Abstract
Purpose:
Enhanced expression and activity of TLR4 in the diabetic retina has been implicated in the pathogenesis of Diabetic Retinopathy (DR). Presently, it is not known how diabetes promotes TLR4 activity in retinal and peripheral blood cells. Here we investigated the role of thioredoxin-dependent peroxidases, peroxiredoxin 1 (Prx1) and 2 (Prx2), in modulating TLR4 signaling in the diabetic retina as well as in retinal cells in culture.
Methods:
Retinas of streptozotocin-induced diabetic rats (STZ-rats, 4 weeks hyperglycemia) and non-diabetic rats (ND rats) as well as post-mortem retinas of control and DR donors (obtained from Georgia Eye Bank), were examined. Interactions between Prx isoforms with thioredoxin-1 (Trx-1) were identified by co-immunoprecipitation and Western blotting in retinal tissues of the experimental groups shown above. The expression and TLR4 interaction of the oxidative stress-induced sulfonic forms of Prx (Prx1-SO3H and Prx2-SO3H) were also measured in retinal tissues, using specific antibodies. Retinal endothelial cells isolated from bovine eyes (BRECs) were transfected with siRNA against Trx-1 and analyzed for the expression of Prx-SO3H and TLR4 association. Following stimulation of ECs with high glucose (HG, 25mM, 48hours), TLR4 expression, interaction with Prx-SO3H, and subcellular localization were analyzed by immunocytochemistry and co-immunoprecipitation.
Results:
Retinas of STZ-rats and donors with DR displayed augmented protein levels of hyperoxized Prx isoforms, and increased TLR4 association to both Prx-SO3H isoforms. HG stimulated the translocation of TLR4 to the plasma membrane as well as an increase in TLR4/Prx-SO3H interaction in ECs. Inhibition of Trx-1, achieved by transfection of the BREC with specific siRNAs, resulted in increased expression of both isoforms of Prx-SO3H and promoted increased TLR4/Prx-SO3H interactions. This effect was partially blunted with blockade of Trx-1.
Conclusions:
We have identified Prx-SO3H as a novel chaperone for TLR4 in the diabetic retina. This correlates with our findings that the anti-oxidant capacities of the Trx and Prx systems are compromised in the diabetic milieu. This study highlights that inhibition of the Trx system leads to an accumulation of stable, oxidized Prx isoforms which crosstalk with TLR4, a striking new pathway which may contribute to the subclinical inflammation seen in DR.
Keywords: 499 diabetic retinopathy •
424 antioxidants •
557 inflammation