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IntroductionCommercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays.Materials and methodsThis is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays.ResultsFor both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays.ConclusionAdoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.

Hydrogen sulfide (Hsub2/subS) emerged as an essential signaling molecule exerting beneficial effects in various cardiovascular, neurodegenerative, or musculoskeletal diseases with an inflammatory component, such as osteoarthritis. These protective effects were initially attributed to protein S-sulfhydration, a posttranslational modification of reactive cysteine residues. However, recent studies suggest that polysulfides and not Hsub2/subS are responsible for S-sulfhydration. To distinguish between Hsub2/subS and polysulfide-mediated effects in this study, we used the slow-releasing Hsub2/subS and persulfide donor P*, which can be decomposed into polysulfides. The effects of P* on IL-1β-induced inducible nitric oxide synthase (iNOS), a pro-inflammatory mediator in osteoarthritis, were determined by nitrite measurement, qPCR, and Western blotting in the murine chondrocyte-like cell line ATDC5. Decomposed P* significantly reduced IL-1β-induced iNOS signaling via polysulfides, independently of Hsub2/subS. In line with this, the fast-releasing Hsub2/subS donor NaHS was ineffective. In RAW 264.7 macrophages, similar results were obtained. P*-derived polysulfides further diminished IL-1β-induced CCAAT/enhancer-binding protein (C/EBP) β and δ expression in ATDC5 cells, which might play a critical role in P*-mediated iNOS decline. In conclusion, our data support the view that polysulfides are essential signaling molecules as well as potential mediators of Hsub2/subS signaling. Moreover, we propose that C/EBPβ/δ might be a novel target involved in Hsub2/subS and polysulfide-mediated anti-inflammatory signaling.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovial inflammation. The major drivers of synovial inflammation are cytokines and chemokines. Among these molecules, TNF activates fibroblast-like synoviocytes (FLSs), which leads to the production of inflammatory mediators. Here, we show that TNF regulates the expression of the transcription factor interferon regulatory factor 1 (IRF1) in human FLSs as well as in a TNF transgenic arthritis mouse model. Transcriptomic analyses of IRF1-deficient, TNF-stimulated FLSs define the interferon (IFN) pathway as a major target of IRF1. IRF1 expression is associated with the expression of IFNβ, which leads to the activation of the JAK-STAT pathway. Blocking the JAK-STAT pathway with the Janus kinase inhibitor (JAKinib) baricitinib or tofacitinib reduces the expression of IFN-regulated genes (IRGs) in TNF-activated FLSs. Therefore, we conclude that TNF induces a distinct inflammatory cascade, in which IRGs are key elements, in FLSs. The IFN-signature might be a promising biomarker for the efficient and personalized use of new treatment strategies for RA, such as JAKinibs.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα. Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Here, the authors identified a previously unknown signaling circuit that contributes to tumor necrosis factor (TNF)-induced activation of FLS. The authors show that FOXO3 and its modulator PIK3IP1 are crucial for the TNF-driven interferon (IFN) response in RA-FLS.

BackgroundThere is a need for biomarker to identify patients ‘at risk’ for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing Protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. ResultshnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-index was found in SLE patients, risk-RA- and early RA-cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC-analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Some effector CD4+ T cell subsets display cytotoxic activity, thus breaking the functional dichotomy of CD4+ helper and CD8+ cytotoxic T lymphocytes. However, molecular mechanisms regulating CD4+ cytotoxic T lymphocyte (CD4+ CTL) differentiation are poorly understood. Here we show that levels of histone deacetylases 1 and 2 (HDAC1-HDAC2) are key determinants of CD4+ CTL differentiation. Deletions of both Hdac1 and 1 Hdac2 alleles (HDAC1cKO-HDAC2HET) in CD4+ T cells induced a T helper cytotoxic program that was controlled by IFN-γ–JAK1/2–STAT1 signaling. In vitro, activated HDAC1cKO-HDAC2HET CD4+ T cells acquired cytolytic activity and displayed enrichment of gene signatures characteristic of effector CD8+ T cells and human CD4+ CTLs. In vivo, murine cytomegalovirus–infected HDAC1cKO-HDAC2HET mice displayed a stronger induction of CD4+ CTL features compared with infected WT mice. Finally, murine and human CD4+ T cells treated with short-chain fatty acids, which are commensal-produced metabolites acting as HDAC inhibitors, upregulated CTL genes. Our data demonstrate that HDAC1-HDAC2 restrain CD4+ CTL differentiation. Thus, HDAC1-HDAC2 might be targets for the therapeutic induction of CD4+ CTLs., Histone deacetylases 1 and 2 restrict naive CD4+ T cell differentiation into CD4+ cytotoxic T lymphocytes and might be targets for the therapeutic induction of CD4+ cytotoxic T lymphocytes.

