S de Roock

Mesenchymal stem cell therapy in proteoglycan induced arthritis

Objectives To explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA). Methods MSC were administered ip or ia after establishment of arthritis. We used serial bioluminescence imaging (BLI) to trace luciferase-transfected MSC. Mice were sacrificed at different time points to examine immunomodulatory changes in blood and secondary lymphoid organs. Results Both ip and local ia MSC injection resulted in a beneficial clinical and histological effect on established PGIA. BLI showed that MSC ip and ia in arthritic mice are largely retained for several weeks in the peritoneal cavity or injected joint respectively, without signs of migration. Following MSC treatment pathogenic PG-specific IgG2a antibodies in serum decreased. The Th2 cytokine IL-4 was only upregulated in PG-stimulated lymphocytes from spleens in ip treated mice and in lymphocytes from draining lymph nodes in ia treated mice. An increase in production of IL-10 was seen with equal distribution. Although IFN-γ was also elevated, the IFN-γ/IL-4 ratio in MSC treated mice was opposite to the ratio in (untreated) active PGIA. Conclusions MSC treatment, both ip and ia, suppresses PGIA, a non-collagen induced arthritis model. MSC are largely retained for weeks in the injection region. MSC treatment induced at the region of injection a deviation of PG-specific immune responses, suggesting a more regulatory phenotype with production of IL-4 and IL-10, but also of IFN-γ, and a systemic decrease of pathogenic PG-specific IgG2a antibodies. These findings underpin the potential of MSC treatment in resistant arthritis.

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.

A2.23 Blockade of increased autophagy in juvenile arthritis synovial fluid T cells reduces cellular activation and cytokine production

Background and objectives Juvenile Idiopathic Arthritis (JIA) is a debilitating autoimmune disease with an important role for aberrant T cell homeostasis in the pathogenesis. Autophagy is a process of lysosomal degradation that is important for (T) cellular homeostasis that has previously been genetically correlated to several auto-immune diseases. We investigated the role of autophagy in JIA synovial inflammation and its effect on T cell activity. Materials and methods PBMC from healthy donors, and PBMC and Synovial fluid cells (SFMC) from 21 JIA patients were used. Cells were cultured overnight with or without hydroxychloroquin to investigate the autophagic flux. Synovial fluid was added to HC cell cultures to investigate the role of inflammatory mediators on autophagy. Autophagy was measured with flow cytometry using the specific dye Cyto-ID or by western blot of LC3-II. The importance of autophagy for inflammatory activity of T cells in the synovium was investigated by blockade of autophagy during anti-CD3 activation and measuring proliferation and cytokine production during 4 day cultures. Results Autophagy was significantly increased in CD4+, and CD8+ T cells from SFMC which was due to a higher basal level of autophagy in SFMC and not to an increase in susceptibility to HCQ. Culturing HC and JIA PBMC and SFMC with SF or inflammatory cytokines did not affect autophagy levels, indicating that the increased autophagy in SFMC is related to a cell intrinsic phenomenon rather than induced by soluble mediators of inflammation. Indeed, cellular activation related to levels of autophagy. Additionally, blocking autophagy reduced proliferation and cytokine production. Conclusions T cells from the inflamed synovium of JIA patients show significantly increased autophagy. This phenomenon is not affected by cytokines in the SF or SF itself, but is related to cellular activation. Moreover, blocking autophagy reduces cellular activity and cytokine production. We conclude that targeting autophagy in JIA has a major potential for future therapy.

Monocytes and neutrophils in the inflammatory cascade of systemic onset Juvenile Idiopathic Arthritis

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

A8.8 Mesenchymal stromal cells suppress synovial fluid-derived T cells from juvenile idiopathic arthritis patients in vitro

Background and objectives Mesenchymal stromal cells (MSC) are multipotent cells with an immunosuppressive capacity. In the last decade the feasibility and safety of MSC therapy has been established in various inflammatory diseases. MSC are known to suppress T cell function and modulate T helper 17 (Th17) cells and regulatory T cells (Tregs), which play an important role in the pathogenesis of juvenile idiopathic arthritis (JIA). MSC may therefore serve as a new treatment option for JIA patients refractory to conventional therapies. However, it is unknown whether MSC are capable of suppressing the highly inflammatory synovial fluid mononuclear cells (SFMC), which have been shown to be resistant to suppression by Tregs. Furthermore, the effect of the inflammatory environment on MSC function is largely unknown, though it has been suggested that proinflammatory cytokines can enhance the suppressive potential of MSC. We aimed to study the in vitroimmunomodulatory effects of MSC on SFMC from JIA patients, with a focus on T cell function. In addition, we assessed the influence of the inflammatory micro-environment, i.e. proinflammatory cytokines, on the suppressive potential of MSC. Materials and methods MSC were cocultured with either peripheral blood mononuclear cells (PBMC) or synovial fluid mononuclear cells (SFMC) from JIA patients (paired samples) or PBMC from healthy controls. We analysed the effect of MSC on T cell proliferation and Th17 and Treg numbers by flow cytometry. Cytokine production was analysed in culture supernatants by Multiplex Immunoassay. Furthermore, we blocked the proinflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 to assess the result on MSC-mediated suppression. Results MSC suppressed proliferation of PB T cells and SF T cells from JIA patients dose-dependently, but patient-derived T cells were less susceptible to suppression than healthy donor-derived T cells. MSC reduced Th17 numbers and increased Treg numbers in PBMC, but not in SFMC. Addition of MSC did not clearly affect IL-17 levels in the supernatant, but TNFα production was suppressed in both PBMC and SFMC. Contrary to previous reports, blockade of TNFα, IFNγ, IL-1β and IL-6 during in vitroculture did not reduce suppression, but rather enhanced suppression in PBMC from JIA patients. Conclusions MSC suppress proliferation of synovial fluid T cells from JIA patients in a dose-dependent manner. However, patient-derived T cells are less susceptible to immunomodulation than healthy donor T cells. We propose that reduction of proinflammatory stimuli in conjunction with MSC therapy may benefit the suppressive effects of MSC in JIA.

