Lgm van Baarsen

Features of the Synovium of Individuals at Risk of Developing Rheumatoid Arthritis

Findings from previous studies have suggested that subclinical inflammation of the synovium does not coincide with the appearance of rheumatoid arthritis (RA)–specific autoantibodies. This study was undertaken to examine the relationship between the presence of autoantibodies, changes in the synovium, and development of arthritis over time in a markedly larger, prospective study.

A7.7 Synovial tissue profiling in autoantibody positive individuals without arthritis reveals gene signatures associated with subsequent development of rheumatoid arthritis

Background Previous work has suggested subtle infiltration of synovial T cells in the absence of overt synovial inflammation in individuals at risk of developing rheumatoid arthritis (RA). Objective To study the molecular changes in synovial tissue preceding arthritis development in preclinical RA. Materials and methods Sixty-one individuals who were IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive and without any evidence of arthritis were included. All individuals underwent mini-arthroscopic synovial biopsy sampling of a knee joint at inclusion and were prospectively followed. An explorative genome-wide transcriptional profiling study was performed on synovial biopsies obtained from 13 individuals using Agilent arrays (test cohort). Survival analysis within the software package Significance Analysis of Microarrays was used to identify transcripts with a significant association with arthritis development. The expression level of differentially expressed genes was validated using quantitative real-time PCR in the total cohort. Results Six of the 13 individuals in the explorative study developed RA after a median follow up time of 20 months (IQR 2 – 44). The 7 individuals who did not develop RA had a median follow up time of 56 months (IQR 56 – 60). Using a False Discovery Rate of < 5% we found that increased expression of 3,151 transcripts correlated with a higher risk of arthritis development, and increased expression of 2,437 transcripts correlated with a lower risk. Gene Set Enrichment Analysis revealed that synovial biopsies of individuals who developed RA after follow up display higher expression of genes involved in several immune response-related pathways (e.g. T cell and B cell receptor pathways, cytokine and chemokine signalling and antigen processing and presentation) compared with biopsies of individuals who did not develop RA. In contrast, lower expression was observed for genes involved in e.g. extracellular matrix receptor interaction, Wnt-mediated signal transduction and lipid metabolism. Subsequently, the expression level of a selection of 26 differentially expressed genes was validated by quantitative real-time PCR in the total cohort (n = 61). Two-way hierarchical cluster analysis classified the individuals into two groups, where those individuals who developed arthritis (n = 16) showed a preference to cluster together in the left arm of the dendogram (Fisher’s exact test P < 0.05). Conclusion This study clearly shows molecular changes appearing in synovial tissues before onset of arthritis in the absence of overt synovitis and demonstrates preclinical synovial activation of immune response genes associated with development of arthritis.

Differential expression of transcription factors predicted to regulate angiopoietin-1 and -2 genes is associated with specific angiopoietin signatures in RA and PsA synovial tissue

The angiogenic factors angiopoietin (Ang)-1 and -2 contribute to inflammation and are differentially expressed in synovial tissue of patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA). The underlying mechanisms for promoting differential expression are unknown. Here, we identified which cells express Ang-1 and Ang-2 in synovial tissue, transcription factors (TFs) predicted to regulate Ang-1 and Ang-2 transcription, and examined expression of these TFs in RA and PsA synovial tissue. Immunofluorescent double stainings of RA and PsA synovial tissue were performed with CD68, CD163, CD3, CD22 and vWF markers, in combination with antibodies against Ang-1 and Ang-2, using confocal imaging. In silico analysis was conducted to identify TF binding sites in the Ang-1 and Ang-2 promoters, using four different TF binding site prediction programs. RA synovial expression of TFs, Ang-1 and Ang-2 was examined in publicly available gene expression data sets. Candidate TFs were characterized by real-time PCR (qPCR). Ang-1 production was detected in CD68 and CD163 –positive macrophage, CD3-positive T lymphocytes, and CD22-positive B cells, while Ang-2 expression was restricted to vWF- positive endothelial cells, as well as macrophages. In silico studies identified 34 TFs that were predicted to regulate Ang-1 expression and 22 TFs which are involved in Ang-2 expression and are upregulated in RA synovial tissue (P< 0.05). Expression of these TF was readily detected by qPCR in RA and PsA synovial tissue biopsies. Our studies suggest that expression of Ang-1 and Ang-2 by different cell types could additional underlie the previously observed specific Ang-1 high signature in RA, compared to PsA. Enhanced expression of Ang-1 and Ang-2 in RA and PsA is strongly associated with expression of specific TFs, and understanding the mechanisms leading to the differential expression of these TFs may give insight into the etiology of RA and PsA.

