Single-cell multiomic analysis of mesenchymal cells reveals molecular signatures and regulators of lung allograft fibrosis
Lu Lu, A. Patrick McLinden, Natalie M. Walker, Ragini Vittal, Yichen Wang, Fatemeh Fattahi, Stephen T. Russell, Michael P. Combs, Joshua D. Welch, Vibha N. Lama
Lu Lu, A. Patrick McLinden, Natalie M. Walker, Ragini Vittal, Yichen Wang, Fatemeh Fattahi, Stephen T. Russell, Michael P. Combs, Joshua D. Welch, Vibha N. Lama
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Research Article Genetics Pulmonology

Single-cell multiomic analysis of mesenchymal cells reveals molecular signatures and regulators of lung allograft fibrosis

Abstract

Survival after lung transplantation is limited by chronic, progressive graft failure, termed chronic lung allograft dysfunction (CLAD). Graft-resident mesenchymal cells (MCs) drive CLAD pathogenesis and exhibit stable dysregulated signaling, yet the transcriptomic and epigenomic drivers underlying this fibrogenic transformation remain elusive. We used single-cell multiomic profiling to characterize gene expression and chromatin accessibility in MCs isolated from bronchoalveolar lavage fluid of lung transplant recipients with and without CLAD, collected early after transplantation or after disease onset. MCs obtained after CLAD onset demonstrated a distinct transcriptomic signature compared with non-CLAD controls, enabling classification of disease status at the single-cell level with greater than 98% accuracy using signature genes. Chromatin accessibility analyses identified enrichment of CCAAT-enhancer-binding protein family transcription factors, specifically CEBPD, in CLAD MCs. MCs early after transplantation showed minimal accessibility differences, suggesting that CEBPD-associated regulatory changes emerge over time. Integration analyses identified 8 MC states and a CLAD-specific shift toward a fibrotic state. CEBPD, SOX4, and FOXP2 were identified as putative regulators of this state with substantial overlap in predicted targets. Targeting CEBPD reversed fibrotic phenotypes of CLAD MCs (decreased ECM expression, contractility, proliferation, and migration). Together, these data provide insights into transcriptomic and epigenomic changes in posttransplant MCs, facilitating the nomination of biomarkers and therapeutic targets.

Authors

Lu Lu, A. Patrick McLinden, Natalie M. Walker, Ragini Vittal, Yichen Wang, Fatemeh Fattahi, Stephen T. Russell, Michael P. Combs, Joshua D. Welch, Vibha N. Lama

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Figure 5

Gene regulatory analysis combining single-cell transcriptomic and epigenomic features nominates regulators of CLAD-enriched fibrotic state.

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Gene regulatory analysis combining single-cell transcriptomic and epigen...
(A) Dot plot showing the state specificity of the predicted targets for each TF. Each row is a TF and each column is a cell state. The color of each square indicates the aggregate expression of predicted target genes for each TF in each cell state. Dot size indicates the area under the recovery curve (AUC) for a classifier trained to predict the cell state from the chromatin accessibility of target peaks. Higher AUC means that the target regions for the TF are more state-specific. (B) Heatmap showing overlap between predicted target regions of each TF. Each square shows the Jaccard similarity index (intersection of target regions divided by the union of target regions) for the pair of TFs in the corresponding row and column. Higher values indicate more overlap between the targets of the pair of TFs. (C) UMAP visualization of CEBPD, FOXP2, and SOX4 and predicted targets. Each dot represents a cell. Dots are colored by TF expression (left) or the sum of target gene expression (right). (D) Visualization of the enhancer-driven gene regulatory network formed by CEBPD, FOXP2, and SOX4. Each circle is a gene; color and size represent the log2(fold change) of the gene’s expression in fibrotic MCs compared with the rest of the cells. Thus, a large, red circle indicates a gene with much higher expression in fibrotic MCs and a large blue circle indicates a gene with much lower expression in fibrotic MCs. Each diamond is a chromatin accessibility peak (putative enhancer) and its color represents the log2(fold change) of the accessibility in fibrotic MCs compared with the rest of the cells (yellow is higher accessibility in fibrotic MCs). Each line indicates a regulatory relationship; line color indicates the strength of the correlation between peak and gene (higher correlation is more yellow). Gene symbols in red letters are part of the CLAD signature from Figure 2E.