December 2015
Volume 56, Issue 13
Free
Immunology and Microbiology  |   December 2015
A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2
Author Affiliations & Notes
  • Qian Shi
    Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
    Research Center for Translational Medicine and Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China
  • Qiu Wang
    Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
  • Jing Li
    Research Center for Translational Medicine and Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China
  • Xiaohui Zhou
    Research Center for Translational Medicine and Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China
  • Huimin Fan
    Research Center for Translational Medicine and Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China
  • Fenghua Wang
    Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
  • Haiyun Liu
    Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
  • Xiangjun Sun
    School of Biology and Agriculture, Shanghai Jiao Tong University, Shanghai, People's Republic of China
  • Xiaodong Sun
    Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
    Shanghai Key Laboratory of Fundus Disease, Shanghai, People's Republic of China
  • Correspondence: Xiaodong Sun, Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, 100 Hai Ning Road, Shanghai 200080, PR China; xdsun@sjtu.edu.cn
Investigative Ophthalmology & Visual Science December 2015, Vol.56, 7728-7738. doi:10.1167/iovs.15-17677
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Qian Shi, Qiu Wang, Jing Li, Xiaohui Zhou, Huimin Fan, Fenghua Wang, Haiyun Liu, Xiangjun Sun, Xiaodong Sun; A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2. Invest. Ophthalmol. Vis. Sci. 2015;56(13):7728-7738. doi: 10.1167/iovs.15-17677.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: Inflammatory status of RPE cells induced by A2E is essential in the development of AMD. Recent research indicated T-cell immunity was involved in the pathological progression of AMD. This study was designed to investigate how A2E suppresses immunoregulatory function of RPE cells in T-cell immunity in vitro.

Methods: Mouse RPE cells or human ARPE19 cells were stimulated with A2E, and co-cultured with naïve T cells under Th1, Th2, Th17, and regulatory T cell (Treg) polarization conditions. The intracellular cytokines or transcript factors of the induced T-cells subset were detected with flow cytometer and qRT-PCR. The ROS levels were detected, and the factors and possible pathways involved in the A2E-laden RPE cells were analyzed through neutralization antibody of IL-1β and inhibitors of related pathways.

Results: The A2E reduced regulatory function of RPE cells in Treg differentiation. The A2E-laden RPE cells promoted polarization of Th1 cells in vitro, but not Th2 or Th17 differentiation. The A2E induced RPE cells to release inflammatory cytokines and ROS, but PGE2 production was inhibited. Through neutralization of IL-1β or inhibition of COX2-PGE2 pathways, A2E-laden RPE cells expressed reduced effect in inducing Th1 cells.

Conclusions: The A2E inhibited regulatory function of RPE cells in suppressing Th1 cell immunity in vitro through production of IL-1β and inhibition of PGE2. Our data indicate that A2E could suppress immunoregulatory function of RPE cells and adaptive immunity might play a role in the immune pathogenesis of AMD.