A1.19 Altered distribution of innate lymphoid cell populations in human LYMPH node biopsies obtained during the earliest phases of systemic autoimmunity

Background and objective Innate lymphoid cells (ILCs) emerge as important mediators of immunity and accumulation of inflammatory ILC populations has been reported in various inflammatory-mediated conditions. We investigated the frequency and distribution of ILCs in lymph node biopsies obtained during the earliest phases of rheumatoid arthritis (RA). Materials and methods Twelve patients with early rheumatoid arthritis (RA), 12 individuals positive for autoantibodies but without arthritis (RA-risk group) and 7 healthy controls underwent ultrasound-guided inguinal lymph node biopsy. Frequencies of ILCs subsets as well as the expression of VCAM and ICAM by lymph node endothelial cells and fibroblasts were analysed by flow cytometry. Results Although no difference in the number of total ILCs (Lin-CD45+/lowCD127+) was found among the three study groups, the distribution of the different ILC populations changed. RA patients showed a relative lower frequency of LTi (c-Kit+NKp44- ILCs) and an increased frequency of ILC1 (c-Kit-NKp44- ILCs) and ILC3 (c-Kit+NKp44+ ILCs) populations compared with controls (p < 0.001, p < 0.05 and p < 0.05, respectively). RA-risk individuals showed a relative increased frequency of inflammatory ILC1 compared with controls (p < 0.01). Frequencies of LTi correlated with the expression of adhesion molecules on endothelial and fibroblastic cells. Conclusions Already during the earliest phases of systemic autoimmunity, the ILC distribution in lymph nodes changes from a homeostatic towards a more inflammatory profile, thereby supporting a role for ILCs in the pathogenesis of RA.

A3.06 Distinct expression pattern of peripheral tissue-restricted antigens in human LYMPH node stromal cells during the earliest phases of rheumatoid arthritis

Background In mice lymph node stromal cells (LNSCs) maintain peripheral tolerance through the presentation of peripheral tissue-restricted antigens (PTAs) and negative regulation of T-cells. However, little is known about PTA expression in human LNSCs. We hypothesise that deregulated expression of PTAs by human LNSCs might underlie the pathogenesis of autoimmune diseases like rheumatoid arthritis (RA). Objective To investigate the expression and possibleregulation of several disease-related PTAsin human LNSCs derived from healthy donors and individuals having systemic autoimmunity. Methods LNSCs were cultured from freshly collected lymph node needle biopsies obtained from 25 patients with rheumatoid arthritis (RA), 24 individuals positive for autoantibodies but without clinical apparent disease (RA-risk group) and 14 seronegative healthy controls. Expression of PTAs and the transcription factors DEAF1 and AIRE possibly regulating PTA expressionwere assessed by qPCR. Aire wasfurther investigated on protein level. LNSCs from five donors of each study group were stimulated with TNFα pluslymphotoxin α1β2 or poly (I:C) to study the regulation of PTA under inflammatory conditions. Results Human LNSCs expressed the PTAs RRAD (Ras-related associated with diabetes), PLP1 (proteolipid protein 1), GAD1(glutamate decarboxylase 1) and the RA-related geneENO1 (Enolase 1(alpha)), but not AFP (alpha-fetoprotein). The expression of RRAD was slightly lower in RA-risk individuals which reached significance inRA patients (P = 0.04), especially in ACPA negativeRA patients (P = 0.0007). In contrast GAD1 mRNA levelswere significantly increased in RA-risk individuals (P = 0.01) with a trend towards increased expression in RA patients. Expression of ENO1 and PLP1 was similar in all donor groups. Invitro stimulation increased the expression of RRADin all donor groups to a similar extent. LNSCs express the transcription factors DEAF1andAIRE with Aire protein detected in the nucleus of LNSCs. Conclusion Here we show for the first time that human LNSCs express disease-related PTAs as well as the transcription factors DEAF1and Aire, which supports their rolein tolerance induction. Already during the earliest phases of RA a differential PTA expression pattern is observed in LNSCs. Future studies are needed to investigate whether the distinct PTA expression pattern in LNSCs has an aberrant effect on T-cell regulation.

