Inhibiting CFTR through inh-172 in primary neutrophils reveals CFTR-specific functional defects

Abstract

The lungs of people with cystic fibrosis (PwCF) are characterized by recurrent bacterial infections and inflammation. Infections in cystic fibrosis (CF) are left unresolved despite excessive neutrophil infiltration. The role of CFTR in neutrophils is not fully understood. In this study, we aimed to assess which antimicrobial functions are directly impaired by loss of CFTR function in neutrophils. In order to do so, we used a specific inhibitor of CFTR ion channel activity, inh-172. CF neutrophils from PwCF harboring severe CFTR mutations were additionally isolated to further discern CFTR-specific functional defects. We evaluated phagocytosis, reactive oxygen species (ROS) production, neutrophil elastase (NE) and myeloperoxidase (MPO) exocytosis and bacterial killing. The inh-172 model identified decreased acidification of the phagosome, increased bacterial survival and decreased ROS production upon stimulation. In PwCF neutrophils, we observed reduced degranulation of both NE and MPO. When co-culturing neutrophils with CF sputum supernatant and airway epithelial cells, the extent of phagocytosis was reduced, underscoring the importance of recreating an inflammatory environment as seen in PwCF lungs to model immune responses in vitro. Despite low CFTR expression in blood neutrophils, functional defects were found in inh-172-treated and CF neutrophils. The inh-172 model disregards donor variability and allows pinpointing neutrophil functions directly impaired by dysfunctional CFTR.

Keywords: CFTR; Cystic fibrosis; Inh-172; Neutrophils; Phagocytosis.

Fig. 1

Detection of CFTR expression in primary non-CF neutrophils. (A) Schematic view of the two PCR products generated by nested PCR to detect CFTR mRNA expression on generated CFTR cDNA. (B)CFTR cDNA from neutrophils of a representative healthy donor, HEK293T-overexpressing CFTR (positive control), and negative control (water) was amplified using Taq Polymerase in the first round of PCR (PCR1). (C) A second PCR (PCR2) was performed on the purified PCR1 amplicon. The obtained PCR2 amplicon was subjected to Sanger sequencing to confirm the correct CFTR sequence (Suppl. Figure 1). (D) CFTR staining of primary neutrophils. Immunofluorescence images show primary neutrophils from a healthy donor (top row) and a PwCF homozygous for the G542X mutation (bottom row). Samples were stained for CFTR (yellow) and counterstained with the nuclear stain DAPI (cyan) in the first two columns. The last two columns show immunofluorescence from the secondary antibody only, serving as a control in the third column, and combined with DAPI in the fourth column. Images from six additional healthy donors are provided in Supp. Figure 2. The scale bar represents 10 μm.

Fig. 2

Effect of CFTR dysfunction on phagocytosis by neutrophils. Representative microscopic picture of healthy donor neutrophils treated with (A) vehicle or (B) inh-172 and incubated for 2 h with Staphylococcus aureus bioparticles. Green color indicates calcein staining; red color indicates pHrodo labelling. The size of the scalebar is 200 μm. (C) Healthy donor neutrophils were pre-incubated for 30 min with inh-172 (CFTR-inhibitor) or DMSO (vehicle) and labelled with calcein, whereupon the cells were primed for 10 min with buffer or fMLP and exposed to pHrodo^TM-labelled S. aureus bioparticles. The cells were subsequently microscopically imaged for 2–3 h. Each data point represents the median of the percentage of pHrodo-positive cells. (D) Healthy donor neutrophils were pre-incubated for 30 min with inh-172 or vehicle (DMSO), followed by exposure to serum-opsonized Flash Red fluorescent beads. Each data point is the median percentage of neutrophils containing at least one bead after 2–3 h of incubation. (E) Neutrophils from healthy donors (CTR) or PwCF were labelled with calcein, whereupon the cells were primed for 10 min with buffer or fMLP and exposed to S. aureus bioparticles. The cells were subsequently microscopically imaged for 2–3 h. Each data point represents the median of the percentage of pHrodo-positive cells. The legend for each symbol of PwCF is displayed in Table 3. Supp. Figure 3 shows the subdivision per mutation class. Graphs represent the data as median ± interquartile range. Statistical differences were determined using the Wilcoxon matched-pairs signed rank test for inh-172 data and the Kolmogorov-Smirnov test for PwCF data.

