actions, which modulates obesity-induced inflammation and insulin resistance inmice and humans.

http://dx.doi.org/10.1016/j.cyto.2013.06.015

13Effects of temperature and pH on IL-1 stability

Adriana Barba-Montoya a,b, Stanislava Panova a,b, Emmanuel Pinteaux b, Alexander P.Golovanov a,b, a Manchester Institute of Biotechnology, University of Manchester,Manchester, UK, b Faculty of Life Sciences, University of Manchester, Manchester, UK

Interleukin-1 (IL-1) is a pro-inflammatory cytokine that plays an important role ininflammatory responses to injury and infection, both, systemically and within thecentral nervous system. There are two IL-1 ligands, IL-1a and IL-1b, which bind tothe interleukin 1 receptor type I (IL-1RI) activating multiple pathways that lead tothe expression of acute phase and pro-inflammatory proteins. Although IL-1a andIL-1b are different at their amino acid sequence (sharing only 26% homology), theyare structurally similar (both protein structures are b-barrel comprised by b-sheets),exert their actions through IL-1RI and are thought to exert similar biological activity.However, in recent years, some differences of action have been observed. Briefly, ithas been suggested that IL-1b is more potent when acting in the brain, whereas IL-1a has been proposed to be more potent when acting systemically. Despite consider-able research efforts, molecular mechanisms responsible for the observed differentialeffects remain unclear. The aim of this work is to carry out a comparative study of theeffects of temperature and pH on the biophysical properties and bioactivities of IL-1aand IL-1b. The thermal stability of both ligands has been investigated using onedimensional [1]H Nuclear Magnetic Resonance Spectroscopy, circular dichroism andfluorescence and all are consistent in that IL-1a and IL-1b retain their folded confor-mation at increased temperature. Additionally, we found that pH also has a significantinfluence in their conformation. In this study, we characterized the biophysical prop-erties and bioactivities of IL-1a and IL-1b under different conditions.

http://dx.doi.org/10.1016/j.cyto.2013.06.016

14Inhibition of P53 by Kaposi’s sarcoma-associated herpesvirus-encoded VIRF3:From transcriptional repression to proteasome-mediated degradation

Petra Baresova, Jana Musilova, Barbora Lubyova, Charles University, First Faculty ofMedicine, Institute of Immunology and Microbiology, Prague, Czech Republic

Kaposi’s sarcoma associated herpesvirus (KSHV/HHV8) is a human herpesvirusassociated with Kaposi’s sarcoma, Non-Hodgkin B-cell lymphoma and Castleman’sdisease. The KSHV genome encodes a cluster of genes known as viral interferon reg-ulatory factors (vIRFs) that were shown to suppress antiviral host immune responsesand provide a growth advantage to infected cells. Our research is focused on under-standing the molecular mechanism by which viral IRF-3 (vIRF-3) can modify the func-tion and protein stability of p53 tumor suppressor. Specifically, we investigated therole of vIRF-3 in (i) transcription repression of genes regulated by p53, (ii) ubiquitina-tion and (iii) subsequent proteasome-mediated degradation of p53. Our data gener-ated by the GST pull-down assay and co-immunoprecipitation indicate that vIRF-3directly interacts with the DNA-binding domain (DBD) (residues 100–292) of p53.Expression of vIRF-3 interfered with binding of p53 to the wild type p21 promoter,formation of p53 tetramers as well as p53-mediated activation of endogenous p21transcription. In addition to direct inhibition of p53 transcription activity, vIRF-3 tar-geted p53 for 26S proteasome-mediated degradation. Expression of vIRF-3 resulted inreduced p53 protein levels, increased p53 polyubiquitination and shortened p53 half-life. The negative effect of vIRF-3 on p53 signaling pathway was also confirmed inKSHV-positive B lymphoma cell line, BC-3, using the method of RNA interference tospecifically knock-down the expression of vIRF-3. Cells with reduced levels of vIRF-3 displayed extended p53 half-life and transcription activity when compared to con-trol cells. P53 was previously shown to exhibit impaired expression and function inmajority of human cancers. Therefore, delineating the precise mechanism throughwhich vIRF-3 modifies the stability and function of p53 will further define the pro-cesses of KSHV pathogenesis and may have implications in future therapy of KSHV-related tumors. This work was supported by grant from the Grant Agency of the CzechRepublic (Project 204/09/0773).

http://dx.doi.org/10.1016/j.cyto.2013.06.017

15An intron of IRF-8 harbors myeloid lineage specific regulatory element that isregulated by dynamic chromatin architecture

