Stinne Ravn Greisen

Increased soluble programmed death-1 (sPD-1) is associated with disease activity and radiographic progression in early rheumatoid arthritis

Objectives: Programmed death-1 (PD-1) is an immunoregulatory molecule functioning by down-regulating immune responses. PD-1 is present on follicular helper T cells (TFH) and is important in the formation of plasma cells. PD-1 exists in a bioactive soluble form (sPD-1) and is thought to be implicated in disease activity in chronic rheumatoid arthritis (RA). Method: We measured sPD-1 at baseline and 9 months after treatment initiation in plasma from early RA patients (n = 34). We tested for correlations with the Disease Activity Score using 28 joint counts (DAS28), the Health Assessment Questionnaire (HAQ) score, immunoglobulin M rheumatoid factor (IgM-RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, C-reactive protein (CRP), interleukin-21 (IL-21), and total Sharp score (TSS). We also measured sPD-1 in plasma from healthy volunteers (HV) (n = 20) and in plasma and synovial fluid (SF) from patients with chronic RA (> 8 years of disease, n = 30). We further investigated the cellular expression of PD-1 and its ligand PD-L1. Results: sPD-1 concentrations in early [median 0.421 ng/mL, interquartile range (IQR) 0.04–2.560 ng/mL] and chronic (median 0.239 ng/mL, IQR 0.184–0.584 ng/mL) RA were increased compared with HV (median 0.04 ng/mL, IQR 0.04–0.04 ng/mL) (all p < 0.005). In early RA the change in sPD-1 was associated with DAS28 (r = 0.363, p < 0.05) and HAQ score (r = 0.554, p < 0.05) and inversely with TSS at 3–5 years (r = –0.468, p < 0.05). sPD-1 concentration correlated with IgM-RF, anti-CCP antibodies, and IL-21 (all p < 0.05). PD-1 was primarily expressed by synovial memory T cells whereas PD-L1 was mainly expressed by synovial monocytes. Conclusions: The significantly elevated plasma levels of sPD-1 in early RA, the association with core disease parameters, and the inverse correlation with TSS suggest that sPD-1 is an important mediator in inflammatory and radiographic disease progression.

Spontaneous generation of functional osteoclasts from synovial fluid mononuclear cells as a model of inflammatory osteoclastogenesis

In osteoimmunology, osteoclastogenesis is understood in the context of the immune system. Today, the in vitro model for osteoclastogenesis necessitates the addition of recombinant human receptor activator of nuclear factor kappa‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). The peripheral joints of patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) are characterized by an immune‐mediated inflammation that can lead to bone destruction. Here, we evaluate spontaneous in vitro osteoclastogenesis in cultures of synovial fluid mononuclear cells (SFMCs) activated only in vivo. SFMCs were isolated and cultured for 21 days at 0.5–1.0 × 106 cells/mL in culture medium. SFMCs and healthy control peripheral blood monocytes were cultured with RANKL and M‐CSF as controls. Tartrate‐resistant acid phosphatase (TRAP) positive multinucleated cells were found in the SFMC cultures after 21 days. These cells expressed the osteoclast genes calcitonin receptor, cathepsin K, and integrin β3, formed lacunae on dentin plates and secreted matrix metalloproteinase 9 (MMP9) and TRAP. Adding RANKL and M‐CSF potentiated this secretion. In conclusion, we show that SFMCs from inflamed peripheral joints can spontaneously develop into functionally active osteoclasts ex vivo. Our study provides a simple in vitro model for studying inflammatory osteoclastogenesis.

Soluble CD206 plasma levels in rheumatoid arthritis reflect decrease in disease activity

Abstract Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and infiltration by activated macrophages. TNFα is a central mediator in this process. The mannose receptor, CD206, is a scavenger receptor expressed by M2A-macrophages and dendritic cells. It is involved in collagen internalization and degradation. The soluble form has been suggested as a biomarker of M2A-macrophage activation. The aim of this study was to investigate sCD206 plasma levels in early RA patients initiating anti-TNFα treatment. Plasma levels of sCD206 were measured by ELISA in samples from 155 early RA patients with an average symptom duration of 3 months. Patients were randomized to 12 months’ methotrexate and placebo (PLA) or methotrexate and adalimumab (ADA) treatment, followed by open-label treatment with disease-modifying anti-rheumatic drugs (DMARD) and if needed, ADA. Disease activity was assessed at baseline and after 3, 6, 12 and 24 months. Baseline plasma level of sCD206 in treatment naïve RA patients was 0.33 mg/L (CI: 0.33–0.38 mg/L) corresponding to the upper part of the reference interval for healthy controls (0.10–0.43 mg/L). In the PLA group, sCD206 levels decreased after 3 months, but did not differ from baseline after 6 months. In the ADA group, however, levels remained lower than baseline throughout the treatment period. In conclusion, initially, plasma sCD206 in early RA patients decreased in accordance with disease activity and initiation of DMARD treatment. Treatment with anti-TNFα preserved this decrease throughout the study period.

