EAU induction and treatment
Female B10.RIII mice, aged 6 to 8 weeks, were obtained from an in-house colony and female C57Bl/6 mice were purchased from Charles River Laboratories (n = 5–8 per experimental group). All studies were conducted according to the UK Home Office Regulations on the Care, Welfare and Treatment of Laboratory Animals and in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement on the Use of Animals in Ophthalmic and Visual Research.
EAU was induced by immunization of naïve mice with subcutaneous 400 µg human IRBP peptides (IRBP161 − 180 for B10.RIII mice; IRBP1 − 20 for C57BL/6 mice) in complete Freund’s adjuvant (CFA; Sigma, Gillingham, UK; 1:1 vol/vol) supplemented with 1.5 mg/ml Mycobacterium tuberculosis H37 Ra (Difco Microbiology, Voigt Global Distribution, Lawrence, KS, USA). All mice simultaneously received 0.4 µg Bordetella pertussis toxin (Sigma-Aldrich, UK) intraperitoneally. Mice receiving CFA in PBS alone were used as controls.
Eyedrops (5µL) containing α4β1 integrin antagonist GW559090 ((S)-3-(4-((4-carbamoylpiperidine-1-carbonyl)oxy)phenyl)-2-((S)-4-methyl-2-(2-(otolyloxy)acetamido)pentanamido) propanoic acid, kindly provided by GSK) solubilized in phosphate buffered saline (PBS, pH = 7) were given twice daily in both eyes either prophylactically (from days 0 to 14 post EAU induction) or therapeutically (from days 10 to 18 post EAU induction). The concentrations of GW559090 were selected based on previously published data [22, 23]. GW559090 was solubilized in PBS and used at a final concentration of 3 mg/ml (GW3), 10 mg/ml (GW10) or 30 mg/ml (GW30) and compared to vehicle treated controls (Veh) or 0.1% dexamethasone eye drops (Maxidex®, Alcon, UK; Dex) as treatment controls. In some experiments, 10 mg/ml GW559090 was applied to the right eye only (GW10R) to evaluate whether it had a sympathetic effect on the untreated left eye (GW10L). Mice were sacrificed for further investigation when those in Veh group reached peak disease (day 14 in B10.RIII mice, day 18–21 for C57BL/6 mice).
Dilated ocular examination was performed using retinal fundoscopy (Micron III; Phoenix Research Laboratories, Pleasanton, CA, USA) on days 10, 14 and/or day 18–21 after disease induction. Pupils were dilated with topical 2.5% phenylephrine and 1% tropicamide, and the corneas protected with 0.2% carbomer eye gel (Bausch & Lomb Ltd, UK). The clinical grading was performed by two independent experts . In summary, optic disc neuropathy, vasculitis, retinitis, and structural damage were scored separately in each eye, from 0 (no disease) to 5 (severe inflammation) with half-point increments and then scores were summed to generate the clinical score of each eye on a scale from 0–20. The clinical score attributed to each mouse corresponds to the mean of the scores from both eyes.
Immunohistochemistry Staining For Cd45 Cells
Eyes were collected and fixed in situ with 4% glutaraldehyde for 1 hour, followed by overnight fixation in 10% formalin, and embedded in paraffin (Sigma Aldrich, UK). A series of four anterior-posterior sections passing through the optic nerve with 4 µm thick sections were prepared and stained with hematoxylin and eosin (H&E). The results were graded by two independent, masked observers .
For immunohistochemistry staining, the sections were blocked with 5% goat serum in PBS for 1 hour at room temperature, and then incubated with primary rabbit polyclonal antibody to CD45 (1:200; ab10558; Abcam, Oxford, UK) at 4°C overnight. After washing, the sections were incubated with biotinylated anti-rabbit IgG Ab (Vectastain Elite ABC HRP Kit, Abcam, UK) for 1 hour followed by visualization with a brick-red indicator product (NovaRED™, Vector Laboratories, UK). Stained sections were scored [27, 28]. In brief, cellular infiltrates (0–30) were scored within the ciliary body, vitreous, vessels, rod outer segments, and choroid, whereas the structural score (0–12) was based on the rod outer segments, neuronal layers, and retinal morphology. Both scores were added for a final score for each eye, and mean scores from both eyes calculated for each mouse (0–42).
Immunofluorescence Staining Locating Immune Cells Within The Eye
Ocular anterior-posterior sections were blocked with 5% bovine serum albumin (BSA) in TBS for 2.5 hours, then incubated overnight at 4°C with the following primary antibodies in different combinations: CD4 (rat; 1:100; Santa Cruz Biotechnology, USA), Tbet (rabbit; 1:100; Santa Cruz Biotechnology, USA), RORγt (goat; 1:100; Santa Cruz Biotechnology, USA), FoxP3 (rabbit; 1:100; Abcam, USA), and Iba-1 ((myeloid cell marker; mouse; 1:100; Santa Cruz Biotechnology), diluted in TBS supplemented with 1% BSA. After three washes in TBS, sections were incubated in the dark for 2 hours with species-specific secondary antibodies coupled to different fluorochromes as indicated in the data: Alexa Fluor 488, 555, and 633-conjugated secondary antibody (1:200; Molecular Probes, Eugene, OR, USA). After three final washings in PBS (20 min each), sections were mounted with Vectashield antifade mounting medium containing DAPI (Thermo Fisher Scientific, UK).
