APOE Isoforms Control Pathogenic Subretinal Inflammation in Age-Related Macular Degeneration

International audienc

Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration

International audiencePhysiologically, the retinal pigment epithelium (RPE) expresses immunosuppressive signals such as FAS ligand (FASL), which prevents the accumulation of leukocytes in the subretinal space. Age-related macular degeneration (AMD) is associated with a breakdown of the subretinal immunosuppressive environment and chronic accumulation of mononuclear phagocytes (MPs). We show that subretinal MPs in AMD patients accumulate on the RPE and express high levels of APOE. MPs of Cx3cr1 À/À mice that develop MP accumulation on the RPE, photoreceptor degeneration, and increased choroidal neovascularization similarly express high levels of APOE. ApoE deletion in Cx3cr1 À/À mice prevents patho-genic age-and stress-induced subretinal MP accumulation. We demonstrate that increased APOE levels induce IL-6 in MPs via the activation of the TLR2-CD14-dependent innate immunity receptor cluster. IL-6 in turn represses RPE FasL expression and prolongs subretinal MP survival. This mechanism may account, in part, for the MP accumulation observed in Cx3cr1 À/À mice. Our results underline the inflammatory role of APOE in sterile inflammation in the immunosuppressive subretinal space. They provide rationale for the implication of IL-6 in AMD and open avenues toward therapies inhibiting pathogenic chronic inflammation in late AMD

Endothelin-1 enhances fibrogenic gene expression, but does not promote DNA synthesis or apoptosis in hepatic stellate cells

BACKGROUND: In liver injury, the pool of hepatic stellate cell (HSC) increases and produces extracellular matrix proteins, decreasing during the resolution of fibrosis. The profibrogenic role of endothelin-1 (ET-1) in liver fibrosis remains disputed. We therefore studied the effect of ET-1 on proliferation, apoptosis and profibrogenic gene expression of HSCs. RESULTS: First passage HSC predominantly expressed endothelin A receptor (ETAR) mRNA and 4th passage HSC predominantly expressed the endothelin B receptor (ETBR) mRNA. ET-1 had no effect on DNA synthesis in 1st passage HSC, but reduced DNA synthesis in 4th passage HSC by more than 50%. Inhibition of proliferation by endothelin-1 was abrogated by ETBR specific antagonist BQ788, indicating a prominent role of ETBR in growth inhibition. ET-1 did not prevent apoptosis induced by serum deprivation or Fas ligand in 1st or 4th passage HSC. However, ET-1 increased procollagen α1(I), transforming growth factor β-1 and matrix metalloproteinase (MMP)-2 mRNA transcripts in a concentration-dependent manner in 1st, but not in 4th passage HSC. Profibrogenic gene expression was abrogated by ETAR antagonist BQ123. Both BQ123 and BQ788 attenuated the increase of MMP-2 expression by ET-1. CONCLUSION: We show that ET-1 stimulates fibrogenic gene expression for 1st passage HSC and it inhibits HSC proliferation for 4th passage HSC. These data indicate the profibrogenic and antifibrogenic action of ET-1 for HSC are involved in the process of liver fibrosis

Otx2 Gene Deletion in Adult Mouse Retina Induces Rapid RPE Dystrophy and Slow Photoreceptor Degeneration

International audienceBACKGROUND: Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells. METHODOLOGY/PRINCIPAL FINDINGS: Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease. CONCLUSIONS: Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE

Characterization of animal models for primary sclerosing cholangitis (PSC)

SummaryPrimary sclerosing cholangitis (PSC) is a chronic cholangiopathy characterized by biliary fibrosis, development of cholestasis and end stage liver disease, high risk of malignancy, and frequent need for liver transplantation. The poor understanding of its pathogenesis is also reflected in the lack of effective medical treatment. Well-characterized animal models are utterly needed to develop novel pathogenetic concepts and study new treatment strategies. Currently there is no consensus on how to evaluate and characterize potential PSC models, which makes direct comparison of experimental results and effective exchange of study material between research groups difficult. The International Primary Sclerosing Cholangitis Study Group (IPSCSG) has therefore summarized these key issues in a position paper proposing standard requirements for the study of animal models of PSC

