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Melanoma-induced suppression of dendritic cells (DC) in the sentinel lymph node (SLN) interferes with the generation of protective antitumor immunity. In an effort to strengthen immune defense against metastatic spread, we performed a three-arm phase II study comprising 28 patients with stage I–II melanoma randomized to receive intradermal injections around the primary tumor excision site of saline or low-dose CpG-B, alone or combined with GM-CSF, before excision of the SLNs. After pathologic examination, 5 patients were diagnosed with stage III melanoma based on the presence of tumor cells in the SLNs. Combined CpG/GM-CSF administration resulted in enhanced maturation of all identifiable conventional (cDC) and plasmacytoid (pDC) DC subsets and selectively induced increased frequencies of SLN-resident BDCA3/CD141+ cDC subsets that also expressed the C-type lectin receptor CLEC9A. Correlative in vivo analyses and in vitro studies provided evidence that these subsets were derived from BDCA3+ cDC precursors in the blood that were recruited to the SLNs in a type I IFN-dependent manner and subsequently matured under the combined influence of CpG and GM-CSF. In line with their reported functional abilities, frequencies of in vivo CpG/GM-CSF–induced BDCA3/CD141+ DCs correlated with increased ex vivo cross-presenting capacity of SLN suspensions. Combined local CpG/GM-CSF delivery thus supports protective antimelanoma immunity through concerted activation of pDC and cDC subsets and recruitment of BDCA3+ cDC subsets with T cell–stimulatory and cross-priming abilities. Cancer Immunol Res; 3(5); 495–505. ©2015 AACR.

Gating strategies for analysis of A) plasmacytoid DC (pDC) and B) skin-migratory conventional DC (cDC) subsets: Dermal DC (DDC) and Langerhans Cells (LC) and C) LN-resident cDC subsets in melanoma Sentinel Lymph Nodes (SLN).

Expression of the cross-priming associated markers BDCA3/CD141 and CLEC9A on the four identified cDC subsets in single-cell suspensions of untreated melanoma Sentinel Lymph Nodes SLN (mean {plus minus} SEM of at least three separate experiments are shown).

OBJECTIVES: Rheumatoid arthritis (RA) is associated with cardiovascular (CV) morbidity and mortality. Interferon regulatory factor 5 (IRF5) gene polymorphisms rs2004640 and rs4728142 have been associated with autoimmune diseases, but also with atherosclerosis. Differences in IRF5 gene expression can lead to the production of different interferons and might play a role in the atherogenic process in RA.METHODS: We investigated the effects of IRF5 gene variants rs2004640 and rs4728142 on clinical parameters related to atherosclerosis, such as cIMT (in subgroup n=101), and new CV events (in whole cohort n=353).RESULTS: For rs2004640, cIMT values at baseline were highest within the group of patients carrying the GG-genotype, followed by GT- and TT- genotypes, which was statistically significant. Over time patients with the TT-genotype had the highest increase in cIMT. For rs4728142 cIMT values were also the highest for patients with the GG-genotype at baseline, but the difference between the groups was not statistically significant. Over time the highest increase in cIMT was in the patients with the AA-genotype. Both rs2004640 and rs4728142 were not associated with new CV events during follow-up.CONCLUSIONS: IRF5 alleles are associated with changes in cIMT, but not with new CV events in RA. Although these findings implicate a role of the IRF5 transcription pathway in atherosclerosis, IRF5 single nucleotide polymorphisms do not appear to increase the risk of future CV events.

BackgroundWe previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8+ T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration.MethodsRe-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles.ResultsSignificant increases in CD83+, CD14+, CD68+, and CD123+ APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123+BDCA2+ plasmacytoid dendritic cells (DCs) and BDCA3/CD141+CLEC9A+ type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14+ APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14+ monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration.ConclusionThe CpG-B-induced concerted recruitment of cDC1 and CD14+ APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites.

Background We previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8+ T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration. Methods Re-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles. Results Significant increases in CD83+, CD14+, CD68+, and CD123+ APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123+BDCA2+ plasmacytoid dendritic cells (DCs) and BDCA3/CD141+CLEC9A+ type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14+ APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14+ monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration. Conclusion The CpG-B-induced concerted recruitment of cDC1 and CD14+ APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites.

Objectives To validate the IFN response gene (IRG) set for the prediction of non-response to rituximab in rheumatoid arthritis (RA) and assess the predictive performance upon combination of this gene set with clinical parameters. Methods In two independent cohorts of 93 (cohort I) and 133 (cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight IRGs was determined, and averaged into an IFN score. Predictive performance of IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28) ≥ 1.8 after 6 months of therapy were considered responders. Results The mean IFN score was higher in non-responders compared to responders in both cohorts, but this difference was most pronounced in patients who did not use prednisone, as described before. Univariate analysis in cohort I showed that baseline DAS28, IFN score, DMARD use and negativity for IgM-RF and/or ACPA were associated with rituximab non-response. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. In cohort II, this model revealed a comparable AUC in PREDN-negative patients (0.78), but AUC in PREDN-positive patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. Conclusions Combination of predictive parameters provided a promising model for the prediction of non-response to rituximab, with possibilities for optimization via definition of the exact interfering effect of prednisone on IFN score. Trial registration (Cohort II, SMART trial) NCT01126541 , registered 18 May 2010.

