(516) Cystic fibrosis sputum attenuates killing of Staphylococcus aureus by neutrophils by impairing phagolysosome fusion

Background: Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of neutrophils (PMNs). In CF, PMNs are unable to kill and clear the main lung pathogen affecting these patients, Staphylococcus aureus (S. aureus). Healthy PMNs eliminate S. aureus primarily via oxidative killing mechanisms. Our published results show that the CF sputum impairs PMNs’ ability to kill S. aureus but it does not affect phagocytosis or formation of neutrophil extracellular traps (NETs). Therefore, we hypothesized that the CF airway environment inhibits S. aureus killing by interfering with phagolysosome fusion in PMNs.

Methods: Human PMNs were isolated from healthy donors, exposed to CF sputum supernatants to mimic the CF airway environment and then infected with CF clinical isolates of methicillin-resistant S. aureus (MRSA). Bacterial killing was measured by a microplate-based photometric assay, bacterial phagocytosis was detected by pHrodo-labelled S. aureus and subcellular localization of myeloperoxidase (MPO) residing in primary granules was detected by fluorochrome-labelled antibody. Cell imaging and quantification was performed by ImageStream (Luminex imaging cytometer) to assess colocalization of lysosomal compartments to the phagosome. Results were subjected to analysis by t-test and were considered significant if p< 0.05.

Results: Our results show that all the CF clinical isolates of S. aureus tested were killed by PMNs by phagocytosis rather than by NET formation. Phagolysosome fusion assessed as co-localization of pHrodo-stained, phagocytosed bacteria and MPO was significantly inhibited by CF sputum pre-treatment of PMNs. Our results suggest that the CF airway environment reduces the ability of PMNs to kill S. aureus at least in part by impairing the fusion of primary granules with the phagosome following bacterial phagocytosis

Conclusions: These results reveal a novel consequence of the CF airway environment on PMN functions allowing S. aureus to survive in the CF airways and identifying a potential new therapeutic target.

Acknowledgements:

Funding: NIH/NHLBI R01 HL136707-01A1 (Rada)

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