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Multimodal evaluation of an interphotoreceptor retinoid-binding protein-induced mouse model of experimental autoimmune uveitis.

ABSTRACT: We aimed to characterize the vascular phenotypes of an experimental autoimmune retinal uveitis (EAU) model induced by interphotoreceptor retinoid-binding protein (IRBP) using multimodal imaging techniques. We systemically administered IRBP or vehicle to adult C57BL/6 mice. Fundus photography, optical coherence tomography (OCT), in vivo live confocal imaging using different tracers, OCT angiography (OCTA), and electroretinography (ERG) were performed after IRBP immunization. Hematoxylin and eosin and immunofluorescence staining were performed to characterize the immune response and vascular permeability. Mice with EAU exhibited perivascular inflammation, vitritis, and superficial retinal inflammation on fundus photography and OCT. H&E revealed immune cell infiltration in the perivascular area of the retina and choroid accompanied by a significant degree of perivasculitis that subsequently damaged photoreceptors 3 weeks postimmunization. Immunofluorescence staining showed subsequent transcytosis induction after local microglial activation followed by neutrophil recruitment in the perivascular area. Transcytosis in the superficial and deep vascular areas was improved by immune cell suppression. Intravital in vivo confocal imaging showed signs of neutrophil infiltration and obstructive vasculitis with perivascular leakage 3 weeks postimmunization. OCTA revealed a significant decrease in vascular flow in the deep capillary layer of the retina. Functional analysis showed that scotopic responses were intact at 2 weeks; however, normal photopic and scotopic responses were hardly detected in mice with EAU mice at 3 weeks postimmunization. Our data suggest that inflammatory cell activation and subsequent transcytosis induction in endothelial cells might be a major pathogenic factor for vascular leakage in uveitis, providing new insights into the pathophysiology of retinal vasculitis in noninfectious uveitis.

SUBMITTER: Yang JM

PROVIDER: S-EPMC8979956 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Multimodal evaluation of an interphotoreceptor retinoid-binding protein-induced mouse model of experimental autoimmune uveitis.

Yang Jee Myung JM Yun KyungA K Jeon Jehwi J Yang Hae Young HY Kim Bora B Jeong Sunhong S Lee Junyeop J Oh Wang-Yuhl WY Uemura Akiyoshi A Song Joon Seon JS Kim Pilhan P Lee Joo Yong JY

Experimental & molecular medicine 20220309 3

We aimed to characterize the vascular phenotypes of an experimental autoimmune retinal uveitis (EAU) model induced by interphotoreceptor retinoid-binding protein (IRBP) using multimodal imaging techniques. We systemically administered IRBP or vehicle to adult C57BL/6 mice. Fundus photography, optical coherence tomography (OCT), in vivo live confocal imaging using different tracers, OCT angiography (OCTA), and electroretinography (ERG) were performed after IRBP immunization. Hematoxylin and eosin ...[more]

PMID: 35264718

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Project description:The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.

Project description:Despite decades of investigation, the function of interphotoreceptor retinoid binding protein (IRBP), the most abundant protein in the interphotoreceptor matrix of vertebrates, remains enigmatic. Roles for IRBP in the visual cycle of rod photoreceptors and in the independent visual cycle of cone photoreceptors have been suggested, yet very little is known of the biology of IRBP in cone-dominant retinas, such as those of diurnal birds. Our aim was to identify and characterize expression of the IRBP of the cone-dominant chicken (Gallus gallus domesticus).Chicken IRBP mRNA was identified by PCR cloning. Primary protein structure, genomic organization, and phylogenies were determined through comparative sequence analyses. Expression of IRBP mRNA was characterized by northern analysis and by in situ hybridization on cryosectioned chicken retina. Expression of the IRBP protein was characterized by western blotting and by indirect immunofluorescence on cryosectioned retina and on retinal whole mounts.The chicken IRBP gene encodes a secreted protein with a predicted 1,252 amino acid length. The gene structure for chicken IRBP resembles that of most other vertebrates, with four homologous, modular repeats and introns within only the fourth module. Each module is more homologous with the corresponding module in other species than it is with the remaining chicken modules. Chicken retinal tissue contains a single IRBP mRNA transcript of approximately 4.8 kb and western analysis of chicken retina shows a single major band of 140 kDa. Chicken IRBP mRNA is expressed exclusively by retinal photoreceptor cells and the intensity of the hybridization signal shows light/dark rhythmicity. The IRBP protein is localized to the interphotoreceptor matrix of the chicken retina and to intracellular regions of photoreceptors, with a spatial distribution indicating an association with cone outer segments.The high degree of conservation of IRBP's primary structure, genomic organization, and cell-specific expression within the retinas of all vertebrates examined to date, including those with cone-dominant retinas, implies a conserved role for IRBP in photoreceptor function and/or health. Expression of chicken IRBP and its mRNA are functionally regulated. This report provides a necessary first step to explore a specific function for IRBP in the cone visual cycle.

Project description:The melanocortin system plays an essential role in the regulation of immune activity. The anti-inflammatory microenvironment of the eye is dependent on the expression of the melanocortin-neuropeptide alpha-melanocyte stimulating hormone (α-MSH). In addition, the melanocortin system may have a role in retinal development and retinal cell survival under conditions of retinal degeneration. We have found that treating experimental autoimmune uveitis (EAU) with α-MSH suppresses retinal inflammation. Also, this augmentation of the melanocortin system promotes immune tolerance and protection of the retinal structure. The benefit of α-MSH-therapy appears to be dependent on different melanocortin receptors. Therefore, we treated EAU mice with α-MSH-analogs with different melanocortin-receptor targets. This approach demonstrated which melanocortin-receptors suppress inflammation, preserve retinal structure, and induce immune tolerance in uveitis. At the chronic stage of EAU the mice were injected twice 1 day apart with 50 μg of α-MSH or an α-MSH-analog. The α-MSH-analogs were a pan-agonist PL8331, PL8177 (potent MC1r-only agonist), PL5000 (a pan-agonist with no MC5r functional activity), MT-II (same as PL5000) and PG901 (MC5r agonist, but also an antagonist to MC3r, and MC4r). Clinical EAU scores were measured until resolution in the α-MSH-treated mice, when the eyes were collected for histology, and spleen cells collected for retinal-antigen-stimulated cytokine production. Significant suppression of EAU was seen with α-MSH or PL8331 treatment. This was accompanied with significant preservation of retinal structure. A similar effect was seen in EAU-mice that were treated with PL8177, except the suppression of EAU was temporary. In EAU mice treated with PL5000, MTII, or PG901, there was no suppression of EAU with a significant loss in whole retina and outer-nuclear layer thickness. There was significant suppression of IL-17 with induction of IL-10 by retinal-antigen stimulated spleen T cells from EAU mice treated with α-MSH, PL8331, PL8177, or PL5000, but not from EAU mice treated with MT-II, or PG901. Our previous studies show the melanocortin system's importance in maintaining ocular immune privilege and that α-MSH-treatment accelerates recovery and induces retinal-antigen-specific regulatory immunity in EAU. Our current results show that this activity is centered around MC1r and MC5r. In addition, the results suggest that a therapeutic potential to target MC1r and MC5r together to suppress uveitis induces regulatory immunity with potentially maintaining a normal retinal structure.

Dataset Information

Multimodal evaluation of an interphotoreceptor retinoid-binding protein-induced mouse model of experimental autoimmune uveitis.

Publications

Multimodal evaluation of an interphotoreceptor retinoid-binding protein-induced mouse model of experimental autoimmune uveitis.

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