POS0001 PHENOTYPE AND FUNCTIONAL CHARACTERISTICS OF ANTIGEN-SPECIFIC, AUTO-REACTIVE B CELL RESPONSES REVEAL DIFFERENTIAL IMMUNOLOGICAL ACTIVITY IN PATIENTS WITH SYSTEMIC SCLEROSIS

Background:Systemic Sclerosis (SSc) is a systemic autoimmune disease that carries the highest mortality burden among the rheumatic diseases. Disease risk and course are difficult to predict in individual patients, and anti-inflammatory and B-cell depleting therapies show varying results. >95% of SSc patients harbor autoantibodies. Among those, anti-topoisomerase antibodies (ATA) and anti-centromere antibodies (ACA) are most prevalent, mutually exclusive in individual patients and associate with distinct disease phenotypes. Despite these associations, the clinical value of both ATA and ACA for patient stratification within these phenotypes is limited. Here, we hypothesized that phenotypic and functional characteristics of the underlying autoreactive B cell responses could allow insights in differential ‘immunological disease activity’ in individual patients, thereby providing indications as to potential drivers of these responses as well as granularity as to which patients may benefit from targeted interventions.Objectives:To assess phenotypic and functional characteristics of anti-topoisomerase and anticentromere specific B cell responses in individual patients with SSc.Methods:Peripheral blood mononuclear cells (PBMC) from ATA- and ACA-positive SSc patients were cultured without stimulation or in the presence of CD40L-expressing fibroblasts, IL-21 and BAFF. Following culture, ATA- and ACA-IgG and -IgA were measured in culture supernatants by ELISA. In addition, PBMC were depleted of circulating plasmablasts by fluorescence activated cell sorting (FACS), and isolated plasmablasts were cultured separately. Furthermore, the presence of antigen-specific plasmablasts was confirmed by ELISPOT. Finally, the degree of spontaneous ATA secretion was correlated to the presence or absence of interstitial lung disease (ILD; based on high-resolution computed tomography). Healthy donors and patients with rheumatoid arthritis served as controls.Results:We observed that individual ATA- and ACA-positive SSc patients harbored circulating B cells that secrete either ATA-IgG or ACA-IgG upon stimulation, depending on their serotype. In addition, we noted spontaneous secretion of ATA-IgG and, more remarkably, extensive secretion of ATA-IgA in ATA-positive patients. This degree of spontaneous, antigen-specific IgA secretion was specific for the ATA response, while spontaneous ACA-IgA secretion was undetectable in patients harboring ACA. FACS experiments and ELISPOT showed that the spontaneous ATA-IgA and -IgG secretion was attributable to circulating plasmablasts. Of note, the degree of spontaneous ATA-IgG secretion was remarkably higher in patients with ILD than in those without.Conclusion:Our findings demonstrate that individual ATA-positive SSc patients harbor activated ATA-IgG and ATA-IgA B cell responses, as indicated by the spontaneous secretion of both ATA isotypes by circulating plasmablasts. Importantly, by taking the presence of plasmablasts as a proxy for recent B cell activation, our data suggest a link between the activity of the antigen-specific B cell response and the presence of ILD. In contrast, the ACA B cell response was far less active and lacked the active IgA component, which suggests a difference in the triggers driving these autoreactive B cell responses in patients. In fact, the remarkable ATA-IgA secretion points towards a potential mucosal trigger of the ATA response, which may be continuously active in individual patients.Disclosure of Interests:None declared.

SAT0297 DIFFERENTIAL PHENOTYPES OF DISEASE-SPECIFIC AUTO-REACTIVE B CELL RESPONSES IN PATIENTS WITH SYSTEMIC SCLEROSIS

Background:Systemic Sclerosis (SSc) carries the highest mortality burden among the rheumatic diseases. >95% of SSc patients harbor autoantibodies. Anti-topoisomerase antibodies (ATA) and anti-centromere antibodies (ACA) are most prevalent, mutually exclusive in individual patients, associate with distinct disease phenotypes and predict disease. Whether and how these auto-reactive B cell responses contribute to disease, however, is currently unclear.Objectives:To delineate phenotypic and functional characteristics of anti-topoisomerase and anti-centromere specific B cell responses in individual patients with SSc.Methods:Peripheral blood mononuclear cells (PBMC) obtained from ATA- and ACA-positive SSc patients were cultured without stimulation or in the presence of CD40L-expressing fibroblasts, IL-21 and BAFF. In addition, PBMC were depleted of circulating plasmablasts (CD19+CD20-CD27++) by fluorescence activated cell sorting (FACS), and isolated plasmablasts were cultured separately. ATA- and ACA-IgG and -IgA were measured in culture supernatants by ELISA. B cell subsets were defined by flow cytometry. Healthy donors and patients with rheumatoid arthritis served as controls.Results:We observed that ATA- and ACA-positive SSc patients harbour circulating B cells that secrete either ATA-IgG or ACA-IgG upon stimulation, depending on their serotype. In addition, we noted spontaneous secretion of ATA-IgG and, more remarkably, extensive secretion of ATA-IgA in ATA-positive patients. This degree of spontaneous, antigen-specific IgA secretion was specific for the ATA response in ATA-positive patients, while spontaneous ACA-IgA secretion was undetectable in the ACA-positive patient group. FACS experiments showed that spontaneously ATA-IgA secreting B cells were primarily present in the plasmablast compartment.Conclusion:Our findings demonstrate that ATA-positive SSc patients harbour an activated ATA-IgG and ATA-IgA B cell response, as indicated by the spontaneous secretion of both ATA isotypes by circulating plasmablasts. Remarkably, the ACA B cell response was far less active and lacked the active IgA component which suggests a difference in the triggers driving these autoreactive B cell responses in patients. Moreover, the remarkable ATA-IgA secretion points towards a potential mucosal origin of the ATA response.Disclosure of Interests:Corrie Wortel: None declared, Nina van Leeuwen: None declared, Maaike Boonstra: None declared, Rene Toes: None declared, Thomas Huizinga Grant/research support from: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Consultant of: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Jeska de Vries-Bouwstra: None declared, Hans Ulrich Scherer Grant/research support from: Bristol Myers Squibb, Sanofi, Pfizer, Speakers bureau: Pfizer, Lilly, Roche, Abbvie