Contains fulltext : 177821.pdf (Publisher’s version ) (Closed access) Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

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

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by antinuclear autoantibodies (ANA) and immunocomplexes, commonly affecting kidneys, skin, heart, lung or even the brain. We have shown that JunB(Δep) mice develop a SLE phenotype linked to increased epidermal Interleukin (IL)-6 secretion. Blocking of IL-6 receptor alpha (IL-6Rα) is considered as therapeutic strategy for the treatment of SLE. JunB(Δep) and wild-type mice were treated for short (5 weeks) or long term (21 weeks) with the IL-6Rα-blocking antibody MR16-1. Skin and kidney of mice were investigated by histology and immunofluorescence, and in addition, kidneys were analysed by electron microscopy. Furthermore, soluble IL-6R (sIL-6R), antihistone and antinucleosome antibodies levels were measured and associated with disease parameters. Treatment with MR16-1 resulted in significant improvement of SLE-like skin lesions in JunB(Δep) mice, compared to untreated mice. The sIL-6R amount upon long-term treatment with MR16-1 was significantly higher in JunB(Δep) versus untreated JunB(Δep) (P = 0.034) or wild-type mice (P = 0.034). MR16-1 treatment over these time spans did not significantly improve kidney pathology of immunoglobulin deposits causing impaired function. Significantly higher antihistone (P = 0.028) and antinucleosome antibody levels (P = 0.028) were measured in MR16-1-treated JunB(Δep) mice after treatment compared to levels before therapy. In conclusion, blockade of IL-6Rα improves skin lesions in a murine SLE model, but does not have a beneficial effect on autoimmune-mediated kidney pathology. Inhibition of IL-6R signalling might be helpful in lupus cases with predominant skin involvement, but combinatorial treatment might be required to restrain autoantibodies.

// Eva Bauer 1 , Michaela Schlederer 1,2 , Ruth Scheicher 3 , Jaqueline Horvath 1,4 , Petra Aigner 1 , Ana-Iris Schiefer 2 , Renate Kain 2 , Heinz Regele 2 , Gregor Hoermann 5 , Gunter Steiner 6,7 , Lukas Kenner 1,2,8 , Veronika Sexl 3 , Andreas Villunger 9,10 , Richard Moriggl 1,5,11 and Dagmar Stoiber 1,4 1 Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria 2 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 3 Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria 4 Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria 5 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria 6 Cluster Arthritis and Rehabilitation, Ludwig Boltzmann Society, Vienna, Austria 7 Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria 8 Unit of Pathology of Laboratory Animals, University of Veterinary Medicine, Vienna, Austria 9 Division of Developmental Immunology, Biocenter, Medical University Innsbruck, Innsbruck, Austria 10 Tyrolean Cancer Research Institute, Innsbruck, Austria 11 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria Correspondence to: Dagmar Stoiber, email: // Keywords : ETV6/RUNX1, BCL2, glomerulonephritis, lymphoma, autoimmunity, Immunology and Microbiology Section, Immune response, Immunity Received : September 11, 2015 Accepted : January 31, 2016 Published : February 23, 2016 Abstract The t(12;21) translocation generating the ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity.

