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Towards Early diagnosis and biomarker validation in Arthritis Management

Final Report Summary - EURO-TEAM (Towards Early diagnosis and biomarker validation in Arthritis Management)

Executive Summary:
The EuroTEAM consortium has catalyzed five key changes in the development of biomarkers and diagnostic kits that are timely, strategically important for European SMEs, and based on a disease (rheumatoid arthritis - RA) in which concrete genetic and environmental risk factors have already been established. These developments have delivered a step change in the way clinicians approach RA, moving from treatment to prevention with major benefits for public health.

Specifically, we have:
• identified novel biomarkers in the preclinical phases of disease which can predict RA development
• looked within the synovium, lymph node, lung and systemic compartments to understand mechanism by which immune tolerance is broken and the onset of RA is triggered
• explored a key cell type (stromal cells) that is almost completely ignored in current biomarker studies and assessed interactions between these cells and leukocytes to understand how this cell type may play a role in disease onset and its characteristics may provide novel biomarkers for patient outcome
• directly involved patients and other specialists, particularly from the fields of genetics, ethics and patient and public involvement in helping to visualize and communicate risk
• provided a model for developing diagnostic, near patient kits in the very early phases of disease

Project Context and Objectives:
Rheumatoid arthritis (RA) is a heterogeneous chronic immune mediated inflammatory disease associated with significant morbidity. RA causes joint pain and stiffness, makes it difficult for patients to use their joints and reduces the patient’s quality of life. Because of the effect of the disease on other areas of the body, including the cardiovascular system and bones, it can also reduce life expectancy. The disease is common, affecting 1 in 100 individuals, and will become more so as the population ages.

An understanding of the mechanisms operating both systemically and in the inflamed joint in the established phase of disease, has led to dramatic advances utilizing biologic therapies that have significantly improved outcomes for patients. These pioneering treatments have provided the rationale for equivalent interventions in other chronic inflammatory diseases e.g. inflammatory bowel disease and psoriasis. However, we remain unable to cure RA. Most patients require lifelong therapy and despite this, many patients still develop progressive joint and extra-articular damage.

An ideal intervention in a chronic inflammatory disease such as RA would be a preventive one. In order to develop preventive strategies and therapies two key step-changes need to occur:

1. Biomarkers need to be identified that can be used to predict an individual’s risk of developing RA with a high degree of confidence
2. Modifiable disease mechanisms need to be identified and characterized in these early pre-RA phases of disease

Work over the last 20 years, to define the phases and processes in the transition from health to established disease, has placed RA in a unique position amongst all immune mediated inflammatory diseases. Very well validated genetic, epigenetic and environmental factors have been identified that interact with immunological and biochemical processes to drive disease pathology. For this reason, the Euro-TEAM consortium focused on RA to characterize the processes involved in the induction and persistence of inflammation during the transition from health to disease.

In the transition to RA, individuals can be described as being in different phases, as defined by the EULAR (European League Against Rheumatism) Study Group on Risk Factors for RA [1]:

(A) Genetic risk factors for RA
(B) Environmental risk factors for RA
(C) Features of systemic autoimmunity associated with the development of RA
(D) Symptoms of RA (e.g. joint pain and stiffness) without clinically apparent arthritis
(E) Clinically apparent arthritis but not yet fulfilling classification criteria for RA
(F) RA

In some cases, individuals will move through these ‘phases’ sequentially from pre-clinical phases (an individual with genetic risk factors (A), will be exposed to environmental risk factors (B), and then develop systemic autoimmunity (C)) through clinical phases (an individual will develop symptoms e.g. joint pain and stiffness (D), then arthritis (E)) and finally a disease classified as RA (F). Although it is often assumed that individuals move sequentially through these phases, the evidence for this is scant.

Importantly, it is known that in some cases individuals may also move ‘backwards’ e.g. with an individual with unclassified clinical arthritis getting better with no further systemic features of disease. Furthermore, it is possible for individuals to “skip” particular phases, and that the phases do not develop in the order represented, for example with evidence of systemic autoimmunity developing after the onset of symptoms. Dissecting these relationships is central to the issue of prognostication in RA.

Transition between phases leading to the development of rheumatoid arthritis (RA) may not always be linear nor unidirectional. ‘Challenging linearity’ illustrates that patients may skip phase C and progress from phase A&B directly to phase D, E or even F. Multiple potential routes to RA are possible and were explored within Euro-TEAM; thus a patient could move from phases A&B to phase C, skip phase D and develop, as their first clinical feature, a clinically apparent arthritis with synovitis (Phase E).

