Methylprednisolone Induces Extracellular Trap Formation and Enhances Bactericidal Effect of Canine Neutrophils

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.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs

“…Intracellular ROS production was measured as described previously with small modifications [54]. Briefly, isolated neutrophils were incubated with MOI = 2 of G. parasuis of three serotypes 7, 13 and 15 from cryostocks.…”

Neutrophil extracellular traps in CSF and serum of dogs with steroid-responsive meningitis-arteritis

“…Furthermore, the effect of several anti-inflammatory drugs, especially glucocorticosteroids, is controversial concerning their influence on NET-formation. Methylprednisolone induces NET-formation and enhances the bactericidal effect on canine neutrophils [49]. On the other hand, NET-formation is inhibited by steroids during bacterial infections [50,51].…”