Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

To establish a culture system for differentiated porcine airway epithelial cells, primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively) were cultured in a transwell filter system. When the cell monolayer had reached confluence, the medium was removed from the apical compartment and -under these ALI conditions -the cells started to differentiate. PTEC and PBEC were maintained at ALI conditions for at least 5 weeks. The presence of cilia, which is considered as an indicator of the extent of differentiation, was determined by immunofluorescence microscopy (Fig. 1A,B) . Interestingly, cilia expression (in red) was more pronounced on PBEC (Fig. 1B) in comparison to PTEC (Fig. 1A) . Immature short cilia were first observed on cells at the border of the filter supports of cultures maintained for 7 days under ALI conditions (data not shown). The amount of cilia further increased until week 7 of cultivation. During the differentiation process, the monolayer adopted the appearance of a pseudostratified epithelium. Mucin production (in green) was demonstrated by mucin-5 AC staining after 5 weeks of culture under ALI conditions in both PTEC (Fig. 1A) and PBEC (Fig. 1B) . Moreover, the presence of tight junctions was demonstrated by a positive staining for occludin (Fig. 1C,D) , reflecting morphological stability and barrier function of PTEC and PBEC. As shown in Fig. 1E , scanning electron microscopy (SEM) revealed mature cilia at a high percentage of the epithelial surface of well-differentiated PBEC 7 weeks after cultivation under ALI conditions. A peak value of transepithelial electrical resistance (TEER) at 6 days of culture under ALI conditions indicated that PTEC and PBEC had adopted a polarized organization (Fig. 1F ). Afterwards TEER values decreased to some extent, but remained constant over the remaining observation period of 40 days. Taken together, we established well-differentiated porcine airway epithelial cells under ALI conditions in vitro, which closely resemble the cells of the airway epithelium.