Interphotoreceptor Retinoid-binding Protein Research Articles

Temporal Regulation of Myopia and Inflammation-Associated Pathways in the Interphotoreceptor Retinoid-Binding Protein Knockout Mouse Model

Purpose Myopia is a complex disorder with etiology involving an interplay between several genetic and environmental factors. Interphotoreceptor retinoid-binding protein (IRBP) is found in the subretinal space and is crucial in the visual cycle. The interphotoreceptor retinoid-binding protein knockout mouse (IRBP KO) was established as a model system to understand myopia and retinal degeneration. The current study investigated genes associated with myopia, retinal homeostasis, and inflammation in IRBP KO. Methods RNA from retinas of congenic IRBP KO and wild-type C57BL/6J (WT) mice at postnatal day 5 (P5), P40, and P213 were subjected to digital droplet PCR (ddPCR) using a Bio-Rad automated droplet generator and QX200 reader. Target genes were selected based on genome-wide association studies, animal models, myopia studies, and other genes associated with retinal homeostasis and inflammation. HPRT, a housekeeping gene, was used for normalization. An average expression ratio (target/HPRT) and standard deviation (SD) were calculated. ANOVA assessed statistical significance, and a p < 0.05 was considered significant. Results The ddPCR data analysis indicated that numerous myopia and inflammation-associated genes were differentially regulated in IRBP KO retinas with distinct temporal variation (upregulated at P5, decreased at P40, and no change at P213 relative to WT). C1qa, Gjd2, Sntb1, and Vsx2 emerged as top genetic candidate pathways. Compared with WT, immunoblotting analysis of C1qa showed no significant differences at P5 but significantly increased protein levels at P7 in IRBP KOs. Vsx2 remained unaltered at P5 and P7 in KO when compared with WT. Conclusions Data analysis indicated significant contributions from C1q, Gjd2, Sntb1, and Vsx2 genes in IRBP deficiency.

Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis.

Crystallin βb2 (crybb2) is upregulated in regenerating retinas and in various pathological conditions of the retina, including uveoretinitis. However, the role of crybb2 in this disease is largely unknown. Therefore, we used recombinant crybb2 (rcrybb2) as intravitreal treatment of B10.RIII mice prior to immunization with human interphotoreceptor retinoid-binding protein peptide 161-180 (hIRBPp161-180) in complete Freund's adjuvant (CFA) and concomitant injection of pertussis toxin (PTX) to induce experimental autoimmune uveoretinitis (EAU). In naïve mice, more beta III-tubulin (TUBB3) + and RNA-binding protein with multiple splicing (RBPMS) + cells were found in the ganglion cell layer of the retina than in EAU eyes, suggesting a loss of retinal ganglion cells (RGC) during the development of EAU. At the same time, the number of glial fibrillary acidic protein (GFAP) + cells increased in EAU eyes. RGCs were better protected in EAU eyes treated with rcrybb2, while the number of GFAP+ cells decreased. However, in retinal flatmounts, both retinal ganglion cells and retinal endothelial cells stained positive for TUBB3, indicating that TUBB3 is present in naïve B10.RIII mouse eyes not exclusive to RGCs. A significant decline in the number of RBPMS-positive retinal ganglion cells was observed in retinal flatmounts from EAU retinas in comparison to naïve retinas or EAU retinas with intravitreal rcrybb2 treatment. Whereas no significant decrease in TUBB3 levels was detected using Western blot and RT-qPCR, GFAP level, as a marker for astrocytes, increased in EAU mice compared to naïve mice. Level of Bax and Bcl2 in the retina was altered by treatment, suggesting better cell survival and inhibition of apoptosis. Furthermore, our histologic observations of the eyes showed no change in the incidence and severity of EAU, nor was the immune response affected by intravitreal rcrybb2 treatment. Taken together, these results suggest that intravitreal injection of rcrybb2 reduces retinal RGC death during the course of EAU, independent of local or systemic autoimmune responses. In the future, treating posterior uveitis with rcrybb2 to protect RGCs may offer a promising novel therapeutic strategy.