Age-related macular degeneration (AMD) is the leading cause of blindness in older people in developed countries.1,2 Although anti-VEGF therapy is useful for the treatment of wet AMD through inhibition of the angiogenesis, unfortunately there are no effective therapies for dry AMD and the underlining etiology mechanisms remain unclear.3,4 The dysfunction of RPE cells lead to immune imbalance and loss of photoreceptors, which was considered to be essential in the pathological progression in AMD.5 
Recent research has indicated AMD as an inflammatory disease in which both environmental and genetic factors are involved.6 The disorder of complement system7 and polymorphisms in complement factor genes have been found to increase statistical risk of AMD.810 Macrophage recruitment in the subretinal space regulated by CXCR3 also represents one of the causes of both wet and dry AMD.11 Moreover, to some degree, AMD was thought of as an autoimmune disease. Hollyfield et al.12 found carboxyethylpyrrole (CEP), a fragment of docosahexaenoic acid from the drusen of AMD donor eyes, could induce geographic atrophy (GA)-like lesions, the end stage characteristic of dry AMD, in mice through oxidative damage and inflammation. Further investigation indicated that NLRP3 mediated inflammasome activation and production of IL-1β and IL-18, which take part in GA lesions.13 
Apart from innate immunity, adaptive immunity was also found essential in the pathophysiology of AMD. In patients, T cells were found in human macular choroid drusen14 and peripheral CD56+CD28T cells were associated with AMD.15 Interleukin-17 production from γδT cells promoted the development of choroidal neovascularization (CNV).16 Moreover, Th1 cells and the proinflammatory macrophage subset M1 were two of the relevant cell types found in CEP-induced GA animal model.17 However, the mechanisms of T-cell immunity in the onset of AMD pathology remains unclear. 
Retinal pigment epithelial cells contribute to immune privilege in the retina and form an essential part of the blood-retinal barrier.18 Normal RPE cells sustain immune balance and remove waste products by modulation of innate and adaptive immunity.18,19 Retinal pigment epithelial cells could induce regulatory CD4+T cells mediated by CTLA-2 and RA20,21 and could suppress effective T subset differentiation such as Th17 cells22 and Th22 cells.23 On the other hand, oxidative stress induced RPE cell injury leading to inflammation and immunity damage in AMD development.24 Vitamin A dimers were the main products formed during visual cycle accumulating with aging. These dimers were the main triggers associated with RPE cell oxidative stress and degeneration in AMD.2527 One of the essential dimers was A2E, which not only induced inflammatory status of RPE cells,28 but also induced AMD-like lesions in animal models.29 However, how A2E impaired the immunoregulatory function of RPE cells in T-cell subset differentiation and triggered inflammation in AMD development should be elucidated. 
This study used A2E as an AMD risk factor to investigate how oxidative stress impaired immunoregulatory function of RPE cells in T-cell immunity in vitro. Our results indicate that A2E inhibits the suppressive function of RPE cells on Th1 cell differentiation through producing IL-1β and inhibiting PGE2. 
Methods
Preparation of Mice Primary RPE Cells
All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the institutional animal care and use committee of Tongji University.30 Primary RPE cells were obtained from 6-week-old wild-type C57BL/6 mice (purchased from Shanghai Laboratory Animal Company, Shanghai, China) and cultivated as reported by Sugita et al.31 Briefly, the eyecups were incubated in 0.25% trypsin (Gibco, Carlsbad, CA, USA) for 1 hour at 37°C in a 5% CO2 atmosphere, and then the RPE sheets were triturated to create a single-cell suspension. Complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% non-essential amino acids (NEAA), 50 μM 2-ME, 1% HEPES (Gibco) was used for the RPE cells cultures. Cultures were passaged by dissociation in 0.25% trypsin (Gibco), and the medium was replaced every 3 days. 
Preparation of A2E and Treatment of RPE Cells
The A2E was prepared as previously described.32,33 Isolated mouse primary RPE cells in passage 2 were collected with or without the stimulation of A2E at a concentration of 10 μM for 6 hours. The RPE cells or the A2E-laden RPE (A2E-RPE) cells were collected and washed three times with PBS, followed by co-culture with naïve T cells under different polarization conditions as indicated. Human ARPE19 cells from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured and treated with A2E as previously described.32 
Isolation and Culture of T Cells
The CD4+CD25CD44low naïve T cells were isolated from spleens of 6-week-old wild-type C57B/L6 mice with a CD4+T cells negative isolation kit (Dynal, AS, Norway) plus anti-CD25 and anti-CD44 antibodies (Biolegend, San Diego, CA, USA); non-naïve T cells were then depleted with anti-rat IgG-coated Dynal Beads. Flow cytometry showed that the purity of CD4+CD25CD44low naïve T cells was more than 95%. The naïve T cells were cultured in complete RPMI 1640 medium containing 10% fetal bovine serum, 1% NEAA, 50 μM 2-ME, and 1% HEPES (Gibco) at a density of 1 million cells per well in 48-well plates or 0.3 million cells per well in 96-well plates. The T cells were activated with plate-coated anti-CD3 (4 μg/mL)/CD28 (4 μg/mL) antibodies. For T-helper cell subset polarization, different cytokines were added as follows: IL-2 50 U/mL for Th0; IL-2 50 U/mL, hTGF-β1 20 ng/mL for induced regulatory T cells; hTGF-β1 10 ng/mL, IL-6 20 ng/mL, anti–IL-4 10 μg/mL, anti–IFN-g 10 μg/mL for Th17; IL-12 10 ng/mL, IL-2 50 U/mL, anti–IL-4 10 μg/mL for Th1; IL-4 20 ng/mL, IL-2 50 U/mL, anti-IFN-g 10 μg/mL for Th2. After 4 days of culture, T cells were collected and analyzed by fluorescence activated cell sorting. 
Human naïve T cells were isolated from peripheral blood of healthy adult volunteers and induced into Th1 and Th17 cells as described previously,34,35 with or without co-culture of ARPE19 or A2E-laden ARPE19 cells. All protocols that involved human blood donors were approved by the Clinical Research Ethics Board at Shanghai Jiao Tong University. Intracellular cytokines of induced T cells were analyzed by flow cytometry. 
T-Cell Proliferation and Suppression Assays
For the suppressive function assay, naïve T cells were stained with 10 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at room temperature and washed three times with PBS-5 as respondent cells. These cells were stimulated with anti-CD3/anti-CD28 antibody-coated beads (Dynal), and co-cultured with RPE or A2E-laden RPE-induced T cells. After 3 days, suspension cells were collected and CD4+CFSE+ cells were detected by flow cytometry. 
Flow Cytometry and Intracellular Staining
The PE/Cy5.5-anti-CD4 and PE-anti-CD25 antibodies (Biolegend) were used as surface staining. For intracellular cytokine analysis, cells were restimulated with 1 μg/mL of ionomycin and 100 ng/mL of PMA (phorbol 12-myristate 13-acetate) in the presence of Golgi Stop (BD Pharmingen, San Diego, CA, USA), BFA (brefelden A), and monencin (eBioscience, San Diego, CA, USA) for 5 hours. The cells were then fixed and permeabilized with a Cytofix/Cytoperm Kit (Biolegend); then AnexFlow488-anti-IL-4, PE-anti-IL-17, FITC-anti-IFN-γ, and PE-anti-IL-10 (Biolegend) antibodies were used to stain these intracellular cytokines. 
For Foxp3 staining, cells were permeabilized and fixed with a Foxp3 staining kit (Biolegend) and stained with AnexFlow 488-anti-Foxp3 antibody. The data were analyzed with FlowJo software (Tree Star, Portland, OR, USA). 
RNA Extraction and PCR Analysis
The RNA was isolated from RPE cells or induced T-cell subsets with TRIzol and were reverse transcribed with PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time quantitative PCR (RT-qPCR) was performed with SYBR Premix Ex Taq (TaKaLa) on 7900HT Fast Real Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Mouse PCR primers sequences are as follows: 
IL-17A, 5′-CCTGGCGGCTACAGTGAAG-3′, 5′-TTTGGACAC GCTGAGCTTTG-3′; RORγt, 5′-CCTCAGCGCCCTGTGTTTT-3′, 5′-GAGAACCAGGGCCGTGTAGA-3′; T-bet, 5′-CAACAACCCCTT TGCCAAAG-3′, 5′-TCCCCCAAGCAGTTGACAGT-3′; IFN-γ, 5′-GCTCTGAGACAATGAACGCT-3′, 5′-AAAGAGATAATCTGGCTC TGC-3′; IL-1β, 5′-AGTTGACGGACCCCAAAAGAT-3′, 5′-GTTGATGTGCTGCTGCGAGA-3′; IL-12b, 5′-CCATCGTTTTGC TGGTGTCTC-3′, 5′-GTCATCTTCTTCAGGCGTGTCA-3′; IL-6, 5′-CAGTTGCCTTCTTGGGACTGA-3′, 5′-TTGCCATTGCACAAC TCTTTTC-3′; IL-18, 5′-CAGGCCTGACATCTTCTGCAA-3′, 5′-TCTGACATGGCAGCCATTGT-3′; VEGFa, 5′-GGTGGACATCTTC CAGGAGT-3′, 5′-TGATCTGCATGGTGATGTTG-3′; COX2, 5′-TGCTCACGAAGGAACTCAGC-3′, 5′-CTCATACATTCCCCACGG TTTTG-3′; NLRP3, 5′-GAGTTCTTCGCTGCTATGT-3′, 5′-ACCTTCACGTCTCGGTTC-3′; IL-10, 5′-TCGGCCAGAGCCACA TG-3′, 5′-TTAAGGAGTCGGTTAGCAAGTATGTTG-3′; GATA3, 5′-AGAACCGGCCCCTTATGAA-3′, 5′-AGTTCGCGCAGGATGTC C-3′. Human PCR primer sequences are as follows: IL-1β, 5′-CATCAGCACCTCTCAAGCAG-3′, 5′-GAGTCCACATTCAGCACA GG-3′; IL-12b, 5′-ACCTGACCCACCCAAGAACT-3′, 5′-GGACCT GAACGCAGAATGTC-3′; IL-18, 5′-AGTCAGCAAGGAATTGTCTC C-3′, 5′-GAAGCGATCTGGAAGGTCTG-3′; VEGFa, 5′-TTCTGAG TTGCCCAGGAGAC-3′, 5′-TGGTTTCAATGGTGTGAGGA-3′; NLRP3, 5′-ACATCTCCTTGGTCCTCAGC-3′, 5′-GCTTCAGTCCC ACACACAGA-3′. 
Enzyme-Linked Immunosorbent Assay
The mouse RPE cells or ARPE19 cells were stimulated with A2E in a 12-well plate and supernatant was collected to measure the levels of PGE2 (R&D Systems, Minneapolis, MN, USA), IL-1β, IL-18, IFN-γ, and IL-12b (eBioscience) by ELISA according to the manufacturer's guidelines. 
Labeling of Reactive Oxygen Species (ROS)
Mouse RPE cells or ARPE19 cells were stimulated with different doses of A2E (0, 2, 10, 25 μM) for 6 hours as indicated in this article. Intracellular ROS were detected using dihydroethidium (DHE; Invitrogen, Eugene, OR , USA) by flow cytometry as reported previously.32 
Western Blot
Western blot was performed as previously described.32 Briefly, total protein was collected from cultured RPE cells as indicated. Proteins were electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) and immunoblotted for COX-2 (Abcom, Cambridge, MA, USA). The blots were developed using Enhanced chemiluminescence (ECL; GE Healthcare Life Sciences, Buckinghamshire, UK) technique. The films were scanned with a flatbed scanner (Epson Stylus NX400; Epson, Suwa, Nagano Prefecture, Japan). 
Statistical Evaluation
Two-tailed unpaired t-test was used for the in vitro experiments; P < 0.05 was defined statistically significant. 
Results
The A2E Suppressed Immunoregulatory Function of RPE Cells in Inducing Regulatory T cells
We induced co-culture system of A2E-laden RPE (A2E-RPE) cells and naïve T cells to investigate how A2E influences immune suppressive function of RPE cells in T cell differentiation. We found that the normal RPE cells induced T cells (TRPE) into Foxp3+ subsets (36.0% positive), while the A2E-RPE–induced T cells (TA2E-RPE) expressed a lower level of Foxp3 (6.9% positive) (Fig. 1A). Furthermore, we analyzed the immunosuppressive function of these induced T cells and found TRPE suppressed responder cells (CFSE-stained CD4+CD25-T cells) proliferation (8.58%) in the co-cultured system. However, TA2E-RPE induced T cells showed lower suppressive function on the proliferation of responder cells (82.2%). This finding indicated that A2E inhibited immunosuppressive effects of RPE cells on regulatory T-cell (Treg) differentiation and function (Fig. 1B). 
Figure 1
 
