Thinning of the RPE and choroid associated with T lymphocyte recruitment in aged and light-challenged mice

International audienceThe choroidal vasculature is essential when it comes to bringing oxygen and nutrients to the functioning retina and evacuating debris resulting from the normal visual cycle. Choroidal thinning is a common feature in many human eye diseases, including high myopia [1,2] and retinitis pigmentosa [3,4], and has been reproducibly observed with age [5-7]. However, the association between choroidal thinning and age-related macular degeneration (AMD) remains controversial. Some authors have reported the loss of choriocapillaries in eyes with exudative AMD [8], and choroidal thinning has been detected in some studies [9-11]. Choroidal thinning has also been associated with geographic atrophy (GA), the dry form of late AMD [12-15]. A morphometric analysis by Ramrattan et al. more than two decades ago showed a decrease in choriocapillary density and diameter with age and in GA, but choroidal thinning was only significant with age [6]. Moreover, it has been reported that the choriocapillaries and choroid are thinner in areas where the RPE has degenerated [8]. However, all studies agree that aging is associated with significant choroidal thinning [16-18]. The exact mechanisms behind choroidal thinning with age or disease are not clear. The RPE is a monolayer of pigmented cells situated between photoreceptors and Bruch's membrane; its plays an essential role in the visual cycle. RPE65, which is also called 11-cis retinol isomerase and is strongly expressed in the RPE, participates in the production of 11-cis retinal [19], which is essential for photoreceptor function [20]. Mutations in the RPE65 gene cause progressive photoreceptor degeneration [21,22] and adult RPE65 −/

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

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CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents

Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.: DLL4's opposing effects in choroidal neovascularization

International audienceInflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. Methods: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8+/− mice expressing ß-galactosidase. Aged Mfge8+/− and Mfge8−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. Results: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8−/− mice as compared to controls. Conclusions: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD

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

Protective efficacy of cross-reactive CD8⁺ T cells recognising mutant viral epitopes depends on peptide-MHC-I structural interactions and T cell activation threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre- existing CD8+ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP336-374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8+ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vb regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2DbNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2Db, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8+ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8+ T cells recognising mutant viral epitopes depend on peptide- MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A. © 2010 Valkenburg et al.Link_to_subscribed_fulltex

Association of choroidal IL-17-producing T lymphocytes and macrophages with geographic atrophy

International audiencePurpose: To evaluate the presence of interleukin-17 (IL-17)-producing cells in patients with geographic atrophy (GA).Methods: In this short report, we analyzed IL-17, CD3 and IBA-1 expression by immunohistochemistry on paraffin-embedded sections from 13 donors with a known history of GA, confirmed by fundus appearance and histology, and 7 age-matched control donors.Results and conclusion: we show that IL-17+ cells are found near areas of retinal pigmented epithelium (RPE) atrophy in eyes of GA patients. IL-17+ cells mainly localized to CD3+ cells, which identifies T lymphocytes, as well as IBA-1+ cells, which identifies mononuclear phagocytes. Therefore IL-17 could be involved in the pathological mechanisms that contribute to degeneration observed in GA

Influence of Radiotherapy Fractionation Schedule on the Tumor Vascular Microenvironment in Prostate and Lung Cancer Models

International audienceBackground. The tumor vasculature acts as an interface for the primary tumor. It regulates oxygenation, nutrient delivery, and treatment efficacy including radiotherapy. The response of the tumor vasculature to different radiation doses has been disparately reported. Whereas high single doses can induce endothelial cell death, improved vascular functionality has also been described in a various dose range, and few attempts have been made to reconcile these findings. Therefore, we aimed at comparing the effects of different radiation fractionation regimens on the tumor vascular microenvironment. Methods: Lewis lung and prostate PC3 carcinoma-derived tumors were irradiated with regimens of 10 × 2 Gy, 6 × 4 Gy, 3 × 8 Gy or 2 × 12 Gy fractions. The tumor vasculature phenotype and function was evaluated by immunohistochemistry for endothelial cells (CD31), pericytes (desmin, α-SMA), hypoxia (pimonidazole) and perfusion (Hoechst 33342). Results: Radiotherapy increased vascular coverage similarly in all fractionation regimens in both models. Vessel density appeared unaffected. In PC3 tumors, hypoxia was decreased and perfusion was enhanced in proportion with the dose per fraction. In LLC tumors, no functional changes were observed at t = 15 days, but increased perfusion was noticed earlier (t = 9-11 days). Conclusion: The vascular microenvironment response of prostate and lung cancers to radiotherapy consists of both tumor/dose-independent vascular maturation and tumor-dependent functional parameters