN-k1 and IFN-k2/3 mRNA. We also suggest that IFN feedback loop is partly responsible for the mechanism of synergy. doi:10.1016/j.cyto.2009.07.558

phosphorylated STATs accumulate in the nucleus, bind DNA and induce gene expression. In addition to this canonical view of nuclear translocation of phosphorylated STATs it is also known that unphosphorylated STATs constitutively shuttle between cytoplasm and nucleus. STAT proteins have been described to dimerize upon tyrosine phosphorylation but also to form dimers in the absence of phosphorylation. We were interested in the structural requirements for dimerization of unphosphorylated STAT3 and its relevance for constitutive nucleocytoplasmic shuttling. We investigated these aspects by the use of chimeric constructs of STAT3 fused to the green fluorescent protein (GFP) or its derivatives. We also introduced the recently developed two-focus fluorescence correlation spectroscopy (2f-FCS) as a technique to analyze the STAT3 population in cell lysates. Our results demonstrate that the N-terminal domain (aa 1–125) of STAT3 is essential for the formation of unphosphorylated STAT3 dimers but not for the assembly of tyrosinephosphorylated STAT3 dimers. Furthermore, in unstimulated cells the monomeric N-terminal deletion mutant shuttles faster between cytoplasm and nucleus than wildtype STAT3 but surprisingly does not accumulate in the nucleus after activation. These results highlight the importance of the N-terminal domain in the formation of unphosphorylated STAT3 dimers and nuclear accumulation upon phosphorylation. Dimer formation is not required for nucleocytoplasmic shuttling of unphosphorylated STAT3. doi:10.1016/j.cyto.2009.07.560

PP2-181 Differential tyrosine phosphorylation of the STAT5A transcription factor by oncogenic kinases Nicolas Chatain, Michael Vogt, Stefan N. Constantinescu, Marc Kerenyi, Matthias Mayerhofer, Richard Moriggl, Veronika Sexl, Gerhard Müller-Newen, Poster Presentation II Differential tyrosine phosphorylation of the STAT5A transcription factor by oncogenic kinases Nicolas Chatain 1, Michael Vogt 1, Stefan N. Constantinescu 2, Marc Kerenyi 3, Matthias Mayerhofer 4, Richard Moriggl 5, Veronika Sexl 6, Gerhard Müller-Newen 1, 1 Institut für Biochemie, Universitätsklinikum RWTH Aachen, Aachen, Germany, 2 Signal Transduction Laboratory, Ludwig Institute for Cancer Research and de Duve Institute, Université Catholique de Louvain, Brussels, Belgium, 3 Department Hematology/Oncology, Children’s Hosptial Boston, Harvard Medical School, Boston, USA, 4 Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria, 5 Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria, 6 Institute of Pharmacology, Medical University of Vienna, Vienna, Austria STAT5A and STAT5B belong to the family of signal transducers and activators of transcription (STATs). These transcription factors modulate gene transcription upon stimulation of cells with cytokines or growth factors. STAT proteins control fundamental cellular processes including survival, proliferation, and differentiation. Uncontrolled activation of STAT factors can lead to pathological conditions such as cancer, chronic inflammation or auto-immunity. In several myeloproliferative neoplasms triggered by constitutively activated kinases, STAT5A is persistently phosphorylated at Y694 and thereby activated. To study the molecular mechanism by which constitutively active kinases activate STAT5A, we cotransfected HEK293-T cells with different STAT5A mutants and constitutively active kinases like v-Src, Flt3-ITD, Kit(D816V), JAK2(V617F) and v-Abl. Phosphorylation at Y694 required the intact SH2 domain of STAT5A with the exception of Flt3-ITD that phosphorylated the SH2 domain mutant R618Q on Y694. By the use of a STAT5A(Y694F) mutant we could show that besides Y694 other tyrosine residues are phosphorylated depending on the cotransfected kinase. The phosphorylation of tyrosine residues different from Y694 did not depend on an intact SH2 domain. Although STAT5A was phosphorylated at tyrosine 694 by vSrc, it did not translocate into the nucleus but seemed to be retained in the cytoplasm. The SH2 domain of STAT5A was critical for the cytoplasmic retention of STAT5A after phosphorylation by v-Src. The TAP-tag methodology will be used to define the additional tyrosine phosphorylation sites and to identify interaction partners of hyperphosphorylated STAT5A. doi:10.1016/j.cyto.2009.07.559