The A2E suppressed immunoregulatory function of RPE cells in inducing Tregs. (A) The CD4+CD25CD44low naïve T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of IL-2 (50 U/mL) alone (T med) or both TGF-β1 (20 ng/mL) and IL-2 (T IL-2+TGF-β), or co-cultured with RPE (T RPE) or A2E-laden RPE (T A2E-RPE) for 4 days. Percentages of induced Foxp3+ cell subsets were analyzed by flow cytometer. (B) Induced T cells were co-cultured with CFSE+CD4+T responder cells at a ratio of 10:1(T:RPE) under the stimulation of coated anti-CD3/CD28(2 μg/mL) for 4 days and the CFSE dilution was analyzed by flow cytometer. Gating on CD4+T cells, similar results were obtained in three independent experiments.
Figure 1
 
The A2E suppressed immunoregulatory function of RPE cells in inducing Tregs. (A) The CD4+CD25CD44low naïve T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of IL-2 (50 U/mL) alone (T med) or both TGF-β1 (20 ng/mL) and IL-2 (T IL-2+TGF-β), or co-cultured with RPE (T RPE) or A2E-laden RPE (T A2E-RPE) for 4 days. Percentages of induced Foxp3+ cell subsets were analyzed by flow cytometer. (B) Induced T cells were co-cultured with CFSE+CD4+T responder cells at a ratio of 10:1(T:RPE) under the stimulation of coated anti-CD3/CD28(2 μg/mL) for 4 days and the CFSE dilution was analyzed by flow cytometer. Gating on CD4+T cells, similar results were obtained in three independent experiments.
The A2E-laden RPE Cells Promoted Th1 Differentiation
To investigate how A2E influenced RPE function in other effector T subset differentiation, we co-cultured A2E-laden RPE cells with naïve T cells in different T subset (Th1, Th2, or Th17) polarization conditions. We found that normal RPE cells dramatically inhibited all these T subsets differentiation (Th1 cells [IFN-γ+ cells, 52.3%–11.6%], Th2 cells [Il-4+ cells, 11.0%–4.32%], and Th17 cells [IL-17+ cells, 26.1%–3.59%] cells) as compared with the T-cell subsets that were induced in each of the polarization conditions alone (Figs. 2A, 2B). When stimulated with A2E, the A2E-laden RPE cells also exhibited suppressive effects on Th2 cells (7.08%) and Th17 cells (5.21%). However, on their own, A2E-laden RPE cells could not reduce Th1 cell differentiation (IFN-γ+ cells, 59.5%) as compared with Th1 differentiation (IFN-γ+ cells, 11.6%) co-cultured with normal RPE cells (Figs. 2A, 2B). Furthermore, we investigated the mRNA levels of the lineage-specific transcription factors and cytokines of the induced T subsets. Normal RPE cells inhibited these Th1-specific transcript factors (T-bet) and cytokines (IFN-γ). But A2E-laden RPE cells increased expression of IFN-γ, T-bet, and IL-12b of the T cells in the Th1 differentiation environment compared with the normal RPE cells. However, A2E did not reduce suppressive function of RPE in reducing specific transcript factors of Th17 (RORγt) and Th2 (GATA3) expression (P > 0.05) (Fig. 2C). These results indicated that A2E inhibited the immunosuppressive effect of RPE cells on Th1 cell differentiation. 
Figure 2
 
The A2E-laden RPE cells promoted Th1 differentiation. (A) Retinal pigment epithelial cells stimulated without (control) or with A2E 10 μM for 6 hours were co-cultured with naïve T cells under the stimulation of coated anti-CD3/CD28 and different T-cell subset polarization conditions for 4 days. Intracellular cytokines (IL-17 for Th17, IFN-γ for Th1, and IL-4 for Th2) were analyzed by flow cytometer; three independent experiments revealed similar results. The percentages of cytokine-positive cells in CD4+ cells were analyzed in (B). (C) The transcript factors or intracellular cytokines of cultured T cells were analyzed by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Figure 2
 
The A2E-laden RPE cells promoted Th1 differentiation. (A) Retinal pigment epithelial cells stimulated without (control) or with A2E 10 μM for 6 hours were co-cultured with naïve T cells under the stimulation of coated anti-CD3/CD28 and different T-cell subset polarization conditions for 4 days. Intracellular cytokines (IL-17 for Th17, IFN-γ for Th1, and IL-4 for Th2) were analyzed by flow cytometer; three independent experiments revealed similar results. The percentages of cytokine-positive cells in CD4+ cells were analyzed in (B). (C) The transcript factors or intracellular cytokines of cultured T cells were analyzed by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
The A2E Inhibited the Immunosuppression Effect of RPE Cells on Th1 Differentiation Rather Than Proliferation
To investigate how RPE cells influenced Th1 cell differentiation, we stained naïve T cells with CFSE and co-cultured with RPE or A2E-RPE cells under Th1 polarization condition. We found that RPE cells did not inhibit Th1 cell proliferation; instead, they reduced IFN-γ expression in induced T cells. However, A2E-RPE cells did not inhibit Th1 cell proliferation or differentiation. This result indicated that A2E suppressed immunosuppressive effect of RPE cells on Th1 differentiation rather than proliferation (Fig. 3A). In the co-culture system of RPE cells and Th17 cells, A2E did not reduced RPE cells' suppressive function on Th17 differentiation or proliferation (Fig. 3B). 
Figure 3
 
