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Abstract / Cytokine 70 (2014) 28–79

by arthritic score, paw volume, paw circumference, joint diameter, body weight, histopathological changes and index of thymus and spleen (mg/g). Biochemical analysis in serum were performed which includes superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), nitric oxide (NO), melonaldehyde (MDA), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT). The levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-aÞ were also determined. Result: PCP (100, 200 and 400 mg/kg) significantly ameliorated the arthritis severity in terms of arthritic score, paw volume, paw circumference and joint diameter. Serum levels of NO, MDA, AST, ALP and ALT were significantly decreased when compared with arthritic rats (p < 0.05). The increased levels of IL-6 and TNF-a were significantly reduced in treatment groups. Conclusion: Our results suggest that, anti-arthritic effect of PCP may be due to immunodepressive effect, cytokine regulation and antioxidant property. http://dx.doi.org/10.1016/j.cyto.2014.07.107

101 Successful combined intra-tumoural immunotherapy of established murine mesotheliomas requires B cell involvement Shruti Krishnan 1, Emily Bakker 1, Cassandra Lee 1, Haydn T. Kissick 2, Demelza J. Ireland 1, Manfred W. Beilharz 1, 1 The University of Western Australia, Crawley, WA, Australia, 2 Department of Surgery, Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, MA, USA Introduction: Cancer immunotherapy aims to stimulate the host’s immune system to overcome immune evasion mechanisms and destroy cancer cells. We had previously developed a Timed Triple Immunotherapy (TTI) in the TGF-b secreting AE17 murine mesothelioma tumour model which targets three aspects of peripheral tolerance [1]. Methods: Subcutaneous growth of non-TGF-b secreting AB1 mesothelioma tumours in BALB/c and B cell knock-out (BKO) mice (BALB/c background). TTI treatment consisted of intra-tumoural (anti-CD25mAb and 7 days daily anti-TGF-bmAb) and intraperitoneal (anti-CTLA4mAb) injections or a novel Triple Immunotherapy Cocktail (TIC) of all three components simultaneously administered as a single intra-tumoural injection. Flow cytometric analysis of spleen and tumour draining lymph nodes (TDLNs). Tumour specific IgG detection in mouse sera by ELISA. Results: TTI conferred tumour clearance in 100% (17/17) of BALB/c mice bearing AB1 tumours. TIC was similarly effective with 88% (15/17) tumour clearance. Cured mice showed elevated levels of tumour specific IgG antibodies at >95 days post treatment. Time-course studies of tumour clearance showed that; (a) IgG levels were not elevated during tumour clearance, (b) B cell numbers were increased in the TDLNs and spleens during tumour clearance. An obligatory role for B cells in tumour eradication was confirmed by the employment of BKO mice in which the TIC treatment was totally ineffective. Conclusions: Successful immunotherapies against mesothelioma tumours require simultaneous targeting of multiple immune mechanisms. Intra-tumoural delivery of monoclonal antibody combinations can effect cures without side effects.

Reference [1] Kissick HT, Ireland DJ, Krishnan S, Madondo M, Beilharz MW. Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy. Immunol. Cell Biol. 2012;90(8):822–6. http://dx.doi.org/10.1016/j.cyto.2014.07.108

102 Immuno-stimulatory effect of Lactobacillus plantarum HY7712 via toll-like receptor 2 signaling pathway Hyung-Keun Ku, Hoyong Lee, Il Dong Choi, Je-Hyeon Ra, Tae-Youl Kim, Ji-Woong Jeong, Sung-Hwan Kim, Jae-Hun Sim, Young-Tae Ahn Ahn, Korea Yakult, Komae, Kiheung, Yongin, Gyeonggi, South Korea The purpose of this study is to investigate the immuno-stimulatory function of the lactic acid bacteria (LAB), Lactobacillus plantarum HY7712, isolated from kimchi. When NF-jB-activating activities of 200 LAB isolated from kimchi were measured in RAW264.7 cells stimulated with or without LPS, L. plantarum HY7712 most potently induced NF-jB activation in RAW264.7 cells, while the presence of LPS induced inhibition of NF-jB activation. HY7712 also induced the expression of IL-12 and TNF-a, transcription factor for NF-jB, in human PBMCs. In order to investigate the effect of toll-like receptor (TLR)-associated HY7712-signaling path-

way, psiTLR2- or psiTLR4-transfected RAW264.7 cells were used to measure the NF-jB activation with the same stimulation with HY7712. HY7712 activated NFjB in wild-type and psiTLR4-knockout cells, but not in psiTLR2-knockout cells, indicating HY7712 can activate Raw264.7 cell via TLR2/NF-jB signaling. In addition, HY7712 induced the expression of IL-12 and IFN-c in mouse splenocytes, and also the activation markers CD80, 86, and MHC class II in mouse bone marrow-derived dendritic cells.

