Joyce Lübbers

The type I interferon signature in leukocyte subsets from peripheral blood of patients with early arthritis: a major contribution by granulocytes.

BackgroundThe type I interferon (IFN) signature in rheumatoid arthritis (RA) has shown clinical relevance in relation to disease onset and therapeutic response. Identification of the cell type(s) contributing to this IFN signature could provide insight into the signature’s functional consequences. The aim of this study was to investigate the contribution of peripheral leukocyte subsets to the IFN signature in early arthritis.MethodsBlood was collected from 26 patients with early arthritis and lysed directly or separated into peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). PBMCs were sorted into CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD14+ monocytes by flow cytometry. Messenger RNA expression of three interferon response genes (IRGs RSAD2, IFI44L, and MX1) and type I interferon receptors (IFNAR1 and IFNAR2) was determined in whole blood and blood cell subsets by quantitative polymerase chain reaction. IRG expression was averaged to calculate an IFN score for each sample.ResultsPatients were designated “IFNhigh” (n = 8) or “IFNlow” (n = 18) on the basis of an IFN score cutoff in whole peripheral blood from healthy control subjects. The difference in IFN score between IFNhigh and IFNlow patients was remarkably large for the PMN fraction (mean 25-fold) compared with the other subsets (mean 6- to 9-fold), indicating that PMNs are the main inducers of IRGs. Moreover, the relative contribution of the PMN fraction to the whole-blood IFN score was threefold higher than expected from its abundance in blood (p = 0.008), whereas it was three- to sixfold lower for the other subsets (p ≤ 0.063), implying that the PMNs are most sensitive to IFN signaling. Concordantly, IFNAR1 and IFNAR2 were upregulated compared with healthy controls selectively in patient PMNs (p ≤ 0.0077) but not in PBMCs.ConclusionsPMNs are the main contributors to the whole-blood type I IFN signature in patients with early arthritis, which seems due to increased sensitivity of these cells to type I IFN signaling. Considering the well-established role of neutrophils in the pathology of arthritis, this suggests a role of type I IFN activity in the disease as well.

B cell signature contributes to the prediction of RA development in patients with arthralgia.

Early recognition followed by treatment of rheumatoid arthritis (RA) helps to maintain joint integrity and functional capacity,1 suggesting it may be beneficial to intervene in patients with arthralgia before RA develops. Anticitrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are established predictive markers,2 ,3 but only 20–40% of ACPA and/or RF positive patients with arthralgia develop RA within 2 years.4 Recently, we have demonstrated that the type I interferon (IFN) signature correctly identifies 52% of patients with arthralgia who will develop RA within 2 years.5 ,6 Our previous study suggested that a B cell related gene signature was associated with protection against arthritis development,6 and could aid in the prediction of arthritis development. We therefore studied the clinical value of the B cell signature, comprising CD19, CD20, CD79α and CD79β, for the prediction of arthritis development in an independent cohort of 115 ACPA and/or RF positive patients with arthralgia followed for median 22 months5 and explored the phenotypical nature of this B cell signature. In total, 44 patients (38%) developed arthritis (defined as one or more swollen joints) within 2 years. Of these, 4 patients had undifferentiated arthritis and 40 patients fulfilled the 2010 American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) criteria for RA. Patients were stratified into B cellhigh …

A1.77 Interferon regulatory factor 5 (IRF5) gene variant RS2004640 is associated with carotid intima media thickness in rheumatoid arthritis patients

Background and Objectives Rheumatoid arthritis (RA) is a chronic inflammatory joint disease and is associated with an increased cardiovascular (CV) risk. Interferons (IFNs), especially IFNβ, might play a role in atherosclerosis as they are known inhibitors of vascular smooth muscle cell proliferation and intimal hyperplasia. We studied whether functional relevant SNPs in the interferon regulatory factor 5 (IRF5) gene are associated with carotid intima media thickness (cIMT), a surrogate maker for CV disease. Materials and Methods In 353 RA patients of the CARRÉ study, IRF5 SNPs rs2004640 and rs4728142 were determined using Taqman Genotyping assay. cIMT was determined in a subgroup of 101 patients by B-mode ultrasonography. Linear regression analyses were used to investigate the association between cIMT and IRF5 genotypes, adjusting for demographic and cardiovascular risk factors. Results Patients homozygous for rs2004640 G-allele have higher cIMT compared to those homozygote for the T-allele (p = 0.019) and a trend towards a higher cIMT was observed (p = 0.103) for patients homozygous for the rs4728142 G-allele versus patients with the AA-genotype. Age was an effect-modifier for this association. Linear regression analysis in patients older than 60 years showed that the rs2004640 GG-genotype was associated with higher cIMT (regression coefficient 0.107 (C. I. 0.008; 0.205), p = 0.035) compared to the TT-genotype. This remained significant after adjustment for traditional risk factors (regression coefficient 0.111 (C. I.0.02; 0.202), p = 0.020). Conclusion We demonstrate that the IRF5 gene variant rs2004640 is associated with preclinical atherosclerosis in RA patients, independent of traditional cardiovascular risk factors. These results might implicate a role for type I IFN in modulating CV disease features in RA.

