ObjectivesTo integrate published single-cell RNA sequencing (scRNA-seq) data and assess the contribution of synovial fibroblast (SF) subsets to synovial pathotypes and respective clinical characteristics in treatment-naïve early arthritis.MethodsIn this in silico study, we integrated scRNA-seq data from published studies with additional unpublished in-house data. Standard Seurat, Harmony and Liger workflow was performed for integration and differential gene expression analysis. We estimated single cell type proportions in bulk RNA-seq data (deconvolution) from synovial tissue from 87 treatment-naïve early arthritis patients in the Pathobiology of Early Arthritis Cohort using MuSiC. SF proportions across synovial pathotypes (fibroid, lymphoid and myeloid) and relationship of disease activity measurements across different synovial pathotypes were assessed.ResultsWe identified four SF clusters with respective marker genes: PRG4+ SF (CD55, MMP3, PRG4, THY1neg); CXCL12+ SF (CXCL12, CCL2, ADAMTS1, THY1low); POSTN+ SF (POSTN, collagen genes, THY1); CXCL14+ SF (CXCL14, C3, CD34, ASPN, THY1) that correspond to lining (PRG4+ SF) and sublining (CXCL12+ SF, POSTN+ + and CXCL14+ SF) SF subsets. CXCL12+ SF and POSTN+ + were most prominent in the fibroid while PRG4+ SF appeared highest in the myeloid pathotype. Corresponding, lining assessed by histology (assessed by Krenn-Score) was thicker in the myeloid, but also in the lymphoid pathotype + the fibroid pathotype. PRG4+ SF correlated positively with disease severity parameters in the fibroid, POSTN+ SF in the lymphoid pathotype whereas CXCL14+ SF showed negative association with disease severity in all pathotypes.ConclusionThis study shows a so far unexplored association between distinct synovial pathologies and SF subtypes defined by scRNA-seq. The knowledge of the diverse interplay of SF with immune cells will advance opportunities for tailored targeted treatments.
Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB plays a critical role in orchestrating the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.
We present an optimized dissociation protocol for preparing high-quality skin cell suspensions for in-depth single-cell RNA-sequencing (scRNA-seq) analysis of fresh and cultured human skin. Our protocol enabled the isolation of a consistently high number of highly viable skin cells from small freshly dissociated punch skin biopsies, which we use for scRNA-seq studies. We recapitulated not only the main cell populations of existing single-cell skin atlases, but also identified rare cell populations, such as mast cells. Furthermore, we effectively isolated highly viable single cells from ex vivo cultured skin biopsy fragments and generated a global single-cell map of the explanted human skin. The quality metrics of the generated scRNA-seq datasets were comparable between freshly dissociated and cultured skin. Overall, by enabling efficient cell isolation and comprehensive cell mapping, our skin dissociation-scRNA-seq workflow can greatly facilitate scRNA-seq discoveries across diverse human skin pathologies and ex vivo skin explant experimentations.
In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor β1 (TGF-β1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation.
Single-cell RNA-sequencing is advancing our understanding of synovial pathobiology in inflammatory arthritis. Here, we optimized the protocol for dissociation of synovial biopsies and created a comprehensive reference single-cell atlas of fresh human synovium in inflammatory arthritis. We derived our protocol from the published dissociation method for cryopreserved synovium (Donlin L. et al. Arthritis Res. Ther. 2019) with modifications to enrich synovial cells and minimize cell loss. These modifications enabled consistently high cell yield and viability, thereby minimizing the rate of synovial tissue sample dropout. Our single-cell atlas of the human synovium comprised more than 100000 unsorted single-cell profiles from 27 synovia of patients with inflammatory arthritis. Synovial cells formed ten lymphoid, 14 myeloid and 17 stromal cell clusters, including IFITM2+ synovial neutrophils. We identified lining SOD2highSAA1+SAA2+ and transitional SERPINE1+COL5A3+ synovial fibroblasts, exhibiting gene signatures linked to cartilage breakdown (SDC4) and extracellular matrix remodelling (LOXL2, TGFBI, TGFB1), respectively. We uncovered synovial endothelial cell diversity and broadened the transcriptional characterization of tissue-resident FOLR2+ COLEC12+ and SLC40A1+ synovial macrophages, inferring their extracellular matrix sensing and iron recycling activities. Our research brings an efficient synovium dissociation protocol for prospectively collected fresh synovial biopsies and expands the knowledge about human synovium composition in inflammatory arthritis.
Single-cell RNA-sequencing is advancing our understanding of synovial pathobiology in inflammatory arthritis. Here, we optimized the protocol for dissociation of synovial biopsies and created a comprehensive reference single-cell atlas of fresh human synovium in inflammatory arthritis. We derived our protocol from the published dissociation method for cryopreserved synovium (Donlin L. et al. Arthritis Res. Ther. 2019) with modifications to enrich synovial cells and minimize cell loss. These modifications enabled consistently high cell yield and viability, thereby minimizing the rate of synovial tissue sample dropout. Our single-cell atlas of the human synovium comprised more than 100’000 unsorted single-cell profiles from 27 synovia of patients with inflammatory arthritis. Synovial cells formed ten lymphoid, 14 myeloid and 17 stromal cell clusters, including IFITM2+ synovial neutrophils. We identified lining SOD2highSAA1+SAA2+ and transitional SERPINE1+COL5A3+ synovial fibroblasts, exhibiting gene signatures linked to cartilage breakdown (SDC4) and extracellular matrix remodelling (LOXL2, TGFBI, TGFB1), respectively. We uncovered synovial endothelial cell diversity and broadened the transcriptional characterization of tissue-resident FOLR2+ COLEC12+ and SLC40A1+ synovial macrophages, inferring their extracellular matrix sensing and iron recycling activities. Our research brings an efficient synovium dissociation protocol for prospectively collected fresh synovial biopsies and expands the knowledge about human synovium composition in inflammatory arthritis.
