Abstract
To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.
Highlights
Understanding the process of attachment of inhaled pathogens to highly differentiated epithelial cells, immune cell transmigration through respiratory epithelia and the removal of airborne particles by dendritic cells (DCs) or macrophages in a spatiotemporal manner proves to be difficult in vivo and in vitro due to lack of appropriate tools
To study infections of the upper (ABPA) and lower (IPA) respiratory tract and to provide a 3D cell culture model that allows the fast investigation of epithelial permeability and immune cell reactions in a realistic and easy to handle in vitro system, we set up a perfused three-dimensional cell culture model
We describe the set-up of a novel fast-track in vitro approach of human primary respiratory tract cells grown in air liquid interphase (ALI) and under perfusion and containing myeloid DCs or macrophages to study airborne infections, allergies or toxic effects of inhaled particles of the upper and lower respiratory tract
Summary
Understanding the process of attachment of inhaled pathogens to highly differentiated epithelial cells, immune cell transmigration through respiratory epithelia and the removal of airborne particles by DCs or macrophages in a spatiotemporal manner proves to be difficult in vivo and in vitro due to lack of appropriate tools. To study infections of the upper (ABPA) and lower (IPA) respiratory tract and to provide a 3D cell culture model that allows the fast investigation of epithelial permeability and immune cell reactions in a realistic and easy to handle in vitro system, we set up a perfused three-dimensional cell culture model. Such perfused and highly differentiated epithelia were used to attach myeloid DCs to the basolateral or macrophages to the apical side and within this system DC and macrophage functions, i.e. DC maturation and migration or macrophage attraction and phagocytosis, were analysed in a three-dimensional space after fungal infection
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