Purpose. To investigate how multipotent retinal stem cells (RSCs) isolated from mice respond to the proinflammatory signaling molecules, IL-1β, IL-18, and IL-17A. Materials and Methods. RSCs were cultured in a specific culture medium and were treated with these cytokines. Cell viability was detected by MTT assay; ultrastructure was evaluated by transmission electron microscopy; expression of IL-17rc and proapoptotic proteins was detected by immunocytochemistry and expression of Il-6 and Il-17a was detected by quantitative RT-PCR. As a comparison, primary mouse retinal pigment epithelium (RPE) cells were also treated with IL-1β, IL-18, or IL-17A and analyzed for the expression of Il-6 and Il-17rc. Results. Treatment with IL-1β, IL-18, or IL-17A decreased RSC viability in a dose-dependent fashion and led to damage in cellular ultrastructure including pyroptotic and/or necroptotic cells. IL-1β and IL-18 could induce proapoptotic protein expression. All treatments induced significantly higher expression of Il-6 and Il-17rc in both cells. However, neither IL-1β nor IL-18 could induce Il-17a expression in RSCs. Conclusions. IL-1β, IL-18, and IL-17A induce retinal cell death via pyroptosis/necroptosis and apoptosis. They also provoke proinflammatory responses in RSCs. Though IL-1β and IL-18 could not induce Il-17a expression in RSCs, they both increase Il-17rc expression, which may mediate the effect of Il-17a.

Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene.

The use of anti-vascular endothelial growth factor (anti-VEGF) therapy, with drugs such as ranibizumab and bevacizumab, to treat neovascular age-related macular degeneration (nAMD) produces an effective but widely variable response. Identifying markers that predict differentiated response could serve as a valuable assay in developing more personalized medicine. This study aimed to identify single nucleotide polymorphisms (SNPs) that influence the outcome of treatment with anti-VEGF therapy for AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.

Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10(-8). These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10(-8) for the first time, near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8, TGFBR1, RAD51B, ADAMTS9 and B3GALTL. A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD.

Animal models of autoimmunity to the retina mimic specific features of human uveitis, but no model by itself reproduces the full spectrum of human disease. We compared three mouse models of uveitis that target the interphotoreceptor retinoid binding protein (IRBP): (i) the "classical" model of experimental autoimmune uveitis (EAU) induced by immunization with IRBP; (ii) spontaneous uveitis in IRBP T cell receptor transgenic mice (R161H) and (iii) spontaneous uveitis in Autoimmune Regulator (AIRE)(-/-) mice. Disease course and severity, pathology and changes in visual function were studied using fundus imaging and histological examinations, optical coherence tomography and electroretinography. All models were on the B10.RIII background. Unlike previously reported, IRBP-induced EAU in B10.RIII mice exhibited two distinct patterns of disease depending on clinical scores developed after onset: severe monophasic with extensive destruction of the retina and rapid loss of visual signal, or lower grade with a prolonged chronic phase culminating after several months in retinal degeneration and loss of vision. R161H and AIRE(-/-) mice spontaneously developed chronic progressive inflammation; visual function declined gradually as retinal degeneration developed. Spontaneous uveitis in R161H mice was characterized by persistent cellular infiltrates and lymphoid aggregation, whereas AIRE(-/-) mice characteristically developed multi-focal infiltrates and severe choroidal inflammation. These data demonstrate variability and unique distinguishing features in the different models of uveitis, suggesting that each one can represent distinct aspects of uveitis in humans.

Phagocytosis and degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components, but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 associated with single-membrane phagosomes containing engulfed POS in an Atg5-dependent manner that required Beclin1, but not the autophagy preinitiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.