PP2-182 The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3 Michael Vogt, Tamas Domoszlai, Dzina Kleshchanok, Swen Lehmann, Walter Richtering, Gerhard Müller-Newen, Poster Presentation II The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3 Michael Vogt 1, Tamas Domoszlai 1, Dzina Kleshchanok 2, Swen Lehmann 2, Walter Richtering 2, Gerhard Müller-Newen 1, 1 Institut für Biochemie, Universitätsklinikum RWTH Aachen, Aachen, Germany, 2 Institut für Physikalische Chemie, RWTH Aachen, Aachen, Germany STATs (signal transducers and activators of transcription) are transcription factors that are activated by phosphorylation on a single tyrosine residue. Tyrosine-

PP2-183 Directed immobilization of fluorescently labelled cytokines for the analysis of their signal transduction by confocal microscopy Tobias Recker, Daniel Haamann, Doris Klee, Stefan Barth, Gerhard Müller-Newen, Poster Presentation II Directed immobilization of fluorescently labelled cytokines for the analysis of their signal transduction by confocal microscopy Tobias Recker 1, Daniel Haamann 2, Doris Klee 2, Stefan Barth 3, Gerhard MüllerNewen 1, 1 Institut für Biochemie, Universitätsklinikum RWTH Aachen, Aachen, Germany, 2 ITMC/DWI, RWTH Aachen, Aachen, Germany, 3 Fraunhofer IME, Aachen, Germany Soluble cytokines signal through receptors on the cell surface to the nucleus resulting in altered gene expression patterns and cell differentiation. Therefore, the immobilization of cytokines on the surface of a biomaterial is a promising approach to improve the biocompatibility. However, cytokine signalling involves internalization of the cytokine–receptor complex which is hardly conceivable for immobilized cytokines. This fact led to the question to what extent signalling from surface-immobilized cytokines differs from soluble cytokines. Moreover, random surface-immobilization of the cytokine can result in impaired cytokine–receptor interaction. To achieve a site-directed covalent coupling of cytokines the SNAPtag technology was used. The SNAP-tag is a derivative of human O6-alkylguanine DNA alkyltransferase that couples itself covalently to benzylguanine-modified substrates. For investigations by confocal microscopy the yellow fluorescent protein (YFP) was also fused to the cytokines of interest. The cytokines interleukin-6 (IL6) and Oncostatin M (OSM) were expressed as SNAP-YFP-fusion proteins in Escherichia coli and in eukaryotic cells. The bioactivities of the cytokine-fusion proteins were shown to be comparable with the non-tagged cytokines. The fusion proteins were successfully coupled to benzylguanine-modified surfaces as detected by the YFP fluorescence bound to the material surface. Cellular responses on the immobilized cytokines were analyzed by confocal microscopy. The SNAP-YFP-cytokines are valuable tools to study the signal transduction of immobilized cytokines. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labelled proteins. doi:10.1016/j.cyto.2009.07.561

PP2-184 Toll-like receptor 3 triggering renders renal epithelial cells sensitive to FAS-induced apoptosis Kirstin M. Heutinck, Jorien Kassies, Eric Eldering, Ajda T. Rowshani, Ineke J.M. ten Berge, Jörg Hamann, Poster Presentation II Toll-like receptor 3 triggering renders renal epithelial cells sensitive to FASinduced apoptosis Kirstin M. Heutinck 1,2, Jorien Kassies 1,2, Eric Eldering 1, Ajda T. Rowshani 2, Ineke J.M. ten Berge 2, Jörg Hamann 1, 1 Department of Experimental Immunology, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands, 2 Department of Internal Medicine, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands Renal tubular epithelial cells (TECs) are resistant to FAS-ligand induced apoptosis despite constitutive FAS expression. Here, we studied the effect of pro-inflammatory cytokines (TNFa, IFNa, IFN-b, IFNc, IL-1b, IL-6) and Toll-like receptor ligands (LTA, LPS, poly(I:C), imiquimod, CpG oligonucleotides) on FAS-mediated apoptosis in primary human TECs. Poly(I:C), a synthetic TLR3-ligand, induced FAS expression in a

