Biological Boundary Conditions Regulate the Internalization of Aspergillus Fumigatus Conidia by Alveolar Cells

Natalia Schiefermeier-Mach ^* Julien Polleux Lea Heinrich Lukas Lechner Oleksandra Vorona Susanne Perkhofer

• Health University of Applied Sciences Tyrol / FH Gesundheit Tirol, Innsbruck, Austria

The lung environment is defined by unique biological boundary conditions, including complex alveolar geometry, extracellular matrix composition and mechanical forces generated during respiration. These factors were shown to regulate alveolar permeability, surfactant secretion, cell contractility and apoptosis, but their role in fungal infections remains unknown. Aspergillus fumigatus is a critical fungal pathogen that causes severe pulmonary infections in immunocompromised individuals. Our study addresses a knowledge gap by investigating how boundary conditions affect A. fumigatus conidia interactions with alveolar epithelial cells.Methods. We applied micropatterned substrates to confine cells into defined shapes and densities, allowing precise control over geometric conditions and extracellular matrix composition. Using cell line stably expressing the phagolysosomal protein Lamp1-NeonGreen and multiplane fluorescent microscopy, we evaluated A. fumigatus conidia binding and internalization efficiency.We observed significantly faster and more efficient A. fumigatus conidia internalization in cells confined on micropatterns compared to previously reported studies using cell monolayers. Altering cell geometry, density and extracellular matrix composition strongly affected conidia binding and localization to Lamp1 + phagolysosomes. Cells on X-shaped or multicellular micropatterns showed higher internalization rates, particularly at the periphery, suggesting spatial heterogeneity in pathogen uptake. Additionally, changes in extracellular matrix composition influenced the intracellular trafficking of A. fumigatus conidia. Discussion. Our findings emphasize the essential role that local mechanical and biochemical cues play in shaping the interactions between fungal pathogens and alveolar cells. Understanding how lung boundary conditions change in disease states will provide important insights into fungal infection outcomes.