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Updated: Oct 27, 2025
In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy
Published on: August 24, 2018
Methylprednisolone Induces Extracellular Trap Formation and Enhances Bactericidal Effect of Canine Neutrophils
Nicole Steffensen1, Rabea Imker2,3, Simon Lassnig2,3
1Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany.
Abstract:
Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.
Insights
Methylprednisolone boosts neutrophil extracellular trap (NET) formation and enhances the killing of Gram-positive bacteria in dogs. This glucocorticoid improves canine neutrophil antimicrobial activity against specific bacterial infections.
Area of Science:
- Veterinary Immunology
- Microbiology
- Pharmacology
Background:
- Glucocorticoids like methylprednisolone can suppress immune responses.
- Neutrophils combat bacterial infections in dogs via mechanisms including neutrophil extracellular trap (NET) formation.
- The impact of methylprednisolone on canine neutrophil function during bacterial infections is not fully understood.
Purpose of the Study:
- To investigate the effect of methylprednisolone on canine neutrophil extracellular trap (NET) formation.
- To assess the influence of methylprednisolone on the efficiency of canine neutrophils in killing Gram-positive and Gram-negative bacteria.
- To determine the concentration-dependent effects of methylprednisolone on neutrophil antimicrobial activity.
Main Methods:
- Canine neutrophils were isolated from blood and treated with varying concentrations of methylprednisolone over time.
- Neutrophil extracellular trap (NET) formation was assessed using immunofluorescence microscopy.
- Bacterial survival rates (Staphylococcus pseudintermedius, Streptococcus canis, Escherichia coli) were determined in the presence of stimulated neutrophils.
- Levels of free DNA and nucleosomes in supernatants were analyzed as markers for NET formation.
Main Results:
- Methylprednisolone (62.5 and 625 µg/mL) enhanced neutrophil killing of Gram-positive bacteria (S. pseudintermedius, S. canis).
- No significant effect on the killing of Gram-negative Escherichia coli was observed.
- Increased free DNA was detected with methylprednisolone stimulation, particularly with S. canis and E. coli.
- Methylprednisolone at 62.5 µg/mL was identified as a NET inducer.
- Nucleosome release varied with methylprednisolone concentration during bacterial infection.
Conclusions:
- Methylprednisolone enhances NET formation in canine neutrophils.
- The drug exhibits a time- and concentration-dependent enhancement of the bactericidal effect against Gram-positive bacteria.
- Methylprednisolone's influence on neutrophil-mediated bacterial killing differs between Gram-positive and Gram-negative pathogens in dogs.
