These findings suggest that IL-22 can function locally within the retina to reduce inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways. in the presence of increasing doses (0.2g/ml, 2.0g/ml, and 20.0g/ml) of IRBP protein in HL-1 medium (Lonza, Walkersville, Inc.) with additives and -MMP (100mg/ml) to neutralize the effect of contaminating Con-A in IRBP preparation. well as WT mice treated systemically or intraocularly with anti-IL-22 antibodies during the expression phase of disease, developed exacerbated retinal damage. Furthermore, IL-22?/? mice were more susceptible than WT controls to Ceftriaxone Sodium Trihydrate glutamate-induced neurotoxicity, whereas local IL-22 supplementation was protective, suggesting direct or indirect neuroprotective effects. Mechanistic studies revealed that retinal glial Muller cells express IL-22r1 IL-22 enhanced their ability to suppress proliferation of effector T cells. Finally, IL-22 injected into the vision concurrently with IL-1, inhibited the (IL-1-induced) expression of multiple proinflammatory and proapoptotic genes in retinal tissue. These findings suggest that IL-22 can function locally within the retina to reduce inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways. in the presence of increasing doses (0.2g/ml, 2.0g/ml, and 20.0g/ml) of IRBP protein in HL-1 medium (Lonza, Walkersville, Inc.) with additives and -MMP (100mg/ml) to neutralize the effect of contaminating Con-A in IRBP preparation. Cells were incubated at 37C and 5% CO2 for a total of 72 hours with H3 pulsing during the last 18 hours of incubation. Level of H3 incorporation was measured using scintillation counting. For cytokine assays cells were cultured with 20g/ml IRBP protein and culture supernatants were collected after 48 hours of incubation. Mouse Muller cells were isolated from eyes of adult mice and were cultured as monolayers in presence of 10% Concanavalin A stimulated spleen cell supernatant as explained previously [18]. Prior to use in experiments, Mller cells were plated in slide chambers or in 96-well plates and allowed to adhere overnight (approximately 18 hours). For T cell suppression assay, Muller cells were cultured with (500ng/ml) or without murine rIL-22 for three days, irradiated (3000 rads), plated in 96-well plate at a Ceftriaxone Sodium Trihydrate concentration of 1 1 104 cells/well, and allowed to adhere for 18 hours. On the day of the assay, freshly isolated splenic cells from naive C57BL/6J mice were enriched for T cells using murine T cell enrichment column (R&D systems, MN) and were labeled with proliferation dye eF670 (eBioscience, CA). Dye labeled T cells (1 105 cells/well) were co-cultured with or without irradiated Muller cells in the presence or absence of anti-CD3 (1g/ml), anti-CD28 (0.5g/ml), and murine rIL-22 (500ng/ml, PeproTech) for 72 hours at 37C and 5% CO2. Inhibition of T cell proliferation was quantified by FACS analysis using the Proliferation tool in FlowJo software TLR3 (Tree Star Inc., OR). 2.6. Analysis of cytokines and infiltrating cells from eyes of EAU mice Enucleated eyes were collected into HL-1 medium made up of 1mg/ml collagenase D (Roche Life science). External tissues (optic nerve and connective tissues) were trimmed and the globes were transferred into 1 ml serum free HL-1 medium (10 eyes from 5 mice per group) or two eyes of individual mouse in 100l PBS (for measuring Tgf) made up of Halt protease inhibitor cocktail (Thermo Fisher Scientific). After cautiously removing the lens by dissection along the limbus, the tissue was minced with scissors, dispersed by vigorous trituration, and centrifuged. The clarified supernatant was collected for cytokine analysis. The pellet was resuspended in 3 ml HL-1 medium made up of 10% FCS, and 1 mg/ml collagenase D and incubated for 1 h in 37C. The cells were dispersed by trituration using a transfer pipette, washed, filtered through 40m strainer, and suspended in 3 ml RPMI medium made up of 10% fetal calf serum for counting. Cells were incubated (4 106 cells/ 3ml/ well of 24 well plate) with or without PMA/ionomycin and monensin and stained Ceftriaxone Sodium Trihydrate for intracellular cytokine analysis and immunophenotyping by FACS, as explained below. Non-inflamed eyes from unimmunized donors did not yield sufficient leukocytes for analysis. 2.7. Cytokine measurement in culture supernatants or vision extracts: IL-22 in culture supernatants and vision extracts was quantitated using mouse-IL-22 Duoset ELISA development system (catalog # DY582, R&D systems, MN). Multiplex cytokine analysis was carried out using SearchLight Assay support (Aushon Biosystems, Billerica, MA) or the BioPlex Pro? Mouse Cytokine assay kit (Bio-Rad Lab, Inc. Hercules, CA) and their Luminex-based system (Bio-Rad Lab, Inc. Hercules, CA). Data from your BioPlex assay were analyzed using BioPlex manager software v6.1 (Bio-Rad Lab, Inc. Hercules, CA). Tgf1 in vision extracts was measured using.