Analysis of the Pathogenesis of Experimental Autoimmune Optic Neuritis

Optic neuritis associated with multiple sclerosis has a strong association with organ-specific autoimmune disease. The goal of our research is to establish an optimal organ-specific animal model to elucidate the pathogenetic mechanisms of the disease and to develop therapeutic strategies using the model. This paper is divided into five sections: (1) clinical picture of optic neuritis associated with multiple sclerosis, (2) elucidation of pathogenesis using animal models with inflammation in optic nerve and spinal cord, (3) clinical relevance of concurrent encephalomyelitis in optic neuritis model, (4) retinal damage in a concurrent multiple sclerosis and optic neuritis model, and (5) development of novel therapies using mouse optic neuritis model. Advanced therapies using biologicals have succeeded to control intractable optic neuritis in animal models. This may ultimately lead to prevention of vision loss within a short period from acute onset of optic neuritis in human. By conducting research flexibly, ready to switch from the bench to the bedside and from the bedside to the bench as the opportunity arises, this strategy may help to guide the research of optic neuritis in the right direction

Expression and Function of Inducible Costimulator on Peripheral Blood CD4 ؉ T Cells in Behçet's Patients with Uveitis: A New Activity Marker?

PURPOSE. Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet's disease (BD) was investigated. METHODS. Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4 ϩ T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb. RESULTS. As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4 ϩ T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4 ϩ T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-␥, IL-17, and TNF-␣ production by PBMCs when stimulated with conA or IRBP in BD with active uveitis. CONCLUSIONS. High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-␥, IL-17, and TNF-␣ production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD. (Invest Ophthalmol Vis Sci. 2010;51:5099 -5104) DOI: 10.1167/iovs.10-5286 E ndogenous uveitis such as Behçet's disease (BD), VogtKoyanagi-Harada syndrome, sympathetic ophthalmia, birdshot retinochoroidopathy, and sarcoidosis is a potentially blinding disease in humans and is responsible for 10% to 15% of the acquired blindness in Japan. 1 Although endogenous uveitis covers a spectrum of clinical entities, all forms are believed to share immunohistologic similarities characterized by the infiltration of mainly T cells. Behçet's disease (BD) is a systemic inflammatory disease characterized by oral and genital ulcers as well as ocular, cutaneous, arthritic, vascular, and neurologic lesions. 3-5 An increasing number of reports indicate that aberrant cellular immunity, such as pathogenic CD4 ϩ T-cell[b]-mediated autoimmunity via the Th1/Th17 pathway, plays a key role in the pathophysiological process in BD with uveitis, 6 -10 although the mechanisms of ocular inflammation in BD remain largely unknown. During recent years, the understanding of immunologic mechanisms involved in uveitis has advanced greatly through investigations of experimental autoimmune uveoretinitis (EAU), an animal model of human uveitis that can be induced by immunization of susceptible animals with interphotoreceptor retinoid-binding protein (IRBP) or with an eye-specific retinal antigen or by adoptive transfer of CD4 ϩ T cells specific for retinal antigens. 11 EAU resembles certain human uveitic conditions in various aspects 14,15 Although CD28 regulation has substantial effects on immunity, its function appears to reside predominantly in the control of primary, but not secondary, immune responses in various autoimmune diseases. 