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Objective: Interleukin-17A (IL-17A) and tumor necrosis factor (TNF) contribute to the pathogenesis of psoriatic arthritis (PsA). However, their functional relationship in PsA synovitis has not been fully elucidated. Additionally, although CD8+ T cells in PsA have been recognized via flow cytometry as a source of IL-17A production, it is not clear whether CD8+ T cells secrete IL-17A under more physiologically relevant conditions in the context from PsA synovitis. This study was undertaken to clarify the roles of IL-17A and TNF in the synovial fluid (SF) from patients with PsA and investigate the impact of CD8+ T cells on IL-17A production. Methods: IL-17A+ T cells were identified by flow cytometry in SF samples from 20 patients with active PsA, blood samples from 22 treatment-naive patients with PsA, and blood samples from 22 healthy donors. IL-17A+ T cells were sorted from 12 PsA SF samples and stimulated using anti-CD3/anti-CD28 or phorbol myristate acetate (PMA) and ionomycin ex vivo, alone (n = 3) or together with autologous monocytes (n = 3) or PsA fibroblast-like synoviocytes (FLS) (n = 5–6). To evaluate the differential allogeneic effects of neutralizing IL-17A and TNF, SF CD4+ T cells and PsA FLS cocultures were also used (n = 5–6). Results: Flow cytometry analyses of SF samples from patients with PsA showed IL-17A positivity for CD4+ and CD8+ T cells (IL-17A, median 0.71% [interquartile range 0.35–1.50%] in CD4+ cells; median 0.44% [interquartile range 0.17–1.86%] in CD8+ T cells). However, only CD4+ T cells secreted IL-17A after anti-CD3/anti-CD28 activation, when cultured alone and in cocultures with PsA monocytes or PsA FLS (each P < 0.05). Remarkably, CD8+ T cells only secreted IL-17A after 4- or 72-hour stimulation with PMA/ionomycin. Anti–IL-17A and anti-TNF treatments both inhibited PsA synovitis ex vivo. Neutralizing IL-17A strongly inhibited IL-6 (P < 0.05) and IL-1β (P < 0.01), while anti-TNF treatment was more potent in reducing matrix metalloproteinase 3 (MMP-3) (P < 0.05) and MMP-13. Conclusion: CD8+ T cells, in contrast to CD4+ T cells, in SF specimens obtained from PsA patients did not secrete IL-17A following T cell receptor activation. Overlapping, but distinct, effects at the level of inflammatory cytokines and MMPs were found after neutralizing IL-17A or TNF ex vivo in a human model of PsA synovitis.

Background Chronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function. Methods Th17/Th22 (CXCR3−CCR4+), Th17.1 (CXCR3+CCR4−), DP (CXCR3+CCR4+), and DN (CXCR3−CCR4−) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry. Results Th17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation. Conclusions Human CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.

AimTo identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients.Materials and methodsDNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months.ResultsBaseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs.ConclusionNo baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.

ObjectivesMethotrexate (MTX) is currently the recommended first-line therapy for treating psoriatic arthritis (PsA), despite lacking clear evidence. No estimates of efficacy of MTX in usual care and no clear MTX responsive clinical or laboratory variables are currently available. This study describes the response to MTX monotherapy in newly diagnosed patients with PsA in usual care. Second, we compared clinical variables and cytokine profiles in patients responding and not responding to MTX monotherapy.MethodsWe used data collected in the Dutch southwest Early Psoriatic Arthritis cohoRt study to select patients with PsA with oligoarthritis or polyarthritis, and at least 1 year follow-up. We analysed disease activity at 6 months of patients who started MTX monotherapy and still used MTX monotherapy 1 year after diagnosis. Cytokine profiles were determined at baseline and after 3 and 6 months with a bead-based multi-immunoassay.ResultsWe identified 219 patients of which 183 (84%) patients started MTX monotherapy within 6 months after diagnosis. 90 patients used MTX monotherapy throughout the first year of which 44 patients (24%) reached minimal disease activity(MDA) at 6 months, decreasing to 33 patients (18%) after 1 year. Non-responders had significantly higher concentrations of interleukin (IL) 23 and IL-10 before and during MTX therapy.ConclusionsOur results showed that only 18% of patients with PsA are in sustained MDA after 1 year of MTX monotherapy and non-responders more often had IL-23-driven disease. Our results indicate the need for more treat-to-target and personalised therapy strategies in PsA.

The IL‐23/Th17 axis has been implicated in the development of autoimmune diseases, such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). RA and PsA are heterogeneous diseases with substantial burden on patients. Increasing evidence suggests that the IL‐23 signaling pathway may be involved in the development of autoimmunity and erosive joint damage. IL‐23 can act either directly or indirectly on bone forming osteoblasts as well as on bone resorbing osteoclasts. As IL‐23 regulates the activity of cells of the bone, it is conceivable that in addition to inflammation‐mediated joint erosion, IL‐23 may play a role in physiological bone remodeling. In this review, we focus on the role of IL‐23 in autoimmune arthritis in patients and murine models, and provide an overview of IL‐23 producing and responding cells in autoimmune arthritic joints. In addition, we discuss the role of IL‐23 on bone forming osteoblasts and bone resorbing osteoclasts regarding inflammation‐mediated joint damage and bone remodeling. At last, we briefly discuss the clinical implications of targeting this pathway for joint damage and systemic bone loss in autoimmune arthritis.

Purpose: After the age of fifty years, the percentage of females with degenerative disorders, including osteoarthritis (OA), increases rapidly. It has been suggested that menopause, and the change in hormones during menopausal transition, influences the development of these disorders. During menopausal transition, not only the hormone levels change, but also metabolism and inflammatory responses. Investigating the influence of menopausal changes, including protein change, on the development of degenerative disorders is difficult, because these changes occur slowly. However, identifying these changes is crucial to develop novel female-specific prevention strategies and therapies for these disorders. Therefore, we propose the use of an unique and novel human model to investigate the influence of menopause on the development of degenerative disorders by modelling a “sudden menopause”, in which we expect that menopausal-related changes will occur faster: the Females discontinuing Oral Contraceptives Use at Menopausal age (FOCUM) model. This model does not have the limitations in generalizability of animal models. Nor does it have the limitations of subsequent hormonal support, malignancy and its associated treatments that are pertinent to human models with a sudden menopause caused by ovariectomy.The aim of our study is to develop the FOCUM model as a disease model for the development of OA. We want to identify when changes, especially in proteins, occur after a “sudden menopause” and which of these changes are associated with the development of OA after two years. Furthermore, we want to explore if the model can also be used to learn more about the development of cardiovascular diseases, diabetes, osteoporosis and tendinopathies. In this abstract we will report on the feasibility of our inclusion. Our aim is to include 50 female participants from the general population in and nearby Rotterdam within one year. Methods: Our study design is a pilot observational prospective cohort study with two years of follow-up. Our main questions for this pilot study are: will the inclusion of participants be feasible and what are the important time points to find changes in proteins? Females between 50 and 60 years of age, who are currently using a combined oral contraceptive and started oral contraceptive use before the age of 45, are recruited from pharmacies and (if necessary) general practices. In general, females are advised to stop oral contraceptive use at the age of 52. Females who are willing to stop oral contraceptive use at short term are invited to participate. Different measurements are performed before (T0 = 0 to 30 days) and after (T1 = 6 weeks;T2 = 6 months;T3 = 1 year;T4 = 2 years) stopping oral contraceptive use. At every time point of measurements a questionnaire is filled in, a normal photograph of both hands is taken and a sample of blood is drawn to measure hormones, proteins, glucose, cholesterol, DNA-methylation and immunological aspects of cells. At the first and final time point of measurements, also a physical examination, an radiograph of both hands, an MRI scan of one knee, a DXA-scan, and an ultrasound tissue characterization of one Achilles tendon are performed. To establish which time points of measurements are most relevant to find changes in proteins after hormone change, we will examine changes in serum levels of approximately 150 different inflammation and cardiometabolic related proteins by using the Olink-technology at different time points of measurements. At the first stage of this study, we will use the Principal Component Analysis on the change in protein levels over time. Thereafter, we will test for the association between principal components and hormone change. At the second stage of this study, we will relate these principal components to various OA outcomes. Results: In January 2020, we sent invitations to 108 local pharmacies to participate in this study. At that time, a total of 55 pharmacies were willing to participate. The participating pharmacies were asked to search in their own information system to identify all possible eligible subjects, based on age and oral contraceptive use, and to send an invitation letter in their name to these subjects. Unfortunately, due to COVID restrictions we started sending these invitation letters by the end of June 2020, four months later than planned. Until now, we have sent 534 invitations from 26 pharmacies to possible eligible subjects. A total of 107 females replied positively and gave permission to provide them more information about the study. After giving information and screening for in- and exclusion criteria, we now have 25 eligible participants who gave informed consent. Of them, 16 participants have had their first (baseline) measurements of 2,5 hours at the Erasmus MC. With the 29 participating pharmacies left, we expect to reach the targeted number of 50 inclusions by February 2021 and to report about the first stage of this study. Conclusions: At this time, we have included half of our study population. So the inclusion of 50 female participants for this study within one year seems to be feasible. By performing different measurements before and after stopping oral contraceptive use, we will be able to investigate when and which menopausal changes occur and which of these changes can be related to OA. With this model we hope to learn more about the influence of menopause on the development of OA, but also of cardiovascular diseases, diabetes, osteoporosis and tendinopathies, in order to develop new prevention strategies and therapies for these diseases.

