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This enhancement was abolished by EP2 and EP4 antagonist, suggesting possibleinvolvement of prostaglandins. We then found the PGE2 secretion and COX-2 expres-sion by BMDMs from WSX-1 KO mice was increased compared to control macro-phages in response to LPS. To explore how IL-27/WSX-1 signal regulate COX-2/PGE2

production, murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) wereestablished. mWSX-1-Raw264.7 showed phosphorylation of both STAT1 and STAT3in response to IL-27 and produced less amounts of PGE2 and COX-2 compared toparental Raw264.7. Finally, to examine whether STAT1 and STAT3 downstream ofIL-27R regulate PGE2 and COX-2 production, siRNA knockdown study was performedand siSTAT1, but not siSTAT3, resulted in increased production of PGE2 and COX-2. Inaddition, BMDMs from STAT1 KO mice showed higher COX-2 expression than thosefrom control mice. Collectively, our result indicated that IL-27/WSX-1 regulatedPGE2 secretion via STAT1-COX-2 pathway in macrophages and affected helper T cellsresponse in a PGE2-mediated indirect fashion.

http://dx.doi.org/10.1016/j.cyto.2013.06.230

228MTOR-signaling pathway plays a role in induction of autophagy by type Iinterferons

Hana Schmeisser a, Samuel B. Fey a, Julie Horowitz a, Elizabeth R. Fischer b, Corey A.Balinsky a, Kotaro Miyake a, Joseph Bekisz a, Andrew L. Snow c, Kathryn C. Zoon a,a National Institutes of Health, National Institute of Allergy and Infectious Disease,Cytokine Biology Section, Bethesda, MD, USA, b National Institutes of Health, NationalInstitute of Allergy and Infectious Disease, Rocky Mountain Laboratories, ResearchTechnologies Section, Hamilton, MT, USA, c Uniformed Services University of the HealthSciences, Department of Pharmacology, Bethesda, MD, USA

Autophagy is a stress-induced cellular recycling mechanism that occurs at a basallevel in all eukaryotic cells. Type I interferons are cytokines that have the ability toinduce antiviral, antiproliferative and immunomodulatory activities in cells. Recently,we found that treatment with IFN-alpha2c and IFN-beta induces autophagy in DaudiB cells, starting at 24 h as indicated by an increase of autophagy markers LC3-II, Atg5-Atg12 complexes, and a decrease of p62. Higher levels of LC3-II were also detected48 h post IFN-alpha2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines.The increase in expression of autophagy markers correlated with inhibition ofmTORC1 activity in Daudi cells. Many signaling pathways play a role in regulationof mTORC1 (e.g. PI3K, AKT). Treatment of Daudi and T98G cells with IFN-alpha2c incombination with either rapamycin (mTORC1 inhibitor) or LY294002 (PI3K inhibitor)increased the level of LC3-II, indicating that PI3K/AKT/mTORC1 signaling pathwaymay affect IFN-induced autophagy in Daudi and T98G cells. The role of mTOR and fac-tors upstream of mTOR in Type I IFN-induced autophagy was confirmed by siRNAknockdown experiments. The presence of autophagosomes was shown using trans-mission electron microscopy. In conclusion, our findings demonstrate a novel func-tion of Type I IFN as inducer of autophagy in a variety of cancer cell lines.


229Dengue virus infection and induction of proinflammatory cytokines in dendriticcells is profoundly inhibited by selective carbohydrate-binding agents (CBAs)

Dominique Schols, Marijke Alen, Dana Huskens, Jan Balzarini, Rega Institute forMedical Research, Department of Microbiology and Immunology, University of Leuven, BE-3000 Leuven, Belgium

We have demonstrated that several carbohydrate-binding agents (CBAs), such asthe plant lectins Hippeastrum hybrid (HHA) and Galanthus nivalis (GNA), have a con-sistent and serotype-independent anti-dengue virus (DENV) activity in DC-SIGNexpressing cells. Because DCs in the skin are the first target cells of this virus we eval-uated monocyte-derived dendritic cells (MDDC) generated from human blood.Remarkably, the potency of the CBAs against DENV in MDDC cultures was signifi-cantly higher (up to 100-fold) than in Raji/DC-SIGN+ cells. The CBAs were also ableto completely prevent the DENV-induction of cellular activation and differentiationmarkers, as measured by flow cytometry.

MDDC were also infected with DENV and then the supernatant was analyzed bythe Bio-Plex human cytokine 27-plex immunoassay for the production of IFN-c, IFN-a, RANTES, MIP-1a, MIP-1b and TNF-a, which all have been demonstrated to play arole in the DENV pathogenesis. We observed an enhanced production of all thesecytokines/chemokines in the supernatant analyzed 48 h after DENV infection. Addi-tion of 10 lg/ml of HHA or GNA profoundly inhibited the induction of RANTES,MIP-1a, MIP-1b, IFN-a and TNF-a, and to a lesser extent the production of IFN-c.