IL-22 has opposing effects in different tissues, from pro-inflammatory (skin, joints) to protective (liver, intestine) but little is known about its effects on neuroinflammation. We examined the effect of IL-22 on retinal tissue by using the model of experimental autoimmune uveitis (EAU) in IL-22-/- mice, as well as by intraocular injections of recombinant IL-22 or anti-IL-22 antibodies in wild type animals. During EAU, IL-22 was produced in the eye by CD4+ eye-infiltrating T cells. EAU-challenged IL-22-/- mice, as well as WT mice treated systemically or intraocularly with anti-IL-22 antibodies during the expression phase of disease, developed exacerbated retinal damage. Furthermore, IL-22-/- mice were more susceptible than WT controls to glutamate-induced neurotoxicity, whereas local IL-22 supplementation was protective, suggesting direct or indirect neuroprotective effects. Mechanistic studies revealed that retinal glial Müller cells express IL-22rα1 in vivo, and in vitro IL-22 enhanced their ability to suppress proliferation of effector T cells. Finally, IL-22 injected into the eye concurrently with IL-1, inhibited the (IL-1-induced) expression of multiple proinflammatory and proapoptotic genes in retinal tissue. These findings suggest that IL-22 can function locally within the retina to reduce inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects of PDGF-C on focal retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)] (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.

Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye.

AMD (age-related macular degeneration) is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. PEDF (pigment epithelium-derived factor) is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in DKO rd8 (Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)]) mice, a model for progressive, focal rd (retinal degeneration). First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and RPE (retinal pigment epithelium) than WT (wild-type, C57BL/6N). Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 μg), followed by a subconjunctival injection of PEDF (3 μg) 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E {[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium} levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. In addition, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both apoptotic and inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.

Tissue factor (TF) is the primary initiator of blood coagulation. In addition to hemostasis, TF can initiate intracellular signaling and promote inflammation and angiogenesis, the key processes underlying the pathogenesis of age-related macular degeneration (AMD). AMD, the leading cause of irreversible blindness among the elderly, involves many genetic and environmental risk factors, including oxidative stress and inflammation. In this study, TF expression was examined in human AMD tissue and in the eyes of a model of AMD, the Ccl2(-/-)/Cx3cr1(-/-) (DKO) mouse, as well as in the ARPE-19 cell line after lipopolysaccharide (LPS) and H(2)O(2) stimulation. Total RNA was extracted from tissue samples and further analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate TF protein expression. In the human retina, a 32-fold increase of TF mRNA expression was detected in AMD macular lesions compared with normal maculae. TF protein expression was also enhanced in human AMD maculae. Similarly, TF transcript and protein expression were moderately increased in retinal lesions, neuroretinal tissue, and cultured RPE cells of DKO mice compared with age-matched wild-type mice. TF expression level correlated with age in both wild-type and DKO mice. In order to better understand how AMD might lead to enhanced TF expression, 1, 5, and 10 μg/ml LPS as well as 100 and 200 μM H(2)O(2) were used to stimulate ARPE-19 cells for 24 and 2 h, respectively. LPS treatment consistently increased TF transcript and protein expression. H(2)O(2) alone or in combination with LPS also moderately enhanced TF expression. These results indicate that upregulated TF expression may be associated with AMD, and inflammatory and oxidative stress may contribute to TF expression in AMD eyes.

Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells is central to the pathogenesis of age-related macular degeneration (AMD). Thus, developing novel strategies to protect these cells is important. We reported previously on the robust antioxidant and therefore cell-protective effects of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) in cultured human RPE cells. New reports citing a novel anti-inflammatory role for OTC in addition to the known glutathione-stimulating and antioxidant properties emerged recently; however, this role has not been evaluated in RPE cells or in intact retina. Given the crucial causative roles of oxidative stress and inflammation in AMD pathogenesis, knowing whether OTC might exhibit a similar benefit in this cell and tissue type has high clinical relevance; thus, we evaluated OTC in the present study.

Blood-retinal barrier (BRB) breakdown and retinal edema are major complications of autoimmune uveitis and could be related to deregulation of aquaporin (AQP) expression. We have therefore evaluated the expression of AQP1 and AQP4 on BRB cells during experimental autoimmune uveitis (EAU) in mice.