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN-inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti-IFNAR or anti-IFN-α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA-binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti-IFNAR or anti-IFN-α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA-binding proteins and with autoantibody multispecificity. These studies identify IFN-α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN-α as a possible target for therapy in these patients.

Background Elevated expression of type I IFN response genes (IRGs) was previously described to be associated with a poor response to rituximab in rheumatoid arthritis (RA). In the present study, we aimed to validate this association and assessed the predictive performance upon combination of the predictive IRG gene set with clinical parameters. Materials and methods In two independent cohorts of 93 (Cohort I) and 133 (Cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight selected IRGs was determined, and averaged into an IFN score. Individual predictive performance of the IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28)≥1.8 after 6 months of therapy were considered responders. Results A higher IFN score was observed in RTX non-responders compared to RTX responders from both Cohort I and II, but this difference was most pronounced in patients who did not use prednisone (PREDN-), as described before. Univariate analysis showed that baseline DAS28, IFN score and DMARD use were associated with non-response to rituximab, whereas positivity for IgM-RF and ACPA was associated with a good response to RTX. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. Validation of this multivariate model in Cohort II revealed a comparable AUC in PREDN- patients (0.78), but the AUC in PREDN+ patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. Multivariate analysis of the PREDN+ subgroup of Cohort II revealed a model containing DAS28 and positivity for IgM-RF and ACPA, which showed an AUC of 0.75. Conclusions The combination of predictive parameters, including IFN score, provided a promising model for the prediction of non-response to rituximab, with possibilities for optimisation via definition of the exact interfering effect of prednisone on IFN score.

To validate the presence and demonstrate the clinical value of the type I interferon (IFN)-signature during arthritis development.In 115 seropositive arthralgia patients who were followed for the development of arthritis (Amsterdam Reade cohort), and 25 presymptomatic individuals who developed rheumatoid arthritis (RA) later, and 45 population-based controls (Northern Sweden cohort), the expression levels of 7 type I IFN response genes were determined with multiplex qPCR and an IFN-score was calculated. The diagnostic performance of the IFN-score was evaluated using Cox regression and Receiver Operating Characteristics (ROC)-curve analysis.In 44 of the 115 at-risk individuals (38%) from the Amsterdam Reade cohort, arthritis developed after a median period of 8 months (IQR 5-13). Stratification of these individuals based on the IFN-score revealed that 15 out of 25 IFN(high) individuals converted to arthritis, compared with 29 out of 90 IFN(low) individuals (p=0.011). In the Northern Sweden cohort, the level of the IFN-score was also significantly increased in presymptomatic individuals who developed RA compared with population-based controls (p=0.002). Cox regression analysis of the Amsterdam Reade cohort showed that the hazard ratio (HR) for development of arthritis was 2.38 (p=0.008) for IFN(high) at-risk individuals after correction for anticitrullinated protein antibodies (ACPA) and rheumatoid factor (RF). The ROC-curve area under the curve (AUC) for the IFN-score combined with ACPA and RF in the prediction of arthritis was 78.5% (p=0.0001, 95% CI 0.70 to 0.87).The results demonstrated clinical utility for the IFN-signature as a biomarker in the prediction of arthritis development.

Background Activation of the type I interferon (IFN) response program is described for several autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS), myositis (IIM) and rheumatoid arthritis (RA). While IFNα contributes to SLE pathology, IFNβ therapy is often beneficial in MS, implying different immunoregulatory roles for these IFNs. This study was aimed to investigate potential diversification of IFNα-and IFNβ-mediated response programs in autoimmune diseases. Methods Peripheral blood gene expression of 23 prototypical type I IFN response genes (IRGs) was determined in 54 healthy controls (HCs), 69 SLE (47 test, 22 validation), 149 IFNβ-treated MS (71 test, 78 validation), 160 untreated MS, 78 IIM and 76 RA patients. Patients with a type I IFN signature were selected for analysis. Results We identified IFNα- and IFNβ-specific response programs (GC-A and GC-B, respectively) in SLE and IFNβ-treated MS patients. Concordantly, the GC-A/GC-B log-ratio was positive for all SLE patients and negative for virtually all IFNβ-treated MS patients, which was confirmed in additional cohorts. Applying this information to other autoimmune diseases, IIM patients displayed positive GC-A/GC-B log-ratios, indicating predominant IFNα activity. The GC-A/GC-B log-ratio in RA was lower and approached zero in part of the patients, implying relative importance of both clusters. Remarkably, GC-A/GC-B log-ratios appeared most heterogeneous in untreated MS; half of the patients displayed GC-A dominance, whereas others showed GC-B dominance or log-ratios near zero. Conclusions Our findings show diversification of the type I IFN response in autoimmune diseases, suggesting different pathogenic roles of the type I IFNs. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0946-9) contains supplementary material, which is available to authorized users.

Identification of biomarkers contributing to disease diagnosis, classification or prognosis could be of considerable utility. For example, primary methods to diagnose multiple sclerosis (MS) include magnetic resonance imaging and detection of immunological abnormalities in cerebrospinal fluid. We determined whether gene-expression differences in blood discriminated MS subjects from comparator groups, and identified panels of ratios that performed with varying degrees of accuracy depending upon complexity of comparator groups. High levels of overall accuracy were achieved by comparing MS with homogeneous comparator groups. Overall accuracy was compromised when MS was compared with a heterogeneous comparator group. Results, validated in independent cohorts, indicate that gene-expression differences in blood accurately exclude or include a diagnosis of MS and suggest that these approaches may provide clinically useful prediction of MS.

