Single-cell analysis reveals fibroblast heterogeneity and myofibroblasts in systemic sclerosis-associated interstitial lung disease

Objectives: Myofibroblasts are key effector cells in the extracellular matrix remodeling 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 4 healthy control and 4 SSc-ILD patients, with findings confirmed by cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) in additional samples. Results: Examination of gene expression in mesenchymal cells identified two major, SPINT2hiand 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 amongst 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 unrecognized 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 pathologic mechanisms driving fibrosis in SSc-ILD.

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