Visual stimulation and brain-derived neurotrophic factor (BDNF) have protective effects in experimental autoimmune uveoretinitis

AimsTo investigate the therapeutic potential of visual stimulation (VS) and BDNF in murine experimental autoimmune uveoretinitis (EAU). Main methodsMice were immunized by subcutaneous injection of interphotoreceptor retinoid-binding protein in Freund's complete adjuvant and intravenous injection of pertussis toxin, and were then exposed to high-contrast VS 12 h/day (days 1–14 post-immunization). EAU severity was assessed by examining clinical score, visual acuity, inflammatory markers, and immune cells in the retina. The transcriptome of activated retinal cells was determined by RNA-seq using RNA immunoprecipitated in complex with phosphorylated ribosomal protein S6. The retinal levels of protein products of relevant upregulated genes were quantified. The effect of BDNF on EAU was tested in unstimulated mice by its daily topical ocular administration (days 8–14 post-immunization). Key findingsVS attenuated EAU development and decreased the expression of pro-inflammatory cytokines/chemokines and numbers of immune cells in the retina (n = 10–20 eyes/group for each analysis). In activated retinal cells of control mice (n = 30 eyes/group), VS upregulated genes encoding immunomodulatory neuropeptides, of which BDNF and vasoactive intestinal peptide (VIP) also showed increased mRNA and protein levels in the retina of VS-treated EAU mice (n = 6–10 eyes/group for each analysis). In unstimulated EAU mice, BDNF treatment mimicked the protective effects of VS by modulating the inflammatory and stem cell properties of Müller cells (n = 5 eyes/group for each analysis). SignificanceVS effectively suppresses EAU, at least through enhancing retinal levels of anti-inflammatory and neuroprotective factors, VIP and BDNF. Our findings also suggest BDNF as a promising therapeutic agent for uveitis treatment.

The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360nm; emission, >420nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Conditional Knockouts of Interphotoreceptor Retinoid Binding Protein Suggest Two Independent Mechanisms for Retinal Degeneration and Myopia.

Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration. IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry. Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs. Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.

Enhanced immunosuppressive capability of mesenchymal stem cell-derived small extracellular vesicles with high expression of CD73 in experimental autoimmune uveitis

BackgroundAutoimmune uveitis is an inflammatory disease triggered by an aberrant immune response. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are emerging as potential therapeutic agents for this condition. CD73, an ectoenzyme present on MSC-sEVs, is involved in mitigating inflammation by converting extracellular adenosine monophosphate into adenosine. We hypothesize that the inhibitory effect of MSC-sEVs on experimental autoimmune uveitis (EAU) could be partially attributed to the surface expression of CD73.MethodsTo investigate novel therapeutic approaches for autoimmune uveitis, we performed lentiviral transduction to overexpress CD73 on the surface of MSC-sEVs, yielding CD73-enriched MSC-sEVs (sEVs-CD73). Mice with interphotoreceptor retinoid-binding protein (IRBP)-induced EAU were grouped randomly and treated with 50 µg MSC-sEVs, vector infected MSC-sEVs, sEVs-CD73 or PBS via single tail vein injection. We evaluated the clinical and histological features of the induced mice and analyzed the proportion and functional capabilities of T helper cells. Furthermore, T-cells were co-cultured with various MSC-sEVs in vitro, and we quantified the resulting inflammatory response to assess the potential therapeutic benefits of sEVs-CD73.ResultsCompared to MSC-sEVs, sEVs-CD73 significantly alleviates EAU, leading to reduced inflammation and diminished tissue damage. Treatment with sEVs-CD73 results in a decreased proportion of Th1 cells in the spleen, draining lymph nodes, and eyes, accompanied by an increased proportion of regulatory T-cells (Treg cells). In vitro assays further reveal that sEVs-CD73 inhibits T-cell proliferation, suppresses Th1 cells differentiation, and enhances Treg cells proportion.ConclusionOver-expression of CD73 on MSC-sEVs enhances their immunosuppressive effects in EAU, indicating that sEVs-CD73 has the potential as an efficient immunotherapeutic agent for autoimmune uveitis.

The wildcat (Felis s. silvestris) in the Mediterranean forest: sighting through photo-trapping and non-invasive hair collection for genetic purposes