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.

Introduction Elevated type I interferon (IFN) response gene (IRG) expression has proven clinical relevance in predicting rituximab non-response in rheumatoid arthritis (RA). Interference between glucocorticoids (GCs) and type I IFN signaling has been demonstrated in vitro. Since GC use and dose are highly variable among patients before rituximab treatment, the aim of this study was to determine the effect of GC use on IRG expression in relation to rituximab response prediction in RA. Methods In two independent cohorts of 32 and 182 biologic-free RA patients and a third cohort of 40 rituximab-starting RA patients, peripheral blood expression of selected IRGs was determined by microarray or quantitative real-time polymerase chain reaction (qPCR), 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) analysis in the rituximab cohort. Patients with a decrease in disease activity score (∆DAS28) >1.2 after 6 months of rituximab were considered responders. Results We consistently observed suppression of IFN-score in prednisone users (PREDN+) compared to non-users (PREDN−). In the rituximab cohort, analysis on PREDN− patients (n = 13) alone revealed improved prediction of rituximab non-response based on baseline IFN-score, with an area under the curve (AUC) of 0.975 compared to 0.848 in all patients (n = 40). Using a group-specific IFN-score cut-off for all patients and PREDN− patients alone, sensitivity increased from 41% to 88%, respectively, combined with 100% specificity. Conclusions Because of prednisone-related suppression of IFN-score, higher accuracy of rituximab response prediction was achieved in PREDN− patients. These results suggest that the IFN-score-based rituximab response prediction model could be improved upon implementation of prednisone use.

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 …

Melanoma-induced suppression of dendritic cells (DC) in the sentinel lymph node (SLN) interferes with the generation of protective antitumor immunity. In an effort to strengthen immune defense against metastatic spread, we performed a three-arm phase II study comprising 28 patients with stage I–II melanoma randomized to receive intradermal injections around the primary tumor excision site of saline or low-dose CpG-B, alone or combined with GM-CSF, before excision of the SLNs. After pathologic examination, 5 patients were diagnosed with stage III melanoma based on the presence of tumor cells in the SLNs. Combined CpG/GM-CSF administration resulted in enhanced maturation of all identifiable conventional (cDC) and plasmacytoid (pDC) DC subsets and selectively induced increased frequencies of SLN-resident BDCA3/CD141+ cDC subsets that also expressed the C-type lectin receptor CLEC9A. Correlative in vivo analyses and in vitro studies provided evidence that these subsets were derived from BDCA3+ cDC precursors in the blood that were recruited to the SLNs in a type I IFN-dependent manner and subsequently matured under the combined influence of CpG and GM-CSF. In line with their reported functional abilities, frequencies of in vivo CpG/GM-CSF–induced BDCA3/CD141+ DCs correlated with increased ex vivo cross-presenting capacity of SLN suspensions. Combined local CpG/GM-CSF delivery thus supports protective antimelanoma immunity through concerted activation of pDC and cDC subsets and recruitment of BDCA3+ cDC subsets with T cell–stimulatory and cross-priming abilities. Cancer Immunol Res; 3(5); 495–505. ©2015 AACR.

Multiple sclerosis (MS) is the most common inflammatory disease of the central nervous system. The disease is characterized by progressive neurological dysfunction due to demyelination of the nerves, which leads to disability. Currently, no curative therapy is available and patients are subjected to a prolonged course of treatment. Interferon-β (IFNβ) was the first agent to show clinical efficacy in the treatment of MS, and is still the best available therapy. Unfortunately, clinical experience indicates that approximately 40% of the patients do not or only poorly respond to IFNβ treatment. Recent advances revealed the presence of an activated type I IFN pathway in a subset of treatment naïve patients with relapsing remitting MS (RRMS), as shown by the presence of an "IFN signature" and type I IFN bioactivity in the blood of these patients. Evidence exists that quantification of the IFN signature in RRMS is informative as a biomarker to predict the clinical response to IFNβ. In this review we summarize the current evidence of type I IFN activation in RRMS and its clinical relevance.

The folate antagonist methotrexate (MTX) is an anchor drug in the treatment of rheumatoid arthritis (RA), but its mechanism of action with regard to the impact on folate metabolism remains elusive. The aim of the present study was to investigate the cellular pharmacologic impact of MTX on peripheral blood cells, by comparing MTX-treated RA patients to MTX-naive RA patients and healthy controls.Gene expression microarray data were used to investigate the expression of 17 folate pathway genes by peripheral blood cells from a cohort of 25 RA patients treated with MTX, 10 MTX-naive RA patients starting treatment with MTX, and 15 healthy controls (test cohort). Multiplex real-time polymerase chain reaction was used to validate the results in an independent cohort, consisting of 151 RA patients treated with MTX, 28 MTX-naive RA patients starting treatment with MTX, and 24 healthy controls (validation cohort).Multiple folate metabolism-related genes were consistently and significantly altered between the 3 groups in both cohorts. Concurrent with evidence of an immune-activation gene signature in MTX-naive RA patients, significant up-regulation of the folate-metabolizing enzymes γ-glutamyl hydrolase and dihydrofolate reductase, as well as the MTX/folate efflux transporters ABCC2 and ABCC5, was observed in the MTX-naive RA group compared to healthy controls. Strikingly, MTX treatment of RA patients normalized these differential gene expression levels to the levels observed in healthy controls.These results suggest that under inflammatory conditions, basal folate metabolism in the peripheral blood cells of RA patients is markedly up-regulated, and treatment with MTX restores folate metabolism to normal levels. Identification of this novel gene signature provides insight into the mechanism of action of MTX, thus paving the way for development of novel folate metabolism-targeted therapies.

