Noncanonical autophagy promotes the visual cycle.

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.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.

Nutrient supplementation with n3 polyunsaturated fatty acids, lutein, and zeaxanthin decrease A2E accumulation and VEGF expression in the retinas of Ccl2/Cx3cr1-deficient mice on Crb1rd8 background.

The Age-Related Eye Diseases Study 2 (AREDS2) clinical trial is assessing the effects of higher dietary xanthophyll (lutein and zeaxanthin) and long-chain n3 polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on progression to advanced age-related macular degeneration (AMD). This study's purpose was to examine the retinal effects of the AREDS2 formulation on Chemokine (C-C motif) ligand 2 (Ccl2(-/-))/CX3C chemokine receptor 1 (Cx3cr1(-/-)) mice on Crumbs homolog 1 retinal degeneration phenotype 8 (Crb1(rd8)) background (DKO), which develop focal retinal lesions with certain features similar to AMD. DKO and C57BL/6N rd8 background mice (WT) were bred and randomized into 4 groups. Two groups, WT mice on AREDS2 diet (A-WT) and DKO mice on AREDS2 diet (A-DKO), were supplemented daily with 1.76 µmol of lutein, 35.1 µmol of zeaxanthin, 215 µmol EPA, and 107 µmol of DHA, and 2 control groups, WT mice on control diet (C-WT) and DKO mice on control diet (C-DKO), were fed an isocaloric diet. All mice had monthly fundus photos and were killed after 3 mo for biochemical and histologic analyses. After 3 mo, 81% of A-DKO mice had lesion regression compared with 25% of C-DKO mice (P < 0.05). Toxic retinal 2-[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 (A2E) concentrations were significantly lower in A-DKO compared with C-DKO mice. The outer nuclear layer thickness in A-DKO mice was significantly greater than that in C-DKO mice. Retinal expression of inducible nitric oxide synthase (iNos) tumor necrosis factor-α (Tnf-α), Cyclooxygenase-2 (Cox-2), interleukin1beta (IL-1ß), and vascular endothelial growth factor (Vegf) was significantly lower in A-DKO compared with C-DKO mice. Xanthophylls and LCPUFAs have antiinflammatory, neuroprotective, and antiangiogenic properties. Our data provide potential mechanisms by which the AREDS2 formula has a protective effect on retinal lesions in DKO mice.

Myeloid suppressor cells induced by retinal pigment epithelial cells inhibit autoreactive T-cell responses that lead to experimental autoimmune uveitis.

PURPOSE: To test whether retinal pigment epithelial (RPE) cells are able to induce myeloid-derived suppressor cell (MDSC) differentiation from bone marrow (BM) progenitors. METHODS: BM cells were cocultured with or without RPE cells in the presence of GM-CSF and IL-4. Numbers of resultant MDSCs were assessed by flow cytometry after 6 days of incubation. The ability of the RPE cell-induced MDSCs to inhibit T cells was evaluated by a CFSE-based T-cell proliferation assay. To explore the mechanism by which RPE cells induce MDSC differentiation, PD-L1-deficient RPE cells and blocking antibodies against TGF-ß, CTLA-2α, and IL-6 were used. RPE cell-induced MDSCs were adoptively transferred into mice immunized with interphotoreceptor retinoid-binding protein in complete Freund's adjuvant to test their efficacy in suppressing autoreactive T-cell responses in experimental autoimmune uveitis (EAU). RESULTS: RPE cells induced the differentiation of MDSCs. These RPE cell-induced MDSCs significantly inhibited T-cell proliferation in a dose-dependent manner. PD-L1-deficient RPE cells induced MDSC differentiation as efficiently as wild-type RPE cells, and neutralizing TGF-ß or CTLA-2α did not alter the numbers of induced MDSCs. However, blocking IL-6 reduced the efficacy of RPE cell-induced MDSC differentiation. Finally, adoptive transfer of RPE cell-induced MDSCs suppressed IRBP-specific T-cell responses that led to EAU. CONCLUSIONS: RPE cells induce the differentiation of MDSCs from bone marrow progenitors. Both cell surface molecules and soluble factors are important in inducing MDSC differentiation. PD-L1, TGF-ß, and CTLA-2α were not measurably involved in RPE cell-induced MDSC differentiation, whereas IL-6 was important in the process. The induction of MDSCs could be another mechanism by which RPE cells control immune reactions in the retina, and RPE cell-induced MDSCs should be further investigated as a potential approach to therapy for autoimmune posterior uveitis.

Couching for cataract in China.

Couching for cataract is one of the most ancient surgical procedures. Maharshi Sushruta, an ancient Indian surgeon, first described the procedure around 600 BCE in Sushruta Samhita. The procedure, also known as jin pi shu in Mandarin, was introduced to China via the Silk Road during the late West Han Dynasty (206 BCE-9 CE), and it spread throughout China during the Tang Dynasty (618-907 CE). As the procedure was combined with the Chinese concept of acupuncture, jin pi shu was integrated into Chinese medical practice until the founding of the Republic of China in 1911. The government of the Republic of China considered jin pi shu to be unscientific. In 1949, the Communists established the People's Republic of China. Jin pi shu was revitalized by Chairman Mao Zedong (1893-1976), who thought that traditional Chinese medicine, including jin pi shu, was a great treasure. After his death and the opening of China to the external world, many Chinese ophthalmologists pointed out that jin pi shu had relatively high complications and a low success rate, compared to various modern techniques for cataract surgery. This procedure is gradually fading away in China. The use of jin pi shu reflects the history, culture, and political transformation of China.

