Search

Objective ‘Treat-to-target principles’ are advised for axial spondyloarthritis (axSpA), although a clear target is not yet defined and targets do not always reflect inflammation. Treat-to-target use and motives for treatment choices in clinics are unknown. Therefore, we studied the presence of residual disease activity according physician’s opinion, patient’s opinion and composite indices and compared them to the subsequent treatment decisions. Methods This cross-sectional multicentre study included 249 patients with a clinical diagnosis of axSpA ≥6 months. Remission and low disease activity according to the BASDAI ( Results A total of 115/249 (46%) patients were in remission according to the physician and 37% (n = 43) of these patients reached remission according to the BASDAI. In 51/83 (60%) of the patients with residual disease activity according to the physician and a BASDAI >3.5 the treatment was left unchanged, either because of low disease activity as rated by the physician [n = 15 (29%)] or because of a combination of low disease activity with non-inflammatory complaints or comorbidities [n = 11 (25%)]. Retrospective treat-to-target evaluations showed that treatments were most frequently intensified in patients with arthritis or inflammatory back pain and less often in patients with other (non-inflammatory) musculoskeletal comorbidities. Conclusion This study shows that physicians do not always strictly apply treat-to-target in case of residual disease activity in axSpA. Usually, they accept low disease activity as satisfactory.

Lymph node stromal cells coordinate the adaptive immune response in secondary lymphoid organs, providing both a structural matrix and soluble factors that regulate survival and migration of immune cells, ultimately promoting Ag encounter. In several inflamed tissues, resident fibroblasts can acquire lymphoid-stroma properties and drive the formation of ectopic aggregates of immune cells, named tertiary lymphoid structures (TLSs). Mature TLSs are functional sites for the development of adaptive responses and, consequently, when present, can have an impact in both autoimmunity and cancer conditions. In this review, we go over recent findings concerning both lymph node stromal cells and TLSs function and formation and further describe what is currently known about their role in disease, particularly their potential in tolerance.

ObjectivesFollicular helper T cells (Tfh cells) provide key B cell help, and are essential in germinal center (GC) formation and (auto) antibody generation. To gain more insight into their role during the earliest phase of rheumatoid arthritis (RA) we analyzed their frequencies, phenotype and cytokine profile in peripheral blood and lymphoid tissues.MethodsUsing flow cytometry, we studied the frequency of Tfh and B cells in peripheral blood and lymph node (LN) needle biopsies. Three donor groups were included and compared: healthy controls (HCs), autoantibody positive individuals at risk for developing RA (RA-risk individuals), and early RA patients. Ex vivo stimulation of lymphocytes with PMA/ionomycin was performed to assess cytokine secretion by Tfh cells.ResultsIn blood, the frequency of circular Tfh cells (cTfh) did not differ between study groups. In lymphoid tissue, the frequency of Tfh cells correlated strongly with the frequency of CD19+ B cells. Compared to healthy controls, LN samples of RA patients and RA-risk individuals showed more CD19+ B cells and more CD4+CXCR5+ and CD8+CXCR5+ Tfh cells. These Tfh cells from LNs expressed less IL-21 upon ex vivo stimulation.ConclusionLN tissue of early RA patients as well as part of RA-risk individuals exhibit increased frequencies of Tfh cells correlating with increased numbers of B cells. Interestingly, IL-21 production is already aberrant in the very early at risk phase of the disease. This suggests that Tfh cells may present a novel rationale for therapeutic targeting during the preclinical stage of the disease to prevent further disease progression.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon &gamma, (IFN&gamma, ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

International audience; During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical auto-immune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

Objectives The exact underlying mechanism of rituximab treatment in patients with RA is poorly defined and knowledge about the effect of B cell depletion on immune cells in secondary lymphoid organs is lacking. We analysed lymphoid tissue responses to rituximab in RA patients. Methods Fourteen RA patients received 2 × 1000 mg rituximab intravenously, and lymph node (LN) biopsies were obtained before and 4 weeks after the first infusion. Tissues were examined by flow cytometry, immunohistochemistry and quantitative PCR. LN biopsies from five healthy individuals (HC) served as controls. Results LN biopsies of RA patients showed increased frequencies of CD21+CD23+IgDhighIgMvariable follicular B cells and CD3+CD25+CD69+ early activated, tissue resident T cells when compared with HCs. After treatment, there was incomplete depletion of LN B cells. There was a significant decrease in CD27−IgD+ naïve B cells, and CD27+IgD+ unswitched memory B cells including the CD27+IgD+IgM+ subset and follicular B cells. Strikingly, CD27+IgD− switched memory B cells persisted in LN biopsies after rituximab treatment. In the T cell compartment, a significant decrease was observed in the frequency of early activated, tissue resident T cells after rituximab treatment, but late activated T cells persisted. B cell proliferation inducing cytokine IL-21 was higher expressed in LN biopsies of RA patients compared with HC and expression was not affected by rituximab treatment. Conclusion Rituximab does not cure RA, possibly due to persistence of switched memory B cells in lymphoid tissues suggesting that factors promoting B cell survival and differentiation need to be additionally targeted.

