T Cell-Mediated Autoimmunity in Glaucoma Neurodegeneration.

Glaucoma as the leading neurodegenerative disease leads to blindness in 3.6 million people aged 50 years and older worldwide. For many decades, glaucoma therapy has primarily focused on controlling intraocular pressure (IOP) and sound evidence supports its role in delaying the progress of retinal ganglial cell (RGC) damage and protecting patients from vision loss. Meanwhile, accumulating data point to the immune-mediated attack of the neural retina as the underlying pathological process behind glaucoma that may come independent of raised IOP. Recently, some scholars have suggested autoimmune aspects in glaucoma, with autoreactive T cells mediating the chief pathogenic process. This autoimmune process, as well as the pathological features of glaucoma, largely overlaps with other neurodegenerative diseases in the central nervous system (CNS), including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. In addition, immune modulation therapy, which is regarded as a potential solution for glaucoma, has been boosted in trials in some CNS neurodegenerative diseases. Thus, novel insights into the T cell-mediated immunity and treatment in CNS neurodegenerative diseases may serve as valuable inspirations for ophthalmologists. This review focuses on the role of T cell-mediated immunity in the pathogenesis of glaucoma and discusses potential applications of relevant findings of CNS neurodegenerative diseases in future glaucoma research.

Dual Function of the IRF8 Transcription Factor in Autoimmune Uveitis: Loss of IRF8 in T Cells Exacerbates Uveitis, Whereas Irf8 Deletion in the Retina Confers Protection.

IFN regulatory factor 8 (IRF8) is constitutively expressed in monocytes and B cells and plays a critical role in the functional maturation of microglia cells. It is induced in T cells following Ag stimulation, but its functions are less well understood. However, recent studies in mice with T cell-specific Irf8 disruption under direction of the Lck promoter (LCK-IRF8KO) suggest that IRF8 directs a silencing program for Th17 differentiation, and IL-17 production is markedly increased in IRF8-deficient T cells. Paradoxically, loss of IRF8 in T cells has no effect on the development or severity of experimental autoimmune encephalomyelitis (EAE), although exacerbating colitis in a mouse colitis model. In contrast, mice with a macrophage/microglia-specific Irf8 disruption are resistant to EAE, further confounding our understanding of the roles of IRF8 in host immunity and autoimmunity. To clarify the role of IRF8 in autoimmune diseases, we have generated two mouse strains with targeted deletion of Irf8 in retinal cells, including microglial cells and a third mouse strain with targeted Irf8 deletion in T cells under direction of the nonpromiscuous, CD4 promoter (CD4-IRF8KO). In contrast to the report that IRF8 deletion in T cells has no effect on EAE, experimental autoimmune uveitis is exacerbated in CD4-IRF8KO mice and disease enhancement correlates with significant expansion of Th17 cells and a reduction in T regulatory cells. In contrast to CD4-IRF8KO mice, Irf8 deletion in retinal cells confers protection from uveitis, underscoring divergent and tissue-specific roles of IRF8 in host immunity. These results raise a cautionary note in the context of therapeutic targeting of IRF8.

Serum and antibodies of glaucoma patients lead to changes in the proteome, especially cell regulatory proteins, in retinal cells.