The identification of similarities and differences between the different phases of disease have informed us of the extent to which treatment strategies currently used in phases (E) and (F) can also be used in the earlier phases of disease that are at present very poorly explored from a therapeutic perspective. For example, it is possible that the biomarkers and therapies that are effective in phases with clinically apparent arthritis (E and F) are ineffective during earlier phases. Furthermore, the study of patients who follow a ‘backwards’ disease course (e.g. patients in a clinically apparent phase who get better) will shed important new light on anti-inflammatory mechanisms and provide novel pro-resolution biomarkers and treatment approaches that can be used in patients at risk to prevent progression. Understanding the processes and associated biomarkers linked to progression versus resolution during the development of RA has been one of several unique features of the EuroTEAM consortium.

Specific aim of the EuroTEAM consortium:
The key aim of the EuroTEAM consortium was to identify biomarkers and disease mechanisms operating during the phases of disease leading up to the development of RA and translate these findings into improved and user appropriate / acceptable diagnostic kits and strategies for the benefit of the EU economy.

In order to achieve this aim, we focussed on the following objectives:

1 To investigate genetic and other risk factors in the transitions between RA risk phases
2 To determine the ability biomarkers to predict transitions between RA risk phases
3 To develop and validate predictive biomarker profiles for RA
4 To investigate the mechanisms by which risk factors influence transition between disease phases
5 To analyse the pathogenic role of immune cells and their interactions in disease initiation
6 To identify novel therapeutic targets based on pathogenic pathways identified in synovial and extra-articular tissues
7 To understand perspective of individuals in different pre-RA phases about the risk of RA and to develop approaches to education about RA risk and risk modification
8 To develop strategies for communication with and education of individuals in different pre-RA phases about RA risk and risk modification

Achieving these objectives required access to considerable technical resource and expertise, as well as large populations of individuals at risk of RA. The EuroTEAM consortium brought together clinical, academic and industrial partners to provide sufficient critical mass to allow us to address these major clinical and scientific questions. Importantly, EuroTEAM also ensured that the patient voice was firmly embedded in the research project – influencing and facilitating the direction, delivery and dissemination of our research findings.

Project Results:
Work package overview:
EuroTEAM had five work packages (WPs). WPs 1, 2 and 3 addressed disease mechanisms and biomarker identification by studying different tissues: genetic material from the blood in WP1, other markers that can be detected in the blood (e.g. antibodies and metabolites) in WP2 and tissue obtained by biopsy (e.g. from the joints and lymph nodes) in WP3.

Ultimately, the aim across all five WPs was to offer predictive tests to individuals without RA - such individuals may be those who are entirely well, but with a family history of RA (Phase A), all the way through to patients with an early but currently unclassified arthritis (Phase E). However, in all cases, potential users of the predictive tests will need to make an informed decision about whether to have the test, having fully understood the advantages and disadvantages of testing. Furthermore, they will need to be provided with resources that allow them to make sense of “risk” information given to them and be supported in making appropriate lifestyle choices decisions about treatment based on the test results. WP4 developed approaches that support patient decision making, understanding and behavioural change in the context of predictive testing.

Co-ordinating activities across 16 institutions and managing a programme of this scale presented a range of challenges, which were effectively met by WP5 (project management).

Central to the EuroTEAM concept was the active involvement of those with the lived experience of RA and of being “at risk” in the development delivery and dissemination of the project. An international team of nine Patient Research Partners supported research activities throughout the work packages and played a key role in dissemination, supported by the project website.

A core element of the EuroTEAM programme was the facilitation of co-ordinated working between partner organisations, and to allow this, an early deliverable was the development of an inventory of patient cohorts and available biological material within the consortium according to RA risk disease phases. The availability of this resource facilitated joint working.

Patient Research Partners in EuroTEAM: A key legacy of the consortium
An important and unique element of EuroTEAM has been the EuroTEAM Patient Research Partner panel. A nine member Patient Research Partners (PRPs) panel was established for the EuroTEAM project. PRPs were from the UK, Sweden, Estonia, Romania and Germany. Eight PRPs had RA, and one PRP had a first degree relative with RA. PRPs were recruited via a number of methods:

• local and national arthritis research / support groups
• volunteers who completed EULAR PARE PRP training
• word of mouth from other PRPs / project staff

PRPs were involved in the development, delivery and dissemination of aspects of the project, particularly in relation to WP4 but also in relation to WPs1,2 and 3.

PRPs attended the annual EuroTEAM meeting, which was comprised of lay and scientific presentations on project progress and work package breakout discussions. The involvement of PRPs at the annual meetings was facilitated by having a half day dedicated to PRPs during which work package leaders present results from the work package in a manner that was accessible to a lay audience. Furthermore, a detailed project guide and glossary was prepared that explained the aims of the project and technical terms used within the project in lay language to PRPs.

