Abstract Rationale Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is a leading cause of morbidity and mortality among SSc patients. A hallmark of SSc-ILD is progressive lung remodeling, which involves, 1) fibrosis of the lungs, leading to scarring and thickening, 2) honeycombing, lined by bronchiolar-like epithelium that includes basal cells, ciliated cells and mucin-producing goblet cells. Basal cells are essential for maintaining epithelial integrity and regeneration following injury by differentiating into other airway epithelial cells. In chronic lung diseases such as COPD and lung cancer, basal cells have been implicated in pathological remodeling. We hypothesize that basal cells via undergoing epithelial to mesenchymal transition (EMT) play an important role in fibrosis in SSc-ILD lungs. Methods The University of Pittsburgh Institutional Review Board approved the study. Single-cell RNA sequencing (10X Genomics) was performed on explanted lungs from SSc-ILD and healthy controls. Data analysis and sub-clustering of epithelial cells was performed using R package Seurat v 5 and pathway analysis using ingenuity pathway analysis. Basal cells were isolated from healthy donor lungs and cultured under submerged conditions. The cells were serum starved and treated with vehicle, 5ng/ml TGFB or 100µg/ml bleomycin, and harvested for RNA analysis. Immunofluorescence analysis of lung tissues was carried out using specific antibodies and imaged using Nikon NI -EI microscope. Results Epithelial cell clustering revealed significant increase in basal cells in SSc-ILD lungs. Other airway epithelial cells, such as goblet and ciliated cells, which originate from basal cells, were also increased significantly. Basal cells from SSc-ILD lungs displayed features of partial EMT, including, upregulation of mesenchymal markers- ITGB6, MMP1 and TNC, downregulation of junction proteins- CLDN1, TJP1, TJP2 and upregulation of transcription factors- ZEB1 and SNAI2. Pathway analysis showed enrichment of genes of activation of MMPs, extracellular matrix degradation, fibrosis and activin-inhibin pathways. Immunofluorescence analysis showed honeycomb cysts lined with basal cells evidenced by expression of KRT17. Treatment of basal cells with TGF-β1, a known inducer of EMT, led to gene expression changes similar to scRNA seq analysis, suggesting TGF-β1-driven EMT phenotype. In contrast, bleomycin treatment did not elicit a similar response, reinforcing the specificity of TGF-β1 in this context. Conclusions Our findings suggest expansion of basal cells and other airway epithelial cell types in SSc-ILD, upregulation of genes and pathways involved in partial EMT and fibrosis, and role of TGF-β1 in inducing basal cell partial EMT. These processes may underlie fibrotic progression and lung remodeling in SSc-ILD. This abstract is funded by: University of Pittsburgh Scleroderma Center
Rajput et al. (Fri,) studied this question.