PURPOSE: Previous studies show significantly specifically changed autoantibody reactions against retinal antigens in the serum of glaucoma and ocular hypertension (OHT) patients in comparison to healthy people. As pathogenesis of glaucoma still is unknown the aim of this study was to analyze if the serum and antibodies of glaucoma patients interact with neuroretinal cells. METHODS: R28 cells were incubated with serum of patients suffering from primary open angle glaucoma (POAG), normal tension glaucoma (NTG) or OHT, POAG serum after antibody removal and serum from healthy people for 48 h under a normal or an elevated pressure of 15000 Pa (112 mmHg). RGC5 cells were additionally incubated with POAG antibodies under a normal pressure. Protein profiles of the R28 cells were measured with Seldi-Tof-MS, protein identification was performed with Maldi-TofTof-MS. Protein analysis of the RGC5 cells was performed with ESI-Orbitrap MS. Statistical analysis including multivariate statistics, variance component analysis as well as calculating Mahalanobis distances was performed. RESULTS: Highly significant changes of the complex protein profiles after incubation with glaucoma and OHT serum in comparison to healthy serum were detected, showing specific changes in the cells (e.g. Protein at 9192 Da (p<0.001)). The variance component analysis showed an effect of the serum of 59% on the cells. The pressure had an effect of 11% on the cells. Antibody removal led to significantly changed cell reactions (p<0.03). Furthermore, the incubation with POAG serum and its antibodies led to pro-apoptotic changes of proteins in the cells. CONCLUSIONS: These studies show that the serum and the antibodies of glaucoma patients significantly change protein expressions involved in cell regulatory processes in neuroretinal cells. These could lead to a higher vulnerability of retinal cells towards stress factors such as an elevated IOP and eventually could lead to an increased apoptosis of the cells as in glaucoma.

Vascular endothelial growth factor-A: a multifunctional molecular player in diabetic retinopathy.

Vascular endothelial growth factor-A (VEGF-A), first described as "vascular permeability factor", is a critical molecule in the pathogenesis of diabetic retinopathy at several levels. Previous studies have outlined the importance of VEGF-A in mediating vascular pathology in both experimental models and clinical diabetic retinopathy, which are characterized by retinal vascular leakage, preretinal neovascularisation and neuronal degeneration. Paradoxically, recent reports have emphasized the potential neurotrophic effects of VEGF-A on the quiescent vasculature, as well as its direct and indirect protective effects on retinal neurons. VEGF-A has also been identified as an important signalling regulator in the normal central nervous system. Consequently, anti-VEGF therapy for diabetic retinopathy has become a controversal issue. This review outlines recently developed concepts relating to the role of VEGF-A in the pathogenesis of diabetic retinopathy, with particular emphasis on its implications for clinical practice.

Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions.

Following retinal degeneration or inflammation that disrupts tissue architecture, there is limited evidence of tissue regeneration, despite evidence of cells with progenitor properties in the adult human retina at all ages. With the prospect of tissue/cell transplantation, redressing homeostasis whilst overcoming glial barrier or gliosis remains key to successful graft versus host integration and functional recovery. Activated human retinal microglia (MG) secrete cytokines, including IL-6, which may suppress neurogenesis or cellular (photoreceptor) replacement. To investigate this hypothesis, adult human retinal explants were cultured in cytokine-conditioned media (TNFalpha, TGFbeta, LPS/IFNgamma) to activate microglia in situ. Following culture of retinal explants for 4 days, supernatant conditioned by resulting migrated microglia was collected after a further 3 days and fed to retinal cell suspensions (RCS). Neurosphere (NS) generation and survival analysis was performed after 7 and 14 days in culture, with or without addition of conditioned media and with or without concomitant IL-6 neutralisation. Neurosphere phenotype was analysed by immunohistochemistry and cell morphology. Migratory MG from retinal explants were activated (iNOS-positive) and expressed CD45, CD11b, and CD11c. LPS/IFNgamma-activated MG conditioned media (MG-CM) contained significant levels of IL-6 (1265 +/- 143) pg/ml, which inhibited neurosphere generation within RCS in the presence of optimal neurosphere generating N2-FGF2 culture medium. Neutralising IL-6 activity reinstated NS generation and the differentiation capacity was maintained in the spheres that formed. Even in the presence of high levels of IL-6, those few NS that did form demonstrated a capacity to differentiate. The data supports activated MG-derived IL-6 influence retinal cell turnover.

Engineering retina from human retinal progenitors (cell lines).

Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell-cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr(2)e(3), expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription-polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell line retains its capacity to differentiate into multiple cell types holds great promise for the use of tissue-specific adult stem cells for therapy.