Some PRPs have written reports on their experiences, which have appeared on the EuroTEAM website and publications such as eBreakthrough, the EULAR PARE newsletter:

A number of PRPs have also assisted with the translation of a EuroTEAM patient video, which will enable the video to reach a larger audience in their home countries:

• Estonian:
• Romanian:
• Swedish:

Key specific activities within WPs 1-4 are described below:

Work package 1: Genetics, epigenetics and the environment
Within this work package, we addressed genetic risk factors for and factors protective against RA, and assessed the influence of epigenetic and environmental factors on transitions to RA. By reclassifying patients in our current cohorts according to phases of pre-RA proposed by the EULAR study group [1], we have been able to study:

• The genetics of progression between phases, allow an understanding of the clinical transitions for which the genetic variants are risk factors
• The genetics of movement backwards towards normality and genetic factors which protect against RA development
• The mechanisms whereby genetic risk factors put individuals at risk of RA remain unclear

WP1.1 Rare Variant Studies
Studies in the Icelandic population identified 100 novel sequence variants potentially associated with RA. However, replication studies in Sweden, The Netherlands and the UK were not able to replicate these findings.

WP1.2 Genetic factors protective against RA
In addition to factors which increase the risk of RA, some factors protect against it. We have shown that the genetic factor DRB1*13:01 is protective against RA development. Although these alleles are associated with protection from ACPA-positive RA, they do not consistently protect against the development of ACPA itself, indicating that HLA-DRB1*13 mainly affects the onset of arthritis once ACPA has already developed. Thus treatments aimed at emulating HLA-DBR1*13’s protective effect may be most effective in ACPA-positive healthy individuals at risk of RA.

WP1.3 Interaction between genetic and environmental risk factors for RA
We found an increased risk of developing RA for smokers compared to those who had never smoked; this effect was strongest in the anti-CCP positive cases. In a UK cohort however, this effect was not so strong.

WP1.4 Investigation of DNA methylation status in RA
DNA methylation was analysed in synovial fibroblasts from patients at different stages of RA and in patients with resolving arthritis. We identified 1,000 differentially methylated genes that were significantly different in synovial fibroblasts of very early RA patients (phase E), compared to synovial fibroblasts from patients with resolving arthritis.

Furthermore, we identified 633 differentially methylated genes (DMGs) when we compared synovial fibroblasts from late RA with early arthritis. Pathway analysis revealed that the most differentially methylated genes were associated with various cellular metabolic pathways such as glycolysis, TCA cycling and fatty acid metabolism. Furthermore, gene ontology (GO) analysis showed glycerol transport and glycerol activity to be significantly affected by DMG in late stage disease. Our data have provided evidence for an epigenetic imprinting of metabolic disturbances in RA synovial fibroblasts.

WP1.5 Investigation of microRNA’s as predictors of transition between disease phases and the effect of RA susceptibility SNPs on microRNA function
Our initial experiments showed a decreased expression of miR-204 in synovial fibroblasts of RA patients with very early as well as established RA, compared to synovial fibroblasts from patients with resolving arthritis or inflammatory arthralgia. Furthermore, miR-204 expression was also reduced in synovial fibroblasts following stimulation with a toll-like receptor 3 ligand, and after prolonged exposure to TGFβ or TNFα, pointing towards a potential influence of the cytokine milieu in changing microRNA expression in early disease. Inhibition of miR-204 resulted in a significant increase in the production of IL-6, IL-8, MMP1 and MMP3. miR-204 inhibition also results in decreased attachment of synovial fibroblasts.

To assess effects of RA susceptibility SNPs on microRNA function, we screened the currently known risk loci for RA (Okada et al, Nature, 2014) for their impact on putative microRNA target sites using MicroSNiPer (Barenboim et al, Hum Mutat, 2010). From 101 SNPs, only two (rs4272 and rs28411352) lie within the 3’UTR of annotated genes and only rs28411352 changes a potential microRNA binding site. The risk variant of this SNP leads to a loss of the first base of the seed region of miR-374a-3p as well as to a loss of the 9th base binding miR-1976 in the 3’UTR of the mRNA of metal regulatory transcription factor 1 (MTF1). This could lead to increased expression of MTF1 in cells carrying the risk SNP. MTF1 was described to increase expression of matrix metalloproteinases in chondrocytes (Kim et al, Cell, 2014). Whether and in which cell type this SNP/microRNA interaction is of relevance still has to be validated in functional experiments.

WP1.6 Exploring RA driven alterations in T-cell epigenetic programing to develop biomarker of RA development
T-cells are deeply involved in the pathogenesis of RA: at the genetic level many susceptibility genes lying in T-cell response pathways; at the cellular level RA is associated with a disruption of normal T-cell differentiation, homeostasis and immune regulation; at the molecular levels, several significant signalling defects have been identified in RA.

