Abstract
Others have previously reported that global loss of toll-like receptor 4 (TLR4) reduced retinal inflammation. To determine cell specific actions of TLR4 in the retina, we generated diabetic endothelial cell specific and Müller cell specific TLR4 knockout mice. Diabetic Cdh5-Cre TLR4 mice, PDGFRα-Cre TLR4 mice, and TLR4 floxed mice were evaluated for retinal permeability, neuronal damage, and numbers of degenerate capillaries, all changes commonly observed in the diabetic retina. We also measured protein levels of key inflammatory mediators. We found that diabetes increased permeability, neuronal, and vascular damage in all mice. Loss of TLR4 in the retinal endothelial cells protected against these changes when compared to diabetic TLR4 floxed mice. In contrast, loss of TLR4 in Müller cells did not reduce diabetes-induced increases in permeability or neuronal and vascular damage. Elimination of TLR4 in either mouse model reduced inflammatory mediators, as well as VEGF levels. Taken together, our findings suggest that loss of TLR4 in endothelial cells is protective against diabetic-induced damage, while Müller cell TLR4 is not involved in the damage.
Keywords: Toll-like receptor 4, diabetic retinopathy, endothelial cells, Müller cells
1.0. Introduction.
There is an increased appreciation of the role of inflammation in the complications of diabetic retinopathy (Joussen et al., 2004; Tang and Kern, 2011). Toll-like receptor 4 (TLR4) is one of many inflammatory pathways reported in diabetic retinopathy. Studies reported that TLR4 polymorphisms occurred in type 2 diabetic patients with non-proliferative diabetic retinopathy (Aioanei et al., 2019). Another study using type 2 diabetic patient monocytes showed increased TLR4 activation and downstream signaling (Dasu et al., 2010).
Since TLR4 was altered in human diabetic patients, a number of studies have investigated the role of TLR4 in animal models. Studies on diabetic TLR4 knockout mice showed that TLR4 drives inflammation in the retina (Devaraj et al., 2011). Work in optic nerve crush models showed that pharmacological TLR4 inhibition led to increased numbers of retinal ganglion cells (RGC)(Nakano et al., 2017). Other studies using pharmacological TLR4 inhibitors showed limited changes in overall retinal thickness, but did find changes in visual acuity (Noailles et al., 2019).
Focusing on endothelial cell or vascular changes due to TLR4 actions, studies on brain microvascular endothelial cells showed that ethyl pyruvate or Tak242, a TLR4 inhibitor, could protect the brain against cerebral ischemia and reduce inflammatory pathways (Wang et al., 2020). Studies on human retinal endothelial cells (REC) exposed to oxidative stress and platelet rich plasma exosomes showed that high glucose mediates inflammatory injury through TLR4 (Zhang et al., 2019). Work in angiogenesis models using human REC showed that LPS, an agonist of TLR4, mediated cell proliferation and migration (Chen et al., 2019). We have reported that TLR4 can inhibit insulin signaling in REC grown in high glucose (Jiang and Steinle, 2018).
In addition to endothelial cells, we previously reported β-adrenergic receptor agonists reduced both the TLR4 MyD88-dependent and -independent pathways in Müller cells cultured in high glucose (Berger et al., 2016). Further, we have also confirmed that specific knockout TLR4 in retinal Müller cells reduced both the MyD88-dependent and independent pathways (Liu and Steinle, 2017a).
Based on all the studies in vivo and in vitro, we generated endothelial cell and Müller cell specific TLR4 knockout mice and made them diabetic for 6 months. We measured neuronal, permeability, and vascular changes in these mice. We also evaluated changes in key protein levels, including tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β), and vascular endothelial growth factor (VEGF). We hypothesized that the loss of TLR4 in the retinal vasculature or in retinal Müller cells would reduce permeability increases, retinal thinning, cell loss in the ganglion cell layer, and formation of degenerate capillaries. These studies provide a compare vs. contrast on endothelial cell vs. Müller cell loss of TLR4 in the diabetic retina.
2.0. Methods.
2.1. Mice.
All animal procedures were done according to the Association for Research in Vision and Ophthalmology requirements and were approved by the Institutional Animal Care and Use Committee of Wayne State University (Protocol 18-08-1575) and conform to NIH guidelines. TLR4 floxed mice (B6(Cg)-Tlr4tm1.1Karp/J mice) and B6 FVB-Tg (cdh5-cre)7Mlia/J Cre mice were purchased from Jackson Laboratories. After 2 generations, the TLR4 floxed mice were bred with the Cdh5-Cre mice to generate conditional knockout mice where TLR4 is knocked out in vascular endothelial cells (Liu et al., 2017).