Abstracts / Cytokine 48 (2009) 91–137 dose dependent manner. TECs pre-treated with poly(I:C) underwent massive cell death after administration of the FAS-specific IgM monoclonal antibody CH11 (46.6 ± 8.4% annexin V+ cells), while poly(I:C) (10.9 ± 0.3%) or CH11 (11.1 ± 0.5%) alone did hardly affect cell viability. Apoptosis was characterized by detachment, phosphatidyl serine exposure analyzed by annexin V staining and loss of mitochondrial membrane potential measured with DiOC6. Western blot analysis demonstrated that CH11 induced full cleavage of pro-caspase-8 and caspase-8 inhibitor FLIP. Active caspase-8 directly cleaves caspase-3 (extrinsic pathway), but also mediates cytochrome C release and mitochondrial fragmentation through cleavage of Bid (intrinsic pathway). Activation of BID and caspase-3 was incomplete after FAS triggering. The combination of poly(I:C) and CH11 resulted in total cleavage of caspase-8, FLIP, BID and caspase-3. Poly(I:C) by itself did not promote cleavage of these proteins, yet the amount of pro-caspase-8 was strongly increased. To test whether poly(I:C) regulates the expression of pro- and anti-apoptotic genes, we performed multiplex ligation-dependent probe amplification of mRNA from TECs stimulated for 2 h. Preliminary data indicated that poly(I:C) induces transcription of the pro-apoptotic BH3-only proteins Puma (5.6-fold compared to unstimulated) and Noxa (5.5-fold). On the other hand, mRNA levels of pro-survival genes, like Bcl-XL( 1.9-fold), showed a downwards trend. Based on these results, we propose that poly(I:C) as a model for viral infection, abrogates the resistance of TECs to killing by FAS-ligand expressing immune cells via modulating gene expression towards apoptosis. doi:10.1016/j.cyto.2009.07.562

PP2-185 Mitochondrial dynamics and innate antiviral responses regulated by RIG-I-like receptor Kazuhide Onoguchi, Mitsutoshi Yoneyama, Takashi Fujita, Poster Presentation II Mitochondrial dynamics and innate antiviral responses regulated by RIG-I-like receptor Kazuhide Onoguchi 1,2,3, Mitsutoshi Yoneyama 1,2,4, Takashi Fujita 1,2, 1 The Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan, 2 The Laboratory of Molecular Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 3 Department of Biology, School of Education, Waseda University, Tokyo, Japan, 4 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan Within hours of infection by pathogens, our body activates an innate immune program which includes the expression of numerous cytokines. Interferons are the important cytokines for the innate antiviral response and confer antiviral activity to the host. RIG-Ilike receptor (RLR; RIG-I, MDA5, and LGP2) senses viral RNA and activates interferon and other cytokine genes to prevent viral replication. Activated RIG-I and MDA5 signal through the adaptor protein, interferon-beta promoter stimulator 1 (IPS-1, also called MAVS/VISA/ Cardif). IPS-1 localizes on the outer membrane of mitochondria, and interacts with RIG-I or MDA5 via its N-terminal caspase recruitment domain (CARD). Although it has been known that the mitochondrial localization of IPS-1 is crucial for its signaling function, precise involvement of mitochondria themselves for the signaling has been elusive. By using specific antibodies, and confocal and electron microscopy, we observed that viral infection induces redistribution of IPS-1 as well as mitochondria. In cells infected with RNA viruses, mitochondria localize in close proximity to viral replication complexes. Furthermore, we found that a mitochondrial factor, which regulates mitochondrial dynamics, plays an essential role for antiviral signaling triggered by RIG-I. We propose a model in which mitochondria provide a platform for the assembly of a multi-molecular signaling complex essential for downstream signaling.

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tosidase activity at low concentration (0.25 mM), whereas high dose of ACR (1.0 mM) has a more pronounced apoptotic effect. Cells treated with ACR showed a dosedependent increase in G0/G1 phase arrest. We also determined the level of cell cycle-and apoptosis-related proteins by using s mall interfering RNA-mediated depletion of ATF3 to investigate the role of ATF3 in apoptosis and senescence induced by ACR. Western blot analysis demonstrated that the levels of p53 and p21 were increased in ATF3 knockdown cells treated with ACR. However, CDK2 and CDK4 levels were decreased, which correlated with G0/G1 phase arrest. In addition, p16 protein level was not altered, indicating that p16-mediated pathway was not involved in the cellular response to DNA-damaging agent. We also observed the increased Bax and decreased Bcl2 protein levels. Overall, these findings suggest that ATF3 may regulate pro-apoptotic and senescence pathway of macrophages in stress response.