We conducted a genome wide methylation analysis of T-cells subsets (naïve versus memory) between health and RA (both ACPA+ and ACPA-). An initial approach used a candidate locus, the IL-17 gene, as a potential biomarker of progression to RA from an early arthritis stage due to the current hypothesis that Th17 cells may drive progression to RA. Data demonstrated significant under-methylation in RA patients’ memory T-cells only (not observed in naïve T-cells and monocytes). Using a Th17 methylation sensitive qPCR assay to evaluate Th17 cell frequency in the blood of 250 patients with early arthritis clinic we showed the value of this assay as a predictor of RA diagnosis. Further data were generated using flow cytometry to demonstrate the significantly higher expression of CXCR4 on TH17 in RA driving cells to the site of inflammation. We are currently assessing the predictive value of specific methylation marks identified within our genome wide methylation analysis.

Work package 2: autoantibodies and metabolites
Considerable evidence suggests that rheumatoid factor (RF) and anti-citrullinated protein / peptide antibodies (ACPA) are prognostically useful in the prediction of the development of RA when measured at phases (C), (D) and (E). Furthermore, the predictive utility of these antibodies in addition to clinical and genetic markers has been assessed. We aimed to progress the field of RA-related autoantibodies in the earliest phases of disease by:

• Determining the ability of recently discovered autoantibodies to predict transition through different phases of pre-RA
• Analysing whether distinct autoantibody characteristics are associated with progression of the disease
• Addressing the mechanisms whereby environmental risk factors are associated with the development of RA specific autoantibodies
• Assessing whether autoantibodies play a pathogenic role in RA development

WP2.1 ACPAs:
We have shown that profiling specific ACPAs in addition to anti-CCP improves the ability to predict RA. ACPAs are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, a multiplex assay has been that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. This custom array contained 16 citrullinated peptides and proteins detected in RA synovial tissues and was used to profiling ACPAs in sera from RA and at risk cohorts. The multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Indeed, the biomarker panel of 16 citrullinated autoantigens identified RA patients with a sensitivity greater than that achieved by the commercial CCP assay in the same cohort. Furthermore, the specificity of this biomarker panel in diagnosing RA was 96%, a specificity comparable with that of the CCP assay. These findings suggest that profiling specific ACPAs has the potential to improve the diagnosis and stratification of ACPA+ RA.

In addition, we have shown that there are changes in ACPA characteristics during preclinical RA, including in relation to isotype avidity, glycosylation and epitope spreading

WP2.2 Novel autoantibodies: anti-carbamylated protein antibodies
We have shown that anti-carbamylated protein (anti-CarP) antibodies are useful biomarkers for RA in phases C and D. Following the initial identification of the presence of anti-carbamylated protein (anti-CarP) antibodies in a cohort of Dutch patients with RA, and the observation that the presence of anti-CarP antibodies is associated with joint damage, we set out to replicate the original findings and to investigate the potential value of testing for anti-CarP antibodies to predict disease onset in individuals in stages that precede RA.

Using large Swedish and Japanese cohorts, we confirmed that anti-CarP antibodies were present in similar frequencies as observed in the Dutch cohort. Using a different assay, anti-CarP antibodies were also shown to be present in sera of RA patients from the UK.

Regarding the possible predictive use of anti-CarP antibodies we observed that in patients with autoantibody positive arthralgia (phase C&D), anti-CarP antibodies were associated with the future development of RA. This effect was independent of the presence of ACPA and RF.

Using samples from healthy blood bank donors who went on to develop RA, we showed that anti-CarP antibodies were present up to 10 years prior to symptom onset in a subset of the individuals who eventually developed RA. Often, ACPA RF and anti-CarP antibodies were observed together, but were also detected as single re-activities. The observation that anti-CarP antibodies can be present many years prior to disease onset has now been replicated in independent international cohorts confirming the potential for this autoantibody to be used as a predictor of RA in at risk populations.

We have addressed mechanisms underlying the formation of anti-CarP antibodies. Carbamylation is a post-translational modification resulting from the conversion of lysine into homocitrulline that requires the presence of cyanate. There are several conditions in which the concentration of cyanate (and therefore carbamylation) is increased, including chronic inflammation and heavy smoking. We therefore addressed the question whether these conditions (using inflammatory bowel disease as an example of on-RA chronic inflammation) of enhanced carbamylation could result in the induction of anti-CarP antibodies. We observed an increase in anti-CarP antibodies in RA patients but not in other conditions with enhanced carbamylation and conclude that increased carbamylation alone is not sufficient for a break of tolerance against carbamylated proteins, but that the presence of carbamylation could predispose to the development of anti-CarP antibodies in susceptible individuals.

WP2.3 Novel autoantibodies: anti- acetylated vimentin antibodies
Acetylation is a reversible enzymatic process where acetyl groups are added to free amines of lysine residues. Citrulline and homocitrulline are structurally very similar, differing only by one carbon atom that makes the homocitrulline side chain longer. Acetylated lysine is identical to homocitrulline except at the side chain terminal amine, which is replaced by a methyl moiety.
We have shown that IgG and IgA anti-acetylated (anti-Ac) vimentin antibodies are present in phase E, suggesting that, like citrullination, carbamylation, acetylation may be involved in the pathogenesis of RA by triggering the generation of autoantibodies and/or by generating targets for antibody responses in the rheumatoid joint. Anti-acetylated vimentin antibodies were found predominantly in patients who were ACPA positive.

