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Abstract / Cytokine 63 (2013) 243–314

inhibition of IRF1 translocation to host cell nuclei and interference with DNA binding of both IRF1 and IRF8 to the Ets2, but not the ISRE, region within the IL-12p40 promoter. Inhibition of IRF1 and IRF8 activity is selective since F. tularensis had no effect on activation of IRF3. These data demonstrate a novel mechanism by which a highly virulent bacterium interferes with cytokines required for protective Th1 type immunity.

H9N2 virus evoked strong pro-inflammatory response in human cells that is in line with the other avian, like H5N1 virus-induced human responses.

http://dx.doi.org/10.1016/j.cyto.2013.06.007

7 Cell-specific action of extracellular HMGB1 in sterile inflammation of rat testis

5 REST negatively and ISGF3 positively regulates the human STAT1 gene in melanoma

Ferial Aslani a, Andreas Meinhardt a, Sudhanshu Bhushan a, Hans-Christian Schuppe b, Monika Fijak a, a Department of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany, b Department of Urology, Pediatric Urology and Andrology, JustusLiebig University of Giessen, Giessen, Germany

James Amalraj a, Samuel J. Cutler a, Ibtisam Ghazawi a, Glen M. Boyle b, Stephen J. Ralph a, a School of Medical Science and Genomics Research Centre, Griffith Health Institute, Griffith University, Gold Coast Campus, Qld, Australia, b Drug Discovery Group, Queensland Institute of Medical Research, Herston, Qld, Australia STAT1 plays a pivotal role in signal transduction and transcriptional activation in response to type I and II interferons (IFNs). Regulation of STAT1 expression has significant consequences in human cancer cells, where STAT1 deficiencies have been associated with cellular resistance to type I IFN. Distinct promoter, enhancer and repressor regions have previously been described in the regulatory part of the human STAT1 gene extending as far as the second intron. A putative ISRE sequence in the STAT1 promoter is inducible by type I IFN and binds the IFN-a/b-induced complex, ISGF3. Together with the previously characterized IRF-E/GAS/IRF-E (IGI) motif, these positive regulatory elements provide a means for intracellular amplification of STAT1 expression which is necessary for increasing cell responsiveness to the IFNs. In contrast, the transcriptional repressor REST binds to an RE-1 element in the STAT1 repressor region and in doing so represses transcription from the STAT1 gene regulatory region in melanoma cells lines. Repression significantly decreased in a REST-null cell line. Altering REST function from a transcriptional repressor into an activator as RESTVP16, increased expression from RE-1-targeted reporters. RNA expression of 65 melanoma cell lines by microarray and selected lines with known IFN responsiveness showed significant inverse correlations between STAT1/REST that were related to cellular responses to IFN. Thus REST, through the intronic RE-1 element, provides a means for down-regulating STAT1 expression, affecting melanoma responsiveness to IFN. Intracellular levels of REST may be a useful marker to test for IFN-resistance and as a novel therapeutic target in IFN-resistant melanomas.

http://dx.doi.org/10.1016/j.cyto.2013.06.009

High mobility group box protein 1 (HMGB1), the non-histone chromosomal protein, plays an important role in onset and chronification of autoimmune diseases once released from the nuclei. In this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo in a rat model of experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates. HMGB1 levels in EAO testis were elevated at late chronic phase of disease as compared to early proinflammatory cytokines such as IL-6 and TNF-a. We found that testicular somatic cells show a cell-specific expression profile of HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as evidenced by p38 MAPK and p65 NF-?B phosphorylation which stimulated an increase in mRNA levels of TNF-a and IL-6. Recent data shows that RAGE induced ERK activation leads to enhanced autophagy levels. In line with these data, extracellular HMGB1 triggered formation of autophagosomes in Sertoli cells. Increased autophagy levels in Sertoli cells might explain how these cells survive the inflammatory environment in EAO testis. Considering HMGB1’s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic intervention in treatment of chronic testicular inflammation. http://dx.doi.org/10.1016/j.cyto.2013.06.010

