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Abstract / Cytokine 70 (2014) 28–79

etic stem cell dysfunction, which is corrected in Bak/Bax/Caspase-9 deficient mice. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

http://dx.doi.org/10.1016/j.cyto.2014.07.204

198 Inflammation and nitric oxide synthase 2 in driving poor prognosis in era(-) breast cancer David Wink, National Cancer Institute, Bethesda, MD, United States Inflammation generates reactive chemical species that induce conditions of oxidative nitrosative stress as emerged as factor in poor outcome of many cancers. Our recent finding show that in the inflammatory protein inducible nitric oxide synthase (iNOS) is a strong predictor of poor outcome in ER(-) patients (Glynn et al. JCI 2010). Furthermore 46 genes, of which 23 were associated with basal like breast cancer, were elevated when iNOS high. In vitro studies using ER(-) cell lines showed that fluxes of nitric oxide (NO) delivered by NO donors surprising mimic this relationship in the patient cohort. Using this model, we show that NO through nitrosation of key thiols and non heme metals at different specific concentration stimulate pro-oncogenic mechanisms such as AKT, ERK, NFkB, AP-1, and HIF-1a that lead to increase of metastatic and cancer stem cells proteins as well as pro-angiogenic and immunosuppressive factors. In addition, we show that tumor suppressor gene BRCA1 and PP2A are inhibited by these NO levels. In the induction of NOS2 was found to dependent inflammatory mediators IFNg, IL6, PGE2, TNFa, and TLR4 agonist (Heinecke et al., PNAS 2014). Since NOS2 and NO also induce these factors, a critical feed-forward autocrine loop is formed that drives poor outcome. These finding provide a novel model that shows how NO can drive numerous inflammatory mechanism that leads to a more aggressive landscape. http://dx.doi.org/10.1016/j.cyto.2014.07.205

199 Cytokine profiles of chronic fatigue syndrome and multiple sclerosis patients Naomi Wong, Ekua Brenu, Sharni Hardcastle, Samantha Johnston, Thao Nguyen, Teilah Huth, Ally Hawthorn, Rachel Passmore, Sandra Ramos, Ali Salajegheh, Simon Broadley, Don Staines, Sonya Marshall-Gradisnik, National Centre for Neuroimmunology and Emerging Diseases, School of Medical Sciences, Griffith University, Gold Coast, Qld, Australia Aims: Chronic Fatigue Syndrome (CFS) and Multiple Sclerosis (MS) are both disorders with severe neuroimmune symptoms, including cognitive impairment, immune dysfunction and abnormal cytokine expression. The purpose of this study was to assess the T helper (Th) 1, Th2 and Th17 cytokine profiles of CFS and MS patients. Methods: This study measured the cytokine profiles of CFS patients (n = 16; mean age 49.88, SD 9.542), MS patients (n = 16; mean age 52.75, SD 12.809) and healthy controls (n = 16; mean age 50.06, SD 11.846). The diagnostic selection method used to identify CFS patients was the International Consensus Criteria. Cytokines were measured from serum using a Bio-Plex ProTM kit for Th1 (IFN-c, TNF-a), Th2 (IL-4, IL-6, IL-10, IL-13) and Th17 (IL-17) cell cytokines. Results: When the three groups were compared, it was found that TNF-a (p = 0.011, p = 0.012), IL-4 (p = 0.000, p = 0.000), IL-6 (p = 0.039, p = 0.031), IL-13 (p = 0.000, p = 0.000) and IL-17 (p = 0.004, p = 0.038) were all significantly higher in MS patients compared with CFS patients and controls respectively. However, in the MS patients and CFS patients, serum levels of IL-13 and IFN-c were significantly higher (p 6 0.001) compared with the controls. IFN-c was significantly different between MS and CFS (p = 0.001), with MS exhibiting higher IFN-c serum levels. Conclusion: Cytokines patterns supported both Th1 and Th2 cytokine profiles in CFS and MS. Similarities in the cytokine profiles of MS and CFS, combined with the already acknowledged similarities in immune cell function and symptoms, suggests a neuroimmune pathology for CFS with parallels to that of MS. Additional studies into the cytokine profiles of both CFS and MS should be conducted, with larger sample sizes, to further expand the understanding of the pathologies of both disorders. http://dx.doi.org/10.1016/j.cyto.2014.07.206