Ofer Barnea-Yizhar, Nitsan Fourier, Aviva Azriel, Sigal Ram, Ben-Zion Levi, Departmentof Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa,Israel

Interferon Regulatory Factor-8 (IRF-8) is an IFN-gamma responsive gene thatserves as a key factor in the hierarchical differentiation of bone marrow derived cellstowards monocyte/macrophage lineage. In attempt to identify the molecular mech-anisms leading to this lineage restricted expression of IRF-8, we are using IRF-8 Bac-terial Artificial Chromosome (BAC) reporter constructs transfected either topermissive macrophage cell line or to restrictive non-hematopoietic fibroblast cellline. We present data showing that the control of lineage specific expression ofIRF-8 is confined to an intronic segment. Deletion of this intron leads to loss of line-age specific expression of BAC IRF-8 reporter. Interestingly, this IRF-8 intron exhibitsevolutionary conserved regions, which are not typical to ‘‘benign” intronicsequences. To gain molecular insight to this restricted expression, we compared his-tone modifications and nucleosome occupancy in this intron between expressionpermissive and restrictive cell lines. For that purpose, we employed ChromatinImmuno-Precipitation (ChIP) and Formaldehyde-Assisted Isolation of Regulatory Ele-ments (FAIRE) analyses. The ChIP data revealed subtle differences in the overall his-tone modification profile (”histone code”). The FAIRE analysis pointed to a significantnucleosome density difference between permissive and restrictive cell lines. Accord-ingly, IRF-8 repression in a restrictive myeloid progenitor cell line was partially alle-viated upon treatment with histone methyltransferase inhibitors. Altogether, ourdata clearly suggests that an intron of IRF-8 harbors myeloid lineage specific regula-tory element that is regulated by dynamic chromatin architecture. Taken together, itis most probable that this intron serves as nucleation core for chromatin condensa-tion in restrictive cells.

http://dx.doi.org/10.1016/j.cyto.2013.06.018

16MIR-17 � 92 promotes T follicular helper cell differentiation and represses sub-set-inappropriate gene expression

Dirk Baumjohann a, Robin Kageyama a, Jonathan M. Clingan b,c, Malika M. Morar d,Sana Patel a, Dimitri de Kouchkovsky d, Oliver Bannard e, Jeffrey A. Bluestone d,f,Mehrdad Matloubian b, K Mark Ansel a,1, Lukas T. Jeker d,f,1, a Department ofMicrobiology & Immunology, Sandler Asthma Basic Research Center, University ofCalifornia San Francisco, San Francisco, CA, USA, b Division of Rheumatology, Departmentof Medicine, and Rosalind Russell Medical Research Center for Arthritis University ofCalifornia San Francisco, San Francisco, CA, USA, c Graduate Program in BiomedicalSciences, University of California, San Francisco, CA, USA, d Diabetes Center andDepartment of Medicine, University of California San Francisco, San Francisco, CA, USA,e Department of Microbiology & Immunology, Howard Hughes Medical Institute,University of California, San Francisco, CA, USA, f Department of Pathology, University ofCalifornia San Francisco, San Francisco, CA, USA

T follicular helper (Tfh) cells are the prototypic T helper cell subset specializedto enable B cells to form germinal centers (GCs) and produce high affinity antibod-ies. Tfh cell differentiation begins very early in the immune response, coincidingwith rapid proliferation that expands the pool of responding cells. Here we showthat T cells lacking all microRNAs completely failed to differentiate into Tfh cellsin vivo. MicroRNA-deficient T cells exhibited impaired proliferation as expected,but they also displayed an independent defect in the induction of the functionallycritical Tfh cell proteins BCL6 and CXCR5. We found that the miR-17 � 92 clusterwas required for robust Tfh cell differentiation and function in a cell intrinsic man-ner that also occurred regardless of changes in proliferation. Conversely, miR-17 � 92 overexpression in T cells promoted Tfh cell differentiation and GC B cellinduction. Early in the response to protein antigen, these effects were partiallydependent on altered expression of the miR-17 � 92 target gene Pten, a knowninhibitor of Tfh cell differentiation. In a viral infection model, Tfh cells dependedon the miR-17 � 92 cluster to restrain the expression of Tfh subset-inappropriategenes, including Ccr6, Il1r2, Il1r1, and the cytokine Il22. Conserved regions in theRAR-related orphan receptor alpha (Rora) 3’ UTR were directly targeted by all 4miRNA families in the cluster. Genetically removing one Rora allele significantlyrescued the inappropriate gene expression signature in miR-17 � 92-deficient Tfh

1 Equal contribution

246 Abstract / Cytokine 63 (2013) 243–314