The mechanisms underlying the generation of these anti-Ac antibodies is unclear, but may relate to environmental factors. Specifically, acetylation profiles can be modulated by certain dietary components and the gut microbiome in vivo.

WP2.4 A pathogenic role for auto-antibodies: A novel sialylation dependent checkpoint
We have shown that ACPA directly induce bone loss by stimulating osteoclast differentiation. Thus these antibodies, present in at risk phases and in established disease, actually play a key role in driving the joint destruction that characterizes RA.

Using a comparative micro-CT analysis of the bone microstructure in the metacarpophalangeal joints of ACPA positive and ACPA negative healthy individuals without clinical signs of arthritis, they have shown that structural bone damage starts before the clinical onset of arthritis in subjects with ACPA. These findings revise the concept that bone damage is an exclusive consequence of synovitis in patients with RA. The concomitant presence of ACPAs and RF was associated with higher erosive disease burden in patients with RA, shown by high-resolution peripheral quantitative CT (HR-pQCT) scans of the metacarpophalangeal joints.

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. As part of our mechanistic work assessing the role of ACPA in osteoclast stimulation we demonstrated that de-sialylated, but not sialylated, immune complexes enhance osteoclastogenesis (i.e. de-sialylated but not sialylated antibodies are pathogenic). Furthermore, Fc sialylation state of random IgG and specific IgG autoantibodies was shown to determine bone architecture in patients with RA. These findings provide a novel mechanism by which immune responses influence the human skeleton.

WP2.5 Moving beyond autoantibodies, we looked at other serum based biomarkers:

WP2.5.1 Micro RNAs (miRNA):
We have shown that certain circulating miRNAs (e.g. miR-146a, miR-155 and miR-16 in the serum) characterize an early stage of RA and may be useful biomarkers of RA development.

WP2.5.2 Metabolite profiles:
Using NMR base metabolomics profiling, we have shown that the serum metabolic fingerprint in established RA was clearly distinct from that of healthy controls. In early arthritis, we were able to stratify the patients according to the level of current inflammation, with C-reactive protein correlating with metabolic differences in 2 separate groups (P < 0.001). Lactate and lipids were important discriminators of inflammatory burden in both early arthritis patient groups. Specifically in relation to outcome, we were able to discriminate between patients with early arthritis and a resolving disease course compared with those with a persistent disease course although sensitivities and specificities were relatively low.

In a separate study, we have shown that individuals at risk of RA have elevated levels of triglycerides (TG), free fatty acids, pancreatic polypeptide (PP) and norepinephrine compared with healthy control individuals.

Work package 3: Cellular and tissue based research
The phase of disease at which the synovium is first involved in patients with RA, and the relationship of pathological processes operating at these earliest phases to those operating in established disease, are unclear. Furthermore, the involvement of extra-articular sites in the earliest phases of disease has not yet been explored. Using minimally invasive techniques, which have been standardized across relevant participating sites in the EuroTEAM consortium, we collected biological material from the synovium in patients at defined pre-RA phases. These samples were interrogated to assess haematopoietic and stromal changes within the synovium during the development of RA. Furthermore we aimed to assess cellular processes at other sites where RA may begin including lymphoid tissues.

The lung is the site where environmental triggers are in direct contact with cells of the immune system, possibly leading to activating of the immune response. Epidemiological studies have proposed smoking as one of the main environmental factors associated with RA and there is the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA.

WP3.1 Synovial tissue in patients with early arthritis:
Assessing synovial tissue cytokine expression in patients with early arthritis, we have identified a transient increase in levels of soluble molecules that attract inflammatory cells (chemokines CXCL4 and CXCL7) as well as the stromal marker FAP levels in patients in phase E who progress to RA.

WP3.2 Synovial tissue changes prior to the onset of clinically apparent synovitis
Looking at individuals in the pre-arthritis phase, we have shown molecular changes appearing in synovial tissues before onset of arthritis in the absence of overt synovitis demonstrating preclinical synovial alterations in immune response genes and lipid metabolism associated with the future development of arthritis. Specifically, we included 61 at risk individuals who underwent synovial biopsy sampling of a knee joint at inclusion and were prospectively followed. 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. This study clearly shows molecular changes appearing in synovial tissues before onset of arthritis in the absence of overt synovitis and demonstrates preclinical synovial alterations in immune response genes and lipid metabolism associated with development of arthritis. Since we previously found no overt immune cell infiltration in synovium of pre-RA individuals, the observed differential gene expression signature most likely originates from mesenchymal stromal cells.

