23 Impaired signaling in memory T-cells during HIV infection

Abstracts / Cytokine 43 (2008) 237–242 of ISGF3 in these cells, and to ascertain its biological significance in the cellular response to IFN-c. doi:10.1016/j.cyto.2008.07.058

19 Simultaneous detection of STAT1, STAT3, and STAT5 activation in response to interferons Mary M. Brodey, Wei Zheng, Jimin Wang, Kevin Reagan, Invitrogen Corporation, Camarillo, CA, USA Interferons (IFNs) are pleiotropic cytokines that regulate a broad range of cellular functions. The major signaling pathways activated by IFNs involve sequential phosphorylation and activation of the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) proteins, providing the primary mechanism through which gene expression is induced. The IFNs are unique among all cytokines in that they can activate all seven known STATs. Once activated, many different STAT homodimer or heterodimer combinations can form. A method to measure various active STAT proteins simultaneously will significantly expedite the drug discovery process targeting JAK/STAT pathways. In this study, cell lines from T cell leukemia, promonocytic cells and erythroleukemia cells were treated with either IFNa, IFN-b, or IFN-c. The activation of STAT1, STAT3 and STAT5 (STAT5a and STAT5b) were monitored concurrently using a newly developed multiplex bead immunoassay. The results demonstrate that each cell line investigated responds differentially to the various interferons, creating unique STAT activation patterns. It is plausible that the differential and unique STAT activation patterns target functionally distinct genes, through which IFNs elicit different biological functions. Our study illustrates that the simultaneous monitoring of STAT activations allows a more complete cell-based analysis of IFN signal transduction. doi:10.1016/j.cyto.2008.07.059

20 Alpha lipoic acid attenuates cadmium-induced inflammatory cytokine expression and apoptosis in HepG2 cells José G. Macías-Barragán 1, Miriam R. Bueno-Topete 1, Juan S. Armendáriz-Borunda 1, Selene G. Huerta-Olvera 2, Fernando R. Siller-López 3, 1 Instituto de Biologı´a Molecular en Medicina y Terapia Ge´nica, Universidad de Guadalajara, Mexico, 2 Doctorado en Ciencias Biome´dicas, Facultad de Medicina, Universidad Auto´noma de San Luis Potosı´, Mexico, 3 Instituto de Investigacio´n en Inmunologı´a y Dermatologı´a, Universidad de Guadalajara, Mexico Inflammation is the basic process whereby cells respond to injury. Alpha-lipoic acid (ALA), exhibits an antioxidant response by Nrf2 activation and anti-inflammatory effects in cells exposed to heavy metals, leading to reduction of apoptotic mediators. Cadmium (Cd) elicits an antioxidant response, but according to the dose also evokes an inflammatory and apoptotic cellular expression. The aims was to analyze the mechanism of protection conferred by ALA pretreatment on Cd cytotoxicity. Gene-expression kinetic of antioxidant, stress, inflammatory and apoptotic, metabolic activity, glutathione, glutamylcysteine ligase activity, cell viability, antioxidant capacity and Nrf2 activation were analyzed from cells treated with 5 mM ALA or 1 lM Cd for 8 h, and then exposed to 2.5 lM Cd. ALA and 1 lM Cd evoked a similar response in GSH content and cellular metabolic activity. GCLM, MTA1 and HMOX1 gene expression was down-regulated by ALA pretreatment, and keep preferentially up-regulated GCLC and GCLM expression by Nrf2 activation. 8 h pretreatment with ALA or low Cd doses protected against 2.5 lM Cd toxicity observed as a recovery of viability and a decrease in caspase-3 regulation, also an elevation of GSH content, and a down regulation of TNFa and IL-6 gene expression was observed. The mechanism of ALA protection is similar to that evoked by a low dose of Cd but without the signs of toxicity. These results suggest that ALA through up-regulation of antioxidant response reduce cellular damage and consequently the inflammatory process elicited by cadmium. doi:10.1016/j.cyto.2008.07.060

