Hypoxia Inhibits Subretinal Inflammation Resolution Thrombospondin-1 Dependently.

Hypoxia is potentially one of the essential triggers in the pathogenesis of wet age-related macular degeneration (wetAMD), characterized by choroidal neovascularization (CNV) which is driven by the accumulation of subretinal mononuclear phagocytes (MP) that include monocyte-derived cells. Here we show that systemic hypoxia (10% O2) increased subretinal MP infiltration and inhibited inflammation resolution after laser-induced subretinal injury in vivo. Accordingly, hypoxic (2% O2) human monocytes (Mo) resisted elimination by RPE cells in co-culture. In Mos from hypoxic mice, Thrombospondin 1 mRNA (Thbs1) was most downregulated compared to normoxic animals and hypoxia repressed Thbs-1 expression in human monocytes in vitro. Hypoxic ambient air inhibited MP clearance during the resolution phase of laser-injury in wildtype animals, but had no effect on the exaggerated subretinal MP infiltration observed in normoxic Thbs1-/--mice. Recombinant Thrombospondin 1 protein (TSP-1) completely reversed the pathogenic effect of hypoxia in Thbs1-/--mice, and accelerated inflammation resolution and inhibited CNV in wildtype mice. Together, our results demonstrate that systemic hypoxia disturbs TSP-1-dependent subretinal immune suppression and promotes pathogenic subretinal inflammation and can be therapeutically countered by local recombinant TSP-1.

Insulin inhibits inflammation-induced cone death in retinal detachment.

BACKGROUND: Rhegmatogenous retinal detachment (RD) involving the macula is a major cause of visual impairment despite high surgical success rate, mainly because of cone death. RD causes the infiltration of activated immune cells, but it is not clear whether and how infiltrating inflammatory cells contribute to cone cell loss. METHODS: Vitreous samples from patients with RD and from control patients with macular hole were analyzed to characterize the inflammatory response to RD. A mouse model of RD and retinal explants culture were then used to explore the mechanisms leading to cone death. RESULTS: Analysis of vitreous samples confirms that RD induces a marked inflammatory response with increased cytokine and chemokine expression in humans, which is closely mimicked by experimental murine RD. In this model, we corroborate that myeloid cells and T-lymphocytes contribute to cone loss, as the inhibition of their accumulation by Thrombospondin 1 (TSP1) increased cone survival. Using monocyte/retinal co-cultures and TSP1 treatment in RD, we demonstrate that immune cell infiltration downregulates rod-derived cone viability factor (RdCVF), which physiologically regulates glucose uptake in cones. Insulin and the insulin sensitizers rosiglitazone and metformin prevent in part the RD-induced cone loss in vivo, despite the persistence of inflammation CONCLUSION: Our results describe a new mechanism by which inflammation induces cone death in RD, likely through cone starvation due to the downregulation of RdCVF that could be reversed by insulin. Therapeutic inhibition of inflammation and stimulation of glucose availability in cones by insulin signaling might prevent RD-associated cone death until the RD can be surgically repaired and improve visual outcome after RD. TRIAL REGISTRATION: ClinicalTrials.gov NCT03318588.

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

IL-1ß induces rod degeneration through the disruption of retinal glutamate homeostasis.

BACKGROUND: Age-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1ß induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1ß but the mechanism is unknown. METHODS: Retinal explants were culture in the presence of human monocytes or IL-1ß and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1ß. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-ß or monocytes. RESULTS: We here show that IL-1ß does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1ß leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1ß or, more importantly, in vivo, in a model of subretinal inflammation. CONCLUSIONS: Our study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1ß-induced rod degeneration.

Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation.

BACKGROUND: The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. METHODS: Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. RESULTS: Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and transepithelial resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and transepithelial-resistance changes were prevented by concomitant Transforming Growth Factor ß inhibition. CONCLUSIONS: Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α.

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14+ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human-induced pluripotent stem cell-derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF-α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF-α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age-related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.

Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells.

PURPOSE: Prostaglandin F2α analogues are the first-line medication for the treatment of ocular hypertension (OHT), and prostanoid EP2 receptor agonists are under clinical development for this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) cells, which could be a causal mechanism of TM dysfunction in glaucoma. METHODS: Human primary TM cells were treated with either latanoprost or butaprost and TGF-ß2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cell-populated collagen gel (CPCG) model. Expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of actin stress fibers and collagen deposition were evaluated by immunocytochemistry. Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific inhibitors. RESULTS: In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, α-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-ß2-induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-ß2-dependent collagen deposition. The latanoprost effects were mediated by p38 pathway. CONCLUSIONS: Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-ß2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT.