Toll-like receptor signaling regulates the differentiation of 3D bronchospheres

Background: Chronic respiratory diseases (e.g. cystic fibrosis) are associated with bacterial infections. Toll-like receptor (TLR) ligands recognize bacterial factors, such as flagellin which is expressed by the cystic fibrosis pathogen Pseudomonas aeruginosa. Aim: Here, we aimed to investigate the effect of flagellin on the differentiation of human 3D bronchospheres. Methods: Human primary epithelial cells were differentiated to bronchospheres in Matrigel. Cells were incubated with control media or media containing TLR ligands (100 ng/ml of flagellin, polyI:C, or Pam3CSK4) during the differentiation period. The appearance of lumina and the presence of cilia were determined. Results: Differentiation of bronchospheres associated with the formation of lumina and ciliation within 30 days after seeding. Incubation with flagellin resulted in a delayed formation of lumina and decreased ciliation compared to control bronchospheres. Incubation with polyI:C did not affect these parameters whereas Pam3CSK4 slightly accelerated the differentiation of bronchospheres. Conclusion: Our results indicate that activation of TLR-signaling cascades modifies the differentiation of bronchospheres. Additional analysis will show whether exposure to flagellin induces the expression of mucus and goblet cell metaplasia. Importantly, bronchospheres can be used for mechanistic studies replacing animal experiments.