16 -18 The CD28 homolog inducible costimulator (ICOS) has recently been identified as a novel member of the CD28 costimulator family and is expressed by activated T cells in both humans and mice. To identify new genes that may cause or contribute to the disease process of ocular BD, we used cDNA microarrays that provide the expression profile of more than 54,000 genes, some of which are immune-related, whereas others are involved in the cell cycle, cell growth, intracellular signaling, cellular adhesion, and transport, and we compared the expression profiles of healthy individuals and patients. One of the genes found to be upregulated in patients with ocular BD is ICOS, an activation marker expressed on activated T cells that binds B7RP-1-expressing monocytes. The engagement of ICOS with B7RP-1 along with an appropriate antigen provide a positive signal that promotes T-cell differentiation, cytokine secretion, and effector function in the absence of CD28. ϩ T cells and B7RP-1 expressed on infiltrating APCs are upregulated directly after disease onset. 30 Therefore, we speculated that in active BD, pathogenic CD4 ϩ T cells that infiltrate the eye express ICOS in the inflamed eye and would be an appropriate target for the treatment of human ocular BD. Moreover, we have demonstrated that blockade of the ICOS/ B7RP-1 costimulatory pathway inhibits ocular inflammation in the effector phase of murine EAU. 30 Therefore, to investigate the role of ICOS in the pathogenesis of ocular BD, we assessed its expression on peripheral blood CD4 ϩ T cells and functional roles in patients with ocular BD. The results suggest that upregulation of ICOS is associated with the pathogenesis of uveitis in BD. MATERIALS AND METHODS PBMC Samples Thirty-five patients with BD (27 men and 8 women, mean age; 37.8 Ϯ 11.1 years) were enrolled in the study Heparinized blood samples were obtained from patients and healthy individuals, and PBMCs were isolated within 2 hours by density gradient centrifugation. The PBMCs were washed twice and resuspended at 1 ϫ 10 6 cells/mL in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 1 mM L-glutamine, 100 U/mL penicillin, and 100 g /mL streptomycin). Cell suspension was dispensed in 24-well plates (Falcon; BD Biosciences, Mountain View, CA) at 1 mL/well and incubated with or without concanavalin A (ConA; 10 g/mL) for 12 hours. Analysis of Gene Expression with cDNA Array Gene expression profile of human PBMCs was analyzed by microarray, as described previously. 37 Briefly, total RNA was isolated from pooled PBMCs obtained from seven patients with BD and active uveitis by an extraction method (Isogen Nippon Gene, Tokyo, Japan), and portions (2 g) of the preparation were subjected to amplification of mRNA with T7 RNA polymerase. Biotin-labeled cRNA (10 g) synthesized from the amplified RNA was subjected to hybridization with the Human Whole Genome Bioarray chip (Amersham Biosciences, Piscataway, NJ) that contains oligonucleotides corresponding to a total of approximately 55,585 human genes. Detection and digitization of hybridization signals were performed (G2565; Agilent Technologies, San Diego, CA) and analyzed (CodeLink Expression Analysis software version 4.0; Amersham Biosciences). The microarray data of various costimulatory molecules are summarized in Reagents FITC-conjugated anti-human CD4 (L3T4) was purchased from eBioscience (San Diego, CA), and anti-human ICOS (DX29) from BD PharMingen (San Diego, CA). ConA was obtained from Vector Laboratories (Burlingame, CA). Preparation of IRBP Fresh swine IRBP was purified according to the method described by Fukai et al. Immunofluorescence and Flow Cytometry The results of ICOS gene expression obtained from microarray were confirmed by flow cytometry. PMBCs were isolated by density gradient centrifugation of heparinized blood samples obtained from patients and healthy individuals. Each PBMC sample was divided into two aliquots: one for direct flow cytometry analysis of ICOS expression and the other for flow cytometry analysis after activation with conA at 10 g/mL for 12 hours. ICOS analysis was performed by using the following procedures: A cocktail of FITC-conjugated anti-CD4 and PE-conjugated anti-ICOS was added to the PBMCs. After the cells were washed with PBS, the stained cells (live-gated based on forward-and sidescatter profiles and propidium iodide exclusion) were passed through a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and the results analyzed (CellQuest program; BD Biosciences). ICOS expression on human CD4 ϩ T cells was calculated by gating for the CD4 ϩ T cell population, and 10,000 cells were analyzed in each experiment. Cytokine Assay Purified PBMCs (5 ϫ 10 5 /well) from BD patients with active uveitis were cultured in 96-well microtiter