TLR4 floxed mice (B6(Cg)-Tlr4tm1.1Karp/J mice) and PDGFRα-Cre (C57BL/6-Tg(Pdgfra-cre)1Clc/J mice were purchased from Jackson Laboratories. TLR4 floxed mice were bred to PDGFRα-Cre mice, generating Müller cell specific TLR4 knockout mice. Genotyping and verification of knockout has been previously published (Liu et al., 2017; Liu and Steinle, 2017a).
At 2 months of age, diabetes was induced in the both Cdh5Cre-TLR4 and PDGFRαCre-TLR4 and TLR4 floxed mice using streptozotocin (60mg/kg for 5 consecutive days). Mice were used at 6 months of diabetes for experiments. Both male and female mice were used for all experiments. Table 1 shows body weight and blood glucose levels for the TLR4-endothelial cell KO mice. Table 2 provides the same information for the TLR4-Müller cell KO mice.
Table 1.
Body weight and blood glucose for TLR4 floxed and Cdh5-Cre TLR4 mice (TLR4 cdh5 cre-lox)
| TLR4 cdh5 floxed | TLR4 cdh5 cre-lox | |||||||
|---|---|---|---|---|---|---|---|---|
| TLR4 floxed | TLR4 floxed +STZ | TLR4cre-lox | TLR4 cre-lox + STZ | |||||
| BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | |
| 2m before STZ | 22.3±1.2 | 116±10 | 22.1±4.9 | 109±6.7 | 22.4±1.1 | 118±10 | 25.2±2.0 | 112±9.1 |
| 2m after STZ | 26.5±1.7 | 112±10 | 20.9±1.3* | 411±15# | 25.8±1.7* | 125±15 | 21.8±1.7* | 425±55# |
| 6m after STZ | 32.6±2.8 | 108±12 | 28.1±3.1* | 429±60# | 36± 3.0* | 122±20 | 26.8±3.3* | 441±45# |
Data are mean ± St. Dev.
p<0.05 vs before STZ for body weight (BW)
p<0.05 Vs. ctrl for blood glucose (BG)
Table 2.
Body weight and blood glucose levels for TLR4 floxed and PDGFRα-Cre TLR4 mice (TLR4 PDGFRα cre-lox).
| TLR4 floxed | TLR4 PDGRa cre-lox | |||||||
|---|---|---|---|---|---|---|---|---|
| TLR4 floxed | TLR4 floxed +STZ | TLR4 pdgf cre-lox | TLR4 pdgf cre-lox + STZ | |||||
| BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | BW (g) | BG (mg/dL) | |
| 2m before STZ | 21.5±1.2 | 110±6.4 | 22.1±4.9 | 109±6.7 | 22.4±1.1 | 118±10 | 22.5±2.0 | 112±9.1 |
| 2 m after STZ | 21.3±1.1 | 116±15 | 20.9±1.3* | 421±21# | 25.8±1.7* | 125±15 | 20.7±1.1* | 421±53# |
| 6 m after STZ | 33.4±1.9 | 114±11 | 28.1±3.1* | 458±56# | 34± 2.4* | 113±9 | 26.4±2.3* | 417±44# |
Data are mean ± St. Dev.
p<0.05 vs before STZ for body weight (BW)
p<0.05 Vs. ctrl for blood glucose (BG).
2.2. Retinal permeability.
At 6 months of age, some mice were subjected to fluorescein angiography. Briefly, the pupil of the mice was dilated with tropicamide ophthalmic solution. Fifteen minutes later, mice were anesthetized by ketamine and xylazine. After mice were deeply anesthetized without toe pinch reflex, 150μl of AK-FLUOR (1% W/V, AKORN, INC, Lake Forest, IL) was injected intraperitoneally. Retinal vessel leakage was analyzed and photographed using the Micron IV (Phoenix Research Labs, Pleasanton, CA).
In addition to fluorescein angiography, we also measured vascular leakage using Evan’s blue. Mice were transfused with 200μl Evans blue (0.5% in saline, Sigma Aldrich) via the tail vein. Forty-five minutes after infusion, mice were euthanized with CO2. The retinas were carefully removed, placed into 100μl formamide, and incubated for 48hours at 55°C. After incubation, the tubes were centrifuged, and the supernatant was transferred to a 96 well plate. The absorbance of the supernatant was measured at 610 (Radu and Chernoff, 2013).
