Uveitis is a group of diseases among the leading causes of visual deficit and blindness. Uveal and retinal layers as well as vitreous cavity could all be affected within disease progression. Clinically, this group of diseases can be divided into infectious and noninfectious causes, the latter one is known as autoimmune uveitis [
Different immune cells subsets play important roles in disease development of EAU. Th1 and Th17 responses and relative cytokines are believed to be pathogenic in tissue damage [
Endogenous metabolite of estrogen, 2-Methoxyestradiol (2ME2), is a promising antiangiogenic and antitumor compound [
In this study, we demonstrated that the endogenous methylated estrogen, 2ME2, could taper autoimmune uveitis development in a time-dependent manner. The disease modifying effect was established on reducing antigen specific T cell proliferation, inhibiting T cell differentiation and related proinflammatory cytokines secretion.
6~8-week-old C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, CAS (SLACCAS), and housed in a specific-pathogen-free condition with water and standard laboratory chow ad libitum. Animal care and use were in compliance with the Institutional Animal Care and Use Committee Guidelines and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Three peptides of IRBP protein were employed to formulate the antigen complex. They were 1–20 (SGIPYIISYLHPGNTILHVD), 461–480 (LRHNPGGPSSAVPLLLSYFQ), and 651–670 (LAQGAYRTAVDLESLASQL) which C57BL/6 mice were sensitive to [
C57BL/6 mice were immunized subcutaneously 0.1 mL at tail and 0.05 mL at both thigh sites with IRBP antigen complex. 500 ng Pertussis toxin was injected concurrently. This day was settled as day 0. Then mice were divided into 4 groups, each group containing 5 mice. 15 mg/kg 2ME2 or vehicle was abdominal injected during 0–13 days, 0–6 days, and 7–13 days. At day 14 eyes or lymphoglandula was collected after euthanasia.
Eyes harvested 14 days after immunization were prefixed in 4% phosphate-buffered glutaraldehyde for 1 h (to prevent artifactual detachment of the retina) and then transferred to 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in methacrylate, and 4 to 6
Eyes’ external tissue was trimmed after enucleation; incision was made along the limbus to remove the lens. The rest of the eye was minced into small pieces and digested in 1 mg/mL collagenase IV in RPMI containing 10% FCS for 1 hour at 37°C. After trituration and filtration, the resultant cell pellet was resuspended in RPMI with 10% FCS and seeded in 48-well plate. Harvested lymph nodes were dispersed by trituration in RPMI with 10% FCS on ice, followed by filtration and washing. Then single-cell suspension was obtained and seeded in 96-well plate for intracellular cytokine evaluation and proliferation assay. PMA (50 ng/mL) and ionomycin (500 ng/mL) together with brefeldin A were added to pulse both the eye-infiltrating cells and lymphocytes for 5 hours. Cells were then fixed with 2% paraformaldehyde, permeabilized in 0.5% saponin, and stained with CD4, IFN-
MTT assay was used to determine the effect of IRBP on cell proliferation. Cells from 4 groups previously described were seeded
Supernatants of lymphocytes pulsed by IRBP antigen complex for 48 hours were harvested and tested for IL-17A and IFN-
All experiments were repeated at least three times. Data were expressed as mean ± SEM. The statistical analysis was carried out using Student’s
To investigate the effect of 2ME2 on uveitis development, C57BL/6 mice were randomly assigned into two groups and immunized with IRBP peptide. 2ME2 group started 2ME2 (15 mg/kg) intraperitoneally from day 0 to day 13 while control group was given with vehicle. On day 14, when inflammation was believed to reach the peak, eyes were harvested for blind histopathological examination. Eyes from control group presented with severe structure destruction, abundant inflammatory cells infiltration both in vitreous body and in retina, vasculitis, photoreceptor layer damage, and moderate to large retinal folds were noticed in almost every case (Figure
Effects of 2ME2 on EAU mice. (a) Retina HE section from vehicle treated EAU mice, scored 3. (b) Retina HE section from 2ME2 treated EAU mice, scored 0. (c) Disease score from 2ME2 D0–D13 group was lower than that from vehicle treated group (
