SSA Autoantibodies Specific for the RING-domain of Ro52 Inhibit E3 Ligase Activity and Polyubiquitination

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OR.56. Anti-Ro/SSA Autoantibodies Specific for the RING-domain of Ro52 Inhibit E3 Ligase Activity and Polyubiquitination Alexander Espinosa, 1 Janosch Hennig,2 Aurélie Ambrosi,1 Filippa Nyberg,1 Maria Sunnerhagen,2 Marie Wahren-Herlenius.1 1Karolinska Institutet, Stockholm, 2 Sweden; Linköping University, Linköping, Sweden Patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) commonly have autoantibodies to the intracellular autoantigen Ro52. Ro52 belongs to the tripartite motif (TRIM) protein family, and was recently identified as a cytoplasmic E3 ubiquitin ligase regulating proliferation and cell death. Although the presence of antiRo52 autoantibodies is used clinically as a diagnostic marker, it is not known whether they directly affect the function of Ro52. To address this issue, we first defined conditions under which Ro52 generates polyubiquitin chains. Using Ro52 mutants, we found that the E3 ligase activity of Ro52 depends on the RING domain and that the coiled-coil and B30.2 domains of the protein are dispensable for ubiquitination. We then screened sera from N50 patients with SS or SLE for anti-Ro52 antibodies and by epitope mapping determined the presence of antibodies to the different domains of Ro52. Sera with different binding profiles were analyzed for inhibition of Ro52 E3 ligase activity. Anti-Ro52 positive sera with autoantibodies binding the RING domain inhibited the E3 ligase activity of Ro52, while anti-Ro52 antibodies against other domains of the protein did not inhibit ubiquitination. The ability of RING-specific patient autoantibodies to inhibit the function of Ro52 was confirmed by using antibodies affinity purified against the RING domain in polyubiquitination assays in vitro. Our data indicate that anti-Ro52 autoantibodies may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52 mediated ubiquitination. doi:10.1016/j.clim.2008.03.062

OR.57. Deficiency of miR-146 Contributes to Abnormal Activation of Type I Interferon Pathway in Systemic Lupus Erythematosus Nan Shen. Shanghai Ren Ji Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by activation of the type I interferon (IFN) pathway. MicroRNAs (miRNAs) are recently identified as the regulators that post-transcriptionally repress target gene expression and involved in shaping of immune responses .Growing evidence has demonstrated the etiological role of miRNAs in various diseases, while it remains elusive whether aberrations in miRNAs contribute to autoimmune disease pathogenesis. Here we test the hypothesis that dysregulation of miRNAs contributes to lupus disease pathways. A miRNA signature of SLE was identified by profiling the expression of 319 miRNAs in blood samples of lupus patients using Taqman

Abstracts assay, which was composed of 17 up-regulated and 13 down-regulated miRNAs. Notably included was miR-146a, which was a negative regulator of innate immunity. Further exploration in enlarged samples revealed underexpression of miR-146a negatively correlated with clinical disease activity and with the activation of type I IFN pathway in lupus patients. To intersect this association we found that ectopic expression of miR-146a reduced, while knockdown of endogenous miR-146 augmented the induction of type I IFNs in primary immune cells. Furthermore, miR-146a repressed the coordinate activation downstream of type I IFNs in vitro and in vivo. To gain insight into the regulatory mechanism we predicted using bioinformatic tools and verified that miR-146 could target key components in the signaling cascade upstream and downstream of type I IFN, including IFN regulatory factor 5 and signal transducer and activator of transcription. These data suggested that deficiency of miR-146 contribute to alterations of the type I IFN pathway in lupus patients and provided potential novel strategies for therapeutic intervention. doi:10.1016/j.clim.2008.03.063

OR.58. Lipids as Autoantigens and Therapeutic Immune Modulators in Autoimmune Demyelination Peggy Ho, Jennifer Kanter, Amanda Johnson, Lawrence Steinman, William Robinson. Stanford University, Stanford, CA The targets of the autoimmune response in multiple sclerosis (MS) remain poorly understood. The myelin sheath is composed of thirty percent proteins and seventy percent lipids, and we hypothesize that lipids could represent important autoantigen targets in MS. We profiled lipid reactive antibodies in spinal fluid from individuals with MS versus other neurological disease controls using our lipid array technology. Although administration of certain lipids exacerbated the experimental autoimmune encephalomyelitis (EAE) model for MS, unexpectedly, some array-identified lipids prevented induction of and treated established EAE. 1-Palmitoyl-2-Glutaroyl-sn-Glycero-3-Phosphocholine (PGPC), an oxidized form of phosphatidyl-choline, was also able to treat ongoing EAE. To understand the mechanism of action, we tested derivatives of PGPC: lipids with modified polar head groups or modified hydrophobic side chains. We found that an accessible phosphate head group, rather than a specific hydrophobic side chain, was necessary for binding to spinal fluid antibodies from individuals with MS. A subset of PGPC derivatives were also able to effectively treat ongoing EAE. In vitro studies have shown that PGPC and these derivatives can specifically bind to T cells and suppress proliferation and the production of several proinflammatory cytokines including interferon-gamma, tumor necrosis factor, and IL-6. Thus, antibodies present in the MS spinal fluid may not only target degrading myelin, but may also serve as negative regulators of inflammation. Further, the lipid targets of these antibodies may prove to be therapeutic. We are currently assessing the abilities of these lipids to act as brakes to the immune system through