ObjectivesWe explored histological and transcriptomic profiles of paired synovial biopsies from rheumatoid arthritis (RA) patients, in order to assess homogeneity in synovial tissue at the individual level.MethodsSynovial biopsies were performed simultaneously in one small and one large joint per patient using needle-arthroscopy for the knee and ultrasound-guided biopsy for the hand or wrist. Synovium from individuals with osteoarthritis was used as controls. Paraffin-embedded samples were stained for CD3, CD20, and CD68. Total RNA was hybridized on high-density microarrays. TCRB variable sequences were obtained from synovial and blood RNA samples.ResultsTwenty paired biopsies from 10 RA patients with active disease were analyzed. Semi-quantification of histological markers showed a positive correlation for synovial hyperplasia, inflammatory infiltrates and CD3-positive T cells between pairs. Pairwise comparison of transcriptomic profiles showed similar expression of RA-related molecular pathways (TCR signaling, T cell costimulation and response to TNFα). T cells clonotypes were enriched in all but one joints compared to blood, regardless of the magnitude of T cell infiltration. Enriched clonotypes were shared between pairs (23–100%), but this was less the case in pairs of joints displaying weaker T cell signatures and more pronounced germinal center-like transcriptomic profiles.ConclusionCellular and molecular alterations in RA synovitis are similar between small and large joints from the same patient. Interindividual differences in magnitude of T cell infiltrates and distribution of enriched T cell clonotypes support the concept of distinct synovial pathotypes in RA that are associated with systemic versus local antigen-driven activation of T cells.
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease targeting the joints. Current treatment strategies are based on clinical, biological and radiological features, yet still fail to reach the goal of early low disease activity in a significant number of cases. Hence, there is a need for refining current treatment algorithms, using accurate markers of response to therapy. Because RA induces histological and molecular alterations in the synovium even before apparition of clinical symptoms, synovial biopsies are a promising tool in the search of such new biomarkers. Histological and molecular characteristics of RA synovitis are heterogeneous. Variations in synovial lining layer hyperplasia, in cellular infiltration of the sublining by immune cells of myeloid and lymphoid lineages, and in molecular triggers of these features are currently categorized using well-defined pathotypes: myeloid, lymphoid, fibroid and pauci-immune. Here, we first bring the plasticity of RA synovitis under scrutiny, i.e., how variations in synovial characteristics are associated with relevant clinical features (disease duration, disease activity, effects of therapies, disease severity). Primary response to a specific drug could be, at least theoretically, related to the representation of the molecular pathway targeted by the drug in the synovium. Alternatively, absence of primary response to a specific agent could be due to disease severity, i.e., overrepresentation of all synovial molecular pathways driving disease activity overwhelming the capacity of any drug to block them. Using this theoretical frame, we will highlight how the findings of previous studies trying to link response to therapy with synovial changes provide promising perspectives on bridging the gap to personalized medicine in RA.
ObjectivesOur goal was to assess for the histological and transcriptomic effects of abatacept on RA synovia, and to compare them with previously published data from four other DMARDs: tocilizumab, rituximab, methotrexate, and adalimumab.MethodsSynovial tissue was obtained using ultrasound-guided biopsy from affected joints of 14 patients, before and 16 weeks after treatment with subcutaneous abatacept 125 mg weekly. Paraffin-sections were stained and scored for CD3+, CD20+, and CD68+ cell infiltration. Transcriptional profiling was performed using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix) and analyzed on Genespring GX (Agilent). Pathway analyses were performed on Genespring GX, Metascape, and EnrichR.ResultsGene expression analysis identified 304 transcripts modulated by abatacept in synovial tissue. Downregulated genes were significantly enriched for immune processes, strongly overlapping with our findings on other therapies. Data were pooled across these studies, revealing that genes downregulated by DMARDs are significantly enriched for both T-cell and myeloid leukocyte activation pathways. Interestingly, DMARDs seem to have coordinate effects on the two pathways, with a stronger impact in good responders to therapy as compared to moderate and non-responders.ConclusionWe provide evidence that the effects of five DMARDs on the RA synovium culminate in the same pathways. This confirms previous studies suggesting the existence of common mediators downstream of DMARDs, independent of their primary targets.
