Modulation of toll-like receptor 4 signaling in human diabetic retina by peroxiredoxin 1

Abstract

Purpose: Toll-like receptor 4 (TLR4) has been shown to promote retinal inflammation in ischemic retinopathies, however its regulation in these pathological conditions still remain unclear and understudied. The redox protein peroxiredoxin 1 (Prx1) is oxidized in response to several oxidative insults and when oxidized promotes TLR4 maturation. More recently our group and others have shown that oxidized Prx-1 can be secreted and can work as an alarmin by activating/phosphorylating TLR4. Due to the potential relevance of TLR4 expression in inflammatory retinal diseases, here we wanted to investigate the potential role of Prx-1 in human diabetic retinopathy (DR).

Methods: Vitreous and retinal samples of post-mortem diabetic (n=9) and not diabetic (n=6) donors were obtained from Georgia Eye Bank. Vitreous of diabetic (n=10) and not diabetic (n=7) patients undergoing pars plan vitrectomy for different ocular pathologies were also analyzed. Western blotting analysis was conducted to assess retinal expression of total and phosphorylated TLR4 as well as Prx-1 levels and oxidation state in retinal and vitreal samples (respectively). Human retinal endothelial cells (HuREC) were subjected to different stressors including hydrogen peroxide (H₂O₂), high glucose (HG, 25mM) and control conditions (5mM glucose and the osmotic control 25mM L-glucose). Secretion of Prx-1 and activation/phosphorylation of TLR4 was also monitored by Western blotting analysis.

Results: Western blotting analysis showed increased TLR4 expression and phosphorylation in diabetic donor retinas as compared to not diabetic donors. Prx-1 expression was also up-regulated, however there was a significant increase in oxidized and oligomeric forms of Prx-1 in the diabetic donors as compared to not diabetic retinas. Furthermore, Prx-1 was present in the vitreous of all the diabetic donors and patients while it was almost absent in the vitreous of not diabetic subjects. Stimulation of HuREC with HG and H₂O₂ promoted secretion of Prx-1 and activation/phosphorylation of TLR4. Blockade of Prx-1 in the cell media with neutralizing antibodies blocked HG and H₂O₂-induced TLR4 phosphorylation/activation.

Conclusions: We have identified Prx-1 as a new contributing factor to the induction of chronic inflammation in human diabetic retinopathy through its modulation of TLR4 phosphorylation/activation.