(488) Chronic Pseudomonas aeruginosa infection in a cystic fibrosis in vitro model

Background: Pseudomonas aeruginosa (Psa) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. Psa infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of Psa infection in vitro commonly observe infections for 6 – 12 hrs. However, these relatively early time points may not encompass downstream airway cell signaling in response to infection. We have established an in vitro model that allows for a Psa infection of CF bronchial epithelial cells for 24 hrs to fill this gap of knowledge.

Methods: We used an immortalized CF cell line (CFBE41o-) and differentiated cells at the air liquid interface (ALI) for 10-14 days. Then, ALI cultures were infected with mucoid and non-mucoid Psa strains at progressively diluted inoculums for 1, 6, 12, or 24 hrs. CFBE survival was measured by LDH assay and microscopic analysis. Fluorescent Psa strains were used to visualize bacteria in fixed ALI cultures. Colony forming units (CFU) were measured to determine Psa inoculum and endpoint numbers. Heat killed Psa and treatment with tumor necrosis factor alpha (TNFα) were used to further characterize the model. Levels of the pro-inflammatory cytokines interleukin (IL)-8, IL-6, and IL-1β were measured by RT-qPCR and ELISA.

Results: Infection of CFBE41o- cells with an inoculum of 10² CFUs of Psa for 24 hrs did not significantly decrease CFBE survival or monolayer confluency. We observed Psa growth increased from approximately 2x10² CFUs at 6 hrs to 4x10³ CFUs at the 24 hr time point. IL-8, IL-6, and IL-1β expression increased at 24 hrs, along with a significant increase in IL-8 secretion into the basal media. Immunofluorescence staining also confirmed IL-8 was increased in Psa treated ALI cultures compared to uninfected controls.

Conclusions: Our data suggest that this newly developed chronic Psa in vitro infection model recapitulates the elevated inflammatory markers observed in CF without compromising cell survival. This extended period of Psa growth on CFBE cells allows for further studies of cell signaling and microbiological studies that were not possible in previous models using shorter Psa exposures.

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