Lumacaftor (VX-809) restores the ability of CF macrophages to phagocytose and kill Pseudomonas aeruginosa

Abstract

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by chronic bacterial lung infection and excessive inflammation, which lead to progressive loss of lung function and premature death. Although ivacaftor (VX-770) alone and ivacaftor in combination with lumacaftor (VX-809) improve lung function in CF patients with the Gly551Asp and del508Phe mutations, respectively, the effects of these drugs on the function of human CF macrophages are unknown. Thus studies were conducted to examine the effects of lumacaftor alone and lumacaftor in combination with ivacaftor (i.e., ORKAMBI) on the ability of human CF ( del508Phe/ del508Phe) monocyte-derived macrophages (MDMs) to phagocytose and kill Pseudomonas aeruginosa. Lumacaftor alone restored the ability of CF MDMs to phagocytose and kill P. aeruginosa to levels observed in MDMs obtained from non-CF (WT-CFTR) donors. This effect contrasts with the partial (~15%) correction of del508Phe Cl^- secretion of airway epithelial cells by lumacaftor. Ivacaftor reduced the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa. Lumacaftor had no effect on P. aeruginosa-stimulated cytokine secretion by CF MDMs. Ivacaftor (5 µM) alone and ivacaftor in combination with lumacaftor reduced secretion of several proinflammatory cytokines. The clinical efficacy of ORKAMBI may be related in part to the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa by macrophages.

Keywords: Pseudomonas aeruginosa; cystic fibrosis; ivacaftor; lumacaftor; macrophage.

Fig. 1.

Lumacaftor corrects phagocytosis of Pseudomonas aeruginosa by cystic fibrosis [CF (Phe508del/Phe508del)] monocyte-derived macrophages (MDMs). CFU, colony forming units; WT, wild-type. A: lumacaftor (VX-809, 3 µM) increases phagocytosis of P. aeruginosa by CF (○, n = 11) and WT (●, n = 6) MDMs. Lines connect paired observations for each donor. **P < 0.01 vs. control in a 1-sided paired t-test. Phagocytosis of P. aeruginosa by untreated CF MDMs is significantly reduced compared with untreated WT MDMs: ###P < 0.001 in a negative binomial regression model. Mean phagocytosis of P. aeruginosa by CF MDMs treated with VX-809 is 78% of phagocytosis by control WT MDMs, and means between groups are not significantly different (P = 0.38). B: ivacaftor (VX-770, 3 nM–5 µM) reduces lumacaftor (VX-809)-stimulated phagocytosis of P. aeruginosa by CF MDMs. Lines connect paired observations for each donor (n = 4). ****P < 0.0001, **P < 0.01, and *P < 0.05 vs. control in a mixed-effect linear model with treatment as a categorical variable. #Phagocytosis is significantly greater in the lumacaftor (VX-809)-only group than in all other groups (P < 0.05). VX-809 and VX-770 concentrations in µM. C: VX-770 alone significantly reduces phagocytosis of P. aeruginosa by CF MDMs in a dose-dependent manner (P < 0.05 in a linear model with VX-770 as a continuous variable, n = 4 for control and 0.003–0.3 µM VX-770 and n = 3 for 1–5 µM VX-770). D: VX-770 alone significantly reduces phagocytosis of P. aeruginosa by WT MDMs in a dose-dependent manner (P < 0.0001 in a linear model with VX-770 as a continuous variable (n = 6 for control and 0.003–0.3 µM VX-770 and n = 4 for 1–5 µM VX-770).

Fig. 2.