Objective Innate lymphoid cells (ILCs) are emerging mediators of immunity, and accumulation of inflammatory ILC populations can occur in inflammatory-mediated conditions. Since early lymph node (LN) activation has been shown in rheumatoid arthritis (RA), we aimed to investigate the frequency and distribution of ILCs in LN biopsy specimens obtained during the earliest phases of RA. Methods Twelve patients with early RA, 12 individuals with IgM rheumatoid factor and/or anti–citrullinated protein antibodies without arthritis (RA risk group), and 7 healthy controls underwent ultrasound-guided inguinal LN biopsy. ILC subsets and the expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) by LN endothelial cells and fibroblasts were analyzed by flow cytometry. Results Although no differences in the frequencies of total ILCs (Lin−CD45+/lowCD127+) were found, the distribution of the ILC subpopulations differed among groups. RA patients showed lower numbers of lymphoid tissue–inducer (LTi) cells (c-Kit+NKp44− ILCs) and increased ILC1 (c-Kit−NKp44− ILCs) and ILC3 (c-Kit+NKp44+ ILCs) numbers compared with controls (P

The balance between proinflammatory and regulatory CD4(+) T cells is tightly controlled in lymphoid organs. In autoimmune diseases this balance is altered in the periphery and target tissue of patients. However, not much is known about the balance initiated in lymphoid organs during the development of disease. Since systemic autoimmunity is present years before the clinical manifestations of rheumatoid arthritis (RA), it is possible to study the immunoregulatory balance during the earliest (preclinical) phases of disease. Here, we report for the first time the frequency and phenotype of proinflammatory and regulatory CD4(+) T cells in lymph node biopsies obtained from autoantibody positive individuals at risk for developing RA, patients with established disease and healthy controls. The frequency of proinflammatory LN Th1 cells was increased in RA patients compared with HCs, while the frequency of regulatory T cells was lower in LN biopsies of RA-risk individuals. Upon in vitro stimulation LN CD4(+) T cells produced lower levels of proinflammatory cytokines, IFN-γ and IL-17A, in both RA-risk individuals and early RA patients. This study shows that already during the earliest phases of systemic autoimmunity the immunoregulatory balance between proinflammatory and regulatory CD4(+) T cells is altered in LN tissue

Tertiary lymphoid structures (TLSs) in chronic inflammation, including rheumatoid arthritis (RA) synovial tissue (ST), often contain high endothelial venules and follicular dendritic cells (FDCs). Endothelial cell (EC)–specific lymphotoxin β (LTβ) receptor signaling is critical for the formation of lymph nodes and high endothelial venules. FDCs arise from perivascular platelet-derived growth factor receptor β + precursor cells (preFDCs) that require specific group 3 innate lymphoid cells (ILC3s) and LTβ for their expansion. Previously, we showed that RA ST contains ECs that express NF-κB–inducing kinase (NIK), which is pivotal in LTβ-induced noncanonical NF-κB signaling. We studied the relation between NIK + ECs, (pre)FDCs, and ILC3s with respect to TLSs in RA ST. TLS + tissues exhibited a significantly increased expression of genes involved in noncanonical NF-κB signaling, including NIK, and immunohistochemical analysis revealed that NIK was almost exclusively expressed by ECs. ILC3s were present in human RA ST in very low numbers, but not differentially in TLS + tissues. In contrast, TLS + tissues contained significantly more NIK + ECs and perivascular platelet-derived growth factor receptor β + preFDCs, which correlated significantly with the quantity of FDCs. We established a strong link between NIK + ECs, (pre)FDCs, and the presence of TLSs, indicating that NIK + ECs may not only be important orchestrators of lymph node development but also contribute to the formation of TLSs in chronic inflammation.

Infections are implicated in autoimmunity. Autoantibodies are produced in lymphoid tissue where lymph node stromal cells (LNSCs) regulate lymphocyte function. Infections can alter the interaction between LNSCs and lymphocytes resulting in defective immune responses. In rheumatoid arthritis (RA) autoantibody production precedes clinical disease allowing identification of at risk individuals. We investigated the ability of human LNSCs derived from RA, RA-risk and healthy individuals to sense and respond to pathogens. Human LNSCs cultured directly from freshly collected lymph node biopsies expressed TLR1-9 with exception of TLR7. In all donors TLR3 triggering induced expression of ISGs, IL-6 and adhesion molecules like VCAM-1 and ICAM-1. Strikingly, T cell guiding chemokines CCL19 and IL-8 as well as the antiviral gene MxA were less induced upon TLR3 triggering in autoimmune LNSCs. This observed decrease, found already in LNSCs of RA-risk individuals, may lead to incorrect positioning of lymphocytes and aberrant immune responses during viral infections.