2.3. Measurement of retinal thickness and cell loss in the ganglion cell layer.
After 6 months of diabetes, mice in each group were sacrificed to measure retinal thickness (Steinle et al., 2009). We have done these measurements at 2 months of diabetes in the past, but due to Covid-19, we were unable to sacrifice mice to get samples at 2 months. Ten micrometer sections were taken from regions throughout the retina. Analyses of retinal thickness and cell numbers for each retinal layer were assessed from the same regions in each retina, as we have done in the past (Steinle et al., 2009; Zhang et al., 2012).
2.4. Measurement of Degenerate Capillaries.
At 6 months of diabetes, we measured numbers of degenerate capillaries as we have done previously (Liu et al., 2019). Briefly, the eyes were enucleated, suspended in 10% buffered formalin for 5 days, and the retina was dissected in 3% crude trypsin solution (Difco Bacto Trypsin 250, Detroit, MI). The retinal vascular tree was dried onto a glass slide and stained with hematoxylin-periodic acid-Shiff.
2.5. Western blotting.
Whole retinal lysates from the mice were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated using pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with a TLR4, IL-1B, TNFa, VEGF, and ICAM1 (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired with an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.
2.6. Statistics.
Statistics were done using Prism 7.0 (GraphPad, San Diego ,CA). Data was analyzed using one-way ANOVA with Tukey’s post-hoc test for all studies. P<0.05 was considered statistically significant. A representative blot is shown for Western blots.
3.0. Results.
3.1. TLR4 in endothelial cells regulates diabetes-induced increases in permeability.
We have previously reported that TLR4 altered permeability in retinal endothelial cells (Liu et al., 2017). We wanted to expand those findings using the Cdh5Cre-TLR4 mice and TLR4 floxed mice. Figure 1 shows that diabetes increased permeability in both sets of mice measured by both Evan’s blue (right) and fluorescein angiography (left). Cdh5Cre-TLR4 mice had reduced permeability in response to diabetes when compared to TLR4 floxed mice, demonstrating that TLR4 regulates permeability in endothelial cells.
Figure 1. Permeability in diabetic TLR4 endothelial cell knockout mice.
Panels in A are fluorescein angiography for control and diabetic TLR4 floxed or cdh5Cre-TLR4 (TLR4 cre-lox) mice. Panel B are levels of Evan’s blue in control and diabetic TLR4 floxed or cdh5Cre-TLR4 (TLR4 cre-lox) mice. *P<0.05 vs. TLR4 floxed. #P<0.05 vs. TLR4 cre-lox. Data are mean ± SEM. N=6–8.
3.2. Loss of TLR4 in the retinal vasculature protected against cell loss in the ganglion cell layer (GCL).
We have previously reported that diabetes causes thinning of the retina and loss of cell numbers in the ganglion cell layer at 2 months of diabetes (Zhang et al., 2012). Unfortunately due to Covid 19, we measured neuronal changes at 6 months of diabetes for these studies. Interestingly, we still observed that loss of TLR4 in the retinal vasculature affected cell numbers. Figure 2A shows that while diabetes caused increase cell loss in the GCL in both sets of mice, the Cdh5Cre-TLR4 mice had less cell loss compared to TLR4 floxed mice. Similar to our recent findings using a TLR4 inhibitor (Liu et al, in submission), there was no difference in diabetes-induced retinal thinning between the TLR4 floxed and Cdh5Cre-TLR4 mice (Figure 2C).
Figure 2. Loss of TLR4 reduces the loss of cells in the ganglion cell layer.
Data are from control and diabetic TLR4 floxed or cdh5Cre-TLR4 (TLR4 cre-lox) mice. A Panels are representative images of the retina from these mice. Panel B is cell number in the ganglion cell layer, while Panel C is retinal thickness. *P<0.05 vs. TLR4 floxed. #P<0.05 vs. TLR4 cre-lox. Data are mean ± SEM. N=10 samples from 5 mice
3.3. Diabetic TLR4 EC-KO mice have less retinal vascular damage compared to diabetic TLR4 floxed mice.
As would likely be expected, diabetes increased vascular damage in both TLR4 floxed and Cdh5-Cre TLR4 mice; however, there was less damage in the diabetic Cdh5Cre-TLR4 mice compared to diabetic TLR4 floxed mice (Figure 3). This data suggests that loss of TLR4 in the vasculature protects the diabetic retina.
Figure 3. Loss of TLR4 in the retinal vasculature protects against diabetes-induced damage.
Data are from control and diabetic TLR4 floxed or cdh5Cre-TLR4 (TLR4 cre-lox) mice. A Panels are representative images of vascular flatmounts from these mice. Panel B is quantitation of degenerate capillaries. *P<0.05 vs. TLR4 floxed. #P<0.05 vs. TLR4 cre-lox. Data are mean ± SEM. N=10 samples from 5 mice.