In a comparative study looking at expression of NF-κB-inducing kinase (NIK), a key regulator of inflammation-induced angiogenesis in RA, in synovial tissue collected from patients with early arthritis (phase E and F) and from individuals at risk of developing RA (phase C and D), NIK was found to be expressed on endothelial cells of small blood vessels in 18.5% of the latter group. Although this expression did not correlate with the development of arthritis in these individuals, this may be indicative of high angiogenic activity in the ST and the NIK+ EC may therefore be playing a role in the persistence of synovitis and might underscore the importance of angiogenesis in synovial inflammation.

In an attempt to explain arthralgia as a symptom preceding the development of synovial inflammation in autoantibody-positive individuals at risk of developing RA, factors of the prostaglandin (PG) E2 pathway in the synovial tissue were investigated in individuals with RA-specific autoantibodies (phase C and D) and in early arthritis patients who developed RA over time compared to disease controls. In all 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. However, in early spondyloarthopathy patients the expression levels of mPGES-1 and COX-1 were significantly increased compared to RA and UA (undifferentiated arthritis) patients.

Therefore, 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.

WP3.3 Extra-articular tissues: lymph nodes
We have developed a technique to allow lymph node biopsy to be performed in a manner which is acceptable to study participants at risk of RA (in phase C and D) and to healthy control individuals. We have studied the lymph nodes of such pre-RA patients and early RA individuals and find altered frequencies of B cells, T cell subsets and innate lymphoid cell subsets in lymph node of RA-risk and early-stage RA patients when compared to healthy controls. Data suggested that epigenetic modifications in lymph node stromal cells may drive the abnormal immune responses in the lymph nodes of individuals at risk of RA.

WP3.4 Extra-articular tissues: the lungs
We have investigated the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status. High-resolution computed tomography (HRCT) was used to examine the lungs of patients with early, untreated RA and healthy individuals and revealed that ACPA-positive RA patients had parenchymal lung abnormalities, compared with ACPA-negative RA patients and of healthy controls. These significant differences remained after adjustment for smoking status. Bronchoscopy with collection of bronchoalveolar lavage (BAL) fluid and mucosal bronchial biopsy specimens was performed in RA patients. Immunohistochemical studies of the bronchial tissue showed increased staining for citrullinated proteins in ACPA-positive RA patients compared with ACPA-negative RA patients. ACPA levels were relatively higher in the BAL fluid as compared with the sera of ACPA-positive RA patients, suggesting that there is local production of ACPAs in the lungs of these patients.

Since immunological events in the lungs might trigger production of ACPA in early RA, the presence of shared immunological citrullinated targets was investigated in joints and lungs of patients with RA. Of note, identical citrullinated peptides were identified in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link. These results from patients with newly diagnosed RA support the notion that early inflammatory events in the lungs may represent a critical initiating factor in the development of ACPA-positive RA.

WP3.5 Extra-pulmonary effects of smoking:
Smoking is one of the most powerful environmental risk factors for RA. By investigating the mechanisms by which smoking drives the development of RA, we have shown that smoke extract enhances the inflammatory behaviour of synovial fibroblasts, a key cell type in the rheumatoid joint. Specifically, cigarette smoke extract enhances the pro-inflammatory and matrix-destructive potential of RASF by inducing the production of pro-inflammatory cytokine interleukin 8 and the matrix-destructive enzyme matrix metalloproteinase 1.

Overall integration and enhanced Interactions between WPs 1, 2 and 3:
An important aim of EuroTEAM was to understand interactions between different types of biomarkers and their utility at different stages of disease. To this end, several highly productive collaborations have developed between different WPs with specific examples listed below:

• WPs1 and 3: DNA methylation was analysed in synovial fibroblasts from patients in phase E who either progressed to RA or whose disease resolved. Several differentially methylated regions were identified between these two groups
• WPs2 and 3: Immunologic events in the lungs may be involved in triggering immunity, in particular production of ACPAs during early phases of RA. We have shown that the presence of ACPAs is associated with lung abnormalities, lung citrullination, and antibody enrichment in the lungs early in the development of ACPA-positive RA

Work package 4: communication, user integration and dissemination
As our understanding of disease mechanisms and predictive and therapeutic strategies evolves into earlier phases of RA, the ethics of research in this area, the ethical implications of research findings and ways of effectively communicating with individuals to allow them to make informed choices, understand the implications of test results and effect appropriate behavioural change need to be researched and developed. This is a particular issue in the field of RA where public knowledge about the disease is limited. European guidelines for the management of RA highlight the importance of early treatment - ideally within the first three months of disease. Multiple studies have shown that this is not achieved - a consequence frequently of the fact that patients do not seek help immediately when their symptoms occur. Central to this is a widespread lack of understanding in the public that RA is a serious disease with significant morbidity and mortality, that effective therapies are available and that rapid reporting and referral is critical to improve outcomes. This poor RA related health literacy in the general public is an important challenge in the context of the development of implementable predictive and preventive strategies. The effective implementation of screening tools and strategies for people with possible pre-RA requires a willingness of individuals to undergo assessments and to understand the implications of test results as a guide for modifying behaviour. This has been specifically highlighted by our SME partners, as a particular problem for musculoskeletal diseases, including RA, where disease specific health literacy is poor amongst the general public. It is important that the information given to people encouraging them to participate in assessments and treatments is designed and tailored to communicate effectively. Such information should communicate risks, the importance of assessment, ways in which lifestyle choice may influence risk, while avoiding psychological distress or misunderstandings (for example, believing that having risk factors for RA is equated to an RA diagnosis). Educational and communication tools should enable decisions about changing behaviour/lifestyle and/or environment in order to diminish the risk of RA (in light of identified risk factors) and encourage appropriate behaviour in the event of symptom development (e.g. early appropriate presentation and medical assessment). An important aim of WP4 was the development of such tools.