plates, with or without stimulation with conA (3 g/mL) or IRBP (10 g/mL) plus anti-ICOS mAb (10 g/mL) or control IgG at 37°C for 48 hours. The unstimulated PBMCs served as negative control samples. After stimulation, cell-free supernatants were collected at 48 hours and assayed for IFN-␥, TNF-␣, IL-2, and IL-6 by cytometric bead array (CBA) kits (BD PharMingen) and IL-17 by ELISA (Human IL-17 Quantikine ELISA kit; R&D Systems, Minneapolis, MN), according to the manufacturers' instructions. The minimum levels detected by CBA kits in either the culture supernatant or serum/plasma were 2.6 pg/mL for IL-2, 3.0 pg/mL for IL-6, 7.1 pg/mL for IFN-␥, and 2.8 pg/mL for TNF-␣; the minimum level in the IL-17 ELISA was 7.8 pg/mL. In a preliminary study, serial concentrations of conA (1, 3, 5, and 10 g/mL) or IRBP (1, 5, and 10 g/mL) were used in the assays. The results showed that optimal concentrations of cytokines were obtained at a concentration of 3 g/mL for conA and 10 g/mL for IRBP, as previously described. Statistical Analysis Data were analyzed (JMP 5; SAS Institute, Inc., Cary, NC) and the results expressed as the mean Ϯ SD. Statistical analysis was performed by using a paired or unpaired t-test. P Ͻ 0.05 was considered significant. RESULTS Analysis of Microarray Results in BD Patients with Active Uveitis To identify the genes involved in the pathogenesis of ocular BD, we compared phosphorescent images of the arrays hybridized with cDNA probes generated from RNA preparations from BD patients with active uveitis and healthy individuals. The array membrane that we used contained 360 positive bacterial controls and 384 negative controls. All positive controls were detected on the microarray. No signals were observed for the negative spots, indicating that the hybridization was highly specific. The DNA screening can evaluate more than 54,000 genes for a single sample. Among these genes, we focused on the immune-related ones, especially those of costimulatory molecules. Among the seven genes of costimulatory molecules, ICOS showed the greatest difference in expression in BD patients with active uveitis compared with that in healthy control subjects ICOS Expression on CD4 ؉ T Cells of BD Patients with Uveitis Flow cytometry was performed to confirm the results of gene array analysis. ICOS expression (median fluorescence intensity [MFI] of ICOS on CD4 ϩ T cells; Comparison of ICOS Expression on CD4 ؉ T Cells in BD Patients with Active or Inactive Uveitis Next, we compared ICOS expression on CD4 ϩ T cells in BD patients with active or inactive uveitis. ICOS expression (MFI of ICOS on CD4 ϩ T cells, Cytokine Production Stimulated with ConA and Uveitogenic Antigen To determine whether ICOS has functional costimulatory activity, we first measured the amounts of pathogenic Th1 and Th17 cytokines (IFN-␥, IL-17, TNF-␣, IL-6, and IL-2) produced by nonspecific stimulation (conA) of PBMCs obtained from BD patients with active uveitis. When ICOS costimulation was blocked with anti-ICOS mAb, the amounts of IFN-␥, IL-17, and TNF-␣ (Figs. 3A-C) produced by PBMCs were both significantly reduced. Since PBMCs from BD patients are known to be sensitized to the two most uveitogenic retina-specific antigens, S-antigen and IRBP, 1 we then examined the amounts of pathogenic Th1 and Th17 cytokines produced by stimulation with swine IRBP protein as the specific antigen. DISCUSSION The precise pathogenesis of uveitis associated with BD is unknown. Accumulated data obtained from animal models such as EAU suggest that Th1 and Th17 cells have major roles IOVS, October 2010, Vol. 51, No. 10 New Activity Marker for Ocular Behçet's Disease 5101 Downloaded from iovs.arvojournals.org on 06/30/2019 in its pathogenesis. We have previously shown that blockade of the ICOS ϩ costimulatory pathway has an ameliorating effect during the effector phase of EAU, by suppressing the expansion and effector function of pathogenic Th1 cells. In conclusion, our data provide additional evidence of the potential utility of ICOS expression on CD4 ϩ T cells as a marker of disease activity and as a promising therapeutic target for ocular BD via its inhibition of Th1 and Th17 cytokines. As the population studied was small and heterogeneous, further studies are needed to confirm the findings