Central nervous system (CNS) autoimmunity such as uveitis and multiple sclerosis is accompanied by Th1 and Th17 responses. In their corresponding animal models, experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE), both responses are induced and can drive disease independently. Because immune responses have inherent plasticity, therapeutic targeting of only one pathway could promote the other, without reducing pathology. IL-27p28 antagonizes gp130, required for signaling by IL-27 and IL-6, which respectively promote Th1 and Th17 responses. We therefore examined its ability to protect the CNS by concurrently targeting both effector responses. Overexpression of IL-27p28 in vivo ameliorated EAU as well as EAE pathology and reduced tissue infiltration by Th1 and Th17 cells in a disease prevention, as well as in a disease reversal protocol. Mechanistic studies revealed inhibition of Th1 and Th17 commitment in vitro and decreased lineage stability of pre-formed effectors in vivo, with reduction in expression of gp130-dependent transcription factors and cytokines. Importantly, IL-27p28 inhibited polarization of human T cells to the Th1 and Th17 effector pathways. The ability of IL-27p28 to inhibit generation as well as function of pathogenic Th1 and Th17 effector cells has therapeutic implications for controlling immunologically complex autoimmune diseases.

Genetic pathways underlying the initiation and progression of age-related macular degeneration (AMD) have not been yet sufficiently revealed, and the correlations of AMD's genotypes, phenotypes, and disease spectrum are still awaiting resolution. We are tackling both problems with systems biology phylogenetic parsimony analysis. Gene expression data (GSE29801: NCBI, Geo) of macular and extramacular specimens of the retinas and retinal pigment epithelium (RPE) choroid complexes representing dry AMD without geographic atrophy (GA), choroidal neovascularization (CNV), GA, as well as pre-AMD and subclinical pre-AMD were polarized against their respective normal specimens and then processed through the parsimony program MIX to produce phylogenetic cladograms. Gene lists from cladograms' nodes were processed in Genomatix GePS to reveal the affected signaling pathway networks. Cladograms exposed a highly heterogeneous transcriptomic profiles within all the conventional phenotypes. Moreover, clades and nodal synapomorphies did not support the classical AMD phenotypes as valid transcriptomal genotypes. Gene lists defined by cladogram nodes showed that the AMD-related deregulations occurring in the neural retina were different from those in RPE-choroidal tissue. Our analysis suggests a more complex transcriptional profile of the phenotypes than expected. Evaluation of the disease in much earlier stages is needed to elucidate the initial events of AMD.

Snowflake vitreoretinal degeneration (SVD, MIM 193230) is a developmental and progressive hereditary eye disorder that affects multiple tissues within the eye. Diagnostic features of SVD include fibrillar degeneration of the vitreous humor, early-onset cataract, minute crystalline deposits in the neurosensory retina, and retinal detachment. A genome-wide scan previously localized the genetic locus for SVD to a 20 Mb region flanked by D2S2158 and D2S2202. This region contains 59 genes, of which 20 were sequenced, disclosing a heterozygous mutation (484C > T, R162W) in KCNJ13, member 13 of subfamily J of the potassium inwardly rectifying channel family in all affected individuals. The mutation in KCNJ13, the gene encoding Kir7.1, was not present in unaffected family members and 210 control individuals. Kir7.1 localized to human retina and retinal pigment epithelium and was especially prevalent in the internal limiting membrane adjacent to the vitreous body. Molecular modeling of this mutation predicted disruption of the structure of the potassium channel in the closed state located immediately adjacent to the cell-membrane inner boundary. Functionally, unlike wild-type Kir7.1 whose overexpression in CHO-K1 cells line produces highly selective potassium current, overexpression of R162W mutant Kir7.1 produces a nonselective cation current that depolarizes transfected cells and increases their fragility. These results indicate that the KCNJ13 R162W mutation can cause SVD and further show that vitreoretinal degeneration can arise through mutations in genes whose products are not structural components of the vitreous.