Autoimmune diseases are characterized by an aberrantly activated immune system, resulting in tissue damage and functional disability in patients. An important therapeutic goal is to restore the deregulated immunological balance between pro- A nd anti-inflammatory T cells. This imbalance is illustrated by elevated levels and activity of memory Th17 cell populations, such as Th17, Th1/Th17, and Th17.1 cells, in various autoimmune diseases. These cells are characterized by the chemokine receptor CCR6, RORC expression and production of IL-17A, IFNγ, and TNFα. Using rheumatoid arthritis (RA) as a model of autoimmune disease, we here demonstrate that pro-inflammatory memory CCR6+ Th cells can switch into anti-inflammatory cells with regulatory capacity using the active vitamin D metabolite 1,25(OH)2D3. Memory CCR6+ Th cells, excluding Tregs, were sorted from healthy controls or treatment-naive patients with early rheumatoid arthritis (RA) and cultured with or without 1,25(OH)2D3. Treatment with 1,25(OH)2D3 inhibited pro-inflammatory cytokines such as IL-17A, IL-17F, IL-22 and IFNγ in memory CCR6+ Th cells from both healthy controls and RA patients. This was accompanied by induction of anti-inflammatory factors, including IL-10 and CTLA4. Interestingly, these formerly pathogenic cells suppressed proliferation of autologous CD3+ T cells similar to classical Tregs. Importantly, the modulated memory cells still migrated toward inflammatory milieus in vitro, modeled by RA synovial fluid, and retained their suppressive capacity in this environment. These data show the potential to reset the pathogenic profile of human memory Th cells into non-pathogenic cells with regulatory capacity.

Defects in Fas function correlate with susceptibility to systemic autoimmune diseases like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE). C57BL/6 lpr (B6/lpr) mice are used as an animal model of ALPS and develop a mild SLE phenotype. Involvement of interleukin-17A (IL-17A) has been suggested in both phenotypes. Since IL-17 receptor A is part of the signaling pathway of many IL-17 family members we investigated the role of IL-17 receptor signaling in disease development in mice with a B6/lpr background. B6/lpr mice were crossed with IL-17 receptor A deficient (IL-17RA KO) mice and followed over time for disease development. IL-17RA KO/lpr mice presented with significantly enhanced lymphoproliferation compared with B6/lpr mice, which was characterized by dramatic lymphadenomegaly/splenomegaly and increased lymphocyte numbers, expansion of double-negative (DN) T-cells and enhanced plasma cell formation. However, the SLE phenotype was not enhanced, as anti-nuclear antibody (ANA) titers and induction of glomerulonephritis were not different. In contrast, levels of High Mobility Group Box 1 (HMGB1) and anti-HMGB1 autoantibodies were significantly increased in IL-17RA KO/lpr mice compared to B6/lpr mice. These data show that lack of IL-17RA signaling aggravates the lymphoproliferative phenotype in B6/lpr mice but does not affect the SLE phenotype.

Throughout life, bone is continuously remodelled. Bone is formed by osteoblasts, from mesenchymal origin, while osteoclasts induce bone resorption. This process is tightly regulated. During inflammation, several growth factors and cytokines are increased inducing osteoclast differentiation and activation, and chronic inflammation is a condition that initiates systemic bone loss. Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease that is characterised by active synovitis and is associated with early peri-articular bone loss. Peri-articular bone loss precedes focal bone erosions, which may progress to bone destruction and disability. The incidence of generalised osteoporosis is associated with the severity of arthritis in RA and increased osteoporotic vertebral and hip fracture risk. In this review, we will give an overview of different animal models of inflammatory arthritis related to RA with focus on bone erosion and involvement of pro-inflammatory cytokines. In addition, a humanised endochondral ossification model will be discussed, which can be used in a translational approach to answer osteoimmunological questions.

Background Despite recent improvements in the treatment of rheumatoid arthritis (RA), an insufficient treatment response and the development of treatment resistance in many patients illustrates the need for new therapeutic strategies. Chronic synovial inflammation could be suppressed by targeting RA synovial fibroblast (RASF) activation by, for example, interleukin (IL)-17A-producing CCR6+ T helper memory (memTh) cells. Here, we modulated this interaction by combining the active vitamin D metabolite 1,25(OH)2D3 with dexamethasone (DEX) and explored the potential therapeutic applications. Methods CCR6+ memTh cells from peripheral blood mononuclear cells (PBMCs) of healthy donors or treatment-naive early RA patients were cultured alone or with RASF from established RA patients for 3 days and treated with or without 1,25(OH)2D3, DEX, or etanercept. Treatment effects were assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Results 1,25(OH)2D3, and to lesser extent DEX, reduced production of the pro-inflammatory cytokines IL-17A, IL-22, and interferon (IFN)γ in CCR6+ memTh cells. Tumor necrosis factor (TNF)α was only inhibited by the combination of 1,25(OH)2D3 and DEX. In contrast, DEX was the strongest inhibitor of IL-6, IL-8, and tissue-destructive enzymes in RASF. As a result, 1,25(OH)2D3 and DEX additively inhibited inflammatory mediators in CCR6+ memTh-RASF cocultures. Interestingly, low doses of mainly DEX, but also 1,25(OH)2D3, combined with etanercept better suppressed synovial inflammation in this coculture model compared with etanercept alone. Conclusion This study suggests that 1,25(OH)2D3 and DEX additively inhibit synovial inflammation through targeting predominantly CCR6+ memTh cells and RASF, respectively. Furthermore, low doses of DEX and 1,25(OH)2D3 enhance the effect of TNFα blockade in inhibiting RASF activation, thus providing a basis to improve RA treatment.