The European wildcat (Felis silvestris silvestris) is a mesocarnivore species widely distributed in Europe, from Eastern Europe to Portugal and from Scotland to Italy. Recent biogeographical studies of wildcat populations have endeavoured to assess in detail the various issues that pose a threat to this species, including hybridization with domestic cats. The use of non-invasive sampling methods supported by photo-trapping and some attractants has made it possible to gather genetic material for the detection of native wildcats in locally threatened populations, some of which live in the Iberian Peninsula. Testimonies of naturalists, hunters and farm workers led our team to choose specific areas in two large territories of Mediterranean forests where the presence of wildcats has been historically attested: the Almonte River basin and the Sierra de San Pedro Mountains. Between 2014 and 2018, non-invasive hair sampling was performed using valerian (Valeriana officinalis) as an attractant and supported by photo-trapping to guarantee the collection of genuine biological material (hair samples). The hair samples were genetically assessed by sequencing the nuclear gene IRBP (interphotoreceptor retinoid-binding protein) and the mtDNA gene ND4 (NADH dehydrogenase subunit 4). Despite the low density of wildcats, this combined protocol proved to be an applicable tool for detecting the presence of elusive wildcats and other mesocarnivore species in this remote region of southern Europe. In addition, non-invasive hair trapping contributes to the collection of genetic material from current wildcat populations. This procedure could enhance future management actions focused on collecting quality individualized biological material.

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1β, and IL-23. Invivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.

Comprehensive Grading System for Experimental Autoimmune Uveitis in Mice.

Experimental autoimmune uveitis (EAU) is the most commonly used animal model to study the progression of chronic uveitis and to test various therapies to treat the disease. However, to accurately evaluate the effectiveness of such treatments, a grading system that combines the latest imaging techniques with definitive quantitative grading thresholds is required. This study aimed to develop a comprehensive grading system that objectively evaluates EAU progression in C57BL/6J mice. EAU was induced following immunisation with interphotoreceptor retinoid-binding protein (IRBP) and pertussis toxin. Weekly fundus and optical coherence tomography (OCT) images were acquired over 12 weeks using a Micron IV imaging system. Each mouse was graded (between 0 to 4) based on changes seen on both the fundus (optic disc, retinal blood vessels and retinal tissue) and OCT (vitreous and retinal layers) images. A total EAU response (with a maximum score of 48) was calculated for each mouse based on the sum of the individual scores each week. Analysis of the clinical scores depicted a gradual increase in inflammatory signs including optic disc and vascular swelling, leukocyte infiltration in the vitreous, lesions in the retina and formation of granulomas and hyper-reflective foci in the retinal layers in EAU mice, with most signs reaching a plateau towards the end of the study period. Development of these signs into sight-threatening complications such as optic disc atrophy, structural damage to the retina and subretinal oedema were noted in 80-90% of mice suggesting consistent disease induction. Overall, a comprehensive and objective grading system encompassing all pathologies occurring in EAU mice was developed to enhance the preclinical evaluation of novel uveitis treatments.

Retinal Responses to Visual Stimuli in Interphotoreceptor Retinoid Binding-Protein Knock-Out Mice.

Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal space bound by the photoreceptor (PR) outer segments and the processes of the retinal pigmented epithelium (RPE). IRBP binds retinoids, including 11-cis-retinal and all-trans-retinol. In this study, visual function for demanding visual tasks was assessed in IRBP knock-out (KO) mice. Surprisingly, IRBP KO mice showed no differences in scotopic critical flicker frequency (CFF) compared to wildtype (WT). However, they did have lower photopic CFF than WT. IRBP KO mice had reduced scotopic and photopic acuity and contrast sensitivity compared to WT. IRBP KO mice had a significant reduction in outer nuclear layer (ONL) thickness, PR outer and inner segment, and full retinal thickness (FRT) compared to WT. There were fewer cones in IRBP KO mice. Overall, these results confirm substantial loss of rods and significant loss of cones within 30 days. Absence of IRBP resulted in cone circuit damage, reducing photopic flicker, contrast sensitivity, and spatial frequency sensitivity. The c-wave was reduced and accelerated in response to bright steps of light. This result also suggests altered retinal pigment epithelium activity. There appears to be a compensatory mechanism such as higher synaptic gain between PRs and bipolar cells since the loss of the b-wave did not linearly follow the loss of rods, or the a-wave. Scotopic CFF is normal despite thinning of ONL and reduced scotopic electroretinogram (ERG) in IRBP KO mice, suggesting either a redundancy or plasticity in circuits detecting (encoding) scotopic flicker at threshold even with substantial rod loss.