Background and objectives Upregulation of type I IFN response genes is described for several autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS), myositis (IIM) and rheumatoid arthritis (RA). While IFNα contributes to SLE pathology, IFNβ therapy is beneficial in MS, implying different immunoregulatory roles for these IFNs. This study aimed to investigate potential diversification of IFNα-and IFNβ-mediated response programs in autoimmune diseases. Materials and methods Peripheral blood gene expression of 23 prototypical type I IFN response genes (IRGs) was determined in healthy controls (HCs, n = 54), SLE (n = 47), IFNβ-treated MS (n = 71), untreated MS (n = 160), IIM (n = 78) and RA patients (n = 76). An IFN score was calculated and patients with a type I IFN signature (IFN score >mean+2SD in HC) were selected for analysis. Results We identified IFNα- and IFNβ-specific response programs by comparing IRG expression in SLE (IFNα-mediated) and IFNβ-treated MS patients. Statistical testing revealed increased expression of 5 IRGs in SLE (Gene cluster (GC-)A, p ≤ 0.006) and 13 IRGs in IFNβ-treated MS (Gene cluster (GC-)B, p ≤ 0.044). Concordantly, the GC-A/GC-B log-ratio was positive for all SLE patients and negative for virtually all IFNβ-treated MS patients. Applying this information to other autoimmune diseases, IIM patients displayed positive GC-A/GC-B log-ratios, indicating predominant IFNα activity. The GC-A/GC-B log-ratios in RA were lower and approached zero in part of the patients, implying relative importance of both clusters. Remarkably, GC-A/GC-B log-ratios appeared most heterogeneous in IFNβ-naive MS; half of the patients displayed GC-A dominance, whereas others showed GC-B dominance or log-ratios near zero. We confirmed these results for SLE, RA and MS using two public microarray datasets containing 22 SLE patients, 112 RA patients, 58 IFNβ-treated MS patients and 62 untreated MS patients. Exploring functional differences between GC-A and GC-B genes revealed enrichment of the ISGF3-binding response element ISRE and IRF8 binding sites only in GC-B, suggesting that IFNβ is more potent in activating these transcription factors than IFNα. Conclusions Conclusively, we demonstrated that type I IFN signatures in autoimmune diseases appear less uniform than generally assumed. The diversification of the type I IFN response in autoimmune diseases suggests different pathogenic roles of the type I IFNs.

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 “IFN high ” (n = 8) and “IFN low ” (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 IFN high patients compared to IFN low patients. This difference was remarkably large for the PMN fraction (mean 25-fold, p 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.

Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease that primarily affects the joints. Aetiology of RA is unknown. Once symptoms are present RA manifests itself as a heterogeneous disease with a clinical spectrum ranging from mild to severe disease, and variable involvement in secondary organ systems. The heterogeneous nature is reflected by variation in responsiveness to treatment. The heterogeneity most likely has its origin in its multifactorial nature, whereby specific combinations of environmental factor(s) and genetic factors are likely to influence not only susceptibility but also the disease severity and prognosis. Unfortunately, our understanding of the molecular complexity of RA is incomplete, and criteria for subtyping of patients, e.g. for prognosis to select those patients who will benefit from a specific treatment, are currently lacking.

Rheumatoid arthritis (RA) is the most common chronic inflammatory disorder of the musculoskeletal system that may cause permanent joint damage. The disease has a major impact on the quality of life of affected individuals, costs for the health care system, and society. Currently, no curative treatment is available, and patients are subjected to a prolonged course of treatment. Due to their role in the pathogenesis of RA, B cells have become an attractive target for therapy. Rituximab (Mabthera®/Rituxan®) is a therapeutic monoclonal antibody against CD20 expressed on B cells, which is effective in depleting B cells and approved worldwide for the treatment of RA. Rituximab was shown to be highly beneficial in decreasing clinical symptoms, safe, and well tolerated. However, clinical experience revealed that approximately 30-40% of RA patients do not respond to it. Given the destructive nature of RA, the risk of adverse effects, and considerable costs for therapy, there is a strong need to make predictions on the clinical outcome before the start of therapy. Since nearly all treated patients experience an effective depletion of circulating B cells, questions have been raised concerning the mechanism of action. In this review, novel developments, in particular the findings on the role of the interferon system, will be highlighted. This may add new and important information to our understanding of the mechanism that underlies the clinical outcome of rituximab treatment and may lead to the identification of biomarkers to predict the response.

Introduction B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX. Methods In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26). Results Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria. Conclusions This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA.

ObjectiveDespite the fact that rituximab depletes B cells in all treated patients with RA, not all patients show a favourable clinical response. The goal of this study was to provide insight into pharmacological changes in peripheral blood that are associated with clinical response to rituximab.MethodsGene expression profiling was performed on peripheral blood RNA of 13 patients with RA (test group) using Illumina HumanHT beadchip microarrays. An independent group of nine patients was used for validation using TaqMan quantitative PCR. Clinical responder status was determined after 6 months using change in 28-joint Disease Activity Score (ΔDAS28) and European League Against Rheumatism (EULAR) response criteria. Significance analysis of microarrays and ontology analysis were used for data analysis and interpretation.ResultsPharmacogenomic analyses demonstrated marked interindividual differences in the pharmacological responses at 3 and 6 months after start of treatment with rituximab. Interestingly, only differences in the regulation of type I interferon (IFN)-response genes after 3 months correlated with the ΔDAS28 response. Good responders (∆DAS>1.2; n=7) exhibited a selective increase in the expression of type I IFN-response genes, whereas this activity was unchanged or hardly changed in non-responders (∆DASConclusionsA good clinical response to rituximab in RA is associated with a selective drug-induced increase in type I IFN-response activity in patients with RA. This finding may provide insight in the biological mechanism underlying the therapeutic response to rituximab.

