Animals

The mice were purchased from the laboratory animal centre of the Academy of Military Medical Sciences (Beijing, China) and housed under standard conditions (22.5 °C and 42.5% humidity, under a 12 h/12 h light–dark cycle, using heated wood chip litter as bedding material) in the SPF animal centre of Wuxi People’s Hospital Affiliated to Nanjing Medical University, and permitted ad libitum consumption of water. The animals were ventilated after being anaesthetized with a mixture of ketamine and xylazine and the effectiveness of the anaesthesia was monitored by observing the parameters like slow breathing, loss of muscular tone, and no response to surgical manipulation. The retina was then harvested for subsequent analyses. All the studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health [NIH], Bethesda, MD, USA) and the ARVO Statement for the Use of Animals in the Ophthalmic and Vision Research. All the animal experiments fulfilled the requirements for humane animal care stated by the Nanjing Medical University.

Laser-induced AMD model

The mice were anaesthetized, and the pupils were dilated by applying Cyclomydril (Alcon, Fort Worth, TX, USA). Using a 532-nm laser, a slit-lamp delivery system, and a sliding glass as a contact lens, 4 spots (laser power, 200 mW; exposure time, 100 ms; hole size, 75 μm) were placed into each eye at a distance of approximately 2 optic disc diameters from the optic nerve head. After the laser-induced injury, the clodronate group mice (> 20 weeks old) received an intravitreal injection with 2 μl/eye of 90 μg clodronate liposomes (including 16 μg of clodronic acid) mixed with 154 mM sodium chloride at a ratio of 1:1 or control liposomes using a 33-gauge needle. The eyes of a selective cyclooxygenase-2 (COX2) inhibitor celecoxib (Cayman Chemical Company #169,590–42-5) injection group were subjected to IVI of celecoxib (10 mM celecoxib dissolved in 154 mM sodium chloride at a ratio of 1:3), or DMSO (vehicle, 25%). In some cases, celecoxib was administered orally at a dose of 100 mg/kg/day. Celecoxib was dissolved in 100% ethanol (v/v) and mixed with the food. After the ethanol was completely volatilized, the food was fed to the mice daily. The control mice were fed with the volatilized ethanol-treated food daily.

Retinal imaging

The animals were anaesthetized and the eyes were dilated and the Optical coherence tomography (OCT) images were obtained using an image-guided OCT system (Micron IV; Phoenix Research Labs, Pleasanton, CA, USA). The fundus fluorescein angiography (FFA) was performed using the Micron IV fundoscopy system (Phoenix Research Labs, Pleasanton, CA, USA). The anaesthetized animals with dilated pupils were intraperitoneally injected with 0.1 ml 0.2% sodium fluorescein (Fluorescite 10%; Alcon, Fort Worth, TX, USA).

Flow cytometry

On day 7 after laser-induced CNV, mice were transcardially perfused with iced-cold PBS. After eyes were quickly removed, the retina and RPE-choroid-sclera complexes were cut into small pieces. The tissue was further mechanically dissociated by trituration and the suspension was applied to 30 um cell strainer. The single cells were pre-incubated with Fc-block followed by stained with FITC-conjugated anti-CD11b (#101,206, BioLegend), APC-conjugated anti-CD80 antibodies (#104,714, BioLegend), PE-conjugated anti-CD80 (#104,708), PE-conjugated anti-CD206 (#141,706, BioLegend) and/or APC-conjugated anti-CD206. Stained cells were processed using LSR-II cytometer (BD Biosciences), and the data were analysed using FlowJo Software (version 7.6.2).

Cell culture and treatments

In our study, the general bone marrow-derived macrophages (BMDMs) were isolated from the C57BL/6 J mice. The isolation and culture of BMDM were performed as described [16]. Briefly, the animals were sacrificed by cervical dislocation and soaked in 75% ethanol. Then, the femurs and tibias were harvested and the bone marrow cells from all the bones were flushed out. After centrifuging for 5 min at 300 × g, the erythrocytes were eliminated using the red blood cell lysing buffer (Sigma-Aldrich, St. Louis, MO, USA). The remaining cells were seeded in the plates and incubated in a complete medium with 50 mg/ml recombinant mouse macrophage colony-stimulating factor (M-CSF, R&D Systems #416-ML) for 7 days to form the proliferative nonactivated cells (also named M0 macrophages).