Patient Research Partners have played a critical role in WP4. By working with individuals within the cohorts under investigation, we have been able to develop approaches to communication and education that have their origins in the target population facilitating the translation of research findings into tests that can be implemented in an ethical manner.

Specific research activities within WP4 included:

WP4.1 Systematic reviews
• The completion of systematic reviews exploring perceptions of predictive testing for diseases such as RA. We have identified several barriers to testing, including concerns about confidentiality of risk information, a lack of motivation for lifestyle change, poor communication of information and a possible negative impact on emotional wellbeing. A number of recommendations to overcome these barriers were identified
• The completion of a systematic review and meta-synthesis of the qualitative literature exploring healthcare professionals’ (HCPs) perceptions of genetic risk in the context of predictive genetic testing. Healthcare professionals’ evaluation of the utility of predictive genetic testing is influenced not only by resource deficits, but may also be interpreted as a response to challenging ethical and social issues associated with genetic risk, that are not well aligned with current medical practice

WP4.2 Perceptions of risk
• We have explored perceptions of risk and predictive testing for RA amongst first degree relatives (FDRs) of patients with RA. Relatives were aware of their susceptibility to RA, but were unsure of the extent of their risk. They were concerned that knowing their actual risk would increase their anxiety, and that of their relative with RA, and would impact on decisions about their future. They were also concerned about the levels of uncertainty associated with predictive testing. Those in favour of knowing their future risk felt that they would need additional support to understand the risk information and cope with the emotional impact of this information. In a similar study of patients of south Asian origin, we have shown that there was interplay between patients’ willingness to communicate risk with relatives, their health and their socio-cultural beliefs about the causes and management of RA. There are clear indications that interventions to prevent RA in multi-ethnic societies require health promotion messages adapted to meet the needs of these diverse populations
• Access to FDRs is dependent on the co-operation of patients with RA, and may not be straightforward. Therefore, it is essential Understanding the views of patients, and their willingness to communicate with their relatives about their risk of developing RA. Some patients described feelings of responsibility or guilt for their relatives’ increased risk of developing RA. Patients generally held positive views of predictive testing, and expressed willingness to communicate with their relatives about their risk of RA

WP4.3 Informational resources
• Informational resources have been produced to educate FDRs and individuals in phases C, D and E about the different stages of RA, the role of interacting biomarkers in prediction of RA and how lifestyle and behaviour factors may affect the risk of developing RA. The content of these resources has been has been developed in collaboration with patient research partners

WP4.4 Patient research partner skills and networks
• We have developed expertise on the involvement of Patient Research Partners in Translational Research projects and have published best practice recommendations in individual countries as well as across Europe
Potential Impact:
1.0 Integration, patient involvement and the development of new networks: The patient focused legacy of Euro-TEAM
EuroTEAM has allowed closer integration and working between academic units, industrial partners (SMEs) and Patient Research Partners, enhancing the deliverability, quality and relevance of the work carried out within the current project. Importantly, it has catalysed future collaborative work on other related projects (e.g. the IMI-PREFER consortium).

The integration of the patient perspective and Patient Research Partners throughout the project has been particularly valuable. For example, they have ensured that the development and use of commercially available kits takes place in an environment where issues of relevance to individuals who are having their “risk” determined are understood and addressed. This has been particularly valuable for our SME partners. Historically, Patient Research Partners have been most involved in clinical research, outcomes research and psychosocial research. The effective integration of Patient Research Partners into this translational research project was an important and positive outcome. Our experiences of PRP involvement in such a project at a European level have helped define best practice in this field. Through PRP involvement we have:

