IADR Abstract Archives
Nupharidine Derivatives Prime Neutrophils Against Aggressive Periodontitis Pathogen – Aggregatibacter actinomicetemcomintans
Objectives: Aggressive periodontitis (AgP) is an infectious condition involved in rapid tissue destruction around teeth, and has been associated with Aggregatibacter actinomycetemcomitans JP2 clone. A major effector cell in the innate immune response to this pathogen is the neutrophil. Furthermore, genetic syndromes with impaired neutrophil function exhibit AgP. Recently, studies showed that the thioalkaloid Nupharidine (6-hydroxythiobinupharidine or 6-hydroxythiobinuphlutine), isolated from Nuphar Lutea (Water Lily), prompts antimicrobial activity in macrophages. The aim of the current study was to test the influence of purified Nupharidine on neutrophil function during JP2 infection.
Methods: Neutrophils were differentiated from HL-60 cells in vitro using DMSO. The mature neutrophils were incubated with Nupharidine (test group) or vehicle only (control group), followed by inoculation with live JP2 for 90min. Following incubation bacterial survival was tested using blood agar viable count. Neutrophil function evaluation included: necrosis/apoptosis (using annexin/PI stain and flow cytometry); Reactive oxygen species (ROS) production (using DCFH and fluorometric plate reader); phagocytosis (using FITC labeled JP2 and flow cytometry) and Neutrophil extracellular traps (NETs) production (using MNase/sytox and fluorometric plate reader). All experiments were repeated at least 3 times and included triplicates of each group.
Results: ROS production after treatment with Nupharidine was significantly higher with (p<000.1) or without (p=0.016) the presence of JP2. NETs doubled in value with Nupharidine and Phagocytosis was tripled. Furthermore, Nupharidine reduced viable count of JP2. Interestingly, Nupharidine without neutrophils had no effect on bacterial viability. On the other hand, higher concentrations than 12.5μg/ml Nupharidine had a cytotoxic effect on neutrophils (both necrosis and apoptosis).
Conclusions: Nupharidine does not have a direct anti-microbial effect on JP2. However, Nupharidine showed a positive effect on the behavior of neutrophils during JP2 infection, showcasing priming properties. Further research is needed in order to evaluate the clinical potential of the effect of Nupharidine on AgP.
Methods: Neutrophils were differentiated from HL-60 cells in vitro using DMSO. The mature neutrophils were incubated with Nupharidine (test group) or vehicle only (control group), followed by inoculation with live JP2 for 90min. Following incubation bacterial survival was tested using blood agar viable count. Neutrophil function evaluation included: necrosis/apoptosis (using annexin/PI stain and flow cytometry); Reactive oxygen species (ROS) production (using DCFH and fluorometric plate reader); phagocytosis (using FITC labeled JP2 and flow cytometry) and Neutrophil extracellular traps (NETs) production (using MNase/sytox and fluorometric plate reader). All experiments were repeated at least 3 times and included triplicates of each group.
Results: ROS production after treatment with Nupharidine was significantly higher with (p<000.1) or without (p=0.016) the presence of JP2. NETs doubled in value with Nupharidine and Phagocytosis was tripled. Furthermore, Nupharidine reduced viable count of JP2. Interestingly, Nupharidine without neutrophils had no effect on bacterial viability. On the other hand, higher concentrations than 12.5μg/ml Nupharidine had a cytotoxic effect on neutrophils (both necrosis and apoptosis).
Conclusions: Nupharidine does not have a direct anti-microbial effect on JP2. However, Nupharidine showed a positive effect on the behavior of neutrophils during JP2 infection, showcasing priming properties. Further research is needed in order to evaluate the clinical potential of the effect of Nupharidine on AgP.