Abstract Introduction Although CFTR modulators have improved lung health tremendously in people with cystic fibrosis (pwCF), there is a plateau in functional benefit that suggests persistence of structural defects. We previously have identified multiple areas of constrictive bronchiolitis and bronchiolar stenosis that likely contribute to the persistent loss of pulmonary function. Here, we employ spatial transcriptomic analysis to identify the primary cells and mechanisms of promoting fibrogenic impingement on the bronchiolar lumen. Hypothesis Discrete fibrogenic pathways are upregulated in areas of bronchiolar stenosis in explanted human CF lungs. Methods CF and failed donor (controls) samples were obtained at the time of lung transplantation through the UAB Airway Tissue Procurement Core. Whole transcriptome spatial transcriptomics were performed using GeoMx Nanostring. Bioinformatics analyses were performed using Geomx software and various R packages. Immunofluorescence analysis for AT1, AT2, ABCs and EMT markers were performed, and images acquired using Leica confocal microscope. Results Differential gene expression analysis suggests dysregulation of several genes related to AT2 cellular function (ABCA3, LAMP3, LRRK2), inflammation and fibrosis (NNMT, CHI3L1) and mucus production (MUC5AC) in remodeled bronchioles in comparison to non-remodeled bronchioles. Receptor-ligand analysis among epithelial, stromal and endothelial cells suggests strong outgoing signaling networks from stromal to epithelial and endothelial zones in remodeled bronchioles. In contrast, a strong outgoing endothelial to epithelial and stromal signaling was observed in non-remodeled airways. Several of the receptor-ligand pairs involved in pro-fibrogenic pathways such as COL1A2-ITGA1, COL1A2-SDC4 were upregulated around remodeled bronchioles. Deconvolution analysis of stromal regions suggests increased levels of activated fibroblast and myofibroblasts in remodeled airways. Similar analysis of remodeled bronchiolar epithelia suggests increased prevalence of aberrant basaloid cells (ABCs), which was further confirmed by IF analysis for its markers such as KRT8, KRT5, Col1A1 and KRT17. ABCs in remodeled regions also expressed myofibroblast markers such as TWIST1 and vimentin (VIM), indicating epithelial-to-mesenchymal (EMT). Conclusion Aberrant basaloid cells are increased in areas of constrictive bronchiolitis in CF in association with fibroblast activation and myofibroblast differentiation. Our results provide initial evidence of EMT in remodeled distal airways. These findings may provide a new therapeutic cellular target of interest and receptor/ligand interaction to ameliorate loss of small airway function in pwCF. This abstract is funded by: KPRI, CYSTIC FIBROSIS FOUNDATION
Kumar et al. (Fri,) studied this question.