Background There is currently a knowledge gap on early pathogenesis prior to Rheumatoid Arthritis (RA) diagnosis. Additionally, current medication available for RA treatment has not been developed for prevention. Collagen induced arthritis (CIA) could aid in extending knowledge on early RA pathogenesis and testing the preventive effects of medicines. Objectives In this study we sought a marker that can differentiate mice prior to clinically manifest disease into their future CIA status with the aim to facilitate research into early disease processes and preventive treatment strategies. Methods Blood was obtained at time points prior (days 12 and 19) and after clinically manifest disease (days 27 and 35) during CIA. Antibodies against bovine and mouse collagen type II (mCII) were measured from plasma by ELISA. CD4+CCR6+ memory Th cells as well as other T cell types were determined in blood. Cytokines and chemokines were detected in plasma by Luminex. Mice were divided into CIA negative and CIA positive groups based on CIA score reached on day 35. Results Antibodies against mCII of the IgG2a isotype differed prior to clinically manifest disease but are not suitable as a differentiation marker. CD4+CCR6+ memory Th cells in blood differed only at day 35. The same holds for IL-6, TNFα and CXCL2. In contrast, CXCL1 differed prior to clinically manifest disease with an AUC significantly better (p=0.003) than random. Conclusions Here we identified CXCL1 as a marker that can differentiate mice prior to clinically manifest disease into CIA positive and CIA negative mice. This might help facilitate research into early disease processes and preventive pre-clinical treatment strategies. Disclosure of Interest None declared

Background Although treatment of rheumatoid arthritis (RA) has significantly improved during the past decades, many patients do not adequately respond or become resistant to current treatments. It is currently unknown why some patients respond well and others do not, and how the response rate could be improved. Vitamin D has strong immunomodulatory properties and it has been shown RA patients have a lower serum 25(OH)D level than healthy individuals. Moreover, vitamin D levels are correlated with disease severity. 1 2 Interestingly, in vitro studies have shown that vitamin D augments the suppressive effects of etanercept in a simplified model for synovial inflammation. 3 This suggests that vitamin D could improve the therapeutic response to etanercept in RA patients. Objectives To investigate if etanercept response is related to serum vitamin D (25(OH)D) levels in RA patients. Methods For this study, data were used from the tREACH trial, a multicenter stratified single blinded randomised clinical trial. RA patients, according to the 2010 classification criteria, who started with etanercept within the first 12 months of the study were included in the analysis. Serum vitamin D (25(OH)D) levels were determined at the start of treatment (Tstart) and 3 months later using the LIAISON 25 OH Vitamin D TOTAL assay. Correlation coefficients between vitamin D levels and the disease activity score (DAS) were calculated. Treatment response was determined with the EULAR response criteria, and difference in response rates was assessed using Chi-Square tests. Results 91 patients started etanercept in the first 12 months of the study, of which 24 did not have serum for 25(OH)D measurements at both start of treatment and three months later. Therefore, a total of 67 patients was included in this study, of which 82% was female. At baseline, 45 (67%) and 48 (73%) were positive for rheumatoid factor and anti-citrullinated protein antibodies, respectively. DAS after etanercept treatment was weakly inversely correlated with serum 25(OH)D after treatment (r=-0.29, p=0.02) and the change in 25(OH)D during treatment (r=-0.25, p=0.04). After correcting for DAS and serum 25(OH)D at the start of treatment the aforementioned correlations were still found. Importantly, EULAR response rate was significantly lower in patients who were vitamin d-deficient at the start of treatment (34.6% vs 59.4%) and in patients with decreasing 25(OH)D levels during treatment (39.2% vs 57.7%) (figure 1). Conclusions RA patients with a serum 25(OH)D level below 50 nmol/L at the start of etanercept treatment or a decreasing level during treatment have a lower EULAR response rate. Therefore, increasing serum 25(OH)D level in vitamin D deficient patients may be important to achieve optimal effects of TNFα blocking therapy. References [1] Lee YH, Bae SC. Clin Exp Rheumatol2016;34(5):827–33. [2] Lin J, Liu J, Davies ML, et al. PLoS One2016;11(1):e0146351. [3] van Hamburg JP, Asmawidjaja PS, Davelaar N, et al. Ann Rheum Dis2012;71(4):606–12. Disclosure of Interest None declared

Background IL-17RD is a member of the IL-17 receptor family. In contrast to the other IL-17 receptors, IL-17RA, -RB, -RC and -RE, little is known about the ligand and function of IL-17RD. Recently, IL-17RD has been described to negatively regulate a selection of IL-17A responsive genes. IL-17RD is therefore proposed to limit IL-17A signalling. Objectives In this study we examined IL-17RD expression in multiple cells types and its role in the development of collagen induced arthritis. Methods Human synovial fibroblasts from Rheumatoid Arthritis (RA) patients were stimulated with tumour necrosis factor α (TNFα), interleukin 1 β (IL-1β) or IL-17A for multiple time points. IL-17RD expression levels were measured via qPCR. Collagen induced arthritis (CIA) was induced in IL-17RD knockout mice and wildtype littermates. At days 1 and 21, mice were immunised intradermally with chicken collagen type II in complete Freund’s adjuvant (CFA). Mice were scored 3 times a week for clinical disease defined as swollen joints with a maximum score of 8. Due to ethical reasons, mice were removed from the experiments when they reached a score of 6. CD4+ memory T cells, CD8+ memory T cells, CD19+ B cells and monocytes were isolated from WT spleens and analysed for IL-17RD expression. Blood neutrophil migration assays were performed in vitro using WT and IL-17RD deficient (IL-17RD KO) mouse synovial fibroblasts. Results Human synovial fibroblasts from RA patients have baseline expression of IL-17RD. Upon stimulation with TNFα a significant downregulation of IL-17RD expression was measured from 24 hours onwards. IL1β stimulation had a similar effect as TNFα on IL-17RD expression. Lack of IL-17RD did not result in differences in CIA severity, but the incidence of CIA was reduced. IL-17RD is mainly expressed in synovial fibroblasts. IL-17RD KO synovial fibroblasts attract less neutrophils likely by lower production of neutrophil attractants. Conclusions An inflammatory environment causes synovial fibroblasts to downregulate IL-17RD expression. Lack of IL-17RD reduces the incidence CIA, which is an IL-17-driven model. The decrease in CIA incidence is likely explained via the reduced attraction of neutrophils to the site of inflammation. Disclosure of Interest None declared

Introduction TNF and IL-17A are proinflammatory cytokines critically involved in the pathogenesis of psoriatic arthritis (PsA). Currently, targeting TNF is the first choice of a biologic disease-modifying antirheumatic drug (bDMARD) in PsA. However, up to 30% of patients receiving anti-TNF monotherapy fail to respond and require switching to a second TNF-inhibitor or bDMARD with different modes of action. Strategies targeted at neutralising IL-17A have shown beneficial effects on skin, enthesitis, dactylitis and joint inflammation. Objectives We explore the effect of neutralising IL-17A versus anti-TNF on the expression of proinflammatory cytokines and metalloproteinases (MMPs) and whether dual-therapy targeting TNF and IL-17A may have superior activity than treatment with either agent alone. Methods An allogeneic co-culture system was used comprising synovial fluid T helper (Th) memory cells and synovial fibroblasts (SF), derived from patients with active PsA. Anti-CD3 and CD28 stimulation was used during culture, and anti-IL-17A antibody (Secukinumab), anti-TNFα antibody (Adalimumab), or the combination were added. PsA unstimulated synovial Th memory cells co-cultured with PsA SF were included as a control group as well as an isotype antibody control group. After 72 hours, supernatants were harvested for ELISA and cells were lysed for qPCR analysis. Results Anti-TNF Ab treatment had no effect on IL-17A levels and neutralisation of IL-17A did not influence the TNF production in co-culture system. Both anti-TNF and anti-IL-17A single treatment significantly inhibited the production of IL-8 and reduced the mRNA expression of IL-1β. Interestingly, neutralising IL-17A resulted in a significant suppression of IL-6 level which was not reduced by anti-TNF. Anti-TNF inhibited the production of MMP-3 and only the combination of anti-TNF and anti-IL-17A resulted in a significant suppression of MMP-1 levels and MMP-9 mRNA expression. MMP-13 mRNA expression was significantly suppressed by anti-TNF but not by anti-IL-17A. However, neutralising both showed a significant improvement in downregulating MMP-13 mRNA expression compared to single treatment. Moreover, anti-IL-17A reduced RANK mRNA expression significantly compared to anti-TNF, but no additive effect was noted for combination blocking. Interestingly, only neutralising both IL-17A and TNF significantly reduced the mRNA expression of RANKL. OPG mRNA expression was not influenced by either treatment or both. Conclusions Neutralising IL-17A or TNF in the PsA synovial T cell–SF co-culture system resulted in overlapping but also distinct effects on proinflammatory cytokine expression. TNF inhibition markedly suppress different MMPs with mostly an additional effect upon neutralisation of IL17A. Neutralisation both IL-17A and TNF is needed to downregulate RANKL expression which changes the RANKL/OPG balance. Together, these data suggest that dual therapy targeting IL-17A and TNF may be superior in their activity to protect against erosive arthropathy in PsA than treatment with either agent alone. Disclosure of interest X. Xu: None declared, N. Davelaar: None declared, A.-M. Otten-Mus: None declared, P. Asmawidjaja: None declared, J. Hazes: None declared, D. Baeten: None declared, M. Vis: None declared, R. Bisoendial: None declared, E. Lubberts Grant/research support from: Novartis