200 Regulation of G-CSF and its receptor in skeletal muscle in response to exerciseinduced damage in vivo and LPS stimulation in vitro Craig R. Wright 1, Erin L. Brown 1, Paul A. Della Gatta 1, Ioannis G. Fatouros 2,3, Leonidas G. Karagounis 4,5, Gerasimos Terzis 6, Gerasimos Matsorakos 7, Yannis Michailidis 2, Dimitris Mandalidis 6, Kontantinos Spengos 8, Athanasios Chatzinikolaou 2, Spiros Methenitis 6, Dimitrios Draganidis 2, Athanasios Z. Jamurtas 3,5, Aaron P. Russell 1, 1 Centre for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria, Australia, 2 Department of Physical Education and Sport Sciences, Democritus University of Thrace, Komotini, Greece, 3 The Institute of Human Performance and Rehabilitation, Center for Research and Technology–Thessaly, Trikala, Greece, 4 Nestlé Research Center, Nestec Ltd, Lausanne, Switzerland, 5 School of Physical Education and Sport Sciences, University of Thessaly, Karies, Trikala, Greece, 6 Athletics Laboratory, School of Physical Education and Sports Science, University of Athens, Athens, Greece, 7 Endocrine Unit, Second Department of Obstetrics and Gynecology, Athens University Medical School, Athens, Greece, 8 The 1st Department of Neurology, University of Athens, School of Medicine, Eginition Hospital, Athens, Greece The cytokine granulocyte-colony stimulating factor (G-CSF) binds to its receptor (G-CSFR) to stimulate haematopoietic stem cell mobilization, myelopoiesis and the production and activation of neutrophils. G-CSF is increased in the circulation in response to exercise-induced muscle damage and the G-CSF receptor (G-CSFR) has recently been identified in skeletal muscle and muscle cells [1]. While G-CSF/G-CSFR activation mediates pro- and anti-inflammatory responses, our understanding of their role and regulation in skeletal muscle is limited. The aim of this study was to investigate in vitro and in vivo the role and regulation of G-CSF and the G-CSFR in skeletal muscle under conditions of muscle inflammation and damage. Firstly, C2C12 myotubes were treated with G-CSF to determine if it modulates the inflammatory response induced by lipopolysaccharide (LPS). Secondly, we measured the regulation of G-CSF and its receptor following eccentric exercise-induced muscle damage and investigated if their expression levels were redox-dependent by administering the antioxidant N-AcetylCysteine. LPS stimulation of C2C12 myotubes resulted in G-CSF production. The addition of G-CSF following LPS stimulation of C2C12 myotubes increased IL-6 mRNA and cytokine release into the media. Following eccentric exercise-induced muscle damage in humans G-CSF levels were either marginally increased in circulation or remain unaltered in skeletal muscle. Similarly, G-CSFR levels remained unchanged in response to damaging exercise while the G-CSF/G-CSFR response was not redox sensitive. Collectively, these findings suggest that G-CSF may cooperate with IL-6 and potentially promote muscle regeneration in vitro, whereas in vivo aseptic inflammation induced by exercise did not change G-CSF and G-CSFR responses. These observations suggest that different models of inflammation produce a different G-CSF responses.

Reference [1] Wright CR, Brown EL, Della Gatta PA, Ward AC, Lynch GS, Russell AP. G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle. Front Physiol 2014;5:175. http://dx.doi.org/10.1016/j.cyto.2014.07.207