Background Forkhead box O (FoxO) transcription factors integrate environmental signals to modulate cell proliferation and survival, and alterations in FoxO function have been reported in rheumatoid arthritis (RA). Objectives To examine the relationship between inflammation and FoxO expression in RA, and to analyse the mechanisms and biological consequences of FoxO regulation in RA fibroblast-like synoviocytes (FLS). Methods RNA was isolated from RA patient and healthy donor (HD) peripheral blood and RA synovial tissue. Expression of FoxO1, FoxO3a and FoxO4 was measured by quantitative PCR. FoxO1 DNA binding, expression and mRNA stability in RA FLS were measured by ELISA-based assays, immunoblotting and quantitative PCR. FLS were transduced with adenovirus encoding constitutively active FoxO1 (FoxO1ADA) or transfected with small interfering RNA targeting FoxO1 to examine the effects on cell viability and gene expression. Results FoxO1 mRNA levels were reduced in RA patient peripheral blood compared with HD blood, and RA synovial tissue FoxO1 expression correlated negatively with disease activity. RA FLS stimulation with interleukin 1β or tumour necrosis factor caused rapid downregulation of FoxO1. This effect was independent of protein kinase B (PKB), but dependent on c-Jun N-terminal kinase (JNK)-mediated acceleration of FoxO1 mRNA degradation. FoxO1ADA overexpression in RA FLS induced apoptosis associated with altered expression of genes regulating cell cycle and survival, including BIM, p27 Kip1 and Bcl-XL. Conclusions Our findings identify JNK-dependent modulation of mRNA stability as an important PKB-independent mechanism underlying FoxO1 regulation by cytokines, and suggest that reduced FoxO1 expression is required to promote FLS survival in RA.

Objectives Rheumatoid arthritis (RA) is a prototypic chronic inflammatory disease with a debilitating course if untreated. A genetic predisposition for RA is known, and its occurrence is associated with the presence of autoantibodies in the serum and with environmental factors. It is unknown if smoking and overweight are contributory factors for developing RA in individuals with RA-specific autoantibodies in the serum. Methods Fifty-five individuals at risk for developing RA, based on the presence of RA-specific autoantibodies in the serum, who never had any evidence of arthritis upon physical examination, were followed over time. Smoking was assessed as being never or ever smoker and body mass index as

ObjectivesTo investigate the expression and activation of mitogen-activated protein kinases in patients with early arthritis who are disease-modifying antirheumatic drug (DMARD) naïve.MethodsA total of 50 patients with early arthritis who were DMARD naïve (disease duration ResultsERK and JNK activation was enhanced at inclusion in patients meeting RA criteria compared to other diagnoses. JNK activation was enhanced in patients diagnosed as having UA at baseline who eventually fulfilled 1987 ACR RA criteria compared to those who remained UA, and in patients with RA fulfilling 2010 ACR/EULAR criteria at baseline. ERK and JNK activation was enhanced in patients with RA developing progressive joint destruction. JNK activation in UA predicted 1987 ACR RA classification criteria fulfilment (R2=0.59, p=0.02) after follow-up, and disease progression in early arthritis (R2=0.16, pConclusionsJNK activation is elevated in RA before 1987 ACR RA classification criteria are met and predicts development of erosive disease in early arthritis, suggesting JNK may represent an attractive target in treating RA early in the disease process.

Objectives Interferon beta (IFN beta) therapy is effective in multiple sclerosis and murine models of arthritis. Surprisingly, systemic IFN beta treatment induces only minimal improvement in rheumatoid arthritis (RA). To explain this paradox, the authors evaluated the mechanism of IFN beta benefit in passive K/BxN arthritis and the effect of IFN beta treatment on RA synovium. Methods Interleukin 10 (IL-10) null, IL-1 receptor antagonist (IL-1Ra) null, IL-1Ra transgenic and wildtype mice were administered K/BxN serum and in some cases treated with IFN beta or normal saline. Clinical response and histological scores were assessed. Gene expression was measured by quantitative PCR. Serum IL-1Ra and IL-6 were measured by ELISA. Paired synovial biopsy specimens from RA patients pre-IFN beta and post-IFN beta treatment (purified natural fibroblast IFN beta (Frone) subcutaneously three times weekly 6 million IU, 12 million IU or 18 million IU) were immunostained for IL-1Ra and IL-10. Results Il1rn transgenic mice had an attenuated course of arthritis, whereas Il1rn(-/-) and Il10(-/-) mice had more severe serum transfer arthritis than wild-type mice. Daily IFN beta treatment significantly decreased arthritis severity in Il10(-/-) but not Il1rn(-/-) mice. IFN beta treatment did not reduce the histological scores in Il1rn(-/-) mice or gene expression of articular cytokines and chemokines. Paired synovial biopsy specimens from RA patients treated with IFN beta demonstrated a trend towards increased IL-1Ra and reduced IL-10 expression on day 85 levels compared with pretreatment specimens. Conclusions The anti-inflammatory effects of IFN beta in passive K/BxN arthritis are dependent on IL-1Ra, but not IL-10. Systemic IFN beta treatment in RA increases synovial IL-1Ra production, but also decreases IL-10 production

Multiple sclerosis (MS) is an inflammatory disease of the CNS. The clinical presentation of MS is heterogeneous. Interferons (IFNs) were the first agents to show clinical efficacy in the treatment of MS and prolonged treatment is still the best available therapy. Although IFN treatment ameliorates immune dysfunction, the response is partial. Clinical experience indicates that there are responders and nonresponders. This distinction suggests that a subset of patients are insensitive or resistant to the action of IFN. This implies that pharmacodynamic responses may differ between patients, leading to interindividual differences in clinical response. Understanding of the factors that underlie the therapeutic response is key to the identification of predictive markers. Here, we describe novel developments in pharmacogenomics research to improve the understanding of the pharmacological effects of IFN therapy, and the identification of biomarkers that allow stratification of MS patients for their response to IFN-β. Ultimately, this information will lead to a personalized form of medicine, whereby a specific therapy will be applied that is best suited to an individual patient.