A1.7 Interferon and B-Cell Gene Signatures Contribute to Diagnosis of Pre-Clinical Rheumatoid Arthritis

Background/Objective Early diagnosis of the preclinical phase of rheumatoid arthritis (pre-RA) allows timely start of treatment with the potential to prevent disease progression. It is known that antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF) have diagnostic value to identify pre-RA. However, since only 20–40% of ACPA+/RF+ arthralgia patients develop arthritis within 5 years, better prognostic markers are needed. Recently, we demonstrated involvement of interferon (IFN) response and B-cell gene signatures in pre-RA. The objective is to demonstrate the value of these signatures to diagnose pre-RA. Methods Peripheral blood (Paxgene) was collected from 115 ACPA+/RF+ arthralgia patients who were clinically followed for arthritis development, one or more swollen joints, with a mean follow-up time of 23 months (IQR 12–30). An IFN and B-cell score was calculated based on 7 Type I IFN response genes and 3 B-cell related genes, respectively, measured by multiplex qPCR. Cox regression analysis and Receiver Operating Characteristic (ROC)-curve analysis were used to demonstrate prognostic and diagnostic significance. Results Out of 115 arthralgia patients 44 developed arthritis after a median time of 8 months (IQR 5–13). Stratification of these individuals based on the IFN score revealed that 60% of the IFNhigh patients converted to arthritis compared to 32% in IFNlow patients (P = 0.011). For the B-cell signature, 58% in B-celllow patients developed arthritis, compared to 33% of B-cellhigh patients (P = 0.020). Combined analysis revealed a significant high risk for arthritis development in IFNhigh/B-celllow patients (80%, hazard ratio (HR) 6.22, P = 0.003) and a low risk for IFNlow/B-cellhigh patients (26%, HR 0.16, P = 0.003). To demonstrate clinical utility a ROC-curve was constructed of ACPA+/RF+ alone and in combination with both signatures. The area under the curve reached 0.619 (P = 0.032, CI 0.514–0.724) for ACPA+/RF+ and increased to 0.803 (P = 0.0001, CI 0.718–0.888) with IFN and B-cell signatures included. The sensitivity to diagnose pre-RA increased from 16% to 52% when both signatures are included, with a cut-off of 94% specificity. Conclusions These findings demonstrate the clinical value of IFN and B-cell gene signatures as biomarkers for the diagnosis of pre-RA. This research was supported by the Center for Translational Molecular Medicine (CTMM) consortium “TRACER”.

08.05 How do glycans affect immune cells in ra

Background Anti-Citrullinated Protein Antibodies (ACPAs) are specific for Rheumatoid Arthritis (RA) and have been implicated in disease pathogenesis. The fragment antigen-binding domain of ACPA was recently shown to be extensively glycosylated. It is known that glycans play a key role in controlling innate and adaptive immunity, however to date there is limited understanding on the mode of action of glycans in RA. We hypothesise that the glycans on ACPA interact with glycan binding receptors and thus modulate immune responses in RA. Therefore, our aim is to elucidate the glycan effect of ACPA and other glycans on immune cells of RA patients to increase our understanding of RA pathogenesis. Materials and methods A whole blood flow assay is used to study glycan interactions with leukocytes. Leukocytes were isolated from blood using Ficoll density centrifugation and lysis of erythrocytes. Cells were incubated for 4 hours with 4 µg/ml glycan at 4°C. Used biotinylated glycans include: sialic acid, Lewis-x, mannose, lactosamine, galactosamine and as a negative control glucitol. Glycan binding and identification of immune cell subsets was assessed with flow cytometry using a whole blood flow antibody panel. Results A whole blood flow assay of four healthy donors showed consistent high binding of mannose to B-cells. Interestingly, no binding of mannose was observed to other immune cells indicating that mannose binds specifically to B cells. At this concentration there was no binding of other glycans to leukocytes. When there is a difference in glycan binding between healthy and RA patients CyTOF3-Helios mass cytometer will be used to have a more in-depth analyse of the interacting leukocyte cell subsets. Conclusion This study examines the glycan-binding capacity of leukocytes in healthy donors and RA patients via the whole blood flow assay. Our preliminary data indicates specific binding of mannose to B cells. This is an important finding because B cells play a key role in the pathogenesis of RA, as they produce ACPA and are very efficient in antigen presentation. Further studies on glycan binding to other key immune cells in RA may aid in elucidating their role in the pathogenesis of RA.