3.4. Diabetes-induced increases in permeability are not altered Müller cell specific TLR4 knockout mice.
We previously reported that TLR4 altered permeability in retinal endothelial cells (Liu et al., 2017). We wanted to expand those findings using the PDGFCre-TLR4 mice and TLR4 floxed mice. Figure 4 shows that diabetes increased permeability in both sets of mice measured using fluorescein angiography (Figure 4a) and Evan’s blue (Figure 4b). No significant differences in permeability were observed between PDGFCre-TLR4 and TLR4 floxed mice, suggesting that loss of TLR4 does not regulate permeability in Müller cells in the diabetic retina.
Figure 4. Permeability in diabetic TLR4 Müller cell knockout mice.
Panel A shows fluorescein angiography for control and diabetic TLR4 floxed or PDGFCreTLR4 (TLR4 cre-lox) mice. Panel B shows levels of Evan’s blue in control and diabetic PDGFCreTLR4 (TLR4 cre-lox) mice. *P<0.05 vs. TLR4 floxed. Data are mean ± SEM. N=6–8.
3.5. Loss of TLR4 does not alter retinal thickness or cell numbers in the GCL with diabetes.
In contrast to the findings in TLR4 endothelial cell specific mice, loss of TLR4 in retinal Müller cells had no effects on diabetes-induced loss of retinal thickness and cell numbers in the GCL (Figure 5)
Figure 5. Ganglion cell layer and retinal thickness in diabetic TLR4 Müller cell knockout mice.
Data are from control and diabetic TLR4 floxed or PDGFCre-TLR4 (TLR4 cre-lox) mice. A Panels are representative images of the retina from these mice. Panel B is cell number in the ganglion cell layer, while Panel C is retinal thickness. *P<0.05 vs. TLR4 floxed. Data are mean ± SEM. N=10 samples from 5 mice. Scale bar is 100um.
3.6. Diabetic TLR4 Müller cell specific KO mice have similar levels of retinal vascular damage compared to diabetic TLR4 floxed mice.
As would be expected, diabetes increased vascular damage in both TLR4 floxed and PDGFCre-TLR4 mice. Little difference in vascular damage was observed between the diabetic PDGFCre-TLR4 mice compared to the diabetic TLR4 floxed mice (Figure 6), suggesting that loss of TLR4 in Müller cells does not protect the diabetic retina against vascular damage.
Figure 6. Loss of TLR4 in the retinal vasculature has little effect against diabetes-induced damage.
Data are from control and diabetic TLR4 floxed or PDGFCre-TLR4 (TLR4 cre-lox) mice. Left panels are representative images of vascular flatmounts from these mice. The right panel is quantitation of degenerate capillaries is shown on the right. *P<0.05 vs. TLR4 floxed. Data are mean ± SEM. N=10 samples from 5 mice. Scale bar is 50um.
3.7. Loss of TLR4 in either endothelial or Müller cells reduced inflammatory factors in the diabetic retina, as well as VEGF and ICAM1 levels.
We measured protein levels of TLR4 (as a control) and other key players often altered in the diabetic retina of the Cdh5-TLR4 and the PDDGFRα-TLR4 mice. Top panels show the results from the endothelial cell TLR4 knockout mice, while the bottom panels show data from the Müller cell knockout mice. In both sets of mice, diabetes increased TNFα, IL-1β, VEGF, and ICAM1 levels (Figure 7). However, loss of TLR4 in each cell type reduced these proteins in the diabetic retina. These data suggest that loss of TLR4 reduced cellular signaling of inflammatory pathways and VEGF in the diabetic retina.
Figure 7. Loss of TLR4 in endothelial cell and Müller cells reduced protein levels of tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β), vascular endothelial cell growth factor (VEGF), and intercellular adhesion molecule (ICAM1).
Top panels (A-E) are from control and diabetic TLR4 floxed or cdh5Cre-TLR4 (TLR4 cre-lox) mice, while bottom panels (F-J) are from control and diabetic TLR4 floxed or PDGFCre-TLR4 (TLR4 cre-lox) mice. Data are representative blots and scatter plots for TLR4 (A, F), TNFα (B, G), IL-1β (C, H), VEGF (D, I) AND ICAM1 (E, J). *P<0.05 vs. TLR4 floxed. #P<0.05 vs. TLR4 cre-lox. Data are mean ± SEM. N=5–8.
