Figure 3

Effects of P. aeruginosa-exposed ALFs on innate immune functions. (a) A representative experiment (of n = 3) of mean fluorescent intensity (MFI) of serum C3 binding to P. aeruginosa, with each ‘n’ value representing a different human neutrophil donor. Non-mucoid PAO1 exposed to CF-ALF show a decrease in serum C3 deposition in 4 of 5 tested CF-ALF samples, with each CF-ALF representing a different human donor. For all three experiments a total of 3 different healthy ALFs (one per each n value) and 7 different CF ALFs (distributed among the n values) were used. (b) Neutrophil activation and degranulation are enhanced when infected with CF-ALF exposed P. aeruginosa. Following infection with H-ALF or CF-ALF exposed P. aeruginosa, CD63 surface expression, a marker of neutrophil activation and primary granule release, on neutrophils was detected by flow cytometry. A representative histogram (of n = 4) showing the CD63 surface expression increase on neutrophils following infection with CF-ALF-exposed P. aeruginosa. Black dashed line represents healthy ALF-exposed P. aeruginosa and solid grey represents CF ALF-exposed P. aeruginosa (left). Percentage and MFI of neutrophils expressing CD63 following infection with H-ALF-exposed or CF-ALF-exposed P. aeruginosa. Data shown are n = 4; M + SEM (right). Each ‘n’ value represents a different human neutrophil donor and a total of 4 healthy and 4 CF ALFs were used in these experiments. Student t test, P. aeruginosa exposed to H-ALF vs. exposed to CF-ALF, *p < 0.05. (c,d) CF ALF exposures enhance ROS burst response of neutrophils exposed to non-mucoid P. aeruginosa. Neutrophils were infected with CF-ALF pre-exposed P. aeruginosa PAO1 strain. Results indicate a significant increase of ROS production, representative experiments performed in triplicate. (e) This phenomenon was not observed in in the mucoid strain (PDO300), representative experiment in triplicate. In (c–e), Student t test, P. aeruginosa exposed to 0.9% NaCl (control) vs. exposed to CF-ALF, *p < 0.05; **p < 0.005. N-PAO1: PAO1 exposed to 0.9% NaCl; H-ALF-PAO1: PAO1 exposed to ALF from a healthy individual; CF-ALF#1- or #2- or #3-PAO1: PAO1 exposed to CF-ALF from different individuals; N-PDO300: PDO300 exposed to 0.9% NaCl; CF-ALF-DPO300: PDO300 exposed to CF-ALF.