201 Casine kinase II is a novel regulator of TBK1/IRF3 and type I interferon induction by various innate receptors Hui Xiao, Min Du, Jinhua Liu, Xia Chen, Jin Zhong, Yu Xiang, Institut Pasteur of Shanghai, Shanghai, China Upon sensing the presence of viral nucleic acids, multiple pattern recognition receptors (PRRs) can trigger the activation of TBK1 and IRF3, eliciting the induction of type I interferon, whereby initiate host defense mechanisms against invading viruses. To better understand the molecular mechanisms underlying the activation of TBK1 and IRF3 by PRR signals, we sought to identify novel protein kinases involved in type I interferon induction. By screening libraries of small molecular inhibitors and shRNAs targeting protein kinases, we identified serine/threonine kinase CK2 as a novel regulator of type I interferon induction in various PRR signals. Inhibition of CK2 activity or knocking-down of CK2 expression resulted in hyperactivation of TBK1 and IRF3 upon the activation of TLR3/4 as well as cytosolic RNA and DNA sensors by their synthetic ligands or RNA/DNA viruses. Moreover, overexpression of the wild-type but not the kinase-inactive mutant of CK2 attenuated type I interferon

Abstract / Cytokine 70 (2014) 28–79 induction upon viral infection, further indicating that its kinase activity is required for tamping IFNs induction. Strikingly, blockade of CK2 activity alone was sufficient to activate TBK1 and IRF3, resulting in type I IFN induction in the absence of PRR stimuli. More importantly, blocking CK2 activity was able to efficiently suppress the infection and replication of VSV and HCV, suggesting a new strategy to overcome viral immune evasion mechanisms which target signal molecules upstream of TBK1. Mechanistically, CK2 regulates the activation of TBK1 and IRF3 through its substrate protein phosphatase PP2A. Indeed, PP2A, but not CK2 forms complexes with TBK1 and IRF3, and was also able to diminish TBK1- and IRF3-induced IFN exprssion. Taken together, our results not only identify CK2 as a novel regulator of TBK1 and IRF3 in PRR signals, but also reveal a new strategy to contain viruses which are capable of interfering type I interferon induction by PRRs.

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However, MMP-9 siRNA had greater effects, compared to MMP-2 siRNA, on promoting apoptosis and suppressing RASF viability, migration and invasion. Suppression/ inhibition of MMP-9 also decreased the production of IL-1b, IL-6, IL-8 and TNF-a, inactivated nuclear factor (NF)-jB, ERK and JNK and suppressed RASF-mediated cartilage degradation. In contrast, Suppression/inhibition of MMP-2 stimulated TNF-a and IL-17 secretion and activated NF-jB, while rMMP-2 inactivated NF-jB and suppressed RASF-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results. Conclusions: Endogenous MMP-2 or MMP-9 contribute to RASF survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RASF-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RASF may directly contribute to joint destruction in RA.

http://dx.doi.org/10.1016/j.cyto.2014.07.208 http://dx.doi.org/10.1016/j.cyto.2014.07.210

202 Interferon regulator factor (IRF) 8 regulates the microglial response to neuronal injury Rui Dan Xie 1, Nàdia Villacampa 2, Beatriz Almolda 2, Berta González 2, Bernardo Castellano 2, Iain L. Campbell 1,2, 1 School of Molecular Bioscience, University of Sydney, Camperdown, NSW, Australia, 2 Medical Histology, Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain

204 Regulatory effect of interleukin-4 in the innate immune and inflammatory responses to Rhodococcus aurantiacus infection in mice Min Yi, Department of Advanced Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-c and is involved in myeloid cell differentiation. We have previously identified IRF8 to be a constitutive and IFN-c-stimulated nuclear factor that regulates the homeostatic properties of microglia. Our study aimed to determine the role of IRF8 in the microglial response to sterile neuronal injury. Facial nerve axotomy (FNA) was performed in wildtype (WT) and IRF8 / (KO) mice and the brains removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) prior to retrieval of the brain. Changes in the facial nucleus (FN) were examined by immunohistochemistry and histochemistry. In brains from IRF8KO mice, nucleoside diphosphatase (NDPase) hisotchemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned FN peaking at day 7 and was accompanied by dense staining for Iba1, lectin, NDPase and CD11b. By contrast, in IRF8KO mice, the microglial response was markedly attenuated with little staining for Iba1, while the density of staining for lectin and NDPase was reduced significantly. The attenuated microglial response in IRF8KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation as monitored by BrdU, phosphohistone 3 and Ki67-positive cells. Furthermore, a decrease in PU.1-positive cells was observed in the FN of IRF8KO mice compared with WT. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absences of IRF8 in the axotomised FN. Quantitative analysis showed that in IRF8KO mice, the degeneration of axotomised motor neurons was significantly increased. These studies extend on our previous finding that IRF8 is a key homeostatic transcription regulator of microglial cell function in the healthy brain, and moreover, has a crucial role in the regulating the response to microglia to neuronal injury.