Although many studies are focused on auto-reactive CD4(+) T cells, the precise role of CD8(+) T cells in autoimmunity is poorly understood. The objective of this study is to provide more insight into the phenotype and function CD8(+) T cells during the development of autoimmune disease by studying CD8(+) T cells in human lymph-node biopsies and peripheral blood obtained during the earliest phases of rheumatoid arthritis (RA). Here, we show that lymphoid pro-inflammatory CD8(+) T cells exhibit a less-responsive phenotype already during the earliest phases of autoimmunity compared with healthy individuals. We found an increase in CD8(+) memory T cells in lymphoid tissue during the earliest phases of autoimmunity, even before clinical onset of RA, accompanied by an increased frequency of non-circulating or recently activated (CD69(+)) CD8(+) T cells in lymphoid tissue and peripheral blood. Importantly, lymphoid pro-inflammatory CD8(+) IL-17A(+) T cells displayed a decreased capacity of cytokine production, which was related to disease activity in early RA patients. In addition, a decreased frequency of regulatory CD8(+) IL-10(+) T cells in peripheral blood was also related to disease activity in early RA patients. Our results suggest that different CD8(+) T-cell subsets are affected already during the earliest phases of systemic autoimmunity

Objectives Epigenetic modifications play an important role in the regulation of gene transcription and cellular function. Here, we examined if pro-inflammatory factors present in the inflamed joint of patients with rheumatoid arthritis (RA) could regulate histone deacetylase (HDAC) expression and function in fibroblast-like synoviocytes (FLS). Methods Protein acetylation in synovial tissue was assessed by immunohistochemistry. The mRNA levels of HDAC family members and inflammatory mediators in the synovial tissue and the changes in HDAC expression in RA FLS were measured by quantitative (q) PCR. FLS were either transfected with HDAC5 siRNA or transduced with adenoviral vector encoding wild-type HDAC5 and the effects of HDAC5 manipulation were examined by qPCR arrays, ELISA and ELISA-based assays. Results Synovial class I HDAC expression was associated with local expression of tumour necrosis factor (TNF) and matrix metalloproteinase-1, while class Ila HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1 beta or TNF stimulation selectively suppressed HDAC5 expression in RA FLS, which was sufficient and required for optimal IFNB, CXCL9, CXCL10 and CXCL11 induction by IL-1 beta, associated with increased nuclear accumulation of the transcription factor, interferon regulatory factor 1(IRF1). Conclusions Inflammatory cytokines suppress RA FLS HDAC5 expression, promoting nuclear localisation of IRF1 and transcription of a subset of type I interferon response genes. Our results identify HDAC5 as a novel inflammatory mediator in RA, and suggest that strategies rescuing HDAC5 expression in vivo, or the development of HDAC inhibitors not affecting HDAC5 activity, may have therapeutic applications in RA treatment

There is growing evidence that rheumatoid arthritis (RA) is a heterogeneous disease. The disease is generally referred to in terms of a group average, which may hamper the progress in understanding its genetic basis, pathogenic mechanisms and the treatment success for subsets of patients. Unfortunately, criteria for subtyping of patients, for example to select those patients who will benefit from a specific treatment, is currently lacking. Since, by definition, nearly every aspect of a disease phenotype should be represented in the pattern of genes and proteins that are expressed in the affected tissues and organs, molecular typing of patients is likely to yield useful classifiers for RA subtyping. This paper will describe novel developments in genomics and proteomics research for the identification of biomarkers for disease subclassification in RA. This information will also improve our understanding of the underlying biology of RA subtypes. Ultimately, this information will help clinicians to select sub...

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1α gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1α gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1α gene expression.

Objective Arthralgia may precede the development of synovial inflammation in autoantibody-positive individuals at risk of developing rheumatoid arthritis (RA). A major pathway involved in pain is the prostaglandin (PG) E2 pathway. We investigated this pathway in the synovium of individuals with RA-specific autoantibodies and in early arthritis patients. Methods Nineteen autoantibody-positive individuals (IgM-rheumatoid factor and/or anti-cyclic citrullinated peptide antibodies) with arthralgia (n=15) and/or a positive family history of RA (n=8), who had been prospectively followed for at least 2 years, were included. In addition, we included early arthritis patients (disease-modifying antirheumatic drug naive) who after 2 years follow up fulfilled classification criteria for RA (n=63), spondyloarthritis (SpA; n=14), or had unclassified arthritis (UA; n=27). In all subjects we assessed pain and performed synovial biopsy sampling by mini-arthroscopy at baseline. Tissue sections were examined by immunohistochemistry to detect and quantify PGE2 pathway enzymes expression levels (mPGES-1; COX-1 and -2; 15-PGDH). Results In both study groups synovial expression of PGE2 enzymes was not clearly related to pain sensation. Expression levels at baseline were not associated with the development of arthritis after follow up (6 out of 19 autoantibody-positive individuals). However, in early SpA patients the expression levels of mPGES-1 and COX-1 were significantly increased compared to RA and UA patients. Conclusion Pain in autoantibody-positive individuals without synovial inflammation who are at risk of developing RA and in early arthritis patients may be regulated by pathways other than the PGE2 pathway or originate at sites other than the synovium. In contrast, in SpA, the PGE2 pathway may be inherently linked to the pathophysiology/etiology of the disease.