A2.7 Interleukin-18 production upon S100 stimulation is reduced in systemic-onset juvenile idiopathic arthritis

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 D1, Vogl T2, Foell D1, Roth J2 1University Hospital Muenster and University Children’s Hospital Muenster, Muenster, Germany 2 University of Muenster, Institute of Immunology, Muenster, Germany

OP0196 The Role of Neutrophils in the Inflammatory Cascade of Systemic Onset Juvenile Idiopathic Arthritis

Background Systemic onset Juvenile Idiopathic Artritis (sJIA) is considered an autoinflammatory disease, with increased circulating neutrophil counts and extremely high serum levels of S100 proteins and interleukin (IL)-18. We have previously shown that upon stimulation with S100 proteins and ATP, peripheral blood mononuclear cells (PBMCs) of healthy donors produce high levels of IL-1β and IL-18; however this cytokine production is decreased in PBMCs of sJIA patients with active disease and partly restored when patients are in remission.1 Given the observed hyporesponsiveness of PBMCs from sJIA patients, we hypothesized that neutrophils might play an important role in the inflammatory cascade of sJIA. Objectives Our objective was to investigate the role of neutrophils in sJIA, with a focus on the relation between neutrophils, S100 proteins and IL-18. Methods We determined ex vivo cell frequencies of patients at disease onset, patients with inactive disease and healthy controls by flow cytometric analysis, using fluorescent beads for absolute counts. Neutrophils from healthy donors were sorted and subsequently primed with S100 proteins in a time series up to 20 hours with or without Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) for the last 10 minutes. Elastase and cytokine levels in supernatant were measured by Luminex, neutrophil activation markers were analysed by flow cytometry. Results Patients with new onset sJIA had significantly elevated neutrophil counts compared to healthy controls and patients with inactive disease. Immature neutrophils (CD16dimCD62L+) and normally matured neutrophils (CD16+CD62L+) were significantly higher in patients with new onset sJIA compared to healthy controls or patients with inactive disease, while hypersegmented neutrophils (CD16+CD62Ldim) were comparable between patients and controls. Given the high serum levels of S100 proteins in active sJIA, we investigated if neutrophils could be activated by these proteins. Priming with S100A9 before stimulation with fMLP increased activation and degranulation of specific granules and secretory vesicles compared to unprimed cells. This was reflected by an increased expression of CD11b, CD66b and CD35. Upon stimulation with S100A9, we observed an increase in elastase secretion in the culture supernatant, as well as a minor increase in IL-6, IL-18 and TNF-α. Conclusions Patients with new onset sJIA have increased neutrophil counts. S100 proteins prime neutrophils and enhance degranulation upon a second stimulus such as fMLP. We are now exploring whether IL-18 or other pro-inflammatory mediators elevated in sJIA plasma can also prime neutrophils. Further, we will investigate whether ex vivo neutrophils from sJIA patients are more activated or respond differently to stimulation compared to patients with inactive disease or healthy controls. References N. Ter Haar, D. Holzinger, W. de Jager, R. Scholman, S. de Roock, B. Vastert. IL-18 production upon s100 stimulation is reduced in active sJIA patients compared to sJIA patients in remission and healthy controls. Pediatric Rheumatology 2014, 12(Suppl 1):O24 Acknowledgements We would like to acknowledge D. Holzinger, T. Vogl, D. Foell and J. Roth for providing the S100 proteins. Disclosure of Interest None declared

Mesenchymal stromal cells suppress synovial fluid-derived t cells from juvenile idiopathic arthritis patients in vitro

Mesenchymal stromal cells (MSC) are multipotent cells with an immunosuppressive capacity. In the last decade the feasibility and safety of MSC therapy has been established in various inflammatory diseases, including graft versus host disease. MSC are known to suppress T cell function and modulate T helper 17 (Th17) cells and regulatory T cells (Tregs), which play an important role in the pathogenesis of juvenile idiopathic arthritis (JIA). MSC may therefore serve as a new treatment option for JIA patients refractory to conventional therapies. However, it is unknown whether MSC are capable of suppressing the highly inflammatory synovial fluid mononuclear cells (SFMC), which have been shown to be resistant to suppression by Tregs. Furthermore, the effect of the inflammatory environment on MSC function is largely unknown, though it has been suggested that proinflammatory cytokines can enhance the suppressive potential of MSC.

PReS-FINAL-2147: Immunological consequences of biologicals in juvenile idiopathic arthritis.

Since biologicals antagonize cytokines or receptors involved in the immune system, one could fear that their (long-term) use might affect the quality of the immune system, leading to a defective defense mechanism against infections and tumors, an insufficient response to vaccinations, or a flawed immunoregulation resulting in autoimmunity or autoinflammation. Finally, a biological agent itself can be handled as an antigen by the immune system, producing antibodies against the biological.

PReS-FINAL-2107: In vitro investigation into mesenchymal stromal cells as a potential therapeutic in juvenile arthritis

Mesenchymal stromal cells (MSC) are multipotent cells with an immune suppressive capacity. In the last decade the therapeutic application of these cells has been tested in inflammatory diseases like graft versus host disease and rheumatoid arthritis. Injection of MSC can be a potential therapy for juvenile idiopathic arthritis patients refractory to conventional therapies.