University of Birmingham Birmingham, United Kingdom
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Christopher Mahony1, Chrissy Bolton2, Charlotte Smith1, Vicky Alexiou3, Huong Nguyen3, Patricia Reis-Nisa1, Søren Lomholt4, Annie Hackland1, Sunit Davda5, Sugrah Sultan6, Charlene Foley5, Catherine Cotter6, Klaudia Kupiec3, Calliope Dendrou2, Elizabeth C Rosser7, Accelerating Medicines Partnership (AMP): RA/SLE8, Fan Zhang9, Soumya Raychaudhuri8, Michael Brenner10, Christopher Buckley2, Manigandan Thyagarajan6, Accelerating Medicines Partnership Program RA SLE Network11, Zishan Shiekh6, Sandrine Compeyrot-Lacassagne5, Samantha Chippington5, Mark Coles2, Eslam Al-Abadi6, Andrew Filer1, Tissue Research in Childhood Onset Inflammatory Arthritis (TRICIA) Consortium12, Lucy R Wedderburn3 and Adam Croft1, 1University of Birmingham, Birmingham, United Kingdom, 2University of Oxford, Oxford, United Kingdom, 3UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 4Aarhus University, Aarhus, Denmark, 5Great Ormond Street Hospital, London, United Kingdom, 6Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, United Kingdom, 7University College London, London, United Kingdom, 8Brigham and Women's Hospital, Boston, MA, 9University of Colorado, Aurora, CO, 10Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 11Cedars-Sinai Medical Center, Los Angeles, CA, 12MRC, Birmingham, United Kingdom
Background/Purpose: The synovial membrane is the primary target tissue during the effector phase of inflammatory arthritis in children and young people with Juvenile Idiopathic Arthritis (JIA). However, due to difficulties accessing synovial tissue in children, little is known of the disease mechanisms that operate locally in the synovium. As part of the UK Tissue Research in Childhood Arthritis Consortium we have generated the first single cell multiomic atlas of the inflamed synovium in JIA.
Methods: Synovial tissues were obtained from inflamed knee joints of 12 children with JIA, who were naïve to disease modifying anti-rheumatic drugs using minimally invasive ultrasound guided biopsy procedures refined for use in children. Where possible, matched synovial fluid and peripheral blood samples were obtained from the same children at the time of the procedure. Tissues were cryopreserved, thawed and disaggregated and viable cells from each sample sorted for single-cell (sc) RNAseq. Multiplex imaging of matched synovial tissue sections (GE Cell Dive) and spatial transcriptomics were performed to generate the first synovial tissue atlas of JIA.
Results: We profiled 250,816 cells from matched tissue, blood and synovial fluid samples in children with newly diagnosed JIA using scRNA sequencing. We resolved 11 major cell types, including stromal (endothelial, pericytes, fibroblasts, lymphatic) and myeloid cells. Analysis of stromal cells revealed 7 transcriptionally distinct fibroblast clusters (Fig. 1A, B). One fibroblast cluster highly expressing Sox5 and Cadherin-11 (and devoid of endothelial/pericyte/lining layer markers) was enriched in JIA synovium when compared to adult synovium of patients with rheumatoid arthritis (RA - analysis from the Accelerated Medicines Partnership (AMP) network, Zhang et al., BioRxiv, 2022). This cluster expresses several transcription factors (Fig. 1B) and ongoing neighbourhood analysis of spatial data will determine the location of these cells in synovial tissue (Fig. 1C,D). Myeloid cells were resolved into 9 clusters that are transcriptionally conserved compared to myeloid cells from adult RA inflamed synovium (AMP: Zhang et al., BioRxiv, 2022, Fig. 2A). Differential gene expression showed heterogeneity of myeloid cells between blood, tissue, and synovial fluid (Figure 2B).
Conclusion: We have generated the first single cell atlas of the inflamed synovium in children with JIA. Through a comparative analysis with adult RA synovium, we have identified a distinct fibroblast subcluster that expresses Sox5 and Cadherin-11 and is enriched in JIA synovial tissue. It is not yet known if this fibroblast subtype represents an age, developmental or disease specific transcriptional program that drives JIA pathology. Fully mapping and characterising the inflamed synovium in children is critical in understanding the cellular pathogenesis of JIA.
References: Zhang, F. et al. (2022) 'Cellular deconstruction of inflamed synovium defines diverse inflammatory phenotypes in rheumatoid arthritis'. bioRxiv, p. 2022.02.25.481990. Available at: https://doi.org/10.1101/2022.02.25.481990
Figure 1. Multiomic characterisation of JIA inflamed synovium. (A) scRNAseq of stromal cells and assigned clusters. (B) Dotplot of key marker genes defining stromal cell subpopulations. (C) example of multiplex cellDIVE imagines. (D) analysis cellDIVE pipeline whereby cells are segmented and fluorescence quantified.
Figure 2. Transcriptional characterisation of JIA myeloid cells. (A) scRNAseq of myeloid cells and assigned clusters. (B) gene module assignment based on co-expression of top 10,000 highly variable genes.
C. Mahony: None; C. Bolton: None; C. Smith: None; V. Alexiou: None; H. Nguyen: None; P. Reis-Nisa: None; S. Lomholt: None; A. Hackland: None; S. Davda: None; S. Sultan: None; C. Foley: None; C. Cotter: None; K. Kupiec: None; C. Dendrou: None; E. Rosser: None; A. Medicines Partnership (AMP): RA/SLE: None; F. Zhang: None; S. Raychaudhuri: AbbVie, 6, Janssen, 1, Mestag, Inc, 2, 8, Pfizer, 1, Sanofi, 1, Sonoma, 1, 8; M. Brenner: 4FO Ventures, 2, GlaxoSmithKlein(GSK), 2, Mestag Therapeutics, 2, 11, Third Rock Ventures, 2; C. Buckley: Bristol-Myers Squibb(BMS), 5, Mestag, 11; M. Thyagarajan: None; A. RA SLE Network: None; Z. Shiekh: None; S. Compeyrot-Lacassagne: None; S. Chippington: None; M. Coles: None; E. Al-Abadi: None; A. Filer: Bristol-Myers Squibb(BMS), 5, GlaxoSmithKlein(GSK), 5, Janssen, 5, Nascient, 5, Sonoma Biotherapeutics, 2; T. Inflammatory Arthritis (TRICIA) Consortium: None; L. Wedderburn: AbbVie/Abbott, 5, GlaxoSmithKlein(GSK), 5, Pfizer, 1, SOBI, 5, UCB, 5; A. Croft: None.
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