Defining Inflammatory Cell States in Rheumatoid Arthritis Joint Synovial Tissues by Integrating Single-cell Transcriptomics and Mass Cytometry

To define the cell populations in rheumatoid arthritis (RA) driving joint inflammation, we applied single-cell RNA-seq (scRNA-seq), mass cytometry, bulk RNA-seq, and flow cytometry to sorted T cells, B cells, monocytes, and fibroblasts from 51 synovial tissue RA and osteoarthritis (OA) patient samples. Utilizing an integrated computational strategy based on canonical correlation analysis to 5,452 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics together revealed cell states expanded in RA synovia: THY1+HLAhigh sublining fibroblasts (OR=33.8), IL1B+ pro-inflammatory monocytes (OR=7.8), CD11c+T-bet+ autoimmune-associated B cells (OR=5.7), and PD-1+Tph/Tfh (OR=3.0). We also defined CD8+ T cell subsets characterized by GZMK+, GZMB+, and GNLY+ expression. Using bulk and single-cell data, we mapped inflammatory mediators to source cell populations, for example attributing IL6 production to THY1+HLAhigh fibroblasts and naïve B cells, and IL1B to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

[1]  Kevin Wei,et al.  Autocrine Loop Involving IL‐6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators , 2017, Immunity.

[2]  A. Rudensky,et al.  Regulatory T cells: mechanisms of differentiation and function. , 2012, Annual review of immunology.

[3]  C. Wijbrandts,et al.  Synovial tissue heterogeneity in rheumatoid arthritis in relation to disease activity and biomarkers in peripheral blood. , 2010, Arthritis and rheumatism.

[4]  M. Monestier,et al.  Efficient Clearance of Early Apoptotic Cells by Human Macrophages Requires M2c Polarization and MerTK Induction , 2012, The Journal of Immunology.

[5]  J. Mesirov,et al.  The Molecular Signatures Database (MSigDB) hallmark gene set collection. , 2015, Cell systems.

[6]  Annette Lee,et al.  Fine-mapping identifies causal variants for RA and T1D in DNASE1L3, SIRPG, MEG3, TNFAIP3 and CD28/CTLA4 loci , 2017, bioRxiv.

[7]  Edda Klipp,et al.  Estimation of immune cell content in tumour tissue using single-cell RNA-seq data , 2017 .

[8]  G. Kolarz,et al.  Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction , 2004, Arthritis research & therapy.

[9]  Michael J. Davis,et al.  Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes. , 2007, Arthritis and rheumatism.

[10]  J. Anolik,et al.  B cells in the pathogenesis and treatment of rheumatoid arthritis , 2010, Current opinion in rheumatology.

[11]  M. Schaub,et al.  SC3 - consensus clustering of single-cell RNA-Seq data , 2016, Nature Methods.

[12]  Andrew Butler,et al.  Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation , 2018, Nature Communications.

[13]  F. Wolfe,et al.  The mortality of rheumatoid arthritis. , 1994, Arthritis and rheumatism.

[14]  G. Tsokos,et al.  Signaling Lymphocytic Activation Molecule Family Member 7 Engagement Restores Defective Effector CD8+ T Cell Function in Systemic Lupus Erythematosus , 2017, Arthritis & rheumatology.

[15]  Erika H. Noss,et al.  The role and therapeutic implications of fibroblast‐like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis , 2008, Immunological reviews.

[16]  Juan Carlos Fernández,et al.  Multiobjective evolutionary algorithms to identify highly autocorrelated areas: the case of spatial distribution in financially compromised farms , 2014, Ann. Oper. Res..

[17]  Sean C. Bendall,et al.  Normalization of mass cytometry data with bead standards , 2013, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[18]  G. Burmester,et al.  Macrophages in rheumatoid arthritis , 2000, Arthritis research.

[19]  Annette Lee,et al.  Fine-mapping and functional studies highlight potential causal variants for rheumatoid arthritis and type 1 diabetes , 2018, Nature Genetics.

[20]  I Petersen,et al.  Grading of chronic synovitis--a histopathological grading system for molecular and diagnostic pathology. , 2002, Pathology, research and practice.

[21]  Thomas Lengauer,et al.  ROCR: visualizing classifier performance in R , 2005, Bioinform..

[22]  H. Jäck,et al.  Stages of Germinal Center Transit Are Defined by B Cell Transcription Factor Coexpression and Relative Abundance , 2006, The Journal of Immunology.

