Treatment with rhDNase in patients with cystic fibrosis alters in-vitro CHIT-1 activity of isolated leucocytes

Abstract

Recent data suggest a possible relationship between cystic fibrosis (CF) pharmacotherapy, Aspergillus fumigatus colonization (AC) and/or allergic bronchopulmonary aspergillosis (ABPA). The aim of this study was to determine if anti-fungal defence mechanisms are influenced by CF pharmacotherapy, i.e. if (1) neutrophils form CF and non-CF donors differ in their ability to produce chitotriosidase (CHIT-1); (2) if incubation of isolated neutrophils with azithromycin, salbutamol, prednisolone or rhDNase might influence the CHIT-1 activity; and (3) if NETosis and neutrophil killing efficiency is influenced by rhDNase. Neutrophils were isolated from the blood of CF patients (n = 19; mean age 26·8 years or healthy, non-CF donors (n = 20; 38·7 years) and stimulated with phorbol-12-myristate-13-acetate (PMA), azithromycin, salbutamol, prednisolone or rhDNase. CHIT-1 enzyme activity was measured with a fluorescent substrate. NETosis was induced by PMA and neutrophil killing efficiency was assessed by a hyphae recovery assay. Neutrophil CHIT-1 activity was comparable in the presence or absence of PMA stimulation in both CF and non-CF donors. PMA stimulation and preincubation with rhDNase increased CHIT-1 activity in culture supernatants from non-CF and CF donors. However, this increase was significant in non-CF donors but not in CF patients (P < 0·05). RhDNase reduced the number of NETs in PMA-stimulated neutrophils and decreased the killing efficiency of leucocytes in our in-vitro model. Azithromycin, salbutamol or prednisolone had no effect on CHIT-1 activity. Stimulation of isolated leucocytes with PMA and treatment with rhDNase interfered with anti-fungal defence mechanisms. However, the impact of our findings for treatment in CF patients needs to be proved in a clinical cohort.

Keywords: fungal; human; inflammation; lung; neutrophils.

Figure 1

Chitotriosidase (CHIT)‐1 is secreted after granulocye–macrophage colony‐stimulating factor (GM‐CSF) and zymosan, but not interkeukin (IL)‐8 stimulation. Neutrophils from non‐cystic fibrosis (CF) donors were seeded at 4 × 10⁶ cells/well in round‐bottomed wells (4 × 10⁶ per well) and stimulated with IL‐8 (100 ng/ml), GM‐CSF (1, 10, 100 ng/ml), opsonized zymosan, or 10 nM phorbol myristate acetate (PMA) for 2·5h, 37°C. Activity of CHIT‐1 following stimulation was measured using 4‐methylumbelliferyl‐beta‐D‐N,N',N''‐triacetylchitotriose (4‐MU‐TACT) in a Fluorstar Omega (BMG Labtech, Ortenberg, Germany) and controlled for basal activity in unstimulated wells; n = 6 *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Chitotriosidase (CHIT)‐1 secretion in response to phorbol myristate acetate (PMA) is comparable in cystic fibrosis (CF) and non‐CF donors. Neutrophils from (a) non‐CF donors and (b) CF patients were seeded in round‐bottomed wells (4 × 10⁶ cells/well) and stimulated with 10 nM PMA for 2·5 h, 37°C. Activity of CHIT‐1 was measured using 4‐methylumbelliferyl‐beta‐D‐N,N',N''‐triacetylchitotriose (4‐MU‐TACT) in a Fluorstar Omega (BMG Labtech, Ortenberg, Germany); n = 16, ***P < 0·001.

Figure 3

Chitotriosidase (CHIT)‐1 secretion increases significantly in non‐cystic fibrosis (CF) donors in response to rhDNase and phorbol myristate acetate (PMA) but not in CF patients. Neutrophils (4 × 10⁶ cells/well) from non‐CF donors (n = 6) or CF patients (n = 10) were stimulated with 10 nM PMA for 2·5h at 37°C in the presence of 0.11M rhDNase or vehicle (milliQ water). Activity of CHIT‐1 in supernatants was measured using 4‐methylumbelliferyl‐beta‐D‐N,N',N''‐triacetylchitotriose (4‐MU‐TACT) in a Fluorstar Omega (BMG Labtech, Ortenberg, Germany); *P < 0·05.

Figure 4

Chitotriosidase (CHIT)‐1 increase in activity is not dose‐related to rhDNase in vitro. Non‐cystic fibrosis (CF) neutrophils (4×10⁶ cells/well) were stimulated with 10 M phorbol myristate acetate (PMA) for 3 h at 37°C in the presence or absence of 0·11×10⁻⁶ M, 0·11 × 10⁻⁷ M or 0·11 × 10⁻⁸ M rhDNase or vehicle (Ringer solution), and CHIT‐1 activity was measured using 4‐methylumbelliferyl‐beta‐D‐N,N',N''‐triacetylchitotriose (4‐MU‐TACT) in a Fluorstar Omega (BMG Labtech, Ortenberg, Germany); n = 5, *P < 0·05.

Figure 5

Number of neutrophil extracellular traps (NETs) is reduced in response to rhDNase. Neutrophils from non‐cystic fibrosis (CF) donors (5 × 10⁴ cells/well) were stimulated with 10nM phorbol myristate acetate (PMA) and increasing concentrations (0·04, 0·4 and 4 µg/ml) of rhDNase or vehicle (0·1, 1 and 10% Ringer solution) were added for 2.5h at 37°C. Propidium iodide was added and fluorescence measured at ex544nm/em590 nm using a FluoStar Omega plate reader (BMG Labtech, Ortenberg, Germany). Standard curves were used to determine NET‐numbers; n = 11; *P < 0·05, ***P < 0·001.

Figure 6

RhDNase reduces neutrophil killing efficiency. Aspergillus fumigatus conidia (5 × 10⁵/well) were left for 6 h at 37°C to form hyphae before non‐cystic fibrosis (CF) neutrophils (1 × 10⁶ cells/well) were added. Neutrophils were given 3 h at 37°C to form neutrophil extracellular traps (NETs) before the addition of rhDNase or vehicle control (10% Ringer solution). After 15 min, supernatant was collected and added to MAE‐agar to examine A. fumigatus growth after 24 h at 37°C. A. fumigatus growth was determined as a percentage of growth in the absence of neutrophils; n = 9. *AF versus AF + neutrophils and vehicle, P < 0·05; # AF + neutrophils + rhDNase, P < 0·05.