ID: 195

Abstract / Cytokine 76 (2015) 66–112 ID: 191 Comparing the stability and activity of recombinant IFNk3 and IFNk4 Hans Henrik Gad *, Kasper Thorhauge Christensen, Ewa Terczyn´ska-Dyla, Rune Hartmann, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark * Corresponding author. Hepatitis C virus (HCV) infection is one of the major causes of liver cancer worldwide and every year 3–4 million people become infected. Whereas some infected people eradicate the virus spontaneously, almost 85% do not and instead develop a chronic infection. Recently, it was shown that people with a functional IFNL4 gene have a lower chance of clearing the virus spontaneously or in response to treatment than people with a non-functional IFNL4 gene. Why it is a disadvantage to have a functional IFNL4 gene during HCV infection is currently not known although a causal relationship between the activity of the IFNk4 protein and poor HCV clearance has been demonstrated. IFNk4 belongs to the type III IFNs together with IFNk1, -2, and -3. However, it differs from the others not only by its low sequence similarity but also by its impaired secretion. This impairment is not due to a weak signal peptide, as swapping the signal peptides between IFNk3 and -4 had no effect on secretion. When we purified IFNk3 and -4, we found that IFNk4 is far more difficult to refold in vitro than IFNk3 suggesting that the poor secretion of IFNk4 could be due to an inherent problem of folding the protein. Because such a problem could also mean that IFNk4 could be far unstable than the other type III IFNs, we decided to compare the stability of recombinant IFNk3 and IFNk4. Our results demonstrate that IFNk4 like IFNk3 is surprisingly stable once it has folded properly. http://dx.doi.org/10.1016/j.cyto.2015.08.195

ID: 192 Role of AhR in IL-22 production by four types of immune cells, including CD4CD8- TCRß T cells, present in psoriasis-like skin after topical imiquimod Perrine Cochez 1,2,*, Camille Michiels 1,2, Emilie Hendrickx 1,2, Astrid Van Belle 1,2, Muriel Lemaire 1,2, Pierre Coulie 2, Jean-Christophe Renauld 1,2, Laure Dumoutier 1,2, 1 Ludwig Institute for Cancer Research, Belgium, 2 de Duve Institute, Belgium * Corresponding author at: Ludwig Institute for Cancer Research, Belgium. IL-22 has a detrimental role in skin inflammatory processes, particularly in the imiquimod-induced psoriasis model. As transcription factor AhR controls IL-22 production by several cell types, we analyzed its role in IL-22 production by immune cells in the inflamed skin. We used a model in which imiquimod is applied on the ears. Our results indicate that IL-22 is still expressed in the skin of imiquimod-treated AhR / mice but in lesser amounts than in wild-type mice. We then studied the role of AhR on each of the three cell populations known to produce IL-22 in the skin: cd T cells, Th17 cells and ILC3. We studied also a new IL-22 producing cell type that we identified in this setting: CD4-/CD8- TCRb+ T cells. In the imiquimod-treated ears, AhR was required for IL-22 production by Th17 but not by the three other cell types. For the latter but not for Th17 cells, AhR had a role in their recruitment into the inflamed skin or in their local proliferation, as their numbers were reduced in AhR / vs wild-type imiquimod-treated ears. http://dx.doi.org/10.1016/j.cyto.2015.08.196

ID: 193 ADAM10 controls a novel IL-11 trans-signalling pathway Juliane Lokau 1,*, Maria Aghte 1, Georg H. Waetzig 2, Stefan Rose-John 1, Christoph Garbers 1, 1 Institute of Biochemistry, Kiel University, Kiel, Germany , 2 Conaris Research Institute AG, Kiel, Germany * Corresponding author. Interleukin (IL)-11 is a member of the IL-6 family which has been initially described to have anti-inflammatory properties. However, recent studies revealed that overshooting IL-11 activity is involved in inflammation and the progression of epithelial cancers. IL-11 binds to the membrane-bound IL-11 receptor (IL-11R), and this complex can then initiate signal transduction by recruiting two molecules of the b-receptor glycoprotein (gp)130, which predominantly leads to activation of the JAK/STAT pathways. Gp130 is ubiquitously expressed, and IL-11R expression was found on several cell types including osteoblasts, osteoclasts, T cells and macrophages. Proteolytic processing of cytokine receptors is an important regulatory element for their signalling capacity, which not only regulates the amount of the receptors on the cell surface, but also creates soluble receptors with distinct biological functions. Here, we show for the first time that the metalloprotease ADAM10, but not ADAM17, is able to cleave the IL-11R and release its ectodomain. Chimeric receptors of the IL-11R and the

