W de Jager

Autologous bone marrow transplantation in autoimmune arthritis restores immune homeostasis through CD4+ CD25+ Foxp3+ regulatory T cells.

Despite the earlier use of potent immunosuppressive or cytostatic drugs and the recent emergence of biologicals as treatment for human autoimmune diseases (AIDs), some patients still remain unresponsive to treatment. To those severely ill patients, autologous bone marrow transplantation (aBMT) is applied as a last resource, leading to disease remission in a majority of patients. The underlying mechanism of action of aBMT is still largely unknown. Here, we showed that regulatory T cells (Tregs) play a role in the natural disease course of proteoglycan-induced arthritis (PGIA) and in disease remission by aBMT. aBMT led to an initial phase of rapid disease improvement corresponding with a relative increase in CD4(+)CD25(+) T cells. At this time, the CD4(+)CD25(+) cells did not yet show an increase in Foxp3 expression and showed less potent suppression. After this initial improvement, disease relapsed but stabilized at a level below the severity before aBMT. This second phase was actively regulated by potently suppressive CD4(+)CD25(+)Foxp3(+) Tregs. This work provided further insight into the role of Tregs in restoration of the immune balance by aBMT and can open the way to explore therapeutic interventions to further improve treatment of AID and disease relapses.

Correlation of CXCL10, Tumor Necrosis Factor Receptor Type II, and Galectin 9 With Disease Activity in Juvenile Dermatomyositis

Juvenile dermatomyositis (DM) is a systemic autoimmune disorder of unknown immunopathogenesis in which the immune system targets the microvasculature of skeletal muscles, skin, and other organs. The current mainstay of therapy is a steroid regimen in combination with other immunosuppressive treatments. To date, no validated markers for monitoring disease activity have been identified, which hampers personalized treatment. This study was undertaken to identify a panel of proteins specifically related to active disease in juvenile DM.

A1.06 Phagocyte involvement in systemic onset juvenile idiopathic arthritis

Background Systemic onset Juvenile Idiopathic Artritis (sJIA) is a systemic autoinflammatory disease, characterised by arthritis, spiking fever and rash and elevation of serum S100-proteins and interleukin (IL)-18. The role of monocytes and neutrophils in the inflammatory cascade of sJIA is still unclear. Objective To study the role of monocytes and neutrophils in the inflammatory cascade of sJIA. Methods We determined neutrophil activation ex vivo (phenotype and cell membrane markers) and after stimulation (ROS-production and degranulation) of cells derived from sJIA with disease onset or in remission, compared to healthy donors (HDs). To investigate the role of monocytes, we assessed cytokine production of PBMCs from sJIA patients and HDs after stimulation with TLR-4 activating S100-proteins (+/- ATP) or other TLR-ligands. In a cohort of sJIA patients at onset and during inactive disease, we evaluated cell counts and serum levels of cytokines, chemokines and other analytes. Cytokine concentrations in supernatant and serum were determined by multiplex immunoassay. Results Twenty-one of 23 patients with onset sJIA had elevated neutrophil counts, while monocyte counts were elevated in only 5/23 patients. Many inflammatory markers were significantly elevated in serum of onset sJIA patients, among which several neutrophil specific proteins indicating the importance of this cell type. Neutrophils from onset sJIA patients showed an activated phenotype, reflected by higher ex vivo cell membraneexpression of FC-gamma receptors (CD32 and CD64), markers of secretory vesicles (CD35) and specific granules (CD66b). ROS production and degranulation were also enhanced in onset sJIA. Neutrophil phenotypenormalized when patients were in remission. In contrast to the hyperactivated status of neutrophils in active sJIA, PBMCs from these patients produced less Il-1b, IL-18, IL-6 and TNF-a upon TLR-stimulation compared to PBMCs from remission patients or HDs, suggesting tolerance after exposure to high TLR4 stimulating S100-levels in vivo. Conclusions We show here that monocytes from onset sJIA patients produce less cytokines upon stimulation, while the neutrophils are hyperactivated, reflected by increased cell membrane activation markers, ROS production and degranulation. The exact role of each cell type and activity and their interaction in sJIA pathology is currently under investigation.

