(489) Cystic Fibrosis airway metabolites contribute to persistent pulmonary infection

Background: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) dysfunction promotes the accumulation of mitochondrial metabolites like succinate and itaconate in response to the major CF airway pathogens, P. aeruginosa and S. aureus. Succinate and itaconate not only regulate macrophage function, but also select for bacterial strains that form biofilms and persist in the airway. Given that neutrophils accumulate in CF airway but fail to clear infection, we postulated that itaconate might be produced by neutrophils and may also inhibit their ability to kill bacteria.

Methods: We used single cell RNA sequencing (scRNA-Seq) to identify cell types that express the itaconate synthetic enzyme (Irg1) and the pathways that are regulated by Irg1 during murine lung infection with S. aureus and P. aeruginosa. We correlated these findings with bacterial clearance and cytokine levels. We also performed in vitro functional assays with WT and Irg1^-/- neutrophils to determine how itaconate affects phagocytosis and killing.

Results: Myeloid cells, especially neutrophils, were the major source of Irg1 expression in the lung in response to CF pathogens. Neutrophil-depleted mice had significantly less airway itaconate than controls. Isolated neutrophils exposed to itaconate in vitro exhibited dose-dependent toxicity which correlated with significantly impaired ability to kill S. aureus. Mice that were unable to make itaconate (Irg1^-/-) demonstrated improved bacterial killing with significantly decreased bacterial loads as well as decreased IL-1b, IL-6 and TNF in the airway fluid. In addition to documenting increased itaconate in CF sputum, we confirmed that DF508 HL-60 cells (made by Guoshong Wong at LSU) exhibited increased Irg1 expression compared with WT controls.

Conclusions: Accumulation of specific airway metabolites in response to CF pathogens has a major effect on neutrophil function and may provide a treatable target to help clear infection.

Acknowledgements: Thank you to the Single Cell Core at the Columbia Genome Center and the Calgary Metabolomics Research Facility for their technical help and raw data processing in the scRNA-seq and metabolomics studies. This project was funded by the National Institutes of Health (NIH 1R35HL135800, 1F30AI161801, S10RR027050) and the Cystic Fibrosis Foundation (RIQUEL9 17F0/PG008837).

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