The Inhibitory Effect of Curosurf® and Alveofact® on the Formation of Neutrophil Extracellular Traps

Abstract

Background: Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.

Methods: The effect of two natural surfactants (Alveofact^® and Curosurf^®) on spontaneous and phorbol-12-myristate-13-acetate-induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.

Results: Both surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.

Conclusions: The described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.

Keywords: alveofact®; anti-inflammatory; curosurf®; neutrophil extracellular traps; neutrophil granulocytes; pulmonary surfactant.

Figure 1

Neutrophils are not altered by animal derived surfactants. (A, B) Treatment with surfactant does not change survival of neutrophils. (A) Cells were treated and stained with PI and Annexin-V. Double negative cells are counted as living cells and displayed as mean+-SD of three independent experiments with no significant changes between untreated and unstimulated cells versus cells treated with surfactant at different timepoints and different doses. (B) Representative blot of 30 min pretreatment with surfactant. (C) Production of reactive oxygen species measured with Dihydrorhodamin-123. Treatment with surfactant does not alter the ability of neutrophils to produce ROS. (D) Neutrophils show no change in activation compared to untreated control. Figure shows 30 min pretreatment and is representative for all other timepoints. Only maximum dose of Curosurf^® shows a decrease of the activation marker CD66b. Significance level was set as p<0.05 (*<0.05, **<0.005, ***<0.0005, ****<0.0001).

Figure 2

Curosurf^® reduces PMA-induced formation of NETs. Neutrophils were incubated with Curosurf^® at three different time points in relation to activation with 100 nm PMA, and then afterwards the NET-specific components were measured in three different assays. Cells that were untreated but stimulated with 100 nm PMA served as positive control in each assay. (A–C) There was a trend toward a dose- and time-dependent decrease in NE, but only the highest dose of 1 mg/ml paired with the longest incubation time of 30 min showed a significant result. (D–F) Proportionate intensity of the blue color emitted by TMB as a chromogenic substrate represented the amount of MPO and showed a significant decrease of NETs for the highest dose at each time point. (G–I) SYTOX Orange assessment of the amount of cfDNA. Significantly less cfDNA was observed in almost every variant of time and dose.

Figure 3

Alveofact^® reduces extracellular NE, MPO, and cfDNA. Neutrophils were incubated with Alveofact^® at three different time points in relation to activation with 100 nm PMA, and then afterwards the NET-specific components were measured in three different assays. Cells that were untreated but stimulated with 100 nm PMA served as positive control in each assay. (A–C) Concentrations of 250 µg/ml and 2.5 mg/ml could reduce the amounts of NE significantly at all time points. (D–F) The amounts of MPO were approximately as low as that in the untreated control when 2.5 mg/ml Alveofact^® was applied at 30 min prior to simultaneous activation with PMA. (G–I) The levels of cfDNA were about as high as that in the untreated and unstimulated control, regardless of timing and concentration of Alveofact^®.

Figure 4

Immunostaining confirms the time- and dose-dependent suppression of NETosis. (A) Neutrophils were activated with PMA 30 min before, simultaneously, or 10 min after treatment with the highest dose of Curosurf^® (1 mg/ml) or Alveofact^® (2.5 mg/ml), and immunostained for H3cit (red) and for DNA with DAPI (blue). Treatment 10 min after stimulation showed no suppression of NETosis and a comparable picture to the positive control (B), while the 0- and 30-min time points showed suppressed NETosis. (B) Negative control with untreated and unstimulated cells had no sign of NETosis. PMA-stimulated cells as positive control showed H3cit formation. (C) Neutrophils were treated with three different concentrations of Curosurf^® (0.001, 0.1, and 1 mg/ml) and Alveofact^® (0.025, 0.25 and 2.5 mg/ml) 30 min before activation with PMA and were immunostained for NE (red) and MPO (green), and stained for DNA with DAPI (blue). The suppression of NETosis was strongest in samples with the highest dose of surfactant and was barely or not detectable at the lower doses. (D) Negative controls for NE/MPO showed intact untreated and unstimulated cells with only slight NE signal; positive controls with PMA-stimulated cells showed massive NET formation with explicit NE and MPO signals co-localized to NET DNA (×40 magnification).

Figure 5

Decrease of P2Y6 Receptor and Calcium mobilization after treatment without change in intracellular PAD4 levels. (A) Neutrophils were treated as described before. P2Y6 was measured by flow cytometry with a FITC-conjugated antibody. Treatment with Alveofact^® decreases extracellular expression of the P2Y6 receptor independent of timing or dosing, while Curosurf^® does not exhibit this effect in every dosing. (B) Calcium levels at 3 h after treatment were used. Data were normalized to stimulated PMA control due to the heterogeneity in basic calcium levels and the response of the neutrophils to stimulation. Results are similar to the findings of the P2Y6 receptor expression. (C) Intracellular PAD4 levels were determined by ELISA using cell lysates of 1×10⁶ cells. Interestingly, PAD4 levels did not change after treatment with surfactant compared to PMA-stimulated neutrophils. Significance level was set as p<0.05 (*<0.05, **<0.005,***<0.0005, ****<0.0001).