99

IL-6R revealed structural traits required for proteolytic processing and showed that a small part of the stalk region is responsible for ADAM17’s ability to discriminate between substrate and non-substrate. Furthermore, we show that a single amino acid mutation within the stalk is sufficient to generate a proteolysis-resistant IL-11R variant. The generated soluble IL-11R binds IL-11, and the resulting complex can then bind to gp130 and thus activate cells even though they do not express the membrane-bound IL-11R. This novel IL-11 trans-signalling pathway can be specifically inhibited by the anti-inflammatory designer protein sgp130Fc. http://dx.doi.org/10.1016/j.cyto.2015.08.197

ID: 194 Role of microRNAs in signal transduction pathways of the inflammatory cytokine Interleukin-6: Relevance for liver diseases Florence Anne Servais 1,*, Mélanie Kirchmeyer 1, Matthias Hamdorf 1, Claude Haan 1, Markus Casper 2, Frank Lammert 2, Petr Nazarov 3, Laurent Vallar 3, Stephanie Kreis 1, Iris Behrmann 1, 1 Signal Transduction Laboratory, University of Luxembourg, 162A Avenue de la Faïencerie, Luxembourg L-1511, Luxembourg, 2 Department of Medicine II, Saarland University Medical Center, Homburg, Germany, 3 Genomics Research Unit, Luxembourg Institute of Health, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg * Corresponding author. IL-6 plays important roles in regulation of liver functions and promotes hepatocarcinogenesis. As the contribution of IL-6-induced miRNAs to these effects is largely unknown, we investigate the effects of IL-6 on the miRNome of hepatoma cells and non-transformed hepatocytes. Moreover, little is known about miRNAs regulating key players of IL-6 signal transduction. Therefore, our aims were to identify such miRNAs in a miRNA mimics screen and to elucidate their effects on this pathway. Integration of the results of both approaches is expected to lead to the identification of regulatory circuits. While IL-6 (and hyperIL-6) induce the differential regulation of thousands of mRNAs in HepG2 and HuH-7 hepatoma cell lines, only selected miRNAs change their expression level significantly, including miR-21, miR-100, miR-145, miR-146b-5p. In contrast, we have observed previously that IFN-g has a profound effect on the miRNome of melanoma cells (Schmitt, Cell Comm. Sign. 2012). Therefore we investigate whether IL-6type cytokines may have generally weaker effects on the miRNome compared to interferons and/or whether our observation is due to cell type-specific differences. To extend our in vitro findings, we analyse and correlate the expression levels of miRNAs and inflammatory cytokines in patients with HCC and advanced nonalcoholic steatohepatitis (NASH), the latter being at high risk to develop HCC. Bio-Plex cytokine immunoassays revealed that the serum level of IL-6 and HGF is higher in HCC patients than in healthy controls and NASH patients. Our work may contribute to the identification of novel biomarkers for the progression of chronic liver diseases. http://dx.doi.org/10.1016/j.cyto.2015.08.198

ID: 195 Identification of cytokine cell sources in a new mouse model of systemic juvenile idiopathic arthritis highlights innate immunity of this disorder Anneleen Avau 1, Maya Imbrechts 1,*, Karen Put 1, Ellen Brisse 1, Tania Mitera 1, Georges Leclercq 2, Carine H. Wouters 3, Patrick Matthys 1, 1 Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium, 2 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium, 3 Pediatric Rheumatology, University Hospital Gasthuisberg, Leuven, Belgium * Corresponding author. Objective: Systemic juvenile idiopathic arthritis (sJIA) is a rheumatic childhood disease, with distinctive systemic inflammatory features and a typical cytokine profile. The etiology of sJIA is largely unknown. Here, we aimed to elucidate the role of specific cell populations and cytokines in the pathogenesis, by means of a new mouse model of sJIA relying on the injection of complete Freund’s adjuvant (CFA) in interferon-gamma deficient (IFN-g KO) mice. Wild type (WT) mice developed a minor inflammation, indicating that IFN-g provides protection in the disease process. Methods: Cytokine mRNA levels were analyzed in organs and purified immune cell populations of CFA-challenged IFN-g KO and WT mice. Cytokine antagonists and cell depleting antibodies were administered to mice. Clinical, laboratory and immune features of sJIA were evaluated. Results: In the diseased IFN-g KO mice, elevated expression of IL 1b, IL-6, IL-17 and IL-22 were found in lymph nodes and – remarkably – also in lung tissue. Gamma-delta T cells were a major cell source for IL-17, and anti-IL-17 antibodies abrogated the disease. In the non-diseased WT mice, expression of IFN-g was almost exclusively found in NK, NKT and gamma-delta T cells. Transient neutralization of IFN-g as well as depletion of NK cells in WT mice both resulted in a fulminant sJIA-like disease.