Lumacaftor corrects the ability of CF (Phe508del/Phe508del) MDMs to kill P. aeruginosa. A: lumacaftor (VX-809, 3 µM) increases the ability of CF MDMs (○, n = 7) to kill P. aeruginosa, while it does not significantly increase killing of P. aeruginosa by WT MDMs (●, P = 0.16, n = 6). Lines connect paired observations for each donor. **P < 0.01 vs. control in a 1-sided paired t-test. Killing of P. aeruginosa by untreated CF MDMs is significantly reduced compared with untreated WT MDMs: ###P < 0.001 in a negative binomial regression model. Mean killing of P. aeruginosa by CF MDMs treated with VX-809 is 73% of killing by control WT MDMs, and means between groups are not significantly different (P = 0.47). B: ivacaftor (VX-770, 3 nM–5 µM) reduces lumacaftor (VX-809)-stimulated killing of P. aeruginosa by CF MDMs. Lines connect paired observations for each donor (n = 4). ****P < 0.0001, **P < 0.01, and *P < 0.05 vs. control in a mixed-effect linear model with treatment as a categorical variable. #Killing is significantly greater in the lumacaftor (VX-809)-only group than in all other groups (P < 0.05). VX-809 and VX-770 concentrations in µM. C: there was a trend for VX-770 alone to reduce killing of P. aeruginosa by CF MDMs in a dose-dependent manner, but it did not reach statistical significance (P = 0.053 in a linear model with VX-770 as a continuous variable, n = 4 for control and 0.003–0.3 µM VX-770 and n = 3 for 1–5 µM VX-770). D: VX-770 alone significantly reduces killing of P. aeruginosa by WT MDMs in a dose-dependent manner (P < 0.0001 in a linear model with VX-770 as a continuous variable, n = 6 for control and 0.003–0.3 µM VX-770 and n = 4 for 1–5 µM VX-770).

Fig. 3.

Cytokine secretion is similar in CF and WT MDMs exposed to P. aeruginosa. Unscaled heat map shows log₁₀-transformed P. aeruginosa-induced cytokine secretion (pg/ml) by CF (n = 8 donors) and WT (n = 7 donors) MDMs under control conditions (DMSO, in the absence of lumacaftor and ivacaftor). Red indicates very low cytokine concentrations in the media, and yellow indicates high cytokine levels in the media. Based on Welch’s t-test, there was no significant difference between CF and WT MDMs in the secretion of any of the 30 detected cytokines. Eleven cytokines were below detection. IL8, interleukin (IL) 8; GRO, growth-regulated oncogene; MIP1a and MIP1b, macrophage inflammatory proteins 1α and 1β; TNFa, tumor necrosis factor-α; MDC, macrophage-derived chemokine; MCP1, monocyte chemoattractant protein 1; GCSF, granulocyte colony-stimulating factor (CSF); IL10, IL-10; GMCSF, granulocyte-macrophage CSF; IL1Ra, IL-1 receptor antagonist; IL1b, IL-1β; IL6, IL-6; RANTES, regulated upon activation, normally T-expressed, and presumably secreted; FRACTALKINE, fractalkine; VEGF, vascular endothelial growth factor; IL12p40, IL-12 p40; MCP3, monocyte chemotactic protein 3; PDGFBB, platelet-derived growth factor BB; IP10, interferon (IFN)-γ-induced protein 10; IFNa2, IFNα₂; IL7, IL-7; FGF2, fibroblast growth factor 2; FLT3L, FMS-like tyrosine kinase 3 ligand; EXOTAXIN, exotaxin; IL12p70, IL-12 p70; IFNg, IFN-γ; EGF, epidermal growth factor; IL1a, IL-1α; IL4, IL-4.

Fig. 4.

Ivacaftor suppresses secretion of proinflammatory cytokines. Heat map presents log₂-fold changes (FC) of treatments compared with DMSO control. Blue/black indicates reduced secretion, green/yellow indicates increased secretion, and cyan indicates no difference. Higher concentrations of ivacaftor, alone or in combination with lumacaftor, reduced P. aeruginosa-stimulated cytokine secretion. N, number in each treatment group. False discovery rate-adjusted P values from mixed-effect linear model are as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. DMSO. VX-809 and VX-770 concentrations in µM.