Introduction Psoriasis is an autoimmune skin disease affecting around 0.6% to 3% of the whole population with detrimental physical and societal impacts. Previously, we established a psoriasis-like skin inflammation model in mice using topical application of imiquimod (IMQ). This model successfully re-captures all critical features of clinical psoriasis such as keratinocyte hyperproliferation, munro’s microabscesses, and shares a similar infiltration profile of various immune cells. Previous data suggest up-regulation of IL-10, but its role in this psoriasiform model is not clear. Objectives To investigate the role of IL-10 in the IMQ-induced psoriasis-like skin inflammation. Methods Psoriasis-like skin inflammation was induced by topical application of imiquimod (Aldara) for 5 or 10 days. Mice were injected intraperitoneally with anti-IL-10 or an isotype control antibody or subcutaneously with dexamethasone. Back skin of mice was scored for up to 10 days using a modified Psoriasis Area and Severity Index (PASI) score system adapted from clinical PASI score. Inflammation and skin thickness were scored histologically. Gene expression and immune cells in the skin were analysed using RT-PCR and flow cytometry, respectively. Results At day 10, both skin thickness and scaling score were significantly higher after neutralising IL-10 compared to isotype control, or either group compared to dexamethasone-treated animals. At days 5 and 10, H and E staining confirmed that epidermal thickness was more prominent in anti-IL-10 treated mice compared to isotype control or dexamethasone-treated mice, with more profound differences at day 10. Ki-67 staining for proliferating keratinocytes showed more proliferation at the epidermal basal layer after neutralising IL-10. In addition, significant more infiltration of neutrophils was found in skin at day 10. At day 5, IL-23/IL-17 pathway cytokines were more significantly upregulated in anti-IL-10 group than the isotype control group, while at day 10, a significant upregulation of IL-19, IL-24 expression were found in anti-IL-10 group compared to isotype control. Conclusions IL-10 regulates skin thickness and scaling during psoriasis-like skin inflammation. Furthermore, our data suggested that IL-10 might influence psoriatic symptoms through dampening of IL-23/IL-17 axis in early phase (day 5) and reducing IL-19 and IL-24 expression at late stage (day 10). The negative feedback signal of IL-10 partially explains the observed decrease of inflammation in imiquimod-induced skin inflammation after day 5. Disclosure of interest X. Xu: None declared, E. Prens: None declared, E. Florencia: None declared, L. Boon: None declared, P. Asmawidjaja: None declared, A.-M. Otten-Mus: None declared, E. Lubberts Grant/research support from: Novartis

It is believed that therapy for rheumatoid arthritis (RA) is the most effective and beneficial within a short time frame around RA diagnosis. This insight has caused a shift from research in patients with established RA to patients at risk of developing RA and recently diagnosed patients. It is important for improvement of RA therapy to understand when and what changes occur in patients developing RA. This is true for both seropositive and seronegative patients. Activation of the immune system as presented by autoantibodies, increased cytokine and chemokine production, and alterations within several immune cells occur during RA development. In this review we describe RA pathogenesis with a focus on knowledge obtained from patients with arthralgia, pre-RA and recently diagnosed RA. Connections are proposed between altered immune cells, cytokines and chemokines, and events like synovial hyperplasia, pain and bone damage.

Interleukin-17-expressing CD4 + T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6 + CXCR3 +), and not Th17 (CCR6 + CXCR3 -) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6 + and CCR6 - CD8 + T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6 + CXCR3 + CCR4 -) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

The discovery that the IL-23-IL-17 immune pathway is involved in many models of autoimmune disease has changed the concept of the role of T-helper cell subsets in the development of autoimmunity. In addition to T(H)17 cells, IL-17 is also produced by other T cell subsets and innate immune cells; which of these IL-17-producing cells have a role in tissue inflammation, and the timing, location and nature of their role(s), is incompletely understood. The current view is that innate and adaptive immune cells expressing the IL-23 receptor become pathogenic after exposure to IL-23, but further investigation into the role of IL-23 and IL-17 at different stages in the development and progression of chronic (destructive) inflammatory diseases is needed. Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are the two most common forms of chronic immune-mediated inflammatory arthritis, and the IL-23-IL-17 axis is thought to have a critical role in both. This Review discusses the basic mechanisms of these cytokines in RA and SpA on the basis of findings from disease-specific animal models as well as human ex vivo studies. Promising therapeutic applications to modulate this immune pathway are in development or have already been approved. Blockade of IL-17 and/or T(H)17-cell activity in combination with anti-TNF therapy might be a successful approach to achieving stable remission or even prevention of chronic immune-mediated inflammatory diseases.

Scope: Hydroxytyrosol acetate (HTy-Ac), an extra virgin olive oil (EVOO) polyphenol, has recently been reported to exhibit antioxidant and anti-inflammatory effects on LPS-stimulated macrophagesand ulcerative colitis. This study was designed to evaluate dietary HTy-Ac supplementation effects on collagen-induced arthritis (CIA) in mice. Methods and results: DBA-1/J mice were fed from weaning with 0.05% HTy-Ac. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrificed 42 days after first immunization. Blood was recollected and paws were histological and biochemically processed. HTy-Ac diet significantly prevent edarthritis development and decreased serum IgG1 and IgG2a, cartilage olimeric matrix protein (COMP) and metalloproteinase-3 (MMP-3) levels, as well as, pro-inflammatory cytokines levels (TNF-alpha, IFN-gamma, IL-1 beta, IL-6 and IL-17A). The activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-kappa B) pathways were drastically ameliorated whereas nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions were significantly up-regulated in those mice fed with HTy-Ac. Conclusion: HTy-Ac improved the oxidative events and returned pro-inflammatory proteins expression to basal levels probably through JAK/STAT, MAPKs and NF-kappa B pathways. HTy-Ac supplement might provide a basis for developing a new dietary strategy for the prevention of rheumatoid arthritis.

Background IL17RD is a member of the IL17 receptor family. In contrast to the other IL17 receptors, IL17RA, -RB, -RC and -RE, little is known about the ligand and function of IL17RD. Recently, IL17RD has been described to negatively regulate a selection of IL17A responsive genes. IL17RD is therefore proposed to limit IL17A signalling. In this study we examined how IL17RD expression could be regulated and whether a reduction of IL17RD contributes to the development of Rheumatoid Arthritis. Materials and Methods Human synovial fibroblasts from Rheumatoid Arthritis (RA) patients were stimulated with tumour necrosis factor α (TNFα), interleukin 1 β (IL1β) or IL17A for 4 and 24 hours. Human synovial fibroblasts were further stimulated with TNFα for 48 and 72 hours. IL17RD expression levels were measured via qPCR. Collagen induced arthritis (CIA) was induced in IL17RD knockout mice and wildtype littermates. At days 1 and 21, mice were immunised intradermally with chicken collagen type II in complete Freund’s adjuvant (CFA). Mice were scored 3 times a week for clinical disease defined as swollen joints with a maximum score of 8. Due to ethical reasons, mice were removed from the experiments when they reached a score of 6. Results Human synovial fibroblasts from RA patients have baseline expression of IL17RD. Upon stimulation with TNFα a significant downregulation of IL17RD expression was measured from 24 hours onwards. IL1β stimulation had a similar effect as TNFα on IL17RD expression, resulting in a significant downregulation at 4 and 24 hours. Lack of IL-17RD did not result in differences in CIA severity, but preliminary data suggest changes in the incidence between IL-17RD knockout mice compared to wild type littermates. Conclusions An inflammatory environment causes synovial fibroblasts to downregulate IL17RD expression. Lack of IL17RD did not influence the severity of CIA.