http://dx.doi.org/10.1016/j.cyto.2014.07.109

103 Identification of a novel interferon-stimulated gene whose product significantly restricts retrovirus replication Yoshinao Kubo, Yuka Yashima, Haruka Kamiyama, Mai Izumida, Hironori Sato, Naoki Yamamoto, Hideki Hayashi, Toshifumi Matsuyama, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Interferons confer cells resistant to retrovirus infection by inducing anti-viral cellular factors, such as tetherin, APOBEC3G, and Mx2. It is believed that many factors inhibiting viral infections among interferon-stimulated genes (ISGs) remain to be identified. On the other hand, we found efficient restriction of retrovirus infection by a compound that inhibits S–S bond formation at acidic pH. Thus, it was postulated that an acidic endosome-localized, S–S bond digesting factor among ISGs may restrict retrovirus replication. We here report for the first time that such a cellular factor efficiently inhibits MLV and HIV-1 replication. When the factor was expressed in target cells, transduction efficiency of HIV-1 vector pseudotyped with ecotropic, amphotropic, xenotropic MLV, or VSV was significantly reduced. In addition, when HIV-1 vectorproducing cells were transfected with the factor, HIV-1 Gag expression and virion production were significantly suppressed. The factor digested S–S bonds of viral envelope proteins and of CD63, which is involved in HIV-1 virion release. A CD63 mutant containing amino acid substitutions at conserved cysteine residues in the second extracellular loop suppressed virion release like GILT. Interferon treatment induced the factor expression, and its knockdown by shRNA abrogated interferon-induced inhibition of HIV-1 vector infection. These results indicate that interferon restricts HIV-1 entry and production at endosomal level by inducing the factor.

http://dx.doi.org/10.1016/j.cyto.2014.07.110

104 Negative regulation of IL-1b through the PI3K pathway is mediated by Akt-dependent inactivation of FoxO1 in TIR-stimulated mast cells Marcel Kuhny 1, Thomas Hochdörfer 2, Samatha Mathew 1,2, Michael Huber 1, University Clinic RWTH Aachen, Aachen, NORDR, Germany, 2 Department of Biotechnology, IIT Kharagpur, Kharagpur, India 1

Mast cells (MCs) are sentinels located in tissues proximal to the outside environment. They alert the immune system to invasion of various pathogens and create an inflammatory milieu. Upon activation via the family of Toll-IL-1-receptors (TIRs), MCs produce pro-inflammatory cytokines. In order to provide a response, which is appropriate to the type of pathogen and the local tissue MCs need to modulate their cytokine profile by integrating input from the surrounding milieu. We have previously shown that activation of the PI3K pathway augments the secretion of TNF-a and IL-6 but attenuates the synthesis of IL-1b in TIR-ligand-stimulated MCs [1]. Thus, the PI3K pathway represents a basic mechanism to differentially modulate NF-jB-driven, pro-inflammatory cytokines. We are particularly interested in the molecular mechanism that negatively regulates IL-1b downstream of the PI3K. To study this we employ genetic and pharmacological approaches as well as co-stimulation experiments with stimuli that elicit a robust PI3K activation e.g. antigen, steel factor (SF), or IGF-1. Our results indicate that PI3K-dependent regulation of IL-1b expression is mainly facilitated via Akt-dependent inactivation of the transcription factor FoxO1. Inhibition of Akt augments the TIR-ligand-induced IL-1b synthesis and mimics inhibition of PI3K. In line with this finding, IL-1b production is attenuated when FoxO1 is inhibited. Furthermore, we are interested in the exact PI3K isoforms that are engaged by the TIR receptors themselves or by the co-stimulatory receptors and whether these depend on the Akt-FoxO1 axis to modulate the TIR-induced cytokines in MCs. These results may reveal a basic PI3K-dependent mechanism that allows MCs to bias their cytokine profile according to the (patho-) physiological milieu. This will be of relevance for inflammatory responses in the context of cancer and allergic diseases, amongst others.

