Fig 1.
IL-22 serum levels in patients with uveitis.
Serum was obtained from patients with uveitis (n = 20) and healthy donors (n = 19). It was separated from whole blood using a serum-separating tube by centrifuging at 600 x g for 10 min. IL-22 serum levels were measured with ELISA kits following the manufacturer’s instructions. P-value was obtained using unpaired two-tailed student’s t-test. ** p<0.001.
Table 1.
Clinical information
Fig 2.
Production of MCP-1 and proliferation in ARPE-19 cells by treatment with rIL-22.
(A) Expression of IL-22Rα in ARPE-19 cells was analyzed by confocal microscopy. Scale bar = 20 μm (B) ARPE-19 cells were treated with 5 ng/ml and 10 ng/ml of rIL-22 for 48 h. For the last 18 h, 1 μCi of [3H]-thymidine was added to each well. Each sample is in triplicates and results are representative of three independent experiments. (C) ARPE-19 cells were incubated with rIL-22 at 10 ng/ml for 24 h and the concentration of cytokines was measured with ELISA. (D) Migration of PBMCs in response to IL-22 was measured by the migration assay. ARPE-19 cells were placed in the lower chamber with ARPE-19 cells with or without rIL-22 and the upper chamber with PBMCs. PBMCs were then allowed to migrate for 72 h. Anti-MCP-1 Ab at 0.2 μg/ml was used for blocking MCP-1. Data were visualized with Diftquick-staining solution. Scale bar = 200 μm.* p<0.05, ** p<0.001, ***p<0.0001.
Fig 3.
The effect of IL-22 on ARPE-19 cells through the regulation of its receptor expression.
(A) ARPE-19 cells were pre-treated with DMSO (vehicle control) and LY294002 at 10 μM for 1 h and then cultured with rIL-22 at 10 ng/ml for another 24 h. (B) ARPE-19 cells were treated with rIL-22 at 10 ng/ml, and western blot for phosphorylation of Akt and Akt1/2 was performed as described in the Materials and Methods. (C) For mRNA expression, ARPE-19 cells were treated with rIL-22 alone or combined with LY294002 at 10 μM, an inhibitor of PI3K, and collected at the indicated time points, followed by RT-PCR.
Fig 4.
IL-22 production in IRBP1-20-induced EAU mice and uveitis patients.
(A) Representative histopathology of mice treated with vehicle (PBS) alone or cysteamine. Negative control (left), mouse treated with PBS (middle), and mouse treated with cysteamine (right). On day 21 post immunization, the eyes from normal and EAU mice were enucleated and scored by examining the histopathological sections (H&E, x100 magnification). (B) Splenocytes from IRBP1-20-treated and IRBP1-20-treated with cysteamine injected mice were re-stimulated with IRBP1-20 (1 μg/ml) in vitro for 24 h. The supernatants from the cultures were collected and assayed for IL-22 by ELISA. P-value was obtained with unpaired two-tailed student’s t-test. *** p<0.0001 (C) IL-22 levels in the CD4+ T cells of uveitis patients (n = 6) and healthy control (n = 6). P-value was obtained with unpaired two-tailed student’s t-test. * p<0.05.
Fig 5.
The regulation of IL-22Rα expression in ARPE-19 cells treated with cysteamine.
(A) Cells were treated with IRBP1-20 at 1 μg/ml for 1, 2, 4, and 6 h and collected for RT-PCR analysis at the indicated time points. (B) Cells were cultured with or without IRBP1-20 and cysteamine at 2.5 μg/ml for 6 h. RT-PCR was performed as described in the Materials and Methods. (C) Eye tissues isolated from each mouse (control vs. IRBP1-20-induced EAU model vs. IRBP1-20-induced EAU model treated with cysteamine) were fixed in 4% PFA at 4°C overnight. Fixed eye tissues were embedded in paraffin and sectioned. The expression of IL-22Rα in eye tissue was assessed by confocal microscopy. Scale bar = 100 μm.
Fig 6.
The effect of cysteamine on EAU.
(A) A diagram of clinical scoring of IRBP1-20-induced uveitis. Clinical score of EAU in mice treated with vehicle (PBS, n = 21) alone or cysteamine (40 mg/kg) (n = 18). EAU was induced as described in the Materials and Methods. Results are presented as the mean clinical score for all eyes for each group of mice, and the significance was determined with the Mann-Whitney test. (B) A diagram of histopathology scores of IRBP1-20-induced uveitis. Histopathological score of EAU was determined by histopathology on day 21 post immunization. Each symbol represents one mouse, showing the higher score of the two eyes for each mouse. Mean EAU score of each group is indicated by a bar. The pathological scores of retinal sections were significantly lower in the cysteamine-treated group (n = 27) than in the control group (n = 26). Results are expressed as the mean ± standard deviation, and significance was determined with the Mann-Whitney test. * p<0.05.