ObjectivesTranscriptomic profiling of synovial tissue from patients with early, untreated rheumatoid arthritis (RA) was used to explore the ability of unbiased, data-driven approaches to define clinically relevant subgroups.MethodsRNASeq was performed on 74 samples, with disease activity data collected at inclusion. Principal components analysis (PCA) and unsupervised clustering were used to define patient clusters based on expression of the most variable genes, followed by pathway analysis and inference of relative abundance of immune cell subsets. Histological assessment and multiplex immunofluorescence (for CD45, CD68, CD206) were performed on paraffin sections.ResultsPCA on expression of the (n=894) most variable genes across this series did not divide samples into distinct groups, instead yielding a continuum correlated with baseline disease activity. Two patient clusters (PtC1, n=52; PtC2, n=22) were defined based on expression of these genes. PtC1, with significantly higher disease activity and probability of response to methotrexate therapy, showed upregulation of immune system genes; PtC2 showed upregulation of lipid metabolism genes, described to characterise tissue resident or M2-like macrophages. In keeping with these data, M2-like:M1-like macrophage ratios were inversely correlated with disease activity scores and were associated with lower synovial immune infiltration and the presence of thinner, M2-like macrophage-rich synovial lining layers.ConclusionIn this large series of early, untreated RA, we show that the synovial transcriptome closely mirrors clinical disease activity and correlates with synovial inflammation. Intriguingly, lower inflammation and disease activity are associated with higher ratios of M2:M1 macrophages, particularly striking in the synovial lining layer. This may point to a protective role for tissue resident macrophages in RA.
BackgroundRheumatoid Arthritis (RA) is a chronic and heterogenous condition characterized by inflammatory involvement of the synovial membrane in multiple joints. Synovial biopsies are used in research setting in order to identify diagnostic and theranostic markers. Many studies have shown a high degree of heterogeneity in histological and transcriptomic profiles between patients.ObjectivesWe wanted to explore histological and transcriptomic profile of synovial biopsies across pairs of joints in the same patients to assess heterogeneity at the individual level.MethodsSynovial biopsies were performed simultaneously in one small and one large joint per patient using needle-arthroscopy for the knee and US-guided needle biopsy for the hand or foot. Synovium from individuals with osteoarthritis (OA) were used as control. Paraffine-embedded samples were stained for CD3, CD 20, CD 68 and CD138. Whole-tissue RNA was extracted and hybridized on GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). The samples were tested for RNA integrity by Bioanalyzer (Agilent). Normalization and statistical analyzes were performed on Genespring. Pathway analysis were performed using DAVID.Results10 RA patients were included (females: 10/10, ACPA/RF positivity: 8/10, mean age (± SEM): 54.4 (± 4.4) years, mean disease duration (± SEM): 13.3 (± 3.7) years, mean DAS28CRP (± SEM): 5.01 (± 0,34), mean HAQ (± SEM): 1.7 (± 0.28)).Quantification of histological markers did not show differences in population of macrophages, plasmocytes, T and B Cells, across pairs of joints.After correction for multiple comparisons, no transcripts were differentially expressed between large and small joints. Similarly, we did not find any significant difference in the expression of transcripts involved in pathways (TCR-activation and cell-division) specifically overexpressed in RA compared to OA synovial tissue.In order to increase our ability to observe pair-wise differences in gene expression profiles, we studied correlations between transcripts significantly overexpressed in RA compared to OA joints with a fold change > 2 (n = 581) and clinical or biological markers of disease activity (DAS28-CRP, CRP, Physician Global Assessment of disease activity). Similar patterns of correlations indicated that disease activity was not driven by different pathways in small versus large joints.ConclusionThis study is an important methodological milestone in the field of synovial biopsies, as it indicates that cellular and molecular alterations occurring in RA synovitis are similar across small and large joints from the same patient. Hence, biopsy of a single joint is representative and can be used to explore pathogenic processes or potential biomarkers in RA.AcknowledgementThis work was funded in part by grants from Cap48 (RTBF), Fonds National de la Recherche Scientifique (FNRS, Communauté française de Belgique) and Fondation Saint-Luc.Disclosure of InterestsClément Triaille: None declared, Louise Vansteenkiste: None declared, Laurent Meric de Bellefon: None declared, Ad...
