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Objectives How the local inflammatory environment regulates epigenetic changes in the context of inflammatory arthritis remains unclear. Here we assessed the transcriptional and active enhancer profile of monocytes derived from the inflamed joints of JIA patients, a model well-suited for studying inflammatory arthritis. Methods RNA sequencing and H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) were used to analyse the transcriptional and epigenetic profile, respectively, of JIA synovial fluid-derived monocytes. Results Synovial-derived monocytes display an activated phenotype, which is regulated on the epigenetic level. IFN signalling-associated genes are increased and epigenetically altered in synovial monocytes, indicating a driving role for IFN in establishing the local inflammatory phenotype. Treatment of synovial monocytes with the Janus-associated kinase (JAK) inhibitor ruxolitinib, which inhibits IFN signalling, transformed the activated enhancer landscape and reduced disease-associated gene expression, thereby inhibiting the inflammatory phenotype. Conclusion This study provides novel insights into epigenetic regulation of inflammatory arthritis patient-derived monocytes and highlights the therapeutic potential of epigenetic modulation for the treatment of inflammatory rheumatic diseases.

Treg cells are critical regulators of immune homeostasis, and environment-driven Treg cell differentiation into effector (e)Treg cells is crucial for optimal functioning. However, human Treg cell programming in inflammation is unclear. Here, we combine transcriptional and epigenetic profiling to identify a human eTreg cell signature. Inflammation-derived functional Treg cells have a transcriptional profile characterized by upregulation of both a core Treg cell (FOXP3, CTLA4, TIGIT) and effector program (GITR, BLIMP-1, BATF). We identify a specific human eTreg cell signature that includes the vitamin D receptor (VDR) as a predicted regulator in eTreg cell differentiation. H3K27ac/H3K4me1 occupancy indicates an altered (super-)enhancer landscape, including enrichment of the VDR and BATF binding motifs. The Treg cell profile has striking overlap with tumor-infiltrating Treg cells. Our data demonstrate that human inflammation-derived Treg cells acquire a conserved and specific eTreg cell profile guided by epigenetic changes, and fine-tuned by environment-specific adaptations.

Treg are critical regulators of immune homeostasis, and increasing evidence demonstrates that environment-driven Treg differentiation into effector (e)Treg is crucial for optimal functioning. However, human Treg programming under inflammatory conditions remains poorly understood. Here, we combine transcriptional and epigenetic profiling to identify the human eTreg core signature. Functional autoimmune inflammation-derived Treg display a unique transcriptional profile characterized by upregulation of both a core Treg (FOXP3, CTLA-4, TIGIT) and effector program (GITR, BLIMP-1, BATF). We identified a specific human eTreg signature that includes the vitamin D receptor (VDR) as predicted key-regulator in eTreg differentiation. H3K27ac/H3K4me1 occupancy revealed pronounced changes in the (super-)enhancer landscape, including enrichment of the binding motif for VDR and BATF. The observed Treg profile showed striking overlap with tumor-infiltrating Treg. Our data demonstrate that human inflammation-derived Treg acquire a specific eTreg profile guided by epigenetic changes. The core eTreg profile is conserved, and fine-tuned by environment-specific adaptations.

Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ., Human regulatory T cells at the maternal-fetal interface show uterine site-specific functional adaptation with late-stage effector differentiation, chronic activation, Th1 polarization, and tumor-infiltrating, Treg-like features.

