Elexacaftor/tezacaftor/ivacaftor corrects monocyte microbicidal deficiency in cystic fibrosis

Abstract

Background: Cystic fibrosis (CF), which is caused by mutations in the CF transmembrane conductance regulator (CFTR), is characterised by chronic bacterial lung infection and inflammation. In CF, monocytes and monocyte-derived macrophages have been shown to display defective phagocytosis and antimicrobial activity against relevant lung pathogens, including Pseudomonas aeruginosa. Thus, we addressed the effect of CFTR triple modulator therapy (elexacaftor/tezacaftor/ivacaftor (ETI)) on the activity of CF monocytes against P. aeruginosa.

Methods: Monocytes from people with CF (PWCF) before and after 1 and 6 months of ETI therapy were isolated from blood and infected with P. aeruginosa to assess phagocytic activity and intracellular bacterial killing. The oxidative burst and interleukin-6 secretion were also determined. Monocytes from healthy controls were also included.

Results: Longitudinal analysis of the clinical parameters confirmed an improvement of lung function and lung microbiology by ETI. Both the phagocytic and microbicidal deficiencies of CF monocytes also improved significantly, although not completely. Furthermore, we measured an exuberant oxidative burst in CF monocytes before therapy, which was reduced considerably by ETI. This led to an improvement of reactive oxygen species-dependent bactericidal activity. Inflammatory response to bacterial stimuli was also lowered compared with pre-therapy.

Conclusions: PWCF on ETI therapy, in a real-life setting, in addition to clinical recovery, showed significant improvement in monocyte activity against P. aeruginosa, which may have contributed to the overall effect of ETI on pulmonary disease. This also suggests that CF monocyte dysfunctions may be specifically targeted to ameliorate lung function in CF.

FIGURE 1

Improvement of forced expiratory volume in 1 s and sweat chloride concentration (SSC) after elexacaftor/tezacaftor/ivacaftor therapy. a) FEV₁ % pred and b) SSC in F508del homozygous (n=14) and heterozygous (n=27) people with cystic fibrosis before (Pre) and after 1 (M1) and 6 (M6) months of therapy. Box plots with median and whiskers pointing to minimum and maximum values. One-way ANOVA with Tukey's multiple comparison test. *: p<0.05; **: p<0.01; ****: p<0.0001.

FIGURE 2

Elexacaftor/tezacaftor/ivacaftor improves the phagocytic activity of cystic fibrosis (CF) monocytes. P. aeruginosa uptake by peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (HD) and people with CF before (Pre) and after 1 (M1) and 6 (M6) months of therapy. a) CFU recovered after the end of infection. Left panel: scattered dot plot with mean±sem. Kruskal–Wallis test with Dunn's multiple comparison. Right panel: longitudinal analysis of data. Friedman test with Dunn's multiple comparison. b) Representative overlaid histograms of monocytes infected with PAO1-GFP. c) Bacterial uptake. Left panel: scattered dot plot with mean±sem. Brown–Forsythe ANOVA with t-test for multiple comparison. Right panel: longitudinal analysis of data. Mixed effect model (restricted maximum likelihood) with Tukey's multiple comparison test. GFP: Green Fluorescent Protein. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 3

Improvement of microbicidal activity by elexacaftor/tezacaftor/ivacaftor. a) Pseudomonas aeruginosa survival in peripheral blood mononuclear cells isolated from healthy donors (HD) and people with cystic fibrosis before (Pre) and after 1 (M1) and 6 (M6) months of therapy. CFU recovered at the end of infection (t₀) and 60 min after infection (t₆₀). The average slopes are reported above the t₀–t₆₀ connecting lines. Slopes were calculated with the linear regression model. Wilcoxon test. b) Killing activity at t₆₀. Scattered dot plot with mean±sem. Kruskal–Wallis test with Dunn's multiple comparison. c) Longitudinal analysis of data. Friedman test with Dunn's multiple comparison. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 4

Microbicidal activity of peripheral blood mononuclear cells (PBMCs) against Pseudomonas aeruginosa PAO1 and clinical isolates. a) PBMCs from healthy donors (HD) and people with cystic fibrosis (PWCF) before elexacaftor/tezacaftor/ivacaftor (ETI) therapy (Pre) infected with PAO1. Demographic and clinical characteristics of PWCF are reported in supplementary table S3. t-test. b) PBMCs from PWCF before ETI therapy infected with PAO1 or the clinical isolates AA2 and AA44. One-way ANOVA test with Tukey's multiple comparison test. Scattered dot plot with mean±sem. *: p<0.05.

FIGURE 5

Elexacaftor/tezacaftor/ivacaftor reduces the exuberant oxidative burst and improves microbicidal activity. a) Superoxide anion O⁻₂ production by infected peripheral blood mononuclear cells (PBMCs) as detected by the luminol assay. i) Representative kinetics of O⁻₂ production (relative luminescence units (RLU)). ii) Quantitative analysis of O⁻₂ produced by the indicated samples (area under the curve (AUC)). Scattered dot plot with mean±sem. Kruskal–Wallis test with Dunn's multiple comparison. iii) Longitudinal analysis of data. Friedman test with Dunn's multiple comparison. b) CFU recovered from untreated or diphenyleneiodonium (DPI)-treated PBMCs. i) CFU recovered from PAO1-infected cells. Wilcoxon test. ii) Fold increase of CFU in DPI-treated cells with respect to untreated cells. Scattered dot plot with mean±sem. Kruskal–Wallis test with Dunn's multiple comparison. iii) Longitudinal analysis of data. Friedman test with Dunn's multiple comparison. HD: healthy donors; Pre: before therapy; M1: month 1; M6: month 6. *: p<0.05; **: 0.01; ***: p<0.001.

FIGURE 6

Elexacaftor/tezacaftor/ivacaftor rapidly reduces the high level of NADPH oxidase 2 (NOX2) activation. Analysis of the p47^phox and phosphorylated p47^phox (p-p47^phox) NOX2 subunits in monocytes from healthy donors (HD; n=6) and people with cystic fibrosis before (Pre; n=6) and after 1 month of therapy (M1; n=6). a) Representative blots of cell lysates from non-infected and PAO1-infected monocytes. b, c) Quantitative analysis of b) p47^phox and c) p-p47^phox. Data are presented as mean±sem. Brown–Forsythe ANOVA test with t-test for multiple comparison. GAPDH: glyceraldehyde 3-phosphate dehydrogenase (loading control). *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 7

Elexacaftor/tezacaftor/ivacaftor decreases interleukin (IL)-6 production by lipopolysaccharide (LPS)-treated cells. IL-6 in supernatants of cystic fibrosis peripheral blood mononuclear cells (PBMCs) before (Pre) and after 1 month of treatment (M1) at baseline (untreated) and after LPS induction. Two-way ANOVA with Bonferroni multiple comparison test. **: p<0.01; ****: p<0.0001.