[23]  R. Darnell,et al.  Identification of Three Rheumatoid Arthritis Disease Subtypes by Machine Learning Integration of Synovial Histologic Features and RNA Sequencing Data , 2018, Arthritis & rheumatology.

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

[25]  R. Irizarry,et al.  Missing data and technical variability in single‐cell RNA‐sequencing experiments , 2018, Biostatistics.

[26]  J. Kriegsmann,et al.  Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. , 1996, The American journal of pathology.

[27]  I. McInnes,et al.  The role of interleukin–15 in T–cell migration and activation in rheumatoid arthritis , 1996, Nature Medicine.

[28]  Shuqiang Li,et al.  High dimensional analyses of cells dissociated from cryopreserved synovial tissue , 2018, bioRxiv.

[29]  S. Pillai.  Now you know your ABCs. , 2011, Blood.

[30]  S. Raychaudhuri,et al.  Mixed Effects Association of Single Cells Identifies an Expanded Th1-Skewed Cytotoxic Effector CD4+ T Cell Subset in Rheumatoid Arthritis , 2018 .

[31]  Matthew Stephens,et al.  Visualizing the structure of RNA-seq expression data using grade of membership models , 2016, bioRxiv.

[32]  Stephen Kelly,et al.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium , 2009, PLoS medicine.

[33]  J. Smolen.  Pharmacotherapy: How well can we compare different biologic agents for RA? , 2010, Nature Reviews Rheumatology.

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

[35]  E. Lee,et al.  Tofacitinib versus methotrexate in rheumatoid arthritis. , 2014, The New England journal of medicine.

[36]  K. Roskin,et al.  Defining antigen-specific plasmablast and memory B cell subsets in blood following viral infection and vaccination of humans , 2016, Nature Immunology.

[37]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[38]  P. Nilsson,et al.  Variability in synovial inflammation in rheumatoid arthritis investigated by microarray technology , 2006, Arthritis research & therapy.

[39]  M. Barbe,et al.  Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo , 2016, Journal of cellular physiology.

[40]  S. Kelly,et al.  New learnings on the pathophysiology of RA from synovial biopsies , 2013, Current opinion in rheumatology.

[41]  I. McInnes,et al.  Cytokine networks—towards new therapies for rheumatoid arthritis , 2005, Nature Clinical Practice Rheumatology.

[42]  D. Tritchler,et al.  Sparse Canonical Correlation Analysis with Application to Genomic Data Integration , 2009, Statistical applications in genetics and molecular biology.

[43]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[44]  Jeff A. Bilmes,et al.  Deep Canonical Correlation Analysis , 2013, ICML.

[45]  H. Khalak,et al.  Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus , 2011, Nature Genetics.

[46]  Edda Klipp,et al.  Estimation of immune cell content in tumour tissue using single-cell RNA-seq data , 2017, Nature Communications.

[47]  U. Wagner,et al.  Functional subsets of CD4 T cells in rheumatoid synovitis. , 1998, Arthritis and rheumatism.

[48]  Song Jiang,et al.  TLR10 Is a B Cell Intrinsic Suppressor of Adaptive Immune Responses , 2017, The Journal of Immunology.

[49]  Paul G. Thomas,et al.  Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination , 2022 .

[50]  Shuqiang Li,et al.  CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq , 2016, Genome Biology.

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

[52]  P. Marrack,et al.  CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs , 2015, The Journal of Immunology.

[53]  Huda Khayrallah,et al.  Deep Generalized Canonical Correlation Analysis , 2017, RepL4NLP@ACL.

[54]  J. Kremer,et al.  A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. , 1999, The New England journal of medicine.

[55]  N. Hacohen,et al.  Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors , 2017, Science.

[56]  A. Carr,et al.  Dickkopf-3 is upregulated in osteoarthritis and has a chondroprotective role , 2016, Osteoarthritis and cartilage.

[57]  R. Satija,et al.  Single-cell RNA sequencing to explore immune cell heterogeneity , 2017, Nature Reviews Immunology.

[58]  S. Rahman,et al.  IL-21 drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+ B cells in SLE , 2018, Nature Communications.

[59]  S. Snelling,et al.  DKK3 CAN PREVENT CARTILAGE DEGRADATION AND MODULATE TGFBETA AND WNT SIGNALLING , 2012 .

[60]  Vanessa M. Peterson,et al.  Multiplexed quantification of proteins and transcripts in single cells , 2017, Nature Biotechnology.

[61]  J. Langhoff‐Roos.  State‐of‐the‐art review , 2016, Acta obstetricia et gynecologica Scandinavica.

[62]  Nir Hacohen,et al.  Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis , 2018, Nature Communications.