A1.20 Lymphoid tissue analyses in autoantibody positive individuals at risk for developing rheumatoid arthritis reveals an important role for CD8+ T cells during the earliest phases of autoimmunity

Background and objective The role of CD8+ T cells in autoimmune disease is poorly understood and may depend on the phase of disease. CD8+ T cells can contribute to autoimmunity by driving inflammation through cytokine release, tissue infiltration and cytotoxic T cell (CTL) function or by dampening the immune response through CD8+ regulatory T cells. To provide more insight into the role of CD8+ T cells during the development of autoimmune disease, we investigated the phenotype and function of CD8+ T cells in lymphoid tissue and peripheral blood during the earliest phases of rheumatoid arthritis (RA). Methods We collected inguinal lymph node biopsies and peripheral blood from 20 individuals with arthralgia but without arthritis, who were positive for IgM rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA; RA-risk individuals); 17 early RA patients; and 19 healthy controls (HCs). CD8+ T cell function and phenotype were analysed using flow cytometry. To measure cytokine production cells were stimulated for four hours with PMA/Ionomycin in the presence of Brefeldin A and Golgi stop. Results We found higher frequencies of lymphoid memory CD8+CD45RO+ T cells in RA-risk individuals (p < 0.05) and early RA patients (p < 0.05) compared with HCs. In addition, we found higher frequencies of activated lymphoid CD8+CD45RA+CD69+ T cells in early RA patients compared with HCs (p < 0.05). Interleukin-(IL-)10 and interferon gamma (IFNg) production in lymphoid CD8+ T cells was decreased in the RA risk individuals compared with HCs (p < 0.01). There was a decrease in both IFNg and IL-17A production in lymphoid CD8+ T cells in early RA patients (p < 0.05). We did not detect changes in frequencies of cytotoxic effector CD8+ T cells in peripheral blood or lymphoid tissue during the earliest phases of RA. In lymphoid tissue of early RA patients there was a negative correlation between the CD8+IL-17A+ mean fluorescent intensity (MFI) and disease activity score in 28 joints (DAS28) (p = 0.005, r = -0.81) and swollen joint count 28 (SJC28; p = 0.01, r = -0.76) and between CD8+IL-10+MFI and DAS28 (p = 0.05, r = -0.67) and tender joint count 28 (TJC28; p = 0.02, r = -0.73). These data indicate that a decrease in lymphoid tissue derived regulatory and pro-inflammatory cytokines was related to disease activity. Conclusion The data present here suggest that CD8+ T cells exhibit an exhaustive phenotype during the RA-risk phase and earliest phases of RA, which might be explained by continuous antigen presentation and CD8+ T cell activation.

A1.8 CD4+ T-helper cell subsets in lymph node biopsies and peripheral blood during the earliest phases of rheumatoid arthritis

Background and objective Recent work has shown that systemic autoimmunity precedes inflammation of the synovium in rheumatoid arthritis (RA). Not much is known about the balance between regulatory and effector CD4+ T-helper cell subsets during the earliest phases of systemic autoimmunity. Therefore, we investigated CD4+ T-helper cell subsets in lymph node biopsies and peripheral blood during the earliest phases of RA. Methods Nineteen individuals with arthralgia but without arthritis, who were positive for IgM rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA; RA-risk individuals) and 17 early RA patients (disease duration less than 1 year and naïve for disease-modifying antirheumatic drugs and biologics) were included. For comparison we included 19 healthy controls (HCs). All study subjects underwent ultrasound-guided inguinal lymph node biopsy. CD4+ T-helper cell subsets were analysed by flow cytometry. Cytokine profiles (frequencies of positive cells and production per cell based on mean fluorescent intensity (MFI)) were determined after stimulation with Phorbol Myristate Acetate (PMA) and Ionomycin in the presence of Brefeldin A and Golgi Stop. Results The frequencies of total lymphoid CD4+ T cells were comparable between early RA patients, RA-risk individuals and HCs. The frequency of lymphoid Th1 cells (CXCR3+CCR6-CCR4-) was significantly higher in early RA patients compared with HCs (p < 0.01). Frequencies of lymphoid CD4+IL-4+ (p < 0.05) and CD4+IL-10+(p < 0.01) T cells were lower in RA-risk individuals compared with HCs. In peripheral blood frequencies of CD4+IL-17A+ T cells were higher in early RA patients compared with RA-risk individuals (p < 0.05) and HCs (p < 0.05). In addition, the frequency of CD4+IL-10+ T cells was lower in peripheral blood of RA-risk individuals compared with HCs (p < 0.05). Of interest, lymphoid CD4+ T-helper cells produced lower levels of cytokines (IFN-y, IL-4, IL-17A and IL-10) per cell (based on MFI) in both RA-risk individuals and early RA patients compared with HCs. Conclusions The decreased frequency of CD4+IL-10+ T cells in RA-risk individuals compared with HCs observed in both peripheral blood and lymphoid tissue indicates that a reduction in the expression of regulatory cytokines by CD4+ T cells precedes the development of clinically manifest RA. These data also suggest that the subsequent development of clinical signs and symptoms is associated with an increase in pro-inflammatory cytokine production by CD4+ T cells, as shown here for IL-17A expression. Changes in pro-inflammatory and regulatory CD4+ T-helper cell subsets during different stages of RA development may provide new preventive and therapeutic targets.

