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CD40 in Retinal Muller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.

ABSTRACT: Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40⁺ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X₇-dependent production of TNF-α and IL-1β by macrophages. P2X₇^-/- mice and mice treated with a P2X₇ inhibitor were protected from diabetes-induced TNF-α, IL-1β, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X₇ pathway.

SUBMITTER: Portillo JC

PROVIDER: S-EPMC5248988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AIMS/HYPOTHESIS:Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygen-induced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1? (also known as Hif1a) knockout (KO) mice. METHODS:Conditional Hif-1? KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1? mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1? and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. RESULTS:While the Hif-1? KO mice showed significantly decreased HIF-1? levels in the retina, they exhibited no apparent histological or visual functional abnormalities under normal conditions. Compared with wild-type counterparts, Hif-1? KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1? in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. CONCLUSIONS/INTERPRETATION:Müller cell-derived HIF-1? is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1? is therefore a promising therapeutic target for diabetic retinopathy.

Project description:The aim of the study was to explore the effect of topically administered sitagliptin, an inhibitor of the enzyme dipeptidyl peptidase-4, on the retinal expression patterns of an experimental model of DR. Background: Synaptic dysfunction and neuronal damage have been extensively associated with diabetic retinopathy (DR). Our group evidenced that chronic hyperglycemia reduces the retinal expression of presynaptic proteins, which are crucial for a proper synaptic function. The aim of the study was to explore the effect of topically administered sitagliptin, an inhibitor of the en-zyme dipeptidyl peptidase-4, on the retinal expression patterns of an experimental model of DR. Methods: Transcriptome analysis was performed comparing the retinas of 10 diabetic (db/db) mice randomly treated with sitagliptin eye drops (10 mg/mL) twice daily, and the reti-nas of 10 additional db/db mice that received vehicle eye drops. 10 non diabetic mice (db/+) were used as control group. Gene Ontology (GO) and Reactome databases were used to assess the Gene Set Enrichment Analysis (GSEA) in order to explore the most enriched biological pathways among groups. The most differentiated genes of these pathways were validated through quantitative RT-PCR. Results: Transcriptome analysis revealed that sitagliptin eye drops have a significant effect on retinal expression patterns and that neurotransmission was the most enriched biological process. Our study evidenced enriched pathways that contain genes involved in membrane trafficking, transmission across chemical synapses, vesi-cle-mediated transport, neurotransmitter receptors and postsynaptic signal transmission with negative regulation of signaling as a consequence of neuroprotector treatment with sitagliptin. This improves the modulation of macromolecule biosynthetic process with positive regulation of cell communication which provides beneficial effects for neuronal metabolism. Conclu-sions: This study suggests that topical administration of sitagliptin ameliorates the abnormali-ties on presynaptic and postsynaptic signal transmission during experimental DR, and that this improvement is one of the main mechanisms behind the previously demonstrated beneficial effects.

Project description:Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial-lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

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CD40 in Retinal Muller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.

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