[63]  S. Raychaudhuri,et al.  Reverse Association of Single Cells to Rheumatoid Arthritis Accounting for Mixed Effects Identifies an Expanded CD27- HLA-DR+ Effector Memory CD4+ T Cell Population , 2017, bioRxiv.

[64]  D. Massart,et al.  The Mahalanobis distance , 2000 .

[65]  B. Volpe,et al.  XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development , 2009, The Journal of experimental medicine.

[66]  Georg Schett,et al.  The pathogenesis of rheumatoid arthritis. , 2011, The New England journal of medicine.

[67]  S. Teichmann,et al.  Exponential scaling of single-cell RNA-seq in the past decade , 2017, Nature Protocols.

[68]  F. Breedveld,et al.  Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis. , 2001, Arthritis and rheumatism.

[69]  N. McGovern,et al.  A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures. , 2016, Immunity.

[70]  R. Tibshirani,et al.  A penalized matrix decomposition, with applications to sparse principal components and canonical correlation analysis. , 2009, Biostatistics.

[71]  Beatriz de la Iglesia,et al.  Clustering Rules: A Comparison of Partitioning and Hierarchical Clustering Algorithms , 2006, J. Math. Model. Algorithms.

[72]  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.

[73]  David A. Knowles,et al.  Batch effects and the effective design of single-cell gene expression studies , 2016, bioRxiv.

[74]  Michael Poidinger,et al.  High-dimensional analysis of the murine myeloid cell system , 2014, Nature Immunology.

[75]  David A. Knowles,et al.  Batch effects and the effective design of single-cell gene expression studies , 2016, Scientific Reports.

[76]  Paul Hoffman,et al.  Integrating single-cell transcriptomic data across different conditions, technologies, and species , 2018, Nature Biotechnology.

[77]  D. Fox,et al.  T cell subsets and their role in the pathogenesis of rheumatic disease , 2014, Current opinion in rheumatology.

[78]  P. Marrack,et al.  Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c⁺ B-cell population is important for the development of autoimmunity. , 2011, Blood.

[79]  P. Tak,et al.  Synovial tissue research: a state-of-the-art review , 2017, Nature Reviews Rheumatology.

[80]  Shuqiang Li,et al.  Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue , 2018, Arthritis Research & Therapy.

[81]  Grace X. Y. Zheng,et al.  Massively parallel digital transcriptional profiling of single cells , 2016, Nature Communications.

[82]  Alain Baccini,et al.  CCA: An R Package to Extend Canonical Correlation Analysis , 2008 .

[83]  Ximeng Zheng.  FOR RNA-SEQ DATA , 2012 .

[84]  A. Regev,et al.  Spatial reconstruction of single-cell gene expression , 2015, Nature Biotechnology.

[85]  Sean C. Bendall,et al.  Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum , 2011, Science.

[86]  R. Jessberger,et al.  Regulation of Age-associated B cells by IRF5 in systemic autoimmunity , 2018, Nature Immunology.

[87]  Bo Wang,et al.  Visualization and analysis of single-cell RNA-seq data by kernel-based similarity learning , 2016, Nature Methods.

[88]  Kamil Slowikowski,et al.  Mixed-effects association of single cells identifies an expanded effector CD4+ T cell subset in rheumatoid arthritis , 2018, Science Translational Medicine.

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

[90]  P. Rousseeuw.  Silhouettes: a graphical aid to the interpretation and validation of cluster analysis , 1987 .

[91]  S. Kummerfeld,et al.  Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics , 2014, Arthritis Research & Therapy.

[92]  Thomas Pap,et al.  Arthritis , 2000, Nature Biotechnology.

[93]  M. Stephens,et al.  Visualizing the structure of RNA-seq expression data using grade of membership models , 2017, PLoS genetics.

[94]  G. Gibson,et al.  Distinct Effector B Cells Induced by Unregulated Toll‐like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus , 2018, Immunity.

[95]  A. Filer.  The fibroblast as a therapeutic target in rheumatoid arthritis. , 2013, Current opinion in pharmacology.

[96]  B. Bresnihan,et al.  Synovial tissue macrophage populations and articular damage in rheumatoid arthritis. , 1996, Arthritis and rheumatism.

[97]  Garry P Nolan,et al.  Single-cell Mass Cytometry for Analysis of Immune System Functional States This Review Comes from a Themed Issue on Host Pathogens Basic Concepts of Mass Cytometry , 2022 .

[98]  Robert M. Plenge,et al.  Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis , 2011, Nature Genetics.