Interleukin-17 receptor C is essential for the pro-inflammatory pathogenicity of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in experimental autoimmune uveitis

Autoimmune uveitis is an inflammatory disorder of the eye triggered by the responses of autoreactive T cells to ocular autoantigens. This study aims to understand the role of granulocyte–macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in the pathophysiology of mouse experimental autoimmune uveitis (EAU). We established an EAU model by immunizing mice with interphotoreceptor retinoid-binding protein (IRBP) 651–670. Splenic or eye-infiltrating ThGM cells were analyzed and enriched by flow cytometry according to the levels of an array of surface markers, transcription factors, and cytokines. Lentiviral transduction was conducted to silence or overexpress the target gene in differentiated ThGM cells. The adoptive transfer was applied to determine the pathogenicity of ThGM cells in vivo. We found that ThGM cells were present in the spleen and the eye after EAU induction. Both splenic and eye-infiltrating ThGM cells were phenotypically CD4+CCR7-CXCR3-CCR6-CCR10hi. Eye-infiltrating ThGM cells up-regulated interleukin-1β (IL-1β), interleukin-6 (IL-6), and IL-17 receptor C (IL-17RC) relative to splenic ThGM cells. IL-17RC overexpression enabled interleukin-17A (IL-17A)-induced up-regulation of IL-1β and IL-6 production in ThGM cells. Adoptive transfer of IL-17RC overexpressing ThGM cells exacerbated EAU severity, as evidenced by a higher histology score as well as increased pro-inflammatory cytokines and inflammatory cells in the eye. However, IL-17RC-silenced ThGM cells did not impact EAU. Therefore, for the first time, this study unveils the essential pro-inflammatory role of IL-17RC-expressing ThGM cells in EAU pathophysiology. We discovered a novel mechanism underlying the pathophysiology of autoimmune uveitis.

Apigenin Alleviates Autoimmune Uveitis by Inhibiting Microglia M1 Pro-Inflammatory Polarization.

Apigenin is a natural small molecule compound widely present in various vegetables and fruits. Recently, Apigenin was reported to inhibit lipopolysaccharide (LPS)-simulated microglial proinflammatory activation. Considering the important role of microglia in retinal disorders, we wonder whether Apigenin could exert a therapeutic effect on experimental autoimmune uveitis (EAU) through reprogramming retinal microglia to a beneficial subtype. EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein (IRBP)651-670, followed by intraperitoneal administration of Apigenin. Disease severity was assessed based on clinical and pathological scores. In vivo, Western blotting was used to quantify protein levels of classical inflammatory factors, microglial M1/M2 markers and the tight junction protein of the blood-retinal-barrier (BRB). Immunofluorescence was used to determine the Apigenin's efficacy on microglial phenotype. In vitro, Apigenin was added in LPS and IFN-γ stimulated human microglial cell line. Western blotting and Transwell assays were used to analyze the phenotype of microglia. In vivo, we found that Apigenin significantly reduced the clinical and pathological scores of EAU. The protein levels of inflammatory cytokines were significantly decreased in retina, and BRB disruption was ameliorated after Apigenin treatment. Meanwhile, Apigenin inhibited microglia M1 transition in EAU mice retina. In vitro functional studies showed that Apigenin decreased LPS and IFN-γ-induced microglial inflammatory factor production and M1-activation via the TLR4/MyD88 pathway. Apigenin can ameliorate retinal inflammation in IRBP induced autoimmune uveitis through inhibiting microglia M1 pro-inflammatory polarization via TLR4/MyD88 pathway.

Gut microbial signatures and their functions in Behcet's uveitis and Vogt-Koyanagi-Harada disease

BackgroundA number of public metagenomic studies reveal an association between the gut microbiome and various immune-mediated diseases including Behcet's uveitis (BU) and Vogt-Koyanagi-Harada disease (VKH). Integrated-analysis and subsequent validation of these results could be a potentially powerful way to understand the microbial signatures and their functions in these two uveitis entities. MethodsWe integrated the sequencing data of our previous metagenomic studies on two major uveitis entities, BU and VKH as well as four other publicly available immune-mediated diseases datasets, including Ankylosing Spondylitis (AS), Rheumatoid Arthritis (RA), Crohn's disease (CD) and Ulcerative Colitis (UC). Alpha-diversity and beta-diversity analysis were used to compare the gut microbiome signatures between both uveitis entities and other immune-mediated diseases and healthy controls. Amino acid homology between microbial proteins and a uveitogenic peptide of the interphotoreceptor retinoid-binding protein (IRBP)161-180 was investigated using a similarity search in the NCBI protein BLAST program (BLASTP). Enzyme-linked Immunosorbent Assay (ELISA) was performed to evaluate the cross-reactive responses of experimental autoimmune uveitis (EAU)-derived lymphocytes and BU patients-derived peripheral blood mononuclear cells (PBMCs) against homologous peptides. The area under the curve (AUC) analysis was used to test the sensitivity and specificity of gut microbial biomarkers. ResultsDepleted Dorea, Blautia, Coprococcus, Erysipelotrichaceae and Lachnospiraceae as well as enriched Bilophila and Stenotrophomonas were identified in BU patients. An enriched Alistipes along with a lower level of Dorea were observed in VKH patients. A peptide antigen (SteTDR) encoded by BU specifically enriched Stenotrophomonas was identified to share homology with IRBP161-180. In vitro experiments showed that lymphocytes from EAU or PBMCs from BU patients reacted to this peptide antigen as shown by the production of IFN-γ and IL-17. Addition of the SteTDR peptide to the classical IRBP immunization protocol exacerbated EAU severity. Gut microbial marker profiles consisted of 24 species and 32 species respectively differentiated BU and VKH from each other as well as from the other four immune-mediated diseases and healthy controls. Protein annotation identified 148 and 119 specific microbial proteins associated with BU and VKH, respectively. For metabolic function analysis, 108 and 178 metabolic pathways were shown to be associated with BU and VKH, respectively. ConclusionsOur study revealed specific gut microbial signatures and their potentially functional roles in BU and VKH pathogenesis that differ significantly from other immune-mediated diseases as well as healthy controls.

