(496) CFTR modulators restore autophagic-dependent bacterial clearance by improving the acidification of autophagolysosomes of F508del macrophages

Background: People with Cystic Fibrosis (pwCF) are more prone to infections by opportunistic pathogens such as Burkholderia cenocepacia (B. cenocepacia) resulting in fatal respiratory infections. Macrophages play critical roles in the process of clearance of lung pathogens. We and others previously reported that CF human and mouse macrophages expressing F508del CFTR are defective in their ability to kill bacteria. Furthermore, the autophagy process in CF macrophages is halted, and the underlying mechanism remains to be elucidated. Autophagy is the main pathway for clearing B. cenocepacia where autophagolysosomal acidification is required. The role of CFTR in the autophagy process in macrophages is yet to be determined. CFTR modulators (Tezacaftor/Ivacaftor) are used for treating CF patients via correction of misfolded CFTR and improving its function in epithelial cells. Yet, their effect on macrophage functions is unclear. Identifying the effect of CFTR modulators’ treatment on the enhancement of the autophagy process and bacterial clearance is the aim of our study.

Methods: Murine bone marrow-derived macrophages from F508del mice as well as human monocyte-derived macrophages from F508del/F508del pwCF were used in our study. Macrophages were treated with CFTR modulators (Tezacaftor/ Ivacaftor) prior to their infection with B. cencepacia. Autophago-lysosomal acidity was measured in non-infected and infected macrophages. CFTR localization was determined using confocal microscopy. After CFTR modulators treatment, the autophagic flux was determined using immunoblotting and confocal microscopy. B. cenocepacia and E. coli clearance ability of macrophages were assayed using colony-forming unit experiments.

Results: Using 3D reconstruction and confocal microscopy we demonstrate that the Cystic Fibrosis transmembrane conductance regulator (CFTR) protein is expressed on autophagosomes and is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Non-autophagosomal cargos such as non-pathogenic E. coli are effectively degraded in acidic vacuoles. On the other hand, CFTR is required for the acidification of B. cenocepacia-containing autophagosomes. CFTR modulators increased F508del CFTR localization to B. cenocepacia-containing autophagosomes and improved their ability to acidify. Additionally, treatment of CF macrophages with CFTR modulators enhanced B. cenocepacia trafficking to the autophagosomes, resulting in their enhanced clearance in CF macrophages. This effect was achieved when the modulators were present before, but not after B. cenocepacia infection.

Conclusions: CFTR plays an important role in autophagy-mediated bacterial clearance in macrophages by regulating the acidification of the autophago-lysosomes. Importantly, CFTR does not contribute to the acidification of other compartments that do not resemble autophagosomes. Therefore, our study describes a new biological location and function for CFTR in macrophages. Acknowledgements: This work was supported by the Cure CF Columbus (C3) RDP Trainee Award.

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