Rôle du gène homéotique Otx2 dans la rétine adulte de souris

Le dysfonctionnement progressif et la dégénérescence des photorécepteurs de la rétine sont à l origine de la majorité des cécités de l adulte dans les pays industrialisés. De nombreuses altérations génétiques à l origine des formes monogéniques et complexes de dégénérescence ont été identifiées, mais n expliquent que la moitié des cas. Certaines concernent des gènes contrôlant le développement, et dont l expression perdure dans la rétine mature. Leurs fonctions tardives sont encore très mal connues. C est le cas du gène Otx2. Ce gène code un facteur de transcription à homéodomaine qui apparaît essentiel depuis la formation de la vésicule optique jusqu à la maturation des cellules bipolaires et des photorécepteurs. Plusieurs mutations associées à des anomalies oculaires graves, dont la rétinite pigmentaire, ont été décrites chez l homme. Pourtant, on ignore tout du réseau génétique qu il contrôle. Les travaux que nous avons menés dans le cadre de cette thèse visent à comprendre les fonctions du gène Otx2 dans la rétine mature de souris. Ce gène est exprimé dans trois types cellulaires : l épithélium pigmentaire rétinien (RPE), les photorécepteurs et les cellules bipolaires. Son ablation, exclusivement restreinte à ses domaines d expression (self-KO), a été induite chez l adulte grâce à des lignées de souris créées au laboratoire. Le système est extrêmement efficace : Otx2 est détruit dans plus de 95% des cellules qui l expriment. Ceci entraîne une dégénérescence progressive et exclusive des photorécepteurs par apoptose, qui commence 20 jours après le KO, et culmine à 30 jours. Au bout de quatre mois, la totalité des photorécepteurs a disparu. Ce modèle génétique est unique, car il offre un contrôle temporel précis. Surtout, i donne la possibilité d observer la phase précédant la mort des photorécepteurs. C est pendant cette phase infra-clinique que se manifestent les conséquences primaires de la perte de fonction d Otx2. Nous avons utilisé cet avantage pour réaliser une étude cinétique du transcriptome rétinien au décours d invalidation. Cette analyse a permis d identifier 71 gènes dont l expression est modifiée par l invalidation d Otx2. La variation de 37 de ces gènes a été validée par RT-qPCR. Parmi eux, une majorité montre une cinétique très fortement corrélée à la disparition d Otrx2, et représente des cibles directes potentielles. Des gènes associés au stresse cellulaire sont induits après un délai de quelques jours, révélant un dépassement des cellules à faire à ce changement. Sur le plan fonctionnel l enregistrement de l activité rétinienne indique que les cellules du RPE présentent rapidement des anomalies de polarisation suivant le self-KO. Cette observation, ajoutée à des données d histologie et d expression, suggère fortement une origine non autonome de la dégénérescence des photorécepteurs, et qui serait liée principalement au dysfonctionnement du RPE. Afin de vérifier cette hypothèse, nous avons déclenché une invalidation du gène Otx2 exclusivement dans les cellules du RE de la rétine adulte à l aide d infections lentivirales topiques. Cette expérience reproduit le syndrome neurodégénératif du modèle de self-KO. Réciproquement, l expression spécifique d Otx2 dans le RPE de rétine self-KO prévient de manière efficace la dégénérescence des photorécepteurs. Ainsi, parmi les fonctions dirigées par Orx2 dans la rétine adulte, celles du RPE sont nécessaires et suffisantes au maintien des photorécepteurs. Le séquençage massif de la chromatine de RPE immunoprécipitée par Otx2 nous a permis d identifier 24 gènes directement régulés. Ces gènes remplissent des fonctions nécessaires à l homéostasie du RPE telles que la mélanogenèse, le métabolisme des rétinoïdes, la régulation du PH ou la concentration de métaux. Ils constituent d excellents candidats pour les formes monogéniques et complexes de dégénérescence des photorécepteurs de type non autonome.The major cause of adult blindness in industrialized countries is the progressive dysfunction and death of retinal photoreceptors. Mutations involved in both hereditary and complex forms of degeneration have been identified. Yet, a significant part of hereditary forms still misses genetic explanation and the number of loci that influence susceptibility to age-related macular degeneration (AMD) is expanding. Several genes involved in photoreceptor degeneration play key functions during development. Although their expression is maintained in the mature retina, their function is poorly known. Among these, Otx2 gene encodes a homeobox transcription factor essential for eye development, from eye territory patterning to photoreceptor and bipolar cell maturation. In human, several mutations in Otx2 locus have been associated to ocular malformation and occasionally to retinitis pigmentosa. In spite of its importance, Otx2 downstream molecular network is poorly defined. This work aimed to elucidate Otx2 functions in the mouse mature retina. In this tissue, Otx2 is found in the nucleus of RPE, photoreceptors, and bipolar cells. To explore its functions at a late stage, we took advantage of a model coined Otx2 self-KO, which allows time-controlled and efficient knockout of Otx2 only in cells that express it. Otx2 deletion leads to a relatively slow, still complete, photoreceptor degeneration by apoptosis starting 20 days after Otx2 ablation, and reaching a peak after 30 days. After 4 months, all photoreceptors have disappeared. Time-monitored gene ablation allows access to the period preceding photoreceptor degeneration, in which primary consequences of Otx2 self-KO occur. To shed light on the early molecular events leading to photoreceptor denegation, we analyzed the retina and RPE transcriptome using DNA microarrays at 0, 2, 4 an d8 days following Otx2 ablation? We identified 71 deregulated genes after Otx2 self-KO among which 37 were confirmed by RT-qPCR. Most of deregulated genes demonstrated a kinetic strongly correlated to Otx2 clearance, likely representing direct targets. After a 4 day delay, genes associated with cell stress signalling were induced, uncloaking cellular response to primary perturbation.NICE-BU Sciences (060882101) / SudocSudocFranceF

