Treatment with the ion channel potentiator ivacaftor corrects impaired Rab27a activation and neutrophil secondary and tertiary granule exocytosis resulting in normalized bacterial killing. (A) Rab27a was immunoprecipitated from neutrophils of HC donors, from CF neutrophils of the ΔF508/ΔF508 (CFΔ) or G551D/ΔF508 (CFG) genotypes, or from G551D/ΔF508 neutrophils donated by PWCF treated with ivacaftor (CFG+iva). Levels of immunoprecipitated GTP-bound Rab27a were enzymatically detected and normalized to precipitated Rab27a levels and the 0 time point value (n = 4 subjects per group). (B) GTP-Rab27a levels at the 30-second time point of TNF-α stimulation were significantly lower in neutrophils from untreated PWCF (CFΔ, CFG) compared with levels in HCs and ivacaftor-treated individuals (CFG+iva) (P = .03 between HC and CFΔ, P = .04 between HC and CFG, P = .4 between HC and CFG+iva, Student t test, n = 4 subjects per group). (C) Neutrophils isolated from healthy volunteers, from PWCF with the ΔF508/ΔF508 or G551D/ΔF508 genotypes, or from G551D/ΔF508 individuals after ivacaftor treatment were stimulated with TNF-α (10 ng/mL) for 0, 5, 10, or 20 minutes. Degranulation was analyzed by measuring up-regulation of CD66b to the cell surface by flow cytometry and was expressed as the percentage increase in MFI. CD66b levels on CFΔ and CFG cells were significantly decreased compared with HCs (P = .01 between HC and CFΔ and P = .02 between HC and CFG, Student t test, n = 4 subjects per group), whereas ivacaftor treatment corrected CD66b levels (P = .03 between CFG and CFG+iva and P = .5 between HC and CFG+iva, Student t test, n = 4 subjects per group). (D) Survival of P aeruginosa after a 4-minute incubation with degranulated proteins obtained from unstimulated control neutrophils of healthy volunteers (ctrl), TNF-α (10 ng/mL, 10 minutes) stimulated control cells (HCs), CF cells of the ΔF508/ΔF508 or G551D/ΔF508 genotypes, or during ivacaftor treatment. The bacterial survival is expressed as percentage survival of the initial bacterial count at time 0. Bacterial survival was significantly decreased following treatment with TNF-α in HC cells (P = .0001 between ctrl and HC, Student t test, n = 3), but was significantly increased in CF samples compared with the HCs or during ivacaftor treatment (P = .0001 between HC and CFΔ and between HC and CFG, P = .2 between HC and CFG+iva, Student t test, n = 3 subjects per group). All measurements are means ± SEM from biological replicates.