Besides, the cells grown in DMEM were incubated with PGE2 (1 μM or 5 μM; Cayman Chemical Company #14,010), H2O2 (100, 200, 400, 800 μM) or recombinant murine IL-4 (20 ng/mL, PeproTech #214–14) or LPS (10 ng/ml #L4391) or recombinant human IL 10 (25, 50 ng/ml, PeproTech #200–10) with different time points. In some experiments, selective celecoxib (10 μM), 17-PT-PGE2 (an EP1R agonist, 10 μM), Butaprost (an EP2R agonist, 10 μM #13,740), Sulprostone (an EP3R agonist, 10 μM #14,765), Cay10598 (an EP4R agonist, 10 μM #13,281), H89 (a PKA inhibitor, 5 μM #10,010,556), LY294002 (a PKB inhibitor, 5 μM #70,920), Bis1 (a PKC inhibitor, 5 μM) obtained from the Cayman Chemical Company (Ann Arbor, MI, USA) were added to cells.

The haematoxylin and eosin (H&E) staining

The mice were anaesthetised, and their eyes were dissected and fixed in 4% paraformaldehyde (wt./vol.) overnight. The retina and the RPE-choroid-sclera complexes were dehydrated in a graded ethanol series and embedded in paraffin. For H&E staining, the 5-μ-thick sections were taken along the vertical meridian. The digital images of H&E staining were observed under an Olympus BX-51 light microscope (Olympus, Tokyo, Japan).

Immunocytostaining

The standard immunofluorescence analysis was performed to indicate the expression of the protein, followed by secondary antibodies (Thermo Fisher Scientific, CA, USA) in the cells or tissue sections, as previously described [17].

The volume of the CNV lesions was measured in the choroidal flat mounts after injury. The anterior segment and retina were removed from the eyecup after fixation in 4% paraformaldehyde in PBS. The remaining RPE-choroid complex was dehydrated in methanol and stained with 7 μg/ml fluorescein-labelled IB4 (Thermo Fisher Scientific #I21411). After relaxing radial incisions, this complex was flat-mounted and coverslips. The images were obtained using a confocal microscope (Leica, Heidelberg, Germany).

For whole-mount analysis, the eyes were enucleated and fixed using 4% buffered neutral formalin fixatives (Biosharp, Beijing, China) at room temperature for 2 h. The connective tissue, muscle, and optic nerve were removed from the back of the eye, and the cornea and lens were removed to form an eyecup. The four radial incisions were made, and the retina was carefully dissected off the RPE/choroid under a dissecting microscope, and its connection to the optic nerve was severed. The RPE-choroid-sclera complexes, now separate samples, were further fixed in the round-bottom microcentrifuge tubes at room temperature for 1 h. The RPE eye-cups were flat-mounted and prepared for immunohistochemistry by blocking them with 10% normal goat serum in 0.3%. The tissues were incubated in triton X-100 in PBS for 1 h at room temperature. They were then incubated overnight at 4 °C with COX1 (Abcam #ab109025), COX2 (Cayman Chemical Company #160,107), CD206 (Abcam, #ab8918), CD80 (Abcam #ab86473), F40/80 (Abcam #ab6640), CD31 (Abcam #ab24590) and EP1R (Cayman Chemical Company #101,740). After washing the RPE flat mounts, they were incubated for 1 h with a secondary antibody (Thermo Fisher Scientific). The RPE flat mounts were then washed and counterstained with DAPI (Sigma, MO, USA) and examined using a confocal microscope (Leica, Heidelberg, Germany).

ELISA

Mouse PGE2 and IL-10 in the peripheral blood of mice was measured using commercial ELISA kits (Cayman Chemical Company #514,010; CUSABIO #CSB-E04594m) according to the manufacturer’s instructions according to the manufacturer’s instructions. The measure of each sample was repeated at least six times.

Dual-luciferase reporter assay

The HEK293T cells were cultured in the RPMI medium 1640 basic DMEM (Gibco, Thermo Fisher) supplemented with 10% (v/v) fetal bovine serum at 37 °C. All the luciferase reporter plasmids were constructed by using a pGL3-Basic Vector. The HEK293T cells were separately transfected with pGL3-I1 (complete promoter IL-10 plasmid, 2000 bp), pGL3-I2 (truncated mutant IL-10 plasmid, 1500 bp), pGL3-I3 (truncated mutant IL-10 plasmid, 1000 bp), pGL3-I4 (truncated mutant IL-10 plasmid, 500 bp) and PGL3 basic (the negative control construct) for 48 h, and then supplemented with 5 μM PGE2. The relative luciferase activities were measured using the Dual-Luciferase® Reporter (DLR™) Assay System (Promega, #E1910) according to the protocol of the manufacturer.

