Effects of ω-3 LCPUFAs and lutein on the development of CNV.

<p>(A) The area of CNV at 7 days after laser photocoagulation was determined by staining of choroidal flat-mount preparations with Alexa Fluor 488–conjugated <i>G</i>. <i>simplicifolia</i> isolectin B4 for mice in the control group (<i>n</i> = 35 lesions), the ω-3 group (<i>n</i> = 23 lesions), the lutein group (<i>n</i> = 34 lesions), and the ω-3 + lutein group (<i>n</i> = 33 lesions). Data are means ± SEM. *<i>P</i> < 0.05, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001 (Tukey’s multiple comparison test). NS, not significant. The data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196037#pone.0196037.g002" target="_blank">Fig 2A</a> are from one of the three experiments performed. (<b>B</b>) Representative staining of CNV lesions for mice in the four groups. Scale bar, 100 μm.</p

Effects of ω-3 LCPUFAs and lutein on the concentrations of representative proinflammatory cytokines and chemokines in the choroid.

<p>The concentrations of IL-1β (<b>A</b>), IL-12 (p40) (<b>B</b>), TNF-α (<b>C</b>), G-CSF (<b>D</b>), MCP-1 (<b>E</b>), MIP-1α (<b>F</b>), MIP-1β (<b>G</b>), and RANTES (<b>H</b>) in the choroid of mice in the four experimental groups were determined with a multiplex assay at 7 days after CNV induction. Data are means ± SEM of triplicate determinations for a pooled sample and are the same as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196037#pone.0196037.s002" target="_blank">S2 Fig</a>. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001 versus the control group (Dunnett’s test). ND, not detected.</p

Effects of ω-3 LCPUFAs and lutein on Nox4 expression in the choroid-retina.

<p>(<b>A</b>) Representative images of Nox4 immunofluorescence (blue) in RPE-choroid sections prepared from mice of the four experimental groups at 7 days after laser photocoagulation. Red ovals enclose the lesion area. Scale bar, 100 μm. (<b>B</b>) The amount of Nox4 in the retina isolated from mice of the four experimental groups at 7 days after laser photocoagulation was determined by densitometric scanning of immunoblots. Data were normalized by the abundance of β-actin and are means ± SEM of triplicate determinations for a pooled sample. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group. (<b>C</b>) Representative immunoblot of Nox4 and β-actin.</p

Effects of ω-3 LCPUFAs and lutein on ROS levels in RPE-choroid sections.

<p>(<b>A</b>) RPE-choroid sections prepared from mice in the four experimental groups at 5 days after laser photocoagulation were stained with DHE for detection of ROS. In lower panels, phase image was taken of corresponding DHE-stained images in upper panel, respectively. Scale bar, 100 μm. (<b>B</b>) Integrated density for DHE staining in the CNV area was measured with Image J software. Data are means ± SEM for four determinations of leasion of each experimental group. *<i>P</i> < 0.05 versus the control group (Dunnett’s test).</p

Experimental design.

<p>Mice maintained on a normal diet up to 6 weeks of age were then fed a diet either supplemented with ω-3 LCPUFAs (<i>n</i> = 80) or free of ω-3 LCPUFAs (<i>n</i> = 80) beginning 2 weeks before the induction of CNV. Half of the mice on each diet received lutein daily via an oral gavage needle beginning 1 week before the induction of CNV. The animals were killed 7 days after laser photocoagulation for measurement of CNV size (<i>n</i> = 15 for each group [<i>n</i> = 5 for each of three separate experiments), serum fatty acid and lutein concentrations (<i>n</i> = 5 for each group from among the 15 mice examined for measurement of CNV size, and one sample was used for the calibration of gas chromatography), and cytokine and chemokine concentrations in the retina and choroid (<i>n</i> = 5 for each group) as well as for immunoblot analysis of Nox4 (<i>n</i> = 5 for each group) and immunohistofluorescence analysis of Nox4 (<i>n</i> = 5 for each group). For detection of ROS (<i>n</i> = 10 for each group [<i>n</i> = 5 for each of two separate experiments]), the animals were killed 5 days after laser photocoagulation.</p

