Dioleoylphosphatidylglycerol (DOPG) inhibits heat shock protein B4 (HSPB4)/toll-like receptor 2 (TLR2) axis inflammatory mediator expression in a macrophage cell line

Abstract

Purpose : The inflammatory response after corneal injury has been shown to be mediated by heat shock protein B4 (HSPB4) released by keratocytes in response to epithelial cell damage and subsequent toll-like receptor 2 (TLR2) activation in macrophages. Dioleoylphosphatidylglycerol (DOPG), a naturally occurring phospholipid, has been shown to accelerate corneal epithelial wound healing. We hypothesize that DOPG will inhibit the production of inflammatory mediators via inhibition of TLR2 activation in a macrophage cell line in vitro.

Methods : RAW 264.7 cells were treated with 0, 0.1, or 1 µg/mL recombinant HSPB4 in the presence or absence of 100 µg/mL DOPG for 2 hours. RNA was isolated and the expression of the inflammatory mediators—IL1α, IL1β, IL6 and TNFα—monitored by quantitative RT-PCR in 3 separate experiments performed in duplicate. A reporter cell line was used to monitor TLR2 activation by HSPB4 and the effect of DOPG.

Results : HSPB4 dose dependently stimulated the expression of the inflammatory mediators, IL1α, IL1β, IL6 and TNFα, with the effect of a high concentration (1 µg/mL) achieving significance (p<0.001). Treatment with DOPG significantly reduced (p<0.001) HSPB4-stimulated cytokine expression. In addition, DOPG inhibited HSPB4-induced TNFα secretion from RAW264.7 cells. These decreases were mediated by DOPG’s ability to inhibit TLR2 activation by HSPB4, since recombinant HSPB4 increased reporter activity in the reporter cell line and DOPG reduced this activity.

Conclusions : Our results are consistent with our hypothesis that DOPG can significantly reduce the in vitro production of inflammatory mediators associated with HSPB4-induced macrophage TLR2 activation. Further work is necessary to demonstrate DOPG’s ability to inhibit TLR2 signaling in corneal epithelial wound healing in vivo. DOPG represents a promising therapeutic agent for sterile corneal inflammation and wound healing.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Figure 1. DOPG inhibits inflammatory mediator expression in a macrophage cell line stimulated with recombinant HSPB4. Expression of the inflammatory mediators, (A) IL1α, (B) IL1β, (C) IL6 and (D) TNFα, reported as percentage of maximal response. Results represent the means ± SEM of 3 separate experiments performed in duplicate; ***p<0.001 versus all other conditions except as indicated where ξp<0.05 and ξξp<0.01.