Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia.

Iron has been implicated in the pathogenesis of age-related retinal diseases, including age-related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria-rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all-trans-retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline-injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD-associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti-inflammatory medications, and choroidal neovascularization inhibitors.

Celastrol Regulates the Secretion of Interleukin-17 in Patients with Sympathetic Ophthalmia Through Signal Transducer and Activator of Transcription 3.

Purpose: The excessive secretion of interleukin (IL)-17 contributes to the pathological process of sympathetic ophthalmia (SO). Celastrol is a naturally active product and exhibits an immunosuppressive effect. However, whether the supplementation of celastrol relieves SO remains unclear. Methods: The peripheral blood mononuclear cells (PBMCs) were extracted from the venous blood samples of 20 SO patients and 20 healthy controls, followed by stimulating with various concentrations of celastrol. The levels of IL-23 and IL-17 were measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction assay. The activation of the signal transducer and activator of transcription 3 (STAT3) in PBMCs of SO patients was detected by Western blot. Results: The levels of IL-23 and IL-17 in PBMCs isolated from SO patients were significantly increased compared with those in PBMCs isolated from healthy controls. Celastrol treatment inhibited the production of both IL-23 and IL-17 in PBMCs of SO patients in a dose-dependent manner. In PBMCs isolated from SO patients and healthy controls, the administration of recombinant human IL-23 (rIL-23) enhanced the production of IL-17, which was then suppressed by co-stimulation with celastrol. Also, celastrol treatment reduced rIL-23-induced phosphorylation of STAT3 in PBMCs isolated from SO patients. Conclusions: Celastrol can reduce the production of IL-17 in PBMCs of SO patients. The mechanism may be related to the reduction of IL-23 secretion, which in turn inhibits the phosphorylation of STAT3.

SYMPATHETIC OPHTHALMIA: Clinicopathologic Correlation in a Consecutive Case Series.

PURPOSE: To correlate the clinical course of sympathetic ophthalmia with the histological and immunohistochemical characteristics of the enucleated inciting eye. METHODS: A consecutive case series with baseline clinical features and subsequent histopathologic findings. RESULTS: Evaluation of the 16 enucleated inciting eyes (blind and painful) disclosed that 9 of the 16 had typical histology, fulfilling the criteria for sympathetic ophthalmia of diffuse granulomatous inflammation. Among the 16, 11 sustained previous penetrating trauma, 4 underwent previous eye surgery, and 1 patient presented with an unknown etiology. Patients with atypical histology (7 of 7) were taking corticosteroids at the time of enucleation. Only 2 of 9 patients with typical histology were taking corticosteroids at the time of enucleation. At 6 months after enucleation of the inciting eye, 4 of the 7 patients with atypical histology had a visual acuity of ≥20/40 compared with 8 of 8 patients (100%) with typical histology. On a 4-point scale (0-3+), the choroidal infiltrate of the 9 histopathologically typical eyes showed an average of 2.5+ CD68 (macrophages), 2.5+ CD20 (B cells), and 1.5+ CD3 (T cells). CONCLUSION: Histopathologic findings had minimal correlation with the clinical course of sympathetic ophthalmia. Corticosteroid treatment before enucleation may influence the pathologic confirmation of sympathetic ophthalmia. The predominance of B lymphocytes and macrophages over T lymphocytes may represent different stages of the disease process.

Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling.

PURPOSE: Recent discovery of microRNAs and their negative gene regulation have provided new understanding in the pathogenesis of inflammatory diseases. This study demonstrated microRNA expression profiling and their likely role in sympathetic ophthalmia, using formalin-fixed, paraffin embedded samples. METHODS: Two groups of four enucleated globes (total eight globes) from patients with clinical and histopathological diagnosis of SO (experimental samples) and one group of four age-matched, noninflamed enucleated globes (control samples) were used. Human genome-wide microRNA PCR array was performed and results were subjected to bioinformatics calculation and P values stringency tests. The targets were searched using the recently published and periodically updated miRWalk software. Quantitative real-time PCR and immunohistochemical staining were performed to confirm the validated targets in the mRNA and in the protein levels, respectively. RESULTS: No microRNA was significantly upregulated in SO, but 27 microRNAs were significantly downregulated. Among these, four microRNAs (hsa-miR-1, hsa-let-7e, hsa-miR-9, and hsa-miR-182) were known to be associated with the inflammatory signaling pathway. Only hsa-miR-9 has the validated targets, tumor necrosis factor-α, and nuclear factor kappa B1, which have been previously shown to be associated with mitochondrial oxidative stress-mediated photoreceptor apoptosis in eyes with SO. CONCLUSIONS: Identification of altered levels of microRNAs by microRNA expression profiling may yield new insights into the pathogenesis of SO by disclosing specific microRNA signatures. In the future these may be targeted by synthetic microRNA mimic-based therapeutic strategies.