A high omega-3 fatty acid diet reduces retinal lesions in a murine model of macular degeneration.

Age-related macular degeneration (AMD) is one of the leading cause of blindness among the elderly; however, current therapy options are limited. Epidemiological studies have shown that a diet that is high in omega-3 polyunsaturated (n-3) fatty acids can slow disease progression in patients with advanced AMD. In this study, we evaluated the effect of such a diet on the retinas of Ccl2(-/-)/Cx3cr1(-/-) mice, a model that develops AMD-like retinal lesions that include focal deep retinal lesions, abnormal retinal pigment epithelium, photoreceptor degeneration, and A2E accumulation. Ccl2(-/-)/Cx3cr1(-/-) mice that ingested a high n-3 fatty acid diet showed a slower progression of retinal lesions compared with the low n-3 fatty acids group. Some mice that were given high levels of n-3 fatty acids had lesion reversion. We found a shunted arachidonic acid metabolism that resulted in decreased pro-inflammatory derivatives (prostaglandin E(2) and leukotriene B(4)) and an increased anti-inflammatory derivative (prostaglandin D(2)). We also measured lower ocular TNF-alpha and IL-6 transcript levels in the mice fed a diet of high n-3 fatty acids. Our findings in these mice are in line with human studies of AMD risk reduction by long-chain n-3 fatty acids. This murine model provides a useful tool to evaluate therapies that might delay the development of AMD.

LX211 (voclosporin) suppresses experimental uveitis and inhibits human T cells.

PURPOSE: To test the therapeutic effectiveness of voclosporin against experimental autoimmune uveoretinitis (EAU) in rats and to evaluate its effect on human T cells. METHODS: EAU was induced by immunization with a uveitogenic protein. Voclosporin administration, by subcutaneous injection, began on day (d) 0 or d7 after immunization. Treatment effectiveness was evaluated in vivo using clinical EAU scoring (d7-d13) and histopathologic evaluation of enucleated eyes after experimental termination. Rodent lymphocytes were harvested from lymph nodes on d14 for antigen-specific proliferation assays. The effect of voclosporin on human T-cell proliferation and cytokine secretion was examined in vitro. RESULTS: Voclosporin prevented EAU development in rats receiving medium and high preventive doses, whereas high-dose voclosporin administration effectively treated EAU. Lymphocytes from animals treated with voclosporin had decreased antigen-specific proliferation in vitro compared with lymphocytes from untreated animals. No evidence of abnormal ocular histopathology was found in the eyes from animals that received high doses of therapeutic voclosporin. Using human T cells, voclosporin inhibited human T-cell proliferation up to 100-fold. Furthermore, voclosporin treatment of human T cells significantly reduced pan T-cell effector responses. CONCLUSIONS: Voclosporin effectively suppressed uveoretinitis in an animal model that imitates the human inflammatory ocular disease by inhibiting lymphocyte proliferation. In addition, voclosporin effectively inhibited human T-cell proliferation and function in vitro. The authors report the first evidence supporting the application of voclosporin to treat intraocular inflammation.

Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23-IL-17 and IL-12-T helper cell (Th)1 pathways in the pathogenesis of EAU. IL-23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-gamma. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-gamma-producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity.

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by "natural" CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freund's adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freund's adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.

Essential role of the MyD88 pathway, but nonessential roles of TLRs 2, 4, and 9, in the adjuvant effect promoting Th1-mediated autoimmunity.

Induction of tissue-specific experimental autoimmune diseases involves an obligatory adjuvant effect to trigger an innate response of a type that will drive a Th1-biased adaptive response. This is achieved by use of CFA containing mycobacteria (Mycobacterium tuberculosis), whose recognition by cells of the innate immune system depends on TLRs that signal through the adaptor molecule MyD88. We examined the role of selected components of the MyD88 pathway in promoting experimental autoimmune uveitis (EAU). Mice deficient in MyD88, TLR2, TLR4, or TLR9 were immunized with the retinal Ag interphotoreceptor retinoid-binding protein in CFA, and their EAU scores and associated immunological responses were examined. MyD88-/- mice were completely resistant to EAU and had a profound defect in Th1, but not Th2, responses to autoantigen challenge. Surprisingly, TLR2-/-, TLR4-/-, and TLR9-/- mice were fully susceptible to EAU and had unaltered adaptive responses to interphotoreceptor retinoid-binding protein. Examination of IL-1R family members, which share the common adaptor MyD88 with the TLR family, revealed that IL-1R-deficient mice, but not IL-18-deficient mice, are resistant to EAU and have profoundly reduced Th1 and Th2 responses. These data are compatible with the interpretation that TLR9, TLR4, and TLR2 signaling is either not needed, or, more likely, redundant in the adjuvant effect needed to induce EAU. In contrast, signaling through the IL-1R plays a necessary and nonredundant role in EAU and can by itself account for the lack of EAU development in MyD88 mice.