The A2E inhibited the immunosuppression effect of RPE cells on Th1 cell differentiation rather than Th1 cell proliferation. The A2E-laden RPE or normal RPE cells were co-cultured with CFSE-stained CD4+T responder cells under the stimulation of bead-coated anti-CD3/CD28 in Th1 or Th17 cell polarization conditions for 4 days; IFN-γ+ Th1 cells (A) and IL-17+ Th17 cells (B) were analyzed by flow cytometer.
Figure 3
 
The A2E inhibited the immunosuppression effect of RPE cells on Th1 cell differentiation rather than Th1 cell proliferation. The A2E-laden RPE or normal RPE cells were co-cultured with CFSE-stained CD4+T responder cells under the stimulation of bead-coated anti-CD3/CD28 in Th1 or Th17 cell polarization conditions for 4 days; IFN-γ+ Th1 cells (A) and IL-17+ Th17 cells (B) were analyzed by flow cytometer.
Interleukin-1β Mediated the Function of A2E-laden RPE Cells on Promoting Th1 Cell Differentiation
To identify which factors contributed to the reduced immunoregulatory function of A2E-laden RPE on Th1 cell differentiation, we analyzed mRNA level of the cytokines related to T-cell subset differentiation in RPE cells. After stimulating with A2E, the A2E-RPE cells expressed a higher level of proinflammatory cytokines, such as IL-1β, IL-12b, IL-6, IL-18, and IFN-γ compared with normal RPE cells (P < 0.05), whereas the level of regulatory factors COX2 and IL-10 were decreased (P < 0.05). The expression of NLRP3 and VEGFa also increased with the stimulation of A2E (Fig. 4). 
Figure 4
 
The A2E-induced RPE produces inflammatory factors. Retinal pigment epithelial cells were stimulated with or without 10 μM A2E for 6 hours; the mRNA levels of cytokines or related factors in RPE were detected with RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
 
The A2E-induced RPE produces inflammatory factors. Retinal pigment epithelial cells were stimulated with or without 10 μM A2E for 6 hours; the mRNA levels of cytokines or related factors in RPE were detected with RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Among these cytokines, IL-1β was essential in modulation of Th1 immunity.28,36 We then neutralized IL-1β in the A2E-laden RPE cells and T-cell co-culture system under the Th1 polarization condition. Neutralization of IL-1β decreased A2E-laden RPE cells induced Th1 differentiation (15.5%) compared with Th1 cells co-cultured with A2E-laden RPE alone (51.1%). This result indicated that IL-1β mediated the effect of A2E on reducing regulatory function of RPE cells in Th1 differentiation (Fig. 5). 
Figure 5
 
The A2E modulated Th1 cell differentiation through IL-1β. The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days, with or without anti–IL-1β antibody; percentage of IFN-γ+ T cells were analyzed by flow cytometer.
Figure 5
 
The A2E modulated Th1 cell differentiation through IL-1β. The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days, with or without anti–IL-1β antibody; percentage of IFN-γ+ T cells were analyzed by flow cytometer.
The A2E Reduced Immunosuppressive Function of RPE Cells Through Inhibition of PGE2
Previous studies have indicated that PGE2 mediated the immunosuppressive function of several types of cells, including mesenchymal stem cells (MSCs), macrophages, and RPE cells.3739 To investigate the mechanisms involved in RPE immunosuppression function in our system, we analyzed PGE2 and COX2 expression in RPE cells by ELISA and Western blot. We found that A2E reduced PGE2 level in RPE cells compared with RPE cells without stimulation (P < 0.01). However, A2E increased IL-1β, IL-18, and IL-12b production (P < 0.01) (Fig. 6A). COX2 expression in RPE cells was also reduced with the stimulation of A2E (Fig. 6B). 
Figure 6
 
The A2E inhibited PGE2, which mediated RPE cell regulatory function on Th1 cell differentiation. (A) Retinal pigment epithelial cells were stimulated with 10 μM A2E for 6 hours, supernatants were collected, and PGE2 and cytokines (IL-1β, IL-18, and IL-12b) were detected by ELISA. (B) Retinal pigment epithelial cells were stimulated with A2E 10 μM; the cells were collected at different times and COX2 expression was detected by Western blot. (C, D) The Th1 cells were induced alone or co-cultured with RPE or A2E-laden RPE cells with or without inhibitors for 4 days; expression of intracellular IFN-γ in T cells was detected by flow cytometer. (E, F) Levels of intracellular ROS in RPE with or without stimulation by 10 μM A2E for 6 hours were measured by flow cytometer. *P < 0.05, **P < 0.01, ns P > 0.05.
Figure 6
 
The A2E inhibited PGE2, which mediated RPE cell regulatory function on Th1 cell differentiation. (A) Retinal pigment epithelial cells were stimulated with 10 μM A2E for 6 hours, supernatants were collected, and PGE2 and cytokines (IL-1β, IL-18, and IL-12b) were detected by ELISA. (B) Retinal pigment epithelial cells were stimulated with A2E 10 μM; the cells were collected at different times and COX2 expression was detected by Western blot. (C, D) The Th1 cells were induced alone or co-cultured with RPE or A2E-laden RPE cells with or without inhibitors for 4 days; expression of intracellular IFN-γ in T cells was detected by flow cytometer. (E, F) Levels of intracellular ROS in RPE with or without stimulation by 10 μM A2E for 6 hours were measured by flow cytometer. *P < 0.05, **P < 0.01, ns P > 0.05.
Furthermore, through blocking COX2-PGE2 pathways in RPE cells with iNOS inhibitor, indomethacin, or EP2/EP4 inhibitors and inhibiting the effects of PGE2, we found that these inhibitors impeded the suppressive function of RPE cells on Th1 cell differentiation. These results indicated that the COX2-PGE2 pathways might mediate regulatory function of RPE in Th1 cell immunity, and be inhibited by A2E (Figs. 6C, 6D). 
We further detected the levels of intracellular mitochondria-associated ROS in mouse RPE cells stimulated with 10 μM A2E for 6 hours. We found that A2E stimulation increased ROS levels in RPE cells (ROS, 85.9% positive) compared with RPE cells without stimulation (0.31%) (Figs. 6E, 6F). 
The A2E Suppressed Immunoregulatory Function of ARPE19 Cells on Regulatory T Cells and Th1 Cells
We used human ARPE19 cells to verify the effects of A2E in the immunoregulatory function in T subsets. We co-cultured ARPE19 cells with naïve T cells, and found that normal ARPE19 cells could induce Foxp3+Tregs (15.4% positive), which suppressed responder T-cell (CFSE-stained CD4+naïve T cells) proliferation (17.7%). However, A2E-laden ARPE19 expressed lower function in inducing Foxp3+Tregs (4.73% positive), which showed lower suppressive function on T-cell proliferation (61.0%) (Figs. 7A, 7B). 
Figure 7
 