The Study Group for Risk Factors for Rheumatoid Arthritis was established by the EULAR Standing Committee on Investigative Rheumatology to facilitate research into the preclinical and earliest clinically apparent phases of rheumatoid arthritis (RA). This report describes the recommendation for terminology to be used to define specific subgroups during different phases of disease, and defines the priorities for research in this area. Terminology was discussed by way of a three-stage structured process: A provisional list of descriptors for each of the possible phases preceding the diagnosis of RA were circulated to members of the study group for review and feedback. Anonymised comments from the members on this list were fed back to participants before a 2-day meeting. 18 participants met to discuss these data, agree terminologies and prioritise important research questions. The study group recommended that, in prospective studies, individuals without RA are described as having: genetic risk factors for RA; environmental risk factors for RA; systemic autoimmunity associated with RA; symptoms without clinical arthritis; unclassified arthritis; which may be used in a combinatorial manner. It was recommended that the prefix ‘pre-RA with:’ could be used before any/any combination of the five points above but only to describe retrospectively a phase that an individual had progressed through once it was known that they have developed RA. An approach to dating disease onset was recommended. In addition, important areas for research were proposed, including research of other tissues in which an adaptive immune response may be initiated, and the identification of additional risk factors and biomarkers for the development of RA, its progression and the development of extra-articular features. These recommendations provide guidance on approaches to describe phases before the development of RA that will facilitate communication between researchers and comparisons between studies. A number of research questions have been defined, requiring new cohorts to be established and new techniques to be developed to image and collect material from different sites.

Personalised medicine has the potential to increase therapeutic effectiveness, reduce side effects and lower cost. This approach has recently taken off in oncology where different malignancies may be treated with specific drugs based on genetic biomarkers or other tumour characteristics. This type of tailored therapy could also be developed for immune-mediated inflammatory disease, for which rheumatoid arthritis (RA) may serve as a prototype. While novel treatments are able to halt or even prevent disease progression, not all RA patients respond, and stratification of patient groups is needed. The identification of biomarkers predictive of the clinical response to specific treatments in subsets of patients may soon become reality in a variety of diseases.

Inflammation of synovium is one of the hallmarks of rheumatoid arthritis (RA). Analysis of synovial tissue has increased our understanding of RA pathogenesis, aided in identifying potential therapeutic targets and has been used in the response and mechanistic evaluation of antirheumatic treatments. In addition, studies are ongoing, aimed at the identification of diagnostic and prognostic biomarkers in the synovium. This paper outlines the currently used procedures for sampling and processing of synovial tissue, and presents a standardised recommendation to support multicentre translational research.

A cross-regulation between type I IFN and TNFα has been proposed recently, where both cytokines are hypothesized to counteract each other. According to this model, different autoimmune diseases can be viewed as disequilibrium between both cytokines. As this model may have important clinical implications, the present review summarizes and discusses the currently available clinical evidence arguing for or against the proposed cross-regulation between TNFα and type I IFN. In addition, we review how this cross-regulation works at the cellular and molecular levels. Finally, we discuss the clinical relevance of this proposed cross-regulation for biological therapies such as type I IFN or anti-TNFα treatment

Objective To identify molecular features associated with the development of rheumatoid arthritis (RA), to understand the pathophysiology of preclinical development of RA, and to assign predictive biomarkers. Methods The study group comprised 109 anti–citrullinated protein antibody (ACPA)– and/or rheumatoid factor–positive patients with arthralgia who did not have arthritis but were at risk of RA, and 25 patients with RA. The gene expression profiles of blood samples obtained from these patients were determined by DNA microarray analysis and quantitative polymerase chain reaction. Results In 20 of the 109 patients with arthralgia who were at risk of RA, arthritis developed after a median of 7 months. Gene expression profiling of blood cells revealed heterogeneity among the at-risk patients, based on differential expression of immune-related genes. This report is the first to describe gene signatures relevant to the development of arthritis. Signatures significantly associated with arthritis development were involved in interferon (IFN)–mediated immunity, hematopoiesis, and chemokine/cytokine activity. Logistic regression analysis revealed that the odds ratio (OR) for developing arthritis within 12 months was 21.0 (95% confidence interval [95% CI] 2.8–156.1 [P = 0.003]) for the subgroup characterized by increased expression of genes involved in IFN-mediated immunity and/or cytokine/chemokine-activity. Genes involved in B cell immunology were associated with protection against progression to arthritis (OR 0.38, 95% CI 0.21–0.70 [P = 0.002]). These processes were reminiscent of those in patients with RA, implying that the preclinical phase of disease is associated with features of established disease. Conclusion The results of this study indicate that IFN-mediated immunity, hematopoiesis, and cell trafficking specify processes relevant to the progression of arthritis independent of ACPA positivity. These findings strongly suggest that certain gene signatures have value for predicting the progression to arthritis, which will pave the way to preventive medicine.