4.0. Discussion.
Studies in retinal endothelial cells grown in high glucose suggested that TLR4 is involved in retinal damage (Rajamani and Jialal, 2014). Diabetic global TLR4 knockout mice showed altered inflammatory markers (Devaraj et al., 2011), yet no studies had focused on cell specific actions of TLR4 in the diabetic retina. To address this gap in the literature, we generated endothelial cell specific (Cdh5-Cre) and Müller cell (PDGFRα-Cre) TLR4 knockout mice (Liu et al., 2017; Liu and Steinle, 2017b). We made these mice diabetic and evaluated permeability, neuronal, and vascular changes in each set of mice.
Diabetes significantly increased permeability in both TLR4 floxed and Cdh5-Cre TLR4 mice, which was reduced in the Cdh5-Cre TLR4 mice at 6 months of diabetes. This suggests that loss of TLR4 in the vasculature protected the retina against leakage. These findings agree with our previous work in retinal endothelial cells and the ischemia/reperfusion model showing that TLR4 altered occludin and zonula occludens 1 (ZO-1) levels (Liu et al., 2017). Similar results were found in the number of degenerate capillaries in the mice. Diabetes significantly increased formation of degeneration of capillaries, which were reduced in the Cdh5-Cre TLR4 mice at 6 months. These vascular findings follow previous literature on the role of TLR4 in the diabetic retina and match what we observed in mice exposed to ischemia/reperfusion (Liu et al., 2017).
We previously reported that ischemia/reperfusion caused retinal thinning and loss of cell numbers in the ganglion cell layer (GCL) at 2 days (Liu et al., 2017), which we have reported in other diabetic animals (Liu et al., 2019). Diabetes did induce both retinal thinning and loss of cell numbers in the GCL in the TLR4 floxed and Cdh5-Cre TLR4 mice. However, knockdown of TLR4 in the retinal vasculature only improved cell number in the GCL, with limited changes in retina thinning. We found similar changes in cell numbers of the GCL, but not retinal thickness in exchange protein for cAMP 1 (Epac1) endothelial cell knockout mice treated with a TLR4 antagonist (Tak242) for 2 months (Liu et al, in submission). Thus, this may be a specific action of TLR4. We will explore this in future experiments. This change in cell numbers in the GCL with TLR4 inhibition matches the literature from studies on optic nerve head damage (Nakano et al., 2017) and work using TLR4 pharmacological inhibition in other models (Ghosh et al., 2018). Thus, our data seems to suggest that loss of TLR4 in the vasculature can alter diabetes-induced cell loss in the GCL, but not retinal thinning.
In contrast to the findings from the Cdh5-Cre TLR4 mice, diabetic PDGFRα-Cre TLR4 mice had limited differences in permeability, neuronal or vascular changes when compared with TLR4 floxed mice. This suggests loss of TLR4 only in Müller cells offered little protection for the retina against diabetes-induced retinal damage.
In contrast to our findings on the retinal histology of the PDGFRα-TLR4 mice, loss of TLR4 in either endothelial cells or Müller cells was able to reduce inflammatory mediators (TNFα, IL-1β), VEGF, and ICAM1 levels in the diabetic retina. This may suggest that the loss of TLR4 in Müller cells alone is not enough to alter histological findings, but can alter cellular signaling. We will pursue possible explanations for these findings in the future.
Future studies will focus on the mechanisms by which TLR4 in endothelial cells protects the cells in the ganglion cell layer. We typically measure neuronal changes at 2 months of diabetes, but we were unable to collect samples at this time point for these mice due to Covid19. We may also use other Cre mice to investigate TLR4 in other retinal cells.
In conclusion, loss of TLR4 in the Cdh5-Cre TLR4 mice demonstrated greater protection against vascular, neuronal, and permeability diabetes-induced changes when compared with TLR4 floxed mice. Loss of TLR4 in Müller cells, in contrast, provided little protection against diabetes-induced permeability and vascular damage compared to TLR4 floxed mice. Loss of TLR4 in either cell type was able to reduce inflammatory mediators and VEGF levels. While previous studies demonstrated lower inflammatory markers in global TLR4 knockout mice, this study suggests cell-specific TLR4 actions are key to global protection to diabetes induced retinal damage.
Highlights.
We generated diabetic cdh5-Cre TLR4 and PDGFRα-Cre TLR4 mice to investigate endothelial cell specific and Müller cell specific TLR4 actions, respectively
Loss of TLR4 in endothelial cells protected against diabetes-induced damage
Loss of TLR4 in Müller cells did not alter retinal permeability, neuronal or vascular changes in response to diabetes
Acknowledgements.
This study was supported by R01EY030284 (JJS), P30EY04068 (Hazlett), and an Unrestricted Grant to the Department of Ophthalmology from Research to Prevent Blindness (Kresge Eye Institute). The funders did not influence the design or execution of these studies.
Footnotes
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