Background The interleukin (IL)−23/IL-17A immune pathway is critical for the development of autoimmune arthritis. Moreover, systemic exposure of IL-23 induced chronic arthritis, increased osteoclast differentiation and systemic bone loss in mice. However, the role of IL-23 on normal and pathologic bone remodelling is not fully elucidated. We studied the role of IL-23R signalling on normal bone phenotype and bone damage during a T cell-mediated inflammatory arthritis in vivo and osteoclast differentiation and activity in vitro. Materials and methods For antigen-induced arthritis (AIA), WT and IL-23RGFP+/GFP+ (IL-23R/-) mice were immunised with methylated bovine serum albumin (mBSA) in supplemented Complete Freund’s Adjuvant. After 7 days mice were injected in the knee joints with mBSA. Mice were macroscopically scored at given time points and knees were used for histological analysis. Naive WT and IL-23R/- bone marrow cells (BM) were cultured towards osteoclasts with MCSF, RANKL and +/- IL-23 and assessed for tartrate-resistant acid phosphatase (TRAP) staining, qPCRs and bone resorption. Femurs were used for MicroCT analysis and three-point bending test. Results During AIA the onset of disease was not prevented in IL-23R-/- mice. However, the progression of arthritis was diminished, resulting in significantly less disease. Histological analysis revealed significantly less inflammation-mediated bone damage in knees of IL-23R/- versus WT mice. In vitro, IL-23 had no direct effect on the number of TRAP+ cells, but increased bone resorption by osteoclasts. Interestingly, microCT data revealed a low bone mass phenotype in IL-23R-/- mice, with lower trabecular bone volume fraction, thickness and number as well as lower cortical volume and thickness. Preliminary three-point bending data show significantly reduced maximum force in IL-23RKO femurs. Conclusions In vitro, IL-23 is involved in the activation of osteoclasts. Whereas T cell-driven inflammatory bone erosion is IL-23R signalling mediated, during normal bone remodelling IL-23R signalling is important for the maintenance of bone mass.

Background In many human autoimmune diseases, including rheumatoid arthritis (RA), vitamin D is associated with disease suppression. Furthermore, experimental collagen-induced arthritis (CIA) is prevented by 1,25(OH)2D3 supplementation, the active form of vitamin D. Since high doses of 1,25(OH)2D3 in humans can cause serious side effects, understanding the mechanism behind the suppression may lead to new therapeutic targets. Although currently this mechanism is not known, it is hypothesised that Th17 cells play an important role in the immunomodulatory effect of vitamin D. Here we study the effect of 1,25(OH)2D3 on the differentiation of Th17 cells and the underlying mechanism in the context of the autoimmune-mediated CIA model. Materials and methods DBA/1 mice were immunised with type II collagen (CII) in complete Freund’s adjuvant. 10 days after immunisation the mice were sacrificed and spleens were harvested. Total CD4+, naive CD4+ or effector CD4+ T cells were isolated and cultured for 3–7 days under Th17 polarising conditions with or without 1,25(OH)2D3. Results 1,25(OH)2D3 inhibits Th17 differentiation in total CD4+ T cells in CII-immunised mice. Interestingly, 1,25(OH)2D3 induces IL-4 and IL-10 in these cells. Therefore we investigated whether the effect of 1,25(OH)2D3 on Th17 differentiation was dependent on IL-4 or IL-10. In both naive and effector CD4+ T cells from CII-immunised mice, 1,25(OH)2D3 inhibited Th17 differentiation as demonstrated by decreased IL-17A, IL-17F and IL-22. Blocking IL-10 did not prevent this effect in either subsets. However, in the effector cells, but not the naive cells, blocking IL-4 partially reduced the effect of 1,25(OH)2D3 on Th17 differentiation. Conclusions 1,25(OH)2D3 similarly inhibits Th17 differentiation of naive and effector CD4+ T cells from CII-immunised mice. However, only the inhibition in effector cells, not in naive cells, is partially dependent on IL-4. These data show that 1,25(OH)2D3utilises different pathways to inhibit Th17 differentiation, depending on the maturation stage of the CD4+ T cell.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease mainly affecting synovial joints. The clinical presentation of RA shows the heterogeneity of this disease with its underlying complex interactions between the innate and adaptive immune system and flare-ups of disease. Different disease models such as collagen induced arthritis, antigen induced arthritis, and Streptococcal cell wall induced arthritis can be exploited to investigate different aspects of the pathogenesis of arthritis. The disease can be monitored macroscopically over time via scoring systems. For histological examination, paraffin embedded knee sections can be used for hematoxylin and eosin staining to visualize cellular infiltration as well as for tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclast-like cells. Cellular infiltration of the synovium by different myeloid cells such as tissue resident macrophages, dendritic cells and neutrophils can be monitored using flow cytometry. Here, we describe the methods for inducing the different mouse models for arthritis, including scoring systems per model, histological and flow cytometric analysis.

ObjectiveBruton's tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.MethodsUsing intracellular flow cytometry, we quantified BTK expression and phosphorylation in subsets of peripheral blood B cells from 30 patients with rheumatoid arthritis (RA), 26 patients with primary Sjogren's syndrome (SS), and matched healthy controls.ResultsIn circulating B cells, BTK protein expression levels correlated with BTK phosphorylation. BTK expression was up-regulated upon BCR stimulation in vitro and was significantly higher in CD27+ memory B cells than in CD27-IgD+ naive B cells. Importantly, BTK protein and phospho-BTK were significantly increased in B cells from anti-citrullinated protein antibody (ACPA)-positive RA patients but not in B cells from ACPA-negative RA patients. BTK was increased both in naive B cells and in memory B cells and correlated with frequencies of circulating CCR6+ Th17 cells. Likewise, BTK protein was increased in B cells from a major fraction of patients with primary SS and correlated with serum rheumatoid factor levels and parotid gland T cell infiltration. Interestingly, targeting T cell activation in patients with primary SS using the CTLA-4Ig fusion protein abatacept restored BTK protein expression in B cells to normal levels.ConclusionThese data indicate that autoimmune disease in humans is characterized by enhanced BTK activity, which is linked not only to autoantibody formation but also to T cell activity.

Over the last three decades, it has become clear that the role of vitamin D goes beyond the regulation of calcium homeostasis and bone health. An important extraskeletal effect of vitamin D is the modulation of the immune system. In the context of autoimmune diseases, this is illustrated by correlations of vitamin D status and genetic polymorphisms in the vitamin D receptor with the incidence and severity of the disease. These correlations warrant investigation into the potential use of vitamin D in the treatment of patients with autoimmune diseases. In recent years, several clinical trials have been performed to investigate the therapeutic value of vitamin D in multiple sclerosis, rheumatoid arthritis, Crohn’s disease, type I diabetes, and systemic lupus erythematosus. Additionally, a second angle of investigation has focused on unraveling the molecular pathways used by vitamin D in order to find new potential therapeutic targets. This review will not only provide an overview of the clinical trials that have been performed but also discuss the current knowledge about the molecular mechanisms underlying the immunomodulatory effects of vitamin D and how these advances can be used in the treatment of autoimmune diseases.