Abstract / Cytokine 70 (2014) 28–79

Reference [1] Hochdörfer T, Kuhny M, Zorn CN, Hendriks RW, Vanhaesebroeck B, Bohnacker T, et al.. Activation of the PI3K pathway increases TLR-induced TNF-a and IL-6 but reduces IL-1b production in mast cells. Cell Signal 2011;23(5):866–75. doi:10.1016/j.cellsig.2011.01.012. http://dx.doi.org/10.1016/j.cyto.2014.07.111

105 The role of IFN-c and IL-27 for Th17 plasticity in mouse CNS inflammation

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restores normal JAK/STAT signaling and IFN-c biological activity. Proteomics analysis indicates that abnormal galectin binding to the mutated IFNGR2 subunit is responsible for these defects. Depletion of galectins restores normal IFNGR conformational change and JAK/STAT signaling in patient cells. Our results provide the first positive evidence for the role of raft lipid nanodomains in IFNGR assembly and JAK/STAT signaling by IFN-c in human cells. They also revealed the key role of receptor glycosylation and galectins in this process.

Reference [1] Vogt G et al.. Gains of glycosylation comprise an unexpectedly large group of pathogenic mutations. Nat Genet 2005;37:692–700. http://dx.doi.org/10.1016/j.cyto.2014.07.113

Florian Wanke 1, Sonja Moos 1, André P. Heinen 1, Stephanie Firmenich 1, Werner Müller 2, Ari Waisman 1, Florian C. Kurschus 1, 1 University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, 2 Faculty of Life Sciences, Manchester, UK Introduction: We and others recently showed that IL-17-producing Th17 cells are highly unstable in their phenotype and swiftly upregulate T-bet and Th1-associated cytokines in the inflamed CNS of mice with EAE [1]. This inherent plasticity was recently associated with IL-23, IFN-c or IL-12 signalling on effector T cells [2,3]. Aim: To understand the role of IFN-c and IL-27 signaling for plasticity of Th17 cells in vivo. Methods: We use mice lacking the IFN-c receptor 2 chain specifically in T cells (CD4cre  IFNcR2FL/FL) as well as blocking antibodies for IFN-c and IL-27-p28 and knockout mice for IL-27-EBI3. Further we use IL-17 reporter mice to sort Th17 cells prior adoptive transfer. We use experimental autoimmune encephalomyelitis (EAE) as in vivo CNS inflammation model in which Th17 cells play an important role and in which plasticity of Th17 cells is predominant found in the CNS. Results: Using CD4cre  IFNcR2FL/FL mice we found that IFN-g signaling on T cells has a suppressive role in transfer EAE but it is negligible for Th17 plasticity in vivo. We therefore screened several cytokines in vitro and found that IL-27 signaling has a strong impact on Th17 plasticity. In vitroTh17 cells shifted strongly to Th1 when restimulated with IL-12 and IL-27 even in absence of IFN-c signaling. To investigate the in vivo effects of IL-28 and IFN-c on Th17 plasticity, experiments are currently performed in CD4cre  IFNcR2FL/FL mice in combination with an IL-27-p28 neutralizing monoclonal antibody or the complete IL-27-EBI3 knockout model. Conclusion: Although IFN-c signaling on T cells has a suppressive role in transfer EAE it does not by itself influence Th17 plasticity in the CNS. We suggest that it may act in a redundant fashion with other cytokines such as IL-27 to induce the Th1 shift of Th17 cells under inflammatory conditions.

References [1] Kurschus et al.. Genetic proof for the transient nature of the Th17 phenotype. Euro J Immunol 2010;40:3336–46. [2] Lexberg et al.. IFN-c and IL-12 synergize to convert in vivo generated Th17 into Th1/Th17 cells. Euro J Immunol 2010:3017. [3] Hirota K et al.. Nat Immunol 2011;12:255–63. http://dx.doi.org/10.1016/j.cyto.2014.07.112

