<i>Mycoplasma hyopneumoniae</i> modulates ciliary function and epithelial integrity in air-liquid interface porcine respiratory epithelial cells (ALI-PRECs)

ABSTRACT Mycoplasma hyopneumoniae ( Mhp ), the causative agent of enzootic pneumonia, disrupts mucociliary clearance by adhering to porcine ciliated airway cells, contributing to chronic respiratory disease. Traditional submerged cell cultures lack airway-like polarity and structural complexity, limiting Mhp in vitro studies. This study utilized a porcine air-liquid interface porcine respiratory epithelial cell (ALI-PREC) model to investigate Mhp infection dynamics, cytopathic effects (CPEs), and host-specific responses. Primary tracheal epithelial cells isolated from three 6-week-old pigs were cultured under air-liquid interface conditions for 4 weeks to form differentiated pseudostratified, ciliated epithelium. ALI-PRECs were inoculated with Mhp strain 232 at 10 5.84 , 10 6.84 , and 10 7.84 CCU/mL for 2 or 5 h and monitored for 144 h. Quantitative microscopy assessed total particle count, area, average size, and percentage area covered by particles, normalized against mock controls. Mhp adhered within 2 h, inducing CPE (cell rounding, clustering, and detachment) and reducing ciliary activity in a dose- and time-dependent manner. Mhp DNA was detected in the epithelium by 24 h post-inoculation (hpi) and in subnatants at higher doses, demonstrating epithelial barrier disruption. Ciliary beating persisted in some replicates, suggesting interindividual variability in host responses. Friis medium had no detectable impact on ALI-PRECs, validating its suitability for infection studies. Gene expression analysis revealed downregulation of ciliary motility genes ( ROPN1L , LRRC51 , CEP162 , and LRRC6 ) at 72 hpi and upregulation of intercellular junction genes ( CLDN1 , CDH1 , and CTNND1 ) by 120 hpi, suggesting a wound healing response. The ALI-PREC model effectively mirrors key aspects of Mhp pathogenesis, providing a robust platform for studying host-pathogen interactions and identifying therapeutic targets. IMPORTANCE Despite its economic impact on pigs, the mechanisms by which Mycoplasma hyopneumoniae ( Mhp ) causes enzootic pneumonia remain poorly understood. Early infection events (bacterial exposure and adherence) are hard to study in vitro because traditional cell cultures lack airway complexity and cellular diversity. We developed an in vitro model replicating porcine airway structure and cell populations. Using this model, we showed that Mhp rapidly adheres to airway cells and disrupts ciliary function and epithelial integrity in a dose- and time-dependent manner. These effects coincide with altered expression of genes governing ciliary motility and cell-cell junctions. Importantly, our model revealed natural variability in cytopathic responses among pigs, indicating that host-specific factors influence disease progression. These insights may guide personalized strategies for preventing and treating swine respiratory infections.