Search

Background Despite enormous efforts to develop new therapeutic strategies for treatment of rheumatoid arthritis (RA), the large number of non responding patients to currently available drugs underlies the unmet need to identify new therapeutic targets. Certain CD4+T cell subsets, especially those polarized toward the T helper (Th) subsets Th1 andTh17, have been shown to be major drivers of inflammation in patients with RA. The expression of their key transcription factors is controlled by histone modifications which includes acetylation of lysine residues mediated by histone deacetylases (HDAC). Indeed, pan HDAC inhibitors have been shown to be a potential therapeutic strategy. However, major side effects limited the clinical use and underline the need of more specific HDAC inhibitors. Objectives We addressed the individual role of HDAC1 on the development of collagen-induced arthritis model (CIA), which partially reflects human RA. Methods Mice with a T cell specific deletion of HDAC1 (HDAC1 cKO) were generated by using the CD4Cre/LoxP system. At week 8 of age arthritis was induced in wild type (WT) and HDAC1 cKO mice by immunizing with chicken collagen II (CII), emulsified in complete Freund’s adjuvant. After 21 days mice received a booster injection of CII. The animals were 3 times per week scored for paw swelling and grip strength. Anti-CII antibody levels were determined by ELISA. Various cell subsets, including Th cells, where detected in the blood, the spleen and the draining lymph node by FACS analysis. To test antigen-specific T cell activation we performed in vitro restimulation of spleen and lymph node cells with collagen II followed by assessment of cytokine production and quantification of the proliferation rate using 3Hthymidine incorporation. After 4 and after 10 weeks mice were sacrificed and paraffin sections of the affected joints were analyzed for histomorphologic signs of inflammation, cartilage and bone destruction. Results Eighty percent of the animals developed serum anti-CII anibodies (IgM and IgG) whereby the antibody levels were a day 21 of disease similar between the HDAC1 cKO and the WT mice. Furthermore, no differences in the production of antibody subclasses, especially of pathogenic IgG2c antibodies, were observed. Enhanced percentages of Th1 and Th17 cells among HDAC1-null CD4+T cells were detected after immunization in the HDAC1 cKO mice. Nonetheless and unexpectedly, these mice did not develop any signs of disease at the clinical level while WT mice developed pronounced paw swelling and loss of grip strength. In accordance with the clinical data, histological analysis revealed no signs of inflammation, no bone erosion and no appearance of osteoclasts in the joints of HDAC1 cKO mice. This appeared to be mainly caused by an impaired migratory capacity of HDAC1 cKO CD4+ T cells, so that they were unable to invade the joints. Conclusion Our data show the importance of HDAC1 as a key immune regulator in the pathogenesis of T cell driven collagen induced arthritis. Therefore it might be considered as an interesting novel therapeutic target in RA. Disclosure of Interests None declared

Background The present study was undertaken to investigate novel anti-hnRNPs autoantibodies in rheumatic diseases. Objectives Novel mutated hnRNP A3 was cloned out of RA synovial tissue linking it directly to Warburg effect and lactate production in RA. Increased lactate production in RA synovia and tumors is klinked to alternative splicing process from PKM1 to PKM2 hnRNPs dependent. Methods After immunobloting and 2D-gel-eletrophoresis of a semipurified hnRNP fraction two protein spots were sequenced and identified to be highly similar to hnRNPA3. The hnRNP A3 variants were cloned from RA synovial tissue, which identified the isoforms found on protein level. 3700 RA sera were screened for the presence of mutated anti-hnRNP A3 autoantibodies using recombinant proteins and peptides thereof. Binding of RNA to hnRNP A3 (MA3) and mutated citrullinated A3 peptides (MCA3) the epitope recognition was investigated. Expression of hnRNP A3 in synovial tissue was analysed by Results Autoantibodies to MA3 protein were detected in 13% of RA (n=215) patients, in 9% SLE (n=154), in 27% of MCTD patients (n=44/10) and in less than 5% of 129 patients with other rheumatic disorders but not at all in healthy controls on immunoblot. When using renaturated MA3 on ELISA 22% of RA patients were detected and 87% of these patients had erosive arthritis. Same modification as in cancer cells were identified in synovial tissue and verified by MS and DNA sequencing. Using 2–3 citrullinated MCA3 peptides up to 81% of patients (n=150) with established and 67% (n=2926) of patients with an early RA with a specificity of 97% were detected. In early RA 27% and 25% in established RA of CCP2 negative and 93% of CCP2 positive patients were identified. By combining with the already established CCP2 and the new MCA3, 72% of early patients are positive. MCA3 autoantibodies predominantly occur (p Conclusions Mutated hnRNP-A3 is as a novel Toll7/9 dependent autoantigen in systemic rheumatic diseases. These mutated proteins are major components of RNA and DNA containing alternative splicing complexes leading to the Warburg effect and and autoantibodies predominantly occurre in an erosive, and severe courses of RA. Disclosure of Interest None declared