The A2E suppressed immunoregulatory function of ARPE19 cells in Tregs and Th1 differentiation through IL-1β. (A, B) The ARPE19 cells stimulated with or without 10 μM A2E for 6 hours were co-cultured with naïve T cells for 4 days. Percentages of induced FOXP3+ T cells and suppressive function of these induced T cells in the CFSE+ responder T cells co-culture system were analyzed by flow cytometer. (C) The ARPE19 or A2E-laden ARPE19 cells were co-cultured with naïve T cells in Th1 or Th17 polarization conditions for 4 days. (D) The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days with anti–IL-1β antibody. Intracellular cytokines were analyzed by flow cytometer.
Figure 7
 
The A2E suppressed immunoregulatory function of ARPE19 cells in Tregs and Th1 differentiation through IL-1β. (A, B) The ARPE19 cells stimulated with or without 10 μM A2E for 6 hours were co-cultured with naïve T cells for 4 days. Percentages of induced FOXP3+ T cells and suppressive function of these induced T cells in the CFSE+ responder T cells co-culture system were analyzed by flow cytometer. (C) The ARPE19 or A2E-laden ARPE19 cells were co-cultured with naïve T cells in Th1 or Th17 polarization conditions for 4 days. (D) The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days with anti–IL-1β antibody. Intracellular cytokines were analyzed by flow cytometer.
We further co-cultured ARPE19 or A2E-laden ARPE19 cells with naïve T cells in Th1 or Th17 differentiation conditions. We found that ARPE19 cells inhibited Th1 (13.3%–2.81%) and Th17 cells (28.2%–8.05%) differentiation. However, A2E-laden ARPE19 cells showed lower suppressive effects on Th1-cell differentiation (9.30%), but not Th17 cells (8.53%) (Fig. 7C). Moreover, through neutralization of IL-1β, the A2E-laden ARPE19 recovered suppressive function on Th1-cell differentiation (2.8%), which indicated the essential role of IL-1β in A2E-mediated inflammation (Fig. 7D). 
The A2E Induced Inflammatory Status and ROS Production in ARPE19 Cells
We analyzed inflammatory status of A2E-laden ARPE19 cells and found that the mRNA levels of IL-1β, IL-12b, IL-18, VEGFa, and NLRP3 in A2E-laden ARPE19 cells, and the cytokine levels of IL-1β, IL-12b, IL-18, and IFN-γ in supernatant were increased compared with ARPE19 cells without stimulation (Figs. 8A, 8B). However, A2E inhibited PGE2 expression in ARPE19 cells (Fig. 8C). We also found that A2E increased ROS levels in ARPE19 cells in a dose-dependent manner (Figs. 8D, 8E). 
Figure 8
 
The A2E induced inflammatory status of ARPE19 cells and ROS production in ARPE19 cells. (A) Intracellular mRNA levels in ARPE19 cells stimulated without or with 10 μM A2E for 6 hours were analyzed by RT-qPCR. (B, C) Cytokines and PGE2 in ARPE19 or A2E-laden ARPE19 supernatants were detected by ELISA. (D, E) The ARPE19 cells were stimulated with different doses of A2E (0, 2, 10, 25 μM) for 6 hours. Levels of ROS were detected by flow cytometer, and mean fluorescence intensity was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Figure 8
 