A1.31 The type I IFN signature in sorted leukocyte subsets from peripheral blood of rheumatoid arthritis patients; a major contribution by granulocytes

Background and objectives A subgroup of rheumatoid arthritis (RA) patients displays elevated type I IFN response gene (IRG) expression in peripheral blood, which has shown clinical relevance in relation to disease onset and therapy response. Identification of the cell type(s) contributing to this IFN signature could provide insight into its functional consequences and pathologic role in RA. This study aimed to investigate the contribution of the major peripheral leukocyte subsets to the IFN signature in RA. Methods Blood was collected from 26 early RA patients and lysed directly or separated into mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). Using flow cytometry, PBMCs were sorted into CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes. mRNA expression levels of three IRGs (RSAD2, IFI44L and MX1), as well as type I IFN receptors IFNAR1 and IFNAR2, were determined in blood and cell subsets by qPCR. IRG expression was averaged to calculate an IFN score for each sample. Results Patients were designated “IFNhigh” (n = 8) and “IFNlow” (n = 18) based on the IFN score cutoff in peripheral blood from healthy controls. As expected, IFN scores were significantly higher in all cell subsets from IFNhigh patients compared to IFNlow patients. This difference was remarkably large for the PMN fraction (mean 25-fold, p < 0.0001) compared to the other subsets (mean 6–9-fold, p ≤ 0.0009). Moreover, the relative contribution of the PMN fraction was significantly higher than expected from its relative abundance in blood alone (3-fold, p = 0.008), whereas this was 3–7-fold lower for the other subsets (p ≤ 0.063). Both IFNAR1 and IFNAR2 expression was highest in the PMN fraction compared to the other subsets, suggesting increased sensitivity of PMNs to type I IFNs. Concordantly, we observed IFNAR1 and IFNAR2 upregulation compared to healthy controls selectively in RA PMNs (p ≤ 0.0077) but not in the PBMCs. Conclusions PMNs are the main contributors to the whole blood type I IFN signature in RA patients, which seems due to increased sensitivity to type I IFN signalling. Considering the well-established role of neutrophils in the pathology of RA, this further supports a pathologic role of type I IFN activity in the disease.

A7.14 Effect of prednisone on type I interferon signature in rheumatoid arthritis: consequences for response prediction to rituximab

Background Elevated type I IFN response gene (IRG) expression has been described to be clinically relevant in predicting the non-response to rituximab in rheumatoid arthritis (RA) patients. Interference between glucocorticoids and type I IFN signalling has been demonstrated in vitro. Since the use and dose of oral GCs is highly variable among patients prior to the start of treatment with rituximab, we aimed to determine what the effect of GC usage is on the IRG expression in relation to the clinical response to rituximab. Methods In two independently recruited cohorts of biologic-free RA patients (n = 32 and n = 182) and a third cohort of 40 RA patients that were candidates for rituximab therapy, peripheral blood gene expression of 8 IRGs was determined by microarray or multiplex quantitative (q)PCR, and an IFN-score was calculated. The baseline IFN-score was tested for its predictive value towards rituximab response in relation to GC use using Receiver Operating Characteristics (ROC) curve analysis in the rituximab cohort. All patients in the cohorts fulfilled the revised American College of Rheumatology (ACR) 1987 criteria for the diagnosis of RA. GC use consisted of oral prednisone in doses varying from 2.5–10 mg/day and occurred in 19%, 29% and 70% of the patients in the three cohorts, respectively. The clinical response to rituximab was determined after 6 months of therapy based on the change in 28 joints Disease Activity Score (∆DAS28); patients with ∆DAS28 > 1.2 were considered responders. Results In all three cohorts, we consistently observed suppression of IRG expression in patients using prednisone compared to patients that were not using prednisone. The suppression appeared to be dose-dependent as it was most pronounced in the highest dose-range (>10 mg/day). In the rituximab cohort, separate ROC analysis on PREDN- patients alone revealed improved prediction of non-response to rituximab based on baseline IRG expression, with an AUC of 0.969 compared to 0.848 when analysed in all patients, whereas prednisone use itself had no predictive value in this cohort. Using a group-specific IFN-score-cutoff for all patients and PREDN- patients alone, sensitivity increased from 41% to 88%, respectively, combined with 100% specificity. Conclusion Because of prednisone-related suppression of the IFN-score, higher accuracy of rituximab response prediction was achieved in PREDN- patients. These results suggest that the IFN-score-based rituximab response prediction modell could be improved upon implementation of prednisone use. Disclosure CLV is an inventor on a patent wherein the predictive value of IFN type I response activity for the prediction of the clinical outcome of B cell depletion therapy via rituximab is claimed. CLV, SV and TdJ are inventors on a patent application wherein the use of the information on the interference of GCs to modulate the IFN system to improve outcome predictions on the use of biologics such as rituximab in chronic inflammatory and other conditions is claimed