Bedside Rapid Flu Test and Zanamivir Prescription in Healthy Working Adults: A Cost-Benefit Analysis

Background: Zanamivir, a neuraminidase inhibitor, reduces the number of days of illness in influenza-positive patients. New bedside rapid flu tests (RFT) should increase the number of influenza-positive patients whom receive zanamivir appropriately. Objective: To estimate the economic effects of implementing RFT and zanamivir among unvaccinated healthy working adults who consult within 2 days of the onset of influenza-like symptoms. Methods: We constructed a decision tree to perform a cost-benefit analysis from a societal perspective. Clinical outcome, i.e. number of influenza days averted, and societal costs were compared for three strategies: 1. RFT and conditional zanamivir prescription; 2. systematic zanamivir prescription; and 3. no zanamivir. A two-way sensitivity analysis was performed including the proportion of influenza-positive patients. Results: During influenza epidemics, systematic zanamivir prescription provided the best health outcome (0.81 influenza days averted) and minimised societal costs (reduced by $US29.80 per person compared with no zanamivir; 1999 values). RFT with conditional zanamivir averted 0.65 influenza days and saved$US14.40 per person. When the proportion of influenza-positive patients was under 39%, the no zanamivir strategy yielded the greatest societal savings; otherwise, systematic zanamivir was the dominant strategy. Medical costs associated with no zanamivir were $US88.70 per patient consulting with influenza-like illness, and increased to$US125.50 with systematic zanamivir and to $US127.60 with RFT and conditional zanamivir. Conclusions: Due to poor sensitivity of current RFT, systematic zanamivir prescription without RFT for unvaccinated healthy working adults should be recommended during influenza epidemics.Antivirals, Cost benefit, Influenza virus infections, Pharmacoeconomics, Zanamivir

Otx2 ChIP-seq Reveals Unique and Redundant Functions in the Mature Mouse Retina.

International audienceDuring mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease

Otx2 and Crx redundancy in the neuroretina.

<p><b>A.</b> Heatmap and dendrogram representation of Diffbind clustering of the indicated ChIP-seq experiments. <b>B.</b> Venn diagrams showing overlapping Crx bound regions (CBRs) and OBRs in the NR and in the RPE. CBRs represented the intersection of both Crx ChIP-seq replicates. <b>C.</b> Venn diagram showing the overlap between the above 415 common OBRs/CBRs in the RPE (grey) and the 426 OBRs common to RPE and NR (purple) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089110#pone-0089110-g004" target="_blank">Fig. 4A</a>. <b>D.</b> Otx2 and Crx transactivation of the <i>RBP3</i> promoter.</p