The A2E induced inflammatory status of ARPE19 cells and ROS production in ARPE19 cells. (A) Intracellular mRNA levels in ARPE19 cells stimulated without or with 10 μM A2E for 6 hours were analyzed by RT-qPCR. (B, C) Cytokines and PGE2 in ARPE19 or A2E-laden ARPE19 supernatants were detected by ELISA. (D, E) The ARPE19 cells were stimulated with different doses of A2E (0, 2, 10, 25 μM) for 6 hours. Levels of ROS were detected by flow cytometer, and mean fluorescence intensity was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Discussion
Previous reports indicated that both T cells and macrophages were involved in pathological progression of AMD.40 But inhibiting the effect of macrophage (M1) was not sufficient for prevention GA.41 Moreover, T-cell subsets could modulate macrophage polarization (such as Th1, which is related to M1 immune response).42 So, in this study we researched the effects of RPE cells in modulating T-cell immunity in the immune pathological progression of AMD through in vitro cell models. 
As an important vitamin A dimer, A2E accumulation was one of the early events in RPE degeneration.26,43 Although the unrelated distribution of A2E with AMD lesions in situ in AMD patients has been previously reported,44 it was also recognized that A2E, as a main product of vitamin A dimer, was the essential early biomarker of RPE damage and AMD development.43 So, in this study, we used A2E as a risk factor of AMD to induce oxidative damage of RPE cells. It was found that A2E could induce ROS both in mice and human ARPE19 cells. Previous research also indicated that A2E and its precursor all-trans retinal could generate ROS inside the RPE cells.4547 The possible mechanisms were related to the C/EBP homologous protein (CHOP) and GRP18 pathways32 and cell damage.48 Moreover, oxidative stress and inflammation are interrelated.49 It was reported that ROS induced by A2E in ARPE19 could promote inflammation.50,51 A recent report indicated that A2E could induce AMD-like lesions through inflammation in an animal model, thereby reinforcing the correlation of A2E, AMD, and inflammation.29 
We have shown that Th1 cells are the main T-cell subsets modulated by RPE cells under A2E. It was previously reported that Th1 cells played essential roles in the development of AMD in a CEP-induced dry AMD model.17 In patients, the expression of Th1-related cytokines, such as IL-1β and IL-18, were also related to the development of dry AMD.14,15 However, the exact T-cell subset immunity in AMD was not clear. Our findings support the previous finding that Th1 immunity was present in AMD. Moreover, our data also show that A2E mainly influences Th1 immunity but not Th17 cells. This result might be controversial with previous reports that IL-17 was essential in the development of AMD.5,52 That might be due to other factors of adaptive immunity, which might also relate to AMD. Recent reports have found that the main source of IL-17 in the development of AMD was from γδT cells rather than Th17 cells.53 
We found that A2E-laden RPE cells expressed a higher level of proinflammatory factors (IL-1β, IL-12b, and IL-18), which was reported increased in ARPE19 cells when stimulated by A2E.28 It was previously reported that ARPE19 cells could produce inflammatory cytokines under the stimulation of TNF-α or IL-1β, through ERK1/2 and JNK pathways,54,55 which indicate possible methods to control inflammatory retinal diseases by inhibiting these inflammatory factors and pathways.55,56 Moreover, previous study indicated that these cytokines were related to Th1 differentiation.36 We found that neutralization of IL-1β inhibited A2E-laden RPE cell function on Th1-cell differentiation. This finding indicated that A2E-induced RPE inflammation was mediated by IL-1β. Therefore, anti-inflammation therapy might be used to reverse the early development of AMD. It was partly verified in the latest research on anti-inflammatory therapy with nucleoside reverse transcriptase inhibitors (NRTIs) in reversing early AMD development in models.57 
We also found that A2E reduced PGE2 production and inhibited COX2-PGE2 pathways in RPE cells. It was previously reported that PGE2 mediated the regulatory function of RPE cells in inflammatory T cells through downregulated CCL4.39 Moreover, the COX2-PGE2 pathways also contribute to the immunoregulatory function of MSCs or regulatory macrophages in modulating other inflammatory diseases.37,38 On the other hand, however, PGE2 and COX2 were thought to be the mediators of CNV; in the LA-induced RPE damage, the expression of NO and PGE2, together with their precursors COX2 and iNOS, were found increasing in a CNV model.58,59 The controversy about the proinflammatory and immune modulatory functions of COX2-PGE2 pathways in AMD might be related to the complex pathological feature of different AMD lesions (CNV or GA), just as the paradox roles of IL-18 in CNV and GA. Recent research has indicated that IL-18 is correlated with the development of GA,13 whereas IL-18 could inhibit the formation of CNV in a laser-induced model.59 In our research, we found that A2E could inhibit COX2-PGE2 pathways to impede the suppressive function of RPE cells in Th1-cell differentiation. This finding indicated that COX2-PGE2 pathways mediated regulatory function of RPE cells in Th1 immunity in vitro. 
In conclusion, this finding indicates the essential role of Th1 immunity in pathological progression of AMD in vitro, and suggests potential therapeutic use of inhibitors of cytokines and pathways of Th1 immunity to attenuate AMD. Although the further pathological mechanisms and the direct relationship of Th1 cells and RPE cells in AMD in situ and in patients need to be further illuminated so as to develop effective clinical therapy. 
Acknowledgments
We thank Yu Chen, PhD (Departments of Pharmacology and Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA), for her valuable suggestion of the manuscript and project. 
Supported by the National Natural Science Foundation of China (Grants 81425006, 81200062, 81271030, 81273263, 81202332, and 81470393), the National Basic Research Program of China “973 Program” (2011CB707506), Shanghai Scholar Leadership Grant (XBR2013081), Shanghai Creative Key Medical Research Grant (1341195400), and Science and Technology Commission of Shanghai Municipality (12ZR1447900). 
Disclosure: Q. Shi, None; Q. Wang, None; J. Li, None; X. Zhou, None; H. Fan, None; F. Wang, None; H. Liu, None; X. Sun, None; X. Sun, None 
References
Javitt JC, Zhou Z, Maguire MG, Fine SL, Willke RJ. Incidence of exudative age-related macular degeneration among elderly Americans. Ophthalmology. 2003; 110: 1534–1539.
Augood CA, Vingerling JR, de Jong PT, et al. Prevalence of age-related maculopathy in older Europeans: the European Eye Study (EUREYE). Arch Ophthalmol. 2006; 124: 529–535.
Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med. 2008; 358: 2606–2617.
de Jong PT. Age-related macular degeneration. N Engl J Med. 2006; 355: 1474–1485.
Ambati J, Atkinson JP, Gelfand BD. Immunology of age-related macular degeneration. Nat Rev Immunol. 2013; 13: 438–451.
Chen Y, Bedell M, Zhang K. Age-related macular degeneration: genetic and environmental factors of disease. Mol Interv. 2010; 10: 271–281.
Khandhadia S, Cipriani V, Yates JR, Lotery AJ. Age-related macular degeneration and the complement system. Immunobiology. 2012; 217: 127–146.
Edwards AO, Ritter RR, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration. Science. 2005; 308: 421–424.
Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005; 308: 385–389.
Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005; 308: 419–421.
Combadiere C, Feumi C, Raoul W, et al. CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration. J Clin Invest. 2007; 117: 2920–2928.
Hollyfield JG, Bonilha VL, Rayborn ME, et al. Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat Med. 2008; 14: 194–198.
Doyle SL, Campbell M, Ozaki E, et al. NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components. Nat Med. 2012; 18: 791–798.
Ezzat MK, Hann CR, Vuk-Pavlovic S, Pulido JS. Immune cells in the human choroid. Br J Ophthalmol. 2008; 92: 976–980.
Faber C, Singh A, Kruger FM, Juel HB, Sorensen TL, Nissen MH. Age-related macular degeneration is associated with increased proportion of CD56(+) T cells in peripheral blood. Ophthalmology. 2013; 120: 2310–2316.
Hasegawa E, Sonoda KH, Shichita T, et al. IL-23-independent induction of IL-17 from gammadeltaT cells and innate lymphoid cells promotes experimental intraocular neovascularization. J Immunol. 2013; 190: 1778–1787.
Cruz-Guilloty F, Saeed AM, Duffort S, et al. T cells and macrophages responding to oxidative damage cooperate in pathogenesis of a mouse model of age-related macular degeneration. PLoS One. 2014; 9: e88201.
Forrester JV, Xu H. Good news-bad news: the Yin and Yang of immune privilege in the eye. Front Immunol. 2012; 3: 338.
Liu B, Wei L, Meyerle C, et al. Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration. J Transl Med. 2011; 9: 1–12.
Sugita S, Horie S, Nakamura O, et al. Retinal pigment epithelium-derived CTLA-2alpha induces TGFbeta-producing T regulatory cells. J Immunol. 2008; 181: 7525–7536.
Kawazoe Y, Sugita S, Keino H, et al. Retinoic acid from retinal pigment epithelium induces T regulatory cells. Exp Eye Res. 2012; 94: 32–40.
Sugita S, Horie S, Yamada Y, Kawazoe Y, Takase H, Mochizuki M. Suppression of interleukin-17-producing T-helper 17 cells by retinal pigment epithelial cells. Jpn J Ophthalmol. 2011; 55: 565–575.
Sugita S, Kawazoe Y, Imai A, Usui Y, Takahashi M, Mochizuki M. Suppression of IL-22-producing T helper 22 cells by RPE cells via PD-L1/PD-1 interactions. Invest Ophthalmol Vis Sci. 2013; 54: 6926–6933.
Nathan C, Cunningham-Bussel A. Beyond oxidative stress: an immunologist's guide to reactive oxygen species. Nat Rev Immunol. 2013; 13: 349–361.
Sparrow JR, Zhou J, Ben-Shabat S, Vollmer H, Itagaki Y, Nakanishi K. Involvement of oxidative mechanisms in blue-light-induced damage to A2E-laden RPE. Invest Ophthalmol Vis Sci. 2002; 43: 1222–1227.
Wu Y, Yanase E, Feng X, Siegel MM, Sparrow JR. Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration. Proc Natl Acad Sci U S A. 2010; 107: 7275–7280.
Karan G, Lillo C, Yang Z, et al. Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: a model for macular degeneration. Proc Natl Acad Sci U S A. 2005; 102: 4164–4169.
Anderson OA, Finkelstein A, Shima DT. A2E induces IL-1ss production in retinal pigment epithelial cells via the NLRP3 inflammasome. PLoS One. 2013; 8: e67263.
Penn J, Mihai DM, Morphological Washington I. and physiological retinal degeneration induced by intravenous delivery of vitamin A dimers in rabbits. Dis Model Mech. 2015; 8: 131–138.
Liu J, Zhou X, Zhan Z, et al. IL-25 regulates the polarization of macrophages and attenuates obliterative bronchiolitis in murine trachea transplantation models. Int Immunopharmacol. 2015; 25: 383–392.
Sugita S, Streilein JW. Iris pigment epithelium expressing CD86 (B7-2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2003; 198: 161–171.
Feng J, Chen X, Sun X, Wang F, Sun X. Expression of endoplasmic reticulum stress markers GRP78 and CHOP induced by oxidative stress in blue light-mediated damage of A2E-containing retinal pigment epithelium cells. Ophthalmic Res. 2014; 52: 224–233.
Parish CA, Hashimoto M, Nakanishi K, Dillon J, Sparrow J. Isolation and one-step preparation of A2E and iso-A2E fluorophores from human retinal pigment epithelium. Proc Natl Acad Sci U S A. 1998; 95: 14609–14613.
Cohen CJ, Crome SQ, MacDonald KG, Dai EL, Mager DL, Levings MK. Human Th1 and Th17 cells exhibit epigenetic stability at signature cytokine and transcription factor loci. J Immunol. 2011; 187: 5615–5626.
Lu L, Zhou X, Wang J, Zheng SG, Horwitz DA. Characterization of protective human CD4CD25 FOXP3 regulatory T cells generated with IL-2 TGF-beta and retinoic acid. PLoS One. 2010; 5: e15150.
Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol. 2010; 10: 89–102.
Nemeth K, Leelahavanichkul A, Yuen PS, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009; 15: 42–49.
Sreeramkumar V, Fresno M, Cuesta N. Prostaglandin E2 and T cells: friends or foes? Immunol Cell Biol. 2012; 90: 579–586.
Wallace CA, Moir G, Malone DF, Duncan L, Devarajan G, Crane IJ. Regulation of T-lymphocyte CCL3 and CCL4 production by retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2013; 54: 722–730.
Cao X, Shen D, Patel MM, et al. Macrophage polarization in the maculae of age-related macular degeneration: a pilot study. Pathol Int. 2011; 61: 528–535.
Espinosa-Heidmann DG, Suner IJ, Hernandez EP, Monroy D, Csaky KG, Cousins SW. Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003; 44: 3586–3592.
Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010; 11: 889–896.
Mihai DM, Washington I. Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells. Cell Death Dis. 2014; 5: e1348.
Ablonczy Z, Higbee D, Anderson DM, et al. Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2013; 54: 5535–5542.
Sparrow JR, Nakanishi K, Parish CA. The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci. 2000; 41: 1981–1989.
Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathobiology. Exp Eye Res. 2005; 80: 595–606.
Li J, Cai X, Xia Q, et al. Involvement of endoplasmic reticulum stress in all-trans-retinal-induced retinal pigment epithelium degeneration. Toxicol Sci. 2015; 143: 196–208.
Bazan NG. Survival signaling in retinal pigment epithelial cells in response to oxidative stress: significance in retinal degenerations. Adv Exp Med Biol. 2006; 572: 531–540.
Bian Q, Gao S, Zhou J, et al. Lutein and zeaxanthin supplementation reduces photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells. Free Radic Biol Med. 2012; 53: 1298–1307.
Fernandes AF, Zhou J, Zhang X, et al. Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8. J Biol Chem. 2008; 283: 20745–20753.
Cao X, Liu M, Tuo J, Shen D, Chan CC. The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice. Exp Eye Res. 2010; 91: 15–25.
Liu B, Wei L, Meyerle C, et al. Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration. J Transl Med. 2011; 9: 1–12.
Zhao Z, Xu P, Jie Z, et al. Gammadelta T cells as a major source of IL-17 production during age-dependent RPE degeneration. Invest Ophthalmol Vis Sci. 2014; 55: 6580–6589.
Wang Q, Qi J, Hu R, Chen Y, Kijlstra A, Yang P. Effect of berberine on proinflammatory cytokine production by ARPE-19 cells following stimulation with tumor necrosis factor-alpha. Invest Ophthalmol Vis Sci. 2012; 53: 2395–2402.
Qiao Y, Hu R, Wang Q, et al. Sphingosine 1-phosphate elicits proinflammatory responses in ARPE-19 cells. Invest Ophthalmol Vis Sci. 2012; 53: 8200–8207.
Watanabe K, Zhang XY, Kitagawa K, Yunoki T, Hayashi A. The effect of clonidine on VEGF expression in human retinal pigment epithelial cells (ARPE-19). Graefes Arch Clin Exp Ophthalmol. 2009; 247: 207–213.
Fowler BJ, Gelfand BD, Kim Y, et al. Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity. Science. 2014; 346: 1000–1003.
Fang IM, Yang CH, Yang CM, Chen MS. Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells. Exp Eye Res. 2007; 85: 667–677.
Doyle SL, Ozaki E, Brennan K, et al. IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration. Sci Transl Med. 2014; 6:230ra44.
Figure 1
 