21 Functional analyses of three human TYK2 variants Josiane Ragimbeau 1, Milica Gakovic 1, Maija L. Eloranta 2, Sandra Pellegrini 1, 1 Cytokine Signaling Unit, CNRS URA 1961, Institut Pasteur, Paris, France, 2 Uppsala University Hospital, Uppsala, Sweden Tyk2 is a tyrosine kinase of the Janus family involved in signaling by a large number of cytokines that orchestrate an immune response (type I IFNs, lambda IFNs, IL-12,

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IL-23, IL-10, IL-6). As of today there are few reports of Tyk2 alterations in human diseases. Tyk2 deficiency was described in one patient with a complex clinical picture of hyper IgE syndrome and susceptibility to multiple pathogens (Minegishi Y et al. Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity. Immunity 2006;25(5):745–55). To refine our understanding of Tyk2 function and address its potential impact in immune response dysfunctions or cancer susceptibility, we have studied a few recently identified human Tyk2 variants. We explored the functional consequence of two Tyk2 SNPs, V362F and I684S, that were analyzed for association with systemic lupus erythematosus, a complex autoimmune disease, and found to display strong signals (Sigurdsson S et al. Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus. Am J Hum Genet 2005;76(3):528–37). We also studied the germline Tyk2 variant P1104A identified using a computational program to distinguish polymorphisms from cancerassociated mutations (Kaminker JS et al. Distinguishing cancer-associated missense mutations from common polymorphisms. Cancer Res 2007;67(2):465–73). P1104A came up also in a high-throughput sequencing approach used to identify variants in TK-encoding genes potentially relevant in de novo acute myeloid leukemia (Tomasson, MH et al. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia. Blood 2008;111(9):4797–808). The Val362 residue is located in the FERM domain, the Ile684 in the kinase-like domain and the Pro1104 is located in a short helix of the TK domain which is uniquely found in all Janus kinases. Human Tyk2-minus cells were reconstituted with the three variants and signaling through the native type I IFN receptor was measured and compared with signaling of the kinase-inactive Tyk2K930R, as well as with the gain-of-function V678F mutants. The impact of Tyk2-P1104A on signaling through an artificial homodimeric EpoR/R1 receptor and a reconstituted IL-12 receptor was also studied. doi:10.1016/j.cyto.2008.07.061

22 The dsRNA binding protein pact in innate immune signaling Christine L. White, Gregory A. Peters, Benjamin K. Dickerman, Avanti Desai, Ganes C. Sen, Department of Molecular Genetics, Cleveland Clinic, Cleveland, OH, USA PACT was originally identified as a dsRNA binding protein capable of interacting with and activating PKR, resulting in eIF2a phosphorylation and translation inhibition. PKR-deficient mice do not show obvious developmental defects. In contrast PACT-deficient mice show growth, reproductive, ear and craniofacial defects. Initial analysis of PACT-deficient mice did not reveal defects in immune system development in these mice. However, in vitro studies revealed production of IL-6 in response to LPS was reduced in PACT-deficient B cells compared with controls. A similar reduction was not seen for other proinflammatory cytokines such as TNFa or IL12-p40 or in other cell types such as dendritic cells or macrophages. Subsequent analysis revealed that both development of spleen B cells and proliferation of B cells in response to LPS was not impaired in PACT-deficient mice. In vivo studies also showed PACT deficient mice were not protected from inflammation or septic shock in response to LPS. As such, PACT deficiency did not confer a generalized defect in inflammation, B cell development or function. To begin investigating the mechanism by which IL-6 production is impaired in response to LPS in PACT deficient B cells an interaction between PACT and TRAF2 and TRAF6 was investigated. Three potential TRAF binding sites were identified in PACT and initial studies have revealed these proteins coimmunoprecipitate when overexpressed. This association was also seen in pkr / MEFs. Mutational analysis is underway to determine which of these putative binding sites are most important for interaction with TRAF family members and the importance of TRAF binding in the known functions of PACT. doi:10.1016/j.cyto.2008.07.062