Background:The heterogeneity of synovial tissues from patients with arthritis could contribute to the interpatient variability in disease course, prognosis and treatment response. Single-cell RNA sequencing (scRNA-seq) permits in-depth analysis of tissue heterogeneity, which could facilitate drug discovery and patient stratification for precision medicine.Objectives:To construct a comprehensive landscape of synovial cell types and molecular pathways in arthritis by integrating our and published scRNA-seq data, generated across different scRNA-seq technologies [Smart-seq2, Drop-seq], cell preparation protocols [dissociated unsorted, sorted cells] and types of arthritis [undifferentiated (UA), rheumatoid arthritis, osteoarthritis].Methods:Synovial tissues were obtained by ultrasound-guided biopsy from patients with UA [not fulfilling the classification criteria for a specific arthritis, n=3]. Biopsies were disintegrated [enzymatic and mechanical disruption] and cell viability assessed with trypan blue. ScRNA-seq libraries [2 per patient] were prepared with 10X Genomics Drop-Seq and sequenced on NovaSeq6000. Bioinformatics analysis of our and published [n=35] datasets1-3was performed using Seurat protocol4with correction for batch effects and filtering low-quality cells. Functional enrichment analysis of marker genes in clusters was done with STRING Protein-Protein networks. Synovitis was assessed with ultrasound and histology.Results:Our tissue disintegration protocol resulted in good cell yield and viability (92%, 72%, 100%). The synovial cellular heterogeneity detected by scRNA-seq reflected the histological findings [Krenn score, pathotype]. These were supported with the ultrasound and clinically assessed disease activity. The integrated analysis of 41 datasets from 38 donors yielded 41845 scRNA-seq cell profiles, 50% contributed by our dataset. An independent analysis of our data and their integration with published data showed that different scRNA-seq methods and protocols can identify all the major synovial cell types and their activation states (Figure 1) with large heterogeneity between donors. We identified a previously undescribed synovial cell population, which was located near the fibroblast cluster, was negative for canonical cell markers, but highly enriched in cell division genes (80% of marker genes). These cells comprised a mixed population of CD34-, podoplanin (PDPN)highor PDPNlowcells that were mostly negative for the sub-lining fibroblast marker THY. Furthermore, they appeared to be highly secretory (extracellular matrix components) and their gene expression profile was inclined towards cell migration, vascular development and insulin growth factor-dependent processes.Figure 1.Heatmap with top 20 cluster gene markers, gene enrichment analysis and UMAP plot of synovial cell clusters.Conclusion:By integrating synovial scRNA-seq data from 41845 cells, we identified a previously undescribed, highly proliferative and secretory synovial cell population in arthritis. We increased the number of known scRNA-seq synovial cell profiles in arthritis by two-fold and demonstrated the robustness of synovial scRNA-seq data outputs across different technologies and protocols. This broadens the current knowledge of synovial tissue heterogeneity and pathology in arthritis.References:[1]Stephenson W. et al. Nat Commun 2017.[2]Mizoguchi F. et al. Nat Commun 2017.[3]Zhang F. et al. Nat Immunol 2018.[4]Stuart T et al. Cell 2019Acknowledgments:This work is supported by Vontobel Foundation and medAlumni University of ZurichDisclosure of Interests:Sam G. Edalat: None declared, Raphael Micheroli: None declared, Tadeja Kuret: None declared, Kristina Buerki: None declared, Chantal Pauli: None declared, Snežna Sodin-Šemrl: None declared, Adrian Ciurea Consultant of: Consulting and/or speaking fees from AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Merck Sharp & Dohme, Novartis and Pfizer., Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Gregor Rot: None declared, Mojca Frank-Bertoncelj: None declared
Most forms of arthritis, have a distinctive topographical pattern of joint involvement. Beyond these differences among diseases, there are also differences in phenotype and response to treatment between joints of the same type of arthritis, suggesting that molecular mechanisms may differ depending on joint location. Here we show that there are joint-specific molecular and tissue changes in the synovium and in local stromal cells (synovial fibroblasts;SF). The long non-coding RNA HOTAIR, expressed only in lower extremities SF, regulates much of this site-specific gene expression in SF. Downregulation of HOTAIR after TNF stimulation regulated relevant inflammatory pathways by epigenetic and transcriptional mechanisms and modified the migratory function of SF, decreased SF-mediated osteoclastogenesis, and increased the attraction of B cells by SF. Since site-specific expression of HOTAIR was also measured in the skin, spine and gastrointestinal tract, we propose HOTAIR as important epigenetic factor that modulates site-specific phenotypes of chronic inflammation.
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