Objective To investigate the clinical relevance of synovial tissue subtypes in rheumatoid arthritis (RA) and to search for peripheral blood (PB) markers that may serve as biomarkers for tissue subtypes. Methods Gene expression analysis using complementary DNA microarrays was applied on paired synovial tissue biopsy and PB samples obtained from 17 RA patients. Molecular tissue subtypes were correlated with histologic parameters (CD3, CD22, CD38, CD68, CD163, tumor necrosis factor α, intercellular adhesion molecule 1, vascular cell adhesion molecule, and E-selectin), disease characteristics, and PB markers. PANTHER classification was used for pathway analysis. Results Genomic subtyping of high- and low-inflammation rheumatoid synovial tissues based on gene expression profiles exactly matched immunohistochemical classification. The patients with the high-inflammation tissue type had higher Disease Activity Scores in 28 joints, higher C-reactive protein levels, higher erythrocyte sedimentation rates, increased numbers of platelets, and shorter disease durations. Comparative analysis of PB gene expression profiles yielded no statistically significant differences between the 2 tissue groups at the single-gene expression level. PANTHER pathway analysis revealed a significant association of increased protein biosynthesis with high-inflammation tissue. Conclusion High-inflammation tissue is associated with more severe disease and shorter disease duration. While pathway-level analysis revealed that coordinate differential expression of genes involved in protein synthesis in PB is associated with high-inflammation tissue types, differential tissue pathology was not reflected in the PB by differential expression of single genes.

BACKGROUND: Although the use of TNF inhibitors has fundamentally changed the way rheumatoid arthritis (RA) is treated, not all patients respond well. It is desirable to facilitate the identification of responding and non-responding patients prior to treatment, not only to avoid unnecessary treatment but also for financial reasons. In this work we have investigated the transcriptional profile of synovial tissue sampled from RA patients before anti-TNF treatment with the aim to identify biomarkers predictive of response. METHODOLOGY/PRINCIPAL FINDINGS: Synovial tissue samples were obtained by arthroscopy from 62 RA patients before the initiation of infliximab treatment. RNA was extracted and gene expression profiling was performed using an in-house spotted long oligonucleotide array covering 17972 unique genes. Tissue sections were also analyzed by immunohistochemistry to evaluate cell infiltrates. Response to infliximab treatment was assessed according to the EULAR response criteria. The presence of lymphocyte aggregates dominated the expression profiles and a significant overrepresentation of lymphocyte aggregates in good responding patients confounded the analyses. A statistical model was set up to control for the effect of aggregates, but no differences could be identified between responders and non-responders. Subsequently, the patients were split into lymphocyte aggregate positive- and negative patients. No statistically significant differences could be identified except for 38 transcripts associated with differences between good- and non-responders in aggregate positive patients. A profile was identified in these genes that indicated a higher level of metabolism in good responding patients, which indirectly can be connected to increased inflammation. CONCLUSIONS/SIGNIFICANCE: It is pivotal to account for the presence of lymphoid aggregates when studying gene expression patterns in rheumatoid synovial tissue. In spite of our original hypothesis, the data do not support the notion that microarray analysis of whole synovial biopsy specimens can be used in the context of personalized medicine to identify non-responders to anti-TNF therapy before the initiation of treatment.

Rheumatic diseases are a diverse group of disorders. Most of these diseases are heterogeneous in nature and show varying responsiveness to treatment. Because our understanding of the molecular complexity of rheumatic diseases is incomplete and criteria for categorization are limited, we mainly refer to them in terms of group averages. The advent of DNA microarray technology has provided a powerful tool to gain insight into the molecular complexity of these diseases; this technology facilitates open-ended survey to identify comprehensively the genes and biological pathways that are associated with clinically defined conditions. During the past decade, encouraging results have been generated in the molecular description of complex rheumatic diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren syndrome and systemic sclerosis. Here, we describe developments in genomics research during the past decade that have contributed to our knowledge of pathogenesis, and to the identification of biomarkers for diagnosis, patient stratification and prognostication.

Background: Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNs in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNs therapy. Methodology: Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNs therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNs. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation. Principal Findings: Pharmacogenomics revealed a marked variation in the pharmacological response to IFNs between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNs therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R= 20.7208; p=0.0016). The negative correlation was maintained after three (R= 20.7363; p=0.0027) and six (R= 20.8154; p=0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n=30; R= 20.4719; p=0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNs indicating that differential responsiveness to IFNs is an intrinsic feature of peripheral blood cells at baseline. Conclusion: These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNs. Citation: van Baarsen LGM, Vosslamber S, Tijssen M, Baggen JMC, van der Voort LF, et al. (2008) Pharmacogenomics of Interferon-s Therapy in Multiple Sclerosis

In the last decade, the analysis of gene expression in tissues and cells has evolved from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and that define the samples' unique biology. By a global analysis of the genes that are expressed in cells and tissues of an individual under different conditions and during disease, we can build up "gene expression profiles (signatures)" which characterize the dynamic functioning of the genome under pathophysiological conditions. This strategy also provides the means to subdivide patients that suffer from a complex heterogeneous disease into more homogeneous subgroups. Such discovery-based research identifies biological processes that may include new genes with unknown function or genes not previously known to be involved in this process. The latter category may hold surprises that sometimes urge us to redirect our thinking. We have used microarrays to disclose the heterogeneity of rheumatoid arthritis (RA) patients at the level of gene expression of the affected synovial tissues. Analysis of the expression profiles of synovial tissues from different patients with RA revealed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One is characterized by genes that indicate an active inflammatory infiltrate with high immunoglobulin production, whereas the other type shows little immune activation and instead shows a higher stromal cell activity. These results confirm the heterogeneous nature of RA and suggest the existence of distinct pathogenic mechanisms that contribute to RA. The differences in expression profiles provide opportunities to stratify patients for intervention therapies based on molecular criteria.

