Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections

Abstract

Rationale: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established.

Objectives: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections.

Methods: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans.

Measurements and main results: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging.

Conclusions: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.

Keywords: Pseudomonas aeruginosa; cystic fibrosis; inflammation; ivacaftor.

Figure 1.

Ivacaftor treatment rapidly improved cystic fibrosis transmembrane conductance regulator activity and lung function. Black lines represent responses of individual subjects, and red lines represent mean values. (A) Ivacaftor reduces sweat chloride levels. The shaded region represents borderline values (41–60 mM); higher values are consistent with cystic fibrosis transmembrane conductance regulator dysfunction, and lower values are considered normal. Effects of ivacaftor on FEV₁ during the first week (B) and year (C) of treatment, with values from the first week removed from C for clarity; **P < 0.005 and ^#P < 0.0001 compared with Day 0. (D) Representative example (from subject 5) of the change in FEV₁ slope calculated from values obtained over the 2 years before and after ivacaftor. Slopes were estimated via linear regression (see Methods). Note that the graph includes 4 years of data, and the x-axis is drawn to scale. Thus it appears that the Day 0, 2, and 7 measurements for FEV₁ all fall near the y-axis. The table lists preivacaftor and postivacaftor subject-specific FEV₁ slope estimates (L/mo), with negative slopes in red, positive slopes in bright green, and slopes that were improved but still negative after ivacaftor in pale green.

Figure 2.

Ivacaftor treatment rapidly reduces sputum Pseudomonas aeruginosa density. (A and C) Changes in the first week. (B and D) Changes between Day 0 and up to Day 975, with values from the first week removed for clarity. (A and B) Culture-based measurements of viable P. aeruginosa CFU/ml. (C and D) Quantitative polymerase chain reaction–based measurements of P. aeruginosa genome copies; *P < 0.05 and **P < 0.005 compared with Day 0. (E) Mean P. aeruginosa CFU/ml increase after 210 days of ivacaftor treatment. CFU values were log transformed and averaged; error bars indicate SEM. Piecewise, mixed-effect linear regression models were used to estimate and test log₁₀ CFU slope changes before and after Day 210; ^#P < 0.0001. CFU = colony-forming units.

Figure 3.

Pseudomonas aeruginosa strains infecting subjects persist after treatment. (A) Multilocus sequence typing (MLST) analysis of consensus sequences before and after treatment. Ninety-six P. aeruginosa isolates collected at the indicated time points were pooled, sequenced, and consensus MLST sequences were generated. The early and late sputum sample of each subject generated the same consensus MLST sequence type. *Subjects in whom the acs loci showed ambiguity (see Figure E1). (B) Changes in MLST allele frequency in P. aeruginosa isolate pools collected before and after treatment. P. aeruginosa isolate pools from before and after treatment from individual subjects (top 8 graphs) showed far fewer differences than pools from different subjects (bottom 2 graphs) (P < 0.0001). See Figure E2 for data from other subjects. (C) Pulsed-field gel electrophoresis results of randomly selected isolates before (left side of each column) and after (right side of each column) treatment from subjects 4, 5, and 9 (see also Figure E3). All isolates from individual subjects were >95% related (see Figure E3).

Figure 4.

Ivacaftor treatment changes the relative abundance of sputum microbiota. The colored segments of each bar represent the proportion of 16S rDNA reads mapping to the indicated bacterial taxa. The key identifies taxa present at greater than or equal to 0.5% average abundance. Lower-abundance taxa are identified in Table E19. *Subjects that were not chronically infected with Pseudomonas aeruginosa based on cultures used for clinical care.

Figure 5.

Decreases in Pseudomonas aeruginosa abundance are accompanied by increases in microbial diversity. Graphs indicate longitudinal changes in subjects with chronic P. aeruginosa infections in (A) P. aeruginosa relative abundance, (B) microbial richness, (C) microbial evenness, and (D) Shannon diversity index. Bars represent average values from subjects at each time point; error bars indicate SEM. Table E2 shows linear regression analysis indicating that richness and Shannon diversity showed positive slopes in the first year, and significant decreases in slopes thereafter (P < 0.05).

Figure 6.

Ivacaftor does not reduce the absolute sputum abundance of Streptococcus and Prevotella. Quantitative polymerase chain reaction measurements of Streptococcus and Prevotella species in sputum (includes all available samples for subjects with chronic Pseudomonas aeruginosa infections, and Day 0–7 samples for subjects without chronic P. aeruginosa infections). (A and C) Changes in the first week. (B and D) Changes between Day 0 and up to Day 975, with values from the first week removed for clarity. No statistically significant changes were detected. (E) Comparison of the extent of decline of total bacterial and P. aeruginosa 16S rDNA in sputum after ivacaftor treatment as represented by percent change in mean values relative to Day 0.

Figure 7.

Ivacaftor treatment reduces airway inflammation. ELISA measurements of sputum inflammatory markers show decreases in (A) IL-1β, (B) IL-8, and (C) neutrophil elastase. Left panels show changes in the first week; right panels show changes between Day 0 and up to Day 600 with values from the first week removed for clarity; *P < 0.05; **P < 0.005 versus Day 0 values. (D) Change in the relative abundance of inflammatory markers in sputum supernatants during the first week, as determined by mass spectrometry. Decreases in all markers at Day 7 were significant with P < 0.05.

Figure 8.

Ivacaftor treatment improves chest computed tomography (CT). Brody scoring of chest CT scans from subjects 1, 3, 4, 5, 6, 9, and 11 obtained before and 1 year after ivacaftor treatment demonstrated a trend toward decreases in the total Brody score (A) and the peribronchial thickening subscore (B). Significant decreases in mucous plugging (C) were observed. (D) Representative images showing decrease in mucous plugging (thin arrow) and peribronchial thickening (thick arrow) in the chest CT scans obtained before (left) and 1 year after (right) ivacaftor treatment in the same subject.