http://dx.doi.org/10.1016/j.cyto.2013.06.008 8 Epigenetic regulation of Th-17 differentiation 6 Innate immune responses in human dendritic cells induced by H9N2 avian influenza virus Veera Arilahti, Ilkka Julkunen, Thedi Ziegler, Pamela Österlund, Virology Unit, National Institute for Health and Welfare (THL), Helsinki, Finland H9N2 influenza viruses have been circulating worldwide in multiple avian species. Occasionally H9N2 influenza viruses have been transmitted to humans. The concern is the adaptation of avian influenza viruses to efficient human transmission, which could lead to a pandemic. In this study we have examined the activation of innate immune responses in low pathogenic avian influenza (LPAI) A/HK/1073/99 (H9N2) and seasonal influenza A/Beijing/353/89 (H3N2) virus-infected human dendritic cells (DCs). In human airways and lungs the influenza virus infects epithelial cells and DCs in close proximity to epithelium which orchestrate the activation of adequate immune responses. The virus infection leads to activation of transcription factors (e.g. IRF3, IRF7 and NF-jB) which regulate the expression of proinflammary cytokines and antiviral interferons. We discovered that the H9N2 virus induced earlier interferon (IFN-a1, -b1 and -k1) gene expression than the H3N2 virus, and the response was switch off in the late time point of H9N2 infection. Nevertheless interferon stimulated gene 15 (ISG15) and antiviral MxA protein were produced in the H9N2 virus-infected cells at least at the same level than in the H3N2 virus-infected cells. For the pro-inflammatory responses CXCL10, IL-1b and TNF-a gene expression was analyzed and it turned out that pro-inflammatory responses were stronger in H9N2 virus-infected cells than in the cells infected with the H3N2 virus. There were no differences seen in the virus-induced phosphorylation of IRF3 transcription factor or phosphorylated p38 MAP kinase between H9N2 and H3N2 viruses. All infection experiments with the H9N2 virus were carried out under biosafety level 3 (BSL-3) conditions. These results indicate that the avian influenza H9N2 virus is inducing strong but transient antiviral interferon response in human DCs. Moreover, avian

Patricia A. Assis, Satoshi Ueha, Matthew Schaller, Steve L. Kunkel, University of Michigan Medical School, Department of Pathology, Ann Arbor, MI, USA Differentiation of Th17 cells requires the combination of IL-6 and TGF-b and is further maintained by IL-23. This T cell subtype is important for both host defense against extracellular pathogens and the pathogenesis of autoimmune diseases. The main cytokines produced by Th17 cells are IL-17, which induces strong proinflammatory effects, and IL-22, an enhancer of epithelial proliferation and healing responses. Previous research has demonstrated that the expression of the transcription factor cMaf is important for inhibition of IL-22 production in Th17 cells. The transcription factor AHR (aryl hydrocarbon receptor) has also been shown crucial for proper Th17 differentiation. The adapter protein PTIP (Pax transactivation domain-interacting protein) has recently been shown to have a role in the maturation of thymocytes. PTIP stably associates with complexes containing the chromatin methyltransferases MLL3 and MLL4, which modify chromatin in order to activate gene transcription. Here we demonstrate that PTIP contributes to Th17 differentiation and function via regulation of both c-Maf and AHR. Using CD4+ T cells from Ptipf/fCD4 Cre+ (PTIP / ) mice and Cre- (WT) controls in a T cell transfer model of chronic colitis, we determined that RAG2 / mice receiving WT cells had aggressive colitis with high expression of IL-17 in the intestine. However, in mice receiving PTIP / cells there was a reduction in IL17 and an increase in IL-22 expression. The change in IL-17 and IL-22 expression in PTIP / mice was confirmed in vitro. Further analysis by chromatin immunoprecipitation (ChIP) assay showed that MLL3 binding and its dependent chromatin modifications at the loci for IL-17, c-Maf and AHR were enhanced in WT Th17 cells when compared to Th0 conditions and reduced in PTIP / Th17 cells. These data highlight, for the first time, that PTIP is essential for proper differentiation of Th17 cells. http://dx.doi.org/10.1016/j.cyto.2013.06.011