Toll-Like Receptor 4 Reduces Occludin and Zonula Occludens 1 to Increase Retinal Permeability Both in vitro and in vivo

Abstract

We reported that β-adrenergic receptors regulate toll-like receptor 4 (TLR4) signaling in the retina of diabetic mice and in retinal endothelial cells (REC) and Müller cells. We hypothesized that TLR4 regulates retinal permeability both in vitro and in vivo in the retinal vasculature. We used REC cultured in normal and high-glucose conditions and TLR4 siRNA treatments for cell culture studies of permeability and protein analyses of tumor necrosis factor α, occludin, and zonula occludens 1 (ZO-1). We used endothelial cell (EC)-specific Cre-Lox TLR4 knockout mice to study retinal permeability and neuronal and vascular changes following exposure to ocular ischemia/reperfusion (I/R) used as a retinal stressor. We found that the loss of TLR4 in the EC led to the reduced permeability following I/R and fewer degenerate capillaries. Retinal permeability was increased in REC grown in high-glucose conditions but was inhibited by TLR4 siRNA treatment. High-glucose culture conditions significantly reduced occludin and ZO-1 levels in REC, and TLR4 siRNA treatment restored levels to baseline. In conclusion, these studies demonstrate that TLR4 in EC strongly regulates retinal permeability and neuronal and vascular changes following exposure to stressors such as I/R.

Keywords: Endothelial cell; Inflammation; Ischemia/reperfusion; Permeability; Retina; Toll-like receptor 4.

Fig. 1

Staining of TLR4 in whole-retina vascular mounts. TLR4 staining in the TLR4 floxed mice (a, c) versus the TLR Cre-Lox mice (b, d). Higher magnification images are provided for TLR4 floxed (c) and TLR4 Cre-Lox (d) mice. n = 3 in each group. Scale bar, 50 μm.

Fig. 2

Loss of TLR4 decreases retinal vascular permeability. Fluorescein angiography results for TLR4 floxed mice (a) or TLR4 floxed mice exposed to ischemia/repferfusion (I/R, b) and for TLR4 Cre-Lox (c) and TLR4 Cre-Lox (d) mice exposed to I/R. n = 4. Arrows point to areas of vascular leakage.

Fig. 3

Loss of TLR4 increases retinal thickness and reduces the numbers of degenerate capillaries. a–d Neuronal thickness measurements in TLR4 floxed control (a), TLR4 floxed + I/R (b), TLR4 Cre-Lox (c), and TLR4 Cre-Lox + I/R (d) mice. e–h Degenerate capillary measurements in TLR4 floxed control (e), TLR4 floxed + I/R (f), TLR4 Cre-Lox control (g), and TLR4 Cre-Lox + I/R (h) mice. i Quantification of retinal thickness in a–d. j The number of cells in the GCL in a–d. k The quantification of the number of degenerate capillaries (caps) in e–h. Scale bar, 100 μm (neuronal data). f, h Arrows point at degenerate capillaries. n = 5 for all analyses. Bar graphs represent mean ± SEM. * p < 0.05 versus TLR4 floxed, ^# p < 0.05 vs. TLR4 floxed + I/R (i, j); ^# p < 0.05 versus TLR4 Cre-Lox Ctrl (k). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Fig. 4

TLR4 regulates occludin and ZO-1 in vivo. Protein levels of ZO-1 (a) and occludin (b) from TLR4 floxed control, TLR4 Cre-Lox control, TLR4 floxed + I/R, and TLR4 Cre-Lox + I/R whole-retina lysates. * p < 0.05 versus TLR4 floxed, ^# p < 0.05 versus TLR4 floxed + I/R, ^§ p < 0.05 versus TLR4 Cre-Lox. n = 5 for all groups. Data are mean ± SEM.

Fig. 5

Loss of TLR4 reduces TNFα and decreases permeability in retinal endothelial cells (REC) grown in high-glucose (HG) medium. REC were cultured in normal-glucose (NG) or HG medium and treated with scrambled siRNA (Sc) or TLR4 siRNA. a TLR4 levels. b TNFα levels. c HG increased REC permeability, which was blocked by TLR4 siRNA. Po, diffusive flux. * p < 0.05 versus NG, ^# p < 0.05 versus HG. n = 4 for all groups. Data are mean ± SEM.

Fig. 6

TLR4 regulates occludin and ZO-1 levels in vitro. Retinal endothelial cells (REC) were cultured in normal-glucose (NG) or high-glucose (HG) medium and treated with scrambled siRNA (Sc) or TLR4 siRNA. a ZO-1 levels. b Occludin levels. * p < 0.05 versus NG, ^# p < 0.05 versus HG. n = 4 for all groups. Data are mean ± SEM.