Retinal inflammation in murine models of type 1 and type 2 diabetes with diabetic retinopathy

Abstract

Aims/hypothesis: The loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival.

Methods: Loss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2^Akita/+ (a model of type 1 diabetes) and Lepr^db/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry.

Results: The essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3.

Conclusions/interpretation: We conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis.

Keywords: Diabetic retinopathy; Gliosis; IFN gamma; IFNγ; Inflammation; PDGFRβ; PKCδ; Pericytes; Platelet-derived growth factor receptor β.

Fig. 1

Pericyte number and inflammation levels are altered in the retinas of Ins2^Akita/+ (Ak) and Lepr^db/db (db) mice. (a) Murine retinal flatmounts from 3-, 6-, 12-, 18- and 24-week-old WT, Ak and db mice were immunolabelled with the pericyte marker NG2 and the number of positive cells per 520 μm² of tissue was quantified. (b, c) Quantification of qRT-PCR data showing the log₂ fold change in mRNA for inflammatory factors in Ak mice (b) and db mice (c) relative to WT levels at 3, 6, 12, 18 and 24 weeks of age. WT levels were set to a value of 1.0, whereas the dotted line indicates a value of 0. Statistical analysis of RNA levels for each marker were compared for WT vs Ak (b) or WT vs db (c) across all ages. Overall, these figures show that pericyte loss was present at 3 weeks of age, with the level of loss plateauing at 12 weeks of age in Ak and db models. Detectable increases in proinflammatory mRNA levels occurred at 6 weeks of age in Ak retinas, but then peaked at 12 weeks and decreased at 18 weeks and 24 weeks of age in comparison vs WT retinas. In comparison, levels of many proinflammatory mRNAs decreased at 3 weeks and 24 weeks of age in retinas from db mice, then increased at 12 weeks and plateaued at 18 weeks of age compared with WT. Data are presented as mean±SEM; for (a), n=5–6 biological replicates, with six technical replicates per retina; for (b) and (c), n=3 biological replicates, with three technical replicates.*p<0.05, **p<0.01, ***p<0.001, ^‡p<0.0001, analysed by one-way ANOVA with Tukey post hoc analysis

Fig. 2

Proinflammatory markers in Ins2^Akita/+ (Ak) and Lepr^db/db (db) mice at 6 and 12 weeks of age. (a, b) Levels of proinflammatory proteins in whole retinal samples were examined in samples from WT, Ak and db mice, obtained at 6 weeks (a) and 12 weeks (b) of age using multiplex ELISA panels. WT levels were set to a value of 1.0, whereas the dotted line indicates a value of 0. At 6 weeks of age, retinas from Ak mice showed an increase in IFNγ, IL-1β and keratinocyte chemoattractant/growth-related oncogene (KC-GRO) in comparison with WT, whereas retinas from db mice showed decreases in IL-5, IL-6 and IL-12p70. At 12 weeks, IFNγ was the only factor that increased in Ak mice compared with WT. Data are presented as mean±SEM; n=4 biological replicates. *p<0.05, analysed by one-way ANOVA. One outlier in the 6-week-old db mice data was identified using Grubbs’ analysis and was omitted from the analysis and graph

Fig. 3

Retinal gliosis accompanies early diabetes in both Ins2^Akita/+ (Ak) and Lepr^db/db (db) mice, with different rates of progression. (a, b) Murine retinal flatmounts from 3-, 6-, 12-, 18- and 24-week-old WT, Ak and db mice were immunolabelled with the myeloid lineage marker IBA1 (a) or the retinal astrocyte, Müller and cholinergic amacrine cell marker SOX2 (b), and the number of positive cells per 520 μm² of tissue was quantified. Statistical analysis of data for IBA1⁺ (a) and SOX2⁺ (b) cells compared WT, Ak and db mice at each age. (c, d) Quantification of qRT-PCR data showing the log₂ fold change in mRNA for factors that are altered during astrogliosis in Ak (c) and db (d) retinas relative to WT levels (WT levels were set to a value of 1.0) at 3, 6, 12, 18 and 24 weeks of age. Dotted line indicates a value of 0. Statistical analysis of RNA levels for each marker were compared for WT vs Ak (c) or WT vs db (d) across all ages. Overall, small changes in the number of IBA1⁺ cells were observed in retinas from db mice at 3 and 12 weeks of age in comparison with WT, but no differences in the number of retinal astrocytes were detected in the retinas of Ak or db mice in comparison with WT. Regarding statistically significant increases in gliosis markers (indicating the presence of gliosis), samples from 12-week-old Ak mice had an increase in Tlr4, Pcan and Mmp14. In samples from db mice, mRNA levels of Gs and Egfr were increased at 12 weeks of age, and Gfap, Egfr and Mmp14 were increased at 18 weeks. Data are presented as mean±SEM; for (a) and (b), n=5–6 biological replicates, with three technical replicates for each biological replicate; for (c) and (d), n=3 biological replicates, with six technical replicates for each biological replicate. *p<0.05, **p<0.01, ***p<0.001, ^‡p<0.0001; analysed by one-way ANOVA with Tukey post hoc analysis