Interferon-β (IFNβ) therapy is effective in approximately half of the patients with relapsing-remitting multiple sclerosis (RRMS). Clinical non-responders were characterized by an increased expression of IFN response genes before the start of therapy, and a lack of a pharmacologically induced increase in IFN response gene activity. Because Interferon Regulatory Factor 5 (IRF5) is a master regulator of IFN-activity, we carried out a candidate gene study of IRF5 gene variants in relation to the pharmacological and clinical response upon IFNβ treatment. We found that patients with the IRF5 rs2004640-TT and rs47281420-AA genotype exerted a poor pharmacological response to IFNβ compared with patients carrying the respective G-alleles (P=0.0006 and P=0.0023, respectively). Moreover, patients with the rs2004640-TT genotype developed more magnetic resonance imaging (MRI)-based T2 lesions during IFNβ treatment (P=0.003). Accordingly, an association between MRI-based non-responder status and rs2004640-TT genotype was observed (P=0.010). For the rs4728142-AA genotype a trend of an association with more T2 lesions during IFNβ treatment and MRI-based non-responder status was observed (P=0.103 and P=0.154, respectively). The clinical relevance of the rs2004640-TT genotype was validated in an independent cohort wherein a shorter time to first relapse was found (P=0.037). These findings suggest a role for IRF5 gene variation in the pharmacological and clinical outcome of IFNβ therapy that might have relevance as biomarker to predict the response to IFNβ in multiple sclerosis.

Systemic lupus erythematosus is a systemic, heterogeneous autoimmune disease. Understanding of its molecular complexity is incomplete and there is a need to identify new therapeutic targets and to optimize criteria for its diagnosis, assessment and prognosis. Recently, Arasappan and colleagues have described a new meta-analysis method that enables data analysis across different DNA-microarray datasets to identify genes and processes relevant to systemic lupus erythematosus. Their study provides a simple and valuable meta-analysis method for the selection of biomarkers and pathways in disease.See related research by Arasappan et al.: http://www.biomedcentral.com/1741-7015/9/65.

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

Objective Despite the fact that rituximab depletes B cells in all treated rheumatoid arthritis (RA) patients, not all patients show a favorable clinical response. The goal of this study was to provide insight into the pharmacological changes in the peripheral blood that are associated with clinical response to rituximab using genome-wide gene expression profiling. Methods Gene-expression profiling was performed on peripheral blood RNA from 13 RA patients (test group) using Illumina HumanHT beadchip microarrays. An independent group of nine patients was used for validation using Taq-man qPCR. Clinical responder status was determined after 6 months using ΔDAS28 and EULAR response criteria. Significance analysis of microarrays, gene set enrichment analysis and metacore ontology analysis were used for data analysis and interpretation. Results Pharmacogenomic studies in 13 RA patients demonstrated marked interindividual differences in the pharmacological responses at 3 months after the start of therapy with rituximab. Interestingly, only differences in the regulation of the type I interferon (IFN) -response genes at 3 months after therapy correlated with the ΔDAS28 response. Good responders (ΔDAS >1.2; n=7) exhibited a selective increase in the expression of type I IFN-response genes, whereas this activity was not or hardly changed in non-responders (ΔDAS Conclusions A good clinical response to rituximab in RA is associated with a selective increase in type I IFN-response activity in RA patients. This finding may provide insight in the biological mechanism underlying the therapeutic response of rituximab.

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 CARRE 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.

Background Although B cell depletion therapy with rituximab is successful in the majority of rheumatoid arthritis (RA) patients, 30–40% of patients do not respond. The mechanism behind this is yet unknown, as B cell depletion generally takes place in all patients. This study aims to explore changes in serum cytokines during rituximab therapy in established RA patients. Methods Serum was collected from 18 established RA patients before and after 1, 3, 6, 9 and 12 months of rituximab therapy. A panel of thirty-four cytokines was measured in a multiplex immunoassay. Patients with a change in disease activity score (∆DAS28) >1.2 after 6 months were considered responders. Results During rituximab therapy, CXCL13 significantly decreased in all patients (median 1.9-fold decrease (IQR 1.4-3.3)), irrespective of responder status. This coincided with B cell depletion, suggesting that CXCL13 reflects B cell levels in the circulation. No differences were observed between responders and non-responders after 1 and 3 months of therapy. However, at 6, 9 and 12 months, CXCL10 dynamics differed significantly between responders and non-responders (R vs. NR, T6/T0 p = 0.004, T9/T0 p = 0.003, T12/T0 p = 0.001). Non-responders showed an increase compared to baseline, whereas responders did not. The change in CXCL10 correlated to the ∆DAS28 at 6 months, indicating that this reflects disease activity rather than a mechanistic effect of rituximab. Most strikingly, IL-12 was undetectable in most non-responders until 6 months, but showed a substantial increase after 9 months of therapy, which did not occur in the responders (R vs. NR T9/T6 p = 0.030, Fisher’s exact p = 0.049). The change in IL-12 did not correlate to ∆DAS28. Conclusions This study shows that during rituximab therapy, cytokine changes occur in the patient serum, both related and unrelated to the clinical response. CXCL13 seems to reflect B cells levels in the circulation, whereas the dynamics of CXCL10 and IL-12 during therapy are regulated differently between responders and non-responders during rituximab treatment. These findings indicate different pharmacological effects of rituximab between responders and non-responders. This research was supported by the Center for Translational Molecular Medicine (CTMM) and the Dutch Arthritis Foundation (TRACER).

