Involvement of B cells in non-infectious uveitis

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Non-infectious uveitis—or intraocular inflammatory disease—causes substantial visual morbidity and reduced quality of life amongst affected individuals. To date, research of pathogenic mechanisms has largely been focused on processes involving T lymphocyte and/or myeloid leukocyte populations. Involvement of B lymphocytes has received relatively little attention. In contrast, B-cell pathobiology is a major field within general immunological research, and large clinical trials have showed that treatments targeting B cells are highly effective for multiple systemic inflammatory diseases. B cells, including the terminally differentiated plasma cell that produces antibody, are found in the human eye in different forms of non-infectious uveitis; in some cases, these cells outnumber other leukocyte subsets. Recent case reports and small case series suggest that B-cell blockade may be therapeutic for patients with non-infectious uveitis. As well as secretion of antibody, B cells may promote intraocular inflammation by presentation of antigen to T cells, production of multiple inflammatory cytokines and support of T-cell survival. B cells may also perform various immunomodulatory activities within the eye. This translational review summarizes the evidence for B-cell involvement in non-infectious uveitis, and considers the potential contributions of B cells to the development and control of the disease. Manipulations of B cells and/or their products are promising new approaches to the treatment of non-infectious uveitis

Recombinant Filaggrin Is Internalized and Processed to Correct Filaggrin Deficiency

This study was designed to engineer a functional filaggrin (FLG) monomer linked to a cell-penetrating peptide (RMR) and to test the ability of this peptide to penetrate epidermal tissue as a therapeutic strategy for genetically determined atopic dermatitis (AD). A single repeat of the murine filaggrin gene (Flg) was covalently linked to a RMR motif and cloned into a bacterial expression system for protein production. Purified FLG+RMR (mFLG+RMR) was applied in vitro to HEK-293T cells and a reconstructed human epidermis (RHE) tissue model. Immunochemistry demonstrated RMR-dependent cellular uptake of FLG+RMR in a dose- and time-dependent manner in HEK cells. Immunohistochemical staining of the RHE model identified penetration of FLG+RMR to the stratum granulosum, the epidermal layer at which FLG deficiency is thought to be pathologically relevant. In vivo application of FLG+RMR to FLG-deficient flaky tail (ft/ft) mice skin demonstrated internalization and processing of recombinant FLG+RMR to restore the normal phenotype. These results suggest that topically applied RMR-linked FLG monomers are able to penetrate epidermal tissue, be internalized into the appropriate cell type, and be processed to a size similar to wild-type functional barrier peptides to restore necessary barrier function, and prove to be therapeutic for patients with AD

Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcriptionquantitative real-time polymerase chain reaction

© 2017 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (Nov 2017) in accordance with the publisher’s archiving policyBackground Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) < 0.2 and coefficient of variation (CV-value) < 0.1. Results Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells

Inhibition of Ocular Tumor and Endothelial Cell Growth with a TEAD4216 Peptide Fragment

Purpose: Transcriptional enhancer factor 1-related (RTEF-1) also known as TEAD4 is expressed in ocular vascular endothelial cells and plays a role in the control of VEGF expression. Alternative processing of TEAD4 hnRNA results in different proteins able to stimulate or inhibit VEGF gene transcription. The purpose of this study is to test whether short peptide fragments (STY-RMR), representing functional domains of the inhibitory TEAD4216 isoform, can inhibit tumor as well as endothelial cell proliferation.Experimental Design: Cell proliferation was assessed using a colorimetric assay in cell lines incubated with STY-RMR, the amount of secreted VEGF within media was determined both in treated and control cell lines.Results: Significant dose dependent inhibition of cell proliferation was observed. Maximal inhibition of ocular melanoma (Mel 202 and Mel 207) cell proliferation was observed at a dose of 30 mg/100ml of STY-RMR (87% and 60% inhibition, respectively). At the same dose, more than 50% inhibition was observed in retinoblastoma and breast cancer cells (P &lt;0.001). Significant inhibition of primate ocular endothelial cell proliferation (42% at 30 mg/100 ml (p &lt; 0.001), and retinal pigment epithelial cells showed also a 75% inhibition (p = 0.007). Secreted VEGF was decreased in the media of all tested cell lines that had been exposed to STY-RMR.Conclusion: Functional short peptide domains derived from the TEAD4216 isoform may prove to be useful for treatment of ocular tumors and other VEGF dependent neovascular disease.Inhibition of proliferation and VEGF production within ocular endothelial cells indicate the potential of this agent to treat age-related macular degeneration (ARMD) and diabetic retinopathy (DR)

Non-Invasive Stem Cell Therapy in a Rat Model for Retinal Degeneration and Vascular Pathology

BACKGROUND: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases

Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells

© 2018 Royal Australian and New Zealand College of Ophthalmologists. This author accepted manuscript is made available following 12 month embargo from date of publication (January 2018) in accordance with the publisher's archiving policy.Background Over‐production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS‐generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Methods Differential Nox enzyme expression was studied by real‐time‐quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2′,7′‐dichlorofluorescein. DMOG‐stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Results Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG‐induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Conclusions Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

© The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Abstract Objective Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. Results We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α

Expression of microRNA in human retinal pigment epithelial cells following infection with Zaire ebolavirus

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Objective: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation—or uveitis—which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. Results: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-α/β activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-γ and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3–RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world’s population, and that siRNAs might induce unanticipated vascular or immune effects