100

Abstract / Cytokine 76 (2015) 66–112

Conclusion: These data pinpoint innate immune cells and non-lymphoid organs as important sources of cytokines in CFA-induced animal models. The results also demonstrate that NK cells provide a regulatory function in the pathogenesis of sJIA, by production of IFN-g.

References [1] Grivennikov, Cancer Cell (2009). [2] Vollmer, Hepatology (2009). [3] Demaria, Aging (2010).

http://dx.doi.org/10.1016/j.cyto.2015.08.199 http://dx.doi.org/10.1016/j.cyto.2015.08.201

ID: 196 Proteolysis of the human Interleukin-6 Receptor in vivo Christoph Garbers 1,*, Steffen Riethmüller 1, Prasath Somasundaram 2, Johanna Ehlers 1, Tomas Koudelka 2, Chien-Wen Hung 2, Andreas Tholey 2, Stefan Rose-John 1, 1 Institute of Biochemistry, Kiel University, Kiel, Germany, 2 Institute for Experimental Medicine, Systematic Proteomics, Kiel University, Kiel, Germany * Corresponding author. Interleukin-6 (IL-6) has been linked to numerous inflammatory diseases and is therefore an attractive therapeutic target in the clinic. Soluble and membranebound forms of the Interleukin-6 Receptor (IL-6R) are responsible for its pro- and anti-inflammatory properties via the membrane-bound b-receptor gp130. Formation of the signaling complex results in activation of the JAK/STAT signaling pathway. In addition, soluble forms of the IL-6R (sIL-6R) have been identified in several body fluids. Interestingly, a coding nonsynonymous single nucleotide polymorphism (SNP) within the IL-6R (rs2228145, Asp358Ala) results in a 2-fold increase in soluble IL-6R (sIL-6R) serum levels and a reduced risk for coronary heart disease. In vitro data show that the sIL-6R is generated either via limited proteolysis of the membrane-bound precursor or via alternative splicing of the IL-6R mRNA, and the latter has been shown to occur also in vivo. In order to identify and analyze sIL-6R that was generated by proteolysis we combined a ‘‘bottomup” proteomics approach with LC-MS. We could determine the cleavage site used in vitro and in vivo, identify novel glycan motifs, and show for the first time that proteolysis contributes to sIL-6R serum levels in humans. Further analysis of N- and O-linked glycosylation revealed different functions in terms of signaling and proteolysis. Mutation of the identified cleavage site resulted in an IL-6R that was resistant towards shedding by ADAM10 and ADAM17. In summary, our data show that limited proteolysis contributes to sIL-6R serum levels in humans, and that glycosylation is an important regulatory modification of the IL-6R.

http://dx.doi.org/10.1016/j.cyto.2015.08.200

ID: 197 Effects of inflammatory cytokines and hypoxia on hepatocytes and HCC cell lines Andreas David Zimmer 1,*, Nadia Battello 2, Karsten Hiller 2, Andre Wegner 2, Iris Behrmann 1, Claude Haan 1, 1 University of Luxembourg, Luxembourg, 2 Luxembourg Centre for Systems Biomedicine, Luxembourg * Corresponding author. Hepatocellular carcinoma (HCC), mostly the result of chronic liver diseases, is ranked as the fifth most common cancer world-wide. To find new treatments, a better understanding of the initiation, the progression and the implication of the cellular metabolism in the development of HCC is needed. Hypoxia and IL6-type cytokines can promote tumour growth, influence cellular metabolism and prevent cancer cells from apoptosis [1]. Furthermore, IL6-type cytokines induce the expression of HIF-1a, one of the key mediators of the cellular response to hypoxia [2]. Constitutively active STAT3 leads to an increased production of lactate under normoxic conditions. This effect was HIF-1a-dependent [3]. We investigated the effects of the IL6-type cytokine Oncostatin M (OSM) on hepatocytes and HCC cell lines under normoxia. Quantitative real-time PCR and quantitative WB analysis was performed focusing on the regulation of glycolytic enzymes, many being direct HIF-1a target genes. Contrary to the HCC lines, in non-neoplastic PH5CH8 hepatocytes an OSM-mediated induction of pyruvate dehydrogenase kinase 1 (PDK1) was found together with an increase in the pyruvate dehydrogenase (PDH) phosphorylation level, indicating that these cells shift to a more glycolytic metabolism. Metabolic analysis is being carried out to validate this finding. We found little OSM effects under normoxia on the induction of glycolytic enzymes (mRNA and protein level). Surprisingly, even hypoxic treatment only affected few genes previously described to be HIF-1a targets. Funded by the University of Luxembourg Tandem project ‘‘Meta-IL6”.

