L G M van Baarsen

Rheumatoid Arthritis subtypes identified by genomic profiling of peripheral blood cells: Assignment of a type I interferon signature in a subpopulation of patients

Background: Rheumatoid arthritis (RA) is a heterogeneous disease with unknown cause. Aim: To identify peripheral blood (PB) gene expression profiles that may distinguish RA subtypes. Methods: Large-scale expression profiling by cDNA microarrays was performed on PB from 35 patients and 15 healthy individuals. Differential gene expression was analysed by significance analysis of microarrays (SAM), followed by gene ontology analysis of the significant genes. Gene set enrichment analysis was applied to identify pathways relevant to disease. Results: A substantially raised expression of a spectrum of genes involved in immune defence was found in the PB of patients with RA compared with healthy individuals. SAM analysis revealed a highly significant elevated expression of interferon (IFN) type I regulated genes in patients with RA compared with healthy individuals, which was confirmed by gene ontology and pathway analysis, suggesting that this pathway was activated systemically in RA. A quantitative analysis revealed that increased expression of IFN-response genes was characteristic of approximately half of the patients (IFNhigh patients). Application of pathway analysis revealed that the IFNhigh group was largely different from the controls, with evidence for upregulated pathways involved in coagulation and complement cascades, and fatty acid metabolism, while the IFNlow group was similar to the controls. Conclusion: The IFN type I signature defines a subgroup of patients with RA, with a distinct biomolecular phenotype, characterised by increased activity of the innate defence system, coagulation and complement cascades, and fatty acid metabolism.

Responsiveness to anti-tumour necrosis factor α therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients

Objective: The response of rheumatoid arthritis (RA) patients to treatment with neutralising antibodies to tumour necrosis factor α (TNFα) is highly variable. The underlying mechanism for therapy responsiveness is currently unknown. We therefore evaluated the relationship between baseline molecular profiles of synovial tissues from RA patients and the clinical response to treatment with infliximab. Methods: Synovial biopsies were obtained by arthroscopy from 18 RA patients with active disease (28 joint count Disease Activity Score (DAS28)⩾3.2) before initiation of treatment with infliximab. All patients were on stable methotrexate treatment. Clinical response at 16 weeks was defined as a reduction in DAS28 of ⩾1.2, non-response as reduction in DAS28 <1.2. Large-scale gene expression profiling using microarrays was performed on synovial tissue samples. To identify biological processes in synovial biopsies that could discriminate between responders and non-responders, we performed pathway analysis on the expression profiles. Results: A total of 12 patients responded to therapy, while 6 patients failed to fulfil the response criteria. We identified several biological processes, related to inflammation, which were up-regulated in patients who responded to therapy, compared to those who did not show clinical improvement. Conclusion: These results indicate that patients with a high level of tissue inflammation are more likely to benefit from anti-TNFα treatment.

A subtype of multiple sclerosis defined by an activated immune defense program.

Given the heterogeneous nature of multiple sclerosis (MS), we applied DNA microarray technology to determine whether variability is reflected in peripheral blood (PB) cells. In this study, we studied whole-blood gene expression profiles of 29 patients with relapsing-remitting MS (RRMS) and 25 age- and sex-matched healthy controls. We used microarrays with a complexity of 43K cDNAs. The data were analyzed using sophisticated pathway-level analysis in order to provide insight into the deregulated peripheral immune response programs in MS. We found a remarkable elevated expression of a spectrum of genes known to be involved in immune defense in the PB of MS patients compared to healthy individuals. Cluster analysis revealed that the increased expression of these genes was characteristic for approximately half of the patients. In addition, the gene signature in this group of patients was comparable with a virus response program. We conclude that the transcriptional signature of the PB cells reflects the heterogeneity of MS and defines a sub-population of RRMS patients, who exhibit an activated immune defense program that resembles a virus response program, which is supportive for a link between viruses and MS.

Features of the Synovium of Individuals at Risk of Developing Rheumatoid Arthritis: Implications for Understanding Preclinical Rheumatoid Arthritis

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

Molecular subtypes of systemic sclerosis in association with anti-centromere antibodies and digital ulcers

The objective of this study was to identify molecular profiles that may distinguish clinical subtypes in systemic sclerosis (SSc). Large-scale gene expression profiling was performed on peripheral blood (PB) from 12 SSc patients and 6 healthy individuals. Significance analysis of microarrays, two-way hierarchical cluster analysis and PANTHER (Protein ANalysis THrough Evolutionary Relationships) ontology classification were used to analyze the data. Quantitative PCR was applied for validation in a cohort of 43 SSc patients. The results show that the expression of genes involved in immune defense, cell cycle and signal transduction was significantly elevated in PB of SSc patients (n=12) compared with healthy individuals (n=6). SSc patients could be stratified into subgroups based on differential expression of genes induced by type I interferon (IFN) and genes involved in antimicrobial (AM) activity. Differential expression of type I IFN or AM signature genes was validated and extended in an independent cohort of 31 patients by quantitative PCR. Low expression of IFN response genes was associated with the presence of anti-centromere antibodies, whereas increased expression was associated with the appearance of digital ulcers. In conclusion, patients with SSc can be classified on the basis of differential expression of immune defense genes. Differences in the activity of the type I IFN response program stratify patients into two clinically relevant subgroups.