Objective: Little is known about the experiences, values and needs of people without arthritis who undergo predictive biomarker testing for the development of rheumatoid arthritis (RA). Our study aimed to explore the perspectives of these individuals and describe their information needs.\ud \ud Methods: A qualitative, multicenter interview study with a thematic analysis was conducted in Austria, Germany and the UK. Individuals who underwent predictive biomarker testing for RA and had a positive test result, but no diagnosis of any inflammatory joint disease, were interviewed. Participants included patients with arthralgia and asymptomatic individuals. Information and education needs were developed from the qualitative codes and themes using the Arthritis Educational Needs Assessment Tool (ENAT) as a frame of reference.\ud \ud Results: Thematic saturation was reached in 34 individuals (76% female; 24 [71%] with arthralgia and 10 [29%] asymptomatic individuals). Thirty‐seven codes were summarized into four themes, namely (i) decision making around whether to undergo initial predictive testing, (ii) willingness to consider further predictive tests and/or (iii) preventive interventions, including medication and (iv) varying reactions after receiving a positive test result. Individuals with arthralgia were more likely to be willing to take preventive action, undergo further testing, and experience psychological distress than asymptomatic individuals. All participants expressed the need for tailored, lay‐understandable information.\ud \ud Conclusion: Individuals at risk of RA are currently the subjects of research aimed at developing better predictive strategies and preventive approaches. Their perceptions and needs should be addressed to inform the future development of interventions combined with education.

Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9−/− mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.

Summary Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA)., Graphical Abstract, Highlights • mTOR regulates fibroblast-like synoviocyte inflammatory transcriptional programs • mTOR limits NF-κB signaling by enhancing IκB-α • mTOR shifts inflammation toward STAT1-dependent genes • mTOR couples amino acid sensing to TNF activation in fibroblast-like synoviocytes, Karonitsch et al. show that TNF signaling co-opts the mTOR pathway in fibroblast-like synoviocytes. mTOR activation is associated with decreased NF-κB-mediated but increased STAT1-dependent gene expression. Thus, the metabolic checkpoint kinase mTOR regulates the synovial tissue response to inflammation in rheumatoid arthritis (RA).

Zusammenfassung 2014 wurden in Zusammenarbeit von zwei großen internationalen Expertengruppen Empfehlungen zur Bestimmung von Autoantikörpern gegen zelluläre Antigene, herkömmlich als antinukleäre Antikörper bezeichnet, herausgegeben. Die Empfehlungen wurden in einer 25 Punkte umfassenden Tabelle zusammengefasst und in einer weiteren Tabelle jene Muster beschrieben, die von einem Autoimmunlabor erkannt werden sollten. Internationale Empfehlungen haben für zertifizierte Labore Gültigkeit und erfordern ein Überdenken des analytischen Vorgehens. Die österreichische Arbeitsgruppe für Autoimmundiagnostik (http://www.easi-network.com) hat sich auf einen Konsens geeinigt, wie im deutschen Sprachraum diese 2014 veröffentlichten Empfehlungen umgesetzt werden könnten.

Multiple sclerosis (MS) is a human neurodegenerative disease characterized by the invasion of autoreactive T cells from the periphery into the CNS. Application of pan-histone deacetylase inhibitors (HDACi) ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model for MS, suggesting that HDACi might be a potential therapeutic strategy for MS. However, the function of individual HDAC members in the pathogenesis of EAE is not known. In this study we report that mice with a T cell-specific deletion of HDAC1 (using the Cd4-Cre deleter strain; HDAC1-cKO) were completely resistant to EAE despite the ability of HDAC1cKO CD4+ T cells to differentiate into Th17 cells. RNA sequencing revealed STAT1 as a prominent upstream regulator of differentially expressed genes in activated HDAC1-cKO CD4+ T cells and this was accompanied by a strong increase in phosphorylated STAT1 (pSTAT1). This suggests that HDAC1 controls STAT1 activity in activated CD4+ T cells. Increased pSTAT1 levels correlated with a reduced expression of the chemokine receptors Ccr4 and Ccr6, which are important for the migration of T cells into the CNS. Finally, EAE susceptibility was restored in WT:HDAC1-cKO mixed BM chimeric mice, indicating a cell-autonomous defect. Our data demonstrate a novel pathophysiological role for HDAC1 in EAE and provide evidence that selective inhibition of HDAC1 might be a promising strategy for the treatment of MS.

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.

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2 S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2 S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1β and treated with the H2 S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1β-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1β-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2 S partially antagonizes IL-1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.