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type–specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell–specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell–specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.

Objective Interleukin-17A (IL-17A) signals through the IL-17 receptor (IL-17R) A/C heterodimer. IL-17RA serves as a common receptor subunit for several IL-17 cytokine family members. Lack of IL-17RA signaling may therefore have additional effects beyond those of lack of IL-17A alone. The present study was undertaken to determine the role of IL-17RA signaling in autoimmune arthritis. Methods Disease incidence and severity were scored in type II collagen–treated wild-type, IL-17RA–deficient, and IL-23p19–deficient mice. T helper cell profiles and humoral immune responses were analyzed at several time points. Pathogenicity of T cells and total splenocytes was determined by in vitro functional assay. IL-17RA signaling was blocked in vivo in mice with antigen-induced arthritis (AIA). Results Comparable to the findings in IL-23p19–deficient mice, IL-17RA–deficient mice were completely protected against the development of collagen-induced arthritis (CIA). However, IL-17RA–deficient mice exhibited an increased number of IL-4–producing CD4+ T cells, distinct from IL-17A+CD4+ T cells. This was associated with fewer plasma cells, lower production of pathogenic IgG2c antibody, and increased production of IgG1 antibody. Both isolated CD4+ T cells and total splenocytes from IL-17RA–deficient mice had a reduced ability to induce IL-6 production by synovial fibroblasts in the setting of CIA, in a functional in vitro assay. Furthermore, blocking of IL-17RA signaling in AIA reduced synovial inflammation. Conclusion These results demonstrate that absence of IL-17RA leads to a Th2-like phenotype characterized by IL-4 production and suggest that IL-17RA signaling plays a critical role in the regulation of IL-4 in CIA and the development of autoimmune inflammation of the joint.

Background Interleukin (IL)-17A and Th17 cells are critically involved in T cell-mediated synovial inflammation. Besides IL-17A, Th17 cells produce IL-22. Recently, Th22 cells were discovered, which produce IL-22 in the absence of IL-17. However, it remains unclear whether IL-22 and Th22 cells contribute to T cell-mediated synovial inflammation. Therefore, we examined the potential of IL-22 and Th22 cells to induce synovial inflammation and whether IL-22 is required for T cell-mediated experimental arthritis. Methods Peripheral and synovial Th17 and Th22 cells were identified and sorted from patients with rheumatoid arthritis (RA). Co-culture experiments of these primary T cell populations with RA synovial fibroblasts (RASF) were performed. The in vivo IL-22 contribution to synovial inflammation was investigated by inducing T cell-mediated arthritis in IL-22 deficient mice and wild-type mice. Results Peripheral Th17 and Th22 cell populations were increased in patients with RA and present in RA synovial fluid. In T cell-RASF co-cultures, IL-22 in the presence of IL-17A had limited effects on IL-6, IL-8, matrix metalloproteinase-1 (MMP-1) and MMP-3 production. Furthermore, primary peripheral blood and synovial Th17 cells were more potent in the induction of these factors by RASF compared with Th22 cells. In line with this, similar synovial inflammation and disease severity was found between IL-22 deficient and wild-type mice in T cell-mediated experimental arthritis. Conclusions These findings show that IL-17A/Th17 cell-mediated synovial inflammation is independent of IL-22 and Th22 cells. This implies that targeting IL-17A/Th17 cells, rather than IL-22/Th22 cells, should be the focus for treatment of T cell-mediated synovial inflammation.

SummaryRecurrent joint bleeding is the most common manifestation of haemophilia resulting in haemophilic arthropathy (HA). The exact pathophysiology is unknown, but it is suggested that arthropathy is stimulated by liberation of fibrinolytic activators from the synovium during haemarthrosis. The aim of this study was to test the hypothesis that haemarthrosis activates the local synovial fibrinolytic system in a murine haemophilia model. The right knees of haemophilic and control mice were punctured to induce haemarthrosis. The left knees served as internal control joints. Synovial levels of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), plasmin, and alpha-2-antiplasmin (A2AP) were compared between the punctured and control knees. In haemophilic mice, an increase in synovial cells expressing urokinase-type plasminogen activator (uPA) in the right punctured knee versus the left unaffected knee was observed: (47% vs 43%) (p=0.03). Additionally, in haemophilic mice, haemar-throsis induced an increase in uPA (0.016 ng/ml vs 0.01 ng/ml) (p=0.03) and plasmin (0.53 μg/ml vs 0.46 μg/ml) (p=0.01) as promoters of fibrinolysis. Synovial levels of PAI-1 (0.32 ng/ml vs 0.17 ng/ ml) (p

Objectives T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) controls Th17-mediated synovial inflammation. Methods Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17-RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)(2)D-3 and/or TNF alpha blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. Results The authors show that the 1,25(OH)(2)D-3, but not TNF alpha blockade, significantly suppressed autocrine IL-17A production in Th17-RASF and synovial biopsy cultures. Combining 1,25(OH)(2)D-3 and TNF alpha blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17-RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. Conclusions These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)(2)D-3. The combination of neutralising TNF activity and 1,25(OH)(2)D-3 controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptor signalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

Objective. To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D-3 (1,25[OH](2)D-3) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology.Methods. Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D-3, dexamethasone (DEX), and 1,25(OH)(2)D-3 and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry.Results. The presence of 1,25(OH)(2)D-3 reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D-3 had favorable effects on tumor necrosis factor alpha (TNF alpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A-and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D-3, in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNF alpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA.Conclusion. These data indicate that 1,25(OH)(2)D-3 may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4+ T cells, CD11c+ dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.

Interieukin-17A (IL-17A) contributes to the pathogenesis of arthritis. Data from experimental arthritis indicate IL-17 receptor signaling as a critical pathway in turning an acute synovitis into a chronic destructive arthritis. The identification of six IL-17 family members (IL-17A-F) may extend the role of this novel cytokine family in the pathogenesis of chronic destructive joint inflammation. Whether the successful anti-IL-17A cytokine therapy in murine arthritis can be effectively translated to human arthritis need to be tested in clinical trials in humans. Interestingly, IL-17A and IL-17F are secreted by the novel T helper subset named Th17. This novel pathogenic T cell population induces autoimmune inflammation in mice and is far more efficient at inducing Th1-mediated autoimmune inflammation in mice than classical Th1 cells (IFN-gamma). In addition to IL-17A and IL-17F, Th17 cells are characterized by expression of IL-6, TNF, GM-CSF, IL-21, IL-22 and IL-26. Th17 cells have been established as a separate lineage of T helper cells in mice distinct from conventional Th1 and Th2 cells. Whether this also applies to human Th17 and whether RA is a Th1 or a Th17 mediated disease is still not clear. This review summarizes the findings about the role of IL-17 in arthritis and discusses the impact of the discovery of the novel Th17 cells for arthritis. Further studies are needed to unravel the role of Th17 cells and the interplay of IL-17 and other Th17 cytokines in the pathogenesis of arthritis and whether regulating Th17 cell activity will have additional value compared to neutralizing IL-17A activity alone. This might help to reach the ultimate goal not only to treat RA patients but to prevent the development of this crippling disease. (C) 2007 Elsevier Ltd. All rights reserved.

Purpose Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods cfDNA was extracted from sera of patients with early and established RA, relapsing-remitting multiple sclerosis patients (RRMS) and healthy subjects, and its concentration was determined by quantitative PCR using two amplicons, Alu115 and β-actin205, corresponding to Alu repetitive elements and the β-actin single-copy gene, respectively. Serum DNase activity was measured by a single radial enzyme diffusion method. Results Reduced levels of cfDNA were observed in patients with established RA in comparison with healthy controls, early RA patients and RRMS patients. There were no significant differences in cfDNA concentration between healthy controls, early RA and RRMS patients. Total DNase activity appeared to be similar in the sera of all tested groups. Conclusions Our results demonstrate that cfDNA levels are strongly reduced in the sera of established RA patients, which is not caused by changes in DNase activity. Measurement of cfDNA can distinguish established RA patients from early RA patients. Thus, cfDNA may serve as a biomarker in RA. Electronic supplementary material The online version of this article (doi:10.1007/s13317-015-0066-6) contains supplementary material, which is available to authorized users.

