Dexamethasone Inhibits S. aureus-Induced Neutrophil Extracellular Pathogen-Killing Mechanism, Possibly through Toll-Like Receptor Regulation

Abstract

Neutrophils release neutrophil extracellular traps (NETs) in a pathogen-killing process called NETosis. Excessive NETs formation, however, is implicated in disease pathogenesis. Therefore, to understand how NETosis is regulated, we examined the effect of dexamethasone (DXM), an anti-inflammatory drug, on this process and the role of toll-like receptors (TLRs). We stimulated human neutrophils with phorbol 12-myristate 13-acetate (PMA) or Staphylococcus aureus (S. aureus) and quantified NETs formation. We also examined the effect of DXM on the bactericidal effect of NETs and the role of reactive oxygen species (ROS) and nuclear factor (NF)-κB in DXM-regulated NETosis. DXM significantly inhibited S. aureus-induced NETosis and extracellular bacterial killing. ROS production and NF-κB activation were not involved in DXM-regulated NETosis. TLR2 and TLR4, but not TLR5 or TLR6, modified S. aureus-induced NETs formation. Neither DXM nor TLRs were involved in PMA-induced NETosis. Furthermore, TLR2 and TLR4 agonists rescued DXM-inhibited NETosis, and neither TLR2 nor TLR4 antagonists could further inhibit NETosis reduction induced by DXM, indicating that DXM may inhibit NETosis by regulating TLR2 and TLR4. In conclusion, the mechanisms of S. aureus- and PMA-induced NETosis are different. DXM decreases NETs formation independently of oxidant production and NF-κB phosphorylation and possibly via a TLR-dependent mechanism.

Keywords: PMA; S. aureus; TLRs; dexamethasone; neutrophil extracellular traps.

Figure 1

Phorbol 12-myristate 13-acetate (PMA) and Staphylococcus aureus (S. aureus) stimulate neutrophil extracellular traps (NETs) formation in human neutrophil. Human neutrophil suspended in media were treated with PMA (50 nM) or S. aureus at MOI of 10. Human neutrophil without treatment (N) was used as control. NETs formation was measured at 2 h. (A) Neutrophils were labelled with 4′, 6′-diamidino-2-phenylindole (DAPI) to identify DNA (blue) and with antibodies to identify neutrophil histone (green) and elastase (red). PMA and S. aureus-induced NETs formation. (B) S. aureus (indicated with arrow) were trapped in NETs and released when NETs structure was degraded by DNase I, as observed by SYTOX green staining Bar: 50 μm.

Figure 2

Dexamethasone (DXM) inhibited neutrophil extracellular traps (NETs) formation induced by Staphylococcus aureus (S. aureus) but not that induced by phorbol 12-myristate 13-acetate (PMA). Human neutrophils suspended in media were pretreated with or without DXM (10 µM) for 2 h, and then NETs formation 2 h after stimulation with PMA or S. aureus was examined using the membrane-impermeable DNA-binding dye SYTOX green and quantified by Quant-iTPicoGreen double-stranded deoxyribonucleic acid assay kit. Neutrophils without any stimulation or treated with DMSO (0.1% v/v) were used as control. (A) DMSO (0.1% v/v) did not affect NETs formation. While dexamethasone did not modify NETs formation induced by PMA, it markedly inhibited that induced by S. aureus. Several typical NETs were indicated with arrows. Bar: 50 μm. (B) Quantification of extracellular DNA confirmed that it inhibited NETs formation induced by S. aureus but not that induced by PMA. Neutrophils (1 × 10⁵cells/well in 100 µl) were stimulated to form NETs, and the mean value of NETs amount in five replicated wells was adopted. The assay was repeated for three times with bloods from three different donors; error bars represent SEM. *p < 0.05 by ANOVA with Bonferroni’s post-test.

