Abstract Rationale Chronic lung diseases, such as asthma and COPD, are characterized by airway mucus plugs that drive morbidity and mortality. Pathological changes in submucosal glands (SMGs), the primary source of mucus in large airways, are key drivers of this pathology. Two major histopathological changes, SMG enlargement (hypertrophy) and increased mucous-to-serous ratio (mucous cell hyperplasia), are thought to be primary drivers of pathological mucus production. However, the mechanisms driving SMG pathogenesis are poorly understood. This knowledge gap persists due to inaccessibility of human SMGs in vivo and a lack of appropriate animal models, as commonly used species (e.g., mice) lack lung SMGs or fail to recapitulate human disease. Here, we characterize SMG pathology in a DNAI1-null pig model of primary ciliary dyskinesia (PCD), which has normal glands at birth and then spontaneously develop SMG pathology in response to chronic muco-inflammation conditions caused by impaired mucociliary transport. Methods Tracheal and bronchial tissues were collected from wild-type (WT) and PCD pigs at newborn, 3-week, and 2-month time points. We utilized quantitative histology to measure gland area and density and immunofluorescence to assess glandular cell proliferation using Ki-67 and differentiation using cell-specific markers including PLUNC and MUC5B. Single-cell RNA sequencing (scRNA-seq) was performed on airway tissues to identify changes in cell populations, gene expression, and intercellular signaling interactions within the glandular microenvironment. Results At birth, PCD pig SMGs were histologically indistinguishable from WT. However, PCD pigs developed progressive and significant SMG hypertrophy; by 2 months, gland area was ∼3-fold larger than in WT (p 0.001). This enlargement was driven by a significant increase in cell proliferation rather than a change in gland density or individual cell size. Immunofluorescence analysis identified PLUNC+ serous cells as the predominant proliferating cell type. However, this serous cell proliferation was coupled with a robust mucous cell hyperplasia, suggesting serous-to-mucous differentiation. By 2 months, the MUC5B+ mucous cell proportion increased from 0.18 in WT to 0.5 in PCD (p 0.001). scRNA-seq analysis of the SMG and niche cell types identified novel pathways correlated with these phenotypes, including upregulation of NRG1-ERBB2/3 pathway that associates with serous cell proliferation, and an accumulation of plasma B cells and elevated BMP6 signaling linked to the mucous cell hyperplasia. Conclusions The PCD pig model spontaneously recapitulates the key SMG pathology seen in COPD, making it as a powerful and clinically relevant model for investigating the pathogenesis of SMG-driven mucus obstruction. This abstract is funded by: Parker B. Francis Foundation, American Lung Association, NIH
Yu et al. (Fri,) studied this question.