The Effect of Different Doses of Zearalenone in Feed on the Bioavailability of Zearalenone and Alpha-Zearalenol, and the Concentrations of Estradiol and Testosterone in the Peripheral Blood of Pre-Pubertal Gilts

The objective of this study was to determine the effect of long-term (48 days), per os administration of specific zearalenone (ZEN) doses (20 and 40 μg ZEN/kg BW in experimental groups EI and EII, which were equivalent to 200% and 400% of the upper range limit of the no-observed-adverse-effect-level (NOAEL), respectively) on the bioavailability of ZEN and the rate of changes in estradiol and testosterone concentrations in the peripheral blood of pre-pubertal gilts. ZEN and α-ZEL levels were similar until day 28. After day 28, α-ZEL concentrations increased significantly in group EI, whereas a significant rise in ZEN levels was noted in group EII. The presence of estradiol in peripheral blood plasma was not observed until day 20 of the experiment. Spontaneous secretion of estradiol was minimal, and it was determined at very low levels of up to 10 pg/mL in EI and EII groups. Testosterone concentrations ranged from 4 to 9 ng/mL in all groups. A decrease in the concentrations of both analyzed hormones was reported in the last stage of the experiment. The results of the experiment indicate that: (i) The bioavailability of ZEN in peripheral blood has low diagnostic value, (ii) exposure to low doses of ZEN induces minor changes in the concentrations of the analyzed hormones, which could lead to situational supraphysiological hormone levels and changes in endogenous hormonal balance.

Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis

ObjectiveTo examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA).MethodsDetection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo.Resultsp-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05).ConclusionsThis is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

Function suggests nano-structure: electrophysiology supports that granule membranes play dice

Cellular communication depends on membrane fusion mechanisms. SNARE proteins play a fundamental role in all intracellular fusion reactions associated with the life cycle of secretory vesicles, such as vesicle–vesicle and vesicle plasma membrane fusion at the porosome base in the cell plasma membrane. We present growth and elimination (G&E), a birth and death model for the investigation of granule growth, its evoked and spontaneous secretion and their information content. Using a statistical mechanics approach in which SNARE components are viewed as interacting particles, the G&E model provides a simple ‘nano-machine’ of SNARE self-aggregation behind granule growth and secretion. Results from experimental work, mathematical calculations and statistical modelling suggest that for vesicle growth a minimal aggregation of three SNAREs is required, while for the evoked secretion one SNARE is enough. Furthermore, the required number of SNARE aggregates (which varies between cell types and is nearly proportional to the square root of the mean granule diameter) affects and is statistically identifiable from the size distributions of spontaneous and evoked secreted granules. The new statistical mechanics approach to granule fusion is bound to have a significant changing effect on the investigation of the pathophysiology of secretory mechanisms and methodologies for the investigation of secretion.

Adaptive and Innate Immune Responsiveness toBorrelia burgdorferi sensu latoin Exposed Asymptomatic Children and Children with Previous Clinical Lyme Borreliosis

Why some individuals develop clinical manifestations in Lyme borreliosis (LB) while others remain asymptomatic is largely unknown. Therefore, we wanted to investigate adaptive and innate immune responsiveness toBorrelia burgdorferi sensu latoin exposedBorrelia-antibody-positive asymptomatic children (n=20), children with previous clinical LB (n=24), and controls (n=20). Blood samples were analyzed forBorrelia-specific interferon (IFN)-γ, interleukin (IL)-4, and IL-17 secretion by ELISPOT andBorrelia-induced IL-1β, IL-6, IL-10, IL-12(p70), and tumor necrosis factor (TNF) secretion by Luminex. We found no significant differences in cytokine secretion between groups, but a tendency towards an increased spontaneous secretion of IL-6 was found among children with previous clinical LB. In conclusion, the adaptive or innate immune responsiveness toBorrelia burgdorferi sensu latowas similar inBorrelia-exposed asymptomatic children and children with previous clinical LB. Thus, the immunological mechanisms of importance for eradicating the spirochete effectively without developing clinical manifestations of LB remain unknown.