Abstract
Purpose :
Dioleoylphosphatidylglycerol (DOPG), a naturally occurring phospholipid that can be produced by the combined action of aquaporin 3 (AQP3) and phospholipase D2 (PLD2), was previously shown to inhibit macrophage inflammatory mediator production in response to heat shock protein B4 (HSPB4) activation of toll-like receptor 2 (TLR2). However, it remains unclear what concentration of DOPG is sufficient to achieve this immunomodulating effect. We hypothesized that lower concentrations of DOPG will also inhibit HSPB4 activation of a TLR2 reporter cell line and that CD14 is involved in the ability of HSPB4 to activate TLR2. Further, we anticipated that AQP3 and PLD2 would colocalize in human cornea.
Methods :
A human TLR2 reporter cell line was used to monitor TLR2 activation by various doses of recombinant HSPB4. Then using 1 µg/mL HSPB4, TLR2 activation was measured in the presence and absence of varying concentrations of DOPG or the negative control phospholipid dioleoylphosphatidylcholine (DOPC). Cells were treated for 24 hours followed by determination of absorbance at 620 nm. These experiments were repeated with anti-CD14 antibody pretreatment for 1 hour. Immunofluorescence was used to stain human cornea for AQP3 and PLD2.
Results :
HSPB4 concentrations between 1 and 5 µg/mL dose-dependently activated TLR2. Treatment with DOPG at concentrations as low as 10 µg/mL significantly reduced HSPB4 activation of TLR2 (p<0.05). A CD14 neutralizing antibody also significantly inhibited HSPB4-induced TLR2 activation (p<0.05); however, this effect was similar to that of DOPG alone and DOPG combined with anti-CD14 antibody pretreatment. Immunofluorescence of human cornea revealed partial co-localization of AQP3 and PLD2, particularly in the limbal region.
Conclusions :
Our results support the hypothesis that lower concentrations of DOPG can significantly reduce HSPB4-induced TLR2 activation in a reporter cell line. Further work is necessary to evaluate DOPG’s interactions with CD14, a known co-receptor for TLR2. Co-localization of AQP3 and PLD2 in the corneal epithelium suggests that DOPG is a naturally occurring inflammatory modulator in an intact cornea. Therefore, DOPG warrants further investigation as a potential therapeutic agent for sterile corneal inflammation and wound healing.
This is a 2020 ARVO Annual Meeting abstract.