Abstract
Purpose :
Neutrophil extracellular traps (NETs), which are released by neutrophils in a pathogen-killing process called NETosis, are protective against the infectious diseases of human tissues including the eye. 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) by using human neutrophils in vitro.
Methods :
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. The role of TLRs in NETs formation were also explored.
Results :
DXM significantly inhibited S. aureus-induced NETosis and extracellular bacterial killing. S. aureus infection elicited significant neutrophil oxidative burst and expression of p-NF-κB (p65), but this effect was not modified by DXM. TLR2 and TLR4 agonists significantly enhanced, while blocking TLR2 and TLR4 with neutralizing antibodies significantly reduced NETs formation induced by S. aureus. However, neither the TLR5/TLR6 agonist nor the antagonist could modulate the formation of NETs induced by S. aureus. Moreover, neither DXM nor TLRs were involved in PMA-induced NETosis. Furthermore, TLR2 and TLR4 agonists rescued DXM-inhibited NETosis, and DXM could not further inhibit NETosis reduction induced by TLR2 or TLR4 antagonists, indicating that DXM may inhibit NETosis by regulating TLR2 and TLR4.
Conclusions :
The mechanisms of S. aureus- and PMA-induced NETosis are different. TLR2 and TLR4, but not TLR5 or TLR6, modified S. aureus-induced NETs formation. DXM decreases NETs formation independently of oxidant production and NF-κB phosphorylation and possibly via a TLR-dependent mechanism.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.