Bacterial origin of a key innovation in the evolution of the vertebrate eye

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer(iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

Molecular assessment of the taxonomic status of pine vole species Microtus subterraneus and Microtus majori in southeastern Europe and Turkey

Intra– and inter–specific phylogenetic relationships and the taxonomic status of pine vole populations have been controversial for years, and cryptic species are thought to exist, especially concering the species' distribution area. To clarify the taxonomic status of Turkish populations we analysed mitochondrial (cytochrome–b and cytochrome oxidase–I) and nuclear (interphotoreceptor retinoid binding protein) gene markers, adding GenBank data from Europe and Caucasia. Considering the data obtained based on mean genetic distance, genetic diversity and fixation index values, Bayesian trees and Median–joining networks, we found that M. subterraneus and M. majori are valid species which have diverged since 1.28 Mya. Findings also suggested that although Anatolian, Thracian (Turkish Thrace and Greece) and European populations of M. subterraneus and Anatolian and Caucasian populations of M. majori have been in the process of divergency since 0.528–0.337 Mya correspond to the Pleistocene glacial periods, these intrapopulations do not appear to be different species.Besides, considering the high intraspecific variation in M. subterraneus, it remains likely that new species could be identified in future studies.

The role of microbiome in uveitis in experimental mice model

AbstractUnlike the individual's genetic makeup, gut microbiota can be influenced, and thus used in the disease therapy or even prevention. We have shown that absence or reduction of microbiota modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). Our further purpose of the project was to test influence of probiotics on intensity of intraocular inflammation in EAU. Uveitis was induced in C57BL/6J mice by interphotoreceptor retinoid binding protein in complete Freund's adjuvant and with pertussis toxin. The grade of uveitis was assessed in vivo by using otoscope connected to camera and post mortem on haematoxylin and eosin stained tissues. Bacteria Escherichia coli were administered orally three times a week and saline solution was administered to control group. Different regimens of administration were tested. Interestingly, mice administered with probiotics reached statistically significant lower grade of uveitis in comparison to the control group in clinical and histological evaluation. This may have valuable implications for the control of sight‐threatening uveitis in patients.Supported by research project AZV MZ CR 17‐31248A.Key words: microbiome, probiotics, Escherichia coli, experimental autoimmune uveitis, EAU.

Pharmacodynamic mechanism of Tibetan medicine Liurui Capsules on experimental autoimmune uveitis in rats based on network pharmacology and animal experiments