Mitochondrial oxidative stress initiates visual loss in sympathetic ophthalmia.

The visual loss that occurs with sympathetic ophthalmia (SO) in the absence of recognizable retinal damage and inflammatory cell infiltration is an enigma. Experimental autoimmune uveoretinitis (EAU) is an animal model used to study human endogenous uveitis. Both innate and adaptive immune responses have been well studied in the photoreceptor damage mechanism of EAU. In our studies, in the early phase of EAU, proinflammatory molecules such as tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) and the subsequent mitochondrial DNA damage, mitochondrial protein alteration, and mitochondrial dysfunction by oxidative stress were observed before retinal inflammatory cell infiltration. Our recent study shows the importance of Toll-like receptors (TLRs) in the production of proinflammatory molecules and the induction of mitochondrial oxidative stress. Thus, the innate immune responses occur first with the activation of TLRs; this activation upregulates proinflammatory molecules, leading to mitochondrial oxidative stress before retinal inflammatory cell infiltration and the subsequent adaptive immune responses. Like EAU, SO also results in photoreceptor mitochondrial oxidative damage without retinal inflammatory cell infiltration. Such damage was associated with TNF-α, TNF-α receptors, and iNOS expression in the photoreceptors, suggesting that this molecular mechanism without retinal inflammatory cell infiltration may initiate photoreceptor damage in SO.

Expression of α-crystallin in the retina of human sympathetic ophthalmia.

Sympathetic ophthalmia (SO) is a bilateral, granulomatous, intraocular inflammation that occurs following a penetrating injury to one eye, and has the potential to cause blindness of both eyes. The aim of this study was to examine the expression of α-crystallin and to detect apoptotic cells in the retina of human eyes with SO. Five globes, including three with SO and two age-matched normal appearing retinae, were examined. Formalin-fixed, paraffin-embedded tissue sections were submitted to hematoxylin and eosin staining and immuno-histochemistry with anti-αA and αB-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, and double-staining immunohistochemistry was conducted together with the TUNEL reaction. In normal-appearing retina, αA-crystallin immunoreactivity was predominantly detected in the cytoplasm of photoreceptors, where αB-crystallin was less marked. In SO globes, granulomatous inflammation was noted in the choroid, whereas the retina and choriocapillaris were preserved. Immunoreactivity for αA-crystallin was detected in the retina, as well as in the cytoplasm and inner/outer photoreceptor segments. By contrast, αB-crystallin was weakly noted in the SO retina. Double-staining immuno-histochemistry revealed no TUNEL-positive photoreceptors in the retina displaying high immunoreactivity for αA-crystallin, but photoreceptor apoptosis was noted where expression of αA-crystallin was relatively low. The present study demonstrated that αA-crystallin was up-regulated in the cytoplasm of photoreceptors in the SO retina. This may play a protective role in the suppression of photoreceptor apoptosis associated with intraocular inflammation.

Inflammatory cytokine and chemokine expression in sympathetic ophthalmia: a pilot study.

Sympathetic ophthalmia is a bilateral uveitis that develops after penetrating injury to one eye. This study aimed to identify the inflammatory cellular sub-phenotypes and expression of pertinent inflammatory cytokines/chemokines in sympathetic ophthalmia (SO). Dalen-Fuchs nodules (DFN), granulomas, and non-granulomatous foci of inflammation were micro-dissected from 15 cases. RNA was extracted, and quantitative PCR was performed to measure IL-17, IL-18, IL-23, IFN-γ, CCL19, CXCL11, CCL17, and CCL22 transcripts. Immunohistochemical methods were used to characterize CD3, CD4, CD8, CD20, CD68, and CD163 expression. Non-granulomatous lymphocytes were predominantly CD3-positive and expressed more IFN-γ than cells within granulomas, consistent with Th1 cells. In contrast, granulomas and DFN contained mainly CD68+, CD163+/- and expressed more IL-17, IL-18, IL-23, CCL19, and CXCL11 than non-granulomatous cells. Our data indicate for the first time that M1 macrophages are the predominant inflammatory cells within granulomas and DFN of SO. We further observed high levels of IL-17 within granulomas and the presence of Th1 and M1 cells.

