La sérine protéase HTRA1 et l'inflammation sous-rétinienne dans le contexte de la dégénérescence maculaire liée à l'âge

Localized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration.Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risque

The serine protease HTRA1 and subretinal inflammation in the context of age-related macular degeneration

Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risque.Localized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration

La sérine protéase HTRA1 et l'inflammation sous-rétinienne dans le contexte de la dégénérescence maculaire liée à l'âge

Localized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration.Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risque

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

International audienceOrthodenticle 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 RPE OTX2 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

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

International audienceBackgroundThe 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.MethodsPrimary 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.ResultsChronic (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 β inhibition.ConclusionsOur 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

Insulin inhibits inflammation-induced cone death in retinal detachment

International audienceRhegmatogenous 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.MethodsVitreous 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.ResultsAnalysis 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 inflammationConclusionOur 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

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

International audienceBackground: 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-1β induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1β but the mechanism is unknown.Methods: Retinal explants were culture in the presence of human monocytes or IL-1β 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-1β. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-β or monocytes.Results: We here show that IL-1β does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1β 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-1β 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-1β-induced rod degeneration

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation

International audienceVariants 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