Electrophoretic mobility shift assay (EMSA)

The analysis of the NF-κB binding activity in the nuclear proteins was performed as described in our previous study [18]. The NF-κB binding activity was examined using a Light Shift Chemiluminescent EMSA kit (Thermo Fisher Scientific #20,148) according to the manufacturer’s instructions. Briefly, the nuclear proteins (5 μg) were isolated and specific unlabeled NF-κB competitors (50- and 100-fold molar excess) were used along with the binding reaction mixture for the competition assay. The biotin end-labelled DNA duplex of sequences containing the NF-κB binding site (5′-AGT TGA GGC GAC TTT CCC AGG C-3′, 3′-TCA ACT CCG CTG AAA GGG TCC G-5′) was incubated with the nuclear proteins at room temperature for 20 min. The reaction mixture was loaded onto 6% non-denaturing polyacrylamide gels and subsequently transferred to a nylon membrane (Hybond N+, Amersham Corp., Arlington Heights, IL). The membranes were exposed to ultraviolet light to cross-link proteins for 1 min and incubated with the conjugate/blocking buffer with the stabilized streptavidin horseradish peroxidase conjugate. The signal on the membranes was detected with the enhanced chemiluminescence system (West Pico kit, Pierce, Loughborough, UK). The membranes were then exposed to X-ray film for 2–6 min and the relative intensities were analysed using the Image J software (National Institutes of Health imaging software).

Viability and proliferation (WST-1) assay

The cell proliferation reagent WST-1 (Beyotime Biotechnology, Shanghai, China) was used to assess the viability in 96-well culture cell plates as described previously [19]. Briefly, the treated cells (5 × 104) were stained with WST at 37 °C for 2 h and quantified by measuring the absorbance at 450 nm and normalized with the control absorbance at 690 nm.

Transwell assay

The cell migration assays were performed in 12-well hanging insert units (Millipore, Billerica, MA, USA). Before the experiment, 1 ml DMEM was added to the lower chamber of the transwell and incubated overnight. HRMECs (5 × 104) were seeded to the upper chamber and 1 ml complete DMEM was added to the lower chamber of the transwell with pharmacological agents at the indicated time. After incubation at 37 °C for 12 h, the cells were fixed with 4% paraformaldehyde (wt/vol.) and then stained with 0.1% crystal violet for 30 min at room temperature. After the washing steps, the cells were removed using a moist cotton swab from the upper surface of the membrane. The cells that migrated to the lower surface of the membrane were solubilized with 300 μl of 10% acetic acid and observed under a fluorescence microscope (EVOS FL Auto Imaging System, Life Technologies).

Wound scratch assay

The cells were seeded in 6-well plates. The wounding was performed by drawing a line with a pipette tip, and cells were washed twice with 1 × PBS. Next, the cells were treated with 25 or 50 ng/ml IL-10 for different times (0, 12, 24, 48 h). The gap size was observed under a microscope and assessed by the Image-Pro Plus software (Silver Spring, MD, USA).

Western blot analysis

Western blotting and immunoprecipitation were conducted as previously described [20]. The cell and tissue proteins were lysed and isolated in the RIPA lysis buffer including protease inhibitor and phosphatase inhibitors for 30 min at 4 °C. The protein was obtained after centrifugation at 12000xg for 10 min, and the protein concentration was detected and quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific, #23,225). An equal amount of protein was added to the 10% SDS-PAGE gel and transferred onto PVDF membranes. The membrane was incubated with the primary antibodies overnight at 4 °C. Then the membrane was washed three times with TBST for 5 min and incubated with the secondary antibodies at room temperature for 1 h. The primary antibodies are detailed in the Additional File 1: Table S1. The secondary antibodies included the HRP-labelled goat anti-rabbit IgG (ZSGB-BIO #ZB-2301, Beijing, China) and HRP-labelled goat anti-mouse IgG (ZSGB-BIO #ZB-2305, Beijing, China). The β-actin antibody (Sigma-Aldrich #A5316) and the Lamin B antibody (Abcam #ab16048) were used to confirm equal protein loading among the samples. The signals were detected with an enhanced chemiluminescence system (West Pico kit, Pierce, Loughborough, UK). The band density was analysed using the Image J software (National Institutes of Health imaging software).

Quantitative real-time PCR

RNA was extracted and collected with RNA iso Plus (Takara, Cat#9109). The mRNA expression of mouse Ep1r, Ep2r, Ep3r, Ep4r, Arg1, Mrc2, Mgl1, Ym1, Inos, Tnfa, Cd16, Cd32, Il-10 was determined by qRT-PCR. Gene expression was analyzed with the 2−ΔΔCt method and normalized by an internal control, Gapdh. More details with primers are presented in Additional File 1: Table S1.

Statistical analysis

The statistical analyses were analyzed with the GraphPad Prism-5 statistical software (Prism v5.0; GraphPad Software, La Jolla, CA, USA). All data are reported as mean ± SEM. We used Student’s t test to compare the mean of two groups. ANOVA with Tukey’s post hoc test was used to compare multiple groups. Results were considered statistically significant at P < 0.05.