Fig. 4

The percentage of the retina covered by IB4⁺ cells and number of branches were altered at various ages in Ins2^Akita/+ (Ak) and Lepr^db/db (db) mice in comparison with WT mice. Flatmounts of retinas from 6-, 12-, 18- and 24-week-old WT, Ak and db mice were immunolabelled with the endothelial cell marker IB4, and digital images from the retinas were subjected to analysis using Angiotool software [30]. (a) Percentage of retinal area covered by IB4⁺ vasculature. (b) Number of branch points per 520 μm² of tissue. Statistical analysis compared WT, Ak and db mice at each age. Retinal area covered by superficial plexus vasculature was increased in retinas from db mice at 6 weeks and in retinas of both Ak and db mice at 24 weeks in comparison with WT mice. The number of branches that were labelled with marker IB4 were increased at 6, 12 and 18 weeks of age in db mice, but no change in branch number was noted at any age in retinas of Ak mice in comparison with WT mice. Data are presented as mean±SEM; n=5–8 biological replicates, with six technical replicates for each biological replicate. *p<0.05, ***p<0.001, ^‡p<0.0001, analysed by one-way ANOVA with Tukey post hoc analysis

Fig. 5

Chronic treatment of murine retinal pericytes with IFNγ reduced PDGFRβ, Akt and ERK signalling, and increased levels of cleaved PKCδ. (a–d) Lysates isolated from murine retinal pericytes incubated for 72 h with vehicle, PDGFBB alone, IFNγ alone, or 1 ng, 25 ng or 50 ng of IFNγ in the presence or absence of 50 ng PDGFBB were immunoblotted for p-PDGFRβ (Tyr751) and total PDGFRβ (a), p-Akt (Ser473) and total Akt (b), pERK1/2 (Thr202/Ty2204) and total ERK1/2 (c), or full-length and activated cleaved PKCδ (d). β-Tubulin and Revert Total Protein Stain were used to normalise readings from PKCδ blots, while β-tubulin was used to normalise the intensity readings of other blots. A representative blot for each label is shown, alongside densitometry data. The densitometry results show that, overall, treatment of isolated murine pericytes with increasing concentrations of IFNγ under chronic conditions decreased the levels of p-PDGFRβ, p-Akt and pERK1/2 as a ratio of the total amount of protein and increased the levels of cleaved PKCδ in comparison with vehicle and control treatments. ND, not detectable; Veh, vehicle. Data are presented as mean±SEM; for (a–c), n=3–4; for (d), n=3. *p<0.05, **p<0.01, ^‡p<0.0001, analysed by one-way ANOVA with Tukey post hoc analysis

Fig. 6

IFNγ increases pericyte death in media containing normal and high glucose levels. Numbers of CC3⁺ pericytes were quantified in media containing normal glucose levels (5.7 mmol/l d-glucose) or high glucose levels (40.7 mmol/l d-glucose), or in an osmolarity control (5.7 mmol/l d-glucose and 35mmol/l l-glucose). Cells were treated with vehicle, or 1 ng, 25 ng or 50 ng IFNγ. The number of CC3⁺ cells is expressed as a percentage of total cells labelled using Hoechst dye (DNA dye). Treating isolated murine pericytes with increasing concentrations of IFNγ increased the percentage of CC3⁺ cells in media containing normal glucose levels or high glucose levels, and in the osmolarity control in comparison with vehicle-treated cells. Under high-glucose conditions, vehicle-treated cells showed an increased percentage of CC3⁺ cells in comparison with vehicle-treated cells under normal glucose and osmolarity control conditions. Data are presented as mean±SEM; n=6–10. **p<0.01, ***p<0.001, ^‡p<0.0001, analysed by one-way ANOVA with Tukey post hoc analysis