ObjectivesRegulatory T cells (Tregs) are crucial for maintaining immune tolerance against the semi-allogeneic fetus during pregnancy. Since their functional profile at the human maternal-fetal interface is still elusive, we investigated the transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy.MethodsBlood and uterine biopsies from the placental bed (=maternal-fetal interface) and incision site (=control), were obtained from women with uneventful pregnancies undergoing primary Caesarean section. Tregs and CD4+non-Tregs (Tconv) were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell level by flow cytometry.ResultsPlacental bed uterine Tregs (uTregs) showed elevated expression of Treg signature markers compared to blood Tregs, including FOXP3, CTLA4 and TIGIT. The uTreg transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation with high expression of immune checkpoints GITR, TNFR2, OX-40, 4-1BB, genes associated with suppressive capacity (CTLA4, HAVCR2, IL10, IL2RA, LAYN, PDCD1), activation (HLA-DR, LRRC32), and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored uTconv Th1 polarization, and characteristics indicating tissue-residency, including high CD69, CCR1, and CXCR6. The particular transcriptional signature of placental bed uTregs overlapped strongly with the specialized profile of human tumor-infiltrating Tregs, and, remarkably, was more pronounced at the placental bed than uterine control site.ConclusionuTregs at the maternal-fetal interface acquire a highly differentiated effector Treg profile similar to tumor-infiltrating Tregs, which is locally enriched compared to a distant uterine site. This introduces the novel concept of site-specific transcriptional adaptation of human Tregs within one organ.

Background: Heterogeneity in response to biological medication is a major challenge in the management of Juvenile Idiopathic Arthritis (JIA) (1). Biomarkers such as cytokines or whole blood RNA expression profiles that predict therapy efficacy prior to treatment pose a solution and could enable precision medicine. It was previously demonstrated that whole blood gene expression profiles can predict response to anti-TNF-α therapy in patients with JIA (2). These findings currently await validation in a multicenter prospective cohort study. Harmonization of data from different cohorts, however, is often hampered by site-dependent differences in biological sample collection and processing (3). For instance, the biological material that is retrospectively available from biobanks depends on what is stored exactly, e.g. whole blood versus peripheral blood mononuclear cells (PBMC). Furthermore, different blood collection tubes containing different additives can affect sample quality at an early stage and make interchangeability or comparability of data impossible. Objectives: We evaluated the comparability of gene expression profiles when whole blood from patients with JIA is collected in different RNA collection tubes and processed accordingly. Next, we will investigate the predictive capacity for therapy response of RNA expression profiles from frozen PBMC of 63 JIA patients. Methods: Peripheral blood from 11 children with non-systemic JIA with active disease was collected in PAXgene (PreAnalytiX) and Tempus (Applied Biosystems) tubes. All tubes were subsequently stored in the freezer at -80 °C. After thawing, RNA from the PAXgene tubes was extracted with use of the PAXgene Blood RNA Kit and RNA from the Tempus tubes was isolated by using the Tempus Preserved Blood RNA Purification Kit I. Gene expression of CSNK1D, C1D, ASAP2, SRRD, PPP1R3B, HLA-DQA1, PDZK1IP1 and MZB1 was determined with qPCR. For our next experiment, frozen PBMC derived from patients with non-systemic JIA who are included in our longitudinal Pharmachild registry biobank will be used. Of the 63 patients, 28 (44.4%) children were diagnosed with oligo articular JIA, 19 (30.2%) with poly articular JIA, 9 (14.3%) with enthesitis-related JIA, 4 (6.3%) with psoriatic JIA and 3 (4.8%) with undifferentiated JIA. The average age at sampling was 11.5 years ± 4.0 and 65% of the patients was female. These samples will be run on a 96-analyte NanoString panel and associated with clinical response at time points 3 and 6 months after start of TNF-α blockade. Results: Both RNA blood collection systems yielded high-quality RNA, with overall higher RNA concentrations for blood collected in Tempus tubes in comparison to PAXgene tubes. qPCR data showed that gene expression of all measured genes is affected by the method of blood collection and processing. However, the inter-individual variation was similar between both collection tubes, indicating that similar RNA profiles were observed. Conclusion: Gene expression profiles derived from whole blood collected in PAXgene or Tempus tubes are comparable, but are not interchangeable. References: [1] Swart JF, de Roock S, & Prakken BJ. Understanding inflammation in juvenile idiopathic arthritis: How immune biomarkers guide clinical strategies in the systemic onset subtype. Eur J Immunol 2016;46(9):2068-2077. [2] Cui A, Quon G, Rosenberg AM et al. Gene Expression Deconvolution for Uncovering Molecular Signatures in Response to Therapy in Juvenile Idiopathic Arthritis. PloS one 2016;11(5):1-17. [3] Yeung RS, Albani S, Feldman BM et al. Enhancing translational research in paediatric rheumatology through standardization. Nature Reviews Rheumatology 2016;12(11):684. Disclosure of Interests: None declared