Vitamin D increases programmed death receptor-1 expression in Crohn’s disease

Background: Vitamin D modulates inflammation in Crohns disease (CD). Programmed death (PD)-1 receptor contributes to the maintenance of immune tolerance. Vitamin D might modulate PD-1 signalling in CD. Aim: To investigate PD-1 expression on T cell subsets in CD patients treated with vitamin D or placebo. Methods: We included 40 CD patients who received 1200 IU vitamin D3 for 26 weeks or placebo and eight healthy controls. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated at baseline and week 26. The expressions of PD-1, PD-L1, and surface activation markers were analysed by flow cytometry. Soluble PD-1 plasma levels were measured by ELISA. Results: PD-1 expression upon T cell stimulation was increased in CD4+CD25+int T cells in vitamin D treated CD patients from 19% (range 10 39%) to 29% (11 79%)(p = 0.03) compared with placebo-treated patients. Vitamin D treatment, but not placebo, decreased the expression of the T cell activation marker CD69 from 42% (31 62%) to 33% (19 - 54%)(p = 0.01). Soluble PD-1 levels were not influenced by vitamin D treatment. Conclusions: Vitamin D treatment increases CD4+CD25+int T cells ability to up-regulate PD-1 in response to activation and reduces the CD69 expression in CD patients.

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis

Introduction Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). Methods Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. Results Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. Conclusion EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.

Divergent effects on macrophage biomarkers soluble CD163 and CD206 in axial spondyloarthritis

Abstract The chronic joint inflammation in axial spondyloarthritis (axSpA) is characterized by infiltration of activated macrophages. The haptoglobin–hemoglobin receptor CD163 and the mannose receptor CD206 are strongly expressed on M2c and M2a macrophages, respectively. We measured the soluble forms of the receptors (sCD163 and sCD206) in plasma (PL) in two axSpA cohorts. All patients fulfil the 2009 Assessment of SpondyloArthritis International Society (ASAS) classification criteria for axSpA and/or the 1984 modified New York criteria for ankylosing spondylitis. The first cohort included anti-TNF-α treated patients with active axSpA (n = 30); the second cohort included patients in early disease stages (n = 38). Plasma sCD163 and sCD206 were both within the reference interval of healthy controls (HC), but sCD163 decreased slightly during anti-TNF-α treatment [baseline: 1.49 mg/L (IQR: 1.22–1.77 mg/L, 12 weeks: 1.29 (IQR: 1.09–1.57) mg/L, 20 weeks: 1.25 (IQR: 0.99–1.75) mg/L, 52 weeks: 1.39 (IQR: 1.15–1.78) mg/L], while sCD206 increased [baseline: 0.17 (IQR: 0.13–0.21) mg/L, 12 weeks: 0.19 (0.16–0.23) mg/L, 20 weeks: 0.20 (0.14–0.24) mg/L, 52: 0.19 (IQR: 0.14–0.23) mg/L], pointing toward a shift in polarization of involved macrophages. Plasma levels of sCD206 proved significantly higher in patients with early disease stages and definite radiological sacroiliitis (n = 10). This was not the case for sCD163. A significant increase in response to anti-TNF-α treatment, could suggest sCD206 as a marker of response to anti-TNF-α treatment, however, the potential for the two macrophage markers as diagnostic and prognostic indicators of disease in axSpA is weak.

T cell co-stimulatory factors.