In the last decade, the analysis of gene expression in tissues and cells has evolved from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and that define the samples' unique biology. By a global analysis of the genes that are expressed in cells and tissues of an individual under different conditions and during disease, we can build up "gene expression profiles (signatures)" which characterize the dynamic functioning of the genome under pathophysiological conditions. This strategy also provides the means to subdivide patients that suffer from a complex heterogeneous disease into more homogeneous subgroups. Such discovery-based research identifies biological processes that may include new genes with unknown function or genes not previously known to be involved in this process. The latter category may hold surprises that sometimes urge us to redirect our thinking. We have used microarrays to disclose the heterogeneity of rheumatoid arthritis (RA) patients at the level of gene expression of the affected synovial tissues. Analysis of the expression profiles of synovial tissues from different patients with RA revealed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One is characterized by genes that indicate an active inflammatory infiltrate with high immunoglobulin production, whereas the other type shows little immune activation and instead shows a higher stromal cell activity. These results confirm the heterogeneous nature of RA and suggest the existence of distinct pathogenic mechanisms that contribute to RA. The differences in expression profiles provide opportunities to stratify patients for intervention therapies based on molecular criteria.

Objective Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues. Methods Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues. Results Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor β/activin A–inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue. Conclusion Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

Background and Objectives Adenosine and ATP are known to have important immunomodulatory properties. Extracellular ATP has multiple roles in inflammation and can act as a damage-associated molecular pattern (DAMP) that can activate the immune system. Conversely, adenosine is primarily anti-inflammatory and can inhibit the production of pro-inflammatory molecules by immune cells. The modulation of ATP and adenosine levels are an essential part of the induction and resolution of an inflammatory response. ENTPD1 (CD39) is a membrane-bound ectonucleoside triphosphate diphosphohydrolase enzyme that converts ATP and ADP to AMP 5NTE1 (CD73) is a 5′ ecto-nucleotidase that dephosphorylates AMP to form adenosine. We investigated the role of genes in the adenosine pathway in patients with rheumatoid arthritis (RA) and determined whether expression of CD39 and CD73 would have an effect in an in vitro inflammation model. Materials and Methods Gene expression analysis using 43k cDNA microarrays (Stanford Functional Genomics Facility) was performed on total RNA extracted from RA synovial tissues obtained by arthroscopy. Adenosine pathway gene expression was compared between high-inflammation versus low-inflammation tissue type synovial biopsies. ATPase levels were measured in synovial fluid (SF) from RA (n = 10) or osteoarthritis (OA) (n = 6) patients. Adeno-associated viral (AAV) vectors expressing CD39 or CD73 were generated and used to transduce HEK 293 cells or RA fibroblast-like synoviocytes (FLS) and these transduced cells were co-cultured with LPS-activated human monocytes (THP-1) in the presence of ATP. Pro-inflammatory cytokine/chemokine (IL-6, CCL2) production was measured by ELISA. Results Genes involved in the ATP:adenosine pathway, including CD73, were differentially expressed in high-inflammation synovial tissues, consistent with the hypothesis that there is skewing of the ATP:adenosine balance during inflammation. The half-life of ATP was significantly increased in SF from RA patients compared with OA (t 1/2 = 8.0 versus 4.5 min., p = 80% reduction p = Conclusions Together, these data suggest that synovial inflammation in RA is characterised by skewing of the ATP:adenosine balance. This could be reversed by overexpression of CD39 or CD73. Thus, these data show that the ATP:adenosine pathway may be a novel therapeutic target for the treatment of RA.

