Single Cell Transcriptomics and Flow Cytometry Reveal Disease-associated Fibroblast Subsets in Rheumatoid Arthritis

Fibroblasts mediate normal tissue matrix remodeling, but they can cause fibrosis or tissue destruction following chronic inflammation. In rheumatoid arthritis (RA), synovial fibroblasts expand, degrade cartilage, and drive joint inflammation. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to disease pathology. Here, we used an integrative strategy, including bulk transcriptomics on targeted subpopulations and unbiased single-cell transcriptomics, to analyze fibroblasts from synovial tissues. We identify 7 phenotypic fibroblast subsets with distinct surface protein phenotypes, and these collapsed into 3 subsets based on transcriptomics data. One subset expressing PDPN, THY1, but lacking CD34 was 3-fold expanded in RA relative to osteoarthritis (P=0.007); most of these cells expressed CDH11. The subsets were found to differ in expression of cytokines and matrix metalloproteinases, localization in synovial microanatomy, and in response to TNF. Our approach provides a template to identify pathogenic stromal cellular subsets in complex diseases.

[1]  Kamil Slowikowski,et al.  Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis , 2017, Nature.

[2]  Lior Pachter,et al.  Near-optimal probabilistic RNA-seq quantification , 2016, Nature Biotechnology.

[3]  J. Mesirov,et al.  The Molecular Signatures Database Hallmark Gene Set Collection , 2015 .

[4]  Hiroshi Takayanagi,et al.  Inhibition of the TNF Family Cytokine RANKL Prevents Autoimmune Inflammation in the Central Nervous System. , 2015, Immunity.

[5]  S. Turley,et al.  Immunological hallmarks of stromal cells in the tumour microenvironment , 2015, Nature Reviews Immunology.

[6]  Sridhar Ramaswamy,et al.  RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance , 2015, Science.

[7]  G. Kollias,et al.  RANKL expressed on synovial fibroblasts is primarily responsible for bone erosions during joint inflammation , 2015, Annals of the Rheumatic Diseases.

[8]  S. Acton,et al.  Lymph node fibroblastic reticular cells in health and disease , 2015, Nature Reviews Immunology.

[9]  Don C Rockey,et al.  Fibrosis--a common pathway to organ injury and failure. , 2015, The New England journal of medicine.

[10]  A. Filer,et al.  The role of the synovial fibroblast in rheumatoid arthritis pathogenesis , 2015, Current opinion in rheumatology.

[11]  Matthew E. Ritchie,et al.  limma powers differential expression analyses for RNA-sequencing and microarray studies , 2015, Nucleic acids research.

[12]  Shawn M. Gillespie,et al.  Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma , 2014, Science.

[13]  A. Gibofsky.  Epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis: A Synopsis. , 2014, The American journal of managed care.

[14]  P. Kharchenko,et al.  Bayesian approach to single-cell differential expression analysis , 2014, Nature Methods.

[15]  Tom C. Freeman,et al.  Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation , 2014, Immunity.

[16]  Åsa K. Björklund,et al.  Full-length RNA-seq from single cells using Smart-seq2 , 2014, Nature Protocols.

[17]  D. Mooney,et al.  Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks , 2012, Nature Immunology.

[18]  G. Watts,et al.  Modulation of matrix metalloproteinase production by rheumatoid arthritis synovial fibroblasts after cadherin 11 engagement. , 2011, Arthritis and rheumatism.

[19]  S. Marsal,et al.  Synovial fibroblast hyperplasia in rheumatoid arthritis: clinicopathologic correlations and partial reversal by anti-tumor necrosis factor therapy. , 2011, Arthritis and rheumatism.

[20]  S. Turley,et al.  Reproducible Isolation of Lymph Node Stromal Cells Reveals Site-Dependent Differences in Fibroblastic Reticular Cells , 2011, Front. Immun..

[21]  Soo Young Lee,et al.  Cadherin-11 regulates fibroblast inflammation , 2011, Proceedings of the National Academy of Sciences.

[22]  N. Karlsson,et al.  The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis , 2011, Arthritis Research & Therapy.

[23]  A. Filer,et al.  Utility of ultrasound joint counts in the prediction of rheumatoid arthritis in patients with very early synovitis , 2010, Annals of the rheumatic diseases.

[24]  K. Nishida,et al.  Inflammatory cytokine-induced expression of vasohibin-1 by rheumatoid synovial fibroblasts. , 2009, Acta medica Okayama.

[25]  S. Vukicevic,et al.  BMP-6 and mesenchymal stem cell differentiation. , 2009, Cytokine & growth factor reviews.

[26]  M. Takeichi,et al.  Cadherin-11 in Synovial Lining Formation and Pathology in Arthritis , 2007, Science.

[27]  U. Müller-Ladner,et al.  Microarrays demystified. , 2004, Environmental health perspectives.

[28]  G. Burmester,et al.  Synovitis score: discrimination between chronic low‐grade and high‐grade synovitis , 2006, Histopathology.

[29]  M. Brenner,et al.  Cadherin-11 induces rheumatoid arthritis fibroblast-like synoviocytes to form lining layers in vitro. , 2006, The American journal of pathology.

[30]  Franz Kainberger,et al.  Musculoskeletal ultrasound including definitions for ultrasonographic pathology. , 2005, The Journal of rheumatology.

[31]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Reimund,et al.  A comparative study of endothelial cell markers expressed in chronically inflamed human tissues: MECA‐79, Duffy antigen receptor for chemokines, von Willebrand factor, CD31, CD34, CD105 and CD146 , 2005, The Journal of pathology.

[33]  D. Patel,et al.  Cadherin-11 Provides Specific Cellular Adhesion between Fibroblast-like Synoviocytes , 2004, The Journal of experimental medicine.

[34]  G. Firestein.  Evolving concepts of rheumatoid arthritis , 2003, Nature.

[35]  N. Bottini,et al.  Duality of fibroblast-like synoviocytes in RA: passive responders and imprinted aggressors , 2013, Nature Reviews Rheumatology.

[36]  Yoshiro Kobayashi.  The role of chemokines in neutrophil biology. , 2008, Frontiers in bioscience : a journal and virtual library.