Spatial Transcriptomics Identify Region-specific Pathogenesis of Bronchiolar and Alveolar Disease in Primary Ciliary Dyskinesia Lungs

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, rare genetic disorder characterized by dysfunctional motile cilia. PCD pulmonary manifestations include impaired airway mucociliary clearance, mucus obstruction, recurrent infection, and bronchiectasis (BE). Notably, thoracic imaging studies indicate that alveolar atelectasis is more prevalent in children with PCD than in age-matched children with cystic fibrosis (CF). However, morphologic and molecular analyses of the site of initiation and progression of PCD airway disease and relationships to PCD alveolar disease have not been reported. In this study, we analyzed region-specific morphologic and molecular characterizations of airway epithelia and alveolar regions in PCD lungs excised at transplantation to address these questions. Methods: Nine PCD lungs and seven non-diseased control lungs were included in the morphometric studies. Bronchi and proximal and distal bronchioles were defined using unique morphologic structures, and distal airway secretory cell (DASC) markers were used to differentiate between proximal and distal bronchioles. Morphologic characteristics of airway epithelia and luminal mucus were quantitated by histology, immunohistochemistry, and RNA in situ hybridization. Spatial transcriptomic technologies (GeoMx) characterized region-specific transcript expression in three airway epithelial regions (bronchi, proximal bronchioles, and distal bronchioles), intraluminal mucus plugs, and alveolar regions of PCD compared to control lungs. Finally, pulmonary function data from a cohort of PCD subjects were analyzed to investigate correlations between alveolar morphologic findings and alveolar gas exchange function, i.e. diffusing capacity for carbon monoxide (DLCO). Results: PCD bronchi and proximal bronchioles exhibited ectasia, characterized by increased MUC5AC and MUC5B-producing cells, mucus plugs containing MUC5AC and MUC5B mucins infiltrated by neutrophils, and epithelial gene signatures associated with ectasia. In contrast, PCD distal bronchioles were not ectatic and exhibited MUC5B-dominant DASCs, mucus plugs containing selectively MUC5B mucin infiltrated by mixture of neutrophils and macrophages, and gene signatures suggestive of impaired ciliogenesis. Molecular analysis revealed upregulation of pathways associated with muco-inflammation, antimicrobial defense, and the innate immune system routinely in PCD airway epithelia. IL-1β gene signatures were observed in both PCD airway and alveolar epithelia, whereas interferon-stimulated gene signatures were restricted to airway epithelia. Transcriptomic and morphologic analyses suggested that PCD alveolar disease was initiated by distal bronchiolar obstruction, associated with chronic microatelectasis, progressive loss of alveolar epithelial type 2 (AT2) cells, and eventual non-resolving fibrosis. A significant percentage (28%) of people with PCD exhibited reduced DLCO. Conclusions: PCD airway disease is manifest by distinct profiles of proximal vs distal bronchiolar disease associated with microatelectatic and fibrotic alveolar disease.

Duke Scholars

Author Shannon Jones McCall Pathology

Author Laurie D. Snyder Medicine, Pulmonary, Allergy, and Critical Care Medicine