38 research outputs found

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

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    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 mug/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

    Assessment of the Composition and Biologic Activity of Platelet Rich Plasma and its Relationship to Clinical Outcomes in Patients with Knee Osteoarthritis

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    Recent studies suggest positive clinical outcomes associated with platelet-rich-plasma (PRP) administration to treat knee osteoarthritis (OA). However, the results remain inconclusive in part because of the high variability in PRP preparations and the limited information regarding the relevant biologically active components of PRP. We hypothesize that the variability in clinical response is driven by the heterogeneous composition of PRP. In this study we evaluated the composition and biological activity of PRP and further correlated our findings to clinical outcomes in patients receiving intra-articular injections for knee OA. After IRB approval and patient consent, we enrolled 51 patients (mean age: 57.9 ± 10.1; mean BMI: 26.0 ± 4.1) with mild-moderate knee OA (Kellgren Lawrence grades 1-3), eligible for intra-articular PRP injection. We obtained MRI at baseline and outcome measures (KOOS JR and PNS) at baseline, 6 weeks, 6 months, and 12 months after PRP injection. Patients were categorized as “good” and “poor” responders based on the outcome measures, corrected using published Minimally Clinically Important Difference (MCID) values. Aliquots of PRP and whole blood from the same patients were used to evaluate composition (CBC with differential and multiplex ELISA) and biologic activity, using a co-culture system of macrophages and fibroblast incubated with TNFa with and without PRP (10% v:v) for 24 hours. Total RNA from cells was used for RNAseq, Nanostring, and RTqPCR analysis. On average, we collected 4.07 ± 01.05 mL of PRP, and 3.24 ± 0.85 mL of PRP were injected intra-articularly. PRP preparations yielded mean fold-changes of 1.60 ± 0.37 platelets and 0.19 ± 0.08v leukocytes, relative to whole-blood from the same patients (set as 1). On average, all patients that reached the 6-month time-point (N = 32) reported improved outcomes at 6-weeks and 6-months after PRP administration (KOOS and PNS p\u3c0.05 vs. baseline). After MCID corrections, we identified “good” (N=17, positive response using both measures) and “poor” responders (N=15, poor response in one or both measures). RNAseq analyses showed PRP-dependent changes in the TNFa-induced modulation of a number of genes, including CXCL7 and CCL5. NanoString and RTqPCR analyses confirmed the RNAseq results. Comparisons of PRP from good and poor responders identified changes in the composition and biologic activity between these groups. This pilot study integrated clinical data with genomic approaches to evaluate variability in the composition and activity of PRP, and how this may influence outcomes in patients with knee OA. We uncovered subsets of genes differentially modulated by co-treatment of PRP with TNFa, in agreement with the concept that the reduced knee OA pain in patients treated with PRP is driven by the ability of PRP to modulate inflammation. Furthermore, we identified changes in the composition and biologic activity of PRP between “good” and “poor” responders

    Cross talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A pathways in Cryptococcus neoformans

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    Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis

    The chromatin landscape of pathogenic transcriptional cell states in rheumatoid arthritis

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    Synovial tissue inflammation is a hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. Here, we examine genome-wide open chromatin at single-cell resolution in 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identify 24 chromatin classes and predict their associated transcription factors, including a CD8 + GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating with an RA tissue transcriptional atlas, we propose that these chromatin classes represent ‘superstates’ corresponding to multiple transcriptional cell states. Finally, we demonstrate the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance, as well as the nomination of classes mediating the effects of putatively causal RA genetic variants.</p

    Regulation of medullary thymic epithelial cell differentiation and function by the signaling protein Sin

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    Medullary thymic epithelial cells (mTECs) play an important role in T cell tolerance and prevention of autoimmunity. Mice deficient in expression of the signaling protein Sin exhibit exaggerated immune responses and multitissue inflammation. Here, we show that Sin is expressed in the thymic stroma, specifically in mTECs. Sin deficiency led to thymic stroma–dependent autoimmune manifestations shown by radiation chimeras and thymic transplants in nude mice, and associated with defective mTEC-mediated elimination of thymocytes in a T cell receptor transgenic model of negative selection. Lack of Sin expression correlated with a disorganized medullary architecture and fewer functionally mature mTECs under steady–state conditions. Additionally, Sin deficiency inhibited the expansion of mTECs in response to in vivo administration of keratinocyte growth factor (KGF). These results identify Sin as a novel regulator of mTEC development and T cell tolerance, and suggest that Sin is important for homeostatic maintenance of the medullary epithelium in the adult thymus

    Automated multi-scale computational pathotyping (AMSCP) of inflamed synovial tissue

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    Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research

    Clonal associations between lymphocyte subsets and functional states in rheumatoid arthritis synovium

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    Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA

    Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes

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    Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments
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