Figure 3

Dexamethasone (DXM) inhibited the bactericidal efficacy of neutrophil extracellular traps (NETs). Neutrophils were pre-incubated with or without DXM for 2 h and then treated with 50 nM phorbol 12-myristate 13-acetate (PMA) or left untreated for another 2 h. One hour after addition of bacteria, colony-forming units (CFU) were determined by overnight incubation at 37°C following serial dilution. Zero killing was defined by control samples consisting of only media. Killing efficacy was determined by subtracting the CFU of indicated treatment from control groups. By using cytochalasin D to abrogate phagocytic killing, dexamethasone was found to significantly inhibit the bactericidal efficacy of NETs. However, dexamethasone could not inhibit PMA-activated bactericidal efficacy of NETs. The assay was repeated for nine times, each case in three wells; error bars represent SEM. *p < 0.05 by ANOVA with Bonferroni’s post-test.

Figure 4

Activation of reactive oxygen species (ROS) or nuclear factor (NF)-κB was not involved in dexamethasone-regulated NETosis. Neutrophils were pretreated with or without dexamethasone (DXM) for 2 h and then stimulated with Staphylococcus aureus (S. aureus) for 1 h. ROS production were determined by dichlorofluorescein diacetate fluorescence and NF-κB activation were determined by Western blot. (A) S. aureus infection elicited significant neutrophil oxidative burst, but DXM treatment neither increased nor decreased this response. Data represent mean ± SEM of triplicate experiments. (B) NF-κB was activated when stimulated with S. aureus but not modified by dexamethasone. (C) Quantification showed that p-NF-κB (p65) expression was significantly higher when the cells were stimulated with S. aureus, but this effect was not modified by dexamethasone. Data represent mean ± SEM of triplicate experiments, *p < 0.05 by Student’s t-test.

Figure 5

Toll-like receptors (TLRs) were involved in Staphylococcus aureus (S. aureus)-induced but not phorbol 12-myristate 13-acetate (PMA)-induced neutrophil extracellular traps (NETs) formation. Neutrophils were pretreated with TLRs agonist or antagonist, followed by PMA or S. aureus stimulation. NETs formation was quantified by Quant-iT PicoGreen double-stranded deoxyribonucleic acid assay kit. (A) None of TLR2 agonist (HKLM), TLR4 agonist (LPS), TLR5 agonist (FSL-ST), and TLR6 (FSL-1) agonist could induce NETs formation. (B) Treatment with TLR2 agonist (HKLM) and TLR4 agonists (LPS) significantly enhanced NETosis, and blocking TLR2 and TLR4 with neutralizing antibodies significantly reduced S. aureus-induced NETs formation. None of TLR5 agonist (FSL-ST), TLR6 agonist (FSL-1), and TLR5 and TLR6 neutralizing antibodies was involved in S. aureus-induced NETs formation. (C) TLRs were not involved in PMA-induced NETs formation. The assay was repeated for three times, each case in five wells, error bars represent SEM. Compared to S. aureus or PMA stimulation, *p < 0.05, ns = p > 0.05 by Student’s t-test.

Figure 6

Dexamethasone (DXM) may modulate Staphylococcus aureus (S. aureus)-induced neutrophil extracellular traps (NETs) formation through toll-like receptor (TLR)2 and TLR4. (A) Neutrophils were pretreated with HKLM (TLR2 agonist) and LPS (TLR4 agonist), followed by DXM treatment and S. aureus stimulation. NETs formation was quantified by Quant-iTPicoGreen double-stranded deoxyribonucleic acid (dsDNA) assay kit. Both HKLM and LPS rescued dexamethasone-reduced NETs formation. (B) Neutrophils were pretreated with TLR2 and TLR4 antagonist, followed by DXM treatment and S. aureus stimulation. NETs formation was quantified by Quant-iTPicoGreen dsDNA assay kit. Neither TLR2 nor TLR4 antagonist could further decrease DXM induced NETosis reduction. The assay was repeated for three times, each case in five wells, error bars represent SEM. *p < 0.05 by Student’s t-test.