Cholera toxin prevents Th1-mediated autoimmune disease by inducing immune deviation.

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, is known for its properties as a mucosal adjuvant that promotes Th2 or mixed Th1 + Th2 responses. In this study, we explore the ability of CT to act as a systemic adjuvant to counteract the Th1 response leading to experimental autoimmune uveitis. We report that susceptible B10.RIII mice immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein could be protected from disease by a single systemic injection of as little as 2 micro g of CT at the time of immunization. The protected mice were not immunosuppressed, but rather displayed evidence of immune deviation. Subsequent adaptive responses to interphotoreceptor retinoid-binding protein showed evidence of Th2 enhancement, as indicated by reduced delayed-type hypersensitivity in the context of enhanced Ag-specific lymphocyte proliferation and IL-4 production. Ag-specific production of several other cytokines, including IFN-gamma, was not appreciably altered. The inhibitory effect of CT was dependent on the enzymatic A subunit of CT, because the cell-binding B subunit alone could not block disease development. Mice given CT displayed detectable IL-4 levels in their serum within hours of CT administration. This innate IL-4 production was critical for protection, as infusion of neutralizing Ab against IL-4 to mice, given a uveitogenic immunization and treated with CT, counteracted immune deviation and abrogated protection. Our data indicate that systemic administration of CT inhibits experimental autoimmune uveitis by skewing the response to the uveitogenic autoantigen to a nonpathogenic phenotype.

Interphotoreceptor retinoid-binding protein (IRBP)-deficient C57BL/6 mice have enhanced immunological and immunopathogenic responses to IRBP and an altered recognition of IRBP epitopes.

Experimental autoimmune uveitis (EAU) and pinealitis (EAP) can be induced in susceptible mice by immunization with immunologically privileged retinal antigens. In the present study, we analyzed the immunologic and immunopathologic responses of mice deficient in the retinal autoantigen interphotoreceptor retinoid-binding protein (IRBP). The consequences of IRBP deficiency on the T-cell repertoire were also investigated. IRBP+/+, IRBP+/- and IRBP-/- mice on the C57BL/6 background were immunized with IRBP or with a pathogenic epitope, IRBP(1-20) peptide in adjuvant, and were evaluated for disease severity and immunological responses. C57BL/6 IRBP-/- mice were completely resistant to EAU and EAP, and had enhanced immunological responses to IRBP and to its pathogenic peptide 1-20, as compared to their IRBP+/+ counterparts. IRBP-/- mice exhibited an altered IRBP epitope recognition. T cell epitope mapping revealed a response to IRBP peptide 271-290 in IRBP-/- mice, that was absent in the wild type. Primed T cells of IRBP-/- mice transferred an exacerbated form of EAU to nai;ve wild type recipients. A gene-dose effect was evident in that C57BL/6 IRBP+/- mice, exhibited intermediate immunological responses and lower disease scores compared to wild type. We conclude that expression of IRBP in target tissues is a necessary prerequisite for disease induction, excluding other retinoid-binding or vision-related proteins as surrogate targets. Furthermore, endogenous expression of IRBP is directly responsible for lowering the threshold of susceptibility to uveitic disease.

RPE65 is highly uveitogenic in rats.

PURPOSE: To examine the hypothesis that RPE65, a protein specific to the retinal pigment epithelium, is uveitogenic in rats. METHODS: Rats of four inbred strains (Lewis, Brown Norway, Fischer, and SHR) were immunized with native or recombinant bovine RPE65, or with S-antigen (S-Ag), emulsified with complete Freund adjuvant, and treated simultaneously with killed Bordetella pertussis bacteria, as indicated. Development of ocular changes was examined and scored both clinically and histologically. RESULTS: Lewis rats immunized with RPE65 showed development of acute and severe inflammatory eye disease that affected most ocular tissues. The minimum uveitogenic dose of RPE65 was similar to that of S-Ag (1 microg per rat), but the changes induced by RPE65 at higher dose ranges were less severe than those induced by S-Ag. Concurrent treatment of the RPE65-immunized rats with B. pertussis bacteria was not critical for disease induction, but enhanced dramatically the pathogenic reaction. Unlike the results with several other retinal proteins, no pinealitis was detected in rats immunized with RPE65. Fischer (F344) rats resembled Lewis rats in being similarly affected by RPE65 or S-Ag. In contrast, Brown Norway (BN) rats developed severe disease when immunized with RPE65, but showed minimal changes in response to S-Ag. SHR rats responded poorly to disease induced by RPE65, and S-Ag-induced disease failed to develop. CONCLUSIONS: RPE65 is highly uveitogenic in rats, thus suggesting that this molecule could be involved in pathogenic autoimmunity in the human eye.