EAU mice immunized using active method undergo priming phase (day 0 to day 6) and effector phase (day 7 to day 13) to develop uveitis. To examine 2ME2 effect on uveitis development in different phases, 2ME2 (15 mg/kg) was intraperitoneally administrated either from day 0 to day 6, from day 7 to day 13, from day 0 to day 13, or vehicle. Compared to control group, HE sections from 2ME2 D0–D13 group remained almost normal retinal structure and exhibited the best curative outcome; those from 2ME2 D0–D6 group had less cell infiltration, milder vasculitis, and less structure disruption, while those from 2ME2 D7–D13 exhibited no much remission in observation (Figure
Time-dependent 2ME2 effects on EAU mice. (a) Retina HE sections from 2ME2 and vehicle treated groups. Vehicle, scored 3. 2ME2 D0–D13, scored 0. 2ME2 D0–D6, scored 1.5. 2ME2 D7–D13, scored 2.5. (b) Disease score from 2ME2 D0–D13 group was the lowest among the four groups, followed by 2ME2 D0–D6 group and 2ME2 D7–D13 group and vehicle group scored the highest (
To assess the inhibitory effect of 2ME2 on Ag-specific immune response, lymphocyte proliferation method was used. Draining lymph node cells from 2ME2 D0–D13, 2ME2 D0–D6, 2ME2 D7–D13, and vehicle groups were pulsed with IRBP for 48 h and cell proliferation was measured using MTT assay. Cells from all 2ME2 treated groups responded significantly weaker than control (
Lymphocytes harvested from 2ME2 treated EAU mice and vehicle mice, seeded
To further investigate the disease modifying mechanism of 2ME2, draining lymph nodes cells and eye-infiltrating cells were collected and analyzed using flow cytometry described above. 2ME2 treated mice developed fewer Th1 cells
Effects of 2ME2 on draining lymph nodes cells differentiation. Flow cytometry figure was from one mouse. Both Th1 (IFN-g) and Th17 (IL-17A) cells percentages were lower in 2ME2 group than those in vehicle group (
Effects of 2ME2 on eye-infiltrating cells differentiation. Flow cytometry figure was from one mouse. Both Th1 (IFN-g) and Th17 (IL-17A) cells percentages were lower in 2ME2 group than those in vehicle group (
Effects of 2ME2 on cytokines secretion. Both IFN-g and IL-17A levels were lower in 2ME2 group than those in vehicle group (
The pathological mechanism during clinical uveitis progression is much more sophisticated than what we have already learnt from EAU models. However, from pathological view, they share lots of similarities including subretinal and choroidal granuloma, retinal vasculitis, retinal detachment, and infiltrating cells both in vitreous cavity and in retina [
The antiproliferative and antimitogenic properties of 2ME2 were widely studied in carcinogenesis and angiogenesis [
EAU underwent priming phase and effector phase to reach the peak of inflammation. To discover whether the efficacy of 2ME2 was time-dependent, 2ME2 was injected on D0–D6, D7–D13, and D0–D13 separately. Besides the best curative effect in 2ME2 D0–D13 group, priming phase drug employment exerted better outcome than those administrated in effector phase. From these results, we could see that it was more important for 2ME2 to perform its inhibitory effect on priming phase. In priming phase, uveitogenic antigen was processed and presented to T cell, resulting in T cell proliferation. The underlying mechanism of superior efficacy of 2ME2 in priming phase might be related to these processes.
In observation of antigen specific lymphocyte proliferation, 2ME2 can significantly inhibit response of IRBP specific lymphocyte harvested from EAU mice. This effect could be maximized in 2ME2 D0–D13 group followed by 2ME2 D0–D6 group, whereas 2ME2 D7–D13 group has the moderate response, only weakened compared to control group. This result is in line with histopathological findings and together proved that a more important effect of 2ME2 is exerted in priming phase. In other researches, 2ME2 can reduce macrophage and NK cells activity, inhibiting T cell activation and proliferation [
By isolating lymphocytes from draining lymph nodes and eye-infiltrating cells, T cell differentiation towards Th1 and Th17 lineages was determined to be inhibited by 2ME2 via flow cytometry. Proinflammatory cytokines, IFN-
Previous studies proved that either Th1 or Th17 cells can independently drive specific effector responses during autoimmune diseases development [
2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism of this effect could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation.
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