Murine precision cut lung slices as a novel 3D-model for studying endocytic pathways in the lung

Abstract

Introduction: Endocytosis plays a pivotal role in cellular and tissue homeostasis. In the lungs, endocytic mechanisms are involved e.g. in phagocytosis, viral infections and receptor-mediated protein uptake. However, the precise lung cell-specific molecular patterns of endocytosis remain poorly understood. Here, we investigated endocytosis in various lung cell types in precision cut lung slices (PCLS), a viable and fully functional 3D lung tissue model system.

Results: By using fluorescent-activated cell sorting (FACS) and confocal microscopy, we studied uptake of different proteins and influenza virus (IV) in PCLS. Our results showed that uptake of albumin, transferrin and IV in epithelial (EpCAM⁺CD31^-CD45^-), endothelial (EpCAM^-CD31⁺CD45^-) and hematopoietic (EpCAM^-CD31^-CD45⁺) cells of PCLS was time- and dose-dependent and of different velocity. Treatment with the endocytosis inhibitor, dynasore, and chlorpromazine, an inhibitor of clathrin-mediated endocytic processes, led to decreased uptake of the cargos and IV in a cell-specific manner. Further differences in endocytic capacity were found, when comparing protein and IV uptake in alveolar epithelial type I and type II cells of PCLS. Moreover, we observed that Ras-related proteins, Rab9 and Rab11, and early endosome antigen-1 (EEA1) played a central role in the uptake of proteins and IV in alveolar epithelial cells from PCLS.

Conclusions: Our studies suggest that PCLS might serve as a novel 3D-model for studying endocytic mechanisms in respiratory cells. Understanding the molecular pathways of endocytosis in the lung may provide new therapeutic approaches for patients with pulmonary diseases.