The A2E suppressed immunoregulatory function of RPE cells in inducing Tregs. (A) The CD4+CD25CD44low naïve T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of IL-2 (50 U/mL) alone (T med) or both TGF-β1 (20 ng/mL) and IL-2 (T IL-2+TGF-β), or co-cultured with RPE (T RPE) or A2E-laden RPE (T A2E-RPE) for 4 days. Percentages of induced Foxp3+ cell subsets were analyzed by flow cytometer. (B) Induced T cells were co-cultured with CFSE+CD4+T responder cells at a ratio of 10:1(T:RPE) under the stimulation of coated anti-CD3/CD28(2 μg/mL) for 4 days and the CFSE dilution was analyzed by flow cytometer. Gating on CD4+T cells, similar results were obtained in three independent experiments.
Figure 1
 
The A2E suppressed immunoregulatory function of RPE cells in inducing Tregs. (A) The CD4+CD25CD44low naïve T cells were stimulated with anti-CD3/anti-CD28 antibodies in the presence of IL-2 (50 U/mL) alone (T med) or both TGF-β1 (20 ng/mL) and IL-2 (T IL-2+TGF-β), or co-cultured with RPE (T RPE) or A2E-laden RPE (T A2E-RPE) for 4 days. Percentages of induced Foxp3+ cell subsets were analyzed by flow cytometer. (B) Induced T cells were co-cultured with CFSE+CD4+T responder cells at a ratio of 10:1(T:RPE) under the stimulation of coated anti-CD3/CD28(2 μg/mL) for 4 days and the CFSE dilution was analyzed by flow cytometer. Gating on CD4+T cells, similar results were obtained in three independent experiments.
Figure 2
 
The A2E-laden RPE cells promoted Th1 differentiation. (A) Retinal pigment epithelial cells stimulated without (control) or with A2E 10 μM for 6 hours were co-cultured with naïve T cells under the stimulation of coated anti-CD3/CD28 and different T-cell subset polarization conditions for 4 days. Intracellular cytokines (IL-17 for Th17, IFN-γ for Th1, and IL-4 for Th2) were analyzed by flow cytometer; three independent experiments revealed similar results. The percentages of cytokine-positive cells in CD4+ cells were analyzed in (B). (C) The transcript factors or intracellular cytokines of cultured T cells were analyzed by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Figure 2
 
