176 Identification of a novel enhancer region in the distal promoter region of the human interferon-lambda1 gene

Abstracts/Cytokine 43 (2008) 280–285 173 ssRNA Viruses inactivated P53 and induce noxa-dependent apoptosis via posttranslational modifications of IRF-1, IRF-3 and CREB C. Lallemand1, B. Blanchard1, P. Lebon2, E. May1, M.G. Tovey1, 1Laboratory of Viral Oncology, FRE2937 CNRS, Institut Andre´ Lwoff, Villejuif, France, 2Laboratory of Virology, Universite´ Rene´ Descartes, Hopital Cohin-Saint Vincent de Paul, Paris, France In order to characterize the mechanisms underlying apoptosis induced by viral infection transcriptional activation of genes encoding members of the BH3 only family of proteins was analyzed during the course of virus infection. Among these genes only NOXA is transcriptionally activated by Vesicular Stomatitis virus (VSV), Sendai virus (SV), Measles virus, Herpes Simplex virus (HSV), or synthetic dsRNA, and at least in the case of VSV infection, is required for efficient virus-induced apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53 but requires the presence of IRF-1, IRF-3 and CREB. Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving dierent pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by TLR3/RIG-1 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection, correlates with a 10 kDa increase in its MW, suggesting a covalent linkage with a previously unknown regulatory polypeptide. doi:10.1016/j.cyto.2008.07.235

174 IRAK2 is critical for LPS-mediated post-transcriptional control Hui Xiao3, , Youzhong Wan1,5, , Tae Whan Kim3, Katarzyna Bulek3, Sujan Chaudhuri4, Barsanjit Mazumder4, George R. Stark1,5, James Thomas2, Xiaoxia Li3, 1Department of Molecular Genetics, Cleveland Clinic Foundation, Cleveland, OH 44195, USA, 2Departments of Pediatrics and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA, 3Department of Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA, 4Department of Biology, Geology and Enviromental Sciences, Cleveland State University, Cleveland, OH 44115, USA, 5Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA IRAK2 is a member of the IL-1 receptor (IL-1R)-associated kinase (IRAK) family and has been implicated in Toll-like receptor (TLR)-mediated signaling. We generated IRAK2-deficient mice to examine the function of IRAK2 in TLR-mediated signaling. The IRAK2-deficient mice were resistant to lipopolysaccharide-induced septic shock, due to impaired TLR4-mediated induction of pro-inflammatory cytokines and chemokines. Macrophages from IRAK2-deficient mice showed reduced cytokine and chemokine production compared to that in wild-type macrophages in response to LPS, indicating an essential role of IRAK2 in TLR4-mediated signaling. While IRAK2 deficiency did not aect the levels of TLR4-mediated NFkB activation, a reduction of LPSmediated mRNA stability contributed to the reduced cytokine and chemokine production observed in bone marrow (BM)-derived macrophages from IRAK2-deficient mice. Furthermore, the ratios of LPS-induced cytokine and chemokine mRNAs in translation-active versus translation-inactive pools were reduced in IRAK2-deficient macrophages compared to wild-type macrophages. Taken together, these results demonstrate that IRAK2 is required for LPS-mediated cytokine and chemokine posttranscriptional control. Importantly, LPS-induced phosphorylation of MKK3/6, p38 and ERK, MnK1 and eIF4E was significantly reduced in IRAK2-deficient macrophages compared to wild-type macrophages. Moreover, LPS stimulation induced interaction of IRAK2 with TRAF6, MKK3/6 and MK2, implicating a critical role of MAP kinase signaling in LPS-induced IRAK2-mediated posttranscriptional control.  

These authors contributed equally.

doi:10.1016/j.cyto.2008.07.236

175 Opposite transcriptional effects of Interferon Regulatory Factor-3 on differential human interferon-A gene expresssion Pierre Génin1, Rongtuan Lin2, John Hiscott2, Ahmet Civas1, 1UPR 2228 - CNRS, Laboratoire de Re´gulation Transcriptionnelle et Maladies Ge´ne´tiques, UFR Biome´dicale des Saints-Pe`res, Universite´ Paris Descartes, Paris, France, 2Lady Davis Institute for Medical Research, and Depts. of Microbiology, Immunology and Medicine, McGill University, Montre´al, QC, Canada In this study, we have determined the expression patterns of human interferon-a (ifn-a) genes in the presence of Interferon Regulatory Factor-3 (IRF-3) and IRF-7, in order to identify the mechanisms accounting for the dierential regulation of these genes. Our data indicated that ifn-a1 gene expression is dependent on three IRF-elements (B, C, and D modules) required for IRF-3 and cAMP-response element binding-