Fig. 3

Effect of CFTR inhibition on actin polymerization by neutrophils. Neutrophils from healthy donors were incubated in the presence or absence of inh-172 (50 µM, 30 min), whereupon the cells were exposed for 30 s to a chemoattractant. The cells were then fixed, permeabilized and stained with fluorescently labelled phalloidin, which binds to polymerized actin. Median fluorescence intensity (MFI) of phalloidin staining after exposure to buffer, CXCL8 (IL-8), fMLP, C5a or LTB4. Statistical differences between treatment groups were determined using Wilcoxon matched-pairs signed rank test.

Fig. 4

ROS production by CFTR-inhibited and PwCF neutrophils. Reactive oxygen species (ROS) production, quantified using a luminol-based chemiluminescence assay, by neutrophils from healthy donors (CTR), with or without CFTR-specific inhibition (‘inh-172’ or ‘DMSO’, respectively) or from people with cystic fibrosis (PwCF) was induced with medium, N-formyl-methionyl-phenylalanine (fMLP), lipopolysaccharide (LPS) or peptidoglycan (PGN). Tumor necrosis factor-alpha (TNF-α) was added as a priming agent to enhance neutrophil function. Peak luminescence is shown for medium, fMLP, LPS, PGN. Baseline ROS production without TNF-α for the (A) inh-172 model and (B) PwCF compared to CTR. ROS production with TNF-α priming for the (C) inh-172 model and (D) PwCF compared to CTR. Statistical differences were determined using Wilcoxon matched-pairs signed rank test for inh-172 data and Kolmogorov-Smirnov test for PwCF data. Supp. Figure 4 shows the subdivision per mutation class. RLU: relative light units.

Fig. 5

Exocytosis of myeloperoxidase and neutrophil elastase in CFTR-inhibited and PwCF neutrophils. (A, C) Healthy donor neutrophils were CFTR-inhibited with inh-172 or DMSO as control or (B, D) neutrophils from CF patients (PwCF) and healthy donors (CTR) were stimulated for 2 h with medium, fMLP, or LPS, whereupon the supernatant was collected and the concentration of myeloperoxidase (MPO – A, B) and neutrophil elastase (NE – C, D) was determined by ELISA. Statistical differences were determined using Wilcoxon matched-pairs signed rank test for inh-172 data and Kolmogorov-Smirnov test for PwCF data. Separation of the results according to homozygous and heterozygous groups is shown in Suppl. Figure 5.

Fig. 6

Bacterial killing by CFTR-inhibited neutrophils. Healthy donor neutrophils were CFTR-inhibited with 50 µM of inh-172 or incubated in 0.5% DMSO as control, and incubated with E. coli at an MOI 3 for 2 h. After incubation, the cell lysates (intracellular) and supernatants (extracellular) were plated to determine the number of bacteria surviving by quantifying the number of colony-forming units (CFU) per mL solution plated on agar plates. Statistical differences were determined using Kolmogorov-Smirnov test.

Fig. 7

Effect of pre-exposure to CF lung micro-environmental stimuli on neutrophil phagocytosis and viability. Phagocytosis Assays (A, B): Wells were pre-treated with either 0.02% (v/v) DTT or 20% CF sputum supernatant (CF sp sup) and then exposed to either inh-172 or DMSO for 2–3 h. Healthy donor neutrophils were pre-incubated with inh-172 or DMSO for 30 min, then added to the treated wells for 2 h. Neutrophils were collected, centrifuged, and replated for the phagocytosis assay, either primed with buffer (A) or fMLP (B) and subsequently exposed to pHrodo^TM-labelled S. aureus bioparticles for 3 h. Co-culture and Phagocytosis Assays (C, D): Epithelial cells were incubated with 0.02% (v/v) DTT or 20% CF sputum supernatant (CF sp sup) and either inh-172 or DMSO for 2–3 h. Healthy donor neutrophils, pre-incubated with inh-172 or DMSO for 30 min, were then added to the epithelial cell cultures for 2 h. After co-culture, neutrophils were collected, centrifuged, and replated for the phagocytosis assay. Neutrophils were primed with either buffer (C) or fMLP (D) and exposed to S. aureus bioparticles for 3 h. Phagocytosis was assessed by imaging and calculating the percentage of pHrodo-positive cells. Statistical differences were determined using paired t tests. Abbreviations: DTT: Dithiothreitol; CF sp sup: cystic fibrosis sputum supernatant.