Abstract Rationale Honeycomb cysts are a defining patho-radiologic feature of usual interstitial pneumonia (UIP), a fibrosing interstitial lung disease associated with increased morbidity and mortality. Honeycomb cysts are composed of excess fibroblasts and extracellular matrix with temporal heterogeneity. However, there are limited studies evaluating the pathobiology of honeycomb cyst formation. We have previously shown that repetitive intratracheal dosing of bleomycin in mice generates multi-lobed cysts in areas of fibrosis that may resemble honeycomb cysts seen in human UIP. Here, we sought to characterize the transcriptional, cellular identities and pathways involved in multi-lobed cysts to develop insights into the pathogenesis of human UIP. Methods C57Bl/6 mice received 3 biweekly intratracheal instillations of bleomycin (1 u/kg) or saline. After 10 weeks, lung tissue was formalin fixed and paraffin embedded for histopathologic and spatial transcriptomic sequencing. Masson’s Trichrome and hematoxylin and eosin staining was performed. Spatial transcriptomic analysis was done on 5μm sections with 6.6 x 6.5 mm capture areas using the Visium HD (10X Genomics) Spatial Gene Expression with CytAssist platform. RNA libraries were sequenced on Illumina NextSeq 2000 and reads mapped to the mouse genome using Space Ranger v3.1.1 with 16 µm binning. Distinct cell clusters were informed by our previously published datasets and downstream analyses were performed using Seurat v5 and R v4.4.1. Results Repetitive bleomycin instillation in mice led to the development of multi-lobed cysts in lung parenchyma surrounded by areas of fibroblast accumulation and fibrosis resembling the honeycomb cysts and fibrosis seen in human UIP. Spatial transcriptomic analysis revealed expression of aberrant epithelial cell markers (Krt8+, Scgb1a1, Sox2) within the cysts coinciding with area of accumulated pro-fibrotic PDGRα+/PDGFRβ+ (Col1a1, Acta2, Tgfb1, Fn) and adventitial (Col14a1+) fibroblasts. Additional transcriptionally distinct fibroblasts populations (mesothelial, pericytes and Wnt2+ alveolar fibroblasts) were not located adjacent to the multi-lobed cysts. Conclusion Our results show that multi-lobed cysts develop in lung parenchyma in response to repetitive bleomycin instillation and exhibit an aberrant epithelial signature similar to epithelial cells found within UIP associated honeycomb cysts. Additionally, they are located adjacently to expanded pro-fibrotic fibroblasts, suggesting cross-talk between these two fibrosis-associated populations. Further pathway analysis may help us understand the involvement of aberrant transitional epithelium, airway epithelium, and fibroblasts in generating the pathobiology of multi-lobed and possibly honeycomb cysts of UIP. This abstract is funded by: NIH P01-HL16267
Schott et al. (Fri,) studied this question.