23 Impaired signaling in memory T-cells during HIV infection Yu Shi, Lydie Trautmann, Yoav Peretz, Joumana Zeidan, Bader Yassine-Diab, Zhong He, Mohamed-Rachid Boulassel, Jean-Pierre Routy, Elias K. Haddad, Rafick-Pierre Sékaly, Laboratory of Immunology, Department of Microbiology and Immunology, Universite´ de Montre´al and CR-CHUM, NSERM U743 and CUSM, Montre´al, Canada Central memory T cells (TCM) have the capacity to self renew and maintain the memory T cell pool whereas effector memory T cells (TEM) are endowed with effector functions. Lately defined transitional memory cells (TTM ) have shown a dual differentiation potential. They can either revert towards a central memory phenotype or differentiate towards an effector phenotype. This study is to further the understanding of functional impairment described in lymphocytes isolated from HIV-infected individuals. Using multiparametric phospho-specific flow cytometry, we investigated phospho-STAT5 signals on CD4+ and CD8+ memory T cells from HIV-infected subjects

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during various stages of disease progression. In this report, we show that signaling profiles in CD4+ and CD8+ memory T cell subsets are heterogeneous following stimulation with cC chain cytokines (IL-2, IL-7 and IL-15). We find that basal levels of pSTAT5 on most CD4+ and CD8+ subsets are lower in chronically HIV infected individuals than on HIV-negative individuals. CD4+ T cells maintain higher basal levels and cytokine-induced levels of pSTAT5 than those from CD8+ T cells. Compared with CD4+ T cells, memory CD8+ subsets from HIV-infected individuals are more frequently impaired. We also find TTM cells have the highest basal pSTAT5 expression in CD4+ T cells and have similar cytokine-induced pSTAT5 expression as effector memory subsets. In addition, the expression of PD-1 negatively correlates with cytokine-induced STAT5 phosphorylation. These data suggest that HIV infection could change memory T cell signaling pathways both at the basal level and during responsiveness to cytokine stimulation. doi:10.1016/j.cyto.2008.07.063

24 OCT-6 (POU3F1, TST-1, SCIP) is an interferon-inducible protein Elisabeth Hofmann 1, Ursula Reichart 1, Christian Gausterer 1, Dies Meijer 2, Mathias Müller 1,3, Birgit Strobl 1, 1 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Austria, 2 Department of Cell Biology and Genetics, Erasmus University Medical Center Rotterdam, The Netherlands, 3 University Center Biomodels Austria, University of Veterinary Medicine Vienna, Austria Octamer binding factor 6 (Oct-6) is a transcription factor of the Pit-Oct-Unc(POU)-domain family. It has been shown to bind to the octamer consensus motif in cellular or viral promoter regions and can either positively or negatively influence gene expression. Oct-6 expression is primarily associated with the developing central and peripheral nervous system. So far, expression of Oct-6 is described to be confined to embryonic stem cells, neuronal subpopulations, myelinating glia, pancreatic islets, and epidermal keratinocytes. Oct-6 is strictly regulated and plays a crucial role in the progression from pro-myelinating to myelinating Schwann cells, whereas its role in the other cell types is less clear. We report here, that expression of Oct-6 is induced by interferon (IFN) in murine embryonal fibroblasts (EFs) as well as bone-marrow derived macrophages (BMMs). We show detailed kinetics of Oct-6 mRNA and protein induction, binding to a consensus-motif containing oligonucleotide, and nuclear localisation upon IFNa/b treatment. Oct-6 expression was also observed upon IFNc, but not IL-6 treatment. We demonstrate that Oct-6 expression upon IFNa/b is dependent on signal transducer and activator of transcription 1 (Stat1) and partially dependent on tyrosine kinase 2 (Tyk2). Oct-6 protein expression is also induced upon vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection and, in both cases, induction is dependent on functional IFNa/b signalling. We are currently aiming to elucidate the role and downstream effects of Oct-6 during viral infection and IFN responses and to identify downstream targets in EFs and BMMs. doi:10.1016/j.cyto.2008.07.064