Photoreceptor oxidative damage in sympathetic ophthalmia.

PURPOSE: To determine photoreceptor oxidative stress and damage in sympathetic ophthalmia (SO). DESIGN: Immunohistologic study. METHODS: Eight formalin-fixed and paraffin-embedded human globes with typical histologic features of SO and five age-matched globes without intraocular inflammation (controls) were retrieved from the Doheny Eye Institute ophthalmic pathology files. Deparaffinized sections of the globes were processed to localize tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor-1 (TNF-R1), acrolein, inducible nitric oxide synthase (iNOS), and nitrotyrosine by immunolocalization method. The latter two were localized to photoreceptor mitochondria using anti-cytochrome C antibody. Apoptotic cells were detected by Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) assay and were localized to the site of oxidative stress using antinitrotyrosine antibody. RESULTS: Increased expression of TNF-alpha can be seen in the photoreceptor nuclear layer in all SO globes, whereas no such expression was observed in control globes. TNF-R1, iNOS, acrolein, and nitrotyrosine were immunolocalized to the inner segments of the photoreceptors in all SO globes, but only mild focal staining was observed in the control retinas. Both nitrotyrosine and iNOS immunolocalization revealed positive staining restricted primarily to mitochondria at the inner segments of the photoreceptors. Most of the TUNEL-positive cells were detected in the photoreceptors at the site of nitrotyrosine staining. In contrast, the age-matched control globes showed negative results. CONCLUSIONS: In SO, photoreceptor mitochondrial oxidative stress occurs in the absence of leukocytic infiltration of the retina and may lead to photoreceptor apoptosis and subsequent vision loss. The oxidative stress seems to be mediated by iNOS and TNF-alpha. The current anti-inflammatory therapy combined with agents that could prevent oxidative stress may prevent photoreceptor damage in SO and may preserve vision.

Expression of chemokines and gelatinase B in sympathetic ophthalmia.

PURPOSE: To examine the expression of gelatinase B (matrix metalloproteinase-9) and the chemokines monocyte chemotactic protein-1 (CCL2/MCP-1) and stromal cell-derived factor-1 (CXCL12/SDF-1) in sympathetic ophthalmia (SO). METHODS: Five enucleated exciting eyes with a clinical diagnosis and typical histopathological findings of SO were studied by immunohistochemical techniques using a panel of monoclonal antibodies directed against gelatinase B, MCP-1, and SDF-1. In addition, a panel of monoclonal and polyclonal antibodies was used to characterize the composition of the inflammatory infiltrate. RESULTS: In all cases, the extensive uveal inflammatory infiltrate was organized as a diffuse infiltrate and as large granulomas consisting of epithelioid cells and multinucleated giant cells. CD20(+) B lymphocytes predominated in the diffuse infiltrate and CD3(+) T lymphocytes were few. The monocyte/macrophage marker CD68 was expressed in scattered inflammatory mononuclear cells and within granulomas and Dalen-Fuchs nodules. Most of the inflammatory cells were HLA-DR(+). Immunoreactivity for gelatinase B, MCP-1, and SDF-1 was observed in cells within granulomas and in scattered epithelioid cells. Immunoreactivity for MCP-1 was noted in retinal pigment epithelial cells. Endothelial cells of choriocapillaries showed weak immunoreactivity for SDF-1. CONCLUSIONS: Gelatinase B, MCP-1, and SDF-1 might have a pathogenic role in the recruitment of leucocytes into the eye in SO.

Adhesion molecule expression in acute and fibrotic sympathetic ophthalmia.

Samples of iris ciliary body, choroid and retina from normal eyes and from 2 cases of sympathetic ophthalmitis (one acute and one late stage fibrosis) were examined for the expression of the VLA integrins beta 1 and alpha 1-6, and the integrin beta 3, in addition to ICAM-1, VCAM-1, ELAM-1 and CD44 using an APAAP staining technique. The expression of VLA-4, VLA-5, VCAM-1, ICAM-1, and CD44 was significantly increased and ELAM-1 was slightly increased in acute sympathetic ophthalmitis in comparison to fibrotic and normal eyes. VLA-6 was moderately increased in acute and fibrotic cases and VLA-2 VLA-3 and beta 3 were moderately expressed on all tissues examined. The differential expression of molecules known to be involved in lymphocyte activation and adhesion in acute sympathetic ophthalmitis suggests that certain adhesion molecules play a role in the pathogenesis of intraocular inflammation and may be suitable targets for immunotherapy.