A8.10 The effect of rituximab treatment on B and T cell subsets in lymphoid tissues of patients with rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterised by the presence of autoantibodies and infiltration of B and T-cells in synovial joints. Accordingly, targeting B-cells by rituximab treatment has been shown to be beneficial for a subset of RA patients. Despite a rapid depletion of B-cells in peripheral blood, B-cells may persist in synovial tissue and bone marrow. Since B-cell differentiation occurs in secondary lymphoid tissue we investigated the effect of rituximab treatment on B and T-cell subsets in lymphoid biopsies of RA patients. Methods Fourteen patients with active RA (baseline Disease Activity Score 28 joint assessment (DAS28) was 5.3 (4.7–6.0 [median, IQR]) were treated with intravenous infusions of 1000 mg of rituximab on day 1 and day 15. Premedication with methylprednisolone was omitted to study the specific effects of rituximab. Ultrasound-guided inguinal lymph node biopsies were obtained before start of rituximab treatment and four weeks after the first infusion and immediately processed for flow cytometry analyses. For comparison 5 lymph node biopsies from healthy controls were analysed. Results Lymph node B-cell subsets in RA patients were not significantly increased compared to healthy controls, while frequencies of early activated T-cells, CD3+CD69+ and CD3+CD25+CD69+ were significantly increased (p = 0.01 and p = 0.02 respectively). The frequencies of switched memory B-cells (CD27+IgD-) correlated significantly with the frequency of early activated T-cells (CD3+CD69+ p = 0.02, r = 0.65; CD3+CD25+CD69+ p = 0.002, r = 0.80). EULAR response after 24 weeks was as follows: 29% good, 50% moderate and 21% non-responders. Four weeks after treatment there was a complete depletion of B-cells in peripheral blood (cut off < 0.0001 × 109/Litre) in 9 of the 13 patients measured. In lymph nodes, 4 weeks after rituximab treatment, total CD19+ B-cells were significantly but incompletely depleted (p = 0.0004). CD19 frequency before treatment was 21.3% (13.8–32.4 [median, IQR]) and CD19 frequency after treatment was 9.7% (1.3–14.9 [median, IQR]). Especially naive (CD27-IgD+; p = 0.0006) and unswitched memory B-cells (CD27+IgD+; p = 0.0012) significantly decreased, while switched memory B-cells (CD27+IgD-) and double negative B-cells (CD27-IgD-) persisted. Also, activated T-cells (CD3+CD25+CD69+ T; p = 0.03) significantly decreased. No correlation was found between lymphoid B or T-cell subset depletion and response to treatment. Conclusion This study provides for the first time detailed insight in the effects of rituximab therapy on lymph node immune cells in RA patients. Of interest, rituximab not only affects the number of lymphoid B-cells but also reduced the number of activated lymphoid T-cells suggesting that lymphoid B-cells contribute to lymphoid T-cell activation in RA.

A7.21 Suppression of HDAC5 Expression by Inflammatory Cytokines is Required to Promote CXCL Chemokine Production in RA FLS