The role of interleukin (IL)-17 in cancer remains controversial. In view of the growing interest in the targeting of IL-17, knowing its cellular sources and clinical implications is crucial. In the present study, we unraveled the phenotype of IL-17 expressing cells in cervical cancer using immunohistochemical double and immunofluorescent triple stainings. In the tumor stroma, IL-17 was found to be predominantly expressed by neutrophils (66%), mast cells (23%), and innate lymphoid cells (8%). Remarkably, T-helper 17 (Th17) cells were a minor IL-17 expressing population (4%). A similar distribution was observed in the tumor epithelium. The Th17 and granulocyte fractions were confirmed in head and neck, ovarian, endometrial, prostate, breast, lung, and colon carcinoma. An above median number of total IL-17 expressing cells was an independent prognostic factor for poor disease-specific survival in early stage disease (p = 0.016). While a high number of neutrophils showed at trend toward poor survival, the lowest quartile of mast cells correlated with poor survival (p = 0.011). IL-17 expressing cells and neutrophils were also correlated with the absence of vaso-invasion (p < 0.01). IL-17 was found to increase cell growth or tightness of cervical cancer cell lines, which may be a mechanism for tumorigenesis in early stage disease. These data suggest that IL-17, primarily expressed by neutrophils, predominantly promotes tumor growth, correlated with poor prognosis in early stage disease. Strikingly, a high number of Th17 cells was an independent prognostic factor for improved survival (p = 0.026), suggesting Th17 cells are part of a tumor suppressing immune response.

The IL-17A producing T-helper-17 (Th17) cell population plays a major role in rheumatoid arthritis (RA) pathogenesis and has gained wide interest as treatment target. IL-17A expressing Th cells are characterized by the expression of the chemokine receptor CCR6 and the transcription factor RORC. In RA, CCR6+ Th cells were identified in peripheral blood, synovial fluid and inflamed synovial tissue. CCR6+ Th cells might drive the progression of an early inflammation towards a persistent arthritis. The CCR6+ Th cell population is heterogeneous and several subpopulations can be distinguished, including Th17, Th22, Th17.1 (also called non-classic Th1 cells), and unclassified or intermediate populations. Interestingly, some of these populations produce low levels of IL-17A but are still very pathogenic. Furthermore, the CCR6+ Th cells phenotype is unstable and plasticity exists between CCR6+ Th cells and T-regulatory (Treg) cells and within the CCR6+ Th cell subpopulations. In this review, characteristics of the different CCR6+ Th cell populations, their plasticity, and their potential impact on rheumatoid arthritis are discussed. Moreover, current approaches to target CCR6+ Th cells and future directions of research to find specific CCR6+ Th cell targets in the treatment of patients with RA and other CCR6+ Th cell mediated autoimmune diseases are highlighted. (C) 2015 Elsevier Ltd. All rights reserved.

Introduction: Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status. Methods: We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA-treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration. Results: ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6-CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA-patients (R-2 = -0.35; p < 0.01) but not in ACPA(+) (R-2 < 0.01; p = 0.94) patients. Conclusions: These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-κB, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

We present a brother and sister with severe rickets, alopecia and highly elevated serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Genomic sequencing showed a homozygous point mutation (A133G) in the vitamin D receptor gene, leading to an amino acid change in the DNA binding domain (K45E), which was described previously. Hereditary vitamin D resistant rickets (HVDRR) was diagnosed. Functional studies in skin biopsy fibroblasts confirmed this. 1,25-(OH)2D3 reduced T helper (Th) cell population-specific cytokine expression of interferon γ (Th1), interleukins IL-17A (Th17) and IL-22 (Th17/Th22) in peripheral blood mononuclear cells (PBMCs) from the patient's parents, whereas IL-4 (Th2) levels were higher, reflecting an immunosuppressive condition. None of these factors were regulated by 1,25-(OH)2D3 in PBMCs from the boy. At present, both patients (boy is 23 years of age, girl is 7) have not experienced any major immune-related disorders. Although both children developed alopecia, the girl did so earlier than the boy. The boy showed complete recovery from the rickets at the age of 17 and does not require any vitamin D supplementations to date. In conclusion, we characterized two siblings with HVDRR, due to a mutation in the DNA binding domain of VDR. Despite a defective T cell response to vitamin D, no signs of any inflammatory-related abnormalities were seen, thus questioning an essential role of vitamin D in the immune system. Despite the fact that currently medicine is not required, close monitoring in the future of these patients is warranted for potential recurrence of vitamin D dependence and diagnosis of (chronic) inflammatory-related diseases.

T cells play a role in the initiation and perpetuation of tissue inflammation that can lead to tissue destruction. With the discovery of a number of T helper subsets and their potential plasticity during disease it became clear that the T cell behaviour in autoimmune diseases is much more complex than the first Th1/Th2 concept. From experimental autoimmune arthritis models it became clear that the IL-23/IL-17 immune pathway is critical in the development of autoimmune arthritis and IL-17A has been recognized to be a key cytokine involved in initiation and perpetuation of chronic destructive arthritis. Functional studies using T cells and stromal cells from patients with RA revealed improvement of anti-TNF effects when combined with agents neutralizing IL-17A or agents suppressing Th17 cytokines production. Clinic trials will be needed to test whether these data from experimental settings can be translated to human arthritis. Different approaches are available or under investigation to target: (1) pathogenic T cell activity by inhibiting RORc or STAT3 or the co-stimulator pathway by CTLA4-Ig; (2) Th17 cytokine production by anti-IL-17A, anti-IL-22, or combination of anti-IL-17A/anti-TNF approaches; (3) Th17 polarization by neutralizing IL-23 using an anti-IL-23 specific antibody, IL-12/IL-23 by an anti-p40 antibody, or the IL-6 signaling pathway; (4) Th17 migration by interfering the CCR6-CCL20 interaction. The challenge is to bring the best to the clinic to further improve current therapy for patients with RA and to reach stable remission or even prevent the development of this disabling disease.

Introduction: A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6(+) memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature. Methods: In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN+) (n = 16) and negative (IFN-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4(+) CD45RO(+) CCR6(+) T cells (CCR6(+) cells) was measured with flow cytometry and compared between IFN+, IFN-patients and HCs. Results: Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6(+) cells were observed in IFN+ patients compared with IFN-patients and HCs. IL-17A and IL-17F expression within CCR6(+) cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)-a factor strongly correlating with IFN type I - and IL-21 producing CCR6(+) cells. Conclusions: We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6(+) memory T-helper cells in IFN+ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.

