C57BL/6 mice (female, 6–8 weeks old; 20–25 g) from the Guangzhou Animal Testing Center (Guangzhou, China) were conducted in specific pathogen-free conditions. The Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center Sun Yat-sen University approved this study, which was performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
EAU Induction and treatment with melatonin
Female mice were subcutaneously immunized with IRBP1−20 (amino acid sequence: GPTHLFQPSLVLDMAKVLLD; GL Biochem, Shanghai, China) emulsified in Freund's Complete Adjuvant (Difco, Detroit, MI, USA) containing Mycobacterium tuberculosis strain H37Ra (Difco; 1:1 v/v), on day 0 and day 2, pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally (17, 18) .The above methods has also been introduced in detail in our previous study(19). from On the third day of immunization, melatonin (Sigma-Aldrich) was administered intraperitoneally (10 mg/kg or 80 mg/kg in solution that contains 4% DMSO and 96% PBS, intraperitoneally). Control group mice were treated with the same volume of vehicle instead of melatonin.
Fundoscopy and histology study in EAU
On day 14 after immunization, the fundus of the mice was examined by Micron IV Retinal Imaging Microscope (PHOENIX, USA) and scored clinically. Clinical scoring was based on the previous study (18). At day 14, the mice were euthanized, and eyes were enucleated and stored in 4% neutral buffered formalin solution for 24 h at room temperature. Then, samples were dehydrated, embedded and stained. Histopathological changes were evaluated and graded in accordance with previously described criteria (18).
Retina-infiltrating cells isolation
This method is consistent with our previous published paper(19).
Surface staining of cells with anti-mouse CD4 Percp-Cy5.5 (clone GK 1.5), anti-mouse CD45 Brilliant Violet 510 (clone 30-F11 ), anti-mouse CD8α PE (clone 53-6.7), anti-mouse CD19 Brilliant Violet 650 (clone 6D5), anti-mouse CD44 antigen-presenting cell (APC) (clone IM7), anti-mouse CD25 PE/Cyanine7 (clone 3C7), anti-mouse CD62L FITC (clone MEL-14), anti-mouse APC/Cyanine7 CD279 (PD-1) (clone 29F.1A12), anti-mouse CD11c PE (clone N418). Above antibodies are from eBioscience (Carlsbad, CA, USA) or BioLegend (San Diego, CA, USA). For intracellular cytokine IFN-γ and IL-17A, the cells were incubated with ionomycin (500 ng/mL), PMA (50 ng/mL), and BFA (1 µg/mL) (Sigma-Aldrich) for 5 h, and after fixation and permeabilization, the cells were stained with anti-mouse IL-17A BV650 (clone TC11-18H10.1) and anti-mouse IFN-γ BV786 (clone XMG1.2). Transcription factor staining kit was used according to the manufacturer's protocol. For total cellular ROS staining, the cells were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (CAS No. 4091-99-0) for 20 min at 37°C in a cell culture incubator, which were analyzed using a BD LSRFortessa instrument (BD Biosciences, Franklin Lakes, NJ, USA), and the acquired data were processed using FlowJo 10.0 (FlowJo Co., Ashland, OR, USA).
T cell polarization
Naïve CD4+T cells (CD4+CD62L+CD25-CD44-) were isolated from the lymph nodes and spleen of wild type mice using a commercial kit (Miltenyi Biotec, Gladbach, Germany). The naïve CD4+T cells (purity: 95%; 2 × 105/well) were incubated with anti-CD3/CD28 beads (one bead to five cells) for three days in a 96-well plate. For Treg cell differentiation, the cultures were supplemented with Treg cell differentiation condition (TGF-β1 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) and recombinant human IL-2 (50 U/mL; PeproTech)).
Enzyme-linked immunosorbent assay (ELISA)
T cells which were isolated from DLNs of EAU mice were preprocessed with melatonin (200 ng/mL) for 4 h, and stimulated with 10 µg/mL IRBP1−20 for three days. The cell culture supernatants were collected, and the concentrations of IFN-γ and IL-17A were assayed using ELISA kits (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative polymerase chain reaction (PCR)
Retinas were isolated from the different treatment groups. Total RNA was extracted using TRIzol reagent (Invitrogen) and quantified using a NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE, USA). Total RNA was used to synthesize cDNA using PrimeScript RT Master Mix (Perfect Real Time, TaKaRa Bio Inc., Kusatsu, Japan). Real-time quantitative PCR was performed using SYBR Premix Ex Taq II (TaKaRa Bio Inc.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. The relative mRNA expression of IFN-γ, IL-17A, and Foxp3 (Sangon Biotech, Shanghai, China) was analyzed using the 2−ΔΔCt method.
Western blot assays
Proteins from cultured cells and DLNs were extracted using whole cell lysis buffer (KeyGen Biotech, Jiangsu, China) and measured the concentration of protein according to the protein assay kit’s instructions, which were separated on polyacrylamide-sodium dodecyl sulfate gels, and electrotransferred onto PVDF membrane. Then, blocking with 5% non-fat dry milk, the PVDF membranes were incubated with TXNIP (rabbit; clone D5F3E; Cell Signaling Technology, Danvers, MA, USA ), HIF-1α (rabbit; clone D1S7W; Cell Signaling Technology) and GAPDH (rabbit; clone D16H11; Cell Signaling Technology) overnight at 4 ℃, followed by incubation with the secondary antibody for 2 h. The western blot signals were imaged using enhanced chemiluminescence (Pierce, Rockford, IL, USA). ImageJ software (NIH, Bethesda, MD, USA) was used to measure the gray scale.
Adoptive transfer assay to induce EAU
T cells isolated from the DLNs of EAU mice (day 14 after immunization) were stimulated using IRBP1−20 (10 µg/mL) under Th17-polarizing conditions, with or without melatonin (200 ng/mL) for 72 h, which were washed three times before administration to C57BL/6 mice (2 × 107 living cells/mouse) via intraperitoneal injection. The extent of retinal inflammation by fundus and HE was evaluated on day 14.
In vitro study, the cells (4 × 105) from the DLNs of EAU mice were cultured in 96-well plates with IRBP1−20 under Th1-polarizing conditions (ImmunoCult 10953; STEMCELL Technologies, Vancouver, Canada) or Th17-polarizing conditions (anti-IFN-γ [10 µg/mL; R4-6A2], anti-IL-4 [10 µg/ml; L11B11], TGF-β [2.5 ng/mL], IL-6 [25 ng/mL], and IL-23 [10 ng/mL]) in the presence or absence of melatonin (0, 2, 20, or 200ng/mL). After three days, the cells were harvested, and flow cytometry was used for the intracellular inflammatory cytokine.
Data were presented as means ± standard deviation(SD). The Student's t-test, Mann-Whitney test, or one-way analysis of variance was selected according to the data sets normality. All data were analyzed by GraphPad Prism 8.2 (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was considered to be statistically significant.