PW02-018 - Impact of PSTPIP1 mutaions on clinical phenotype

Hyperzincaemia and hypercalprotectinaemia (Hz/Hc), a rare condition within the spectrum of autoinflammatory diseases, is associated with hepatosplenomegaly, arthritis, anemia, cutaneous inflammation, and failure to thrive. So far, no genetic cause has been identified. While the clinical appearance is heterogeneous, all affected individuals present with extremely elevated MRP8/MRP14 (calprotectin) serum concentrations (0.9-12.0 g/l (normal range < 0.001 g/l)).

OP0303 The salivary gland secretome as a potential new tool to identify biomarkers of dryness and immunopathology in primary sjÖgren's syndrome and non-autoimmune sicca patients

Background Salivary gland biopsy is essential in primary Sjögrens syndrome (pSS) diagnostics. However, tissue analysis using traditional methodology has several limitations including inaccurate quantification of lymphocytic infiltration and poor correlation with dryness. To perform biomarker identification in the target organ, tissue would have to be sacrificed. By performing saliva proteomics the biopsy tissue can be saved, but hitherto, this technique has not yielded consistent biomarkers and is limited by the absence of saliva production by many sicca patients. Objectives We aimed to explore whether Luminex analysis of a broad panel of cytokines in salivary gland biopsy supernatants (secretome) could provide biomarkers to stratify sicca patients and could give insights into pathogenesis. Methods Labial salivary gland (LSG) tissues were rinsed after biopsy and incubated in 200μL of saline for 1h at room temperature. Tissue supernatants were rendered cell-free, frozen in liquid nitrogen and stored at -80°C. In supernatants from pSS and non-Sjögrens sicca (nSS) patients 104 targets were measured by Luminex. Eight pSS and 8 nSS patients were selected for analysis based on matched biopsy weights. Results from this discovery cohort were validated in an additional cohort (n=18 nSS, n=16 incomplete SS: iSS, n=26 pSS) and correlations with clinical parameters were assessed. Non-SS were defined as sicca patients without lymphocytic infiltration in the salivary gland biopsy or anti-SSA/SSB autoantibodies. Incomplete SS patients were defined as sicca patients having lymphocytic infiltration (lymphocytic focus score (LFS)>0) and/or anti-SSA/SSB autoantibodies but do not fulfill the AECG classification criteria and are not diagnosed as pSS. Results Levels of 20 cytokines were significantly different between the nSS and pSS patients in the discovery cohort (p≤0.05). These 20 and 13 additionally selected cytokines based on a trend towards statistical significance and/or literature, were measured in a validation cohort. Weights of the biopsies did not significantly differ: 59.8±48.1mg in nSS vs 72.7±45.2mg in iSS vs 67.4±28.6mg in pSS. Fifteen out of these 20 cytokines were validated. From the 13 cytokines 7 were significantly elevated in pSS vs nSS. In iSS CXCL10 (IP-10) and CCL19 (MIP-3β) were significantly elevated. Cytokines correlating with LFS, ESSDAI, ESSPRI, % IgG and IgM+ plasma cells in LSG, Schirmer and/or serum IgG with Spearman r≥0.4 and p≤0.05 in pSS were selected for classification tree analysis, these were IL-2, IL-3, IFN-β, IL-21, CXCL13 (BLC), CXCL10 and CCL19. Using CXCL13 and IL-21 levels, 87.5% of pSS patients could be classified correctly. Based on the used cut off levels, 5 nSS and 9 iSS patients would be classified as pSS. Follow up of these patients may reveal development of pSS. Conclusions Elevated levels of numerous cytokines were found in LSG biopsy secretomes from pSS patients versus non-autoimmune sicca patients correlating with clinical parameters. This method represents a novel tool to provide insights in pSS immunopathology and to identify therapeutic targets and biomarkers for diagnosis, prognosis and treatment response. Disclosure of Interest None declared

OP0091 S100a8 triggers a novel immune-regulatory mechanism in developing dendritic cells