Background:Increasing evidence indicates that synovial tissue analysis can deliver pathophysiological insights but also individual clinically-relevant information in adult-onset inflammatory arthritides. Little is known about synovial pathology in juvenile idiopathic arthritis, especially regarding inter-patient variability of histopathological features.Objectives:To assess the heterogeneity of main synovial features (synoviocyte hyperplasia and immune cells infiltration) in juvenile idiopathic arthritis (JIA) patients and a cohort of young adults (<30 years old) with early rheumatoid arthritis (RA).Methods:Synovial biopsies were sampled using needle arthroscopy or ultra-sound (US) guided biopsy during intra-articular joint injection. Tissue was embedded in paraffin then sections were stained with hematoxylin and eosin. Synoviocyte hyperplasia (SH) and immune cells infiltration (ICI) was assessed by an experienced pathologist on a 0 – 3 scale where 0 represents the absence of the feature and 3 the highest level.Results:34 JIA patients (age (median ±SD): 15.5±6.47 years, oligo-articular JIA n=28/34, polyarticular JIA n=6/34, ANA-RF-ACPA positivity=56%-10%-3%) and 22 RA (age (median ±SD): 24.3±2.6 years, ANA-RF-ACPA positivity=10%-36%-32%) patients were included. Synovial tissue was obtained from knee (n=49/56), wrist (n=4/56) or metacarpophalangeal/intercarpophalangeal joints (n=3/56), using US guided biopsy in 27% of patients and needle arthroscopy in 73%.Individual scores of SH and ICI were correlated in both JIA (Spearman’s r=0.503, p value=0.0024) and RA (Spearman’s r=0.636, p value=0.0015). There was no significant difference in SH and ICI scores between the 2 groups (SH score (Q25-Q50-Q75) in JIA= 0.5-1.125-2 and in RA = 0.75-2-2; ICI score (Q25-Q50-Q75) in JIA= 1-2-2 and in RA = 0.75-2-2.25). Intra-group variability of the two assessed features was comparable between the 2 groups (SH coefficient of variation: 72.2% for JIA and 68.2% for RA; ICI coefficient of variation: 52.2% for JIA and 71.2% for RA). Within JIA patients, there was no significant difference in SH/ICI scores between groups based on ANA positivity, oligo or polyarticular involvement nor ongoing treatment.Conclusion:Studying main histological features of synovitis, we found no difference between JIA and young RA patients. Furthermore, we report a similar degree of inter-patient heterogeneity in synovial pathological features of JIA and RA patients. These variations were not explained by common clinical characteristics. Whether they relate to different molecular signatures as suggested in adult RA will be further investigated using bulk tissue RNA sequencing.Acknowledgements:This work was funded in part by Cap48 (RTBF). Clément Triaille is funded by the Fonds National de la Recherche Scientifique (FNRS, Communauté française de Belgique) and Fondation Saint-Luc (Cliniques Universitaires Saint-Luc).Disclosure of Interests:Clément Triaille: None declared, Cécile Boulanger: None declared, Tatiana Sokolova: None declared, Laurent Meric de Bellefon: None declared, Adrien Nzeusseu Toukap: None declared, Christine Galant: None declared, Nisha Limaye: None declared, Bernard Lauwerys Employee of: currently employed at UCB Biopharma, Patrick Durez: None declared.