Objectives The aims of this study were to investigate the extent of MRI-determined joint disease (erosion and synovitis) in SLE and to link this to autoantibody profiles known to be relevant to SLE, including ACPA, RF and anti-RA33 antibodies. Methods Contrast-enhanced MRI of the hand and wrist was performed in 34 symptomatic SLE patients and in 15 RA patients with similar disease duration. Images were scored by two observers using the OMERACT rheumatoid arthritis MRI scoring (RAMRIS) system. Findings were correlated with clinical examination and autoantibody status. Results Erosions were present at the wrist in 93% of SLE patients and at the MCP joints in 61% of SLE patients. Despite the high prevalence of MRI-determined erosion, only 8.8% of SLE patients were ACPA positive, although these patients had a higher burden of erosive disease. There was no positive correlation with anti-RA33 titres and erosion scores in the SLE patients, but there was a negative correlation with anti-RA33 titres and total bone oedema scores in the SLE patients. Ninety-three per cent of SLE patients had at least grade 1 synovitis at one or more MCP joints, and wrist joint synovitis was present in all the SLE patients. Conclusion An MRI-determined joint erosive phenotype is common in SLE, even in ACPA-negative cases. The conventional radiographic observation that anti-RA33 is not positively associated with erosion in patients with RA was also found to be the case in SLE patients.

Hydrocarbon oils such as pristane or hexadecane induce arthritis and lupus in rodents sharing clinical and pathological features with the human diseases rheumatoid arthritis and systemic lupus erythematosus, respectively. In pristane-induced lupus in the mouse induction of apoptosis and augmentation of type-I Interferon signalling by pristane have been suggested to contribute to pathology, whereas in pristane-induced arthritis (PIA) in the rat the pathological mechanisms are still elusive. Here we show that pristane induces cell death in rat and human cells. Increased numbers of apoptotic cells were found in draining lymph nodes of pristane-injected rats and increased percentages of apoptotic and necrotic cells were observed in peripheral blood. In addition, neutrophil extracellular trap formation was triggered by pristane and hexadecane in neutrophils. Because levels of interleukin (IL)-1β were elevated in sera of pristane-injected rats, with levels mirroring the course of PIA, we examined the effect of pristane at single cell level in vitro, using rat splenocytes and the human monocytic cell line THP-1. Pristane and other hydrocarbon oils induced IL-1β secretion in THP-1 cells as well as in rat splenocytes. The potassium channel inhibitor glibenclamide partly inhibited IL-1β induction, suggesting involvement of the inflammasome. Elevated levels of IL-1α were also found in supernatants of cells treated with pristane and hexadecane. In conclusion, autoimmunogenic hydrocarbon oils induce various forms of cell death in rat and human cells. The higher serum IL-1β levels in pristane-injected animals might be caused by both inflammasome-dependent and -independent mechanisms, such as passive release from dying-cells and probably extracellular maturation of pro-IL-1β.

Autoimmunerkrankungen sind eine klinisch heterogene Gruppe von Erkrankungen, die den Hausarzt oft vor differentialdiagnostische Herausforderungen stellen. Mit Ausnahme der rheumatoiden Arthritis, die mit einer Pravalenz von ca. 1 % der Bevolkerung zu den haufigen Autoimmunerkrankungen zahlt, sind systemische Autoimmunerkrankungen in der allgemeinmedizinischen Praxis so selten, dass der niedergelassene Arzt fur Allgemeinmedizin meist wenig Erfahrung mit den einzelnen Krankheitsbildern hat. Bei entsprechender klinischer Symptomatik und mit gezielten Fragestellungen konnen bestimmte Autoantikorper die Verdachtsdiagnose stutzen. Die osterreichische Gruppe der Europaischen Autoimmun Standardisierungsinitiative (EASI, European Autoimmunity Standardisation Initiative) hat es sich zum Ziel gesetzt, im vorliegenden und 2009 publizierten Leitfaden, dem Allgemeinmediziner einen Wegweiser durch den Dschungel der Biomarker fur Autoimmunerkrankungen zu bieten.