25 Role of Syk in IL-4-induced human neutrophils Jamila Ennaciri, Denis Girard, Universite´ du Que´bec, INRS-Institut Armand-Frappier, Que., Canada IL-4 is a Th2 cytokine that is produced mainly by activated T cells, mast cells and NK cells. IL-4 is involved in many inflammatory disorders in which polymorphonu-

clear neutrophils (PMNs) play an important role. Previous studies from our laboratory have demonstrated that IL-4 is a human PMN agonist that stimulates PMN phagocytosis and delays apoptosis. We have recently reported that IL-4 activates Syk in PMNs and that these cells express only the IL-4 type I receptor which is composed of IL-4Ra and CD132 subunits. Syk is known to associate with IL-2R and IL-15R, but such an association has never been investigated in the IL-4/IL-4R system. The protein tyrosine kinase Syk is known to play a crucial role in a variety of functions other than phagocytosis, including adhesion and spreading in different hematopoietic cells. We hypothesise that Syk is involved in PMN functions other than phagocytosis in response to IL-4 and that Syk can associate with IL-4Ra. Here, we demonstrate for the first time that Syk is constitutively associated with IL-4Ra and that this physical association is increased by IL-4. Moreover, upon IL-4 treatment, a pool of phosphorylated Syk is associated with IL-4Ra. Stimulation of PMNs by IL-4 was found to activate specific substrates of Syk, including PLC-c2, Akt and tublin a and b. Pretreatment of cells with the Syk-selective inhibitor piceatannol was found to significantly inhibit the ability of IL-4 to enhance phagocytosis, delay PMN apoptosis and increase adhesion. These results are explained by the fact that the pre-treatment of PMNs with piceatannol reduced Syk activation and its association with IL-4Ra. We conclude that Syk is involved in various PMN functions and that IL-4Ra is a new component that has to be added to the growing list of components that can physically associate with Syk. doi:10.1016/j.cyto.2008.07.065

26 Molecular and genetic analysis of the resistance of the mouse strain SPRET/Ei to endotoxemia and gram-negative sepsis Lien Dejager, Iris Pinheiro, Filip Van Hauwermeiren, Claude Libert, Molecular Mouse Genetics Unit, DMBR, VIB and Ghent University, Ghent, Belgium The mouse strain SPRET/Ei (S), derived from the species Mus spretus, displays an extreme resistance to the lethal effects of several notorious inflammatory triggers, such as lipopolysaccharide (LPS). Linkage analysis showed that this dominant LPS resistance is a complex trait, controlled by multiple loci, namely on chromosomes 2, X, 10 and 13. However, these loci interact in an epistatic fashion. Some signaling pathways are normal in S macrophages, but others are not. Thus, several genes are only slightly induced by LPS in S mice and the functions of some of these genes (e.g. encoding type I interferons or matrix metalloproteinases) in endotoxemia are now under investigation. We are currently studying the gene expression patterns in S and C57BL/6 (B) macrophages in detail. After generating the necessary tools to manipulate the genome of S mice, we are also following a reverse genetic way to identify the critical anti-inflammatory genes of SPRET/Ei. We believe that these genes can be interesting target molecules in sepsis since SPRET/Ei mice not only resist the toxic effects of LPS, but they still retain the capacity of phagocytosis of gram-negative bacteria. S mice exhibit a huge and dominant resistance to Salmonella typhimurium bacteria. In order to identify the genes that confer this protection, an interspecific backcross was set up. Currently we are performing a genome scan and linkage analysis will show which loci are important in this phenotype. Clearly, SPRET/Ei is a very important tool, not only because it resists the toxic effects of LPS, but it can also clear infection of a gram-negative pathogen. Hence, by making use of this model, we hope to contribute to the development of a new therapeutic approach in the treatment of endotoxemia and sepsis. doi:10.1016/j.cyto.2008.07.066