Title: Increased Pseudomonas aeruginosa population diversity elevates IL-8 production in CF bronchial epithelial cells

Abstract: Chronic infection in CF with Pseudomonas aeruginosa (Pa) is associated with increased morbidity and mortality. This is despite the use of aggressive antimicrobial interventions against Pa, and the reason for this poor outcome remains unclear. It is now recognized that there is an emergence of phenotypically and genotypically diverse populations of Pa in CF lungs. However, the impact of this heterogeneity on lung pathophysiology remains unknown. We aimed to determine whether the degree of heterogeneity of Pa populations obtained from CF patients was associated with worse outcomes. Based on the literature, we hypothesized that Pa populations with greater diversity would increase inflammatory responses in CF airways. To begin to address this, we first determined diversity levels of Pa populations isolated from the freshly expectorated sputum samples of six CF patients aged 21 to 28 years. We determined diversity levels using bacterial phenotypic assays and DNA-deep sequencing. To assess the impact of Pa population diversity on host responses, we infected CF-derived primary bronchial epithelial cells (CF-BECs DF508/DF508) in an air-liquid interface (ALI) in vitro model, with diverse Pa populations and also single colonies isolated from each population. In this model, we used a synthetic sputum media (SCFM2), which captured the chemical environment of CF sputum. We measured levels of IL-8 in the supernatants by ELISA after 24 hours of infection. We observed Pa populations with higher levels of diversity (i) showed significantly decreased protease activity in vitro; (ii) induced higher IL-8 production in the ALI model after infection. We also found a significant correlation between in vitro induction of IL-8 and the ppFEV1 score of patients at the time of sputum sample collection (r=0.8264; p< 0.001); suggesting that highly diverse Pa populations, lead to increased levels of IL-8 that can cause long-term damage to the airways. Further transcriptomic analysis of both Pa populations and CF-BECs, will allow us to determine whether changes in Pa population diversity modulate signaling pathways in CF-BECs, leading to higher inflammation. Investigating interactions between genotypically and phenotypically diverse Pa populations will help inform new strategies for efficient antimicrobial treatments and prevention of the emergence of highly adapted populations of Pa in CF lungs.