A5.30 Systemic Inflammation and B-Cells in Rheumatoid Arthritis

Background and Objectives Rheumatoid arthritis (RA) is heterogeneous in clinical symptoms, clinical parameters, pathogenesis and gene expression levels. Previously, we demonstrated variation in B-cell related gene expression between RA patients. The aim was to explore the relation of B-cell related gene expression to clinical parameters of disease severity in early arthritis (EA) and established RA (esRA). Methods B-cell related gene expression (B-cell score) was determined in peripheral blood cells of 26 EA and 180 esRA patients, using multiplex real-time PCR. For the EA cohort, B-cell counts were also measured using flow cytometry. The esRA cohort was (randomly) divided into test and validation group of each 90 RA patients, with a mean DAS28 of 5.0 and 5.2, respectively. Associations were assessed between B-cell scores and the clinical disease parameters DAS28, CRP, RF, anti-CCP, nodules and erosions in all cohorts and B-cell counts only in EA cohort. Statistical testing was executed according to a bootstrap method which randomises the esRA group a 1000 times into two equally sized groups. Results We demonstrated that the B-cell score reflected the peripheral blood B-cell count (p < 0.0001, r = 0.7463). In EA, the B-cell score revealed a significant negative correlation with CRP levels (p = 0.0175; r = –0.4618). In the esRA group we also observed a negative correlation between the B-cell score and CRP levels (p = 0.0006, r = –0.3542; p = 0.0096 after Benjamini-Hochberg multiple testing correction). This result was confirmed in the independent validation group (p = 0.0356; r = –0.2218). Additionally, we performed a randomisation with the bootstrap method, which showed the same significant correlations in almost all cases. However, no correlations were found between B-cell score and DAS28, RF, anti-CCP, nodules or erosions. Conclusions The B-cell score reflects the B-cell count in RA and a low B-cell count is associated with an increased marker of systemic inflammation in RA.

OP0020 Validation of gene signatures to predict rheumatoid arthritis development

Background Early recognition of development of rheumatoid arthritis (RA) allows timely start of treatment. It is known that antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF) have a predictive value for development of RA within 5 years. Since only 20-40% of ACPA+ and/or RF+ seropositive arthralgia patients develop arthritis, better prognostic markers are required. Recently, we reported gene signatures in the peripheral blood (PB), involving interferon (IFN) response gene activity and B-cell related genes, which are associated with the development of arthritis. Objectives The objective of this study is to validate the signatures of IFN response gene activity and B-cell related genes in independent cohorts. Methods PB samples of an independent group of 40 seropositive arthralgia patients from the Amsterdam Reade cohort who are clinically followed for arthritis development were analyzed for their IFN-response gene activity, defined as a score based on the average expression of 7 IFN-response genes. In addition PB mononuclear cells (MCs) of 22 pre-onset RA, 25 RA patients and 48 population based controls (PC) from the Medical Biobank of Northern Sweden (Umeå) were studied. FACS-analysis for B-cell markers expression was performed on PB from 87 seropositive arthralgia patients and 30 healthy controls (HC) from the Amsterdam Reade cohort. qPCR for B-cell markers expression was performed in 45 seropositive arthralgia patients and 6 HC from the Amsterdam Reade cohort. Results Of the 40 seropositive arthralgia patients analyzed for INF-scores, 19 developed arthritis within a median period of 11 months (IQR 9-28). Comparative analysis of INF-score high and low patients confirmed that an increased IFN-score is associated with conversion to arthritis (p=0.025). In addition, the IFN-score was measured in PBMC from RA, pre-onset RA patients and PC from the Medical Biobank of Northern Sweden. Comparative analyses between the groups revealed that both pre-onset RA patients as well as RA patients had significantly increased IFN-scores compared to HC (Mann-Whitney U test p=0.006 and p=0.008, respectively). Of the 87 seropositive arthralgia patients analyzed for B-cell marker expression, 22 developed arthritis within a median period to conversion of 11 months (IQR 5-13). Low B-cell markers expression was significantly associated with conversion to arthritis (p=0.01). An interim analysis with qPCR showed a strong trend towards decreased B-cell transcript markers and arthritis conversion (p=0.07). Conclusions These results provide evidence for the validation for a role of IFN-activity and low B-cell presence in the preclinical phase of RA. Further studies are aimed to demonstrate the diagnostic value of these markers. Acknowledgements This research was supported by the Center for Translational Molecular Medicine (CTMM) consortium “TRACER”. Disclosure of Interest None Declared