OBJECTIVE: Resistance of effector T cells (Teff) to regulatory T cell (Treg)-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients. However, an unanswered question is whether this resistant phenotype is self-sustained and whether CD8(+) and CD4(+) Teff share the same mechanism of resistance to suppression. Here, we investigated CD8(+) Teff intrinsic resistance to suppression and how this can be targeted therapeutically. METHODS: CD8(+) or CD4(+) Teff were cultured with or without antigen presenting cells (APC) in Treg-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff were cross-cultured with peripheral blood (PB) Treg from JIA patients or healthy controls. TNF-α or IFN-γ blocking agents were used to restore Teff responsiveness to suppression. RESULTS: Suppression of cell proliferation and cytokine production by CD8(+) Teff from the SF of JIA patients was severely impaired compared to PB of JIA patients, regardless of APC and CD4(+) Teff cell presence. Similarly to CD4(+) Teff, impaired suppression of CD8(+) Teff was shown to be an intrinsic feature of this cell population. Whereas TNF-α blockade rescued both CD8(+) and CD4(+) Teff resistance, autocrine release of IFN-γ selectively sustained CD8(+) Teff resistance, which could be relieved by IFN-γ blockade. CONCLUSION: Unlike CD4(+) Teff, resistance of CD8(+) Teff to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFN-γ and blockade of IFN-γ restores CD8(+) Teff responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFN-γ to restore immune regulation in JIA. This article is protected by copyright. All rights reserved.

Mutations in STAT3 have recently been shown to cause autoimmune diseases through increased lymphoproliferation. We describe a novel Pro471Arg STAT3 mutation in a patient with multiple autoimmune diseases, causing hyperactivation of the Th17 pathway. We show that IL-17 production by primary T cells was enhanced and could not be further increased by IL-6, while IL-10 reduced Th17 cell numbers. Moreover, specific inhibition of STAT3 activation resulted in diminished IL-17 production. We show that the Pro471Arg STAT3 mutation yields both increased levels of IgA and IgG, probably due to high IL-21 levels. When remission was reached through medical intervention, IL-17 levels normalized and the clinical symptoms improved, supporting the idea that STAT3 gain-of-function mutations can cause hyperactivation of the Th17 pathway and thereby contribute to autoimmunity.

Background: Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children. Methods: 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed. Results: Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction. Conclusions: First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

Objective Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to investigate the role of neutrophils in systemic-onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation. Methods Fifty patients with systemic-onset JIA who were receiving treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence-activated cell-sorted neutrophils from 3 patients with active systemic-onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic-onset JIA and 15 healthy controls. Results Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL-1Ra therapy. RNA-sequencing analysis revealed a substantial up-regulation of inflammatory processes in neutrophils from patients with active systemic-onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic-onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL-1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL-1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL-1Ra. In vitro, rIL-1Ra antagonized the priming effect of IL-1β on neutrophils from healthy subjects. Conclusion These results strongly support the notion that neutrophils play an important role in systemic-onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic-onset JIA are both susceptible to IL-1 blockade.

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was

We identified a novel Q27W FcγRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcγRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcγRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcγRIIa variants. The Q27W FcγRIIa mutation does not disrupt FcγRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcγR, precluding careful analysis of Q27W FcγRIIa functional deviation. For functional analysis of FcγRIIa function, we therefore overexpressed the Q27W FcγRIIa and common FcγRIIa variant in IIA1.6 cells that are normally deficient in FcγR. We show that FcγRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcγRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.