We would like to reply to the letter by Dolff and colleagues [1] regarding our article in a recent issue of Arthritis Research & Therapy[2]. We thank the authors for their interest in our work and the critical reading of our article. The aim of our study was to investigate a possible association between the IFN type I signature and memory T helper 17 (Th17) cells and their cytokines in systemic lupus erythematosus (SLE). Since CCR6 can be expressed by regulatory T (Treg) cells, we have excluded CD25high cells to discriminate between CD4+CD45RO+CCR6+CD25− (primary Th17 cells) and Treg cells. Concerning the observation on co-expression of IL-17A with IFN-γ, we would like to remark that IFN-γ is an IFN type II, and not an IFN type I, cytokine and does not bind to the IFN type I receptor. Detection of IFN type I activity is hampered by the difficulty to assess the different subtypes of this cytokine. Therefore, analysis of IFN type I-induced gene expression in RNA from peripheral blood cells, the so-called IFN type I signature, is used as a measure for IFN type I activity. Although we fully agree with the authors that the relationship between Th cells and IFN type I deserves further study, their remark on the ‘genetic’ signature is confusing and probably refers to the IFN type I-induced gene expression signature, which is detected at the RNA level. The relationship between CD25+ Tregs and IFN type I is certainly of interest for further study as these Tregs are carrying the IFN type I receptor and thus respond to increased systemic IFN type I activity in SLE. Also, follow-up studies with a focus on adaptive and innate cells producing IL-17, including the relation with IL-21 and IL-22 and plasticity, will be of interest [3,4]. In our article, we presented data supporting a potential co-activity between IFN type I and CD4+memory CCR6+ Th17 cytokines. Further studies are needed to confirm this co-activity with a focus on revealing the mechanism of this dangerous link in SLE. Abbreviations IFN: Interferon; IL: Interleukin; SLE: Systemic lupus erythematosus; Th17: T helper 17; Treg: Regulatory T. Competing interests The authors declare that they have no competing interests.

Background and objectives CCR6 + T-helper (Th) cells and their pro-inflammatory cytokines, including IL-17A, are implicated in the pathogenesis of rheumatoid arthritis (RA). However, within CCR6 + Th cells various subpopulations are present and their clinical relevance in RA is unclear. Therefore, we characterised CCR6 + Th subpopulations with regard to pathogenic potential and disease-modifying antirheumatic drugs (DMARDs) and glucocorticoid (GC) therapy outcome in RA. Material and methods Within total CCR6 + Th cells from patients with RA, CCR4 + CXCR3 - (Th17), CCR4 - CXCR3 + (Th17.1), CCR4/CXCR3 double-positive (DP) and double-negative (DN) cells were distinguished and/or sorted by flow cytometry. These subpopulations were: analysed for Th17/Th1-associated factors; co-cultured with RA-derived synovial fibroblasts (RASF); related to disease-modifying antirheumatic drugs (DMARDs) and glucocorticoid (GC) therapy response; analysed regarding the expression of multidrug transporters MDR1 and MRP1 and drug efflux potential. Results All these populations expressed the transcription factor RORC and were present in RA peripheral blood and synovial fluid. Despite differential IL-17A, IL-17F, IFNg and TBX21 expression, all subpopulations, including the IL-17A low-producing Th17.1, DP and DN cells, showed pathogenic activity in the induction of IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression by synovial fibroblasts. MDR1 (ABCB1) and MRP1 (ABCC1) are cellular efflux transporters of glucocorticoids and the DMARD methotrexate. In particular Th17.1 and DN cells expressed relatively high levels of MDR1, whereas MRP1 was expressed at similar levels among the subpopulations. Interestingly, increased drug efflux potential by CCR6 + Th cells, as measured by rhodamine123 and calcein transport, was associated with the lack of DMARD/GC therapy response. Conclusions Therefore, we conclude that in addition to IL-17- high Th17 cells, IL-17- low CCR6 + Th cells display pathogenic activity in the context of RA. Moreover, we identified efflux transporter expression and efflux activity by RA CCR6 + Th cells. These findings suggest that pathogenic and multidrug resistant CCR6 + Th cells are associated with the lack of DMARD/GC therapy response in patients with RA.

Background and objectives Rheumatoid arthritis (RA) is preceded by a period where the patients experience pain but no clinically apparent arthritis can be detected yet. Without treatment RA can lead to severely inflamed joints and bone damage. Early treatment has shown to be beneficial in limiting joint damage. Therefore early detection of patients at risk of developing RA is needed. Anti–citrullinated protein antibodies and Rheumatoid factor levels in the blood does not predict RA development for all patients. A betterunderstanding of the immunological processes ongoing during arthralgia could aid in early detection and prevention of joint damage. The time period between first immunisation and booster in the collagen induced arthritis (CIA) model might reflect the arthralgia stage in rheumatoid arthritis development. In this study we examined whether the CIA model could be used to investigate immunological processes during arthralgia. Materials and methods DBA/1 mice were subjected to CIA. Blood was drawn at different time points via submandibular vein puncture during CIA development. Antibodies directed against bovine collagen type II (bCII) and mouse collagen type II (mCII) of the IgG1 and IgG2a isotype were measured by ELISA. Clustered analysis was performed to correlate CIA development with antibody levels. Results Significantly higher antibody levels of the IgG2a isotype directed against mCII (anti-mCII-IgG2a antibodies) were found at day 12 in mice who developed CIA compared to those who did not (P = 0.037). The antibody levels directed against bCII and mCII further increased during CIA development. Anti-mCII-IgG2a antibody levels did not reflect clinical disease score reached at day 35. Conclusions The detection of antibodies against mouse type II collagen indicates an early auto-immune process in the CIA model prior to clinical detectable disease.

Fc gamma RIIB-deficient mice generated in 129 background (Fc gamma RIIB(129)(-/-)) if back-crossed into C57BL/6 background exhibit a hyperactive phenotype and develop lethal lupus. Both in mice and humans, the Fc gamma r2b gene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named Sle16, causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of Fc gamma RIIB deficiency to the development of lupus in Fc gamma RIIB(129)(-/-) mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of "loss of function" mutations in the Fc gamma r2b gene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that Fc gamma RIIB(-/-) mice generated by gene targeting in B6-derived ES cells (Fc gamma RIIB(B6)(-/-)), lacking the 129-derived flanking Sle16 region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in Fc gamma RIIB(129)(-/-) mice, not Fc gamma RIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived Fc gamma r2b flanking region cause loss of tolerance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of Fc gamma RIIB, culminating in lethal lupus. In the presence of the Yaa lupus-susceptibility locus, Fc gamma RIIB(B6)(-/-) mice do develop lethal lupus, confirming that FcgRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci. The Journal of Immunology, 2011, 187: 1304-1313.

Contains fulltext : 98492.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis factor (TNF)-induced joint pathologic processes in vivo, and to analyze the surplus ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis (CIA). METHODS: Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. RESULTS: Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of S100A8, IL-1beta, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not merely the result of enhanced inflammation, but also was associated with a direct synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected against IL-17/TNF-induced expression of cartilage NITEGE neoepitopes. During established arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine treatments alone, and significantly inhibited further joint inflammation and cartilage destruction. CONCLUSION: Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in parallel with a synergistic up-regulation of S100A8, IL-1beta, and erosive MMPs. Moreover, neutralizing IL-17 in addition to TNF further improves protection against joint damage and is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination blocking of TNF and IL-17 may have additional therapeutic value for the treatment of destructive arthritis.

Studies with IL-23-deficient mice revealed the critical role of IL-23 and in particular IL-17-producing cells, including TH17 cells, in the initiation of experimental auto-immune arthritis. Interestingly, the recognition of TH17 cells and TH17 cytokines at the very early stage of rheumatoid arthritis (RA) fits with the concept that the IL-23/TH17 immune pathway is important in the development of this auto-immune disease. However, the exact biological role of this immune pathway in the development of RA and other arthropathies needs further examination. The first data from clinical trials using anti-IL-17A antibody treatment in psoriatic arthritis and RA is promising. Whether the regulation of TH17 cell activity or specific combinations of TH17 cytokines will have additional value compared to neutralizing IL-17A activity or TNF needs to be elucidated. However, first evidence from in vitro studies showed that the inhibition of the TH17 pathway in addition to anti-TNF suppressed the pro-inflammatory feedback loop between TH17 cells and synovial fibroblasts of patients with early RA. This approach may help to reach the ultimate goal of permanent remission or even prevent the development of this crippling disease. In addition, further understanding of the plasticity of T cell subsets in the pathogenesis of chronic destructive arthritis especially at different stages of the disease will be essential to understanding the T cell biology in RA. Moreover, understanding T cell plasticity due to different therapies may further improve treatment of patients with chronic destructive arthritis.