The cellular composition of lymph nodes in the earliest phase of inflammatory arthritis

Objectives Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease of unknown aetiology. Recent work has shown that systemic autoimmunity precedes synovial inflammation, and animal models have suggested that changes in the lymph nodes may precede those in the synovial tissue. Therefore, we investigated the cellular composition of the lymph node in the earliest phases of inflammatory arthritis. Methods Thirteen individuals positive for immunoglobulin M (IgM) rheumatoid factor and/or anticitrullinated protein antibodies without arthritis were included. Additionally, we studied 14 early arthritis patients (arthritis duration ≤6 months, naïve for disease-modifying antirheumatic drugs), and eight healthy controls. All subjects underwent ultrasound-guided inguinal lymph node biopsy. Different T- and B-lymphocyte subsets were analysed by multicolour flow cytometry. Results There was an increase in activated CD69 CD8 T cells and CD19 B cells in early arthritis patients compared with healthy controls. We also observed a trend towards increased CD19 B cells in autoantibody-positive individuals without arthritis compared with healthy controls. Conclusions This exploratory study suggests that there is increased immune cell activation within lymph nodes of early arthritis patients as well as in autoantibody-positive individuals at risk of developing RA. This method provides a unique tool to investigate immunological changes in the lymph node compartment in the earliest phases of inflammatory arthritis.

Pharmacogenomics of infliximab treatment using peripheral blood cells of patients with rheumatoid arthritis.

To provide insight into the pharmacological changes in the peripheral blood (PB) molecular profile induced by tumor necrosis factor (TNF)-blockade in patients with rheumatoid arthritis (RA), blood was obtained in PAXgene tubes from 33 RA patients before and 1 month after TNF-blocking therapy (infliximab). From 15 randomly chosen patients pre- and post-treatment gene expression profiles were determined. The remaining 18 RA patients served as validation cohort. A group-based paired analysis of the gene expression profiles from the post- vs pre-treatment samples revealed a signature of genes significantly regulated by TNF-blockade. Downregulated genes reflected several biological pathways such as inflammation, angiogenesis, B- and T-cell activation. Further analysis revealed that the pharmacological response signature was significantly regulated in all treated patients, irrespective of clinical response, which is indicative for the presence of an active TNF pathway in all RA patients. The data imply that all patients carried features of TNF bioactivity irrespective of clinical response. These results favor a model for the parallel presence of TNF-dependent and TNF-independent pathways in the individual RA patient. Clinical response status to TNF-blockade may be dependent on the relative contribution of TNF-independent effector pathways.

Investigating the cellular composition of lymph nodes in preclinical and early inflammatory arthritis: a feasibility study

Backgroundand objectives Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease of unknown aetiology. To ultimately cure or prevent this chronic disease, it is critical to understand the earliest changes in the immune system that cause RA. Recent work has shown that systemic autoimmunity precedes inflammation in the synovium of RA patients. Animal models have suggested that changes in the lymph nodes may precede those in the synovial tissue. To provide insight into the immunological mechanisms involved in the pathogenesis of RA, the authors developed a method to obtain lymph node biopsies under local anaesthesia and investigated the lymph node cellular composition and distribution in individuals at risk of developing RA, early arthritis patients and healthy controls. Materials and methods Six individuals without any evidence of arthritis upon physical examination who were positive for IgM rheumatoid factor and/or anticitrullinated protein antibodies were included. For comparison 12 early arthritis patients (1× systemic lupus erythematosus, 1× psoriatic arthritis, 1× gout, 5× undifferentiated arthritis and 4× RA; disease duration <1 year, disease-modifying antirheumatic drug naïve), and four autoantibody negative healthy controls without joint complaints were included in the study. All study subjects underwent ultrasound-guided inguinal lymph node biopsy. Different T lymphocyte subsets were analysed by multi-colour flow cytometry using labelled antibodies specific for CD3, CD4, CD8, CD45 and CD69. Results The procedure was well tolerated; no complications occurred. Different T cell subsets could be distinguished and differences between autoantibody positive individuals at risk of developing RA, early arthritis patients and healthy controls could be observed. Interim analysis indicate an increase of activated CD69+ T cells in the early arthritis as well as in the at risk group compared to the control group. Interestingly, the CD4/CD8 distribution within the activated T cells was significantly changed in the early arthritis patients compared to healthy controls and the same trend was observed for the at risk group. Conclusions Flow cytometry analysis of ultrasound-guided inguinal lymph node biopsies is a feasible method for investigating the cellular composition of lymph nodes in the earliest phases of inflammatory arthritis. These preliminary results suggest increased CD8+ T cell activation within lymph nodes of early arthritis patients as well as in autoantibody positive individuals at risk of developing RA. This method provides a unique tool to investigate the immunological changes in the lymph node compartment in the earliest phases of inflammatory arthritis. These data support the rationale for larger studies using more extensive panels of cellular markers.