The A2E-laden RPE cells promoted Th1 differentiation. (A) Retinal pigment epithelial cells stimulated without (control) or with A2E 10 μM for 6 hours were co-cultured with naïve T cells under the stimulation of coated anti-CD3/CD28 and different T-cell subset polarization conditions for 4 days. Intracellular cytokines (IL-17 for Th17, IFN-γ for Th1, and IL-4 for Th2) were analyzed by flow cytometer; three independent experiments revealed similar results. The percentages of cytokine-positive cells in CD4+ cells were analyzed in (B). (C) The transcript factors or intracellular cytokines of cultured T cells were analyzed by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Figure 3
 
The A2E inhibited the immunosuppression effect of RPE cells on Th1 cell differentiation rather than Th1 cell proliferation. The A2E-laden RPE or normal RPE cells were co-cultured with CFSE-stained CD4+T responder cells under the stimulation of bead-coated anti-CD3/CD28 in Th1 or Th17 cell polarization conditions for 4 days; IFN-γ+ Th1 cells (A) and IL-17+ Th17 cells (B) were analyzed by flow cytometer.
Figure 3
 
The A2E inhibited the immunosuppression effect of RPE cells on Th1 cell differentiation rather than Th1 cell proliferation. The A2E-laden RPE or normal RPE cells were co-cultured with CFSE-stained CD4+T responder cells under the stimulation of bead-coated anti-CD3/CD28 in Th1 or Th17 cell polarization conditions for 4 days; IFN-γ+ Th1 cells (A) and IL-17+ Th17 cells (B) were analyzed by flow cytometer.
Figure 4
 
The A2E-induced RPE produces inflammatory factors. Retinal pigment epithelial cells were stimulated with or without 10 μM A2E for 6 hours; the mRNA levels of cytokines or related factors in RPE were detected with RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
 
The A2E-induced RPE produces inflammatory factors. Retinal pigment epithelial cells were stimulated with or without 10 μM A2E for 6 hours; the mRNA levels of cytokines or related factors in RPE were detected with RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
 
The A2E modulated Th1 cell differentiation through IL-1β. The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days, with or without anti–IL-1β antibody; percentage of IFN-γ+ T cells were analyzed by flow cytometer.
Figure 5
 
The A2E modulated Th1 cell differentiation through IL-1β. The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days, with or without anti–IL-1β antibody; percentage of IFN-γ+ T cells were analyzed by flow cytometer.
Figure 6
 
The A2E inhibited PGE2, which mediated RPE cell regulatory function on Th1 cell differentiation. (A) Retinal pigment epithelial cells were stimulated with 10 μM A2E for 6 hours, supernatants were collected, and PGE2 and cytokines (IL-1β, IL-18, and IL-12b) were detected by ELISA. (B) Retinal pigment epithelial cells were stimulated with A2E 10 μM; the cells were collected at different times and COX2 expression was detected by Western blot. (C, D) The Th1 cells were induced alone or co-cultured with RPE or A2E-laden RPE cells with or without inhibitors for 4 days; expression of intracellular IFN-γ in T cells was detected by flow cytometer. (E, F) Levels of intracellular ROS in RPE with or without stimulation by 10 μM A2E for 6 hours were measured by flow cytometer. *P < 0.05, **P < 0.01, ns P > 0.05.
Figure 6
 
The A2E inhibited PGE2, which mediated RPE cell regulatory function on Th1 cell differentiation. (A) Retinal pigment epithelial cells were stimulated with 10 μM A2E for 6 hours, supernatants were collected, and PGE2 and cytokines (IL-1β, IL-18, and IL-12b) were detected by ELISA. (B) Retinal pigment epithelial cells were stimulated with A2E 10 μM; the cells were collected at different times and COX2 expression was detected by Western blot. (C, D) The Th1 cells were induced alone or co-cultured with RPE or A2E-laden RPE cells with or without inhibitors for 4 days; expression of intracellular IFN-γ in T cells was detected by flow cytometer. (E, F) Levels of intracellular ROS in RPE with or without stimulation by 10 μM A2E for 6 hours were measured by flow cytometer. *P < 0.05, **P < 0.01, ns P > 0.05.
Figure 7
 
The A2E suppressed immunoregulatory function of ARPE19 cells in Tregs and Th1 differentiation through IL-1β. (A, B) The ARPE19 cells stimulated with or without 10 μM A2E for 6 hours were co-cultured with naïve T cells for 4 days. Percentages of induced FOXP3+ T cells and suppressive function of these induced T cells in the CFSE+ responder T cells co-culture system were analyzed by flow cytometer. (C) The ARPE19 or A2E-laden ARPE19 cells were co-cultured with naïve T cells in Th1 or Th17 polarization conditions for 4 days. (D) The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days with anti–IL-1β antibody. Intracellular cytokines were analyzed by flow cytometer.
Figure 7
 
The A2E suppressed immunoregulatory function of ARPE19 cells in Tregs and Th1 differentiation through IL-1β. (A, B) The ARPE19 cells stimulated with or without 10 μM A2E for 6 hours were co-cultured with naïve T cells for 4 days. Percentages of induced FOXP3+ T cells and suppressive function of these induced T cells in the CFSE+ responder T cells co-culture system were analyzed by flow cytometer. (C) The ARPE19 or A2E-laden ARPE19 cells were co-cultured with naïve T cells in Th1 or Th17 polarization conditions for 4 days. (D) The T cells induced in the Th1 polarization condition were cultured alone or with A2E-laden RPE for 4 days with anti–IL-1β antibody. Intracellular cytokines were analyzed by flow cytometer.
Figure 8
 
The A2E induced inflammatory status of ARPE19 cells and ROS production in ARPE19 cells. (A) Intracellular mRNA levels in ARPE19 cells stimulated without or with 10 μM A2E for 6 hours were analyzed by RT-qPCR. (B, C) Cytokines and PGE2 in ARPE19 or A2E-laden ARPE19 supernatants were detected by ELISA. (D, E) The ARPE19 cells were stimulated with different doses of A2E (0, 2, 10, 25 μM) for 6 hours. Levels of ROS were detected by flow cytometer, and mean fluorescence intensity was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
Figure 8
 
The A2E induced inflammatory status of ARPE19 cells and ROS production in ARPE19 cells. (A) Intracellular mRNA levels in ARPE19 cells stimulated without or with 10 μM A2E for 6 hours were analyzed by RT-qPCR. (B, C) Cytokines and PGE2 in ARPE19 or A2E-laden ARPE19 supernatants were detected by ELISA. (D, E) The ARPE19 cells were stimulated with different doses of A2E (0, 2, 10, 25 μM) for 6 hours. Levels of ROS were detected by flow cytometer, and mean fluorescence intensity was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×