106 Gain of glycosylation in MSMD patients reveals the key role of galectins and lipid nanodomains in IFN-c receptor assembly and JAK/STAT signaling Christophe Lamaze 1, Hai-tao He 2, Jean-Laurent Casanova 3, 1 Institut Curie, Paris, France, 2 Immunology, CIML, Marseille, France, 3 St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY, USA Several patients with Mendelian susceptibility to mycobacterial diseases (MSMD) were shown to present a common mutation T168N creating an additional N-glycosylation site in the extracellular domain of the interferon c receptor subunit IFNGR2 [1]. This inherited modification of the IFNGR2 subunit caused a complete lack of gene response to IFN-c, a key cytokine for host defense, and resulted in children death. In patient cells, IFN-c-induced JAK/STAT signaling was fully inhibited. Spot variation fluorescence correlation spectroscopy (SvFCS) in live cells revealed that the IFNGR complex associates with membrane lipid nanodomains of the raft type upon IFN-c stimulation. In contrast, the IFNGR2 T168N mutant did not associate with lipid nanodomains. Bioluminescence resonance energy transfer (BRET) experiments found a lack of IFNGR conformational change induced by IFN-c binding and defects in JAK kinase association with IFNGR complex in patient cells. Removal of the extra glycan

107 STAT3 interacts with Cyclophilin D in cancer cells to regulate the mitochondrial permeability transition pore Jeremy Meier, Ali Raza, Akimitsu Yamada, Yun Dai, Shuang Chen, Steven Grant, Kazuaki Takabe, Andrew Larner, Virginia Commonwealth University, Richmond, VA, United States Reactive oxygen species (ROS) have been closely associated with cellular transformation, and promoting and sustaining tumorigenesis. Elevated ROS levels have been shown to trigger opening of the mitochondrial permeability transition pore (MPTP), which can lead to mitochondrial dysfunction and ultimately, cell death. In cancer cells the MPTP appears to be less susceptible to opening, which may account for their ability to proliferate in a more oxidative environment. It is well established that signal transducer and activator of transcription 3 (STAT3), is constitutively activated in numerous human cancers. Recent studies indicate that STAT3 is also present in the mitochondria where it controls the activity of the electron transport chain, the production of ROS, Ras transformation of cells and growth of breast cancer cells. We now provide evidence that in response to oxidative insult, mitochondrial STAT3 interacts with Cyclophilin D (CypD), the key regulator and activator of the MPTP. Treatment of a variety of cells with hydrogen peroxide (H2O2) induces a rapid cycling of mitochondrial STAT3 where we have observed both its loss and re-accumulation in the mitochondria. We hypothesize that binding of CypD with STAT3 protects against oxidative stress induced cell death in cancer cells by preventing activation of the MPTP. We also provide evidence that mitochondrial STAT3 may play an important role in cancer cell resistance to chemotherapy. Consistent with this possibility, we observe a robust and selective increase in mitochondrial STAT3 levels following chemotherapeutic treatment of cancer cells. While prior reports have focused on the nuclear function of STAT3 in mediating tumorigenesis and therapeutic resistance, these results suggest a previously unappreciated and analogous role of STAT3 in the mitochondria. http://dx.doi.org/10.1016/j.cyto.2014.07.114

108 The role of nucleotide-binding oligomerization domain 1 (Nod1) in cytokine production by macrophages in response to Mycobacterium tuberculosis Jun-young Lee 1, Eun-ha Hwang 1, Dong-jae Kim 1, Sung Jae Shin 2, Jong-Hwan Park 1, Kon-yang University, Daejeon, South Korea, 2 Microbiology, Yon-sei University, Seoul, South Korea 1

Mycobacterium tuberculosis infection is one of the leading causes of death worldwide. Recognition of pathogen by pattern recognition receptors is crucial for the activation of both innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are cytoplasmic receptors which detect unique muropeptide from bacterial peptidoglycan. Although Nod2 is critical for initiation of host immune responses against M. tuberculosis infection, the role of Nod1 remains largely unknown. In this study, we investigated the role of Nod1 on cytokines production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis. We found that pro-inflammatory cytokines such as IL-6, TNF-a, and IL-1b were induced in BMDMs infected with M. tuberculosis, which was not affected by Nod1 deficiency. Activation of NF-jB and MAPKs by M. tuberculosis was also comparable between WT and Nod1-deficient BMDMs. Interestingly, IL-6 and IL-1b production was reduced in Nod1/Nod2-double deficient BMDMs, as compared with Nod2 single deficient cells. Furthermore, when TLRs signaling was inhibited by LPS pretreatment, such cytokines production was diminished in Nod1-deficient BMDMs, as