• ensured that patients are involved in shaping the research agenda, and therefore that our research is of value to patients
• facilitated the production of efficient, patient centred research, thus speeding up the clinical translation of our research into new treatments and clinical services that are of direct benefit to patients
• involved patients in the development of the design of research procedures and patient facing materials, thus enhancing their experience of participating in clinical research
• promoted collaboration between researchers and patients that is mutually advantageous, where researchers learn from patients and vice versa. Many of our patient research partners are appreciative of opportunities to contribute to research. Feedback on our activities from PRPs has been very positive (e.g. “We were welcomed and included in the team. We were listened to with respect. We were included in setting recommendations for future work”; “The staff team are all incredibly encouraging and make you feel like your opinions and thoughts are of real importance. This warmth that comes from the staff means that it is easier to be a more effective PRP.”). Furthermore, many of our researchers, both from clinical and non-clinical backgrounds have benefitted greatly from understanding the patient perspective and therefore adapting their future research to better fit genuine needs and priorities

2.0 Advancing scientific knowledge for the benefit of patients and the public:
EuroTEAM has provided a platform through which signatures for risk assessed in a variety of different materials and tissues can be integrated together to provide an optimal risk signature. Work within EuroTEAM has led to identification of new and robust biomarkers / biomarker combinations to predict RA development including novel autoantibodies.

The increased collaboration on biomarker development between academia and industry, and across various European countries has had the following impact:

• new information is now available to inform the diagnosis of patients at risk of developing RA
• the prediction of disease development can be determined at an individual patient level

3.0 Allowing SMEs to develop products that are of benefit to patients and the public:
The involvement of SMEs has allowed the development of commercially available kits to allow the biomarkers / biomarker combinations to be tested for in at risk populations.

The long term impacts include the identification of novel therapeutic approaches to the management of chronic inflammatory diseases.

The integration of the patient perspective and Patient Research Partners throughout the project (and in particular with our SME Orgentec) has ensured that the development and use of commercially available kits takes place in an environment where issues of relevance to individuals who are having their “risk” determined are understood and addressed. This has been particularly valuable for our SME partners.

4.0 Enhancing patient and public knowledge about RA, RA risk and current research in this area:
We have developed educational resources for use by individuals at risk of RA to inform them about the disease, what it means to be at risk, and how risk can potentially be modulated – this will help raise public awareness of this important phase of RA. It will also help individuals make informed decisions in the context of undergoing predictive testing and making lifestyle changes / taking medications to reduce the risk of RA. The booklets have been translated into a number of languages, including English and German, are freely available via the EuroTEAM website, and are under review by national patient organisations (e.g. the UK’s National Rheumatoid Arthritis Society) who are keen to take them on an print and promote them – facilitating the uptake and impact after the end of the EuroTEAM project.

Through extensive public engagement by academic and industrial partners (including via health fairs and national events to promote medical science) we have raised awareness of issues related to risk of RA, current research in this area and the valuable contribution of the EU in supporting this research.

5.0 Dissemination activity and exploitation of results
The EuroTEAM consortium has resulted in over 85 publications, with further articles currently in submission which will be published in 2017. 175 individual dissemination activities have taken place over the 4 year duration of the project, ranging from:

• informal talks to small patient groups, and conferences with regional patient groups
• posters and oral presentations given at the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR), two of the largest congresses on rheumatology (see section A on the portal for further information)
• promotional videos
• a project website which has been accessed by individuals in over 120 countries

Whilst difficult to quantify the impact this has had on the general public, the spreading of awareness about RA as a disease, who is most at risk, and which steps can be taken to reduce risk and improve symptoms has been immense.

The immediate exploitation of results has stemmed mainly from the work undertaken in work packages 2 and 4. Finger prick tests for RA related autoantibodies have been developed by Orgentec (WP2). The development of the at risk brochures has resulted in interest from NRAS, who see them as being of benefit to patients and their families. As more brochures are distributed, this should increase the likelihood of the uptake of predictive testing, patients seeking help sooner when symptoms develop, as well as behavioural changes on end users following test results (WP4).

Work package 4 has also informed and influenced other EU funded projects, such as Mind the Risk (concerned with how genetic risk information is communicated to patients) and IMI PREFER (using patient preferences to determine when benefits may outweigh harms in drug development). The involvement of patient research partners in EuroTEAM has been extremely fruitful, and both the patients and the researchers agreed that patient involvement would be key in future projects (which is already underway with Karolinska’s new IMI RTCure, concerned with tolerance induction in the early stages of disease). Our patients have already identified EuroTEAM as the gold standard for patient involvement in projects, and EuroTEAM should be held as an exemplar for studies addressing prediction and prevention in chronic inflammatory disease. Researchers involved with EuroTEAM have confirmed that moving forward in future projects, they will seek to actively involve patients at every stage, as they have seen the benefits of involving the end user in research. The impact of this could be extremely significant, both in terms of the numbers of patients becoming involved in research, and more research outputs being designed with the patient in mind, therefore being more appropriate, beneficial and more likely to be adopted in clinical practice.

List of Websites:
Project Co-ordinator: Professor Christopher Buckley, University of Birmingham +44 121 371 3240
Deputy Co-ordinator: Professor Karim Raza, University of Birmingham, +44 121 371 3242
Project Manager: Rebecca Birch, University of Birmingham +44 121 371 8115