8th International Congress of Familial Mediterranean Fever and Systemic Autoinflammatory Diseases

Resistance of Teff cells to Treg cell-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self-sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.CD8+ or CD4+ Teff cells were cultured with or without antigen-presenting cells (APCs) in Treg cell-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff cells were crosscultured with peripheral blood (PB) Treg cells from JIA patients or healthy controls. Tumor necrosis factor (TNF) or interferon-γ (IFNγ) blocking agents were used to restore Teff cell responsiveness to suppression.Suppression of cell proliferation and cytokine production in CD8+ Teff cells from the SF of JIA patients was severely impaired compared to that in CD8+ Teff cells from the PB of JIA patients, regardless of the presence of APCs and CD4+ Teff cells. Similar to CD4+ Teff cells, impaired suppression of CD8+ Teff cells was shown to be an intrinsic feature of this cell population. While TNF blockade restored both CD8+ and CD4+ Teff cell susceptibility to suppression, autocrine release of IFNγ selectively sustained CD8+ Teff cell resistance, which could be relieved by IFNγ blockade.Unlike CD4+ Teff cells, resistance of CD8+ Teff cells to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFNγ, and blockade of IFNγ restores CD8+ Teff cell responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFNγ to restore immune regulation in JIA.

OBJECTIVE: The balance between Treg and effector T cells (Teff) is crucial for immune regulation in JIA. How MTX, the cornerstone treatment in JIA, influences this balance in vivo is poorly elucidated. The aim of this study was to investigate quantitative and qualitative effects of MTX on Treg and Teff in JIA patients during MTX treatment. METHODS: Peripheral blood samples were obtained from JIA patients at the start of MTX and 3 and 6 months thereafter. Treg numbers and phenotypes were determined by flow cytometry and suppressive function in allogeneic suppression assays. Teff proliferation upon stimulation with anti-CD3, activation status and intracellular cytokine production were determined by flow cytometry. Effector cell responsiveness to suppression was investigated in autologous suppression assays. Effector cell cytokines in supernatants of proliferation and suppression assays and in plasma were measured by cytokine multiplex assay. RESULTS: MTX treatment in JIA did not affect Treg phenotype and function. Instead, MTX treatment enhanced, rather than diminished, CD4(+) and CD8(+) T cell proliferation of JIA patients after 6 months of therapy, independent of clinical response. Effector cells during MTX treatment were equally responsive to Treg-mediated suppression. MTX treatment did not attenuate Teff activation status and their capacity to produce IL-13, IL-17, TNF-α and IFN-γ. Similarly to Teff proliferation, plasma IFN-γ concentrations after 6 months were increased. CONCLUSION: This study provides the novel insight that MTX treatment in JIA does not attenuate Teff function but, conversely, enhances T cell proliferation and IFN-γ plasma concentrations in JIA patients.

CXCL8 is a potent neutrophil recruiting chemokine. CXCL8 is produced by several innate immune cells, including neutrophils, macrophages, as well as epithelial cells. Although previously considered only to be produced as a result of TLR signaling in these cells, recent reports show that T cell–derived cytokines also induce CXCL8 in epithelial cells. Likewise, we observed that T cell inhibition diminished intestinal production of functional mouse homologs of CXCL8 in the early phase of enterocolitis. In this study, we specifically investigated whether adaptive cells contribute to innate cxcl8 expression in the intestines. To this end, we used the zebrafish as our model system. Unlike murine models that lack CXCL8, zebrafish have two CXCL8 chemokines that are both elevated after an acute inflammatory stimulus and recruit neutrophils. Furthermore, zebrafish develop innate and adaptive immunity sequentially, enabling analysis of intestinal cxcl8 expression in the absence (3 wk of age) of adaptive immunity. In this study, we show that intestinal cxcl8-l1 but not cxcl8-l2 expression is regulated by T lymphocytes under homeostatic conditions. In contrast, during intestinal inflammation especially, cxcl8-l1 expression is upregulated independent of T lymphocyte presence. Furthermore, we show that human CXCL8 is able to induce intestinal zebrafish neutrophil recruitment and cxcl8-l1 expression, demonstrating that zebrafish can be used as a model to study CXCL8 function and regulation. In conclusion, these data provide evidence that Cxcl8-l1 and Cxcl8-l2 are differentially regulated via T lymphocyte–dependent and –independent mechanisms during homeostasis and inflammation.