Recent discoveries elucidating the complex mechanisms of T cell activation have provided important novel therapeutic targets for both autoimmune and oncological disease. The coordinated sequence of immune checkpoints that take place before the Th cell becomes a fully activated memory T cell are orchestrated by T cell costimulatory factors. These co-stimulatory factors can either activate the T cell or inhibit the activation process. Inhibition of T cell activity has clinical relevance in treatment of rheumatological disease, whereas enhancement of T cell activity has gained increasing use in oncological disease. CD28 is the best-characterized T cell co-stimulatory pathway. CD28 is the receptor for the canonical B7 ligands expressed by antigen presenting cells, CD80 (B7.1) and CD86 (B7.2), as well as for inducible T cell co-stimulator. CD80 and CD86 can alternatively signal through cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to inhibit T cells. CTLA-4 is up-regulated after antigen recognition, and can also be expressed by regulatory T cells. CTLA-4 binds to CD80 and CD86 with higher affinity than CD28, thus competitively inhibiting activation of T cells. In autoimmune disease, this system has been targeted by CTLA-4 fused to the Fc part of IgG1 (abatacept). In people with RA, abatacept decreases disease activity and slows progression, with the highest benefit seen in autoantibody-positive individuals [1]. Potential limitations of Fc:CTLA-4 treatment include that it compete with Programmed Cell Death ligand-1’s binding to CD80 (see below), which could interfere with regulatory T cell function. Studies are therefore ongoing in RA using anti-CD28 antibody treatment as an alternative modality. Antigen recognition and T cell activation are often aberrant, not only in chronic inflammation but also in malignancy, leading to generation of exhausted T cells. The CD28 system has been exploited to enhance immunemediated tumour recognition. Anti-CTLA-4 antibodies have been used to release the break on T cell activation and to block regulatory T cells, resulting in enhanced immune-mediated eradication of malignant tumours [2]. Given that inhibition of CTLA-4 also weakens self-tolerance, anti-CTLA-4 therapy causes immune-related adverse events. The most common adverse events are rash, colitis/diarrhoea, inflammatory endocrine diseases, arthralgia and arthritis [3, 4]. Surprisingly, pre-existing autoimmune joint diseases are not aggravated by antiCTLA-4 or anti-programmed cell death 1 (PD-1) treatment [3, 4]. It appears that steroids can be used concomitantly with CTLA-4 blockade to mitigate immune-related adverse events; studies thus far have shown that the beneficial effects of CTLA-4 blockade are not diminished by steroid use [3]. Interestingly, although antibody blockade of CTLA-4 is used to treat cancer, treatment with Fc:CTLA-4 in RA does not appear to increase the risk of developing malignancy [5]. However, studies are needed to elucidate further the arthritic condition described in connection with blocking T cell co-stimulatory factors. This is especially important when it comes to the clinical picture of the arthritic condition and mechanistic biomarkers, including autoantibodies. A detailed description of these patients could give us a better understanding of which immunological factors leads to arthritis, thereby also helping to identify patients at risk for developing future joint diseases. The CD28 family is not the only T cell co-stimulatory pathway. Several members of the TNF superfamily also have co-stimulatory activity. The best described are CD40, OX40 and 4-1BB, each of which has important roles in autoimmunity. CD40 is expressed by T cells and is a receptor for CD154. The CD40 CD154 pathway is essential for optimal interaction in the lymphoid follicle. Defects in this pathway result in elevated IgM levels, because the CD40 CD154 interactions are necessary to induce immunoglobulin class switching. CD154 also binds integrins involved in thrombosis. Although anti-CD154 antibody treatment reduces anti-dsDNA antibodies in lupus, it was also associated with increased thrombotic incidents [6]. OX40 is expressed after antigen recognition and is necessary for optimal T and B cell response. The OX40 ligand is expressed by antigen presenting cells. Activation of T cells by OX40 ligand makes these less responsive to the inhibitory signals from regulatory T cells. 4-1BB is expressed rapidly after antigen recognition. 4-1BB requires stimulation by both galectin-9 and its ligand (4-1BBL) to induce a signal. Therefore, 4-1BBexpressing T cells first become fully activated when they enter the arthritic joint, where galectin levels are high [7]. As for the other members of the TNF superfamily, CD40, OX40, 4-1BB and their ligands are present in a soluble, biologically active form. Anti-PD-1 has recently received much attention in oncological diseases. PD-1 is expressed by activated T cells, B cells and myeloid cells. It has two known ligands (PD-L1 and PD-L2). While PD-L1 is expressed by antigen presenting cells in addition to many other cell types, PD-L2 expression is restricted to antigen presenting cells. Both PD-L1 and PD-L2 bind additional receptors; PD-L1 binds CD80, and PD-L2 binds repulsive guidance

OP0070 Pd-L2 – A New Link between Inflammation and Bone Modulation in Rheumatoid Arthritis