281

binding protein (CBP) recruitment and synergistic activation by IRF-3. High expression levels of ifn-a1 are also observed at maximal occupancy of the three modules, when high amounts of IRF-7 are stimulated by TANK-Binding Kinase-1 (TBK-1) or IkB kinase-e (IKK-). Naturally occurring nucleotide substitutions in the C module of other ifn-a genes disrupt their IRF-3-mediated transcription, and a G/A substitution in the D module considerably enhances their IRF-7-mediated expression. When both IRF-3 and IRF-7 were activated, IRF-3 exerted opposite eects on ifn-a gene expression. A synergistic eect of both factors was observed on ifn-a1 transcription, whereas the synergism was downregulated by recruitment of IRF-3 and CBP on ifn-a gene promoters. These regulatory mechanisms that tightly control dierential expression of IFN-A genes could be determinant for IFN-a production in dierent cell types by Toll-like receptor-dependent or Retinoic-acide-Inducible Gene I-dependent signallings.

doi:10.1016/j.cyto.2008.07.237

176 Identification of a novel enhancer region in the distal promoter region of the human interferon-lambda1 gene Scott J.P. Thomson1, FuiGoon Goh1, Helen Banks1, Brian Foxwell1, Sergei V. Kotenko2, Irina A. Udalova1, 1Kennedy Institute of Rheumatology, Imperial College London, London, United Kingdom, 2Department of Biochemistry and Molecular Biology, New Jersey Medical School, Newark, New Jersey, USA Interferon (IFN)-lambdas are new class II cytokines, evolutionary related to both type I IFNs and interleukin-10. Activation of the IFN-lambda receptor leads to induction of anti-viral and anti-tumour activities, while additional immuno-regulatory functions remain ill-defined. Like type I IFNs, IFN-lambda1 production is mainly regulated at the level of transcription, and is induced in response to viruses, as well as specific toll-like receptor ligands, such as lipopolysaccharide (LPS). Here we systematically examined the organisation of the genomic region encompassing the IFNlambda1 gene and generated a deletion set of luciferase reporter constructs based on the evolutionary conservation of the region. Expression of this set in both human cell lines and primary myeloid cells indicated the presence of a strong enhancer region in the distal promoter of the gene. Over-expression, RNA interference and chromatin immuno-precipitation studies showed that the LPS-induced enhancer activity is highly dependent on NF-kappaB Rel-A. Moreover, our bioinformatic analysis revealed the presence of a homogenic cluster of NF-kappaB sites within this enhancer region. The functional interaction of the novel enhancer region with the previously described proximal promoter region mainly induced by interferon regulatory factor-3 will be discussed in the context of stimulus-specific induction of the IFNlabmda1 gene. doi:10.1016/j.cyto.2008.07.238

177 Multiple intracellular signaling pathways contribute to synergistic TLR ligandinduced cytokine gene expression in human monocyte-derived dendritic cells Sanna Mäkelä, Mari Strengell, Taija Pietilä, Pamela Österlund, Ilkka Julkunen, Department of Viral Diseases and Immunology, National Public Health Institute, FI-00300 Helsinki, Finland Toll-like receptors (TLRs) are pattern-recognition receptors of the innate immune system that recognize various pathogen-associated molecules. Binding of ligands to dierent TLRs can induce a synergistic production of pro-inflammatory cytokines. In the present study, we have analyzed the molecular mechanisms of synergy in TLR ligand stimulated human monocyte-derived dendritic cells (moDCs). Stimulation of moDCs with TLR8 ligand R848 together with TLR3 or TLR4 ligands poly(I:C) or LPS, respectively, led to a synergistic IL-6, IL-10, IL-12p70 and TNF-alpha mRNA expression and cytokine production, while no such synergistic eect was observed with combinations of TLR2 (synthetic lipopeptide, PamCSK), TLR3, TLR4 or TLR5 (bacterial flagellin) ligands. To clarify the mechanisms behind the synergistic eect, DNA binding assay was used to study TLR ligand-induced binding of interferon regulatory factor (IRF), signal transducer and activator of transcription (STAT) and nuclear factor (NF)-B family transcription factors to the respective binding sites on IL-12p35 gene promoter. TLR3 and TLR8 stimulation induced the binding of multiple IRF and STAT family members to the proximal promoter interferon stimulated response element (ISRE) site in moDCs. We also show that mitogen activated protein kinases (MAPKs) and phosphor inositol 3 kinase (PI3K) have an important role in TLR induced cytokine production. Regulation of phosphorylated forms of p38, cJun, p44/42 and Akt was detected in TLR3 and TLR8 ligand-stimulated moDCs. Moreover, inhibitors of MAPK p38, JNK and ERK signaling pathways inhibited TLR3, TLR4 and TLR8 ligand-induced cytokine expression. Furthermore, the synergistic IL-12p70 production was abolished in NF-B, p38 and PI3K inhibitor treated moDCs. Our data suggests that TLR-dependent synergistic cytokine gene expression