Backgroundand objectives Forkhead box O (FoxO) transcription factors, regulated by phosphatidlyinositol 3-kinase and reversible acetylation, integrate environmental signals to orchestrate inflammatory responses, cell cycle and apoptosis. Here, the authors examined the relationship between inflammation and FoxO expression and activity in rheumatoid arthritis (RA) synovial tissue, determined the effects of histone deacetylase inhibitors (HDACi) on FoxO expression and activity in RA fibroblast-like synoviocytes (FLS), and identified genes regulated by FoxO1 in RA FLS. Materials and methods Total RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients. FoxO1, FoxO3a, FoxO4, TNFα, MMP-1 and IL-6 expression were measured by quantitative PCR (qPCR) in synovial tissue and RA and osteoarthritis (OA) FLS. RA FLS were stimulated with IL-1β or TNFα, in the absence or presence of the HDACi trichostatin A, and FoxO family member expression and FoxO1 DNA binding activity were measured by qPCR and ELISA-based assays, respectively. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on RA FLS gene expression using low density qPCR arrays. Results Negative correlations were observed between RA synovial tissue expression of FoxO1 and the levels of serum C reactive protein (CRP) (R=−0.771, p=0.0008), erythrocyte sedimentation rate (ESR) (R=−0.739, p=0.0003), and disease activity score 28 (DAS28) (R=−0.575, p=0.01). A strong negative correlation was also observed between synovial FoxO1 and IL-6 mRNA levels (R=−0.628, p=0.004), but not TNFα or MMP-1. FoxO1, FoxO3a and FoxO4 mRNA were each detected in RA and OA FLS, and FoxO1 mRNA levels were significantly reduced in RA FLS compared to OA FLS (p Kip1 . FoxO1ADA also suppressed IL-1β-mediated induction of inflammatory mediators, including CCL2, CXCL6, PDGF and ICAM1. Conclusions The authors demonstrate that inflammatory stimuli decrease FoxO1 expression and DNA binding activity, effects reversed by exposure of RA FLS to HDACi. Restoration of FoxO1 activity suppresses a subset of genes regulated by HDACi during FLS activation, identifying the enhancement of FoxO1 function as a novel mechanism by which HDACi might mediate their anti-inflammatory effects in RA synovial cells.

Background Fms-like tyrosine kinase 3 ligand (Flt3L) is a potent endogenous growth factor for myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). Its administration to mice and humans leads to dramatic increases of various DC subsets while Flt3L−/− mice show reduced DC numbers. Flt3L and its receptor (CD135) have been poorly studied in the setting of autoimmune diseases in general. Typically, CD135 is expressed on early myeloid and lymphoid progenitors and is activated by its soluble ligand, Flt3L. The highly differentiated cellular pattern in rheumatoid arthritis (RA) synovium made the authors hypothesise that Flt3L, with its ability to induce proliferation and differentiation, could be of importance in induction and/or progression of arthritis. Material and methods Patients with active RA, psoriatic arthritis (PsA), spondyloarthritis (SpA), osteoarthritis (OA), gout and healthy donors (HD) were included in this study. Soluble (s)Flt3L levels in synovial fluid (SF) and serum were determined by ELISA. Expression of membrane-bound (m)Flt3L and Flt3L receptor (CD135) in peripheral blood mononuclear cells (PBMC) and SFMC were assessed by FACS. In addition, immunohistochemical analysis of Flt3L and CD135 was performed in RA, PsA, gout, OA and HD synovial tissues. Results SF levels of Flt3L in RA (n=103), PsA (n=33), and SpA (n=32), OA (n=8) and gout (n=43) were significantly higher compared to paired serum. In addition, Flt3L levels were significantly higher in RA, PsA and SpA SFs compared to gout SF. In peripheral blood (PB) monocytes, B cells and mDC the expression of mFlt3L in RA was higher compared to HD. Flt3L receptor expression was confined to monocytes and mDC and higher in RA SF compared to PB. Immunohistochemistry and immunofluorescence data showed the presence of Flt3L and CD135 in RA ST. Interestingly, microarray data of RA synovial tissue showed that CD135 expression is increased in patients with high inflammatory gene profile compared to low inflammatory gene profile. There is no different in Flt3L expression between RA and OA confirming the ELISA data. In addition, anti-TNF therapy reduced Flt3L serum levels in RA patients. Conclusion The data presented in this study point to inflammatory role for Flt3L/CD135 system. Moreover, as the Flt3L/CD135 system is implicated in the generation of DC and B cells, inflammatory cells important in RA pathogenesis, this system might be of importance in RA. Achieving a detailed understanding of Flt3L function(s) in arthritis may lead to the development of novel immunotherapies for RA and other immune-mediated inflammatory diseases.

Keratinocytes are continuously in contact with external stimuli and have the capacity to produce several soluble mediators. Pathogen-associated molecular patterns (PAMPs) are recognized, among others, by Toll-like receptors (TLRs). The functional responses of keratinocytes to different PAMPs have not yet been fully established. Here we show that keratinocytes constitutively express TLR1, 2, 3, 4, 5, 6, 9, and 10 mRNA, but not TLR7 and 8. Stimulation of keratinocytes with TLR3, 4, 5, and 9 ligands resulted in differential immune-associated responses. Tumor necrosis factor-alpha, CXC chemokine ligand 8 (CXCL8), CCL2, and C chemokine ligand 20 (CCL20) release was enhanced in response to all PAMPs tested, in a time- and dose-dependent manner. Only TLR9 ligand CpG-oligodeoxynucleotides (ODNs) and TLR3 ligand poly-I:C could additionally induce type I IFNs. CCL27 production was selectively induced by poly-I:C and flagellin, whereas CXCL9 and CXCL10 were exclusively induced by CpG-ODNs and/or poly-I:C. Upregulation of ICAM-1, HLA-DR, HLA-ABC, FasR, and CD40 was mainly observed in response to poly-I:C, flagellin, and lipopolysaccharide. Furthermore, PAMP triggering resulted in the phosphorylation of phosphorylated-IkappaB alpha and in the nucleus translocation of NF-kappaB p65. Altogether, these findings stress an unexpectedly multifaceted role of keratinocytes in innate immunity as evident by their differential, TLR-mediated responses to PAMPs associated with different classes of pathogens.