Background and Objectives Histone deacetylases (HDACs) are important regulators of gene expression and protein function in the immune system. HDAC inhibitors (HDACi) display anti-inflammatory properties in animal and in vitro models of rheumatoid arthritis (RA), as well as initial safety and efficacy in the treatment of systemic onset juvenile idiopathic arthritis. However, as most of the currently available HDACi display little selectivity or specificity for class I (HDAC 1–3, 8) and class II (HDAC 4–6, 9, 10) HDACs, the role of specific HDACs in RA is unclear. Here we examined the relationship between HDACs and inflammation in RA synovial tissue and fibroblast-like synoviocytes (FLS). Materials and Methods RNA was isolated from arthroscopic synovial biopsies from 19 RA patients. MMP-1, TNFα, IL-6, and HDAC 1–10 expression was measured by quantitative PCR (qPCR). RA FLS were stimulated with IL-1β, TNFα and LP, S and HDAC expression was measured by qPCR RA FLS were transduced with adenovirus encoding control GFP or GFP-HDAC5 or transfected with control siRNA or siRNA targeting HDAC5. Effects of HDAC5 modulation on RA FLS gene expression were analysed by custom qPCR array. Results Positive correlations were observed between RA synovial tissue expression of TNFα and HDAC1 (R = 0.651, P = 0.003) HDAC2 (R = 0.523, P = 0.022) and HDAC3 (R = 0.570, P = 0.011) and between MMP-1 and HDAC1 (R = 0.501, P = 0.029) and HDAC2 (R = 0.512, P = 0.025). A significant negative correlation was observed between synovial tissue expression of IL-6 and HDAC5 (R = –0.477, P = 0.039) and between clinical parameters of disease activity and HDAC5 (CRP: R = –0.664, P = 0.007; ESR: R = –0.556, P = 0.013: DAS28: R = –0.567, P = 0.011). HDAC5 mRNA expression was significantly and selectively reduced after RA FLS stimulation with TNFα and IL-1β, but not LPS. Of 84 genes regulated in RA FLS by IL-1β or TNFα, mRNA expression of CXCL9, CXCL10, and CXCL11 was selectively upregulated following silencing of HDAC5 expression. Conversely, mRNA expression of these chemokines was suppressed by overexpression of HDAC5 in RA FLS. Conclusions RA synovial expression of HDAC 1 and 2, but not class II HDACs, positively correlates with local inflammatory mediators, while HDAC5 expression negatively correlates with IL-6 mRNA expression and with disease activity. HDAC5 mRNA is decreased after inflammatory stimulation, and silencing of HDAC5 leads to an increase of CXCL chemokine expression in RA. FLS, an effect reversed by HDAC5 overexpression. Our results suggest a protective role for HDAC5 in RA, and that HDACi which fail to target HDAC5 may be more promising for therapeutic applications.

A1.5 Exploring the Role of the Lymph Node Microenvironment in Health and Disease

Background and Objective Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease of unknown aetiology. Changes in the lymph nodes could precede those in the synovial joints. Recent studies reported the importance of lymph node stromal cells (LNSCs) in lymphoid homeostasis, peripheral tolerance and the adaptive immune response. Therefore, we characterised the functional capacities of LNSCs in human lymph nodes during health and different phases of RA. Materials and Methods Individuals with arthralgia but without any evidence of arthritis upon physical examination who were positive for IgM rheumatoid factor and/or anti-citrullinated protein antibodies were included (n = 12; RA risk group). In addition, we included patients with early arthritis (disease duration <1 year; n = 6), RA (n = 15) and healthy controls (n = 8). All study subjects underwent ultrasound-guided inguinal lymph node biopsy. LNSCs were isolated and cultured from freshly collected lymph node needle biopsies. LNSCs originating from different culture passages were studied to investigate the effects of in vitro culture on the expression level of stromal cell associated genes, including VCAM-1, ColIVa and IL-6. In addition, we analysed the expression of Deaf1 and Aire, transcriptional regulators involved in peripheral tolerance. Functional capacities of LNSCs were studied by analysing STAT-1 and MxA mRNA induction and interleukin (IL)-6 and IL-8 production after TLR-3 triggering by PolyI:C. Results Cells with a fibroblast-like morphology started to grow out from the stromal part of lymph node biopsies within a few weeks of culture. Passage 0 consisted of a mixture of adherent cells, resulting in a lower expression of the measured stromal genes. From passage 1 gene expression levels for VCAM-1, ColIVa and IL-6 increased and stabilised. All LNSCs cell lines expressed Deaf1 while the expression of Aire was only detected at very low levels. LNSCs were sensitive for TLR-3 ligation by PolyI:C resulting in upregulated STAT-1 and MxA, downregulated Deaf1 and high levels of IL-6 and IL-8. Chemokines CCL19 and CCL20 were only expressed after stimulation with PolyI:C. There was a high variability between donors and interim analysis showed no clear differences between LNSCs cultured from healthy individuals, RA risk or RA patients. Conclusions We developed a culture system for human LNSCs to facilitate research on the role of the lymph node microenvironment in the pathogenesis of RA. Cultured human LNSCs express typical stromal cell markers and are responsive for TLR-3 triggering. Interestingly, the LNSCs express the transcriptional regulator Deaf1 which may indicate peripheral tissue antigen expression by LNSCs.