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 (Umea) 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

Backgroundand objectives The interferon (IFN) signature has been described to be present in many autoimmune diseases, including myositis. The extent of this signature appears to be related to disease activity, however, the underlying mechanism resulting in such a signature has not been revealed yet. Detailed insight into such a mechanism would increase the understanding of role of the IFN systems in myositis and autoimmunity. In this study, the authors investigated the association between (the extent of) the IFN signature and autoantibody profiles in myositis. Materials and methods RNA samples, obtained from whole blood from 94 patients with polymyositis, dermatomyositis, and inclusion body myositis recruited from the Karolinska University Hospital and Prague University, were assessed for expression levels of 9 IFN related genes using BioMark Dynamic Arrays. IFN score was determined as the average gene expression level of these genes. Median IFN score (4.38) was used to define patients as IFN high or IFN low . ANA positivity was determined by indirect immunofluorescence test and myositis specific autoantibodies against Jo-1 and SRP, and myositis associated autoantibodies PM-Scl, Ro52, Ro60 (SSA), La and U1RNP were detected by a line blot and in-house immunoblot assay. Results IFN signature in peripheral blood was present in a subgroup of patients with myositis. In order to determine the relevance of the differential expression of IFN type I activity in myositis the authors studied the association between IFN score and the presence of ANA or specific autoantibodies. No significant difference in the extent of the IFN score was observed between ANA negative (n=44) and ANA positive (n=50) nor in the number of IFN high or IFN low patients. Comparison of IFN score in patients positive for myositis associated or specific autoantibodies revealed that the highest IFN score was found in U1RNP positive patients (mean IFN score=23.66, 7/9 were IFN high ), followed by La positive patients (mean IFN score=11.94, all were IFN high n=4). In addition, a majority of Jo-1(16 out of 23), Ro-52 (15 out of 22), Ro-60 (8 out of 10) positive patients were characterised by an IFN high expression profile. Detailed analysis revealed that IFN high patients were characterised by multi-autoantibody specificity, that is, 17 out of 23 patients positive for 2 or more autoantibodies were IFN high (70%) versus 30 out of 71 of the patients positive for 1 or none of the specific autoantibodies (45%) (Pearson Chi square p=0.008). Conclusions An IFN score in myositis patients is associated with the presence of myositis specific or associated autoantibodies Jo-1, U1RNP, Ro-52, Ro-60 or La and the association was stronger when 2 or more autoantibodies were present. Based on these data and on previous studies the authors hypothesise that these autoantibodies could act as an endogenous trigger of the type I IFN pathway and contribute to the chronicity of these diseases.

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 IFN high patients converted to arthritis compared to 32% in IFN low patients (P=0.011). For the B-cell signature, 58% in B-cell low patients developed arthritis, compared to 33% of B-cell high patients (P=0.020). Combined analysis revealed a significant high risk for arthritis development in IFN high /B-cell low patients (80%, hazard ratio (HR)(6.22, P=0.003) and a low risk for IFN low /B-cell high 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

Background A role for type I interferon (IFN) activity is suggested in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). While the mechanism of induction of the IFN activity in SLE is rather known, this remains to be determined for RA. This study aims to characterise the source of IFN activity in RA serum. Methods Healthy PBMCs were exposed to serum from RA (n = 18) or SLE (n = 25) patients. IFNα protein production was measured in an immunoassay after 20 h incubation with 5% patient serum. Samples were also co-cultured with apoptotic or necrotic cell material, as this has proven to enhance IFNα production by SLE serum. Moreover, expression of IFN response genes (IRGs), IFNα and IFNβ mRNA was determined by qPCR after 4 h and 8 h incubation with 25% patient serum. To study the involvement of new protein synthesis, part of the samples was co-cultured with 2 µg/ml cycloheximide. All cultures were performed with healthy donor serum (NHS) as a negative control. Results As expected, SLE serum induces more IFNα protein production compared to NHS (p = 0.0006). This increases even further in the presence of dead cell material. RA serum does not show more IFNα protein inducing activity than NHS, although a small, nonsignificant increase was observed in the absence of dead cell material. With respect to IRG induction, both RA and SLE sera induced higher levels compared to NHS. SLE serum showed IRG induction at 4 h, which remained high after 8 h. The IRG induction at 8 h was not decreased by CHX treatment, indicating that it occurs independently of new protein synthesis, supporting a proposed direct effect by IFNα. RA serum induced IRG induction only after 8 h, which was inhibited upon CHX treatment, suggesting an indirect induction process. The IRG induction by RA serum was positively correlated with IFNβ mRNA induction at 4 h and 8 h (p = 0.0023 and p = 0.0130, respectively), but not with IFNα mRNA induction. Conclusions Altogether, these results indicate different mechanisms underlying the induction of type I IFN activity between SLE and RA.