FRI0101 The value of gene signatures in the diagnosis of pre-clinical ra

Background Diagnosis of the preclinical phase of rheumatoid arthritis (RA) allows timely start of treatment with the potential to prevent disease progression. It is known that antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF) have diagnostic value to identify pre-clinical RA. However, since only 20-40% of ACPA+/RF+ arthralgia patients develop arthritis within 5 years, better prognostic markers are needed. Recently, we demonstrated involvement of Interferon (IFN) response and B-cell gene signatures in pre-clinical RA. Objectives The objective of this study is to demonstrate the diagnostic value of the IFN and B-cell gene signatures in the diagnosis of pre-clinical RA. Methods Peripheral blood (Paxgene) was collected from 115 ACPA+/RF+ arthralgia patients from the Jan van Breemen Research Institute | Reade Amsterdam. Patients where clinically followed for arthritis development, one or more swollen joints, with a mean follow-up time of 23 months (IQR 12-30). IFN response and B-cell related gene expression was measured by multiplex qPCRs. An IFN score was calculated based on 7 highly correlating Type I IFN response genes. A B-cell score was calculated based on three highly correlating B-cell related genes. Cut-off levels for the IFN and B-cell high or low definition were determined by the 85% specificity of the individual IFN and B-cell Receiver Operating Characteristics (ROC)-curves. Cox regression analysis and ROC-curve analysis were used to demonstrate prognostic and diagnostic significance. Results Out of 115 arthralgia patients 44 developed arthritis after a median time of 8 months (IQR 5-13). Stratification of these individuals based on the IFN score revealed that 60% of the IFNhigh patients converted to arthritis compared to 32% in IFNlow patients (P=0.011). For the B-cell signature, 58% in B-celllow patients developed arthritis, compared to 33% of B-cellhigh patients (P=0.020). Combined analysis revealed a significant high risk for arthritis development in IFNhigh/B-celllow patients (80%, hazard ratio (HR)(6.22, P=0.003) and a low risk for IFNlow/B-cellhigh patients (26%, HR 0.16, P=0.003). To demonstrate clinical utility a ROC-curve was constructed of ACPA+/RF+ alone and in combination with both signatures. The area under the curve reached 0.619 (P=0.032, C.I. 0.514-0.724) for ACPA+/RF+ and increased to 0.803 (P=0.0001, C.I. 0.718-0.888) with IFN and B-cell signatures included. The sensitivity to diagnose pre-clinical RA increased from 16% to 52% when both signatures are included, with a cut-off of 94% specificity. Conclusions These findings demonstrate the clinical value of IFN and B-cell gene signatures as biomarkers for the diagnosis of pre-clinical RA. Acknowledgements This research was supported by the Center for Translational Molecular Medicine (CTMM) consortium “TRACER”. Disclosure of Interest J. Lubbers Grant/research support from: CTMM TRACER, L. van de Stadt Grant/research support from: Dutch Arthritis Foundation, S. Vosslamber: None Declared, J. G. Wesseling Grant/research support from: CTMM TRACER, D. van Schaardenburg Grant/research support from: Dutch Arthritis Foundation, C. Verweij Grant/research support from: CTMM TRACER