Background:Modes of action of DMARDs (disease-modifying antirheumatic drugs) in rheumatoid arthritis (RA) are not completely understood at the level of the synovium. Studying treatment-induced modifications in RA synovial tissue can provide unique insights into the pathways modulated downstream of different DMARDs.Objectives:Our goal was to assess histological and transcriptomic effects of Abatacept (ABA) on RA synovium, and to compare them with previously published data obtained by our group using the same study design on other DMARDs: Tocilizumab (TCZ), Rituximab (RTX), Methotrexate (MTX) and Adalimumab (ADA).Methods:Synovial tissue was obtained using ultrasound-guided biopsy from affected joints before (W0) and 16 weeks (W16) after treatment with subcutaneous Abatacept 125mg per week on a MTX background. Paraffin-sections were stained for CD3, CD20 and CD68 and scored by a pathologist for T cell, B cell and macrophage infiltration. Transcriptional profiling was performed using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), and analyzed on Genespring GX (Agilent). Pathway analyses were performed on Genespring GX, Metascape (https://metascape.org/) and EnrichR (https://maayanlab.cloud/Enrichr/). Protein-Protein Interaction (PPI) networks were generated on STRING (https://string-db.org/).Results:14 RA patients were included (female: 9, ACPA/RF positive: 8, erosive disease: 12, median disease duration in years (± SD): 11.7 (± 8.1), median DAS28CRP (± SD): 4.78 (± 1.11)). Median DAS28CRP significantly decreased between W0 and W16, as did US GS score. Evaluation of histological slides (n=11 pairs of samples) showed no significant effect of Abatacept on T cell, B cell or macrophage infiltration. Gene expression analysis (n=10 pairs of samples) identified 304 transcripts differentially expressed (129 downregulated, 175 upregulated) between W0 and W16 (FC≥1.5 and p<0.05, paired Mann-Whitney). Downregulated genes were significantly enriched for immune processes and included several key T cell regulatory genes (IL2RA, CD28, IL7, IL7R), strongly overlapping with data from previous studies on TCZ (n= 12 pairs), RTX (n=12 pairs), MTX (n=8 pairs) and ADA (n=8 pairs). Thus, each treatment shares 31 to 48% of its downregulated genes with the others, with genes downregulated by at least three involved in key RA-associated pathways such as leukocyte activation, NF-kappa B signaling, TNF signaling and JAK-STAT signaling. Given their seemingly overlapping effects, data were pooled across these studies, markedly improving power thanks to their paired-design. This revealed that genes downregulated by DMARDs (n=573, Benjamini-Hochberg corrected p-value<0.05, paired Mann-Whitney) were significantly enriched for both T cell and myeloid leukocyte activation pathways. Interestingly, DMARDs seem to have a coordinate effect on the two pathways (correlation of mean Log2FC: r=0.8558, p<0.0001), with a stronger impact (Log2FCW16-W0) in good responders to therapy (n=17) as compared to moderate (n=20) and to non-responders (n=13) (p<0.0001, Mann-Whitney). Finally, Transcription Factor enrichment and PPI network analyses point to a central role for molecules including JAK/STATs as mediators of all studied therapies.Conclusion:We provide evidence that the effects of five DMARDs on RA synovium culminate in the same pathways (namely, T cell and myeloid leukocyte activation). This confirms previous studies suggesting the existence of common mediators downstream of DMARDs, independent of their primary targets, and suggests attractive new therapeutic targets.Acknowledgements:This work was funded in part by unrestricted grants from Cap48 (RTBF) and Bristol-Myers Squibb. Clément Triaille is funded by the Fonds National de la Recherche Scientifique (FNRS, Communauté française de Belgique) and Fondation Saint-Luc (Cliniques Universitaires Saint-Luc).Disclosure of Interests:Clément Triaille: None declared, Tilman Gaelle: None declared, Tatiana Sokolova: None declared, Laurent Meric de Bellefon: None declared, Christine Galant: None declared, Patrick Durez Grant/research support from: unrestricted research grant from Bristol-Myers Squibb, Bernard Lauwerys Employee of: currently employed at UCB Biopharma, Nisha Limaye: None declared
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