Background S100A8/A9 heterodimers are well-known alarmins that, upon release from activated or necrotic phagocytes, promote inflammation by binding to Toll-like receptor 4 (TLR4). These proteins are highly expressed in synovial phagocytes during arthritis and proved to be reliable biomarkers for monitoring disease activity in RA. Interestingly, we now identify a novel immune-regulatory mechanism of S100A8 in human monocyte-derived dendritic cells (moDCs). Objectives This study aims to analyze immune-regulatory functions of S100 proteins in human DCs. Methods MoDCs are differentiated with or without exposure to S100A8 prior to maturation with LPS. After characterization of the activation status using flow cytometry, the ability of these cells to induce autologous CD4+, CD8+, and γδ T-cell proliferation is investigated. Cytokines, secreted during development are analyzed by Luminex cytokine arrays. The metabolic state of DCs is examined by using Seahorse XFp Analyzer assays. Finally, to identify molecular mechanisms leading to an immune-regulatory phenotype, the mRNA expression of moDCs is analyzed by genome-wide gene expression arrays. Results Our results demonstrate that early exposure to S100A8 interferes with in-vitro differentiation of moDCs. Compared to controls S100A8-exposed moDCs show dramatically reduced surface expression of co-stimulatory molecules upon LPS-induced maturation. In addition, early treatment of moDCs with S100A8 alters the secretion of immune-regulatory cytokines and chemokines depending on the developmental state of moDCs. S100A8-induced effects on moDC maturation are not limited to TLR4 stimulation but rather trigger a common state of unresponsiveness. Furthermore, mitochondrial respiration and glycolytic function is diminished in S100A8-treated moDCs. As a consequence, S100A8-exposed moDCs have a reduced potential to induce autologous T-cell proliferation. We can show that these differences are mainly caused by reduced surface expression of co-stimulatory molecules on S100A8-treated moDCs. Mechanistically, genome-wide gene expression analysis reveals dramatic differences in gene expression between S100A8-exposed and conventionally differentiated moDCs. We demonstrate that S100A8 pre-treatment of moDCs significantly blocks LPS-induced gene expression during moDC activation. Interestingly, in-silico analysis of transcription factor networks predicts NFκB and C/EBPδ as master regulators of S100A8-induced effects in developing moDCs. C/EBPδ on protein level, indeed, shows reduced expression in S100A8-differentiated moDCs prior and after LPS-induced maturation when compared to conventionally differentiated moDCs. Conclusions Taken together, our results demonstrate a novel regulatory mechanism of innate immunity to prevent overwhelming immune responses. Dysregulated repression of detrimental adaptive immune responses might very well contribute to the disease phenotype in auto-immune disorders with high systemic S100A8/A9 levels. Therefore, S100A8-differentiated immune-suppressive DCs potentially represent a promising therapeutic tool to treat auto-immune diseases in the future. Disclosure of Interest None declared

AB0202 Galectin-9 is a Robust Biomarker for Disease Activity in Juvenile Dermatomyositis and Acts as a T Cell Activator

