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Single Cell RNA-Seq Analysis of Idiopathic Pulmonary Fibrosis Reveals Alterations in Lung Cell Populations and in Selective Cell Expression of the Biomarkers MMP-7, CCL-18, and MUC1
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A4356 - Single Cell RNA-Seq Analysis of Idiopathic Pulmonary Fibrosis Reveals Alterations in Lung Cell Populations and in Selective Cell Expression of the Biomarkers MMP-7, CCL-18, and MUC1
Author Block: E. Valenzi1, T. Tabib2, C. Morse2, M. Rojas1, A. L. Mora1, R. Lafyatis2; 1Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, United States.
RATIONALE: Many biomarkers have been proposed in idiopathic pulmonary fibrosis (IPF) for diagnosis, prognostication, and treatment response. To date, the cells of origin for these markers have been described primarily by immunohistologic imaging and sequencing of in vitro cell cultures. In this study, we utilize single cell RNA-seq to confirm the origin cell identities for matrix metalloproteinase 7 (MMP7), CC motif chemokine ligand 18 (CCL18), and human transmembrane mucin 1(MUC1), as well as explore the shifting cell populations in IPF upper and lower lobes compared to normal controls.
METHODS: Deidentified lung tissue was obtained from upper lobe and lower lobes of patients with IPF (n=2) at the time of transplantation. Lung tissue from adult donors designated unsuitable for lung transplant were used as normal controls (n=2). A droplet-based instrument (10X Genomics) was used to create single cell gel bead-in-emulsions, followed by library preparation and RNA sequencing. Transcripts were mapped, assigned to individual cells by barcodes using Cell Ranger, and analyzed using Seurat. Cell populations were identified with t-distributed stochastic neighbor embedding and clustering, followed by examination of gene markers.
RESULTS: We examined gene expression in a total of 30,865 cells. Average gene expression of MMP7 was upregulated 29.36 fold in club cells, 17.89 fold in goblet cells, and 5.45 fold in basal cells of patients with IPF, compared to gene expression in normal controls. CCL18 was upregulated 2.75 fold in macrophages of patients with IPF compared to normals. MUC1 was upregulated 4.19 fold in type I pneumocytes, 1.85 fold in type II pneumocytes (AT2), 1.91 fold in club cells, and 1.33 fold in ciliated cells of patients with IPF compared to normals. Macrophage numbers were similar between normal control lungs (26.56%), IPF upper lobes (45.5%), and IPF lower lobes (30.26%). AT2 cells decreased dramatically in IPF lower lobes (0.30%) compared to normals (5.11%), and IPF upper lobes (4.09%). In contrast, fibroblasts dramatically increased from 2.08% of cells in normals to 4.50% of cells in IPF upper lobes, and 13.54% of cells in IPF lower lobes. Significant changes were also seen in the populations of natural killer, ciliated, endothelial, mast, and basal cells between groups.
CONCLUSIONS: Our data demonstrate that biomarkers associated with progressive IPF likely indicate pathogenic processes from several different cell types in the lung, suggesting that AT2 cells, macrophages, and fibroblasts all play important roles in pathogenesis.
Author Block: E. Valenzi1, T. Tabib2, C. Morse2, M. Rojas1, A. L. Mora1, R. Lafyatis2; 1Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, United States.
RATIONALE: Many biomarkers have been proposed in idiopathic pulmonary fibrosis (IPF) for diagnosis, prognostication, and treatment response. To date, the cells of origin for these markers have been described primarily by immunohistologic imaging and sequencing of in vitro cell cultures. In this study, we utilize single cell RNA-seq to confirm the origin cell identities for matrix metalloproteinase 7 (MMP7), CC motif chemokine ligand 18 (CCL18), and human transmembrane mucin 1(MUC1), as well as explore the shifting cell populations in IPF upper and lower lobes compared to normal controls.
METHODS: Deidentified lung tissue was obtained from upper lobe and lower lobes of patients with IPF (n=2) at the time of transplantation. Lung tissue from adult donors designated unsuitable for lung transplant were used as normal controls (n=2). A droplet-based instrument (10X Genomics) was used to create single cell gel bead-in-emulsions, followed by library preparation and RNA sequencing. Transcripts were mapped, assigned to individual cells by barcodes using Cell Ranger, and analyzed using Seurat. Cell populations were identified with t-distributed stochastic neighbor embedding and clustering, followed by examination of gene markers.
RESULTS: We examined gene expression in a total of 30,865 cells. Average gene expression of MMP7 was upregulated 29.36 fold in club cells, 17.89 fold in goblet cells, and 5.45 fold in basal cells of patients with IPF, compared to gene expression in normal controls. CCL18 was upregulated 2.75 fold in macrophages of patients with IPF compared to normals. MUC1 was upregulated 4.19 fold in type I pneumocytes, 1.85 fold in type II pneumocytes (AT2), 1.91 fold in club cells, and 1.33 fold in ciliated cells of patients with IPF compared to normals. Macrophage numbers were similar between normal control lungs (26.56%), IPF upper lobes (45.5%), and IPF lower lobes (30.26%). AT2 cells decreased dramatically in IPF lower lobes (0.30%) compared to normals (5.11%), and IPF upper lobes (4.09%). In contrast, fibroblasts dramatically increased from 2.08% of cells in normals to 4.50% of cells in IPF upper lobes, and 13.54% of cells in IPF lower lobes. Significant changes were also seen in the populations of natural killer, ciliated, endothelial, mast, and basal cells between groups.
CONCLUSIONS: Our data demonstrate that biomarkers associated with progressive IPF likely indicate pathogenic processes from several different cell types in the lung, suggesting that AT2 cells, macrophages, and fibroblasts all play important roles in pathogenesis.
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