Dietary Omega-3 Fatty Acids Suppress Experimental Autoimmune Uveitis in Association with Inhibition of Th1 and Th17 Cell Function

<div><p>Omega (ω)–3 long-chain polyunsaturated fatty acids (LCPUFAs) inhibit the production of inflammatory mediators and thereby contribute to the regulation of inflammation. Experimental autoimmune uveitis (EAU) is a well-established animal model of autoimmune retinal inflammation. To investigate the potential effects of dietary intake of ω-3 LCPUFAs on uveitis, we examined the anti-inflammatory properties of these molecules in comparison with ω-6 LCPUFAs in a mouse EAU model. C57BL/6 mice were fed a diet containing ω-3 LCPUFAs or ω-6 LCPUFAs for 2 weeks before as well as after the induction of EAU by subcutaneous injection of a fragment of human interphotoreceptor retinoid-binding protein emulsified with complete Freund’s adjuvant. Both clinical and histological scores for uveitis were smaller for mice fed ω-3 LCPUFAs than for those fed ω-6 LCPUFAs. The concentrations of the T helper 1 (Th1) cytokine interferon-γ and the Th17 cytokine interleukin-17 in intraocular fluid as well as the production of these cytokines by lymph node cells were reduced for mice fed ω-3 LCPUFAs. Furthermore, the amounts of mRNAs for the Th1- and Th17-related transcription factors T-bet and RORγt, respectively, were reduced both in the retina and in lymph node cells of mice fed ω-3 LCPUFAs. Our results thus show that a diet enriched in ω-3 LCPUFAs suppressed uveitis in mice in association with inhibition of Th1 and Th17 cell function.</p></div

Abundance of T-bet and RORγt mRNAs in the retina of EAU mice.

<p>The amounts of T-bet (A) and RORγt (B) mRNAs in the retina of EAU mice were measured by RT and real-time PCR analysis at 21 days after injection of hIRBP(1–20) and maintenance on an ω-6 or ω-3 LCPUFA diet. Data are means ± SD (<i>n</i> = 2 for both ω-6 and ω-3 groups). **<i>P</i> < 0.01.</p

Production of IFN-γ and IL-17 by CD4⁺ T cells isolated from EAU mice.

<p>CD4⁺ T cells isolated from lymph nodes of EAU mice at 14 days after injection of hIRBP(1–20) and maintenance on an ω-6 or ω-3 LCPUFA diet were incubated in the absence or presence of hIRBP(1–20) for 48 h, after which the concentrations of IFN-γ (A) and IL-17 (B) in the culture supernatants were measured with ELISAs. The cytokine concentrations for the stimulated cells are presented as means ± SD (<i>n</i> = 4 for both ω-6 and ω-3 groups). ***<i>P</i> < 0.001.</p

Fatty acid concentrations (ng/ml) in serum of EAU mice.

<p>Fatty acid concentrations in serum of EAU mice were measured at 14 days after injection of hIRBP(1–20) and maintenance on a control, ω-6 LCPUFA, or ω-3 LCPUFA diet. Values in parentheses represent the percentage of total fatty acids by weight. Data are means (<i>n</i> = 5). PA, palmitic acid; SA, stearic acid; LA, linoleic acid; AA, arachidonic acid; DTA, docosatetraenoic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.</p><p>Fatty acid concentrations (ng/ml) in serum of EAU mice.</p

Concentrations of inflammatory cytokines and chemokines in intraocular fluid of EAU mice.

<p>The concentrations of IL-1β (A), IL-6 (B), TNF-α (C), MCP-1 (D), RANTES (E), IFN-γ (F), IL-17A (G), and IL-12(p40) (H) in intraocular fluid of EAU mice were determined with a multiplex assay at 14 days after injection of hIRBP(1–20) and maintenance on an ω-6 or ω-3 LCPUFA diet. Data are means ± SD (<i>n</i> = 10 for both ω-6 and ω-3 groups). ***<i>P</i> < 0.001, **<i>P</i> < 0.01 versus ω-6 mice. ND, not detected.</p