Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses

Abstract

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr^-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca²⁺ homeostasis led to increased intracellular concentrations of Ca²⁺ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca²⁺ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca²⁺ homeostasis (via CFTR potentiation or by the regulation of Ca²⁺ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Figure 1. Neutrophils are essential to control B. cenocepacia infection, but CF neutrophils show defective bacterial killing

(A) C57BL/6 (WT) or Cftr^−/− mice were i.t. infected with 5×10⁶ CFU of B. cenocepacia, the infection was followed up to 8 days post-infection, and graph shows survival of mice in each group (n=6 per group). (B) WT mice were pretreated intravenously with 250 µg of anti-Ly6G (PMN) or anti-Gr1 (PMN/Mo) purified antibody 24 h before bacterial infection. Animals were infected with B. cenocepacia and survival was monitored for 8 days. (n=10 mice per group). (C) WT or Cftr^−/− bone marrow neutrophils were infected with B. cenocepacia (Bc), P. aeruginosa (Pa01), Staphylococcus aureus (Sa) or nontypeable Haemophilus influenzae (NTHI) (MOI of 10), and CFU were enumerated at 3 h (n≥4 mice). (D) Purified neutrophils from non-CF or CF patients were infected with Bc, Pa01, Sa or NTHI (MOI=10), then CFU were enumerated at 3 h (Bc n=11, Pa01 n=7, Sa n=8, NTHI n=6). Asterisks indicate p value for statistical significance (**p<0.01, ***p<0.001, ****p<0.0001).

Figure 2. ROS-dependent antimicrobial mechanisms are impaired in CF neutrophils

(A) Neutrophils purified from healthy donors were stained with APF, treated with 10⁻⁵ M of PPQ-102 for 20 minutes at 37 °C and stimulated with 5×10⁻⁸ M PMA or 1µg/ml LPS. hROS products including hypochlorite (HClO⁻), hydroxyradical (^•OH), and peroxynitrite (ONOO⁻) were measured with a fluorescence plate reader (488/515 nm). (B) hROS was measured in CF or non-CF neutrophils stimulated with PMA or LPS or by using (C) 2.5×10⁻⁴ M of pyocianin, S. aureus or NTHI (MOI=10) as indicated. (D) CF and non-CF neutrophils were pretreated with 10⁻⁵ M DPI for 10 minutes followed by the infection with B. cenocepacia (MOI=10). CFU were quantified by serial dilutions and plating. Graphs are representative of 3 independent experiments. Asterisks indicate p value for statistical significance (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001).

Figure 3. NETosis is partially impaired in neutrophils from CF patients

Non-CF or CF neutrophils were stimulated with (A) 5×10⁻⁸ M PMA or (B) 1µg/mL LPS for 3 h. Slides were fixed and stained with rabbit anti-NE plus goat anti-rabbit IgG Alexa Fluor 568 and DAPI. The images were acquired with a fluorescence microscope at 40× (scale bars represent 50 µm). Images are representative of five independent experiments.

Figure 4. CF neutrophils show distinct pattern of activation and inability to form NETs

Non-CF or CF neutrophils were stimulated with 5×10⁻⁸ M PMA for 1–3h, then processed for scanning electron microscopy (SEM). The scale bars represents 10 µm. Images are representative of 3 independent experiments.

Figure 5. CF neutrophils fail to release NETs when infected with B. cenocepacia

Non-CF or CF neutrophils were infected with B. cenocepacia expressing red fluorescence protein (RFP) (MOI=10) for 3 h, (A) The slides were fixed and stained with rabbit anti-NE plus chicken anti-rabbit IgG Alexa Fluor 488 and DAPI. White arrows indicate bacteria that co-localize with NETs (scale bars represent 20 µm). (B) Neutrophils were infected with B. cenocepacia for 3 h, then fixed and processed for visualization by SEM. Bacteria are pseudo colored red; neutrophils are pseudo colored green; and DNA is pseudo colored blue. Images were taken from one out of three representative experiments. Scale bars indicate 20 µm.

Figure 6. Calcium homeostasis is dysregulated in Cftr^/− neutrophils

WT, Cftr^−/− or p47^phox−/− mouse bone marrow neutrophils were loaded with Fluo-4, cells were aliquoted in HBSS buffer containing Ca²⁺, (A) WT or Cftr^−/− neutrophils were stimulated with 10⁻⁷ M of IL-8, (B) 10⁻⁷ M of C5a, (C) 10⁻⁷ M of PAF or (D) 10⁻⁷ M of fMLP. (E) Neutrophils were stimulated with fMLP in free Ca²⁺ media. (F) Cells were pretreated with 10⁻⁵ M of PPQ-102 for 20 minutes, then stimulated with fMLP. WT or p47^phox−/− neutrophils were stimulated with 10⁻⁷ M of fMLP (G) with Ca²⁺ or (H) without Ca²⁺, the intracellular free Ca²⁺ levels were measured by FACS for 250 seconds, each second of the kinetics represents around 10³ events. The graphs show MFI±SEM of 3 mice each one, data representative of 3 independent experiments.

Figure 7. Potentiation of CFTR channel reduces intracellular Ca²⁺ levels, which results in improved antimicrobial killing of CF neutrophils

Human CF and non-CF neutrophils (A-C) were loaded with fluorescent Ca²⁺-detecting dye Fluo-4, in Ca²⁺ and Mg²⁺ containing media, and then stimulated with 10⁻⁸M fMLP, in the presence of EGTA (B) or DPI (C). The accumulation of intracellular free Ca²⁺ was assessed by flow cytometry for 340 seconds by measuring the kinetic of fluorescence emission of Fluo-4. The graphs shown are representative of 3 independent experiments. (D) Non-CF and CF neutrophils were infected with B. cenocepacia (MOI=10) for 45 minutes and seeded in 24-well plates. Infected neutrophils were treated with 3×10⁻⁶M EGTA or 10⁻⁴M 2-APB for 3 hours at 37 °C. Cells were lysed and CFU were calculated (n=5 donors). (E) Non-CF and CF neutrophils were pretreated with VX-770 before loading with Fluo-4 in HBSS media containing Ca²⁺ and Mg²⁺. Cells were stimulated with fMLP and the kinetic of Ca²⁺ measurement recorded by flow cytometry. Graphs are representative of 3 independent experiments. (F) Non-CF and CF neutrophils were treated with VX-770 for 60 minutes, following by the infection with B. cenocepacia (MOI=10) for 45 minutes, then infected cells were incubated 3 hours and CFU enumerated (n=3 donors). Asterisks indicate statistically significant differences between groups (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001).