ID: 198 Discovery of a novel chemokine binding activity in varicella zoster virus Victor Gonzalez-Motos 1, Birgit Ritter 1, Tihana Lenac 2, Stipan Jonjic 2, Fernando Arenzana-Seisdedos 3, Ulrich Kalinke 4, Abel Viejo-Borbolla 1,*, 1 Institute of Virology, Hannover Medical School, Hannover, Germany, 2 Center for Proteomics and Department of Histology and Embryology, University of Rijeka, Rijeka, Croatia, 3 Viral Pathogenesis Unit, Institute Pasteur, Paris, France, 4 Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany * Corresponding author. Varicella zoster virus (VZV) is a highly prevalent human neurotropic herpesvirus that establishes latency in sensory ganglia. During primary infection it causes varicella and, upon reactivation, herpes zoster, which may be followed by extreme pain. Following replication in epithelial cells of the respiratory mucosa VZV infects T cells at the regional lymph node and spreads systemically. Colonization of sensory ganglia and modulation of the immune system, including T cell migration, are required for VZV mediated pathogenicity. The viral and cellular factors involved in these processes are not well characterized. Chemokines orchestrate the migration of leukocytes to the site of infection playing essential roles in the antiviral response. To carry out their action chemokines are presented to their receptors through binding to glycosaminoglycans (GAGs). Some related viruses express proteins that act as secreted or transmembrane chemokine receptors interfering with chemokine activity, sometimes through GAG interaction. However, until present, none of these proteins have been described in VZV. We have addressed the immunomodulatory role of VZV glycoprotein C (gC). Our data show that VZV gC interacts with GAGs and chemokines with nanomolar affinity as shown by surface plasmon resonance. This binding results in enhanced migration of T cells. The domains involved in the interaction are currently being identified. This constitutes the discovery of the first VZV protein that modulates chemokine activity and points to VZV gC as a relevant factor in the modulation of T cell migration by VZV. http://dx.doi.org/10.1016/j.cyto.2015.08.202

ID: 199 Expression of IL-20-related cytokines in PPD-induced allergic contact dermatitis and their role in a mouse model Astrid Van Belle 1,2, Magali De Heusch 1,2, Marie Baeck 3, Perrine Cochez 1,2, Emilie Hendrickx 1,2, Guy Warnier 1,2, Jean-Christophe Renauld 1,2, Laure Dumoutier 1,2,*, 1 Ludwig Institute for Cancer Research, Belgium, 2 De Duve Institute, UCL, Belgium, 3 Department of Dermatology, Cliniques Universitaires Saint-Luc, Brussels, Belgium * Corresponding author at: Ludwig Institute for Cancer Research, Belgium. Para-Phenylenediamine (PPD) is an aromatic amine used in hair dyes and in temporary black henna tattoos, which is a frequent cause of allergic contact dermatitis (ACD). ACD is a skin inflammatory disease characterized by modifications such as spongiosis, exocytosis and acanthosis. The aim of this study is to characterize the expression and the role of IL-20-related cytokines, including IL-19, IL-20, IL-22 and IL-24, in ACD. In 11 patients with a history of positive patch-test reaction to PPD, patch tests with PPD 1% in petrolatum were applied and skin biopsies were performed after 8, 24 and 48 h. The mRNA level of IL20-related cytokines is increased in affected vs uninvolved skin from PPD allergic patients (positive patch test to PPD). In order to assess the role of these cytokines, we set up a mouse model of PPD-induced allergic contact dermatitis and we showed that IL-22R / or IL-20R2 / mice are partially protected against development of PPD-induced contact hypersensitivity. These mice presented a lower ear thickening than WT treated mice. This was confirmed at histological level, because, after PPD treatment, IL-22R / mice showed less acanthosis than WT mice. In addition, we showed that IL-22R plays a role in induction of chemokine expression as well as neutrophil marker. Along the same line, after PPD treatment, proportion of neutrophils is lower in the skin of IL22R / mice compare to WT mice. We conclude that IL-22R associated