Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration

Abstract

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.

Keywords: Elastase; Neutrophil extracellular traps; Resveratrol.

Figure 1

Resveratrol decreases the release of NETs by neutrophils stimulated with PMA, LPS, Leishmania amazonensis, and L. major. Neutrophils (5 × 10⁴/well) were pretreated with different concentrations of resveratrol (RSV) for 30 min and then stimulated with (a) 100 nM PMA, (b) 10 µg/ml LPS, (c) Leishmania amazonensis, (d) L. major or (e) fixed L. major (1NØ: 1 Leishmania ratio) for 4 h. The amount of NET-DNA in the supernatant was quantified using PicoGreen and normalized to the control. Results expressed as n fold related to control, mean ± SEM of at least 3 donors. (f) Immunofluorescence of neutrophils labeled with anti-elastase antibody (green) and DAPI (blue). Bar: 50 µm. *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 2

Resveratrol inhibits the release of NETs induced by ROS-independent stimuli. Neutrophils (5 × 10⁴/well) pretreated with 50 µM of resveratrol (RSV) for 30 min were then stimulated for 4 h with (a) 100 ng/ml IL-8 or (b) 10 min with Leishmania amazonensis promastigotes (1NØ: 1 Leishmania ratio). The amount of NET-DNA in the supernatants was quantified using PicoGreen. Results shown as n-fold related to control, expressed as mean ± SEM of 4 (a) and 6 (b) donors. *p < 0.05; ***p < 0.0001.

Figure 3

The effect of resveratrol on the modulation of NETs is not related to signaling through A2A, A2B adenosine receptors, or PKA. Neutrophils (5 × 10⁴/well) were pretreated with 100 nM of the selective antagonists to A2A receptor (SCH 58261) or A2B receptor (MRS 1754) for 15 min, followed by treatment with 50 µM of RSV for 30 min and stimulation with (a, e) 100 nM PMA, (b) Leishmania major for 4 h or (c) L. amazonensis for 10 min (1NØ: 1 Leishmania ratio). (d) Neutrophils were pretreated with RSV and PKA inhibitor for 30 min and then stimulated with 10 µg/ml of LPS. NET-DNA quantification was performed in the supernatant by PicoGreen. Results are shown as n-fold related to control, expressed as mean ± SEM of 3 (a, b), 7 (c), 4 (d), and 5 (e) donors. *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 4

Modulation of the H₂O₂-MPO axis in human neutrophils treated with resveratrol. (a) Kinetics of hydrogen peroxide detection after pretreating neutrophils (5 × 10⁵/well) with 50 µM of RSV for 30 min followed by the addition of Amplex Red probe and stimulated or not with 100 nM of PMA or 10 µg/ml of LPS. Result from a representative donor out of 4 tested. (b) Area under the curve (AUC) of the amount of ROS released by neutrophils stimulated from 4 donors. (c) The activity of intracellular MPO was quantified in neutrophils upon the 1 h addition of RSV. Results shown as Amplex Red fluorescence and MPO activity/mg protein, expressed as mean ± SEM of 4 (b, c) donors. *p < 0.05; **p < 0.001, ***p < 0.0001.

Figure 5

Resveratrol modulates the localization of neutrophil elastase. Adherent neutrophils were pretreated or not with 50 μM RSV for 30 min and subsequently stimulated with 100 nM PMA for (a) 1 h or (b) 2 h. Cells were stained with anti-elastase antibody (green) and DAPI (blue). (a) Maximum intensity projection (MIP) of 12 optical slices along the Z axis. (b) Orthogonal view of 9 optical slices along the Z axis. Bar: 20 μm.