doi:10.1016/j.cyto.2009.07.564

PP2-187 Apoptosis resistance in HIV-exposed monocytes is modulated by interferon gamma and CCR5-inducible p53 expression Bethsebah N. Gekonge, Luis J. Montaner, Poster Presentation II Apoptosis resistance in HIV-exposed monocytes is modulated by interferon gamma and CCR5-inducible p53 expression Bethsebah N. Gekonge, Luis J. Montaner, HIV-1 Immunopathogenesis Laboratory, The Wistar Institute, Philadelphia, PA, USA Unlike CD4+ T lymphocytes, circulating monocytes and tissue macrophages are resistant to HIV-induced apoptosis. We previously characterized the steady-state gene expression profile of circulating monocytes from HIV+ donors, and identified a predominantly anti-apoptotic signature. Here we present evidence in support of p53 activation as a central feature of apoptosis resistance in monocytes exposed to HIV-1. Furthermore, stimulation of cells with Interferon gamma (IFN-c) or CCR5 activation using macrophage inflammatory protein 1 beta (MIP-1b), are sufficient to reproduce this effect. Peripheral blood mononuclear cells (PBMC) were obtained from HIV donors and untreated HIV+ donors. Monocytes were isolated by negative selection, analyzed for apoptosis induction, and for expression levels of p53 and p53-target genes p21 and PAI-1, by flow cytometry or Western blot. Effect of exposure to R5- or X4-tropic HIV-1, IFN-c or MIP-1b for 36 h was measured. In vitro, contrary to X4-isolates, R5-exposed monocytes had lower Caspase-3 activation in association with higher p53 mRNA and protein levels. Increases in p21 and PAI-1, both linked to enhanced survival of cells, were noted. IFN-c and MIP-1b offered partial protection from cadmium chloride (CdCl2)-induced apoptosis; moreover, MIP-1b was sufficient to increase p53 and PAI-1 expression. In vivo, circulating monocytes from HIV+ donors showed similar profiles; lower inducible Caspase-3 activation and higher expression of p53 and PAI-1 when compared to uninfected controls. Our data suggest; first, a correlation between CCR5 activation, IFN-c receptor activation and p53 expression that corresponds to apoptosis resistance in HIV-1-exposed monocytes. Secondly, a potential for CCR5-utilizing HIV-1 isolates, within immune-activated microenvironments, to modulate monocyte/macrophage pools, thereby increasing cell viability, and thus contributing to viral persistence.

doi:10.1016/j.cyto.2009.07.565

doi:10.1016/j.cyto.2009.07.563

PP2-186 The role of activating transcription factor 3 in stress-induced macrophage apoptosis and senescence Kyung-Ho Kim, Eunhwa Sohn, Dong Kwon Rhee, Suhkneung Pyo, Poster Presentation II The role of activating transcription factor 3 in stress-induced macrophages apoptosis and senescence Kyung-Ho Kim 1, Eunhwa Sohn 2, Dong Kwon Rhee 1, Suhkneung Pyo 1, 1 College of Pharmacy, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea, 2 Department of Herbal Medicine Resource, Kangwon National University, Samcheok, Gangwon-do, Republic of Korea Activating transcription factor (ATF) 3, a member of the ATF/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (ATF/CREB) family of transcription factors, is known to play a pleiotropic role in various biological processes. However, the mechanism and regulation by which cell choose either apoptosis or senescence in DNA damage-mediated pathways remains unclear. This study examines the hypothesis that ATF3 is involved in the apoptosis and senescence in response of macrophages to acrylamide (ACR). Treatment of macrophages with ACR resulted in senescence-like morphology and the increase in senescence – associated beta – galac-

PP2-188 Monocyte to macrophages differentiation confers resistance to HIV–Vpr induced apoptosis: Role of anti-apoptotic Bcl2 and inhibitors of apoptosis (IAP) proteins Aurelia Busca, Ashok Kumar, Poster Presentation II Monocytes to macrophages differentiation confers resistance to HIV–Vpr induced apoptosis: Role of anti-apoptotic Bcl2 and inhibitors of apoptosis (IAP) protein Aurelia Busca, Ashok Kumar, Children’s Hospital of Eastern Ontario, Research Institute, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ont., Canada Latently infected cells represent the main barrier to curing HIV infection and a unique therapeutic challenge. Persistently infected macrophages are long-lived viral reservoirs, but the molecular mechanisms of macrophage survival during HIV infection are poorly understood. In this study, we investigated the role of Bcl2 and IAPs (inhibitors of apoptosis), the main families of antiapoptotic proteins, and the upstream signalling molecules in the survival of HIV infected macrophages by using the HIV accessory protein, viral protein R (Vpr), as a model. HIV–Vpr causes apoptosis in primary monocytes and THP1 promonocytic cells. However, after differentiation, monocyte-derived macrophages are resistant to Vpr induced apoptosis. In contrast, PMA differentiated THP-1 macrophages exhibit low level of apoptosis to HIV–Vpr. Therefore, THP-1 macrophages provide an excellent model to study how monocyte