Background Inflammatory conditions affect bone turnover, as is seen in rheumatoid arthritis (RA). Inflammatory cytokines in RA stimulate osteoclasts and shift the balance between bone formation and resorption. Programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2, are important for maintaining self-tolerance, and this pathway plays a prominent role in RA. Recently, repulsive guidance molecule b (RGMb) and bone morphogenetic protein (BMP) was identified as a novel receptor complex for PD-L2 (1). We previously showed the PD-1 pathway to be associated with radiographic progression in RA (2). The interaction of PD-L2 with the RGMb/BMP receptor complex could potentially provide a link between bone homeostasis and the PD-1 pathway. Objectives To investigate the possible association between bone homeostasis and the PD-1 pathway in RA. Methods We generated osteoclasts from RA synovial fluid mononuclear cells (SFMCs) (3), stimulated them with recombinant (rec) PD-1 and PD-L2, and subsequently evaluated these osteoclasts for TRAP activity, and expression of PD-1, PD-L2 and RGMb. We induced RA-like disease in PD-1-and PD-L2-KO mice by immunization with Freunds adjuvant and chick collagen, and evaluated clinical arthritis. After sacrifice, paws were paraffin embedded and stained for TRAP and osteocalcin. The femur was subjected to ex vivo DXA scanning. In addition, we stimulated normal mouse calvarial cells with the recombinant proteins in a BMP reporter assay to evaluate the potential of PD-1 and PD-L2 to affect BMP signaling. Results Recombinant PD-1 and PD-L2 increased TRAP activity in osteoclasts generated from RA SFMCs, at day 21 (p=0.046). Pre-osteoclasts in the culture expressed both PD-L2 and RGMb. The KO mice did not develop clinical signs of arthritis, ensuring intact mobility and constant bone load. DXA scan revealed that immunized PD-L2 KO mice had a higher bone mass density than other KO mice, WT and non-immunized PD-L2 KO (p=0.03). These mice did not differ histologically in TRAP and osteocalcin expression. Rec PD-L2 significantly decreased BMP signaling in the BMP reporter assay. Conclusions The clinical association between the PD-1 pathway and radiographic progression in early RA points to its importance in RA bone homeostasis. The increase in TRAP activity after incubation with recombinant PD-L2 supports this association. The increased bone mass density observed in immunized PD-L2 KO mice further supports a potential role for PD-L2 in bone homeostasis. The expression of PD-L2 in pre-osteoclasts also suggests a role for PD-L2 in bone homeostasis. These data provide a new insight into the close relation between the immune system and bone, as well as suggest a potential therapeutic target in diseases involving inflammatory bone changes. References Xiao Y, et. al Journal of Exp Med. 2014 Apr 21;3(1):81. Greisen SR, et al. Scand J Rheum. 2013 Nov 1;43(2):101–8. Greisen SR, et al. APMIS. 2015 Sep;123(9):779–86. Disclosure of Interest None declared

SAT0003 The IL-20 Receptor Axis in Early Rheumatoid Arthritis: Novel Inflammation-Independent Links Between Rheumatoid Arthritis-Associated Autoantibodies and Radiographic Progression

Background Rheumatoid arthritis (RA) is characterized by high occurrence of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs) and bone erosions. Successful treatment with biologic drugs such as anti-tumor necrosis factor alpha compromises the normal immune response increasing the risk of infections. The interleukin 20 receptor (IL-20R) axis comprising IL-19, IL-20, and IL-24 (“the IL-20R cytokines”) and their shared receptors IL-20R2/IL-20R1 and IL-20R2/IL-22R activates tissue homeostasis processes but not the immune system (Figure 1). Objectives The objective of this study was to determine the role of the IL-20R axis in early RA (eRA) with focus on associations of the three cytokines with clinical disease activity and prognosis. Methods The IL-20R cytokines were measured in plasma samples from treatment naïve early RA patients during 24 months of treatment with methotrexate, adalimumab/placebo and intra-articular glucocorticoid injections (the OPERA trial) (n=152)1. The IL-20R1 and IL-22R expression was studied in paired peripheral blood and synovial fluid cells from a different cohort of RA patients (n=15) with at least one swollen joint (for obtaining synovial fluid) by flow cytometry and confocal microscopy. Heat aggregated human gamma globulins (HAGGs) and immune complexes containing citrullinated fibrinogen (cFb-ICs) were used to stimulate myeloid cells. Osteoclasts (OCs) derived from synovial fluid cells were used to assess the effect of the IL-20R cytokines. Results The plasma concentrations of IL-20 and IL-24 (but not IL-19) were increased in eRA patients compared with HCs (both P<0.002) and decreased after 6 months of treatment (both P<0.0001). The expression of IL-22R (but not IL-20R1) was increased on monocytes from RA synovial fluid compared with RA and HC peripheral blood. The plasma concentrations of IL-20 and IL-24 were increased in RF and ACPA positive compared with negative eRA patients (all P<0.0001). HAGGs and cFb-ICs stimulated the production of the IL-20R cytokines by myeloid cells. Increased baseline plasma concentrations of IL-20 and IL-24 associated with radiographic progression (evaluated by the total Sharp-van der Heijde score) after 12 months (Spearmans rho=0.27 and 0.23, both P<0.005) and 24 months (Spearmans rho=0.19 and 0.26, both P<0.05) in the eRA patients. No associations were observed between concentrations of the IL-20R cytokines and disease activity. The IL-22R was expressed by RANK+ OC precursors and in multinucleated TRAP+ OCs and these cells were activated by IL-20 and IL-24. Conclusions Inflammation-independent associations of the IL-20R axis with RA-associated autoantibodies and radiographic progression in eRA were detected. Our data support that dual inhibition of IL-20 and IL-24 or attenuation of the shared IL-22R subunit could attenuate radiographic progression without compromising the normal immune response especially in seropositive RA. References Hørslev-Petersen et al. Ann Rheum Dis. 2013. Disclosure of Interest None declared