Background Systemic onset Juvenile Idiopathic Artritis (sJIA), also known as Still’s disease, is characterised by arthritis with symptoms of systemic inflammation such as spiking fever, rash and serositis. It is considered an autoinflammatory disease with a major role for the innate immune system. The S100-proteins S100A8, S100A9 and S100A12 interleukin (IL)-18 in plasma are extremely elevated in sJIA patients and have been proposed to be useful biomarkers for diagnosis, disease activity and response to therapy. However, the relationship between S100-proteins and IL-18 in sJIA and the role of S100 proteins in pathogenesis is still unknown. Objective To study the relation between S100-proteins and IL-18 in sJIA. Materials and methods Peripheral blood mononuclear cells (PBMCs) from sJIA patients during active disease and during remission and PBMCs from healthy controls were stimulated with S100A8, S100A9, S100A12 or LPS (positive control) for four hours. As a second signal for NLRP3 inflammasome activation, ATP was added during the last 30 min. Cytokine levels in supernatant were measured by Luminex; cell frequencies and TLR-expression were analysed by flow cytometry. Results Upon stimulation with S100-proteins, PBMCs from healthy donors produced inflammasome dependent IL-1b, IL-18 as well as inflammasome independent IL-6 and TNF-a; IL-18 and IL-1b production was further enhanced when ATP was added. S100 proteins are known to bind to TLR4 and RAGE; blocking TLR4 by adding CLI-095 decreased cytokine production to near normal levels, while blocking RAGE did not have an effect on cytokine production. When compared to healthy controls, PBMCs from sJIA patients produced less IL-18 upon S100 stimulation. The addition of ATP enhanced this differential effect. PBMCs from patients with active disease were less responsive to S100 stimulation than PBMCs from the same patient in remission. The same trend of hyporesponsiveness was seen when PBMCs from sJIA patients were stimulated with TLR2 or TLR7/8 ligands, suggesting cross-tolerance in these patient cells. Conclusions S100-proteins serve as a first signal via TLR4 to establish NLRP3 inflammasome activation and thereby induce production of IL-1b and IL-18. Interestingly, PBMCs from sJIA patients with active disease are hyporesponsive to S100-proteins compared to sJIA patients in remission and healthy controls. We are currently investigating whether this hyporesponsiveness can be explained by stress tolerance of PBMCs after prolonged elevation of serum S100-proteins. Acknowledgments for providing S100 proteins Holzinger D 1 , Vogl T 2 , Foell D 1 , Roth J 2 1 University Hospital Muenster and University Children’s Hospital Muenster, Muenster, Germany 2 University of Muenster, Institute of Immunology, Muenster, Germany

Systemic onset Juvenile Idiopathic Artritis (sJIA) is considered to be an autoinflammatory disease. The S100-proteins S100A8, S100A9 and S100A12 in serum and interleukin (IL)-18 in plasma are extremely elevated in sJIA patients and have been proposed to be useful biomarkers for diagnosis, disease activity and response to therapy. Moreover, sJIA patients seem to have a defective IL-18 NK cell axis as NK cell lytic function is impaired in spite of high IL-18 levels. However, it is still unknown how the inflammatory S100 proteins and IL-18 relate to each other, and to the defective NK cell lytic function.

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.

The balance between regulatory (Treg) and effector T cells (Teff) is crucial for immune regulation in juvenile idiopathic arthritis (JIA). How methotrexate (MTX), the cornerstone treatment in JIA, influences this balance in vivo is poorly elucidated.