Guidance on noncorticosteroid systemic immunomodulatory therapy in noninfectious uveitis: fundamentals of care for uveitis (focus) initiative

Topic: An international, expert-led consensus initiative to develop systematic, evidence-based recommendations for the treatment of noninfectious uveitis in the era of biologics. Clinical Relevance: The availability of biologic agents for the treatment of human eye disease has altered practice patterns for the management of noninfectious uveitis. Current guidelines are insufficient to assure optimal use of noncorticosteroid systemic immunomodulatory agents. Methods: An international expert steering committee comprising 9 uveitis specialists (including both ophthalmologists and rheumatologists) identified clinical questions and, together with 6 bibliographic fellows trained in uveitis, conducted a Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocol systematic reviewof the literature (English language studies from January 1996 through June 2016; Medline [OVID], the Central Cochrane library, EMBASE,CINAHL,SCOPUS,BIOSIS, andWeb of Science). Publications included randomized controlled trials, prospective and retrospective studies with sufficient follow-up, case series with 15 cases or more, peer-reviewed articles, and hand-searched conference abstracts from key conferences. The proposed statements were circulated among 130 international uveitis experts for review.Atotal of 44 globally representativegroupmembersmet in late 2016 to refine these guidelines using a modified Delphi technique and assigned Oxford levels of evidence. Results: In total, 10 questions were addressed resulting in 21 evidence-based guidance statements covering the following topics: when to start noncorticosteroid immunomodulatory therapy, including both biologic and nonbiologic agents; what data to collect before treatment; when to modify or withdraw treatment; how to select agents based on individual efficacy and safety profiles; and evidence in specific uveitic conditions. Shared decision-making, communication among providers and safety monitoring also were addressed as part of the recommendations. Pharmacoeconomic considerations were not addressed. Conclusions: Consensus guidelines were developed based on published literature, expert opinion, and practical experience to bridge the gap between clinical needs and medical evidence to support the treatment of patients with noninfectious uveitis with noncorticosteroid immunomodulatory agents

Streilein, “In vitro generation of regulatory CD8+ T cells similar to those found in mice with anterior chamber-associated immune deviation

PURPOSE. When injected intravenously into naïve mice, peritoneal exudate cells (PECs) incubated with ovalbumin (OVA) in the presence of transforming growth factor (TGF)-␤2 induce immune deviation similar to that evoked by injection of OVA into the anterior chamber of the eye. Intraocular antigen injection elicits two distinct populations of regulatory T cells that impair delayed hypersensitivity (DH) by two different mechanisms: a CD4 ϩ T cell that suppresses the induction of DH (afferent) and a CD8 ϩ T cell that inhibits DH expression. In an effort to understand the origin and mechanism of action of these regulatory cells, CD8 ϩ T cells from OVA-specific T cell receptor (Tcr) transgenic mice (OT-1) were used

Suppressive effects of a novel compound on interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis in rats

The immunosuppressive effect of ethyl O-(N-(pcarboxyphenyl)-carbamoyl)-mycophenolate(CAM) was examined in interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveoretinitis (EAU) in rats. Lewis rats immunized with bovine IRBP were treated with various oral doses of CAM postimmunization. The degree of inflammation was assessed clinically each day and histologically on day 14 or day 20. Production of various cytokines and IRBP-specific antibody, as well as IRBP-specific proliferation response, was assessed. Complete inhibition of EAU in rats, both by clinical and histologic criteria, was achieved with 50 mg/kg CAM when administered daily for 14 days following IRBP immunization. Partial inhibition was observed at lesser doses of CAM. This CAM-mediated response was accompanied by diminished production of cytokines interleukin-2, interferon-γ and tumor necrosis factor-α, as well as a reduction in IRBP-specific antibody production. Furthermore, administration of CAM either in the induction phase only (days 0–7) or in the effector phase only (days 9 or 11 to day 20) was also capable of suppressing EAU, as assessed histopathologically on day 20. We conclude that CAM is effective in suppressing EAU in rats and its mechanism of action appears to involve modulation of T cell function