AB0057 Soluble pd-1 and spd-l2 are unaffected by adalimumab treatment in early ra and associated with presence of anti-ccp and igm-rf

Background Disturbance of the balance in the immune system plays a major role in rheumatoid arthritis (RA). Programmed Death 1 (PD-1) expressed by activated T cells and its ligand (PD-L2) expressed by antigen presenting cells, are involved in negative regulation of T cell receptor induced activation 1. Both PD-1 and PD-L2 exist in soluble forms (sPD-1 and sPD-L2). Soluble PD-1 is up-regulated in RA2, but sPD-L2 has to our knowledge not been investigated. Objectives To investigate sPD-1 and sPD-L2 in early RA patients and their association with disease. Methods In a longitudinal set of steroid- and DMARD-naïve RA patients (n=76, <3 months’ disease) who initiated treatment with methotrexate (MTX) or MTX and adalimumab, we measured plasma levels of sPD-L2 and sPD-1 by ELISA, at baseline (0 month) and after 3 and 12 months. To remove un-specific binding, plasma samples were saturated with mouse, bovine and human IgG. We examined for correlations (Spearman’s rho) with disease parameters (DAS28, doctors VAS, HAQ, CRP), anti-CCP and IgM-RF. Data are expressed as median values (IQR). Results Plasma levels of sPD-L2 were 3.88ng/ml (2.8-5.2) at baseline, had increased 78% after 3 months, and stayed high at 12 months (6.44ng/ml (5.5-8.1)), (both p<0.05). In contrast, plasma levels of sPD-1 showed no significant changes. Addition of adalimumab to MTX had no influence on sPD-1 or sPD-L2 plasma levels. Baseline sPD-1 and sPD-L2 plasma levels were associated with the presence of anti-CCP antibodies (rho=0.4 and 0.3, respectively, both p<0.05) and IgM-RF (rho=0.64, p<0.0001 and rho=0.4 p<0.05). Interestingly, sPD-L2 relative to sPD-1 (sPD-L2/sPD-1) showed inverse correlation with anti-CCP and IgM-RF (all rho= -0.40, p<0.05). Changes in plasma levels of sPD-L2 (0-12 months) and sPD-1 correlated with disease parameters. Conclusions Plasma levels of sPD-L2 and sPD-1 in RA are unaffected by addition of adalimumab to MTX treatment. The association between plasma levels of sPD-L2 and disease parameters suggests that sPD-L2 plays a vital role in disease progression. Also considering the close correlations with anti-CCP and IgM-RF, sPD-1 and sPD-L2 are possible new indicators of disease activity and progression. Because PD-1 is closely connected to the regulation of follicular T cells, our findings support the involvement of these T cells in the early phase of RA. References Messal, N., Serriari, N.-E., Pastor, S., Nunès, J. A. & Olive, D. PD-L2 is expressed on activated human T cells and regulates their function. Molecular Immunology 48, 2214–2219 (2011). Wan, B. et al. Aberrant regulation of synovial T cell activation by soluble costimulatory molecules in rheumatoid arthritis. J. Immunol. 177, 8844–8850 (2006). Disclosure of Interest None Declared