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

Predicting the paths of peripherals: The interaction of identification and future possibilities

Two studies investigated how both degree of identification and the individual's position within the group influence aspects of group loyalty. The authors considered ingroup position in terms of both the individual's current position within a group and expectations concerning the likelihood that one's position might change in the future. Peripheral group members learned that their acceptance by other group members would improve in the future or that they could expect rejection by other group members. Various indices of group loyalty (ingroup homogeneity, motivation to work for the group, and evaluation of a motivated group member) showed that when group members anticipated future rejection, the lower the identification the less loyal they were. In contrast, those who expected future acceptance were more loyal (more motivated to work for the group) the lower their identification. Current group behavior depends on both intragroup future expectations and level of identification

DHEA metabolism in arthritis - A role for the p450 enzyme Cyp7b at the immune-endocrine crossroad

For dehydroepiandrosterone (DUEA) both immunosuppressive and immuno-stimulating properties have been described. The immunosuppressive effects may be explained by the conversion of DHEA into androgens and/or estrogens. The described immuno-stimulating effects of DHEA may be due to the conversion of DHEA into 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA) by the activity of the p450 enzyme, Cyp7b. 7 alpha-OH-DHEA is thought to have anti-glucocoticoid activity preventing the anti-inflammatory action of endogenous glucocorticoids. To investigate a putative role of Cyp7b in the arthritic process, tissues from both the murine collagen-induce arthritis (CIA) model and from patients with rheumatoid arthritis (RA) were studied. We determined the Cyp7b expression levels in synovial tissue and the level of 7 alpha-OH-DHEA in both serum and arthritic joints of mice with CIA. Our studies showed that the severity of arthritis correlates with increased Cyp7b activity. Next, we investigated Cyp7b expression and activity in RA patients where the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are known to control the disease process. Fibroblast-like synoviocytes (FLS), isolated from RA synovial biopsies were found to express Cyp7b mRNA. In addition, Cyp7b enzymatic activity was detected in these cells. We also investigated whether Cyp7b activity is regulated by cytokines. Proinflammatory (e.g., TNF-alpha and IL-1 beta) cytokines were found to stimulate Cyp7b activity and the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) was found to suppress Cyp7b activity in FLS. Next, we studied which signal transduction pathway is involved in the TNF-alpha-mediated induction of Cyp7b activity in human FLS. The results show a role for nuclear factor kappa B (NF kappa B) and activator protein-1 (AP-1) in the regulation of Cyp7b expression. Finally, we established that the effects of DHEA or 7 alpha-OH-DHEA on the immune system can not be explained by glucocorticoid receptor (GR) engagement. The role of the p450 enzyme Cyp7b in DHEA metabolism and its relevance in the arthritic process will be discussed.</p