Background Release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions important in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). Nucleic acid sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, but their role in RA is less clear. To gain more insight into the role of TLR9 in autoimmune arthritis, rats with pristane-induced arthritis (PIA) were treated with a TLR9 antagonist. To further investigate TLR9 involvement, serum transfer arthritis was induced in TLR9 knock-out mice. Materials and methods Arthritis was induced in mice with arthritogenic K/BxN serum and in rats with the mineral oil pristane. Rats were treated with a TLR9 antagonist every other day, starting one day before disease induction. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantified by histological analysis. Furthermore, the role of TLR9 in osteoclast differentiation and activation was investigated in vitro . Results In the serum transfer model, which is independent of the adaptive immune system, arthritis severity was not changed in mice lacking a functional tlr9 gene. In PIA, which is T cell-dependent, the TLR9 antagonist reduced arthritis severity by ~50%. TLR9 inhibition led to reduced inflammation, bone erosion and cartilage degradation. However, when treatment was started after the onset of arthritis TLR9 inhibition did not influence disease severity. Treatment with the TLR9 antagonist influenced downstream signalling, mainly via NFκBp65, in lymph nodes. Remarkably, mRNA levels of TLR7 and TLR9 strongly differed in the course of in vitro osteoclastogenesis. Whereas TLR7 expression did not change throughout osteoclastogenesis, expression of TLR9 was higher in precursor cells than in mature osteoclasts and stimulation with a TLR9 agonist (CpG) completely inhibited osteoclastogenesis. Conclusions Taken together, the results suggest a role for TLR9 in the T cell-dependent initiation phase of PIA and thus an important involvement of the DNA (CpG) recognising TLR9 in the initiation of autoimmune arthritis and during osteoclastogenesis. The precise role(s) of TLR9 in the different stages of arthritis needs to be further elucidated in future experiments.

Objective The transcription factor (TF) FOXO3 is known to integrate information from multiple upstream signals (eg, cytokines, oxidative stress, growth factors) in order to maintain tissue homeostasis during stress. Interestingly, although an association between a FOXO3 genotype (SNP) and the severity of rheumatoid arthritis (RA) was recently reported, the role of this TF in rheumatoid fibroblast-like synoviocytes (FLS) has not yet been investigated. Methods With approval by the ethics committee synovial tissues from patients fulfilling the ACR/EULAR classification criteria for RA were obtained as discarded specimens following synovectomy. RA-FLS were isolated according to standard procedures. FOXO3 phosphorylation was determined by western blots. MK2206 was used to inhibit AKT. Nuclear-cytoplasmatic shuttling of FOXO3 was visualised and quantified by confocal immunofluorescence microscopy. FLS were transfected either with control or FOXO3 siRNA pools in order to investigate the role of FOXO3 in the TNF-induced inflammatory response. The expression of cytokines, chemokines and proteases, that are all known to be involved in RA pathogenesis, was assessed by ELISA, qPCR and western blots. Results TNF, which is well known to be at the apex of the inflammatory cytokine network in RA, promoted the phosphorylation and nuclear export (inhibition) of FOXO3 in FLS. FOXO3 phosphorylation by TNF was inhibited by the AKT-inhibitor MK2206, demonstrating that TNF induces AKT phosphorylation to subsequently control FOXO3 activity in FLS. To further investigate the role of FOXO3 in the TNF-induced inflammatory response we silenced FOXO3 expression by using specific siRNA pools. Interestingly, while IL6 and IL8 expression was not affected by FOXO3 knockdown, a significant reduction in MMP3 expression was observed. In contrast, FOXO3 knockdown by siRNA promoted the TNF-induced expression of BAFF, TNFSF10 and CXCL11, suggesting that FOXO3 is a negative regulator of these genes. Conclusions Our data reveal differential regulation of arthritis-associated genes by FOXO3 in FLS and thus support the idea that FOXO3 plays a crucial role in rheumatoid synovitis.