Objective Performance of the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) rheumatoid arthritis (RA) criteria was analysed in an internationally recruited early arthritis cohort (≤16 weeks symptom duration) enrolled in the ‘Stop-Arthritis-Very-Early’ trial. This sample includes patients with a variety of diseases diagnosed during follow-up. Methods Two endpoints were defined: Investigators’ diagnosis and disease-modifying antirheumatic drug (DMARD) treatment start during the 12-month follow-up. The 2010 criteria were applied to score Patients’ baseline data. Sensitivity, specificity, predictive values and areas under the receiver operating curves of this scoring with respect to both endpoints were calculated and compared to the 1987 criteria. The optimum level of agreement between the endpoints and the 2010 classification score ways estimated by Cohen’s ϰ coefficients. Results 303 patients had 12-months follow-up. Positive predictive values of the 2010 criteria were 0.68 and 0.71 for RA-diagnosis and DMARD-start, respectively. Sensitivity for RA-diagnosis was 0.85, for DMARD-start 0.8, whereas the 1987 criteria’s sensitivities were 0.65 and 0.55. The areas under the receiver operating curves of the 2010 criteria for RA-diagnosis and DMARD-start were 0.83 and 0.78. Analysis of inter-rater-agreement using Cohen’s ϰ demonstrated the highest ϰ values (0.5 for RA-diagnosis and 0.43 for DMARD-start) for the score of 6. Conclusions In this international very early arthritis cohort predictive and discriminative abilities of the 2010 ACR/EULAR classification criteria were satisfactory and substantially superior to the ’old’ 1987 classification criteria. This easier classification of RA in early stages will allow targeting truly early disease stages with appropriate therapy.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting various tissues. Involvement of B and T cells as well as increased cytokine levels have been associated with disease manifestation. Recently, we demonstrated that mice with epidermal loss of JunB (JunB Δep ) develop a myeloproliferative syndrome (MPS) due to high levels of G-CSF which are secreted by JunB-deficient keratinocytes. In addition, we show that JunB Δep mice develop a SLE phenotype linked to increased epidermal interleukin 6 (IL-6) secretion. Intercrosses with IL-6-deficient mice could rescue the SLE phenotype. Furthermore, we show that JunB binds to the IL-6 promoter and transcriptionally suppresses IL-6. Facial skin biopsies of human SLE patients similarly revealed low JunB protein expression and high IL-6, activated Stat3, Socs-1, and Socs-3 levels within lupus lesions. Thus, keratinocyte-induced IL-6 secretion can cause SLE and systemic autoimmunity. Our results support trials to use α-IL-6 receptor antibody therapy for treatment of SLE.

To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes.Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes.STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

Objectives:Autoantibody formation and T cell reactivity against the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been observed in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Since no differences in epitope recognition were reported and the usefulness of anti-hnRNP-A2 antibodies as diagnostic markers of SLE is unknown, it was our objective to characterise linear B cell epitopes of hnRNP-A2 and to relate the anti-hnRNP-A2 antibody responses to disease activity and clinical features of SLE.Methods:Sequential serum samples from 15 patients with SLE and sera from patients with other rheumatic diseases and healthy subjects were investigated by ELISA for autoantibody reactivities against a set of 13 overlapping peptides spanning the RNA-binding region of hnRNP-A2. Antibody reactivity against the complete protein was determined by western immunoblotting and ELISA. SLE disease activity was assessed by European Consensus Lupus Activity Measure scores, by SLE Index scores and the British Isles Lupus Assessment index.Results:Anti-peptide antibody reactivities were found in 60% of SLE sera but in only 5% of control samples, and were mainly directed to four peptides, one of which (p155–175) appeared to be immunodominant. Antibodies to p155–175 were exclusively seen in patients with SLE and correlated with clinical disease activity as well as kidney and skin involvement. No correlations were found for the other anti-peptide antibody responses.Conclusion:Peptide p155–175 encompasses a disease-specific immunodominant epitope of hnRNP-A2. Since antibodies to p155–175 correlate with disease activity and nephritis, they may be useful as markers for active SLE.

