Investigating the role of mechanical stress in pulmonary fibrosis on epithelial-fibroblast crosstalk

Introduction: Idiopathic pulmonary fibrosis (IPF), a chronic lung disease affecting three million worldwide, is characterized by extensive extracellular matrix deposition and mechanical tissue stress. Epithelial-mesenchymal trophic unit injury and subsequent cell-cell signaling may promote IPF pathogenesis. Despite the separate study of epithelial-fibroblast crosstalk and mechanical dysregulation in IPF, the role of mechanical stress on modulating this crosstalk remains underexplored. Here, we establish an in vitro model to investigate epithelial-fibroblast dysregulation under mechanical stress.

Methods: A custom 3D co-culture collagen hydrogel model was established using freshly FACS sorted primary pulmonary fibroblasts from bleomycin-challenged mice and cultured mouse primary alveolar epithelial cells. Cells were encapsulated (bottom layer 0.8 mL fibroblasts 200,000 cells/mL, top layer 0.2 mL epithelial cells 800,000 cells/mL), allowed to equilibrate, applied a 10% biaxial mechanical strain, and examined using immunofluorescence and bulk RNA sequencing.

Results: Stretched monoculture fibroblast hydrogels demonstrate increased α-SMA expression, with maximal α-SMA expression occurring after 48 hours of equilibration and 36 hours of applied strain (p < 0.05). Monoculture hydrogels of epithelial cells exhibit decreased E-cadherin expression, potentially indicating epithelial-mesenchymal transition. Co-culture hydrogels with in vitro cultured fibroblasts maintain bilayer separation and recapitulate these results. Bulk RNA sequencing reveals the transcriptional activation of genes previously implicated in lung disease (Kcnk2, Gsta1, Ccl6) and common mechanosensing markers (Col1a2, Col3a1, Acta2) after stretching.  

Conclusion: Our engineered 3D co-culture model demonstrates robust mechanical activation, providing an in vitro platform that allows for an investigation of mechanobiological factors on epithelial-fibroblast dysregulation and that preserves in vivo fibroblast heterogeneity and spatially-segmented fibroblast-epithelial cell populations.