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 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.

Background/purpose Early recognition of development of rheumatoid arthritis (RA) allows timely start of treatment. Recently, the authors reported gene signatures in the peripheral blood (PB), involving interferon (IFN) activity and B cell related genes, which are associated with the development of arthritis. The objective of this study is to validate these findings in independent cohorts. Method The authors analysed IFN-response gene expression in PB samples from 40 anticitrullinated protein antibodies (ACPA)+/rheumatoid factor (RF)+arthralgia patients from the Amsterdam Reade cohort who are clinically followed for RA development. In addition, PB mononuclear cells (MCs) of 22 preonset RA, 25 RA patients, and 48 healthy controls (HC) from the Northern Sweden Population Repository (Umea University) were studied. Fluorescence-activated cell sorting (FACS)-analysis for B cell marker expression was performed on PB from 87 ACPA+/RF+ arthralgia patients and 30 HC from the Amsterdam Reade cohort. Quantitative PCR (qPCR) for B cell marker was performed of 45 ACPA+/RF+ arthralgia patients and six HC from the Amsterdam Reade cohort. Results The IFN-score (average of seven IFN-response genes) was measured at inclusion in an independent group of ACPA+/RF+ arthralgia patients at risk for RA (n=40). A total of 19 patients developed arthritis within a median period of 11 months (IQR 9–28). Comparative analysis of IFN-score high and low patients confirmed that the high IFN-score is associated with conversion to RA (p=0.025). In addition, the authors measured the IFN-score in PBMC from RA and preonset RA patients, and HC from the Northern Swedish Repository. Comparative analyses between the groups revealed that both preonset RA patients as well as RA patients had significantly increased IFN-scores compared to HC (Mann–Whitney U test p=0.0064 and p=0.0075, respectively). For the analysis of the role of B cells in development of RA the authors analysed the FACS profile of 87 ACPA+/RF+ at risk patients at inclusion, of who 22 developed RA within a median period to conversion of 10.5 months (IQR 4.5–13). The results confirmed that low B cell marker expression is significantly associated with RA conversion (p=0.01). An interim analysis with qPCR showed a trend towards the association between decreased B cell transcript marker and RA conversion (p=0.0714). Conclusion These results provide evidence for a role of IFN-activity and B cell abundance in the preclinical phase of RA. Further studies are aimed to demonstrate the diagnostic value of these markers.

Background and objectives B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumour necrosis factor (TNF) blocking agents. However, approximately 40–50% of rituximab (RTX) treated RA patients have a poor response. The authors investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX. Materials and methods In 14 consecutive RA patients starting with RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina HumanHT beadchip microarrays. Supervised cluster analysis (patients ranked on difference in 28 joints disease activity score (DAS28) after 6 months RTX) identified genes expressed differently at baseline in case of non-response (both ΔDAS28 Results Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, the authors demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (p=0.0074 and p=0.0599, respectively). Based on the 8 IRGs an IFN-score was calculated that reached an AUC of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using ROC curves analysis according to ΔDAS28 Conclusions This study demonstrates clinical utility for the use of baseline IRG expression levels as predictive biomarker for non-response to RTX in RA.

Backgroundand objectives The folate antagonist methotrexate (MTX) is an anchor drug in the treatment of rheumatoid arthritis (RA). Many attempts have been undertaken to predict response to MTX treatment, in particular by evaluating possible correlations with polymorphic variations of methotrexate/folate-related genes. Thus far, however, many of these studies were not elusive in providing robust predictive markers. The aim of this study was to explore whether analysis of expression of methotrexate/folate-related genes provide information on the pharmacodynamics and (non)responsiveness of RA patients for MTX therapy. Methods Gene expression profiling was performed on peripheral blood from 25 MTX-treated (MTX + ) and 10 untreated (MTX − ) RA patients, and 15 healthy controls (HC). Statistical analysis was performed for 25 genes involved in the methotrexate/folate pathway using student9s t test or Mann–Whitney U test, p values of ≤0.05 were considered to be statistically significant. Results Several folate/MTX-related genes were markedly and significantly altered between the three study groups. Interestingly, the metabolic enzymes FPGS and GGH were significantly up-regulated in the MTX − RA group compared to the healthy control group (HC group), whereas GART expression was markedly down-regulated. Following MTX treatment, these alterations in expression levels were normalised to those observed in the HC group. Furthermore, the MTX-efflux transporters multidrug resistance protein-2 (MRP2) and MRP3 showed an increased expression in the MTX + group compared to the MTX − group and the HC group, suggesting that cellular extrusion may contribute to a diminished MTX response in the MTX + group. Conclusions Collectively, these results indicate that, under inflammatory conditions, basal folate metabolism is altered in blood cells of RA patients versus HC. Treatment with MTX restores expression of these genes to the levels within the range of the HC group. Finally, our results provide the first indication that multidrug resistance protein efflux transporters could contribute to an attenuated MTX response in MTX-based RA treatment. Acknowledgements Dr Y G Assaraf (Technion, Haifa, Israel) was a recipient of a visiting professor fellowship provided by the Dutch Arthritis Foundation to the VU University Medical Center Amsterdam. This study is partly supported by the ‘TRACER’ consortium of the Center for Translational and Molecular Medicine (CTMM), and the Dutch Arthritis Foundation.

B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX. In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26). Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria. This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA.

