Differential Transcriptomic Signatures of Small Airway Cell Cultures Derived from IPF and COVID-19-Induced Exacerbation of Interstitial Lung Disease
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Lung Explant Samples
2.2. Isolation and Propagation of Distal Small Airways
2.3. RNA Isolation and Sequencing
2.4. Differential Expression Analysis
2.5. cDNA Synthesis and Real-Time PCR
2.6. Protein Extraction and Western Blotting
2.7. Immunohistochemistry
2.7.1. Immunostaining and Quantification of Patient-Derived Cell Cultures for Epithelial Cell Markers
2.7.2. Immunofluorescent Detection of pSMAD1/5/8 in Patient Lung Tissue
3. Results
3.1. Patient-Derived Small Airway Cell Cultures Maintain Cell Markers and Characteristics Found in Fibrotic Lung Tissue
Patient-Derived Small Airway Cell Cultures Show Evidence of Ciliated Cells
3.2. Determination of IPF Transcriptomic Signature
3.2.1. RNA-Sequencing of IPF and Non-IPF Control Small Airway Cells Demonstrate Unique Transcriptomic Signatures
3.2.2. Analyzing the Canonical Pathways in Small Airway Cultures of IPF Compared to Non-IPF Controls Reveals a Suppression of Pathways Associated with IPF
3.2.3. Upstream Regulators Associated with Common IPF Genes Predict the Inhibition of the TGF-β1-Signaling Pathway and Host Immune Response
3.2.4. Gene Ontology Results for Common IPF Genes Show Activation of Ciliary Movement and Cell Cycle Regulation and a Downregulation of Genes Associated with ECM Components and Immune Response
3.2.5. Transcriptomic Signatures of Cultured IPF Patient Cells Reflect Gene Expression Changes Seen in Single-Cell RNA Sequencing Data
3.2.6. RT-PCR Shows an Increase in FOXM1 in the IPF Cell Cultures
3.3. Determination of Post-COVID Fibrosis Transcriptomic Signature
3.3.1. Gene Expression Differences Are Observed between Post-COVID Fibrosis Cells and Non-IPF Control Small Airway Cells Cultured from Human Patients
3.3.2. Ingenuity Pathway Analysis of Post-COVID Fibrosis vs. Non-IPF Control Small Airway Cultures Demonstrates a Trend toward Host Immune Response and Interferon Signaling
3.3.3. Upstream Regulator Results for Post-COVID Fibrosis Common Genes Show Predicted Activation of the Immune Response but Not Cytokine-Related Molecules
3.3.4. Post-COVID Fibrosis Gene Ontology Results Show Activation of Host Immune Response and Decrease in Cilium Movement and DNA Repair
3.4. Identification of Crucial Differences between IPF and Post-COVID Fibrosis Small Airway Cell Cultures
3.4.1. Differential Gene Expression Analysis of IPF vs. Post-COVID Fibrosis Shows Common Genes between Baseline Cultures and TGF-β1 Culture Conditions
3.4.2. Reactome Pathway Results for the IPF vs. Post-COVID Fibrosis Common Genes Show Upregulation in Interferon Signaling, Microtubule Organization, and Cell Cycle in the IPF Cell Cultures Compared to the Post-COVID Fibrosis
3.4.3. Reactome Pathway Results for the IPF vs. Post-COVID Fibrosis Common Genes Show Downregulation in Extracellular Matrix Assembly, Cell-Surface Interactions, and Integrin Signaling in the IPF Cell Cultures
3.4.4. Comparing Gene Expression in Small Airway Cell Cultures from IPF and Post-COVID Fibrosis Reveals Distinctions in Baseline TGF-β1 Signaling
3.4.5. Differences in BMP Signaling Are Observed between Post-COVID Fibrosis and IPF Cell Cultures
3.4.6. pSMAD1/5/8 Expression and Nuclear Localization in Normal, IPF, and Post-COVID Fibrosis Tissues
4. Discussion
4.1. Patient-Derived Small Airway Cell Cultures Maintain Tissue-Specific Markers and Reflect Gene Expression Patterns Observed in Fibrotic Lung Tissue
4.2. Transcriptomic Signatures of IPF Cell Cultures Include the Activation of Cell Cycle Regulators, the Upregulation of Ciliary Movement, and the Predicted Inhibition of the Pulmonary Fibrosis Idiopathic and TGF-β1 Signaling Pathways
4.3. The Disease Signature of Post-COVID Fibrosis Illustrates a Distinct Interplay of Activation and Inhibition within Components of the Immune System
4.4. Comparison of IPF vs. Post-COVID Fibrosis DEGs Highlights Mucociliary Dysfunction in Post-COVID Fibrosis Cell Cultures Compared to IPF
4.5. IPF Cell Cultures Demonstrate a Decrease in Extracellular Matrix-Associated Pathways and Collagen Fiber Biosynthesis Compared to Post-COVID Fibrosis Cell Cultures
4.6. IPF Small Airway Cell Cultures Demonstrate Lower Levels of Baseline TGF-β1 Signaling
4.7. Higher Levels of BMP Signaling in IPF Samples Decrease TGF-β1 Signaling and Contribute to the Predicted Inhibition of Key Fibrosis-Related Pathways
5. Study Limitations
6. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ILD | Interstitial lung disease |
IPF | Idiopathic pulmonary fibrosis |
COPD | Chronic obstructive pulmonary disease |
SARS-CoV-2, COVID-19 | Severe acute respiratory syndrome coronavirus 2 |
TGF-β1 | Transforming growth factor beta |
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Uhl, K.; Paithankar, S.; Leshchiner, D.; Jager, T.E.; Abdelgied, M.; Dixit, B.; Marashdeh, R.; Luo-Li, D.; Tripp, K.; Peraino, A.M.; et al. Differential Transcriptomic Signatures of Small Airway Cell Cultures Derived from IPF and COVID-19-Induced Exacerbation of Interstitial Lung Disease. Cells 2023, 12, 2501. https://doi.org/10.3390/cells12202501
Uhl K, Paithankar S, Leshchiner D, Jager TE, Abdelgied M, Dixit B, Marashdeh R, Luo-Li D, Tripp K, Peraino AM, et al. Differential Transcriptomic Signatures of Small Airway Cell Cultures Derived from IPF and COVID-19-Induced Exacerbation of Interstitial Lung Disease. Cells. 2023; 12(20):2501. https://doi.org/10.3390/cells12202501
Chicago/Turabian StyleUhl, Katie, Shreya Paithankar, Dmitry Leshchiner, Tara E. Jager, Mohamed Abdelgied, Bhavna Dixit, Raya Marashdeh, Dewen Luo-Li, Kaylie Tripp, Angela M. Peraino, and et al. 2023. "Differential Transcriptomic Signatures of Small Airway Cell Cultures Derived from IPF and COVID-19-Induced Exacerbation of Interstitial Lung Disease" Cells 12, no. 20: 2501. https://doi.org/10.3390/cells12202501