Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Generalized Bone Loss as a Predictor of Three-Year Radiographic Damage in African American Patients With Recent-Onset Rheumatoid Arthritis

Objective. To examine the association between baseline bone mineral density (BMD) and radiographic damage at 3 years of disease duration in a longitudinal cohort of African Americans with recent-onset rheumatoid arthritis (RA). Methods. African American RA patients with a disease duration of <2 years (n = 141) were included in the study. All patients underwent baseline BMD measurements (femoral neck and/or lumbar spine) using dual x-ray absorptiometry. T scores were calculated using normative data from the general population of African Americans. Patients were categorized as having osteopenia/osteoporosis (T score less than or equal to -1) or as being healthy. Hand and wrist radiographs, obtained at baseline and at 3 years of disease duration, were scored using the modified Sharp/van der Heijde method. The association between baseline BMD and total radiographic score at 3 years of disease was examined using multivariable negative binomial regression. Results. At baseline, the mean age and the mean disease duration were 52.4 years and 14.8 months, respectively; 85.1% of the patients were women. The average total radiographic scores at baseline and at 3 years of disease were 2.4 and 5.7, respectively. In the final reduced multivariable model, adjusting for age, sex, anti-cyclic citrullinated peptide antibody positivity, and the presence of radiographic damage at baseline, the total radiographic score at 3 years disease in patients with osteopenia/osteoporosis of the femoral neck was twice that in patients with normal bone density, and the difference was statistically significant (P = 0.0084). No association between lumbar spine osteopenia/osteoporosis and radiographic score was found. Conclusion. Our findings suggest that reduced generalized BMD may be a predictor of future radiographic damage and support the hypothesis that radiographic damage and reduced generalized BMD in RA patients may share a common pathogenic mechanism.Pathophysiology and treatment of rheumatic disease

High Expression of Genes in the Toll-Like Receptor and Interferon Pathways Are Associated with Radiographic Damage in African-Americans with ACPA-Positive RA

Pathophysiology and treatment of rheumatic disease