Objectives Tumour necrosis factor α (TNF-α) and interferons (IFNs), which are abundantly present in the rheumatoid synovium, are thought to exert two opposing effects on RA pathology. We studied the opposing effects of IFNs and TNF on the proliferative capacity of rheumatoid fibroblast-like synoviocytes (FLS). Methods IFN and/or TNF induced changes in FLS gene expression were measured by DNA-microarray analysis and data was analysed using Significance Analysis of Microarrays (SAM) and pathway analysis using PANTHER. Activation of STAT-1 was determined by immunofluorescence and real-time PCR. FLS proliferation was monitored by 3 H-thymidine incorporation. Apoptosis was determined by staining with Annexin V and propidium iodide (PI). Staining with PI was also used to analyse cell cycle distribution. Expression of GADD45β mRNA was measured by real-time PCR. Results Gene expression profiling of IFN-stimulated rheumatoid FLS provided evidence for inhibition of cell cycle regulation. Accordingly, IFN stimulation resulted in a >50% decrease in DNA synthesis. The inhibitory effect was not due to induction of apoptosis but was shown to be the consequence of limited cell cycle progression due to accumulation of cells in G0/1 phase. The inhibitory effect overruled TNFα-induced cell cycle progression to G2/M phase and proliferation. This effect correlated with expression of the Growth-Arrest and DNA Damage-Inducible gene GADD45β. Conclusion These results indicate that IFN-induced inhibition of cell cycle progression in rheumatoid FLS overrules TNFα-induced proliferative effects. Funding This work has been supported by the EU Marie Curie Training Network (EURO-RA), the EU integrated program AUTOCURE, and CMSB (Netherlands Centre of Excellenc on Genomics Research)

B cells are supposed to play an important role in rheumatoid arthritis (RA). Accordingly, rituximab, a chimeric monoclonal antibody against CD20, effectively depletes B cells ameliorates disease although a subset of patients don't respond. We aimed to identify biomarkers and biological …

Background Multiple lymphocyte subsets like T and B cells have been connected to joint infiltration and inflammation in rheumatoid arthritis (RA). Identification of leucocyte subsets that are dysregulated in arthritis development could provide insight into the aetiology of RA. This study aimed to investigate the composition of the peripheral blood components, i.e. CD14+ monocytes, CD4+ and CD8+ T lymphocytes (CD3+), CD80+, C-X-C chemokine receptor 3 (CXCR3)+ and CD27+ B lymphocytes (CD19+), CD16+CD56+CD3− natural killer (NK) cells and activated CD56+CD3+ T cells, for association with arthritis development in patients with arthralgia. Methods Peripheral blood was collected from 89 patients with early RA (disease duration 1 year and 73 did not develop arthritis). Absolute numbers of monocytes and lymphocyte subsets in whole heparinized blood were determined with flow cytometry using quantification beads in combination with fluorescent labelled antibodies for T cells, B cells, monocytes, NK cells and activated T cells. Results In patients with early RA, significant decreases in numbers of (activated) T cells, CD80+ and memory B cells and a trend towards smaller numbers of CD8+ T cells was observed compared to HC. Similar differences were seen in patients with arthralgia who developed or did not develop arthritis (non-converters), with significantly decreased CD8+ T cells and memory B cells. Patients with arthralgia who developed arthritis were split into groups that developed arthritis within 1 year (early converters) or after 1 year (late converters). Late converters had a significantly decreased number of CD8+ T cells compared to non-converters; early converters had a decreased number of memory B cells. Longitudinal analysis of converters showed a significant relative increase in CD80+ B cells towards the conversion time point compared to 24 months prior to conversion. Conclusions This study revealed that patients with arthralgia who develop arthritis demonstrate a change in cellular immune parameters apparent in the periphery, starting with a decrease in cytotoxic T cells 24 months prior to arthritis development, followed by a decrease in the number of memory B cells 12 months prior to disease onset. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1102-2) contains supplementary material, which is available to authorized users.

Background The 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. Methods Blood 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. Results Patients 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. Conclusions PMNs 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. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1065-3) contains supplementary material, which is available to authorized users.

Background A peripheral blood interferon (IFN) signature (i.e., elevated type I interferon response gene [IRG] expression) has been described in a subset of patients with rheumatoid arthritis (RA). In the present study, we systematically assessed the association between this IRG expression and clinical parameters. Methods Expression of 19 IRGs was determined in peripheral blood from 182 consecutive patients with RA and averaged into an IFN score per individual. Correlation and unpaired analyses were performed on the complete patient group. The analyses were internally validated by using an algorithm to randomize the patient group 1000 times into two equally sized sets, and then analyses were performed on both sets. Results Associations were assessed between IFN score and disease duration, 28-joint Disease Activity Score and its components, the occurrence of erosions and nodules, autoantibody positivity, and immunosuppressive treatment. This analysis revealed lower IFN scores in patients using hydroxychloroquine, prednisone, and/or sulfasalazine, but it did not show significant associations between the other parameters and the IFN score. Selecting patients who were not treated with hydroxychloroquine, prednisone, and/or sulfasalazine (n = 95) did not reveal any significant associations either. Conclusions IRG expression in RA is affected by immunosuppressive treatment with prednisone, hydroxychloroquine, and/or sulfasalazine, but it is not evidently associated with other clinical parameters. Hence, the IFN signature appears to describe a subgroup of patients with RA but does not seem to reflect disease activity. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1191-y) contains supplementary material, which is available to authorized users.