Aim: Inoculation of wild-type (WT) mice with gram-positive bacterium Rhodococcus aurantiacus induces Th1-type granulomatous inflammation, in which interleukin (IL)-4 promotes the regression of granulomas during the late phase of infection. As IL-4 plays diverse roles in disease pathogenesis, we investigated the effect of IL-4 in the innate response to this bacterium infection by using IL-4-deficient mice. Methods: After intravenous inoculation with 1  108 CFU of bacteria, mouse survival rates, bacteria load in organs, local and systemic cytokine production, and morphological changes in liver were assessed. Following stimulation with heat-killed R. aurantiacus, cytokine production in mouse peritoneal macrophages was also examined. Results: Compared to WT mice, IL-4-deficient mice showed the decreased production of TNF-a and IL-6 in the spleen, liver and blood during the early phase of infection, as well as low bacterial load in the liver and improvement in survival rate. IL-4deficient mice also showed diminished IL-10 secretion in the spleen and blood, whereas their hepatic IL-10 levels were similar to those observed in WT mice, which were concomitant with the augmented hepatic IFN-c production. Moreover, histological studies revealed reduction in hepatic granuloma formation at day 14 post-infection in IL-4-deficient mice. Upon stimulation with heat-killed R. aurantiacus, macrophages from IL-4-deficient mice showed lower expression of TNF-a, IL-6, and IL-10 at both the gene and protein levels than WT mouse cells. Conclusions: These findings indicate that IL-4 deficit attenuates cytokine responses in macrophages but augments the IFN-c response during the initial stage of R. aurantiacus infection, which together contribute to amelioration of mouse survival, rapid bacterial elimination in liver at the early phase, and reduction of granulomatous inflammation. We previously demonstrated that the early IFN-c response to R. aurantiacus is induced in mouse natural killer cells. This study thus identifies the essential effect of IL-4 on regulating the activation and function of immunocompetent cells, including macrophages and natural killer cells, during the initial phase of systemic R. aurantiacus infection.

http://dx.doi.org/10.1016/j.cyto.2014.07.209

http://dx.doi.org/10.1016/j.cyto.2014.07.211

203 Endogenous matrix metalloproteinase (MMP)-9, and not MMP-2, promotes rheumatoid synovial fibroblast survival, inflammation and cartilage degradation

205 A role for protein kinase R in regulating NLRP3 inflammasome assembly

Meilang Xue, Kelly McKelvey, Kaitlin Shen, Lyn March, Christopher J. Jackson, University of Sydney, St. Leonards, NSW, Australia Aim: To investigate the effect of endogenous matrix metalloproteinase (MMP)-2 and MMP-9 on the invasive characteristics of rheumatoid synovial fibroblasts (SF). Methods: SF isolated from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were treated with MMP siRNAs, inhibitors and recombinant (r) proteins or TNFa, with or without cartilage explants. Cell viability and proliferation were measured by MTT and BrdU proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelectTM invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; inflammatory mediators and MMPs by ELISA, western blot and zymography. Results: MMP-2 was expressed by both OASF and RASF, whereas only RASF expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RASF proliferation equally.

Howard C.H. Yim 1,2, Liang Yu 3, Die Wang 1, Arindam Chakrabarti 4, Robert Silverman 4, Bryan Williams 1,2, Anthony Sadler 1,2, 1 Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria, Australia, 2 Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia, 3 Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia, 4 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, USA Aim: NLR family pyrin domain containing-3 (NLRP3) has been associated with inflammatory disorders. In response to exogenous and endogenous molecules, NLRP3 assemble an inflammasome containing the apoptosis-associated speck like protein containing a CARD (ASC) for activating caspase-1, inducing pyroptosis and processing interleukin (IL)-1b and IL-18. A recent model proposes that the assembly of the inflammasome is dependent on the cytoskeleton. As the protein kinase R (PKR) has been shown to regulate actin dynamics, we examined whether PKR regulates inflammasome activity.