Expression levels of interleukin-17a, interleukin-17f and their receptors in synovium of patients with rheumatoid arthritis, psoriatic arthritis and osteoarthritis: a target validation study

Background Accumulating evidence suggests an important role for interleukin (IL)-17 in the pathogenesis of several inflammatory diseases, including rheumatoid arthritis (RA). IL-17A has been well studied in models of arthritis, but little is known about the relative expression and cellular source(s) of IL-17A, IL-17F, and their receptors in human synovial tissue. Objective To determine the origin and expression of IL-17A, IL-17F and their receptors IL-17RA and IL-17RC in synovial tissues of patients with RA, psoriatic arthritis (PsA) and inflammatory osteoarthritis (OA). Methods Synovial biopsy specimens were obtained by arthroscopy from patients with RA (n=13), PsA (n=15) and inflammatory OA (n=14). For comparison synovial tissues from non-inflammatory controls (n=7) were included. Immunohistological analysis was performed on frozen sections using monoclonal antibodies specific for IL-17A, IL-17F, IL-17RA and IL-17RC and evaluated by digital image analysis. Immunofluorescence analysis was used to determine which cells in the synovium produce IL-17A and IL-17F using antibodies specific for T cells (CD4, CD8), neutrophils (CD15), macrophages (CD68, CD163), B cells (CD19), endothelial cells (CD31, Von Willebrand Factor, PNAd), lymphatics (Lyve-1) and mast cells (mast cell tryptase). Stained sections were evaluated by confocal microscopy. Results Levels of IL-17A, IL-17F, IL-17RA and IL-17RC were abundantly present in synovial tissues of all patient groups and highly variable between patients. Whereas IL-17RA was mostly present in the synovial sublining, IL-17RC was abundantly expressed in the intimal lining layer. Digital image analysis showed a significant increase of IL-17A but not of IL-17F, IL-17RA and IL-17RC in patients with arthritis compared to non-inflamed control tissues, while the expression of IL-17A, IL-17F and IL-17RA was similar between the different patient groups. Expression of IL-17RC in the intimal lining layer was significantly increased in PsA compared to OA patients. IL-17A was found to be expressed by CD4 and CD8 positive cells, while CD15, CD19 and mast cell tryptase (MCT) positive cells were negative. IL-17F was not expressed by CD15 positive cells and only occasionally by CD4, CD8 and MCT positive cells in the synovial tissues of arthritic patients. Interestingly, IL-17A and IL-17F staining was also observed in some macrophages as well as in endothelial cells and lymphatics.. Conclusions Increased expression of IL-17A is not restricted to synovial tissues of RA patients but also observed in other forms of inflammatory arthritis. In inflamed synovium various cell types contribute to the production of IL-17A and IL-17F.

A8.34 CD1C + dendritic cells are overrepresented in lymph nodes of early arthritis patients and related to B cell responses

Background and Objectives The immune system is composed of different cell populations that circulate in our body through lymphoid organs. We have recently shown that the numbers of CD69+ T cells and B cells are increased in lymph node (LN) biopsies from early arthritis patients. DCs are fundamental in initiating and modulating T and B cell responses and may therefore play an essential pathologic role in autoimmune diseases. We hypothesize that an in-depth analysis of DCs within LNs of at risk individuals and early arthritis patients can provide a better understanding of immune function and dysregulation during the earliest phases of arthritis. Materials and Methods We included 22 individuals with arthralgia without any evidence of arthritis who were positive for IgM rheumatoid factor and/or anti-citrullinated protein antibodies (ACPA; RA risk group), 20 DMARD and biological naïve early arthritis patients (2010 criteria < 1 year) and 8 seronegative control individuals. All study subjects underwent ultrasound-guided inguinal LN biopsy. DC subsets (CD1a, CD1c, and CD304) were analysed by multi-colour flow cytometry. Results All DC subsets included in this study could be detected in LN biopsies. However, CD1c+ DC were the major subset present in LNs of early arthritis patients compared to both CD304+ DCs (p < 0.0001) or CD1a+ DCs (p < 0.002). In line with this, there was a significant increase of CD1c+ DCs in the early arthritis group compared to the at risk group (p = 0.009) and compared to control individuals (p = 0.01). Moreover, in early arthritis patients the frequencies of CD1c+ DCs were significantly increased in ACPA+ compared to ACPA- patients (p = 0.03). The frequencies of CD304+ DC were also significantly increased in early arthritis compared to the at risk group (p = 0.009) but did not correlate with ACPA status. Conclusions This is the first study that reports the presence of DC subsets in LNs during the earliest phases of arthritis. As CD1c+ DCs are the main DC subset present in early arthritis and the only subset related with ACPA status, this study supports the notion that in addition to the well-known capacity of CD1c+ DCs to activate naïve T cells, CD1c+ DC might also contribute to B cell responses.