Images from at least three separate fields per retinal section (X 400) were captured using a Zeiss LSM 700 (or 710) confocal microscope. Staining patterns were observed to be consistent between consecutive single-, double-, or triple-stained sections using ZEN 2.1 software (Carl Zeiss, Oberkochen, Germany). ImageJ software (http://imagej.nih.gov/ij/) was used for counting cells in the images. These images were used for counting populations of CD4+Tbet+, CD4+RORγt+, CD4+Tbet+RORγt+, CD4+Foxp3+ and Iba-1+ cells in eye tissue. For cell scoring within the anterior chamber, total cells from three non-overlapping images were counted; for scoring cells in inner retina and vitreous, cell counts were from 5 images to cover the whole eye. Cells were verified with DAPI and counted within the anterior chamber, ciliary body, vitreous, vessels, retinal layers and choroid, and were summed up for a final score. The mean percentages of CD4+ T cell subsets in different experimental groups were calculated.
Identification of T cell subsets and myeloid populations by flow cytometry
Single-cell suspensions were prepared from retinal tissues, dLNs and blood and investigated by flow cytometric staining for their expression of CD4-FITC (RM4-5) and CD11b-PE (M1/70) (myeloid cell marker; eBioscience); CD11c-BV786 (HL3), Ly6G-PE-CF594 (IA8), CD45-BV605 (30-F11), FoxP3-PE/Cy5 (FJK-16 s), Ly6C-PerCP/Cy5.5 (HK 1.4), IFNγ-PE-Cy7 (XMG1.2), IL-17A-APC (TC11-18H10.1) and CD64-BV421 (X54-5/7.1; all BD Biolegend). In experiments investigating the myeloid population, data were obtained from 2–3 eyes pooled from the same treatment group in order to have sufficient cell numbers for analysis. For intracellular cytokine staining, cultures were treated for 4 hours with PMA (50 ng/mL) and ionomycin (1 µg/mL), and additional brefeldin A (1 µg/mL; all Sigma-Aldrich) for final 1 hour. Cells were incubated with relevant surface antibodies and a Live/Dead fixable dye (Molecular Probes; Life Technology, Paisley, U.K.) for 30 min at 4°C, before resuspending in FACS buffer and fixed. Cells were then permeabilized using fixation/perm buffer (Fix and Perm cell permeabilization kit; eBioscience, La Jolla, CA, USA) for 10 min. Transcription factor expression was determined using the Foxp3 cell Staining Kit (BD Biosciences), according to the manufacturer’s instructions. Relevant isotype control mAbs (BD Biosciences and eBioscience) were combined as fluorescence minus one (FMO) controls. Up to one million live cells per sample were acquired on a BD FACSCalibur using CellQuest (BD Cytometry Systems, Oxford, UK) or a BD LSR Fortessa-x20 using FACSDiva (BD Cytometry Systems). Isotypes and FMO controls were used for accurate gating. Compensation matrices were performed using OneComp Beads (eBioscience, UK). Flow cytometric data were analyzed using FlowJo software (Version 9.6.4; Tree Star). Debris and doublets were excluded, and live cells were gated before further analysis.
In vitro leukocyte migration assays
Human microvascular endothelial cells (HMEC; a gift from Professor P. Turowski UCL Institute of Ophthalmology, London, UK) were cultured with EGM2-MV (Endothelial cell growth medium-2 bullet kit, Lonza, USA) with supplements and the culture medium replaced weekly. Transmigration of lymphocytes was assayed using 6.5 mm Transwell inserts with a 3 µm pore size in a 24 well plate (Corning, Sigma-Aldrich, UK). HMEC cells were seeded on fibronectin-coated filters at a density of 3 × 105 cells and stimulated with human IFNγ (200 IU/ml, PeproTech, UK) 3 days before the assay .
Venous blood was obtained from healthy volunteers and collected in EDTA (3 mmol/L). Human buffy coat leukocytes were isolated by density gradient centrifugation by layering over Histopaque (Sigma Aldrich, UK) and centrifugation at 300 g to obtain mononuclear leukocytes. Mononuclear lymphocytes were cultured at 1 × 106/ml in T cell medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 2 mM L-glutamine, and 50 µM β-mercaptoethanol (all Sigma Aldrich). Lymphocytes were stimulated weekly with phytohaemagglutinin (PHA; 3 µg/ml) and human rIL-2 (50 IU/ml; PeproTech, UK) added every 3 days for a week before the assay , and lymphocytes were re-stimulated with PHA 24 hours before the assay. This methodology has proven to allow to keep the effector T cells in culture for a maximum of 4 weeks, when their viability is reduced [31, 32]. Cells were pre-treated with 10, 100, 1000 µg/ml GW559090 or Saline (Sal) as controls for 2 hours prior to assay.
To assess migration, 2 × 106 lymphocytes were pelleted by centrifugation, re-suspended in 100 µl fresh T cell medium and placed into each Transwell insert. The lower wells were filled with 500 µl of T cell medium. Following incubation for 18 hours at 37˚C, not adherent, adherent and migrated lymphocytes were carefully harvested from each compartment and stained for CD4-FITC (OKT4), RORγt-APC (AFKJS-9), Tbet-PE (eBio4B10) (all from eBioscience), and FoxP3-BV711 (236AIE7; BD Biosciences) and acquired for flow cytometry, as described above. In some experiments, additional markers for CXCR3-BUV395 (1C6/CXCR3), CCR6-BV421 (G034E3), IFNγ-BV605 (4S.B3; BD Biosciences) and IL-17A -PE (eBio64DEC17; eBioscience) were used. BD Liquid Counting Beads (BD Biosciences) was used to evaluate the cell numbers within each chamber, according to the manufacturer’s protocol.
Results are presented as the mean ± SEM for at least 5 data points and mean ± SD for 3 data points. For comparing means from two groups, P values were calculated by the unpaired two-tailed Student t-test. For comparison of means from three or more groups for two factors, two-way ANOVA was used to reveal differences in the data sets, followed by Dunn’s or Tukey’s multiple comparison post hoc test. Statistical analyses were performed using GraphPad Prism 6 software. P values < 0.05 were considered statistically significant.