title: Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.
abstract: BACKGROUND
  Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described.
  
  
  OBJECTIVE
  We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes.
  
  
  METHODS
  Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE.
  
  
  RESULTS
  Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.
  
  
  CONCLUSIONS
  Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif.
authors.forename: William E
authors.initials: WE
authors.lastname: O'Gorman
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif; Department of Pediatrics, Division of Allergy, Immunology and Rheumatology, Stanford University, Stanford, Calif.
authors.forename: Elena W Y
authors.initials: EW
authors.lastname: Hsieh
authors.affiliation: Cancer Biology Program, Stanford University, Stanford, Calif.
authors.forename: Erica S
authors.initials: ES
authors.lastname: Savig
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif.
authors.forename: Pier Federico
authors.initials: PF
authors.lastname: Gherardini
authors.affiliation: Department of Pediatrics, Division of Allergy, Immunology and Rheumatology, Stanford University, Stanford, Calif; Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Hernandez
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif.
authors.forename: Leo
authors.initials: L
authors.lastname: Hansmann
authors.affiliation: Department of Pediatrics, Division of Allergy, Immunology and Rheumatology, Stanford University, Stanford, Calif.
authors.forename: Imelda M
authors.initials: IM
authors.lastname: Balboni
authors.affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University, Stanford, Calif; Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, Calif.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Utz
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Bendall
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif; Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Stanford University, Stanford, Calif.
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Fantl
authors.affiliation: Department of Pediatrics, Division of Allergy, Immunology and Rheumatology, Stanford University, Stanford, Calif.
authors.forename: David B
authors.initials: DB
authors.lastname: Lewis
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif; Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, Calif. Electronic address: gnolan@stanford.edu.
authors.forename: Garry P
authors.initials: GP
authors.lastname: Nolan
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, Calif; Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, Calif; Howard Hughes Medical Institute, Stanford University, Stanford, Calif. Electronic address: mmdavis@stanford.edu.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Davis
mesh_terms: D002478:Cells, Cultured; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology*; D008213:Lymphocyte Activation; D009000:Monocytes / Q000276:immunology*; D016328:NF-kappa B / Q000378:metabolism; D009928:Organ Specificity; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology*; D051193:Toll-Like Receptors / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D018925:Chemokines; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; D051193:Toll-Like Receptors
keywords: Mass cytometry; Toll-like receptors; inflammation; monocyte chemotactic protein 1; monocytes; systemic lupus erythematosus