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Single-cell analysis reveals fibroblast heterogeneity and myofibroblasts in systemic sclerosis-associated interstitial lung disease
  1. Eleanor Valenzi1,
  2. Melissa Bulik2,
  3. Tracy Tabib3,
  4. Christina Morse3,
  5. John Sembrat1,
  6. Humberto Trejo Bittar4,
  7. Mauricio Rojas1,
  8. Robert Lafyatis3
  1. 1 Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  2. 2 Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  3. 3 Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  4. 4 Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  1. Correspondence to Dr Eleanor Valenzi,Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; valenzie{at}upmc.edu

Abstract

Objectives Myofibroblasts are key effector cells in the extracellular matrix remodelling of systemic sclerosis-associated interstitial lung disease (SSc-ILD); however, the diversity of fibroblast populations present in the healthy and SSc-ILD lung is unknown and has prevented the specific study of the myofibroblast transcriptome. We sought to identify and define the transcriptomes of myofibroblasts and other mesenchymal cell populations in human healthy and SSc-ILD lungs to understand how alterations in fibroblast phenotypes lead to SSc-ILD fibrosis.

Methods We performed droplet-based, single-cell RNA-sequencing with integrated canonical correlation analysis of 13 explanted lung tissue specimens (56 196 cells) from four healthy control and four patients with SSc-ILD, with findings confirmed by cellular indexing of transcriptomes and epitopes by sequencing in additional samples.

Results Examination of gene expression in mesenchymal cells identified two major, SPINT2hi and MFAP5hi, and one minor, WIF1hi, fibroblast populations in the healthy control lung. Combined analysis of control and SSc-ILD mesenchymal cells identified SPINT2hi, MFAP5hi, few WIF1hi fibroblasts and a new large myofibroblast population with evidence of actively proliferating myofibroblasts. We compared differential gene expression between all SSc-ILD and control mesenchymal cell populations, as well as among the fibroblast subpopulations, showing that myofibroblasts undergo the greatest phenotypic changes in SSc-ILD and strongly upregulate expression of collagens and other profibrotic genes.

Conclusions Our results demonstrate previously unrecognised fibroblast heterogeneity in SSc-ILD and healthy lungs, and define multimodal transcriptome-phenotypes associated with these populations. Our data indicate that myofibroblast differentiation and proliferation are key pathological mechanisms driving fibrosis in SSc-ILD.

  • systemic sclerosis
  • fibroblast
  • pulmonary fibrosis
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Footnotes

  • Handling editor Professor Josef S Smolen

  • Contributors RL and EV conceived the project. EV wrote the manuscript and RL edited the manuscript. JS and MR provided patient samples. TT, CM and EV performed the experiments, and TT performed the single-cell RNA-seq. EV performed the RNA-seq analysis. HTB performed the histological assessment of samples. MB and CM performed the immunological staining. All authors provided editorial commentary of the manuscript.

  • Funding Research reported in this publication was supported by the National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases under award number 2P50AR060780 (RL) and the National Heart, Lung, and Blood Institute under award numbers R01HL123766 (RL) and 2T32HL007563-31 (EV). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • Competing interests RL has received consulting fees from PRISM BioLab, Merck, Bristol Myers Squibb, Biocon, Formation, Genentech/Roche, UCB and Sanofi, and grant support from Elpidera, Kiniksa and Regeneron, outside the submitted work.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement For scRNA-seq data, raw counts in sparse matrix format for all samples are available at the public, open access repository Gene Exppresion Omnibus (GEO)- GSE 128169.

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