Objective Synovial fluid single-cell immunoprofiling is a powerful tool for investigating joint arthropathies. However, cell isolation protocols are yet to be standardised. We compared different cell-isolation protocols across diverse synovial fluid samples, evaluating cell recovery, sample inclusion rate and immune-cell composition.Methods From 48 synovial fluid samples with volumes>2 mL, we performed paired cell isolation of whole synovial fluid cells (WSFCs), with and without hyaluronidase and synovial fluid mononuclear cells (SFMCs) by density-gradient centrifugation. Of these, nineteen paired samples underwent deep immunophenotyping using a 31-marker spectral flow cytometry panel with dimensionality reduction and unsupervised clustering. Immune cell frequencies were quantified across protocols. Synovial fluid rheology was assessed in an additional 23 samples.Results WSFC isolation yielded higher cell recovery and lower sample loss than SFMC isolation, with differences varying with inflammation level. SFMC isolation reduced the sample inclusion rate (the proportion of samples with sufficient cell numbers for downstream profiling) by 50% in low-grade and 10% in high-grade inflammatory samples. Cell recovery correlated with synovial fluid viscosity. Repeated centrifugation alone matched the cell recovery seen with hyaluronidase treatment. Deep immunophenotyping revealed systematic protocol-dependent differences: WSFCs were enriched for CD8+, CD4+ and γδ T cells, while SFMCs contained higher proportions of dendritic cells and classical monocytes. Both methods stratified samples by inflammation, but WSFCs provided superior cellular resolution.Conclusions SFMC isolation excluded low-grade inflammatory samples, introducing bias and reducing statistical power. WSFC-based approaches with hyaluronidase or repeated centrifugation improved recovery and are therefore recommended for synovial fluid immunoprofiling studies.
Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by synovial inflammation and osteoclast-driven bone erosion, leading to joint destruction. T cells play a central role in driving inflammation and contribute to the pathogenesis of RA. This study aims to identify T cell subsets in RA using single-cell RNA sequencing (scRNA-seq) technology.Methods An in silico analysis of publicly available scRNA-seq datasets was conducted to identify a T cell population enriched in RA synovial tissue. To generate this population in vitro, peripheral blood T cells were stimulated with various cytokines. Gene expression was measured by quantitative PCR, and bulk RNA sequencing was used to define the transcriptomic profile of this population. Proteomic analysis was also performed to characterise its protein expression profile and functional pathways.Results A population of T cells expressing immunoglobulin genes was identified in RA synovial tissue in silico. Among the various cytokines tested, interleukin-4 specifically upregulated immunoglobulin gene expression in peripheral blood lymphocytes, particularly in naive CD8+ T cells. Transcriptomic analysis showed increased osteoclast differentiation but reduced pathways related to osteoclast differentiation regulation, RA and inflammatory responses. In contrast, proteomic analysis showed consistent downregulation of the osteoclast differentiation and inflammatory response pathways.Conclusions These findings suggest a T cell population in RA exhibiting immunoglobulin gene expression at the transcript level and reduced inflammatory and osteoclast-related pathways at the protein level.
Objectives To report population-level data describing long-term trends in remission, sustained remission and relapse among patients with rheumatoid arthritis (RA).Methods We conducted a population-based inception cohort study of adults with incident RA in Olmsted County, Minnesota, USA, meeting 1987 or 2010 ACR criteria between 1990 and 2019. Patients were followed through 31 December 2023. We estimated cumulative incidence of first remission, sustained remission (≥6 months) and relapse after remission, accounting for competing risk of death. Cox models comparing calendar periods were adjusted for age, sex, baseline body mass index and serostatus.Results Among 1179 patients followed for a median of 9.4 years, 816 achieved remission and 630 achieved sustained remission. One-year remission, defined by our prespecified criteria, increased from 12.8% in 1990–1999 to 26.4% in 2010–2019. Median time to remission shortened from 6.4 years to 2.8 years. Despite these gains, relapse within 5 years after remission increased from 62.8% in 1990–1999 vs 79.2% in the 2010–2023 period.Conclusion Remission in RA has become earlier and more frequent over time, but durability has not improved. Strategies to sustain remission remain a critical unmet need.
Objectives To examine associations between patient-reported outcome measures (PROMs) for disease activity, physical function and well-being and clinical measurements for mobility as well as C reactive protein (CRP) in axial spondyloarthritis (axSpA). A secondary aim was to explore cut-off values that may serve as indicators to prompt further mobility assessment or consideration of treatment adjustment.Methods This cross-sectional study used data from 22 105 visits among 5095 individuals with axSpA, registered in the Swedish Rheumatology Quality Register. Variables for PROMs were Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Global Score. Variables for clinical measurements were Bath Ankylosing Spondylitis Metrology Index (BASMI) and CRP. Associations were tested with Pearson’s correlations. Receiver operating characteristic (ROC) curve analysis assessed discrimination of impaired spinal mobility in BASMI or elevated CRP for variables with r ≥0.5.Results Associations with r ≥0.5 were found only between BASFI and BASMI (r=0.51), resulting in an area under the curve of 0.74 (95% CI 0.71 to 0.76) in the ROC analyses. The strongest associations were found between BASFI subquestions 5 (getting up off the floor without help from lying on your back) and 8 (looking over your shoulder without turning your body) and BASMI sum (r=0.5 and r=0.63, respectively).Conclusions BASFI and its subquestions 5 and 8 may be helpful in identifying individuals with axSpA who require further mobility measuring. Neither PROMs associate with CRP to any higher degree (r <0.5), which reinforces that PROMs cannot replace objective inflammatory markers.
Objective To evaluate feasibility, tolerability and acceptability of performing minimally invasive synovial tissue biopsies for the purpose of research in children and young people diagnosed with inflammatory arthritis.Methods Synovial tissue biopsies were obtained from the joints of children with arthritis undergoing intra-articular procedures performed under general anaesthetic, and embedded within routine clinical care pathways. Questionnaires pertaining to the tolerability of the procedure were completed. Tissue samples were processed for histological assessment of tissue quality and graded for synovitis.Results Synovial tissue biopsies were obtained from 62 children, aged between 1 and 17 years of age (median 7.1 years). While most children had a single joint biopsied, seven procedures involved biopsy of two different joints. At baseline, 65 knees, two ankles and two wrist joints were biopsied; seven children underwent repeat knee biopsies later in their disease. Almost 63% of biopsies (n=39/62 participants) were performed in children with oligoarticular juvenile idiopathic arthritis (JIA). Postprocedure questionnaires revealed a majority of families (80%, n=32/40) would be ‘very likely’ or ‘somewhat likely’ to consent to a repeat biopsy in the future. Histological assessment of tissue using an adapted Krenn synovitis score confirmed differences between oligoarticular and polyarticular JIA. No serious adverse events were reported.Conclusions Ultrasound-guided synovial tissue biopsies in children undergoing intra-articular procedures under general anaesthetic are feasible, safe and well-tolerated, and can be embedded within routine clinical care pathways for the procurement of tissue samples for the purpose of research.