By integrating network pharmacology and animal experiments, we studied the pharmacodynamic mechanism of the Tibetan medicine Liurui Capsules in the treatment of experimental autoimmune uveitis(EAU). The active ingredients and targets of Liurui Capsules were searched against the Encyclopedia of Traditional Chinese Medicine(ETCM), Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM), and relevant literatures. The EAU-related targets were obtained from Gene Expression Omnibus(GEO), GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). The common targets shared by Liurui Capsules and EAU were identified, and the protein-protein interaction(PPI) network was established via STRING. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were conducted via g: Profiler. The rat model of EAU was induced by interphotoreceptor retinoid-binding protein(IRBP) and treated with Liurui Capsules. The inflammatory response of anterior segment and the pathological morphology of retina were observed. The mRNA and protein levels of delta-like ligand 4(DLL4), Notch1, interleukin-17(IL-17), and tumor necrosis factor-alpha(TNF-α) were determined by real-time quantitative PCR(q-PCR) and Western blot, respectively. The network pharmacology analysis predicted 51 common targets of Liurui Capsules and EAU, which were mainly involved in IL-17, TNF, and nuclear factor-kappa B(NF-κB) signaling pathways, as well as liposome receptors and other biological processes. Compared with the control group, the modeling of EAU caused inflammatory changes in the anterior segment and retina and up-regulated mRNA and protein levels of DLL4, Notch1, IL-17, and TNF-α in ocular tissue. Compared with the model group, Liurui Capsules reduced the inflammatory reaction of anterior segment and retina and down-regulated the mRNA and protein levels of DLL4, Notch1, IL-17, and TNF-α. Liurui Capsules can down-regulate the expression of the proteins involved in DLL4/Notch1/IL-17 signaling pathway in ocular tissue and alleviate the ocular inflammation, which may be one of the mechanisms of Liurui Capsules in the treatment of EAU.

Genetic Ablation of Nrf2 Exacerbates Neuroinflammation in Ocular Autoimmunity.

Experimental autoimmune uveoretinitis (EAU) is an animal model of non-infectious uveitis and is developed by immunization with retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). Nuclear factor erythroid 2- (NF-E2-) related factor 2 (Nrf2) is responsible for regulating antioxidant and inflammatory responses. In this study, we investigated the role of Nrf2 on the development of EAU. Clinical and pathological examination demonstrated that retinal inflammation was exacerbated in Nrf2 knockout (Nrf2 KO) mice compared to wild type (WT) mice, and the expression of inflammatory cytokines (IFN-γ, IL-6, and IL-17) in the retina was significantly elevated in Nrf2 KO mice. GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia cells) in the retina were more numerous in Nrf2 KO mice compared to WT mice. Furthermore, we examined the suppressive effect of the Nrf2 activator CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline) on the development of EAU. The treatment with CDDO-Im significantly reduced the clinical and pathological score of EAU compared to those of vehicle-treated mice. These findings suggest that Nrf2 plays a regulatory role in the pathogenesis of autoimmune uveoretinitis and the activation of the Nrf2 system may have therapeutic potential for protecting vision from autoimmune neuroinflammation.

Antigen-Specific Intraocular Cytokine Responses Distinguish Ocular Tuberculosis From Undifferentiated Uveitis in Tuberculosis-Immunoreactive Patients

To compare antigen-specific intraocular immune responses between different clinical phenotypes of tuberculin skin test (TST)-positive and TST-negative uveitis. Single center, retrospective cross-sectional study. Patients requiring diagnostic or therapeutic vitrectomy for the management of intraocular inflammation were divided into 3 groups based on Standardization of Uveitis Nomenclature (SUN) classification criteria for tubercular uveitis. Group 1 included patients with ocular tuberculosis (OTB; n=23) who were TST-positive patients, met the SUN criteria, and/or had a polymerase chain reaction (PCR)-positive test for TB. Group 2 included patients with uveitis of unknown origin (UNK; n=24) who were undifferentiated TST-positive patients who had not met SUN criteria. Group 3 included non-TB uveitis patients (n=24) who were TST-negative either with or without a well-defined non-TB diagnosis. Total vitreous cells were activated with Mycobacterium tuberculosis-specific Early Secreted Antigenic Target-6 (ESAT-6) or the retinal autoantigen, interphotoreceptor retinoid-binding protein peptide (pIRBP 1-20), stained forintracellular interferon gamma (IFNγ), tumor necrosis factor-alfa (TNFα), and interleukin 17 (IL-17), and analyzed by flow cytometry. Antigen-specific single and dual (polyfunctional) cytokine responses to ESAT-6 and IRBP were compared between the 3 groups. All cytokine responses to ESAT-6 were higher in the UNK group compared with the non-TB control subjects, while all except IL-17 were comparable between the OTB and non-TB groups. Polyfunctional responses-IFNγ/IL-17 (P=.002), TNFα/IL-17 (P=.02), and TNFα/IFNγ (P=.01)-were significantly greater for UNK than the OTB group. Polyfunctional cells also produced more cytokine per cell than respective monofunctional cells. IRBP cytokine responses were comparable between different groups and were not affected by the clinical phenotype or duration of disease. The intraocular polyfunctional cytokine response is stronger in undifferentiated TST-positive uveitis than in OTB patients, likely representing an exaggerated anti-TB immune response rather than active infection.