(415) Bacteriophages that Treat Pseudomonas aeruginosa Infections Induce Pro-Inflammatory Immune Responses in the CF Airway Epithelium

Background: Bacteriophages are a promising treatment alternative for multidrug resistant bacterial infections. People with Cystic Fibrosis (CF) are often chronically colonized with Pseudomonas aeruginosa. In clinical trials and compassionate use cases, phage therapy has proven effective at reducing P. aeruginosa titers and improving health outcomes in people with CF. While effective at treating the infection, the consequences of high doses of foreign viruses on the mucosal surface of the airways has not been studied. We hypothesized that interactions between the lung epithelium and phages occur during therapy that could have unintended outcomes.

Methods: To understand the effects of phage therapy in the CF airways, we utilized a panel of P. aeruginosa phages that are effective at killing clinical isolates both in vitro and in clinical use. We used these phages to examine interactions with CFBE41o- cells, a CF airway epithelial cell line that when grown at air-liquid interface, polarizes and forms a pseudostratified epithelium. Cytokine secretion following phage treatments was evaluated in apical and basolateral media by ELISA.To determine factors that account for differences in cytokine induction, we quantified phage internalization in the airway epithelium by plaque assay. To visualize phage interactions with the respiratory epithelium and intracellular trafficking, we conducted confocal microscopy assays using fluorescently labeled phages.

Results: We found that some P. aeruginosa phages in our panel induce pro-inflammatory cytokines, suggesting that phages differentially activate pattern recognition receptors (PRRs) and the innate immune response. In phage internalization assays, we detected that very low levels of phage enter airway epithelial cells and these levels do not correlate with pro-inflammatory cytokine induction. Our imaging results indicated that phages that induce more pro-inflammatory cytokines associate better with the plasma membrane, suggesting that an extracellular PRR is responsible for phage detection. Current experiments are focused on identifying possible phage PRRs using various immunomodulatory ligand screens.

Conclusions: The information obtained from our research will help identify phage pathogen-associated molecular patterns (PAMPs) and will be crucial to personalize phage therapy to the specific immunological milieu that could maximize therapeutic benefit. Overall, our studies suggest that the airway epithelium recognizes phage particles and demonstrates the need for more studies when considering the use of phage therapy to ensure its safety in people with CF.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (ZAMORA20F0, ARMBRU19F0, BOMBER19P0, and BOMBER21P0); and the National Institutes of Health (T32AI060525, T32HL129949).

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