Background Juvenile dermatomyositis (JDM) is a rare, but severe chronic systemic autoimmune disease in children, characterized by muscle weakness and a typical skin rash. Clinical evaluation of disease activity remains challenging. Recently, we identified a protein that highly correlates with disease activity in a Dutch JDM cohort: galectin-9 (gal-9). The immunobiological role of gal-9 in autoimmune diseases is still controversial. On the one hand it is known for its immunosuppressive effects by inducing apoptosis in T-helper (Th) 1 and Th17 cells and activating regulatory T cells, on the other hand it has been implicated in T cell activation and Th1 skewing. Objectives To validate the potential of gal-9 as a biomarker in JDM and investigate its immunobiological effects on T cell skewing and activation. Methods Gal-9 was measured in patients serum of an independent JDM cohort by multiplex immunoassay. For functional experiments, naive CD4 T cells were isolated and stimulated with different concentrations of gal-9, plus anti-CD3 and antigen presenting cells. To test specificity, a gal-9 blocking agent (TIM-3 fusion protein) as well as a control from the galectin family (galectin-8) were included. Flow cytometric analysis of proliferation and T cell activation markers was performed on day 3 and 5 of culture. Cytokines TNFα, IFNγ, IL-13, IL-17 and IL-10 were measured in the culture supernatants of days 3, 5 and 7 by multiplex immunoassay. Results Measurement of gal-9 in serum confirmed its high discriminative value for active disease versus remission (P=.0001; AUC 0.894; OR 9.17) even under medication, as well as a strong correlation with the clinical disease activity scores CMAS and Physicians Global VAS. On a functional level, the presence of gal-9 induced a slight but significant increase in naive CD4 T cell proliferation after 3 days of culture. The T cell activation markers CD25, CD69 and TIM-3 showed the same pattern. Gal-9 also increased production of IFNγ, TNFα and IL-10, mainly at day 7. These effects were not seen in the control conditions. Conclusions We confirmed the potential use of a very robust biomarker, galectin-9, that highly correlates with disease activity in juvenile dermatomyositis. Introduction of this biomarker into clinical practice will help to personalize treatment. Functionally, we found that gal-9 is a T cell activator, causing increased proliferation, cytokine production and expression of T cell activation markers. The high levels of circulating gal-9 in JDM patients may therefore contribute to the immunopathogenesis of JDM. References Bellutti Enders et al. Correlation of CXCL10, TNFRII, and Galectin-9 With Disease Activity in Juvenile Dermatomyositis. Arthritis Rheumatol. 2014 Aug;66(8):2281-9. Disclosure of Interest None declared

PReS-FINAL-1016: Micro vesicles as a magnifying glass; uncovering potential biomarkers in juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is a common chronic inflammatory diseases in childhood. Despite remission as a result of a plethora of treatment techniques, the chronic and relapsing nature of the disease requires continuous treatment which causes adverse side effects. It is important to uncover a biomarker that can efficiently predict patient responses to therapy as well as determine if patients will progress or regress as a result of treatment. Micro vesicles are key messengers containing many immune signaling molecules including cytokines, molecules known to play a major role in JIA.

PReS-FINAL-2131: Hunting for biomarkers in juvenile dermatomyositis

Juvenile Dermatomyositis (JDM) is a systemic autoimmune disorder in which the immune system targets the microvasculature of skeletal muscles, skin and other organs, with for the most part an unknown immunopathogenesis. Moreover, evaluation of disease activity remains challenging in juvenile dermatomyositis as muscle enzyme levels and inflammatory markers, routinely used in clinics, are no reliable biomarkers in JDM, especially for monitoring the disease.

Increased stimulatory capacity of antigen presenting cells at the site of autoimmune inflammation interferes with regulatory T cell function

Background FOXP3+ regulatory T cells (Treg) are critical in maintaining self tolerance and are therefore considered important targets for the treatment of autoimmune disease. However, environmental factors at the site of autoimmune inflammation, such as enhanced costimulatory potential of antigen presenting cells (APC) and increased proinflammatory cytokine production, can negatively affect Treg function, thereby limiting effectiveness of these Treg targeted approaches. Aim Here we studied the phenotype of APC present at the site of inflammation in patients with Juvenile Idiopathic Arthritis (JIA) and investigated whether these cells can interfere with Treg mediated suppression. Methods Mononuclear cells were isolated from peripheral blood (PB) of healthy controls (HC) and from paired PB and synovial fluid (SF) of JIA patients. The phenotype of APC was analysed using flow cytometry. In vitro suppression assays were performed to study T cell activation and Treg mediated suppression in the presence of SF and PB derived APC. Results Monocytes from the site of inflammation displayed a more pro-inflammatory phenotype, with significantly increased costimulatory molecule expression, compared to monocytes from PB. In line with this pro-inflammatory phenotype, SF APC induced enhanced proliferation of effector cells and decreased suppression of effector cell proliferation in the presence of Treg. Conclusions APC from the site of inflammation have an enhanced stimulatory capacity that interferes with Treg mediated suppression. Therefore, this increased stimulatory potential should be targeted as well, in order for a Treg enhancing approach to be fully effective in the treatment of autoimmune inflammation.