Objective T cell intracytoplasmic antigen 1 (TIA-1) and TIA-1–related protein (TIAR) are involved in posttranscriptional regulation of the expression of tumor necrosis factor α (TNFα) and other proteins. Given the pivotal role of TNFα in chronic inflammatory diseases, this study was undertaken to analyze sera from patients with systemic autoimmune diseases for the presence of autoantibodies to TIA proteins and to investigate the expression of these proteins in inflamed tissue. Methods The presence of autoantibodies to TIA proteins in sera from 385 patients with rheumatic diseases and healthy controls was determined by immunoblotting using recombinant antigens. Expression of TIA proteins in skin and kidney tissue was analyzed by immunohistochemistry. Serum levels of TNFα were measured by enzyme-linked immunosorbent assay. Results Autoantibodies to TIA-1 and/or TIAR were detected in 61% of patients with systemic lupus erythematosus (SLE), 42% of patients with systemic sclerosis (SSc), 15–31% of patients with other rheumatic diseases, and 6% of healthy controls. Compared with patients negative for anti-TIA antibody, anti-TIA antibody–positive SLE patients had higher disease activity (P = 0.01), elevated antibodies to double-stranded DNA (P = 0.0003), and increased serum TNFα levels (P = 0.018). In SLE patients, anti-TIAR antibodies were associated with lupus nephritis (P = 0.02), while in patients with SSc, anti–TIA-1 was associated with lung involvement (P = 0.02). Immunohistochemical analysis of skin and kidney tissue revealed aberrant expression of TIA proteins in skin lesions from SLE and SSc patients, as well as in glomerular cells from SLE patients. Conclusion Aberrant expression of TIA proteins in inflammatory tissue may lead to systemic autoantibody responses, particularly in SLE and SSc. Increased occurrence of anti-TIA autoantibodies in patients with severe organ involvement may point to a possible pathogenetic role.

To study the effects of short-term intermediate dose glucocorticoid (GC) therapy in patients with active rheumatoid arthritis (RA) on circulating endothelial progenitor cells (EPC), which are known to influence cardiovascular risk, and to elucidate mechanisms potentially responsible for the reduction of EPCs in patients with active RA.EPCs were quantified in 29 patients with active RA by flow cytometry, colony forming unit (CFU) and circulating angiogenic cell (CAC) assays before and after 7 days of intermediate dose GC therapy. CFU from patients with RA and from healthy referents (HR) were cultured in vitro in the absence or presence of dexamethasone (Dex) and/or TNF.After 1 week of GC therapy, EPC increased from 0.026 (SD 0.003)% to 0.053 (SD 0.010)% (p0.01), and from 12 (SD 4) to 27 (SD 7) CFU/well (p0.02); CAC also increased from 7 (SD 2) to 29 (SD 8) cells/high power field (p0.05). In parallel, disease activity decreased significantly after GC treatment. TNF serum levels also decreased from 36 (SD 10) to 14 (SD 6) pg/ml (p0.0001). Addition of Dex to the RA CFU led to a significant increase of mean CFU counts, whereas addition of TNF induced a decrease of CFU.Our data indicate that TNF may be at least partly responsible for the reduction of EPC seen in patients with RA. Intermediate doses of GCs for a short period of time, apart from reducing disease activity, significantly increase circulating EPC.

OBJECTIVE Abatacept (CTLA-4Ig) blocks CD28-mediated T cell activation by binding to the costimulatory B7 ligands CD80/CD86 on antigen presenting cells. Costimulatory molecules, however, can also be expressed on T cells upon activation. Therefore, the aim of our study was to investigate direct effects of CTLA-4Ig on distinct T cell subsets in RA patients. METHODS Phenotypic and functional analyses of CD4(+) T cells, including CD4(+) FoxP3(+) CD25(+) regulatory T cells (Treg), from RA patients were performed before and during CTLA-4Ig therapy. In addition T cells from healthy volunteers were analysed on in vitro culture with CTLA-4Ig or anti-CD80 and anti-CD86 antibodies. Apoptotic DNA fragmentation in CD4(+) and CD4(+) FoxP3(+) T cells was measured by TUNEL staining. RESULTS We observed an increase in T cells, including Treg cells, after initiation of CTLA-4Ig therapy, which was linked to a downregulation of activation-associated marker molecules and CD95 on CD4(+) T cells and Treg cells. CTLA-4Ig decreased CD95-mediated cell death in vitro in a dose-dependent manner. Functional analysis of isolated Treg cells from RA patients further revealed a diminished suppression of responder T cell proliferation. This was found to be due to CTLA-4Ig-mediated blocking of CD80 and CD86 on responder T cells that led to a diminished susceptibility for Treg cell suppression. CONCLUSION CTLA-4Ig therapy in RA patients exerts effects beyond the suppression of T cell activation, which has to be taken into account as an additional mechanism of CTLA-4Ig treatment.