The cytokines/chemokines production was also evaluated in uninfected MDDC incu-bated with CBAs. These agents, by themselves, had no effect on the cytokine/chemo-kine production profile compared to the uninfected MDDC. Thus, we demonstratedthat the CBAs, in addition to their consistent antiviral activity, inhibited the secretionof several pro-inflammatory cytokines and chemokines important in the DENV path-ogenesis, rendering CBAs as attractive protein lead structures to combat DENV trans-mission and infection.


230Unraveling the role of Adam17 in IL-6 trans-signaling

Jeanette Schwarz a, Isabell Yan b, Olga Will c, Paul Saftig a, Stefan-Rose-John a, Hans-Willi Mittrücker b, Athena Chalaris a, a Institute for Biochemistry, CAU, Kiel, Germany,b Section Biomedical Imaging, UKSH, Kiel, Germany, c Institute for Immunology, UKE,Hamburg, Germany

IL-6 is a key regulator of immune responses after bacterial infection. IL-6 signal-ing is mediated via the receptor subunits IL-6R and gp130. The signal transducingsubunit gp130 is ubiquitously expressed whereas IL-6R expression is restricted tohepatocytes and some leukocytes. The IL-6R can be cleaved from the cell surface byADAM proteases. The soluble form of the IL-6R (sIL-6R) has the same IL-6 bindingaffinity as the membrane-bound receptor. The resulting IL-6/sIL-6R complex activatescells, which only express gp130 on their cell surface, a process called trans-signaling.Thus, IL-6-transsignaling following IL-6R proteolysis essentially renders all cells of thebody responsive to IL-6. The proteolytic web controlling IL-6R shedding in vivo underpathophysiological conditions is poorly characterized. Recently, it was demonstratedthat human IL-6R is a substrate for the protease ADAM17 in contrast to the murineADAM17, which is mainly cleaved by ADAM10. However, these data were raised incell culture systems. We analyzed IL-6R shedding in vivo on leukocytes after infectionwith Listeria monocytogenes or LPS-challenge of ADAM17 and ADAM10-deficientmice. ADAM17 and ADAM10 knockout mice are not viable. For this reason we gener-ated a hypomorphic ADAM17 mouse model which expresses only 5% of the normalADAM17 levels. Additionally we use conditional ADAM10 mice with ADAM10 deletedeither in monocytes or Tcells. Listeria infection led to massive shedding of the IL-6R onTcells and inflammatory monocytes. Further we could demonstrate that Listeria-induced IL-6R cleavage is abrogated in hypomorphic ADAM17ex/ex mice. Additionallywe can show that serum levels of sIL-6R are elevated after LPS challenge of wildtypebut not ADAM17ex/ex mice. These results strongly suggest that ADAM17 is the mainIL-6R sheddase under pathophysiological conditions. In future experiments we willevaluate the contribution of the protease ADAM10 in mediating IL-6R shedding touncover the proteolytic steps involved in IL-6 trans-signaling.


231Type I interferon controls gammaherpesvirus latency

Johannes Schwerk a, Kendra Bussey b, Stefan Lienenklaus c, Siegfried Weiss c, MelanieBrinkmann b, Hansjörg Hauser a, Mario Köster a, a Department of Gene Regulation andDifferentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany,b Research Group of Viral Immune Modulation, Helmholtz Centre for Infection Research,Braunschweig, Germany, c Department of Molecular Immunology, Helmholtz Centre forInfection Research, Braunschweig, Germany

Murine gammaherpesvirus 68 (MHV-68) serves as a murine model for the humangammaherpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and EpsteinBarr virus (EBV). Like all herpesviruses, MHV-68 establishes latency with spontane-ously recurring reactivation events. While it is known that type I interferon (IFN) isessential for survival of acute MHV-68 infection, its role during latency and reactivationis unclear. To define the contribution of the type I IFN system in control of MHV-68 acuteinfection and latency in vivo, we employed bioluminescent reporter mice (mx2-Lucifer-ase and ifnb-Luciferase) to visualize IFN-b induction and type I IFN action. Splenocytesfrom latently infected wild type and inducible type I IFN receptor knockout (IFNARflox/

flox) mice were adoptively transferred into wild type and IFNAR-deficient (IFNAR�/�)mice. Survival, virus burden and reporter gene induction was determined as a measurefor the extent of in vivo reactivation. Our data show that (i) acute MHV-68 infectionevokes an IFN response in vivo. (ii) MHV-68 latency is controlled by type I IFN signaling.(iii) An IFN response is induced upon MHV-68 reactivation in vivo. (iv) IFN controls thefrequency of reactivation by acting on latently infected cells directly, thus suppressingreactivation. Overall, our findings demonstrate a substantial impact of the type I IFNsystem on the control of MHV-68 latency and upon its reactivation.

Abstract / Cytokine 63 (2013) 243–314 297

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